1 
 
                DEPARTMENT OF HEALTH AND HUMAN SERVICES 
 
                      FOOD AND DRUG ADMINISTRATION 
 
             CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

           BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE  
 
                XENOTRANSPLANTATION SUBCOMMITTEE MEETING

                      Wednesday, December 17, 1997 
 
                             9 o'clock a.m.

                           Double Tree Hotel 
                                Plaza I 
                          1750 Rockville Pike 
                       Rockville, Maryland 20852 

 
                                                                 2 
 
                              PARTICIPANTS 
 
      Hugh Auchincloss, Jr., M.D., Chairperson 
      Gail Dapolito, Executive Secretary 
 
      MEMBERS 
 
      Jonathan S. Allen, D.V.M. 
      John M, Coffin, Ph.D. 
      Ronald C. Desrosier, Ph.D. 
      Martin S. Hirsch, M.D. 
      Richard Kaslow, M.D., M.P.H. 
      Nicholas W. Lerche, D.V.M. 
      Abbey S. Meyers 
      Claudia A. Mickelson, Ph.D. 
      David Onions, BVSC, Ph.D., MRCVS, FRSE 
      Prem S. Paul, D.V.M., Ph.D. 
      Daniel R. Salomon, M.D. 
      Harold Y. Vanderpool, Ph.D., Th.M. 
      Leroy Walters, Ph.D. 
 
      GUESTS 
 
      Antonio Benedi 
      William G.Lawrence, J.D. 
      Marian Michaels, M.D., M.P.H. 
      Robin A. Weiss, Ph.D., FRCPath, FRS 
 
      FDA 
 
      David Feigal, M.D. 
      Philip Noguchi, Ph.D. 
      Any P. Patterson, M.D. 
      Mary K. Pendergast 
      Jay P. Siegel, M.D. 
      Carolyn Wilson, Ph.D. 
      Kathryn C. Zoon, Ph.D. 

 
                                                                 3 
 
                            C O N T E N T S 
 
      Administrative Remarks: William Freas, Ph.D.               5 
 
      Opening Remarks:  
                Kathryn C. Zoon, Ph.D.                          11 
                Mary K. Pendergast                              13 
 
      Open Public Hearing 
 
                Dr. Achilles Demetriou                          18 
                Dr. Barbara Potts                               21 
                Corinne Savill                                  26 
 
      Introduction of Focus Groups and Presentation of 
      Questions for Subcommittee Discussion: 
                Hugh Auchincloss, Jr. M.D.                      32 
 
      Endogenous Retroviruses: An Overview: 
                John Coffin, Ph.D.                              34 
 
      Data Demonstrating Capacity of Porcine Endogenous 
      Retrovirus (PERV) to Infect Human Cells: 
                Carolyn Wilson, Ph.D.                           57 
 
      Data Demonstrating Capacity of PERV to Infect 
      Human Cells: 
                Robin Weiss, Ph.D.                              69 
                David Onions, Ph.D.                             81 
 
      Data from Clinical Studies 
      Diacrin/Genzyme Assay and Clinical Data: 
                Michael Egan, Alan Moore and Dr. Steven Fink   101

      Circe Biomedical Assay and Clinical Data: 
                Barry Solomon, Ph.D.                           120 
 
      CDC Collaborations and Surveillance: 
                Walid Heneine, Ph.D.                           136 
 
      Subcommittee Discussion of Presentations  
 
      Open Public Hearing (Continued) 
                Marlon F. Levy, M.D.                           147 
 
      Presentation of Focus Group Analysis of Question 1 
      Development of Strategies for the Detection of  
      Porcine Endogenous Retrovirus: 
                John M. Coffin, Ph.D.                          150 
 
      Discussion of Question 1 by Subcommittee                 172 

 
                                                                 4 
 
                       C O N T E N T S(Continued) 
 
      Presentation of Focus Group Analysis of Question 1 
      Development of Programs for Monitoring and  
      Clinical Follow up: 
                Claudia A. Mickelson, Ph.D.                    240

      Summary Points by Subcommittee Chair:                    304 
                Hugh Auchincloss, Jr., M.D. 
 
      Adjournment                                              305 

 
                                                                 5 
 
  1                      P R O C E E D I N G S

  2                      Administrative Remarks 
 
  3             DR. FREAS:  Good morning.  I am Bill Freas.  I am 
 
  4   the acting chief of the CBER advisory committee program.  I 
 
  5   would like to welcome all of you here to this meeting this 
 
  6   morning.

  7             The entire meeting today will be open to the 
 
  8   public and everyone is welcome to attend.  I would like to 
 
  9   explain to those present in the audience that, despite the 
 
 10   hard work of the organizer of this meeting today, Dr. Amy 
 
 11   Patterson, she tried to make it an official advisory

 12   committee meeting. 
 
 13             However, to charter and establish a full advisory 
 
 14   committee takes a considerable period of time.  Therefore, 
 
 15   today's meeting will be conducted as a subcommittee meeting 
 
 16   of the Biological Response Modifiers Advisory Committee. 

 17   This will be a subcommittee on xenotransplantation.  This 
 
 18   was listed in the Federal Register Notice. 
 
 19             There are two notable differences between a 
 
 20   subcommittee meeting and a full advisory committee meeting, 
 
 21   and they are that there are only two standing advisory

 22   committee members present at the table today.  As I go 
 
 23   around and introduce the members at the table, I will point 
 
 24   them out to you. 
 
 25             The next difference is the report that will be 

 
                                                                 6 
 
  1   furnished today by the subcommittee will be approved by the

  2   parent committee, the Biological Response Modifiers Advisory 
 
  3   Committee.  Other than that, you will not be able to tell 
 
  4   any difference between a subcommittee meeting and a full 
 
  5   advisory committee meeting. 
 
  6             At this time, I would like to go around the table

  7   and introduce to you the members seated at the table.  Would 
 
  8   the subcommittee members please raise their hand as their 
 
  9   name is called so the people in the audience can identify 
 
 10   you. 
 
 11             Starting at the end of the table on the audience's

 12   right‑hand side is Dr. Prem Paul, Associate Dean for 
 
 13   Research and Graduate Studies, Iowa State University.  Next 
 
 14   is Dr. Ronald Desrosier, Professor of Microbiology and 
 
 15   Molecular Genetics, Harvard Medical School.  Next is Dr. 
 
 16   Richard Kaslow, Professor of Epidemiology, Medicine and

 17   Microbiology, University of Alabama at Birmingham. 
 
 18             Next is Dr. Nicholas Lerche, Associate Professor, 
 
 19   California Regional Primate Research Center, Davis.  Next is 
 
 20   Dr. Martin Hirsch, Professor of Medicine, Harvard Medical 
 
 21   School.  Next is Dr. Jonathan Allan, Adjunct Associate

 22   Professor, University of Texas Health Science Center.  Next 
 
 23   is Dr. David Onions, Professor of Veterinary Pathology, 
 
 24   University of Glasgow. 
 
 25             Next is Dr. Harold Vanderpool, Professor of 

 
                                                                 7 
 
  1   Preventive Medicine and Community Health, University of

  2   Texas.  Next is Dr. Claudia Mickelson, Director, Biosafety 
 
  3   Office of Massachusetts Institute of Technology.  Next is 
 
  4   Dr. Leroy Walters, Director, Kennedy Institute of Ethics, 
 
  5   Georgetown University. 
 
  6             Next is our Chairman and representative from the

  7   Biological Response Modifiers Advisory Committee, Dr. Hugh 
 
  8   Auchincloss, Associate Professor of Surgery, Harvard Medical 
 
  9   School.  Next is the Executive Secretary for this Committee, 
 
 10   or the designated federal official, Gail Dapolito. 
 
 11             Next is Dr. John Coffin, Professor of Molecular

 12   Biology, Tufts University School of Medicine.  Next is Dr. 
 
 13   Daniel Salomon, Director of Transplantation Research and 
 
 14   Graduate Studies, the Scripps Research Institute.  Next is 
 
 15   Abbey Meyers, President and Executive Director, National 
 
 16   Organization for Rare Diseases, New Fairfield, Connecticut.

 17             Also seated at the table this morning are several 
 
 18   FDAers who are here to coordinate presentations this 
 
 19   morning.  They are Dr. Kathy Zoon, Director, Center for 
 
 20   Biologics Evaluation and Research; Mary Pendergast, Deputy 
 
 21   Commissioner at FDA; Amy Patterson, Medical Reviewer,

 22   Division of Cellular and Gene Therapy; Dr. Jay Siegel, 
 
 23   Director, Office of Therapeutics, Research and Review; and 
 
 24   Dr. Phil Noguchi, Director, Division of Cellular and Gene 
 
 25   Therapy. 

 
                                                                 8 
 
  1             I would like to welcome everyone again.  I would

  2   like to make sure that the members of the audience 
 
  3   understand that Gail Dapolito, the designated federal 
 
  4   official, will be your point of contact should you need to 
 
  5   communicate with members of the committee.  Please do not 
 
  6   directly approach the table but, during a break or during

  7   the lunch period, see the Executive Secretary, Gail 
 
  8   Dapolito, and she will relay your messages to the committee. 
 
  9             I now will read into the public record the 
 
 10   conflict of interest statement for this meeting.  The 
 
 11   following announcement addresses the issue of conflict of

 12   interest issues associated with this meeting and is made a 
 
 13   part of the record to preclude even the appearance of a 
 
 14   conflict of interest at this meeting. 
 
 15             The following announcement is made part of the 
 
 16   public record to preclude even the appearance of a conflict

 17   of interest at this meeting.  Based on the agenda made 
 
 18   available, it has been determined that all financial 
 
 19   interests in firms regulated by the Center for Biologics, 
 
 20   Evaluation and Research which have been reported by the 
 
 21   participating members and consultants, as of this date,

 22   present no potential for an appearance of a conflict of 
 
 23   interest at this meeting. 
 
 24             Based on the agenda made available, it has been 
 
 25   determined that all financial interests in firms regulated 

 
                                                                 9 
 
  1   by the Center for Biologics, Evaluation and Research which

  2   have been reported by the participating members and 
 
  3   consultants as of this date present no potential for an 
 
  4   appearance of a conflict of interest at this meeting with 
 
  5   the following notations to preclude even the appearance of a 
 
  6   conflict.

  7             Dr. Hugh Auchincloss; a waiver was approved by the 
 
  8   agency permitting his full participation in committee 
 
  9   discussions and deliberations.  Dr. Martin Hirsch reported 
 
 10   that he consulted with a regulated firm on virological 
 
 11   issues including testing materials for porcine retroviruses. 

 12   He received a fee for his services. 
 
 13             Dr. David Onions; a waiver was approved by the 
 
 14   agency permitting his participation in the committee 
 
 15   discussions and deliberations.  It was also disclosed that 
 
 16   Dr. Onions reported that he is director of Q‑One Biotech

 17   Limited. Q‑One is negotiating with two regulated firms to 
 
 18   conduct safety testing provided by Sanzino Transplantation. 
 
 19             Dr. Prem Paul disclosed that he consulted with a 
 
 20   regulated firm on retrovirus screening in 
 
 21   xenotransplantation.  He received a fee for his services. 

 22   In addition, he reported a consulting agreement under 
 
 23   negotiation with another regulated firm.  Also, members of 
 
 24   the subcommittee had disclosed consulting relationships with 
 
 25   firms not on the agenda that may, in the future, be 

 
                                                                10 
 
  1   regulated by FDA.  

  2             The following participants have no financial 
 
  3   interests to disclose; they are Mrs. Abbey Meyers, Dr. 
 
  4   Jonathan Allan, Dr. Ronald Desrosiers, Dr. John Coffin, Dr. 
 
  5   Richard Kaslow, Dr. Nicholas Lerche, Dr. Claudia Mickelson, 
 
  6   Dr. Daniel Salomon, Dr. Harold Vanderpool and Dr. Leroy

  7   Walters. 
 
  8             With regards to FDA's invited guests, the agency 
 
  9   has determined that the service of these guests is 
 
 10   essential.  Mr. Anthony Benedi, Dr. Walid Heneine, Mr. 
 
 11   William Lawrence, Dr. Marian Michaels disclosed no financial

 12   interests relevant to today's topics. 
 
 13             The following interest is being made public to 
 
 14   allow the meeting participants to objectively evaluate any 
 
 15   presentation and/or comments made by Dr. Weiss.  Dr. Weiss 
 
 16   reported that he received consultant fees from a regulated

 17   firm. 
 
 18             In the event that the discussions involve other 
 
 19   products or firms not already on the agenda for which FDA 
 
 20   participants have a financial interest, the participants are 
 
 21   aware of the need to exclude themselves from such a

 22   discussion and this exclusion will be noted on the public 
 
 23   record. 
 
 24             A copy of the waivers are available by written 
 
 25   request under the Freedom of Information Act.  With respect 

 
                                                                11 
 
  1   to all other meeting participants, we ask, in the interest

  2   of fairness, that they address any current or previous 
 
  3   financial involvement with any firm upon whose product they 
 
  4   may wish to comment. 
 
  5             So ends the reading of the word. 
 
  6             Dr. Auchincloss, I turn the microphone over to

  7   you. 
 
  8             DR. AUCHINCLOSS:  Thank you, Bill. 
 
  9             We are going to begin today's meeting with some 
 
 10   opening remarks from Dr. Kathryn Zoon.  As you have just 
 
 11   heard, Dr. Zoon is the Director of FDA's Center for

 12   Biologics Evaluation and Research. 
 
 13                         Opening Remarks 
 
 14             DR. ZOON:  Good morning and welcome.  I would like 
 
 15   to particularly extend my thanks to the subcommittee to 
 
 16   address this very important issue today and thank you to the

 17   public for attending. 
 
 18             Xenotransplantation raises important questions 
 
 19   regarding potential public‑health benefits and risks of 
 
 20   across‑species transplantation.  The recent reports by 
 
 21   patients et al. in Nature Medicine and Le Tissier in Nature

 22   describe the capacity of porcine endogenous retrovirus to 
 
 23   infect human cells in vitro. 
 
 24             These reports exemplify the rapidly evolving 
 
 25   nature of our understanding of the transmissibility of 

 
                                                                12 
 
  1   infectious agents across species barriers.  The dynamic

  2   nature of the potential public‑health risks and benefits 
 
  3   call for the implementation of the Xenotransplantation 
 
  4   Advisory Subcommittee constituted with members possessing 
 
  5   the expertise to address risk assessment and risk management 
 
  6   and calls for continuing public awareness of new scientific

  7   data in xenotransplantation. 
 
  8             During the meeting today, the subcommittee is 
 
  9   asked to address to following public‑health issues 
 
 10   concerning xenotransplantation.  One is the development of 
 
 11   appropriate assays for detection and identification of

 12   porcine endogenous retrovirus. 
 
 13             Second, limitations of current screening and 
 
 14   diagnostic tools.  Third, diagnostic testing and clinical 
 
 15   care of patients post‑transplant.  Fourth, impact of test 
 
 16   results on clinical‑trial development and, fifth, patient

 17   education, informed consent, and eligibility. 
 
 18             The meeting is intended to provide an open public 
 
 19   forum for discussion between members of the scientific and 
 
 20   biomedical community and to provide advice and 
 
 21   recommendations to the FDA regarding public‑health issues

 22   raised by xenotransplantation.  The FDA and the Center for 
 
 23   Biologics, in particular, appreciate the time, the insights 
 
 24   and the perspectives of the committee members in addressing 
 
 25   these important public‑health issues. 

 
                                                                13 
 
  1             At this point, I would like to introduce Deputy

  2   Commissioner Mary Pendergast. 
 
  3             MS. PENDERGAST:  Good morning.  I would like to 
 
  4   join Kathy Zoon in welcoming you to this new FDA advisory 
 
  5   committee and in thanking you for participating in the 
 
  6   committee's work.  The FDA cannot do its job of assessing

  7   the safety and effectiveness of new therapies without the 
 
  8   dedication and commitment of outside experts such as 
 
  9   yourselves. 
 
 10             Our need for advise is, perhaps, nowhere greater 
 
 11   than in the area of xenotransplantation. 

 12   Xenotransplantation offers the hope of treatments to many 
 
 13   desperately sick persons.  Yet, while we are mindful of its 
 
 14   promise, we must be equally mindful of its risks and we must 
 
 15   think of those risks quite broadly to include not just the 
 
 16   patient being treated but the larger society in which that

 17   patient lives. 
 
 18             It is never easy to regulate a new technology.  
 
 19   Indeed, the FDA has often led a new technology progress for 
 
 20   a while before imposing any type of regulations.  This 
 
 21   hesitancy is understandable.  Many promising new

 22   technologies fade from view after a very short time and, for 
 
 23   other new technologies, agency staff are hesitant to 
 
 24   regulate when very little is known and sensible standards 
 
 25   are difficult, if not impossible, to set. 

 
                                                                14 
 
  1             It goes without saying that most physicians and

  2   companies would prefer to have as unfettered a field as 
 
  3   possible so agency staff know that any efforts to regulate 
 
  4   will not be met with universal approval. 
 
  5             These same factors apply here.  There was no FDA 
 
  6   regulation of the Baby Fay baboon heart transplant or of

  7   other xenotransplantation efforts that took place in years 
 
  8   past.  It is a technology that could have, but did not, fade 
 
  9   away.  Rather, because of recent advances in immunology, 
 
 10   genetics and biochemistry, we are at the cusp of a possible 
 
 11   explosion of new xenotransplantation efforts.

 12             Yet, as is painfully evident from the porcine 
 
 13   endogenous retrovirus issue you are going to discuss today, 
 
 14   the science of xenotransplantation is still evolving and 
 
 15   there is much we do not know about the risks from 
 
 16   transplantation, thereby making the creating of a regulatory

 17   system different. 
 
 18             As usual, there are some who would prefer to have 
 
 19   the agency leave their xenotransplantation efforts alone.  
 
 20   However, the field will not stand still as we wonder what to 
 
 21   do and we believe that the public will be disadvantaged if

 22   we do nothing. 
 
 23             This disadvantage can come in several ways.  
 
 24   First, if we are too afraid to regulate and, so, ban the 
 
 25   technology outright, thereby depriving patients of possibly 

 
                                                                15 
 
  1   effective therapy.  Second, if we do not properly regulate

  2   and either personal or public health disasters occur.  
 
  3   Third, if we extensively overregulate, thereby unnecessarily 
 
  4   slowing down a promising technology. 
 
  5             We need your help in finding a middle path.  But 
 
  6   recognizing all the while that if the science is not there

  7   to let this technology go forward safely, then we have to 
 
  8   have a courage to say no. 
 
  9             Our staff took a very important step towards 
 
 10   finding that middle path when they spearheaded the efforts 
 
 11   to create a public health service guideline on

 12   xenotransplantation.  It is now our job to build on those 
 
 13   efforts. 
 
 14             Because the science an public‑health issues 
 
 15   concerning xenotransplantation are so complicated, this is a 
 
 16   technology where we will expect the very best from industry

 17   and physicians.  In our jobs, we hear a lot of quibbling.  
 
 18   Isn't a small trial sufficient?   Do we really have to run 
 
 19   so many tests?  Does it really matter that we have 
 
 20   invalidated this assay? 
 
 21             In this arena, we are going to expect and demand

 22   the best.  The stakes are too high not to.  Similarly, this 
 
 23   is not a field where science and technology will be able to 
 
 24   go forward behind closed doors.  We cannot regulate 
 
 25   xenotransplantation without full and open discussion by all 

 
                                                                16 
 
  1   interested parties.  And that will require a level of

  2   openness about the science and study plans that may be 
 
  3   uncomfortable to some. 
 
  4             My advice is if you don't want your work in 
 
  5   progress to be publicly discussed and assessed, then you had 
 
  6   best find a new field.  This is also going to be an area

  7   where extensive public discussion and debate will be needed.  
 
  8   Patients will want treatments but we also have a duty to the 
 
  9   public at large. 
 
 10             We will have to work collectively to figure out 
 
 11   new ways to engage the public and get their ideas and

 12   concerns on the table as we move forward. 
 
 13             Finally, I think that today's meeting, when we 
 
 14   discuss what to do in the face of uncertainty about porcine 
 
 15   endogenous retroviruses is only the first of a series of 
 
 16   discussions we will need to have as we face the inevitable

 17   challenge of creating and regulating science and medicine in 
 
 18   the face of uncertainty. 
 
 19             I, for one, am incredibly pleased that we are able 
 
 20   to call upon all of you to help us as we think through these 
 
 21   very difficult issues.  There is an enormous amount of fire

 22   power around this table and it is hard not to be in awe of 
 
 23   your expertise in so many scientific disciplines all of 
 
 24   which are needed to discuss xenotransplantation issues. 
 
 25             Again, I would like to thank you for your 

 
                                                                17 
 
  1   willingness to help us.

  2             DR. AUCHINCLOSS:  Thank you very much. 
 
  3                       Open Public Hearing 
 
  4             DR. AUCHINCLOSS:  We would now like to move on to 
 
  5   the open public hearing.  I would like to initiate that by 
 
  6   asking Gail Dapolito if she would introduce us to the

  7   responses to the Federal Register announcement of this 
 
  8   meeting. 
 
  9             MS. DAPOLITO:  As part of the FDA Advisory 
 
 10   Committee meeting procedure, we hold an open public hearing 
 
 11   for those members of the public who are not on the agenda

 12   and would like to make a statement concerning matters 
 
 13   pending before the committee. 
 
 14             We have two sessions scheduled on today's agenda 
 
 15   for public comment, one in the morning and one in the 
 
 16   afternoon.  In response to the Federal Register announcement

 17   regarding this open public hearing, we have received 
 
 18   requests for written submissions and requests for oral 
 
 19   presentations.   
 
 20             Imuntran Inc. Novartis has provided a written 
 
 21   submission of public comment to the committee and that can

 22   be found in your blue folders.  We have also received four 
 
 23   requests for oral presentations. 
 
 24             Our first public speaker is Dr. Achilles Demetriou 
 
 25   from Cedar Sinai Medical Center.  Dr. Demetriou would you 

 
                                                                18 
 
  1   please come to the microphone.  I would like to remind those

  2   individuals who are speaking to please keep your comments to 
 
  3   five or six minutes.  We will be using the three‑light 
 
  4   system to help you keep track of your time.  Also, we ask, 
 
  5   in the interest of fairness, that anyone addressing the 
 
  6   committee please state any affiliation that you may have

  7   with sponsors and products or competing firms and products 
 
  8   under discussion today 
 
  9             Thank you. 
 
 10             DR. DEMETRIOU:  Thank you very much and good 
 
 11   morning.  I am Achilles Demetriou.  I am the chairman of the

 12   Department of Surgery at Cedars Sinai.  I am a professor of 
 
 13   surgery at UCLA.  I work with Circe Biomedical who is the 
 
 14   industry sponsor of the clinical trial that I participate 
 
 15   in. 
 
 16             We have developed a bioartificial liver which

 17   utilizes porcine hepatocytes and we have used it to treat 37 
 
 18   patients at our institution with severe acute liver failure.  
 
 19   What I would like to do is put the clinical situation into 
 
 20   perspective and urge the committee to take into 
 
 21   consideration the disease which is being treated and the

 22   risks of the disease and assess the risk/benefit, taking 
 
 23   that in mind, and recognizing that there may be a real risk 
 
 24   with PERV transmission in the clinical setting. 
 
 25             Patients with fulminant liver failure, without 

 
                                                                19 
 
  1   transplants, have a 90 percent mortality.  With

  2   transplantation, survival is approximately 70 percent.  
 
  3   However, 50 percent of patients never make it to transplant.  
 
  4   These are patients who are candidates for transplantation 
 
  5   but waiting around and they cannot get a liver.  So we lose 
 
  6   half of our transplant candidates while they are waiting for

  7   a liver to treat fulminant liver failure. 
 
  8             90 percent, if they are not candidates for liver 
 
  9   transplantation will actually die.  So we are dealing with a 
 
 10   disease which is certainly lethal.  So the theoretical risk, 
 
 11   at this point, of PERV side effects or infection in the

 12   clinical setting needs to be put in that perspective. 
 
 13             I am not aware of a single clinical instance of 
 
 14   transmission of disease from pig to human.  There have been 
 
 15   over 100 whole‑liver perfusions in the literature.  Again, 
 
 16   there has never been a single report of transmission of the

 17   disease. 
 
 18             Secondly, if we are going to regulate future 
 
 19   studies using porcine tissue, maybe we need to take a step 
 
 20   back and look at the instances where porcine tissues have 
 
 21   been used to treat patients.  For example, pig skin has been

 22   used for years to treat thousands of patients.  This is 
 
 23   cryopreserved tissue which is place on top of open wounds 
 
 24   with direct contact between cellular elements. 
 
 25             Are we now going to notify patients who have been 

 
                                                                20 
 
  1   treated with porcine skin grafts, thousands of patients over

  2   a period of years, or are we going to restrict notification 
 
  3   to patients who participate in future studies. 
 
  4             So these are issues that are not simple and I 
 
  5   think we need to address.  Similarly, slaughterhouse workers 
 
  6   who work with porcine tissues; is the USDA going to issue

  7   directives that these workers have to be following 
 
  8   essentially HIV precautions in that setting.  Again, that is 
 
  9   something that needs to be considered. 
 
 10             All patients that we have tested that we have 
 
 11   treated in our study tested positive for porcine antibodies

 12   in their plasma suggesting that there is massive contact, at 
 
 13   least, with porcine tissues.  So porcine tissues are all 
 
 14   around us in things we eat and in things we touch or come 
 
 15   into daily contact.  Therefore, I think we need to put this 
 
 16   whole thing in perspective.  

 17             Finally, gene‑therapy trials are using 
 
 18   retroviruses in which we deliberately utilize a retrovirus 
 
 19   to correct a specific genetic defect of treated disease.  
 
 20   Are we now going to expand the warnings to include 
 
 21   retroviral use in gene‑therapy trials?  

 22             This is certainly a very complex problem.  I think 
 
 23   our major responsibility is to notify our patients regarding 
 
 24   the risks of PERV transmission.  I think patients and 
 
 25   families need to have informed consent when they participate 

 
                                                                21 
 
  1   in the studies.

  2             I think the papers in Nature and Nature Medicine 
 
  3   are important because they outline the risks and we need to 
 
  4   follow it and we need to inform our patients and their 
 
  5   families.  However, that risk needs to be placed in 
 
  6   perspective considering the very high morbidity and

  7   mortality of the disease that we are trying to treat. 
 
  8             Thank you very much. 
 
  9             MS. DAPOLITO:  Thank you, Dr. Demetriou. 
 
 10             Our next speaker will be Dr. Barbara Potts from 
 
 11   Tektagen.

 12             DR. POTTS:  Good morning.  Tektagen is a contract 
 
 13   service lab and we do do work for companies that have 
 
 14   xenotransplant products. 
 
 15             [Slide.] 
 
 16             The list of animal retroviruses that may pose a

 17   health hazard to humans is really quite lengthy.  I have 
 
 18   just organized them here for you according to their 
 
 19   morphology and family.   
 
 20             [Slide.] 
 
 21             The way to detect these viruses in humans, we need

 22   to amplify the virus.  It is either by PCR or cocultivation.  
 
 23   I am going to approach the cultivation assay and the  
 
 24   possibilities and limitations of this system today detecting 
 
 25   it in human PBLs.  That is going to be my focus. I am 

 
                                                                22 
 
  1   detecting the virus either by a reverse‑transcriptase assay

  2   using either magnesium or manganese, which I am going to Mg 
 
  3   and Mn from now on because I get them mixed up and also 
 
  4   antigen‑capture ELISA assay. 
 
  5             Just to look quickly at this, this is an assay 
 
  6   that was developed by Michael Martin at the NIH because it

  7   gave us a high signal and a low noise level for HIV and, 
 
  8   also, for Mn, there is a high signal and a low noise and 
 
  9   fairly good reproducibility. 
 
 10             For HIV, we know that two times this negative 
 
 11   control was ten infectious units and I know that two times

 12   this negative control for simian foamy virus is 20 plaque‑ 
 
 13   forming units.  So I set two times the negative controls as 
 
 14   the arbitrary cutoff on this assay.  It is very 
 
 15   conservative. 
 
 16             [Slide.]

 17             I am just going to march through the alphabet in 
 
 18   these viruses real quickly.  Porcine endogenous retroviruses 
 
 19   are in all of the tissues, in porcine tissues.  This is just 
 
 20   a titration of the cells times 103 versus counts‑‑this is a 
 
 21   P32 RT assay.  You can see that for the fetal porcine test

 22   that there is a nice dose response. 
 
 23             At PK15, there is a nice dose response.  Minipig 
 
 24   kidney is a little bit lower and there is a nice dose 
 
 25   response.  If these cells are frozen and thawed more than 

 
                                                                23 
 
  1   three times, this goes flat.  So it is very unstable.

  2             However, the supernatant, especially in the 
 
  3   minipig soup, remains stable after many freeze‑thaws.  If we 
 
  4   look at primary culture, we have 107 here porcine PBLs.  We 
 
  5   have about 800 counts.  Again, this goes flat after three or 
 
  6   four freeze thaws and porcine hepatocytes, really, this,

  7   perhaps, is noise. 
 
  8             Again this negative control cutoff is really 
 
  9   arbitrary.  It hasn't been established for this system.  
 
 10   This is using Mn in an RT assay.   
 
 11             [Slide.]

 12             If we go to Mg, you see that almost all of them 
 
 13   are negative.  However, what we have here is we have this 
 
 14   really high signal in the minipig soup. 
 
 15             I have repeated, I might add, that all of these 
 
 16   tests are a replicate of eight separate assays done by two

 17   analysts on at least three different days.  This, I did ten 
 
 18   separate assays because I didn't believe it but this is 
 
 19   real.  There is definitely an Mg signal coming out of the 
 
 20   minipig soup and it is stable after many freeze‑thaws. 
 
 21             I would like to hear some comments about that

 22   later.  Again, you have your porcine hepatocytes are just 
 
 23   really bouncing around here with the noise.  Here you have 
 
 24   your porcine PBLs. 
 
 25             [Slide.] 

 
                                                                24 
 
  1             Just quickly mention the HTL‑V group.  There is

  2   P19 antigen ELISA that works for both PLV and STLV.  I just 
 
  3   wanted to point out this nice low noise and the nice dose 
 
  4   response for P12 versus cell concentration. 
 
  5             [Slide.] 
 
  6             For the type D's, we have a good RT signal here,

  7   in negatives, and it is very low. 
 
  8             [Slide.] 
 
  9             I will just quickly go through this.  The 
 
 10   lentiviruses are all really very closely related in their RT 
 
 11   and, consequently, we were unable to detect all of these

 12   viruses.  Using these as positive controls, we were able to 
 
 13   detect all of these lentiviruses in a fairly good reverse‑ 
 
 14   transcriptase assay. 
 
 15             I wanted to present this first so that you believe 
 
 16   that I can grow these viruses and detect them.

 17             [Slide.] 
 
 18             The negative controls are nice and quiet. 
 
 19             [Slide.] 
 
 20             Simian foamys have nice signal, a little bit of 
 
 21   noise with the M. dunnis here but that is the murine

 22   retrovirus.  But still we have a good signal here. 
 
 23             [Slide.] 
 
 24             So the real key question is can any of these 
 
 25   animal retroviruses replicate and produce infectious progeny 

 
                                                                25 
 
  1   in human PBMCs.  The assay system, we used both select and

  2   random human PBMC donors because we know that that makes a 
 
  3   difference for HIV‑1 and HTLV‑1.   
 
  4             I enriched for monocyte macrophages because some 
 
  5   of these viruses replicate exclusively in monocyte 
 
  6   macrophages and PHA stimulate for three days, maintained and

  7   IL‑2.  And virus infection was either by inoculation or by 
 
  8   cocultivation with gamma‑irradiated virus‑infected cells.  
 
  9   This was 10,000 rads. 
 
 10             Additional PBMCs were added at day 13.  Cultures 
 
 11   were maintained, splitting feeding two to three times a week

 12   for 20 days for inoculation, 30 days for cocultivation.  
 
 13   Cells and supernatant were harvested at different times.  I 
 
 14   am going to show you the data from 20 days for the infection 
 
 15   and 30 days for the cocultivation. 
 
 16             Samples were frozen and thawed only once in either

 17   an Mg or an MNRT assay were run on them. 
 
 18             [Slide.] 
 
 19             So the bottom line is that all of our positive 
 
 20   controls replicated nicely.  These are logs, TDIC 50 per ml, 
 
 21   and all of these were negative in both systems.  The BIV, I

 22   saw a lot of CPE. It either may be non‑protective infection 
 
 23   or it may be another virus.  Just because I think it is BIV, 
 
 24   there may be something else in there but there is definitely 
 
 25   CPE. 

 
                                                                26 
 
  1             [Slide.]

  2             Probably the most important is if you look at the 
 
  3   simian foamy viruses, they all replicated to greater than 
 
  4   2 logs in the human PBMCs.  I took this culture, then, back 
 
  5   and titered it back into M. dunnis, which is the parental 
 
  6   cell‑line that I used to grow the simians, and I had 20

  7   plaque‑forming units. 
 
  8             So even though the RT was very low, we definitely 
 
  9   could detect the virus.  So this, I think, is a good 
 
 10   circular result because we know that simian foamys can 
 
 11   infect humans.  Now we know they can infect PBLs at a very

 12   low level. 
 
 13             Also, BLV and STLV, I did detect transformation.  
 
 14   I won't present the P19 antigen ELISA data today.  All of 
 
 15   the PERV are irradiated to PK15 cells, minipig cells, STs.  
 
 16   And the porcine PBLs were all negative at 30 days.

 17             Thank you. 
 
 18             MS. DAPOLITO:  Thank you, Dr. Potts. 
 
 19             Our next speaker for open public comment was Dr. 
 
 20   Bob Brown, University of North Carolina, Chapel Hill.  Is 
 
 21   Dr. Brown here?   I guess he didn't show up today.

 22             We have a speaker who was scheduled for this 
 
 23   afternoon.  I wonder if Dr. Savill‑‑are you prepared for 
 
 24   this morning.  Thank you.  Dr. Corinne Savill from Imutran. 
 
 25             DR. SAVILL:  Good morning. 

 
                                                                27 
 
  1             [Slide.]

  2             What I am going to try and address is one of the 
 
  3   subcommittee's remits which is the impact of diagnostic 
 
  4   screening on clinical‑trial development in 
 
  5   xenotransplantation. 
 
  6             [Slide.]

  7             In order that the undoubted benefits of 
 
  8   xenotransplantation can be weighed against the risk of 
 
  9   retrovirus infection, we are proposing a risk analysis of 
 
 10   this infection in the context of limited, closely monitored, 
 
 11   clinical trials of xenotransplantation.

 12             The risk assessment falls into three categories: 
 
 13   firstly, to look at and characterize the expression and the 
 
 14   release of the virus from our herd of transgenic pigs; 
 
 15   secondly, to determine the infectious status of human 
 
 16   recipients who have been subjected, as we have just heard,

 17   to treatment with living porcine tissue; and, thirdly, to 
 
 18   look at our archive of primates who have been treated with 
 
 19   solid‑organ xenografts, either hearts or kidneys. 
 
 20             [Slide.] 
 
 21             Looking at the expression of the retrovirus in our

 22   herd of pigs, we are characterizing the variance which were 
 
 23   actually released from our pig herd in terms of the tropism 
 
 24   of each variant, whether it is infected for human cells of 
 
 25   whether it is a pig‑to‑pig species to determine which type 

 
                                                                28 
 
  1   of cell in these pigs expresses the retrovirus and, perhaps,

  2   most importantly, determining whether PERV‑containing 
 
  3   virions are released from cells from the organs both pre‑ 
 
  4   and post‑transplant into non‑human primates. 
 
  5             If virions are detected, we will also look to 
 
  6   determine whether they contain xenotropic variants of the

  7   retrovirus. 
 
  8             [Slide.] 
 
  9             As has been said already, living pig tissue has 
 
 10   been used to treat human disease.  We are looking into this 
 
 11   and we have found many more than 100 patients already

 12   worldwide have been exposed to living porcine tissue.  We 
 
 13   are in the process of sampling these patients, collecting 
 
 14   blood and cells for analysis and testing for potential PERV 
 
 15   transmission, looking at the DNA level, the RNA level by RT‑ 
 
 16   PCR on plasma and also testing to see whether these patients

 17   have made an anti‑PERV‑specific immune response. 
 
 18             The numbers in the brackets indicate the number of 
 
 19   patients so far that we have identified and are in the 
 
 20   process of drawing blood from. 
 
 21             [Slide.]

 22             We now have an archive of well over 200 non‑human 
 
 23   primates who have received a solid‑organ xenograft.  We are 
 
 24   testing those primate recipients by PCR for PERV and, if 
 
 25   positive, we will have to look and determine whether this 

 
                                                                29 
 
  1   represents a true infection or whether the test is picking

  2   up potential contamination by pig cells in microchimerism in 
 
  3   these primates. 
 
  4             We will look by RT‑PCR, if we find any positive 
 
  5   samples, and we will screen all the primates for an anti‑ 
 
  6   PERV antibody response.

  7             [Slide.] 
 
  8             I think it is important to consider, and it may be 
 
  9   something that is discussed later today, that not all 
 
 10   xenotransplantation approaches and not all clinical trials 
 
 11   may be equivalent in terms of retroviral risk.  One should

 12   look at the length of exposure, the presence and the nature 
 
 13   of any immunosuppression, the type of tissue, whether or not 
 
 14   that tissue is encapsulated, for instance, and the number of 
 
 15   patients who are proposed to be treated and, of course, 
 
 16   distinguish between risk to individual patient and that

 17   which may present a public‑health hazard. 
 
 18             [Slide.] 
 
 19             In our estimation, the studies that we are 
 
 20   performing which will be key for the initiation of 
 
 21   extracorporeal trials, is the retrospective study of

 22   patients who have already been treated in similar manners 
 
 23   with living porcine tissue and also looking to see, in our 
 
 24   pigs, whether we actually see virion release. 
 
 25             In our opinion, if we detect positive PERV genome 

 
                                                                30 
 
  1   in human cells in these patients or if we detect virion

  2   release containing xenotropic variants of the PERV 
 
  3   retrovirus, we would consider that this is important to 
 
  4   think again about our approach to this trial and to perform 
 
  5   further risk analysis. 
 
  6             [Slide.]

  7             When one considers the initial trials closely 
 
  8   monitored of solid‑organ xenotransplants in addition to the 
 
  9   two studies I just stated, also looking at our archive with 
 
 10   xenografted non‑human primates is important to be 
 
 11   considered.  Again, we feel that if we detect positive‑PERV

 12   genome in the primate cells, so true transmission of the 
 
 13   infection, we would do more work and restudy these archives 
 
 14   before we would actually approach a trial in humans. 
 
 15             [Slide.] 
 
 16             We also have a longer‑term approach under way.  We

 17   are characterizing the loci of the retrovirus in transgenic 
 
 18   animals and mapping those loci which are associated with the 
 
 19   production of the xenotropic virus.  By carrying out this 
 
 20   work, one will be able to analyze and make an assessment of 
 
 21   whether such approaches to either breed out or, if possible,

 22   if the technology advances, to genetically knock out the 
 
 23   virus from such animals is technically feasible. 
 
 24             [Slide.] 
 
 25             So, overall, in summary, moving towards more 

 
                                                                31 
 
  1   extensive trials in launch, we would propose a stepwise and

  2   cautious approach to the development, to take each step as 
 
  3   it comes and with a safety follow‑up of initial trials 
 
  4   before proceeding. 
 
  5             This will enable us, and anybody else working in 
 
  6   the field, to actually gain an increased understanding of

  7   the PERV infection as one proceeds towards a more extensive 
 
  8   development of this technology and, also, in parallel, to 
 
  9   work on longer‑term approaches toward selective breeding or 
 
 10   genetic knockout of the xenotropic form of the virus. 
 
 11             Thank you.

 12             MS. DAPOLITO:  Dr. Savill, for the public record, 
 
 13   would you please state your affiliation. 
 
 14             DR. SAVILL:  Sorry.  Imutran Limited Novartis 
 
 15   Pharma. 
 
 16             DR. DAPOLITO:  Thank you.

 17             At this time, I would like to ask if there is 
 
 18   anyone else in the audience who would like to make a public 
 
 19   statement to the committee.  If you would, please raise your 
 
 20   hand.  I see no one. 
 
 21             Dr. Auchincloss, I turn it over to you.

 22             DR. AUCHINCLOSS:  Thank you very much. 
 
 23             We will move now to the open committee discussion. 
 
 24   In preparation for doing that, I would like to introduce 
 
 25   several specially invited guests of the Xenotransplantation 

 
                                                                32 
 
  1   Subcommittee.  These guests are Dr. Marian Michaels, Mr.

  2   Antonio Benedi, Mr. William Lawrence and Dr. Robin Weiss. 
 
  3             At this time, I would like to invite them to the 
 
  4   table to participate in the day's discussions. 
 
  5             The next item on the agenda is the Chairman's 
 
  6   introductory remarks, which I promise to keep very brief. 

  7   Introduction of Focus Groups and Presentation of Questions  
 
  8                    for Committee Discussion   
 
  9             DR. AUCHINCLOSS:  First of all, I am not an expert 
 
 10   on today's subject.  I think I know a fair amount about 
 
 11   xenotransplantation but I am not a virologist and,

 12   therefore, am here to learn about a subject that I do think 
 
 13   is of enormous importance, of such importance that I really 
 
 14   want to stress how much I appreciate the members of the 
 
 15   subcommittee and the effort they have made to come here. 
 
 16             I know many of you have traveled long distances

 17   and, for every one of you, it is an inconvenience, at least, 
 
 18   and we really appreciate it.  I also want to call attention 
 
 19   and offer our thanks to a number of people from the FDA.  In 
 
 20   particular, I think it is the drive and energy of Amy 
 
 21   Patterson who has kept this going, the FDA's effort, really,

 22   to grapple with the problems presented by 
 
 23   xenotransplantation. 
 
 24             She is quite an extraordinary woman.  They tell me 
 
 25   that she puts together these reading books for us in just an 

 
                                                                33 
 
  1   evening's worth of work, which is a terrifying thought

  2   because it is hardly an evening's worth of work to go 
 
  3   through them. 
 
  4             But it is a tremendous effort that you have made 
 
  5   and we really do appreciate it, Amy. 
 
  6             There are three other people who, every time we

  7   come to the FDA, make our lives so much easier including 
 
  8   Bill Freas and Gail and Rosanna Harvey, and we really do 
 
  9   appreciate, again, the efforts that you make on our behalf. 
 
 10             You understand, then, the content of my 
 
 11   introduction otherwise is simply to introduce the fact that

 12   there will be questions that will be addressed to this 
 
 13   committee during the course of the afternoon after hearing 
 
 14   some data and presentations during the morning. 
 
 15             In essence, and I am not going to go through these 
 
 16   questions in detail now, those questions have to do with

 17   first developing the assays for detection of porcine 
 
 18   endogenous retrovirus; second, what kind of screening for 
 
 19   patients is appropriate; and, third, questions related to 
 
 20   the aspect of informed consent and selection of patients. 
 
 21             What I do want to do at this point is to simply

 22   highlight a process that we have used in preparation for 
 
 23   this meeting and that is that members of the subcommittee 
 
 24   and, in addition, other participants in this group have been 
 
 25   assigned to "focus groups" in order to have a preliminary 

 
                                                                34 
 
  1   discussion on each of these questions prior to this meeting.

  2             So that has happened and you will hear brief 
 
  3   presentations this afternoon from these focus groups.  What 
 
  4   I want to emphasize, however, about that process is that it 
 
  5   was not designed to limit discussion; it was designed to 
 
  6   initiate discussion.  Whether you are a member of a

  7   particular focus group or not, we still invite every member 
 
  8   of the committee to offer their views on every subject that 
 
  9   we are discussing this afternoon and it is very important 
 
 10   that we understand that process. 
 
 11             If I have not forgotten anything else, I think it

 12   is time to move on with the morning's agenda with an initial 
 
 13   presentation from John Coffin on endogenous retroviruses 
 
 14   providing us with the overview. 
 
 15               Endogenous Retroviruses: an Overview 
 
 16             DR. COFFIN:  Thank you very much.  I am very

 17   pleased and honored by the invitation to give this talk on 
 
 18   this important subject.  A little personal note; the study 
 
 19   of endogenous retroviruses has been, really, a fairly major 
 
 20   aspect of my career for the last 25 years and actually of a 
 
 21   few other people at the table as well, that was undertaken

 22   because it is really a fascinating aspect of how 
 
 23   retroviruses interact with their host and provide some very 
 
 24   interesting models for disease processes and, also, for 
 
 25   understanding host virus evolution. 

 
                                                                35 
 
  1             When I started off doing it, and I never imagined

  2   that we would be discussing these in the context of real 
 
  3   applications and potential risks. 
 
  4             That said, let me move on to the first slide. 
 
  5             [Slide.] 
 
  6             The focus of the whole meeting today really is the

  7   risk that endogenous pig viruses might present in the 
 
  8   context of essentially cocultivation of human pig tissues 
 
  9   that occurs during xenotransplant procedures. 
 
 10             Let me just set the context of that quickly and 
 
 11   then I will get on to talk about endogenous viruses in

 12   general and give some examples of things that we have 
 
 13   uncovered from our experience, particularly with the mouse 
 
 14   endogenous viruses. 
 
 15             I come from the position that at least some kind 
 
 16   of infection of the recipient of a xenotransplant, albeit

 17   limited, perhaps, to some very small numbers of cells, is 
 
 18   virtually an inevitable consequence of introducing tissue 
 
 19   from any species that can, potentially, express infectious 
 
 20   endogenous viruses into humans. 
 
 21             There is precedent for that based on experiments

 22   where human tissues have been transplanted into mice and 
 
 23   where mice are capable of expression endogenous viruses, 
 
 24   although they don't.  But if those viruses can infect human 
 
 25   tissue, then, in fact, very often, the tumor that comes out 

 
                                                                36 
 
  1   of new mice that are transplanted in this way ends up

  2   producing infectious virus. 
 
  3             When I had my hat as chair of the IBC at Tufts, 
 
  4   several times people came running around to me with human 
 
  5   tumors that were producing these large numbers of tissues or 
 
  6   human cell lines, and my first question was has it ever

  7   passed through a nude mouse. 
 
  8             The answer always is yes, actually, when you see 
 
  9   that.  So it is a very famous phenomenon that actually Robin 
 
 10   Weiss wrote about very nicely in an earlier version of the 
 
 11   retrovirus book.

 12             [Slide.] 
 
 13             Retroviruses are really remarkable in their 
 
 14   ability to form a permanent genetic association with their 
 
 15   host.  This is the only infectious agent of higher organisms 
 
 16   that we know that is capable of doing this.  Over the years,

 17   over our evolutionary history, our germ lines and that of 
 
 18   all our mammalian, avian and probably other vertebrate 
 
 19   relatives as well, have been under fairly constant assault 
 
 20   by infection with these agents. 
 
 21             In fact, it is amazing that we tolerate that.  A

 22   substantial fraction of our DNA, a half percent or more, 
 
 23   thousands of elements, has come from infection in the more 
 
 24   or less distant past of germ‑line tissue. 
 
 25             Endogenous retroviruses form several distantly 

 
                                                                37 
 
  1   related groups, as I will show you in a minute.  About half

  2   of the extant retrovirus groups have been know to leave 
 
  3   endogenous rows in their germ line and most groups are 
 
  4   almost certainly derived‑‑if you look at the patterns that 
 
  5   these are in in species‑‑from exogenous viruses that have 
 
  6   infected the same species.

  7             So endogenous viruses are not a precursor to the 
 
  8   infectious retroviruses that we see.  Perhaps, there are 
 
  9   some elements that are such precursors, but all of the ones 
 
 10   that we can see almost certainly are the consequence of 
 
 11   infection of the germ line with infectious retroviruses and

 12   can often be induced. 
 
 13             A useful distinction is whether the endogenous 
 
 14   viruses were recently introduced into the germ line or, in 
 
 15   fact, entered there in the very distant past.  Humans have, 
 
 16   as I said, thousands of elements.  But, as far as we know,

 17   every single one was introduced more than a million years 
 
 18   ago. 
 
 19             So there is no genetic polymorphism among humans 
 
 20   in any of these elements that have been discovered.  
 
 21   Furthermore, their infectivity and their ability to even

 22   make particles, in most instances, has been severely eroded 
 
 23   just by the accumulation of random mutations in the absence 
 
 24   of any counterselection over the evolutionary history. 
 
 25             [Slide.] 

 
                                                                38 
 
  1             As I said before, this slide shows sort of what we

  2   now take as the correct retrovirus taxonomy or at least the 
 
  3   current retrovirus taxonomy.  Of the seven groups of virus 
 
  4   genera that are defined here, four of these have been known 
 
  5   to leave endogenous relatives in the germ line. 
 
  6             The one that is of the most concern today is the

  7   probably the largest of these that is in the most species, 
 
  8   and these are the MLV‑related or C‑type retroviruses, 
 
  9   mammalian C‑type retroviruses. 
 
 10             These are all viruses that are fairly closely 
 
 11   related in sequence to murine leukemia virus and much more

 12   closely so than they are to retroviruses from any other 
 
 13   group.  This taxonomy is based on sequence relationships of 
 
 14   reverse‑transcriptase. 
 
 15             [Slide.] 
 
 16             So, over the years, endogenous retroviruses have

 17   been associated with quite a number of rather interesting 
 
 18   and famous genetic phenomena.  I will just list a few here 
 
 19   just to get us into tune of what we are talking about. 
 
 20             There is the famous human rumor viruses.  Again, 
 
 21   it is very nicely defined by Robin in his chapter about

 22   fifteen years ago, often due to expression of defective 
 
 23   human proviruses that are endogenous to cells, although no 
 
 24   infectious agent and no definitive genetic association of 
 
 25   any of these with actual tumor causality has ever been 

 
                                                                39 
 
  1   established and these are probably sort of epiphenomena.

  2             Endogenous retroviruses can recombine with 
 
  3   infecting exogenous viruses to create new antigens, and the 
 
  4   famous feline virus cell‑membrane antigen is an example of 
 
  5   that.  That appeared in the study of cats some years ago. 
 
  6             Endogenous proviruses can often masquerade as

  7   developmental or strain‑specific antigens.  These are some 
 
  8   famous examples in mice.  Some endogenous viruses, although 
 
  9   they not the ones we are most interested in, encode 
 
 10   superantigens which are necessary for the efficient 
 
 11   replication of the exogenous virus in the immune system.

 12             This is the case of mammary‑tumor virus.  These 
 
 13   superantigens, when expressed in endogenous viruses, 
 
 14   actually inhibit the replication of the corresponding 
 
 15   endogenous virus and probably make the animal somewhat 
 
 16   resistant.

 17             Endogenous viruses are actually a problem in gene 
 
 18   therapy because they can be very efficiently transmitted 
 
 19   with retroviral vectors.  In fact, a few studies have shown 
 
 20   that there are often more of these VL30s or MLV‑related 
 
 21   sequences in vector preparations than there are of your

 22   vector.  These can be very efficiently transmitted, and are 
 
 23   not a matter of our concern today, but are something that 
 
 24   should be kept in mind in gene‑therapy applications. 
 
 25             They can repair defects by recombination with 

 
                                                                40 
 
  1   vectors or other species or other defective infecting

  2   viruses and a lot of replication.  They have come to the 
 
  3   attention of the FDA before in the context of monoclonal 
 
  4   antibodies and other types of biological preparations as 
 
  5   possible contaminants. 
 
  6             I mentioned before the phenomenon where human

  7   tumor cells, grown in nude mice, very frequently come back 
 
  8   producing very large amounts of virus.  This is not a subtle 
 
  9   phenomenon.  If you take a thin section of these cells, 
 
 10   these cells are loaded with virus. 
 
 11             Then the question at hand is can these be

 12   transmitted to xenotransplant recipients and, if so, of 
 
 13   course, what are the consequences. 
 
 14             [Slide.] 
 
 15             I want to focus, now, on what I would like to 
 
 16   refer to as the recent endogenous proviruses.  These are

 17   ones that have been put into the species, into the germ 
 
 18   line, of some individuals in a species subsequent to 
 
 19   speciation.  They are not found in all species.  We know of 
 
 20   none in humans, for example. 
 
 21             They are, however, common in some birds but not

 22   other closely related birds.  The distribution of these is 
 
 23   taxonomically erratic suggesting that this is a horizontal 
 
 24   transmission effect.  They are found in most strains of mice 
 
 25   and most subspecies of wild mice.  They are found in cats 

 
                                                                41 
 
  1   but not dogs, as far as we know.  And they can be found in

  2   some primates such as baboons and also in other animals, of 
 
  3   course, such as pigs. 
 
  4             These are inserted recently.  So if you look from 
 
  5   one individual to another, there is polymorphism in the 
 
  6   genetic location although different individuals and

  7   different locations can be very, very similar to one 
 
  8   another. 
 
  9             Different proviruses at different sites can be 
 
 10   more than 99 percent identical to one another.  Many of 
 
 11   these can be replication‑competent or potentially

 12   replication‑competent if they are allowed to replicate in an 
 
 13   appropriate environment and if they are induced by some 
 
 14   inducing agent.  Endogenous proviruses are generally 
 
 15   expressed at a very low level because of germ‑line 
 
 16   methylation but can be efficiently induced by agents that

 17   reverse DNA methylation. 
 
 18             It actually might be worth including this sort of 
 
 19   a treatment in protocols to try to viruses out to give one 
 
 20   the best chance in vitro to see what is there. 
 
 21             [Slide.]

 22             So the recent endogenous proviruses of mice‑‑mice, 
 
 23   like all other species, also have a load of old ones that 
 
 24   are like the human ones that I have mentioned that are 
 
 25   largely defective.  The recent ones include a fairly large 

 
                                                                42 
 
  1   group of C‑type viruses of which, per animal or inbred

  2   strain, there are about 40 to 60 members; the B‑type 
 
  3   viruses, the mammary‑tumor virus, which have just a few 
 
  4   members; and the A‑type sequences which are not necessarily 
 
  5   viruses in that there is no evidence that these get out and 
 
  6   infect cells, but they are closely related to some groups of

  7   retroviruses and, genetically, it has been shown they can 
 
  8   move around by a mechanism resembling retrovirus infection.  
 
  9             The numbers of these can be quite large, up to 
 
 10   1,000 proviruses in a given strain, and so the genetics of 
 
 11   this, not surprisingly, has not been as well worked out as

 12   it has in some of these other groups. 
 
 13             [Slide.] 
 
 14             I want to focus the attention now on the murine 
 
 15   leukemia virus related, the C‑type ones, of which there are 
 
 16   about 50 to 60 members, or 40 to 60, I guess, in a given

 17   inbred strain.  Some years ago, Jonathan Stoye, when he was 
 
 18   a post‑doc in my lab, discovered that one can conveniently 
 
 19   divide these into four classes based on what a really subtle 
 
 20   sequence difference is and also differences particularly 
 
 21   focus on the enveloped gene which provide these viruses with

 22   potentially different host ranges, meaning they use 
 
 23   different receptors. 
 
 24             A small class are the so‑called ecotropic viruses 
 
 25   which can use a receptor that is limited to mouse cells.  

 
                                                                43 
 
  1   Then the larger groups are these others which use a

  2   receptor‑‑actually, they use what is probably the same 
 
  3   receptor on different species, but the receptor, itself, has 
 
  4   been altered in mice so that this particular class of 
 
  5   proviruses, the xenotropic proviruses, although it is 
 
  6   present in mice, can no longer infect the species that it is

  7   found in. 
 
  8             This is almost certainly an adaption of the 
 
  9   species to having this provirus, to protecting it from 
 
 10   infection.  In term, probably, we visualize the evolution.  
 
 11   Some descendants of these viruses probably, then, evolved an

 12   ability to use now the receptor as it has been modified 
 
 13   genetically in mice. 
 
 14             So these now can see the receptor in mice and in 
 
 15   other species.  These are limited to the receptor as it is 
 
 16   found in other species but not in mice.  Unfortunately, we

 17   don't have this receptor cloned yet so we can't flesh this 
 
 18   speculation out with some actual hard data. 
 
 19             There are, also, some restriction‑site 
 
 20   polymorphisms and some sequence polymorphisms which allow 
 
 21   the synthesis of small simple oligonucleotide probes which

 22   allow these all to be distinguished in simple southern 
 
 23   blotting or in PCR experiments. 
 
 24             So, using southern blotting with these sequence 
 
 25   probes, one can identify the provirus composition of a given 

 
                                                                44 
 
  1   animal or a given strain of animal.

  2             [Slide.] 
 
  3             This is just the seven most popular strains of 
 
  4   inbred mice probed with each one of these different probes.  
 
  5   Each of these detects a unique set.  There is no overlap 
 
  6   between these maps.  What these bands are junction fragments

  7   composed of virus sequence joined to cell sequence. 
 
  8             Almost all the viruses have the restriction 
 
  9   enzymes that are used here.   
 
 10             We have to go back, actually. 
 
 11             [Slide.]

 12             They have, say, this echo‑R1 site in common.  What 
 
 13   makes the fragment different is the appearance of the next 
 
 14   echo‑R1 out here.  So these detect proviruses not by their 
 
 15   internal sequence but by their location in the genome.  This 
 
 16   is a very important point that people often still don't

 17   quite follow with these. 
 
 18             [Slide.] 
 
 19             So each of these represents the insertion of a 
 
 20   provirus at a specific point and some ancestor of the 
 
 21   particular animal that you are looking at here.  If you

 22   extend this analysis to about fifteen different inbred 
 
 23   strains, what you actually find is the polymorphism, which 
 
 24   is obvious here, is such that there is only one provirus, in 
 
 25   fact, that is present in all inbred strains of mice. 

 
                                                                45 
 
  1             So it would, in fact, at least in theory, be

  2   possible to design a series of genetic crosses which take 
 
  3   out of mice‑‑all of these, despite the fact that any given 
 
  4   strain, if you add up all of these lanes, you have, as I 
 
  5   say, 40 to 60 proviruses‑‑it would, at least in theory, to 
 
  6   design a series of crosses that removed all but one of them

  7   from mice. 
 
  8             It would take quite a long time and some fancy 
 
  9   genetics, but at least it is a theoretical possibility. 
 
 10             Each of these proviruses, as I say, has a certain 
 
 11   sequence that defines it and each of them probably is

 12   associated with certain kinds of biological properties.  We 
 
 13   don't know, for the most part, what they are. 
 
 14             Only a few of these, particularly one of the XMVs, 
 
 15   are known to give rise to infectious virus and we don't know 
 
 16   whether that is because a very large number of these are

 17   inherently defective or because they have some other 
 
 18   properties where they simply don't replicate well in the 
 
 19   host cell. 
 
 20             This really hasn't been investigated very well.  
 
 21   There has been very little work of cloning full‑length

 22   proviruses and testing them for infectivity and things like 
 
 23   that. 
 
 24             It is known, however, and I will come back to 
 
 25   this, that if you take a virus that is of a type I haven't 

 
                                                                46 
 
  1   shown here, an ecotropic virus, and the mouse expresses

  2   that, in the course of replication in the lifetime of an 
 
  3   animal, you can get a series of recombination events which 
 
  4   join sequences from these various elements and lead to a 
 
  5   pathogenic virus. 
 
  6             [Slide.]

  7             These proviruses are widely distributed.  This 
 
  8   actually doesn't even show all of the proviruses.  It just 
 
  9   shows the proviruses that different between two strains of 
 
 10   mice, and you can see, however, they provide markers and 
 
 11   they were actually useful for genetic purposes for this for

 12   a while‑‑that are distributed across the entire mouse 
 
 13   genome. 
 
 14             As far as we can tell, they are randomly 
 
 15   distributed.  There is a noteworthy cluster here near the 
 
 16   FV1 locus, but we don't know the significance of that.  No

 17   test for significant clustering has ever yielded anything 
 
 18   with any statistical value. 
 
 19             So these are randomly distributed in the provirus.  
 
 20   This may or may not reflect what really goes on in real‑life 
 
 21   species because inbred mice are somewhat special.  Inbred

 22   mice are derived by taking mice that have evolved somewhat 
 
 23   independently and then recombining them.  
 
 24             So one of the things about these individual 
 
 25   proviruses is that probably, individually, they are fairly 

 
                                                                47 
 
  1   benign to the animal but, by bringing strains of mice that

  2   evolved independently back together again, we set up 
 
  3   situations where you get pathogenic interactions that may 
 
  4   not occur in the wild. 
 
  5             We don't know that for sure.  In fact, we 
 
  6   presently have a study going on where we are looking at the

  7   genetics of proviruses in wild strains of mice. 
 
  8             [Slide.] 
 
  9             As I said, endogenous proviruses probably often 
 
 10   evolve in the direction of being fairly benign to their 
 
 11   host.  But, even in the mouse, there are several tumor

 12   pathogenic models that have been pretty well studied.  The 
 
 13   first deals with the fact that the insertion of a provirus 
 
 14   into a particular locus is a genetic event that sometimes 
 
 15   leads to a detectable mutation. 
 
 16             Several known mouse mutations were found to be due

 17   to the insertion of endogenous proviruses.  Particularly 
 
 18   these coat mutations, hairless and dilute, were caused 
 
 19   originally by the insertion of a provirus into the gene and 
 
 20   disrupting, in some way, the activity of that gene. 
 
 21             The expression of endogenous proviruses can also

 22   give rise to specific effects.  I mentioned the 
 
 23   superantigens before which lead to the deletion of specific 
 
 24   subsets.  Expression or replication of endogenous proviruses 
 
 25   can give rise to tumors as in the model of the Akr mouse. 

 
                                                                48 
 
  1             The Akr mouse was originally bred as a mouse that

  2   had a high incidence of thymic lymphoma at a fairly 
 
  3   reproducible age.  When that was studied in detail, it was 
 
  4   found that this mouse, what one had selected for in 
 
  5   generating this strain of mouse was a pattern of proviruses 
 
  6   which gave rise to these so‑called mink‑cell focus‑forming,

  7   or MCF, recombinant viruses that arise. 
 
  8             As a regular evolutionary event, and I will show 
 
  9   you this in a second, in every single one of these mice, 
 
 10   virtually in lock‑step, you have this evolution of viruses 
 
 11   that involves at least four genetic changes to the virus

 12   plus that insertion of the proviruses next to the "myc" gene 
 
 13   and a few other genes in these cells. 
 
 14             Similarly, the mammary‑tumor virus which doesn't 
 
 15   need to undergo this kind of complicated recombination 
 
 16   events but can, in some strains of mice, be expressed,

 17   infect mammary epithelium and give rise to mammary carcinoma 
 
 18   in a fairly high frequency in certain strains of mice. 
 
 19             Then, as I said, there is the genetic alterative 
 
 20   of provirus that goes on in the course of these events. 
 
 21             [Slide.]

 22             This just shows an example of what the 
 
 23   recombination events in the course of this evolution that 
 
 24   occurs of viruses in mice.  As I say, this occurs in every 
 
 25   single Akr mouse virtually in lock‑step during the early 

 
                                                                49 
 
  1   life of this mouse, shortly after birth.

  2             You can get, first, the expression and infection 
 
  3   of mouse tissue with an ecotropic provirus of which there 
 
  4   are only a couple in the mouse that is capable of giving 
 
  5   rise to a full‑length infectious virus and that, then, 
 
  6   spreads around the animal which becomes viremic.

  7             It is noteworthy, by the way, that the mouse, of 
 
  8   course, inherits these proviruses and these proviruses are 
 
  9   capable of giving rise to a virus which will affect, 
 
 10   probably, a large fraction of the tissue of the mouse but 
 
 11   yet you don't actually see that happening until about the

 12   time that the mouse is born. 
 
 13             In cell cultures from embryos, it takes a while 
 
 14   even for cell cultures to start to show this virus.  That 
 
 15   indicates that the methylation that holds the virus in check 
 
 16   is actually pretty tight because if one cell expressed a lot

 17   of virus, you would imagine that the whole animal would get 
 
 18   infected. 
 
 19             But it doesn't happen for a while.  But once that 
 
 20   appears, you again get a rather rapid series of events in 
 
 21   which you acquire first either a new enveloped gene or

 22   partial enveloped gene or new LTR.  This gives the virus, 
 
 23   probably, a unique ability both to replicate in thymus 
 
 24   tissue and to give some stimulation to the tissue.  
 
 25             This enveloped gene actually has site‑stimulatory 

 
                                                                50 
 
  1   effect on cell replication.  You then get a reduplication

  2   event where the enhancer sequence is actually reduplicated 
 
  3   or triplicated in some cases.  Then, finally, you see 
 
  4   proviruses that are integrated next to the proto‑oncogene. 
 
  5             I will give you the source for this picture right 
 
  6   at the very end.

  7             [Slide.] 
 
  8             In addition to causing diseases, the integration 
 
  9   of genetic virus, as I said, can have genetic effects.  I 
 
 10   think I have probably covered most of these already.  You 
 
 11   have the insertional inactivation of genes.

 12             You actually can have insertional activation of 
 
 13   expression.  A famous case, one of my favorite endogenous 
 
 14   provirus stories, and it is not really relevant, but 
 
 15   salivary amylase in humans‑‑the expression of amylase in the 
 
 16   salivary glands in humans is driven by an endogenous virus

 17   LTR which is why starches, of course, taste slightly sweet 
 
 18   to us and may well have had something to do with our 
 
 19   cultural evolution.  Most other species do not have that. 
 
 20             You can get, as I said, spontaneous malignancy due 
 
 21   to virus expression and replication.  You can new antigens

 22   and superantigens.  Endogenous viruses actually also provide 
 
 23   very useful genetic markers and, also, markers for 
 
 24   evolution.  That is another thing we are doing in the lab. 
 
 25             [Slide.] 

 
                                                                51 
 
  1             So, to get back closer to the chase here, one of

  2   the potential hazards of endogenous viruses in 
 
  3   xenotransplantation‑‑this is my own personal view, now, of 
 
  4   course and this will be a lot of the focus of what the 
 
  5   discussion will be later on, but I think it is virtually 
 
  6   certain that at least some recipient cells will become

  7   infected in some significant fraction of patients. 
 
  8             This could be a very limited infection where you 
 
  9   just get a few cells.  It is likely that an immunocompetent 
 
 10   recipient will reject the virus and the infection will go no 
 
 11   further in many cases.  In immunosuppressed individuals, or

 12   perhaps even in some competent individuals, one might get a 
 
 13   spreading infection that, again, might or might not be 
 
 14   resolved after immune restoration, the infection could, 
 
 15   conceivably, lead to‑‑malignancy is the most likely outcome 
 
 16   because models of immunosuppression and other diseases

 17   really require extensive virus replication for long periods 
 
 18   of time. 
 
 19             So that is a lower‑order event whereas in models 
 
 20   where you get malignancy, you have an infection early on.  
 
 21   Even if the virus is cleared, you can still have cells which

 22   have an inactivation that are waiting around.  So this can 
 
 23   occur long after signs of the virus are no longer present, 
 
 24   at least in theory.   
 
 25             I think recombination‑‑although the issue of 

 
                                                                52 
 
  1   recombination of human endogenous viruses is often raised in

  2   this, I think it unimportant in the sense that, in the first 
 
  3   place, it is likely to be a secondary issue.  The important 
 
  4   issue here is whether infection is occurring. 
 
  5             If infection is occurring, then recombination is 
 
  6   actually a pathogenic mechanism.  So one wants to know, A,

  7   is infection occurring and B, is there pathogenesis.  If 
 
  8   there is, then you can go back and ask about mechanism.  
 
  9   This is a mechanistic issue.  It is not an issue which 
 
 10   should be right at the top of our thinking. 
 
 11             Furthermore, actually the recombination events I

 12   showed you in the mice are not essential in other animals 
 
 13   for these things to occur.  You can accomplish some of the 
 
 14   same kinds of things, this is with point mutations of the 
 
 15   virus, if the recombination partners are not available. 
 
 16             Of course, the thing that everybody really worries

 17   about is transmission of the virus particularly to contacts 
 
 18   and so immunocompetent individuals.  This is a very, I would 
 
 19   argue, unlikely outcome but it is obviously where much of 
 
 20   our thinking and discussion should focus. 
 
 21             Finally, I want to change the subject a little bit

 22   for those of you who are interested in learning more about 
 
 23   this, and many of you asked me this question.  I want to 
 
 24   point out that there new Cold Spring Harbor book on 
 
 25   retroviruses is actually coming out.  It is being printed 

 
                                                                53 
 
  1   right now.  The first copies will be mailed out around the

  2   first of the year.  This is really happening for those of 
 
  3   you that have wondered about this over the last how many 
 
  4   years. 
 
  5             Thank you very much. 
 
  6             DR. AUCHINCLOSS:  We do have time later in the

  7   morning before lunch for a general discussion of the 
 
  8   morning's presentations, but I would be perfectly willing to 
 
  9   take a few questions at this point, if you like. 
 
 10             DR. HIRSCH:  John, can you just briefly go over 
 
 11   the history of transmission and disease production with

 12   either polytropic or xenotropic viruses from species to 
 
 13   species?  What is the example when these have been 
 
 14   intentionally transmitted, say, a mouse virus to the pig or 
 
 15   whatever. 
 
 16             DR. COFFIN:  There has been relatively little

 17   work, actually, on that done of intentional infection.  
 
 18   Maybe Robin has more insights into that than I do.  I know 
 
 19   Robin, some years ago, performed experiences with endogenous 
 
 20   avian viruses where, in fact, he could show some 
 
 21   pathogenicity in other species.

 22             I can't think of any examples where introduction 
 
 23   of endogenous, say, mouse virus into other species‑‑of the 
 
 24   straight endogenous viruses, not necessarily recombinant 
 
 25   virus, has actually given rise to any kind of pathogenesis.  

 
                                                                54 
 
  1   But maybe I am blanking on something I should remember.

  2             DR. WEISS:  I agree with John Coffin.  There is 
 
  3   very little data on cross species, pathogenicity of pure 
 
  4   endogenous viruses.  But we do know that viruses such as 
 
  5   mouse leukemia virus will cause disease in other animals.  
 
  6   We had an example of this fairly recently in that

  7   replication‑competent recombinant virus that emerged from a 
 
  8   retroviral vector being tested for gene therapy delivery 
 
  9   gave use to leukemia in monkeys. 
 
 10             Not only that, it happened to transfer some 
 
 11   endogenous AL30‑like elements, too.  So I think if the virus

 12   grows in a foreign species to a high enough load, then there 
 
 13   is no particular protection of that species. 
 
 14             The gibbonate leukemia virus which is reported as 
 
 15   a natural virus of gibbons also has a recent mouse origin. 
 
 16   The transfer probably occurred in recent historical times in

 17   gibbons in captivity.  Gibbons are apes and it causes 
 
 18   leukemia because it grows to a high viral load in those 
 
 19   animals. 
 
 20             So I think there is no question that these types 
 
 21   of the C‑type retrovirus that could emerge from rodents or

 22   other animals can be pathogenic in primates if they take off 
 
 23   and become a rip‑roaring infection in primates. 
 
 24             DR. ONIONS:  I concur with Robin's last comment.  
 
 25   There are data from another model in the cat leukemia virus 

 
                                                                55 
 
  1   which is actually a naturally transmitted virus from cats to

  2   cats.  These actually shed viruses in their saliva that are 
 
  3   infectious both for canine and for human cells. 
 
  4             There is no evidence that these viruses, 
 
  5   nevertheless, have infected either people or dogs.  But 
 
  6   there is a difference.  If you take that virus that is

  7   capable of infecting canine cells and inject it parentally 
 
  8   into newborn dogs that are also immunosuppressed, it is 
 
  9   capable of causing disease. 
 
 10             I think this is important to bear in context of 
 
 11   the route of transmission is important and reflects the

 12   comments that I think came from the floor earlier that one 
 
 13   should be cautious of drawing analogies that we have been 
 
 14   exposed to, to pig tissue in other ways, just in, say, 
 
 15   slaughterhouses and so on, that this is a group that would 
 
 16   be at risk.

 17             That is not necessarily so.  There is a vast deal 
 
 18   of difference between casual exposure and true parental 
 
 19   delivery of these viruses. 
 
 20             DR. COFFIN:  FLV is one of the very few viruses of 
 
 21   this type for which there is a good model for horizontal

 22   transmission.  But all of these viruses can be transmitted 
 
 23   vertically from mother to offspring with some efficiency. 
 
 24             DR. AUCHINCLOSS:  Any more questions? 
 
 25             DR. MICHAELS:  Dr. Onions, is there any data, 

 
                                                                56 
 
  1   then, or has it been looked at to see whether those newborn

  2   dogs which are now infected could then transmit from more 
 
  3   casual types of contact with other dogs? 
 
  4             DR. ONIONS:  These experiments were conducted in 
 
  5   the 1970s by Rikard here in Cornell.  If I remember 
 
  6   correctly, the interesting feature was this was really

  7   before we had good molecular tools. 
 
  8             If I remember correctly, these dogs did not become 
 
  9   viremic.  Some of them developed tumors but most of them 
 
 10   eventually became latently infected and there was no 
 
 11   evidence‑‑I can't be sure but I don't believe there was a

 12   tracer dog in there to check for horizontal transmission.  
 
 13   But, certainly, these dogs were not shedding virus in their 
 
 14   saliva. 
 
 15             DR. SALOMON:  I apologize if I got this wrong, but 
 
 16   you suggested that in the human that there was no evidence

 17   of recent endogenous viral integration which raises a very 
 
 18   interesting point, then.  Are you saying that something that 
 
 19   seems to have been a very powerful evolutionary mechanism 
 
 20   going on for millions of years for some reason, now, we have 
 
 21   reached some point in evolution in the human species that

 22   this is not happening anymore? 
 
 23             DR. COFFIN:  I don't view it that way.  There are 
 
 24   many bird species for which there is also no evidence for 
 
 25   recent endogenous virus infection.  I view it as being more 

 
                                                                57 
 
  1   a chance thing that the appearance of endogenous viruses in

  2   the germ line depends on an endemic infection in the 
 
  3   species.  That has occurred in some species, as in mice, for 
 
  4   example, which probably do transmit exogenous virus around 
 
  5   in the wild even now although we don't know for sure that is 
 
  6   true.

  7             DR. SALOMON:  So you don't believe that would be 
 
  8   an argument, then, that we would be more resistant? 
 
  9             DR. COFFIN:  No; I don't think it is justifiable 
 
 10   at all to argue from the absence of these infections.  Human 
 
 11   cells can be infected with viruses of this kind perfectly

 12   well if they have the right receptor utilization. 
 
 13             DR. AUCHINCLOSS:  Thank you very much.  We will 
 
 14   have a chance, again, to return for discussion on these 
 
 15   overall issues for the morning. 
 
 16             I would now like to ask Dr. Carolyn Wilson from

 17   CBER to talk to us about data that she has demonstrating the 
 
 18   capacity of the porcine endogenous retrovirus to infect 
 
 19   human cells. 
 
 20   Data Demonstrating Capacity of Porcine Endogenous Retrovirus 
 
 21                   (PERV) to Infect Human Cells

 22             DR. WILSON:  Thank you.  I also want to thank Amy 
 
 23   Patterson for providing the opportunity for me to present 
 
 24   some of the data that we have generated here at CBER on 
 
 25   porcine endogenous retrovirus specifically. 

 
                                                                58 
 
  1             [Slide.]

  2             When we first started thinking about what the 
 
  3   safety concerns are in the use of porcine xenografts 
 
  4   regarding the presence of porcine endogenous retrovirus, it 
 
  5   was already well known at that time that these agents 
 
  6   existed, that they were capable of coding infectious

  7   retroviruses. 
 
  8             Therefore, because, as John already pointed out, 
 
  9   this is an endogenous element that is integrated into the 
 
 10   genome of the pigs that there is a potential to generate 
 
 11   infectious porcine endogenous retrovirus in all pig cells.

 12             What wasn't know was what conditions are required 
 
 13   for activation of this virus. 
 
 14             [Slide.] 
 
 15             The approach that we took is outlined on this 
 
 16   slide.  This has been in collaboration with a number of

 17   investigators at CBER.  What we decided to do was look in 
 
 18   primary cells.  We looked a peripheral blood mononuclear 
 
 19   cells from two different breeds.  I will be talking only 
 
 20   about the NIH minipig studies today. 
 
 21             We mitogenically stimulated these cells and then

 22   looked for activation of viral expression as measured by the 
 
 23   retroviral enzyme reverse transcriptase, abbreviated RT. 
 
 24             To then correlate any RT activity with the 
 
 25   presence of an infectious virus, we then did a coculture 

 
                                                                59 
 
  1   assay with different target cells and then looked in these

  2   target cells again for RT activity as a measure of spreading 
 
  3   productive infection as well as a more sensitive and 
 
  4   specific RT‑PCR assay which we have developed for detecting 
 
  5   porcine endogenous retrovirus. 
 
  6             [Slide.]

  7             These are the activation results which we found.  
 
  8   These are NIH minipig PBMCs which we cultured either with 
 
  9   PHA and PMA, shown in blue, or PMA and a calcium ionophore, 
 
 10   shown in red.  On the Y axis is tritiated thymidine 
 
 11   incorporation.  This is a measure of reverse‑transcriptase

 12   activity. 
 
 13             As you can see, at five days there is a clear peak 
 
 14   of RT activity but this is transient.  By day 8, it returns 
 
 15   to baseline levels. 
 
 16             [Slide.]

 17             In the coculture experience that we did, we 
 
 18   directly exposed a pig cell line, ST, which is swine testis, 
 
 19   and a human cell line, 293, which is human embryonic kidney 
 
 20   cells, to either live PBMCs‑‑again, these are NIH minipigs 
 
 21   which have been activated, mitogenically activated, or

 22   irradiated PBMCs. 
 
 23             In either case, we were able to see an increase in 
 
 24   RT activity in the ST cells after about a two to three‑week 
 
 25   lag time.  It took a bit longer going out to more like 40 

 
                                                                60 
 
  1   days before we saw an increase in RT activity.

  2             Actually, it is not shown in this slide, but if we 
 
  3   go out to day 56, RT activity is as high as it is in ST 
 
  4   cells.  If we look at earlier timepoints with more sensitive 
 
  5   RT‑PCR assay, these are the results we have found in the 
 
  6   human 293 cells.

  7             At these early timepoints where we don't see 
 
  8   detectable RT activity, we clearly can see evidence for 
 
  9   transfer and expression of the retrovirus. 
 
 10             [Slide.] 
 
 11             So, because of the delay or the difference in the

 12   time course in the ST and 293 cells after exposure to PBMCs, 
 
 13   we wanted to look at whether or not this represented 
 
 14   different populations of virus that were being activated out 
 
 15   of the primary cells.  So the strategy which we have 
 
 16   developed to study that question is, again‑‑this is what I

 17   have already shown you‑‑mitogenic stimulation of the porcine 
 
 18   PBMCs, exposure to either a pig‑cell line or a human‑cell 
 
 19   line. 
 
 20             These two cell lines are then used as chronic 
 
 21   producers of the virus‑‑they are called here ST/NIH and

 22   293/NIH since they are from NIH.  They have virus in the 
 
 23   mice later from NIH minipig cells‑‑and then going back and 
 
 24   reexposing ST and 293 cells‑‑this is by a coculture assay 
 
 25   using irradiated, lethally irradiated, virus producer cells‑ 

 
                                                                61 
 
  1   ‑and then looking at the time course of viral infection by

  2   RT assay.  So we are looking at the spread of virus in these 
 
  3   cultures. 
 
  4             [Slide.] 
 
  5             On the left side are the results that we found 
 
  6   when we used the pig cell line, ST/NIH, as the source of

  7   chronic virus producer.  As you can see, by two weeks‑‑ 
 
  8   again, this is tritiated thymidine incorporation on the Y 
 
  9   axis, we see evidence for a very productive infection.  It 
 
 10   is pretty much maximal output by two weeks in the pig cell 
 
 11   line.

 12             Human cells become infected but the kinetics is 
 
 13   somewhat delayed.  293 cells, when we use human cells as a 
 
 14   source of virus, shown in the right panel, human cells 
 
 15   become infected and, although it looks like this is more 
 
 16   rapid, I want to just point out this is a different scale

 17   and, actually, if you overlay these curves, they are 
 
 18   actually fairly similar. 
 
 19             But what is quite striking is the pig cells are 
 
 20   infected with much slower delayed kinetics.  At day 32, this 
 
 21   is about 8,000 CPMs compared to 100,000 at day 15.  This

 22   suggested to us that, in fact, we are selecting for 
 
 23   different populations and viruses, that the phenotype of the 
 
 24   viruses coming out of the pig cells and the human cells is, 
 
 25   in fact, different. 

 
                                                                62 
 
  1             [Slide.]

  2             Just to sort of summarize what I have shown you so 
 
  3   far, what we think we are seeing is when we activate primary 
 
  4   cells, that what comes out is actually a population of 
 
  5   naturally occurring varying viruses, that we are not 
 
  6   activating a single porcine endogenous retrovirus, that

  7   depending on the cell line that you put that virus into, 
 
  8   that there is a process of selection, perhaps adaptation, of 
 
  9   accumulation of mutations and we can identify a human tropic 
 
 10   virus. 
 
 11             Again, the issues of concern here are whether or

 12   not that would cause disease in the recipient, whether or 
 
 13   not it would be transmitted to others. 
 
 14             In an initial effort to get a handle on whether or 
 
 15   not this virus can cause disease in the recipient, we have 
 
 16   extended our host‑range analysis of this virus into a

 17   variety of human and non‑human primate cell lines to try to 
 
 18   look at what types of cells or tissues are susceptible to 
 
 19   infection. 
 
 20             [Slide.] 
 
 21             This is the first table.  I know this isn't a very

 22   pretty slide, but what we did is, again, using virus‑ 
 
 23   producer cells, we did lethal irradiation and coculture with 
 
 24   a variety of different target cells.  We then looked at 
 
 25   various time points after coculture by the sensitive RT‑PCR 

 
                                                                63 
 
  1   assay which is specific for the porcine endogenous

  2   retrovirus as well as by the more generic and less sensitive 
 
  3   RT assay. 
 
  4             But the advantage of doing an RT assay is it 
 
  5   allows you to actually determine whether or not it is a 
 
  6   productive spreading infection.

  7             In all cases where you see a positive and the 
 
  8   number of days, that is the earliest time point where we see 
 
  9   a positive signal.  When you see a negative in all these 
 
 10   tables I will be showing you, that is the latest time point 
 
 11   where we tested where it was still negative.  If it is

 12   negative, it has always been negative.  It is not positive 
 
 13   and then goes negative. 
 
 14             In this slide, these are a variety of non‑ 
 
 15   hematopoietic human cell lines.  We looked in cervical 
 
 16   carcinoma cell line, colon adenocarcinoma, liver

 17   hepatoblastoma, astrocytoma, so a variety of different 
 
 18   tissue types, as well as normal skin fibroblasts. 
 
 19             In all cases, at some time, we were able to detect 
 
 20   an RT‑PCR signal suggesting that the virus, in fact, does go 
 
 21   in and, perhaps, is replicating at a low level.  At least it

 22   is getting expressed. 
 
 23             However, only in some of the cell lines were we 
 
 24   able to detect an RT result, in the HeLA cell line and 
 
 25   HepGW, the liver hepatoblastoma.  All other cells at this 

 
                                                                64 
 
  1   time, even in some cases being carried out for almost two

  2   months, are still negative by RT assay suggesting that these 
 
  3   RT‑PCR‑positive results do not necessarily reflect a 
 
  4   productive spreading infection. 
 
  5             [Slide.] 
 
  6             Then we looked at a variety of hematopoietic human

  7   cells.  In this case, we looked at primary monocyte 
 
  8   macrophage and primary lymphocytes.  These are human.  In 
 
  9   both of these cases, we never saw a signal by RT‑PCR or by 
 
 10   RT assay.  This is at day 38 these cultures were carried 
 
 11   out.

 12             In a promonocytic leukemia cell line, THP 1, we 
 
 13   have gotten a signal by RT‑PCR but, again, out to day 46, no 
 
 14   RT activity.  A T‑cell line, PM‑1, we see a signal by RT‑PCR 
 
 15   but never by an RT assay. 
 
 16             [Slide.]

 17             In our analysis of non‑human primate cells‑‑and I 
 
 18   apologize because we have also done this in cos cells and I 
 
 19   forgot to put this on the slide‑‑the results are the same as 
 
 20   vero.  But if we look at rhesus monkey, in this case it is 
 
 21   primary lymphocytes, and we see an RT‑PCR signal.  But this

 22   didn't translate into a positive RT result. 
 
 23             In a rhesus monkey kidney cell line, again, we see 
 
 24   RT‑PCR but never an RT.  In African green‑monkey cells, 
 
 25   either the vero or the cos, we never see an RT‑PCR or an RT. 

 
                                                                65 
 
  1             [Slide.]

  2             So, to summarize what I have shown you, then, we 
 
  3   have demonstrated that mitogenic activation of primary pig 
 
  4   PBMCs releases a type C retrovirus which infects human cells 
 
  5   and we have done EMs.  I am just not showing them to you for 
 
  6   the sake of time.

  7             We think that different virus populations are 
 
  8   selected when primary isolates of this virus are passaged 
 
  9   through pig or human cell lines.  Some human tumor cell 
 
 10   lines can be productively infected with porcine endogenous 
 
 11   retrovirus but it is certainly not true for all we have

 12   looked at.  
 
 13             Cells of human hematopoietic lineages as well as 
 
 14   rhesus monkey cells are susceptible to infection but not a 
 
 15   productive infection.  
 
 16             [Slide.]

 17             So what are the implications of these results?  
 
 18   What I have done here is I have tried to highlight some of 
 
 19   the issues that we think these results are important in the 
 
 20   context of the questions that you will be discussing this 
 
 21   afternoon.

 22             The first, obviously, is whether or not these 
 
 23   findings with PBMCs can be generalized to other porcine 
 
 24   tissues since, obviously, a variety of different porcine 
 
 25   xenograft tissues are being examined for clinical use. 

 
                                                                66 
 
  1             Secondly, what cell substrate is most appropriate

  2   for virus detection.  It is clear that not all human cells 
 
  3   are permissive.  Also, going back to one of the earlier 
 
  4   slides I showed you where the ST cells became positive 
 
  5   before 293s, primary isolates may more efficiently infect 
 
  6   porcine cells.

  7             Third, we don't fully understand the implications 
 
  8   of our positive RT‑PCR but negative RT assay results, but 
 
  9   one hypothesis we have is that this may allow for low‑level 
 
 10   replication, perhaps resulting in accumulation of mutations 
 
 11   and development of a strain which may be more fully adapted

 12   for human cell lines. 
 
 13             Finally, our results with the hematopoietic cell 
 
 14   lineages really beg the question whether human PBMCs are the 
 
 15   best sample for patient monitoring and, if not, what type of 
 
 16   cells would be more appropriate.

 17             I want to thank the committee for their attention. 
 
 18             DR. AUCHINCLOSS:  Thank you very much. 
 
 19             Questions?  A small number of questions for Dr. 
 
 20   Wilson? 
 
 21             DR. COFFIN:  Carolyn, have you used PERT assays at

 22   all? 
 
 23             DR. WILSON:  No; we haven't. 
 
 24             DR. AUCHINCLOSS:  I'm sorry; could you explain 
 
 25   that question just a bit. 

 
                                                                67 
 
  1             DR. COFFIN:  Yes; there is a very much more

  2   sensitive assay for reverse‑transcriptase which is fairly 
 
  3   easy to do which is being studied here. 
 
  4             DR. WILSON:  I have talked to Keith Peden who is 
 
  5   in our center who has been doing a lot of work on those 
 
  6   assays.  In his hands‑‑now, this is more specifically for

  7   HIV.  He has done direct comparisons of an RT‑PCR assay 
 
  8   versus a PERT assay.  He claims that they are relatively 
 
  9   similar in sensitivity. 
 
 10             DR. COFFIN:  But you are comparing it with an RT‑ 
 
 11   PCR assay.  That is different.  What you are looking for is

 12   RT activity in the soup. 
 
 13             DR. WILSON:  Right; these are conventional RT 
 
 14   assays.  I purposely, actually, in this case, want to do 
 
 15   conventional RT assays because I want to see a full‑flown 
 
 16   infection.

 17             DR. COFFIN:  But then that does get to the 
 
 18   question that you got to at the end of whether there is any 
 
 19   spread at all.  For that, it would be a good idea to go to a 
 
 20   very sensitive assay. 
 
 21             DR. WILSON:  But I think that is what the RT‑PCR‑‑

 22             DR. COFFIN:  Oh; that is in soup.  Sorry. 
 
 23             DR. HIRSCH:  Have you passed our cells that are 
 
 24   RT‑PCR‑positive and RT‑negative several times?  I mean, 
 
 25   sometimes it comes out only after many passages. 

 
                                                                68 
 
  1             DR. WILSON:  Right.  Well, some of these cultures,

  2   as you saw, I have carried out 30 to 40 days.  What I plan 
 
  3   to do sort of after the holidays is to continue growing 
 
  4   these cells for longer periods of times. 
 
  5             Also, what I have done now is I have taken sort of 
 
  6   a secondary 293 passage virus and I am repeating the

  7   experiment I have showed you with the ST and 293s.  If you 
 
  8   passage it a second time on 293s, now, actually, the 
 
  9   kinetics on 293 actually does increase.  Those curves are no 
 
 10   longer overlaid. 
 
 11             DR. AUCHINCLOSS:  At this point, the agenda calls

 12   for a coffee break.  I think it is really more like a coffee 
 
 13   gulp, since you have exactly ten minutes to have some coffee 
 
 14   and be back to reconvene at 10:45. 
 
 15             DR. AUCHINCLOSS:  There is an announcement from 
 
 16   Gail.

 17             MS. DAPOLITO:  I would just like to make a short 
 
 18   announcement about an upcoming xenotransplantation workshop 
 
 19   that is going to be held at the NIH in January.  There are 
 
 20   handouts regarding that meeting on the table outside, 
 
 21   handouts and registration forms, for your convenience, if

 22   you would like to fill out the registration forms today and 
 
 23   leave them with Ms. Harvey at the registration table. 
 
 24             Thank you very much. 
 
 25             [Break.] 

 
                                                                69 
 
  1             DR. AUCHINCLOSS:  We are going to resume the

  2   morning session with a presentation from Dr. Robin Weiss, 
 
  3   again on data demonstrating the capacity of the porcine 
 
  4   endogenous retroviruses to infect human cells. 
 
  5    Data Demonstrating Capacity of PERV to Infect Human Cells 
 
  6             DR. WEISS:  Thank you for inviting me.  It has

  7   been a short trip for me because I was in Washington 
 
  8   downtown giving a lecture earlier this morning at the 
 
  9   American Society of Cell Biology.  If I start to talk about 
 
 10   pig herpes viruses instead, it will show I am mixed up. 
 
 11             [Slide.]

 12             Most of our studies have been published already.  
 
 13   The committee has the papers in their big blue folders.  So 
 
 14   I won't go over too much of it and I will try and be brief 
 
 15   so that David Onions has plenty of time, too. 
 
 16             Our work is a collaboration between members of my

 17   laboratory at the Institute of Cancer Research in London, 
 
 18   and collaboration with Jonathan Stoye's lab.  John Coffin 
 
 19   has already mentioned him, a great expert in endogenous 
 
 20   viruses in mammals, and Paul Le Tissier at the National 
 
 21   Institute of Medical Research in London, too.

 22             [Slide.] 
 
 23             We published some eight months ago in Nature 
 
 24   Medicine observations on pig retroviruses that were produced 
 
 25   from two well‑established cell lines in culture, so differs 

 
                                                                70 
 
  1   from Carolyn Wilson's because she was looking at virus that

  2   came straight out of primary cells in normal pigs.   
 
  3             These are established cell lines, but it seemed, 
 
  4   at the time we started this a couple of years ago or so, a 
 
  5   sensible starting point because it has been known for over 
 
  6   20 years that these two cell lines, pig kidney line 15 and

  7   minipig kidney line, released viruses, retroviruses, into 
 
  8   culture and they turn out to be endogenous retroviruses. 
 
  9             We found that the virus released from PK15 pig 
 
 10   kidney cells would grow in another indicator line that 
 
 11   Carolyn has already mentioned, the ST cell line of pigs,

 12   would grow in a mink cell line that is a favorite of 
 
 13   retrovirologists and in kidney 293 cells which is why these 
 
 14   have become a favorite cell for looking at transmission to 
 
 15   human cells. 
 
 16             We did not get a take in a variety of other human

 17   cells including diploid fibroblasts.  However, if we 
 
 18   cocultivated the producer cell line with these human target 
 
 19   cell lines, many of them became positive for productive 
 
 20   infection‑‑that is, they released particles that were 
 
 21   positive by reverse‑transcriptase assays, though we use a

 22   PCR amplified one, what John Coffin called PERT or Walid 
 
 23   would call something else, which is more sensitive. 
 
 24             There were a number of cell lines which were in 
 
 25   our paper in the footnotes but not on the slide that were 

 
                                                                71 
 
  1   not productively infected.  But, among them, several had

  2   been infected.  We could detect DNA provirus there and, by 
 
  3   RT‑PCR, which is, again, very sensitive for RNA 
 
  4   transcription, you can show that there is some expression. 
 
  5             So if you grow cells that are actively producing 
 
  6   the virus, pig cells, together with human cells, some virus

  7   transfers across and, in a variety of cell types, you can 
 
  8   get a productive infection. 
 
  9             In the case of this particular cell line here, we 
 
 10   only got infection of pigs amongst the ones we have tested.  
 
 11   So, using John Coffin's terminology, we would say that this

 12   virus is ecotropic, that it is probably infectious for pigs 
 
 13   and not for humans, whereas this one appears to be 
 
 14   amphitropic, can infect both pigs and foreign species. 
 
 15             In fact, we suspected that this virus was a 
 
 16   mixture and it might be a mixture of a pig‑tropic virus and

 17   a xenotropic virus because, in early experiments where we 
 
 18   used the PK15 virus to rescue a retroviral infector 
 
 19   containing a marker gene, we found that there was no 
 
 20   infectivity for pigs. 
 
 21             It turns out we were correct in suspecting that

 22   the virus produced from the pig kidney cells is a mixture, 
 
 23   but we were incorrect in that it was a mixture of an 
 
 24   ecotropic and a xenotropic virus.  It turns out it is a 
 
 25   mixture of two amphitropic viruses, both of which have the 

 
                                                                72 
 
  1   capacity to enter human cells, one a little more strongly

  2   than the other and one of which is very efficient at 
 
  3   reinfecting pig cells and the other is less efficient. 
 
  4             So we think we have three that are biologically 
 
  5   active‑‑that is, infectious viruses‑‑from these cell lines.  
 
  6   Two viruses from PK15 that we provisionally called PERV‑A

  7   and B, and the PERV‑C from minipig kidney cell line which is 
 
  8   ecotropic. 
 
  9             They have different properties.  They are all very 
 
 10   closely related in genome sequence, if you look at the 
 
 11   reverse‑transcriptase or protease parts of the genome, which

 12   we have characterized as virtually indistinguishable.  But 
 
 13   when you look at the envelope, which is probably the major 
 
 14   determinant of host range, their infectivity for different 
 
 15   cells in culture, they turn out to be different. 
 
 16             [Slide.]

 17             So lets look at the PERV‑A and B, the viruses with 
 
 18   envelopes that can deliver the virus to human cells.  A 
 
 19   sequence analysis indicates that they have a very common 
 
 20   sequence across the transmembrane part of the envelope gene 
 
 21   as we would expect and the bottom part of the surface

 22   glycoprotein.  These are very closely related in mouse and 
 
 23   cat leukemia viruses, too. 
 
 24             But the two subtypes of the pig endogenous virus 
 
 25   differ particularly in the regions that, by analogy to what 

 
                                                                73 
 
  1   is already known about the mouse leukemia viruses, are the

  2   regions that interact with cell surface receptors and 
 
  3   determine host range at domain A and B, the variable 
 
  4   sequences in the protein‑rich the domain here where they are 
 
  5   quite widely divergent. 
 
  6             This divergence here has allowed us to prepare

  7   specific probes, PCR probes and the hybridization probes, 
 
  8   that allow the differentiation between PERV‑A and B and C so 
 
  9   that we can distinguish these different virus infections 
 
 10   molecularly and can distinguish how many genomes of each 
 
 11   there are.

 12             [Slide.] 
 
 13             If we look at the host range in culture, the 
 
 14   susceptibility of cells in culture which may not be the same 
 
 15   as in vivo susceptibility, we can show you here just A and 
 
 16   B.  In this case, we have made an artificial virus.  We have

 17   taken a fairly standard murine leukemia virus vector for the 
 
 18   gag and pol genes and have supplied it with the envelope 
 
 19   genes cloned from the pig virus subtype A or subtype B and 
 
 20   have then allowed it to rescue a betagalactosidase gene as a 
 
 21   marker.

 22             What we find is that PERV‑A has a high efficiency.  
 
 23   This envelope will deliver with high efficiency into pig 
 
 24   cells and mink cells but poorly into human cells.  But is 
 
 25   positive. 

 
                                                                74 
 
  1             PERV‑B delivers with fairly high efficiency to

  2   mink cells but poorly to pig cells and rather poorly to 
 
  3   human cells, although the titer is twice that of pig cells.  
 
  4   This is a positive control.  This is an endogenous virus of 
 
  5   cats. 
 
  6             So one of the conclusions here is that, in fact,

  7   these viruses, at least as cloned recombinant vectors, their 
 
  8   envelopes are rather inefficient at delivering the virus to 
 
  9   human cells.  We think the suspectibility of human cells to 
 
 10   these pig viruses is positive but is low. 
 
 11             However, if you keep recycling the virus in human

 12   293 cells, the whole live virus, take off the supernatant 
 
 13   and put it back on new cells and do that for six or seven 
 
 14   passages, then the efficiency replication does increase.  
 
 15   But it is not going up remarkably.  So we think there is a 
 
 16   low susceptibility but a real susceptibility for human cell

 17   infection as gauged in culture. 
 
 18             [Slide.] 
 
 19             We have also, and this took a lot of work, 
 
 20   biologically separated the A and B viruses because they were 
 
 21   growing as a mixture in the 293 cells.  So we took

 22   supernatant and we made serial dilutions on new human 293 
 
 23   cells and the PERV‑B is growing at a higher titer. 
 
 24             But, by making a whole series of serial dilutions, 
 
 25   a lot of microtiter wells, we were able to obtain 293 cells 

 
                                                                75 
 
  1   that were singly infected with PERV‑A or singly infected

  2   with PERV‑B.   
 
  3             [Slide.] 
 
  4             If we took those cells that became chronically 
 
  5   infected, so all the cells are infected, and we took a range 
 
  6   of laboratory C‑type viruses, mouse leukemia viruses,

  7   gibbon‑8 leukemia virus, the cat endogenous virus, we could 
 
  8   then ask whether these two viruses used common receptors to 
 
  9   enter cells.  This is a classical retroviral interference 
 
 10   test where the cells are chronically infected with these 
 
 11   viruses, each singly and a passage until all their receptors

 12   are saturated, you then come in and challenge them with a 
 
 13   retroviral vector with a reporter gene that has the 
 
 14   envelopes of the same viruses. 
 
 15             A plus means you get positive blocking of 
 
 16   preinfection with the whole retrovirus blocks the entry of

 17   the marker virus, the vector. 
 
 18             What we can see here‑‑I mean, a message that may 
 
 19   or may not be important for health purposes, is that these 
 
 20   two pig viruses that do have the capacity to place on human 
 
 21   cells, appear to be getting in by different cell surface

 22   receptors, as we might have expected from the sequence 
 
 23   analysis, at least Jonathan Stoye said it was perfectly 
 
 24   obvious but it is also nice to back obvious suppositions 
 
 25   with actual fact. 

 
                                                                76 
 
  1             [Slide.]

  2             If we take the specific probes that distinguish 
 
  3   between PERV‑A and B and all other C‑type viruses, and look 
 
  4   at normal pig tissues‑‑here are some abattoir organs and 
 
  5   some ordinary peripheral blood lymphocytes from a standard 
 
  6   Landroce Duroc F1 hybrid pigs, we find that both the genomes

  7   of the A subtype virus and the B subtype virus are expressed 
 
  8   in normal tissues. 
 
  9             I just give them as pluses and minuses here.  In 
 
 10   fact, the PBLs and the spleens are richest for expression 
 
 11   and we think that expression is mainly in blood cells and,

 12   of course, hearts and kidneys have blood cells in them.  But 
 
 13   we haven't done really careful cell separations. 
 
 14             This is just showing that there is some expression 
 
 15   at the RNA level.  It is not showing that these normal 
 
 16   animals destined for the butchers and your kitchens are

 17   releasing live virus.  We don't have that evidence.  We are 
 
 18   just saying is they have the sequences in them and those 
 
 19   sequences can be expressed. 
 
 20             [Slide.] 
 
 21             If we take normal pig DNA from a variety of

 22   different strains of pigs, including these meat‑strain pigs, 
 
 23   the minipigs that Carolyn Wilson has already mentioned, 
 
 24   Meishan Chinese pigs that were probably separately 
 
 25   domesticated thousands of years ago, separately from the 

 
                                                                77 
 
  1   European wild pigs, we find that there aren't many copies.

  2             This is a southern blot along the lines that John 
 
  3   Coffin showed from murine leukemia viruses where each band 
 
  4   essentially represents a different exogenous viral genome 
 
  5   somewhere in the pig DNA.  Again, they are probably 
 
  6   scattered amongst different chromosomes and the people

  7   interested in pig genomics are actually quite excited about 
 
  8   using these as markers just as they were used for mouse 
 
  9   genetics. 
 
 10             Our analysis would indicate that there are more 
 
 11   copies of subtype A than subtype B and that certain bands

 12   are common across these widely different strains of pigs.  
 
 13   Now, that is significant if we want to pose the question, 
 
 14   can we eliminate these endogenous viral genomes by 
 
 15   classical, conventional breeding.  Can we take distantly 
 
 16   related pigs and breed them and get out virus‑free animals.

 17             That may be possible but it doesn't look as if it 
 
 18   is going to be easy.  It is going to be a hard, long job.  
 
 19   It has been done with chickens that have had not quite as 
 
 20   many copies as these but, certainly, several copies and it 
 
 21   was successful.

 22             So I think what we need now is an analysis of 
 
 23   those pigs that are being used by the pharmaceutical biotech 
 
 24   companies of these different strains of viruses and the 
 
 25   systemic, systematical, careful cloning of these different 

 
                                                                78 
 
  1   proviruses to see which ones have the potentiality to give

  2   rise to live virus. 
 
  3             Again, as John Coffin said, many of these may be, 
 
  4   so to speak, dead genomes.  They may be defective and not be 
 
  5   able to give rise to live virus.  It is going to be 
 
  6   important to find which of them can give rise to live virus

  7   and can give rise to viruses with these envelopes that can 
 
  8   infect human cells. 
 
  9             These specific probes, as well as specific probes 
 
 10   we have developed in the protease region of the genome, we 
 
 11   have tested out for sensitivity on human cells and primate

 12   cells.  We have very sensitive nested PCR techniques that 
 
 13   will only pick up pig sequences and not endogenous primate 
 
 14   sequences. 
 
 15             We can get down to between 1 and 3 copies of the 
 
 16   pig genome.  I think Walid Heneine will speak on the CDC's

 17   experience with this, too.  So, with these probes that are 
 
 18   all in the public domain, one can use them to probe exposed 
 
 19   humans and to see whether these genomes have got across and 
 
 20   whether pig viruses can be detected in human tissue. 
 
 21             I think that will be useful for later analysis by

 22   a number of labs investing in xenotransplantation. 
 
 23             I will stop at this point.  Thank you. 
 
 24             DR. AUCHINCLOSS:  Thank you very much. 
 
 25             Any particular questions for Dr. Weiss before we 

 
                                                                79 
 
  1   move on?

  2             DR. HIRSCH:  Robin, when you looked at the spleen, 
 
  3   heart, kidney and PBL, does that mean you didn't look at 
 
  4   other tissues or that other tissues were negative, is the 
 
  5   first question.   
 
  6             The second question is we heard earlier this

  7   morning that several hundred people have been exposed to pig 
 
  8   tissues in one form or another.  Is it possible to use fixed 
 
  9   tissue from some of these preparations with your probe? 
 
 10             DR. WEISS:  Yes; it is possible to use our probe‑‑ 
 
 11   well, let me answer the first question first.  This is Dr.

 12   Marty Hirsch asking.  We went down to the abattoir and got a 
 
 13   job lot of organs, and we have not done a more systematic 
 
 14   analysis.  I think David has so he might speak to that. 
 
 15             Do you want to wait for your talk or answer that 
 
 16   one now?

 17             DR. COFFIN:  It is not my work.  It is the work of 
 
 18   Gilian Langford at Imutran.  She has shown expression in 
 
 19   very many different tissues including, I think, the ear.  
 
 20   Our working assumption is that this expression is coming 
 
 21   probably from polymorphic nuclear cells which are positive

 22   wherever you look. 
 
 23             So we think that these are probably infiltrating 
 
 24   cells.  But we don't have formal evidence for that. 
 
 25             DR. WEISS:  After analyzing human material, the 

 
                                                                80 
 
  1   only experience we have had, and this is rather preliminary,

  2   concerned material from two kidney dialysis patients in 
 
  3   Sweden who were exposed for a very short period by dialyzing 
 
  4   their blood through a pig kidney on a slab rather than 
 
  5   through a machine. 
 
  6             We have looked at prebleeds and serial blood

  7   samples afterwards up to two years out.  Using our most 
 
  8   sensitive techniques that get down to about 1 to 3 copies of 
 
  9   pig genomes and using mitochondrial probes is, again, 
 
 10   absolutely specific for the pig mitochondrial DNA rather 
 
 11   than human mitochondrial DNA.

 12             So far, we have struck a negative.  We got a 
 
 13   terrible shock because one of the patients, two years out, 
 
 14   turned out to be positive.  It was contamination in the lab.  
 
 15   Even though the extractions were made in a different medical 
 
 16   school halfway across London and then brought to our lab, we

 17   still, somehow, got contamination.  It was both positive for 
 
 18   mitochondrial DNA and viral DNA so it was pig DNA that got 
 
 19   across. 
 
 20             So that is just a cautionary tale that you cannot 
 
 21   be too careful.  Of course, in academic labs like ourselves,

 22   we are not as clean as sort of quality‑controlled companies.  
 
 23   But you can never be too careful.  You have got to do it 
 
 24   twice or three times in separately isolated systems. 
 
 25             But, in fact, from those two patients, the studies 

 
                                                                81 
 
  1   have been entirely negative to date once we cleaned up our

  2   act.  But I think the similar techniques, and many labs now 
 
  3   have them down to this level of sensitivity, can be used for 
 
  4   probing.  Perhaps that could be taken up later in the day. 
 
  5             DR. COFFIN:  A quick point.  One has to be a 
 
  6   little bit cautious about interpreting the same‑size bands

  7   with one single enzyme as indicating the same provirus.  
 
  8   Probably a few of those are not, based on our experience 
 
  9   with the mouse. 
 
 10             DR. WEISS:  Yes; John Coffin is absolutely right.  
 
 11   These are bands in a gel and they don't necessarily mean

 12   they are exactly the same sequence. 
 
 13             DR. AUCHINCLOSS:  Thank you very much.   
 
 14             One of the things I appreciate from our speakers 
 
 15   is that you have all stayed so closely within your time 
 
 16   allotments.  Our next speaker, however, was given a time

 17   allotment longer than what we have listed him on the sheet, 
 
 18   so when he speaks longer, he is still doing what we asked 
 
 19   him to do. 
 
 20             David Onions will, again, be talking about data 
 
 21   demonstrating the capacity of PERVs to infect human cells.

 22             DR. ONIONS:  Thank you very much indeed.  I would 
 
 23   like to thank the FDA and, particularly, Amy Patterson.  I 
 
 24   think this is a very important meeting and I think it is 
 
 25   going to be very important in our progression to the safe 

 
                                                                82 
 
  1   introduction of xenotransplantation.

  2             [Slide.] 
 
  3             This is the virus we are concerned about and, as 
 
  4   has already been articulated, the real concern with 
 
  5   xenotransplantation is not so much the concern for the 
 
  6   individual patient, although that is a matter of concern,

  7   but the issue of whether the endogenous retrovirus from the 
 
  8   pig could infect human cells and, either through a selection 
 
  9   process that Robin mentioned in vitro or, possibly, less 
 
 10   remotely, through recombination with human sequences, 
 
 11   commented on by John Coffin, could develop viruses that

 12   could be transmitted on to the general public. 
 
 13             [Slide.] 
 
 14             About two years ago, now, Q‑One Biotech, at the 
 
 15   request of Imutran, sponsored by Imutran, looked at the 
 
 16   expression of human retroviruses from the porcine cell line,

 17   PK15, and these were cocultivated with a variety of cells. 
 
 18             The way this was done is, perhaps, quite relevant.  
 
 19   It was not using cell‑free virus nor was it, in all 
 
 20   instances, direct cocultivation, but used a system which 
 
 21   separates the two cells with a permeable membrane.  I say

 22   this because it falls in between in terms of sensitivity 
 
 23   cell‑free virus and cocultivation. 
 
 24             These were, then, passaged for five passages in 
 
 25   the usual way.  If I could just summarize on the next slide. 

 
                                                                83 
 
  1             [Slide.]

  2             This just summarizes some of the data.  We looked 
 
  3   at a large number of cell lines and I am not going to dwell 
 
  4   on this because the data that has been presented by Robin 
 
  5   and by others and Carolyn are very, very similar.  First of 
 
  6   all, we found that would could get the virus from PK15 cells

  7   into Raji cells.  These were positive by reverse‑ 
 
  8   transcriptase and also positive by electron microscopy and 
 
  9   by PCR, of course. 
 
 10             We found, however, that a large number of cells, 
 
 11   like 293 cells, in our hands, using this transwell system,

 12   we could, in fact, but usually they were RT‑negative; that 
 
 13   is, it was an infection.  The provirus was there, but the 
 
 14   provirus was transcriptionally silent. 
 
 15             This is actually the norm out of our range of 
 
 16   cocultivations we have now done with human cells.  We

 17   normally see the second situation rather than the first 
 
 18   situation.  We are also, of course, all interested in 
 
 19   finding a cell line that might be permissive for the range 
 
 20   of these viruses to do cocultivation studies on patients. 
 
 21             Like Robin, we have found that mink cells can be

 22   infected reasonably efficiently by several of these viruses 
 
 23   but, so far, in our hands, the feline cell line, an in‑house 
 
 24   cell line, has been the most useful. 
 
 25             [Slide.] 

 
                                                                84 
 
  1             What we then did was to try and characterize

  2   molecularly these viruses that were getting into the Raji 
 
  3   cell line.  First of all, we sequenced, obtained the 
 
  4   complete sequence‑‑sequenced the complete genome, the 
 
  5   consensus sequence of the virus that has been released from 
 
  6   PK15 cells.  You must remember, of course, however, these

  7   are a mixture of viruses but we are sequencing the commonest 
 
  8   sequence in that population. 
 
  9             We independently sequenced the genomes of the two 
 
 10   proviruses that have infected the human Raji cells.  To 
 
 11   preempt the next comments, this is identical in sequence to

 12   PERV‑B commented by Robin.  For a variety of reasons, we 
 
 13   call this, under our nomenclature, PoEV2 and this one PoEV1.  
 
 14   These two sequences, we will see in a moment, are distinct. 
 
 15             We then wanted to confirm that the envelope gene 
 
 16   sequence that was present in this provirus in the human

 17   cells did, indeed, confer the ability to infect human cells.  
 
 18   This is important because viruses can get into cells by 
 
 19   wrapping their genome in the envelope of another virus so 
 
 20   that genome A, for instance, could get into a human cell 
 
 21   being wrapped by genome B.

 22             But, as Robin has shown, in fact, both viruses do 
 
 23   get into human cells. 
 
 24             What we did was we took the envelope gene.  We 
 
 25   cloned the envelope gene and then mixed it with a variety of 

 
                                                                85 
 
  1   murine leukemia virus genes to form a pseudotype virus. 

  2   When this pseudotype virus is released, we can then ask the 
 
  3   question how efficiently does this envelope gene which is 
 
  4   derived from the pig‑cell line confer the ability of this 
 
  5   murine virus to infect human and other cells. 
 
  6             [Slide.]

  7             So, this infers an ability to infect about 10,000 
 
  8   infectious units per ml on a standard 293 cell.  On the 
 
  9   other hand, the PERV‑B, as it is, or as we would call it 
 
 10   PoEV‑2 envelope gene only allowed about 10 infectious units 
 
 11   per ml on the same cell line.

 12             So, again, these data, as Robin has already said, 
 
 13   show that this particular genome, at least, confers the 
 
 14   ability to infect human cells but it is very inefficient at 
 
 15   getting into human cells. 
 
 16             [Slide.]

 17             We also have looked at the potential receptor 
 
 18   usage by PERV‑B or PoEV‑2, and, essentially, this is the 
 
 19   same data as Robin with slightly different viruses.  FeLV‑B 
 
 20   uses the same receptor as gibbonate leukemia virus as, 
 
 21   indeed, Robin showed, some years ago and, in fact, this data

 22   just shows that we can infect either the clone‑4 cells that 
 
 23   contain the FeLV‑B genome or control cells that don't 
 
 24   contain the FeLV‑B genome.  They can all be infected by 
 
 25   amphitropic murine leukemia virus, xenotropic murine 

 
                                                                86 
 
  1   leukemia virus, FeLV‑B or FeLV‑C. 

  2             This demonstrates that probably these are using 
 
  3   different receptors to get into cells than the range of 
 
  4   viruses shown here.  So it looks like these virus is using a 
 
  5   receptor distinct from these viruses. 
 
  6             [Slide.]

  7             This is a Tom and Jerry cartoon that sums up many 
 
  8   years of John Coffin and Jonathan Stoye's very distinguished 
 
  9   work on the envelope genes of retroviruses.  Just to make 
 
 10   the point that upstream of‑‑this is the major surface 
 
 11   envelope of protein.  As Robin has already pointed out,

 12   there are two domains, this VRA region and the VRB region, 
 
 13   which are associated with diversity in a whole range of 
 
 14   viruses, whether you talk about murine or feline viruses or, 
 
 15   indeed, the pig virus. 
 
 16             These are domains that are variable and are

 17   believed to be associated or, indeed, one can demonstrate 
 
 18   this is the case, are associated with envelope interaction. 
 
 19             [Slide.] 
 
 20             We now look here, what I have done here is‑‑I am 
 
 21   sorry for the quality of this overhead.  We have now

 22   sequenced four different viruses.  I should say that these 
 
 23   data are a concatenation of data from several groups.  They 
 
 24   are from my own group in the university, from Q‑One Biotech, 
 
 25   from Biotransplant, Pipia Bannerjee, and also from Jay 

 
                                                                87 
 
  1   Fishman at Massachusetts General Hospital.

  2             We put our collection of data together.  These 
 
  3   data have been in the public domain since the spring of this 
 
  4   year, but we just pulled it altogether.  This shows four 
 
  5   distinct envelope genes that we can identify associated with 
 
  6   pig cells.

  7             These are called PoEV‑1, PoEV‑2, PoEV‑3 and PoEV‑ 
 
  8   4.  The important point is that these are distinguished in 
 
  9   two domains, both in the variable region A and the variable 
 
 10   region B.  PoEV‑1 is the virus that we sequenced coming out 
 
 11   of the PK15 cells and is identical to PoEV‑2‑‑that is, PERV‑

 12   B‑‑ through much of its sequence in the envelope gene. 
 
 13             But, in fact, there is a frame shift just upstream 
 
 14   of the proline hinge which may well be associated with their 
 
 15   distinct receptor usage of this virus. 
 
 16             PoEV‑3 and PoEV‑4 are, in fact, very distinct

 17   viruses.  PoEV‑4 is the same as PERV‑A that Robin has 
 
 18   commented on whereas PoEV‑3 is a virus that has been 
 
 19   sequenced by Jay Fishman from an inbred pig cell line.  This 
 
 20   may or may not be the same as the MPK virus.  We must not 
 
 21   jump to conclusions and assume that they are the same, but

 22   it is possible that it is the same virus. 
 
 23             [Slide.] 
 
 24             I just wanted to comment on the general grouping 
 
 25   of these viruses.  These viruses are tightly group and this 

 
                                                                88 
 
  1   group of viruses are related to murine, feline, baboon and,

  2   in fact, the gibbonate leukemia virus.  If we look at the 
 
  3   pulse sequences, this virus is closely related to gibbonate 
 
  4   leukemia virus as it is in its envelope gene as well. 
 
  5             This is important because this group of viruses 
 
  6   biologically behave in a very distinct way than the viruses

  7   like the lentiviruses or, indeed, the foamy viruses or the 
 
  8   bovine and human T‑cell leukemia viruses.  Biologically, 
 
  9   these are a very tight group in terms of their behavior. 
 
 10             [Slide.] 
 
 11             This now just puts these four different sequences

 12   in perspective.  Down here is PERV‑B, which is the virus 
 
 13   commented on by Robin, which we know gets, from our data and 
 
 14   from Robin's data, into human cells.  This is PoEV‑1.  This 
 
 15   is about 10 percent divergence in the envelope gene region. 
 
 16             PoEV‑4, which is the PERV‑A virus which is

 17   identically sequenced to PERV‑A, is more distantly related 
 
 18   but, as Robin has commented, also gets into human cells.  
 
 19   PoEV‑3 and the secuba isolate which has also been widely 
 
 20   used, consists of two characterized isolates, secuba and the 
 
 21   virus isolated by Jay Fishman which is PERV‑MSL.

 22             The complete genome sequences of this virus and 
 
 23   this virus are available.  We have the complete genome 
 
 24   sequence of both of these. 
 
 25             There are some quite, perhaps, important points 

 
                                                                89 
 
  1   here.  PoEV‑1 is actually very abundant in our hands in PK15

  2   cells in terms of its real experience.  We have not yet 
 
  3   demonstrated that this virus is able to get into human 
 
  4   cells.  You have to be careful when using southern probes 
 
  5   because it is very difficult to distinguish this virus and 
 
  6   this virus using a southern blot strategy.

  7             But we have not yet shown this virus actually gets 
 
  8   into human cells. 
 
  9             [Slide.] 
 
 10             Just to summarize a few features of these viruses.  
 
 11   PoEV‑1 is present in virions released from PK15 cells.  It

 12   differs from PoEV‑2‑‑that is, PERV‑B‑‑in the VRB region 
 
 13   through a frame ship.  There is no evidence thus far that 
 
 14   this virus infects human cells.  There is no evidence thus 
 
 15   far that this virus infects human cells.  I am not saying 
 
 16   that it could not but, so far, we have been unable to show

 17   that. 
 
 18             [Slide.] 
 
 19             PoEV‑3, remember, is the most distantly related of 
 
 20   these viruses, at least in the envelope gene sequence.  It 
 
 21   differs from PoEV‑1 in both its VRA and its VRB domains.  It

 22   is a very different virus.  It also differs in another 
 
 23   interesting way for the retrovirologists.  PoEV‑3, I should 
 
 24   say here, utilizes a proline transfer on A primer whereas 
 
 25   PoEV‑1 uses a glycine transfer on A primer.  These are 

 
                                                                90 
 
  1   sometimes features that are used to classify retroviruses

  2   and we normally put them in slightly different groups. 
 
  3             Ironically, these viruses actually have different 
 
  4   tRNA primers.  The reason, I don't know. 
 
  5             The U3 regions which are in the area of the 
 
  6   transcriptional machinery of the virus‑‑that is, the

  7   enhancer region of these viruses‑‑are also very, very 
 
  8   distinct.  We have reported previously that PoEV‑1 has an 
 
  9   LTR enhancer that contains a number of motifs that look like 
 
 10   it would be reasonably well expressed in lymphoid tissue. 
 
 11             PoEV‑3, however, has a very different LTR

 12   structure from PoEV‑1.  We have not yet determined the host 
 
 13   range of PoEV‑3 and we must be careful in making analogies 
 
 14   that this is the same virus as PERV‑C or the MPK virus.  We 
 
 15   don't yet know that and Robin and I will have to compare 
 
 16   some sequences and see if they are the same virus.

 17             But, so far, we don't have any host‑range data.  
 
 18   It is also worth commenting that the MPK virus was reported 
 
 19   by Lieber and Todaro to infect mink cells.  So it is not 
 
 20   entirely clear in my mind, Robin, whether that virus really 
 
 21   is strictly ecotropic.  I think we need to maybe talk about

 22   that. 
 
 23             [Slide.] 
 
 24             But the important point is that PoEV‑1 and 3 
 
 25   display high identity in other regions.  For instance, in 

 
                                                                91 
 
  1   the gag and pol regions, there is 90 percent identity in the

  2   gag region and 96.8 percent identify in the pol region.  So, 
 
  3   in certain domains, these viruses are very closely related. 
 
  4             [Slide.] 
 
  5             The relevance of this is this means that we can 
 
  6   now use strategies to distinguish these viruses, first of

  7   all by using RT‑PCR because the envelope gene is a spliced 
 
  8   product.  This means by using a primer down here, and we can 
 
  9   find conserved primers or primers up here, which are 
 
 10   distinct, we can actually distinguish between all of these 
 
 11   viruses using an RT‑PCR strategy

 12             So we can distinguish four different variants 
 
 13   using an RT‑PCR strategy.  We can use a similar strategy to 
 
 14   identify virions in plasma or in other tissues to identify 
 
 15   specific subgroup viruses. 
 
 16             [Slide.]

 17             However, we can also use these criteria to ask 
 
 18   questions what is going on in terms of pigs, in terms of 
 
 19   primates, and in terms of people because these are the three 
 
 20   groups of data we are going to use to determine the safety 
 
 21   of xenotransplantation, what of these four viruses are being

 22   expressed and is there data in primates that these 
 
 23   proviruses are being expressed in the transplanted organ. 
 
 24             [Slide.] 
 
 25             Rather like Carolyn Wilson, using a very similar 

 
                                                                92 
 
  1   protocol, and I think her data were very important in

  2   demonstrating you could isolate virus from primary cells, we 
 
  3   have looked at the isolation of virus from Imutran's pigs 
 
  4   using stimulated PBNCs.  there is some difference to 
 
  5   Carolyn.  We see P production at day 3, but I don't think 
 
  6   that is important.  

  7             You see the kind of stochastic nature sometimes of 
 
  8   infecting cells.  For instance, so far, we have not infected 
 
  9   293 cells.  On the other hand, we have got the virus into 
 
 10   Raji cells but, this time, there doesn't seem to be RT 
 
 11   positivity and the virus is not spreading through the

 12   culture. 
 
 13             This time, however, we have managed to get into 
 
 14   MRC‑5 cells, which we haven't done before.  We have also 
 
 15   looked at number of primates because these may be useful 
 
 16   models in terms of determining whether we can use these as

 17   models for the safety of xenotransplantation. 
 
 18             So far, and I should say these data are 
 
 19   preliminary, we have not demonstrated infection in vitro in 
 
 20   cynomolgus cells using virus from immunotrans pigs, but we 
 
 21   have demonstrated infectivity of a baboon cell line, the

 22   2C1B cell line.  This cell line is positive by PCR but is 
 
 23   not positive by reverse‑transcriptase. 
 
 24             [Slide.] 
 
 25             I mentioned earlier that the gag regions were very 

 
                                                                93 
 
  1   similar.  We have now expressed recombinant gag protein, the

  2   major capsid protein, and also taken whole virus to try and 
 
  3   develop a western blot which is just under development at 
 
  4   the moment.  It is not yet complete. 
 
  5             This is a marker lane.  I made this slide and I am 
 
  6   at fault; this is the wrong position.  It is not 30

  7   kiloDaltons.  It is actually lower than that.  This is a 
 
  8   peptide antisera that we have raised which is common to all 
 
  9   the viruses that we so far have sequenced.  This is a 
 
 10   negative control which is a supernatant from a non‑infected 
 
 11   Raji cell culture.

 12             This is a Raji‑infected culture and it picks up a 
 
 13   P30‑like protein in this Raji cell line.  We are now doing 
 
 14   mass spec to sequence this.  We are doing mass‑spec 
 
 15   sequencing to confirm that it is the virion protein. 
 
 16             This is actually using recombinant P30.  Again,

 17   this peptide antisera picks up the recombinant sera.  We are 
 
 18   now beginning to go through a whole series of sera from 
 
 19   people who might be in risk occupations.  This is a normal 
 
 20   serum, normal human serum.  It is not entirely normal.  It 
 
 21   is my Ph.D. student and she is far from normal.

 22             But these are three sera that are quite 
 
 23   interesting.  They come from patients who have acute 
 
 24   lymphoblastic leukemia, and they are a group of eight 
 
 25   patients who were identified by the Leukemia Research Fund 

 
                                                                94 
 
  1   Epidemiology Center in the U.K.  These are all butchers or

  2   abattoir workers from the South Wales region of the United 
 
  3   Kingdom and all developed leukemia in a relatively short 
 
  4   time frame as well.  Unusually, they were acute 
 
  5   lymphoblastic leukemias.  None of these are positive for the 
 
  6   P30.

  7             [Slide.] 
 
  8             I just want very briefly at the end‑‑how do we put 
 
  9   all these data together to think of the testing strategy.  I 
 
 10   just want to comment in two minutes about models that we 
 
 11   have.  This is a model from the cat which is a natural

 12   model.  These viruses are transmitted from cat to cat.  We 
 
 13   know a lot about this virus.  Many hundreds of thousands of 
 
 14   samples, literally, have been screened from cats. 
 
 15             We have been able to identify‑‑I say "we;" I mean 
 
 16   the whole collective field has been able to identify a

 17   number of patterns of infection.  The commonest is actually 
 
 18   recovery.  Most animals infected by this virus recover.  
 
 19   They have a transient viremia with antigen, virus antigen 
 
 20   which usually the capsid protein can detect and plasma 
 
 21   virus.

 22             But, after a period of time, that declines and the 
 
 23   animals recover with neutralizing and antibody to that P30 
 
 24   protein.  That is the commonest pattern of infection.  The 
 
 25   worst kind of infection is a persistent infection which 

 
                                                                95 
 
  1   virus antigen and, also, of course, plasma viremia persist

  2   for life.  It is these groups of animals that die. 
 
  3             This is also true in the primates that have been 
 
  4   infected with amphitropic murine leukemia virus, the studies 
 
  5   Robin referred to.  They fell into this pattern of infection 
 
  6   before dying of leukemia or lymphoma.

  7             We see two other modes of infection, however.  One 
 
  8   we call sequestered infection in which you get an apparent 
 
  9   transient infection.  But this time the virus is eliminated 
 
 10   from the bone marrow in the blood stream but is confined to 
 
 11   epithelial glands like the salivary gland or, perhaps, the

 12   mammary gland.  We can detect in the circulation antibody; 
 
 13   no virus, but we can, sometimes, not always, detect antigen 
 
 14   of the virus which seems to leak back from the epithelial 
 
 15   surface. 
 
 16             Finally, there is a true latent infection which,

 17   again, looks like a recovery infection but there is no virus 
 
 18   in the plasma.  But if we look in the bone marrow or we look 
 
 19   in the lymph nodes, we can find viral sequences which tend 
 
 20   to disappear through clonal extinction over a period of 
 
 21   time.

 22             It is this pattern of infection that is probably 
 
 23   the kind of infection that was seen by Cornetta in his 
 
 24   studies when he put amphitropic murine leukemia virus into 
 
 25   primates.  I think, if I had to make a bet, and we shouldn't 

 
                                                                96 
 
  1   in these kinds of meetings, this is the kind of infection

  2   that we most likely might see in patients exposed to PoEV, 
 
  3   if we see an infection. 
 
  4             [Slide.] 
 
  5             Just to show the difference between these; this is 
 
  6   a cat's mucous membrane.  This is the nasal mucous membrane

  7   and the submucosal glands stained with an antibody to that 
 
  8   P30.  This is cat virus, I emphasize.  These animals are 
 
  9   throwing out 105 infectious units into saliva and mucous 
 
 10   secretions. 
 
 11             This just shows that effectively all the

 12   epithelial cells become infected in these florid infections.  
 
 13   This persistent viremic type of infection is very different 
 
 14   from the kind of latent infection that is seen in other 
 
 15   systems. 
 
 16             [Slide.]

 17             What does this mean in terms of a testing 
 
 18   strategy?  This is the last overhead.  Just to emphasize 
 
 19   that if we are going to talk about using these collective 
 
 20   data in terms of testing patients, then I think we have got 
 
 21   to account for all those types of infection; transient,

 22   persistent, latent and sequestered.  What I want to draw 
 
 23   your attention to is that we tend to jump looking at this 
 
 24   population, peripheral blood mononuclear cells, but there 
 
 25   are occasions when this is not the most useful site. 

 
                                                                97 
 
  1             In a persistent infection, it is plasma viremia

  2   that is the critical determining event of disease.  We 
 
  3   occasionally see tumors developing in these latent animals 
 
  4   that don't have a plasma viremia but it is uncommon.  Tumors 
 
  5   and other conditions normally develop in these animals that 
 
  6   have high‑titer plasma viremias.

  7             But I also want to draw your attention to this 
 
  8   group of infections, the sequestered infections, where there 
 
  9   is neither plasma virus nor provirus in the peripheral blood 
 
 10   mononuclear cells and we have to look for other criteria of 
 
 11   infection like antibody or plasma antigen.

 12             Thank you.   
 
 13             DR. AUCHINCLOSS:  Questions for Dr. Onions? 
 
 14             DR. LERCHE:  My question would be you said you 
 
 15   expect the latent or the sequestered pattern to be most 
 
 16   likely in human recipients.  Would you expect the pattern‑‑

 17   or how would the distribution of these outcomes be 
 
 18   influenced by the immunosuppressive regimens. 
 
 19             DR. ONIONS:  That is an important point.  In fact, 
 
 20   studies in cats can switch the start of a latent infection 
 
 21   back into a productive viremic phase.  That is certainly

 22   true and studies done in this country by Ed Hoover, I think, 
 
 23   have very elegantly shown that. 
 
 24             I perhaps shouldn't speculate like this but I just 
 
 25   think from the data that we are all collectively getting 

 
                                                                98 
 
  1   from Carolyn and Robin and ourselves that this virus is not

  2   very infectious for human cells.  It does get into human 
 
  3   cells, clearly, but it is a sort of grumbling type of 
 
  4   infection. 
 
  5             That, to me, would suggest that the patterns of 
 
  6   infection we might see are those kinds of infection.  Of

  7   course, there is a balance between what will happen in 
 
  8   immunosuppression and I don't know. 
 
  9             DR. COFFIN:  In the cases where there has been 
 
 10   more than one sequence determination of a given type of 
 
 11   virus, are those sequences identical to the base, because

 12   this addresses the number of possible infectious proviruses 
 
 13   there are. 
 
 14             DR. ONIONS:  Yes.  We have two clones of PoEV‑‑I 
 
 15   could be wrong about this.  One of them is identical in the 
 
 16   envelope sequence to the sequence Robin has, the available

 17   sequence of PoEV.  The other one has got one nucleotide 
 
 18   difference but it is conserved. 
 
 19             DR. COFFIN:  That is actually not good news 
 
 20   because that suggests the likelihood of a second provirus 
 
 21   that is very, very similar but with similar biological

 22   activity. 
 
 23             DR. ONIONS:  We don't know whether that has 
 
 24   occurred during‑‑ 
 
 25             DR. COFFIN:  Right; it obviously could be‑‑in the 

 
                                                                99 
 
  1   worst case‑‑it is good news to see the identical sequence,

  2   potentially good news to see the identical sequence, but 
 
  3   potentially to not see it. 
 
  4             DR. ONIONS:  The PoEV sequence; I'm sorry, I just 
 
  5   can't remember.  The sequence that Robin, of course‑‑PoEV‑3.  
 
  6   They are very, very similar.  I think they are almost

  7   identical.  I honestly can't remember.  I will obviously 
 
  8   talk to Robin about that and to Jonathan. 
 
  9             I should add these data are all available.  They 
 
 10   are all in public domain and have been since the spring in 
 
 11   case people are wondering.

 12             DR. SALOMON:  There is certainly no quick answer 
 
 13   to the question I have but a critical question that is on 
 
 14   the table for this group right now is what kind of screening 
 
 15   strategy we use, and that is relevant even to these 
 
 16   retrospective studies that we have heard today are going on,

 17   for example, from Corinne Savill at Imutran. 
 
 18             So, as I listen to this, there are so many 
 
 19   subtleties here.  I want to make sure that I make sure that 
 
 20   I understand what the different speakers are saying.  What 
 
 21   cell and what screening strategy should we be using.  I am

 22   not talking about PCR, now, or something but just what 
 
 23   target.  Is it peripheral blood mononuclear cells? 
 
 24             It seems to be pretty good, but how good is it? 
 
 25             DR. ONIONS:  I think Carolyn Wilson made a point 

 
                                                               100 
 
  1   that, perhaps, those cells were not particular set to an

  2   infection.  That is a very important point.  But you are 
 
  3   also limited by what you can access. 
 
  4             My own view is that we certainly should look at 
 
  5   peripheral blood mononuclear cells for infection, and that 
 
  6   might be a latent infection.  It might not be productive. 

  7   It could be just the proviruses, though. 
 
  8             I believe we should also look at the plasma for 
 
  9   the virus.  Let me make just a suggestion.  Perhaps this 
 
 10   pigs might produce virions but they might fall into one of 
 
 11   the classes of virions that do not infect human cells at

 12   all, but we would like to know whether they are there or 
 
 13   not. 
 
 14             So we need to able to look at plasma and be able 
 
 15   to distinguish which virus is present in the plasma.  I 
 
 16   would reemphasize that where we normally see disease, that

 17   is usually associated with a high‑titer plasma viremia‑‑not 
 
 18   always, but usually. 
 
 19             I think that is the real worry I have is when you 
 
 20   get that situation.  So I would always want to look at 
 
 21   plasma.  And I would also want to look at antibody.  I know

 22   these patients are going to be immunosuppressed, but I would 
 
 23   still want to look at antibody because that helps you define 
 
 24   certain categories of infection, particularly infections 
 
 25   where the virus might be lurking somewhere that you are not 

 
                                                               101 
 
  1   sure.  You can't find the virus in the blood stream but it

  2   might be in some other site. 
 
  3             DR. AUCHINCLOSS:  Thank you very much. 
 
  4             I think we will move on to the next presentation 
 
  5   by Michael Egan from Diacrin/Genzyme describing their data 
 
  6   from clinical studies with their assays for detection of

  7   porcine endogenous retroviruses. 
 
  8        Data from Clinical Studies: Diacrin/Genzyme Assay  
 
  9                       and Clinical Study  
 
 10             MR. EGAN:  Good morning.  We also want to thank 
 
 11   the committee and the FDA for the opportunity to present.

 12   Our objective today is to give an overview of our program 
 
 13   but, unlike what is listed on the sheets you have, I will 
 
 14   not be able to do this alone. 
 
 15             I am going to have help in the form of Alan Moore 
 
 16   who is going to present our assay systems and our clinical

 17   data using those assay systems and then Dr. Steven Fink who 
 
 18   will review our medical data from our phase I program. 
 
 19             My objective is to give you an overview of the 
 
 20   program as well as to take you through some of the 
 
 21   manufacturing and quality‑control issues so that you get an

 22   understanding of what we are trying to achieve. 
 
 23             [Slide.] 
 
 24             Our focus is the use of porcine field tissue for 
 
 25   the treatment of Parkinson's and Huntington's disease.  Both 

 
                                                               102 
 
  1   of these diseases are neural degenerative diseases where

  2   very specific cell types are lost and, therefore, you have 
 
  3   the capability of replacing them. 
 
  4             The current treatments for these diseases, in the 
 
  5   case of Parkinson's disease is L‑dopa which is clearly 
 
  6   effective but only for a certain period of time and then it

  7   is efficacy wears off.  There is no such treatment for 
 
  8   Huntington's disease to date. 
 
  9             A lot of the basic science in this area was done 
 
 10   using human fetal tissue and it has been shown that human 
 
 11   fetal tissue used in Parkinson's disease patients can

 12   reverse their symptoms.  Similarly, in our own preclinical 
 
 13   studies, we have been able to show that porcine fetal tissue 
 
 14   can work in Parkinson's and Huntington's disease models. 
 
 15             [Slide.] 
 
 16             To give you a bit of history, we began this

 17   process in our first meetings with the FDA in April of '94 
 
 18   and then it was approximately a year later where we treated 
 
 19   our first Parkinson's disease patient. 
 
 20             I want to emphasize here that the Parkinson's 
 
 21   disease patients that we are focusing on are these late‑

 22   stage patients.  These are patients whose L‑dopa is no 
 
 23   longer effective.  Our first Huntington's disease patient 
 
 24   was treated in May of 1966. 
 
 25             [Slide.] 

 
                                                               103 
 
  1             We continued this program and concluded the

  2   recruitment in October of 1996 for the Parkinson's disease 
 
  3   and March of 1997 for Huntington's disease.  There were 12 
 
  4   patients treated under each of these conditions so there is 
 
  5   a total of 24 patients that have now been treated. 
 
  6             Since that time, we have continued meetings with

  7   the FDA and have also clearly, over the past several months, 
 
  8   spent a fair amount of effort in collaboration with them and 
 
  9   their scientists addressing the porcine endogenous 
 
 10   retrovirus issue. 
 
 11             [Slide.]

 12             The manufacturing here clearly has a number of 
 
 13   parts to it.  I am going to focus on the four major aspects 
 
 14   to give you, again, the sense of what we are trying to 
 
 15   achieve from the manufacturing and quality perspective. 
 
 16             [Slide.]

 17             The first aspect is clearly the qualification of 
 
 18   the animals.  We go through an extensive screening that is 
 
 19   done on an animal‑by‑animal basis in order to be admitted 
 
 20   into the program and use as a source of tissue. 
 
 21             The screening consists of screens for parasites,

 22   bacteria, mycoplasm and viruses.  These viruses do not 
 
 23   include the porcine endogenous retrovirus because the data 
 
 24   that is available now to look for that was developed later.  
 
 25   We will talk about that in a moment. 

 
                                                               104 
 
  1             [Slide.]

  2             They are then transferred to a biomedical animal 
 
  3   facility. [Slide.] 
 
  4             The objective of a biomedical animal facility is 
 
  5   to maintain the health status of the animals once they have 
 
  6   been screened.  This biomedical animal facility has filtered

  7   air.  You will see, through some of the photographs I am 
 
  8   going to show you, that the personnel are required to gown 
 
  9   as entry into that facility. 
 
 10             [Slide.] 
 
 11             Animals have extensive checks from the veterinary

 12   staff and then, very importantly, all of the procedures that 
 
 13   are done on those animals in preparation for harvest are 
 
 14   done under aseptic conditions. 
 
 15             [Slide.] 
 
 16             Here you will see an animal being prepped for

 17   surgery.   
 
 18             [Slide.] 
 
 19             Clearly, the staff is dressed appropriately.  This 
 
 20   is done by a veterinary surgeon where the intact uterus is 
 
 21   removed at the appropriate time during gestation.  It is

 22   then double bagged and is ready to be transferred to an 
 
 23   isolation facility. 
 
 24             [Slide.] 
 
 25             This isolation facility is run under GMP.  That is 

 
                                                               105 
 
  1   good manufacturing practice.  These are a set of standards

  2   that are in place in the pharmaceutical industry to which we 
 
  3   adhere. 
 
  4             [Slide.] 
 
  5             This isolation facility is dedicated.  As I said 
 
  6   earlier, we have all the GMP controls and, very importantly,

  7   the quality control and quality assurance is in place all 
 
  8   during this process.   
 
  9             [Slide.] 
 
 10             Dedicated room.   
 
 11             [Slide.]

 12             Again, staff in full aseptic dress. 
 
 13             [Slide.] 
 
 14             There they are unloading the uterus.  This is the 
 
 15   beginning of the process.  This is done inside a biological 
 
 16   cabinet where the fetuses are isolated.

 17             [Slide.] 
 
 18             It is then moved into a cell isolation facility 
 
 19   where, again, we have biocontainment.  You will see the 
 
 20   technician is appropriately dressed.  This is a microscope‑ 
 
 21   outfitted hood so that all the microdissections are done

 22   within the biological cabinet. 
 
 23             [Slide.] 
 
 24             All of the quality assurance is in place such that 
 
 25   every piece of equipment and every reagent that is used is 

 
                                                               106 
 
  1   tracked.

  2             [Slide.] 
 
  3             Air monitoring goes on continuously, both in the 
 
  4   room as well as in the hood during the process. 
 
  5             [Slide.] 
 
  6             As far as final release is concerned, once the

  7   cells have been isolated, and you will see them right here, 
 
  8   beginning with that intact uterus through all the 
 
  9   processing.  
 
 10             [Slide.] 
 
 11             This is basically what you end up with.

 12             [Slide.] 
 
 13             There is a quality‑control step prior to their 
 
 14   release. 
 
 15             [Slide.] 
 
 16             Then it is through a pass‑through such that you

 17   never get the crossing of incoming product and outgoing 
 
 18   product as part of that quality‑control process. 
 
 19             [Slide.] 
 
 20             Therefore, our objective, overall, in this program 
 
 21   clearly is the production, the quality production of porcine

 22   fetal tissue.  We have treated 12 patients in our phase I 
 
 23   Parkinson's disease program and 12 as well in our 
 
 24   Huntington's disease program.  We have and we will continue 
 
 25   to work with the FDA on the issue of porcine endogenous 

 
                                                               107 
 
  1   retrovirus, and that gives me the opportunity to introduce

  2   Alan Moore, who has actually done all of our assay 
 
  3   development and patient testing. 
 
  4             DR. AUCHINCLOSS:  It is obviously very important 
 
  5   that we try and stay in our time allotments here as we come 
 
  6   towards the end of the morning.  Clearly, this is the

  7   important part of the presentation in terms of the assays, 
 
  8   so we want to focus on that. 
 
  9             MR. MOORE:  I will try to cover it thoroughly but 
 
 10   succinctly.  I apologize for the overheads but some of these 
 
 11   were wet as of last night.

 12             [Slide.]  
 
 13             Our laboratory has been involved in the 
 
 14   characterization of the porcine endogenous retrovirus of the 
 
 15   porcine neural tissue.  One of the things that marked this 
 
 16   program, at least from our perspective, was the opportunity

 17   to explore samples from patients post‑treatment.  So it is 
 
 18   not an opportunity that we have in a QC laboratory very 
 
 19   often. 
 
 20             So our program included an in vivo component which 
 
 21   was the analysis of patient samples from the Parkinson's

 22   disease trial with the DNA PCR assay systems that were 
 
 23   published by Robin and which Robin referred to.  The 
 
 24   development and validation of that DNA PCR was discussed in 
 
 25   quite some detail with the CEBR scientists as well as some 

 
                                                               108 
 
  1   of the advisory members of the scientific group.

  2             Additionally, we launched an effort to conduct an 
 
  3   in vitro characterization, a cocultivation study.  That 
 
  4   employed endpoints such as reverse‑transcriptase and RT‑PCR 
 
  5   assays as referred to by some of the other speakers like 
 
  6   Carolyn Wilson.

  7             The goal was to look for potential virus capable 
 
  8   of infecting human cells. 
 
  9             [Slide.] 
 
 10             Just to refresh the knowledge about the assay 
 
 11   systems, the DNA PCR system was described by Robin Weiss and

 12   his coworkers.  It was conducted in a QC setting following 
 
 13   validation.  The sensitivity of the assay system was between 
 
 14   0.02 and 0.1 porcine cell per 105 human PBMC.  The 
 
 15   specificity was confirmed by testing fresh PBMCs as well as 
 
 16   cells integrated human retrovirus sequences.

 17             The RT‑PCR system was validated along the same 
 
 18   lines.  The sensitivity was shown to be capable of detecting 
 
 19   10‑3 dilution of unconcentrated PERV material in a 5 
 
 20   microliter reaction sample.  The specificity was confirmed 
 
 21   as previously described.

 22             [Slide.] 
 
 23             The results using the DNA PCR assay for, it turned 
 
 24   out to be 11 Parkinson's disease patients who are presented 
 
 25   here.  That patients ranged in post‑transplant interval from 

 
                                                               109 
 
  1   six months to 24 months.  In all cases, we turned up

  2   negative results. 
 
  3             [Slide.] 
 
  4             We tested 15 samples.  Some of those samples 
 
  5   turned out to be Diacrin employees that were included as a 
 
  6   laboratory control.  I do have to echo Robin's comments

  7   about the care that must be taken in performing the PCR 
 
  8   analysis.  We utilized a five‑room protocol and were able to 
 
  9   prevent cross contamination with specific carry‑over 
 
 10   prevention mechanisms. 
 
 11             So, basically, the DNA PCR results for the

 12   patients were negative and the PBMCs were negative using the 
 
 13   probe. 
 
 14             [Slide.] 
 
 15             The cocultivation study, and I apologize that this 
 
 16   is a very busy slide, employed LGE and VM which are the

 17   porcine fetal neural cells.  This was actually conducted in 
 
 18   duplicate.  The porcine cells were cocultivated with human 
 
 19   293 cells or STIowa cells, as Carolyn Wilson had mentioned. 
 
 20             In addition, monocultures of the neuronal cells 
 
 21   were maintained and the appropriate controls along with

 22   those including human 293 cells, STIowa infected with PERV, 
 
 23   293 cells infected with PERV, and PK15.  In all cases, the 
 
 24   cells were carried out for a period of 35 days which 
 
 25   represented a significant population doubling level. 

 
                                                               110 
 
  1             Then supernatants were passaged onto 293 or STIowa

  2   cells and then those cells carried out for an additional 
 
  3   interval.  Each time there is a chevron or an arrow as a 
 
  4   sampling point, we were collecting samples for a reverse‑ 
 
  5   transcriptase assay, RT‑PCR analysis of cell supernatants 
 
  6   and, where these may be negative, DNA PCR analysis of cells.

  7             [Slide.] 
 
  8             The results obtained with the cocultivation study 
 
  9   are presented in this slide.  The porcine fetal neural cells 
 
 10   cocultivated for intervals of 35 days with the human 293 
 
 11   cells were negative when tested using the RT PCR assay

 12   system. 
 
 13             Also, at day 49 in the supernatant passage 
 
 14   component, these cells were negative using the DNA PCR so 
 
 15   there did not appear to be integration of the PERV in those 
 
 16   human cell populations.  STIowa was employed and positive

 17   results were obtained with the RT‑PCR and the DNA PCR 
 
 18   analysis.  This is, I believe, by virtue of the fact that 
 
 19   the STIowa cells were, in fact, expressing porcine 
 
 20   endogenous retrovirus. 
 
 21             The appropriate controls were employed.  PERV plus

 22   human 293 cells did give positive results so infection was 
 
 23   transmitted using the porcine endogenous retrovirus 
 
 24   preparations.  The same is true with the STIowa. 
 
 25             The monoculture cells of human 293, VM1, the 

 
                                                               111 
 
  1   porcine neuronal cells were negative in both assay systems

  2   and, as noted, STIowa and PK15, as expected, would be 
 
  3   positive. 
 
  4             [Slide.] 
 
  5             Additionally, the same samples were analyzed using 
 
  6   the enzymatic RT assay.  The human 293 cells cocultivated

  7   with porcine neural cells were negative after that 
 
  8   cocultivation interval of 35 days.  Positives were obtained 
 
  9   with the cells cocultivated with STIowa.  I think it is 
 
 10   important to point out that these levels of incorporation 
 
 11   were less than or equal to the STIowa control cultures, so

 12   those monocultures that are shown here were positive. 
 
 13             These were not any more positive than that.  
 
 14   Again, the controls gave expected results PERV plus STIowa 
 
 15   and PERV plus the human 293 cells. 
 
 16             [Slide.]

 17             So, in summary, we can demonstrate, at least from 
 
 18   the data in these studies, that the post‑transplant patient 
 
 19   samples which were from six to 24 months post‑transplant 
 
 20   were negative using the DNA PCR assay system.  In the 
 
 21   cocultivation study, supernatant samples from the

 22   cocultivation were negative for PERV using the RT‑PCR assay 
 
 23   system and all of the human 293 cells from the cocultivation 
 
 24   study with, of course, the exception of those spiked with 
 
 25   the PERV, were negative for the DNA PCR. 

 
                                                               112 
 
  1             I should point out as well that the RT assay

  2   results were conducted using the same setting as Dr. Carolyn 
 
  3   Wilson presented before.  Lastly, the assay systems would 
 
  4   seem to be very relevant for appropriate monitoring of 
 
  5   patients and the analysis of samples from clinical 
 
  6   administration.

  7             Thanks. 
 
  8             DR. AUCHINCLOSS:  Before you step away from the 
 
  9   microphone, are there any results on the patient that we 
 
 10   know died at eight months from tissue‑‑ 
 
 11             MR. MOORE:  No; we have not received samples for

 12   patient No. 12 for analysis. 
 
 13             MS. MEYERS:  Were they collected?  Were those 
 
 14   samples collected?  Was there an autopsy?  Was there 
 
 15   anything? 
 
 16             MR. MOORE:  This is Dr. Steven Fink.

 17             DR. AUCHINCLOSS:  Dr. Fink, now, you have got sort 
 
 18   of two or three minutes left in the 20 minutes.  I have 
 
 19   looked through your slides and they are largely the clinical 
 
 20   data.  Is there, in fact, information that will add to our 
 
 21   understanding of the assays?

 22             DR. FINK:  There is some safety and efficacy data 
 
 23   from the Parkinson's phase I trial. 
 
 24             DR. AUCHINCLOSS:  Particularly related to viral 
 
 25   issues? 

 
                                                               113 
 
  1             DR. FINK:  No; this is all clinical data.  It is

  2   general adverse event, safety reporting and clinical 
 
  3   information from the neurological rating scales. 
 
  4             DR. AUCHINCLOSS:  I guess what I am asking is for 
 
  5   the purpose of this meeting, can you shorten the talk so 
 
  6   that you stay within the 20 minutes and move on?

  7             DR. FINK[:  Yes. 
 
  8             DR. AUCHINCLOSS:  Because I think we are 
 
  9   concentrating on the viral issue here today. 
 
 10             DR. FINK:  Okay.  Let me just highlight a couple 
 
 11   of slides.

 12             [Slide.] 
 
 13             I would just like to highlight some of the safety 
 
 14   and efficacy parameters in the xenotransplant trial that was 
 
 15   initially started in Parkinson's disease.  This is a trial 
 
 16   in which 12 patients received 12 million ventral mesen

 17   cephalic cells implanted in three sites in the striatum.   
 
 18             Six of the patients received standard 
 
 19   immunosuppression with cyclosporine and six patients 
 
 20   received cells that had been treated with monoclonal 
 
 21   antibody to complex 1 of MHC.  These patients were followed

 22   at three‑month intervals for safety and efficacy testing. 
 
 23             [Slide.] 
 
 24             This slide summarizes all of the adverse events 
 
 25   seen in the two groups.  There were 232 adverse events.  

 
                                                               114 
 
  1   There were more serious and non‑serious adverse events in

  2   the cyclosporine group.  The profile of these adverse events 
 
  3   was often consistent with known side effects of 
 
  4   cyclosporine. 
 
  5             [Slide.] 
 
  6             In terms of relationship to study treatment,

  7   investigators felt that in the antibody‑treated group, there 
 
  8   were no adverse events that were probably or definitely 
 
  9   related to study treatment.  The three adverse events in the 
 
 10   cyclosporine group so judged related probably or definitely 
 
 11   were all laboratory deviations in cyclosporine levels.

 12             [Slide.] 
 
 13             This is the serious adverse events.  I can just 
 
 14   summarize this briefly by saying that with the exception of 
 
 15   the one patient that was already mentioned who died of a 
 
 16   pulmonary embolus, all of the other serious adverse events

 17   were resolved without sequelae. 
 
 18             [Slide.] 
 
 19             Regarding the patient that died, there was an 
 
 20   autopsy performed and the opportunity was available to 
 
 21   perform a neuropathological analysis on that patient.  

 22             [Slide.] 
 
 23             From the safety and efficacy standpoint, I think 
 
 24   two points are important.  First is that using a specific 
 
 25   porcine genomic marker using a repetitive element, specific 

 
                                                               115 
 
  1   cells that were of porcine origin were seen within the

  2   putamen and those are indicated here in the purple. 
 
  3             [Slide.] 
 
  4             These are dopaminergic cells that were seen in the 
 
  5   graft. 
 
  6             [Slide.]

  7             When markers for inflammatory response were looked 
 
  8   at, either for T‑cells or MHC class II upregulation, there 
 
  9   was an minimal or mild upregulation and infiltration of T‑ 
 
 10   cells at the border of the graft site. 
 
 11             [Slide.]

 12             This is the efficacy data.  The column on the left 
 
 13   is where you should look.  At 12 months, the ten evaluable 
 
 14   patients have improved in their Parkinson scores of 20 
 
 15   percent.  This level of improvement has been seen in both 
 
 16   the group that has received cyclosporine immunosuppression

 17   and in the antibody‑treated group. 
 
 18             I was going to show a brief video of one of the 
 
 19   patients but, with respect to Dr. Auchincloss' guidance 
 
 20   document, perhaps I will pass on that. 
 
 21             DR. ALTER:  That would be terrific.  Thanks very

 22   much. 
 
 23             Back to Abbey's question. 
 
 24             MS. MEYERS:  You evaluated for the virus in twelve 
 
 25   Parkinson's patients, but did you look at the tissues of the 

 
                                                               116 
 
  1   Huntington's patients?

  2             DR. FINK:  The Huntington's patients have had 
 
  3   blood sampled and saved but we have not had the resources 
 
  4   given to the other testing.  We felt it was high priority, 
 
  5   but we have not run those samples on these patients yet but 
 
  6   we intend to do so.

  7             MS. MEYERS:  When do you intend to do so?  Soon?  
 
  8   A year from now? 
 
  9             DR. FINK:  Soon.  It is very high priority. 
 
 10             DR. AUCHINCLOSS:  And the post‑mortem tissue? 
 
 11             DR. FINK:  The post‑mortem tissue was saved and is

 12   available.  That has not been looked at.  I think we do need 
 
 13   to consider some of the technical aspects of whether the 
 
 14   assays that have been talked about can be actually applied 
 
 15   to that tissue. 
 
 16             DR. DESROSIER:  I am surprised that nobody has

 17   said anything about antibody responses to the pig virus.  
 
 18   Can you tell us what is being done along those lines?  Is 
 
 19   that a fair question? 
 
 20             DR. FINK:  Currently, we do not have that kind of 
 
 21   assay available.  I think that there is a belief that this

 22   would be important and very useful in not only analyzing the 
 
 23   patients that have been transplanted but in subsequent 
 
 24   monitoring of these patients and others.   
 
 25             DR. SALOMON:  We got into this at the Cross‑ 

 
                                                               117 
 
  1   Species Infectivity meeting a few months ago.  In this

  2   particular case, for example, we are seeking reassurance 
 
  3   that we are not getting the infection in the clinical 
 
  4   setting and these are very important studies. 
 
  5             The use of the UT93 kidney‑cell line, though, is 
 
  6   not biologically, perhaps, the most reassuring cell line

  7   used as a target.  It does have cell receptors for some of 
 
  8   the known PERVs, but there are multiple PERVs.  We don't 
 
  9   know that they have the same cell receptors of the others. 
 
 10             Moreover, in the clinical sense, neural cell lines 
 
 11   or lymphocytes, would be the two kinds of cells most likely

 12   having access to tissue in a clinical setting would be more 
 
 13   appropriate, I think, targets for testing these things. 
 
 14             So I think before we make conclusions like, well, 
 
 15   this data shows that there is no risk, I think that we have 
 
 16   to be very cautious about the studies that we are

 17   interpreting. 
 
 18             DR. ONIONS:  Could I just extend that comment?  
 
 19   One of the things that I am concerned about is if we rely on 
 
 20   cell cultures to detect infectious virus‑‑or, sorry, plasma 
 
 21   viremia on infectious virus, there are mutations where we do

 22   see this stochastic nature with the stocks of virus we know 
 
 23   have high amounts of particles but their infective cells can 
 
 24   be very stochastic on experiences on several occasions. 
 
 25             Say on four out of five occasions we get 

 
                                                               118 
 
  1   infectivity and on the fifth occasion, we don't, with the

  2   same amount stuff.  I think if we are going to look for 
 
  3   plasma viremia, you look for the virus in other situations 
 
  4   and you need to consider using electrical techniques which 
 
  5   are not ideal, but, for instance, for plasma, by using RT‑ 
 
  6   PCR.

  7             DR. LERCHE:  I have just a question on, again, the 
 
  8   tissues that are being examined.  If I understood the 
 
  9   presentation correctly, it was buffy‑coat cells in the 
 
 10   patients?  I was wondering if any opportunity was available 
 
 11   to look at, for example, by RT‑PCR or other methods in, say,

 12   spinal fluid, some other tissue or fluid. 
 
 13             DR. FINK:  So your question is whether there is 
 
 14   utility in looking for PERV in cerebrospinal fluid which is, 
 
 15   perhaps, the most readily accessible fluid adjacent to the 
 
 16   graft site.  We have given this some though and I think,

 17   still, we are in discussions of the technical applicability 
 
 18   of that in that presumably one would need to get cells from 
 
 19   cerebrospinal fluid and there may be very few there. 
 
 20             DR. AUCHINCLOSS:  I think it is going to be a very 
 
 21   important question for us to get back to this afternoon and

 22   definitely plan on doing so. 
 
 23             DR. COFFIN:  I had a question regarding the 
 
 24   sensitivity of the PCRs.  The slide indicated a detection 
 
 25   rate of 0.02 cells.  I assume that that was based on pig 

 
                                                               119 
 
  1   cells where there are 50 copies per cell or so for viral

  2   DNA.  Is that where that comes from? 
 
  3             DR. MOORE:  Yes. 
 
  4             DR. COFFIN:  But an infected human cell can only 
 
  5   be detected at 150, although at that sensitivity, you may 
 
  6   only have one provirus.  You have got to be a little careful

  7   here about what it is that you are actually seeing.  That is 
 
  8   a real problem, though, if you are thinking about detection 
 
  9   of these, this differential sensitivity between the host 
 
 10   cell that you would really like to be looking at and the 
 
 11   much, much more sensitive detection by the same assay in the

 12   donor cells where there might be traces of microchimers and 
 
 13   may make the assay impossible. 
 
 14             DR. AUCHINCLOSS:  Again, I think this is a 
 
 15   critical issue that we can come back to this afternoon as we 
 
 16   talk about what assay is good enough.

 17             DR. COFFIN:  Could I ask one specific question?  
 
 18   What was the denominator in those assays?  You said the 
 
 19   sensitivity is 0.02.  You didn't see anything.  I didn't 
 
 20   catch how many cells were actually tested. 
 
 21             DR. MOORE:  That was in 105 human cells.

 22             MS. MEYERS:  Did you save the tissue from the rest 
 
 23   of the pig?  Can you go back to that tissue? 
 
 24             MR. EGAN:  We covered it briefly but part of this 
 
 25   is that there is a regular sampling of all the various 

 
                                                               120 
 
  1   tissues that are part of an archive system.  This actually

  2   is part of a registry that is being developed.  So the 
 
  3   answer is yes, we do have those tissues available.  So, it 
 
  4   being an endogenous virus, the DNR PCR obviously is there.  
 
  5   It is a question of whether it is expressed by the 
 
  6   particular tissue that we are transplanting.

  7             MS. MEYERS:  Have you gone back to test that 
 
  8   tissue? 
 
  9             MR. EGAN:  It is there. 
 
 10             MS. MEYERS:  It is there? 
 
 11             MR. EGAN:  It is there because it is an endogenous

 12   virus.  So you will find it in all the tissue.  The question 
 
 13   is whether it is active or expressed as people have been 
 
 14   talking about. 
 
 15             DR. AUCHINCLOSS:  We are going to move on to our 
 
 16   next speaker, but I appreciate your flexibility in response

 17   to the Chairman's prompting. 
 
 18             From Barry Solomon, The Circe Biomedical assay and 
 
 19   clinical data. 
 
 20            Circe Biomedical Assay and Clinical Data  
 
 21             DR. SOLOMON:  Thank you, Mr. Chairman and advisory

 22   committee, for inviting me here.  I am going to try to stay 
 
 23   in the 20 minutes, I promise. 
 
 24             [Slide.] 
 
 25             We are going to be talking today about a liver‑ 

 
                                                               121 
 
  1   support system that we have developed which utilizes porcine

  2   hepatocytes for the treatment of acute liver failure.  Liver 
 
  3   failure is not  
 
  4   small disease.  There were about 250,000 patients last year 
 
  5   in the United States.  About 50,000 of those died, most 
 
  6   deaths from complications of chronic liver disease.

  7             [Slide.] 
 
  8             With liver transplantation, we have been able to 
 
  9   transplant about 4,100 liver transplants last year, but 
 
 10   there are about 9,000 patients in the United States 
 
 11   currently waiting for liver transplant and, last year, over

 12   1,000 patients died while waiting for a donor liver.  About 
 
 13   1,700 of these 4,100 liver transplants were emergency liver 
 
 14   transplants which were required because the patient would 
 
 15   die without that. 
 
 16             [Slide.]

 17             There are two specific types of patients in which 
 
 18   there is an acute need for liver support.  One is the 
 
 19   fulminant hepatic failure which is a very rapid onset of the 
 
 20   liver failure leading to encephalopathy and coma.  The liver 
 
 21   potentially can regenerate to provide necessary liver

 22   function.  The morality of this group without 
 
 23   transplantation is over 80 percent.  Even with 
 
 24   transplantation, considering all comers with FHF into the 
 
 25   hospitals, it ranges from a mortality of about 40 to 50 

 
                                                               122 
 
  1   percent.

  2             The challenge is to provide time for liver 
 
  3   regeneration of transplantation.  Another group of primary 
 
  4   non‑function patients occurs after liver‑transplant surgery 
 
  5   in which the liver has not been able to kick in.  There is 
 
  6   no liver activity.  There is no sustainable liver function.

  7             These patients, essentially, have a 100 percent 
 
  8   mortality without a subsequent retransplantation.  
 
  9             [Slide.] 
 
 10             From the basis of the National Center for Health 
 
 11   Statistics in UNOS, we estimate about 2,000 of the acute

 12   liver patients last year fell in the FHF and the PNF range. 
 
 13             [Slide.] 
 
 14             Our solution has been to come up with an 
 
 15   extracorporeal system which allows us to be able to treat 
 
 16   liver failure patients with isolated normal porcine

 17   hepatocytes that are encased in a hollow‑fiber reactor such 
 
 18   that the plasma from the patient, once separated from the 
 
 19   blood using a plasmapheresis system, is recirculated in an 
 
 20   extracorporeal loop so the plasma passes through the center 
 
 21   of the hollow fibers and the hepatocytes are sequestered

 22   from the plasma outside the hollow fiber in a situation in 
 
 23   which the membrane, which has about a 100 nanometer pore 
 
 24   size, allows proteins, nutrients and oxygen to be able to 
 
 25   perfuse those cells and allow those hepatocytes to function 

 
                                                               123 
 
  1   and provide missing metabolic function for a period of time.

  2             Our current protocol utilizes a six‑hour treatment 
 
  3   approximately once a day until the patient has been able to 
 
  4   either recover, regenerate or, in certain instances, die. 
 
  5             [Slide.] 
 
  6             We have put together a machine, our HepatoAssist

  7   machine, which allows to be able to treat patients with the 
 
  8   hollow‑fiber cartridge in the intensive‑care system.  All 
 
  9   the tubing sets, all of the ancillary materials are attached 
 
 10   to the system so that they can be utilized the same way 
 
 11   throughout all the centers in our study.

 12             [Slide.] 
 
 13             Working with the FDA, we were able to begin a 
 
 14   phase I clinical investigation in which we targeted severe 
 
 15   acute‑liver failure patients, end‑stage with stage III or 
 
 16   stage IV hepatic encephalopathy.

 17             We have, in this phase I trial, treated 41 
 
 18   patients, 27 FHF, three PNF, nine chronic patients where 
 
 19   they had an acute exacerbation of a chronic disease, and two 
 
 20   cancer patients, these in compassionate opportunities.  We 
 
 21   have utilized three clinical sites, the fundamental one with

 22   Cedars Sinai Medical Center which we have treated now 24 
 
 23   patients in our clinical trial under the direction of Dr. 
 
 24   Demetriou, whom you heard from this morning. 
 
 25             Dr. Demetriou and his group were key in developing 

 
                                                               124 
 
  1   this system, and the system that we are using today is based

  2   principally on the work that was done at Cedars Sinai 
 
  3   Medical Center. 
 
  4             We have also involved the UCLA Medical Center with 
 
  5   six patients with six patients were treated and the Paul 
 
  6   Brousse Hospital in Paris where an additional eleven

  7   patients have been treated. 
 
  8             [Slide.] 
 
  9             All in all, we have had 96 treatments over six 
 
 10   hours, as I said.  The average number of treatments per 
 
 11   patient was approximately 2.3 treatments.  In addition to

 12   those 41 patients, an additional 12 patients were treated at 
 
 13   Cedars Sinai prior to the IND under local IRB approval with 
 
 14   a previous generation of this system, seven FHF, one PNF and 
 
 15   four on chronic patients. 
 
 16             [Slide.]

 17             All in all, these are the results of the 53 
 
 18   patients that we have seen.  We have been able, bottom line, 
 
 19   looking at survival of 30 days, to see about a 90 percent, 
 
 20   89 percent, survival of 30 days for these targets FHF and 
 
 21   PNF patients.  For the acute liver patients, we see about a

 22   47 percent survival. 
 
 23             Interestingly, there are six patients who are 
 
 24   listed status 1 for emergency liver transplant that 
 
 25   recovered and left the hospital without a transplant after 

 
                                                               125 
 
  1   our bioassist treatment.  Also, no patients died while

  2   awaiting a transplant. 
 
  3             [Slide.] 
 
  4             To put this in perspective, we compared this data 
 
  5   to data that was generated at the Paul Brousse hospital by 
 
  6   Dr. Henri Bismuth, where he looked at the same stage 3,

  7   stage 4, coma patients in FHF and PNF patients.  He looked 
 
  8   at 175 patients in his hospital where the average waiting 
 
  9   time for a liver was about 1.3 days.  He still saw an 
 
 10   overall survival of only 67 percent. 
 
 11             However, when we look over here, we see that only

 12   1 percent, or two of those patients, recovered and about 24 
 
 13   patients, or 14 percent actually died having been listed and 
 
 14   waiting for transplant. 
 
 15             [Slide.] 
 
 16             How do we this?  We deliver to the hospital our

 17   machine, the cells.  Our hepatocytes are delivered in a 
 
 18   cryopreserved state.  The hollow‑fiber biocartidges in a 
 
 19   sterilized manner are also delivered to the hospital and the 
 
 20   other disposables are ready at the hospital. 
 
 21             [Slide.]

 22             Once the patient arrives, the cells go through a 
 
 23   cell‑preparation step, are seeded into the cartridge, put on 
 
 24   the machine and the treatment can commence. 
 
 25             [Slide.] 

 
                                                               126 
 
  1             But, to do this, we have had to develop a

  2   significant quality‑control process that involves our animal 
 
  3   facility where serological testing on the herd occurs prior 
 
  4   to bringing the animals into our system.  The animals, just 
 
  5   prior to the excise of the organ, are anesthetized and 
 
  6   weighed, blood and stool samples are taken.

  7             We screen the herd for the series of viral 
 
  8   contaminants before they are even brought into our facility.  
 
  9 
 
 10             [Slide.] 
 
 11             The excise suite, which occurs under a

 12   hepafiltered air shower‑‑the preparations occur, and 
 
 13   additional samples are taken. 
 
 14             [Slide.] 
 
 15             This is example of the surgical removal of the 
 
 16   liver which at the time is filled with a transport solution

 17   and the liver is doubled back to the animal facility and 
 
 18   brought into the cell‑preparation facility in our GMP 
 
 19   facility. 
 
 20             [Slide.] 
 
 21             There, we have perfusion, digestion, dissociation

 22   of the cells.  The cells are washed.  The cells are pooled, 
 
 23   when appropriate and a precryopreservation suspension is 
 
 24   used and they are filled into the bag. 
 
 25             [Slide.] 

 
                                                               127 
 
  1             This is the teasing of the tissue bag in the GMP

  2   room.  We have gowned people working under a hood.   
 
  3             [Slide.] 
 
  4             It is interesting to note that once the cells have 
 
  5   been digested and are placed into a bag, the cells never are 
 
  6   exposed to the environment again.  Throughout the rest of

  7   the process, the cells are in bags and a completely closed 
 
  8   system has been developed which allows us to transport these 
 
  9   cells, wash the cells to the clinical site and put them into 
 
 10   the final reactor without ever exposing the cells to air 
 
 11   again.

 12             In this particular case, we are looking at a cell 
 
 13   washing.  We have put them into the cryopreserved state. 
 
 14             [Slide.] 
 
 15             A very important part of our process is the 
 
 16   cryopreservation of the cells.  All of our cells are

 17   cryopreserved in order for us to be able to carry out 
 
 18   extensive sterility endotoxin, standard viral for culture, 
 
 19   mycoplasma, viability and function tests.  It is interesting 
 
 20   to note that from the time the organ is excised from the 
 
 21   animal until it is actually released to the clinical sites

 22   is at least 45 days for all of these data to come back. 
 
 23             [Slide.] 
 
 24             The cells are placed in special cryobags at this 
 
 25   point.  They are put into a specially designed cooling 

 
                                                               128 
 
  1   chamber.

  2             [Slide.] 
 
  3             The cells are stored after controlled cooling in 
 
  4   liquid nitrogen for long‑term storage.  We have now checked 
 
  5   the stability of these cells for a period of over three 
 
  6   years, now, for function and viability and the cells are

  7   performing the same as the freshly isolated systems. 
 
  8             [Slide.] 
 
  9             So, in an instance, we have the animal, the 
 
 10   hepatocyte as well as the final product all quality 
 
 11   controlled prior to their being delivered to the clinical

 12   site.  At the clinical site, they are thawed.  When the 
 
 13   cells are used, again, we do a cell account and viability.  
 
 14   They are added to the microcarrier, seeded into the 
 
 15   cartridge and there sterility and endotoxin samples are 
 
 16   taken.

 17             [Slide.] 
 
 18             At this point in time, we are ready to treat the 
 
 19   patient.  This shows the cells on the outside of the 
 
 20   cartridge and the system is ready to go. 
 
 21             [Slide.]

 22             We have looked at a strategy for our risk analysis 
 
 23   for the curve.  We see this in two particular areas; the 
 
 24   patients, we have now begun a retrospective analysis for the 
 
 25   evidence of PERV in the blood cells of patients treated 

 
                                                               129 
 
  1   prior to or with the HepatoAssist system.

  2             Blood samples are now taken of serum samples that 
 
  3   we have stored so we are now in the process of identifying 
 
  4   and contacting all of our patients that have been treated 
 
  5   and looking back and seeing if we can get blood samples from 
 
  6   them, samples which I will talk about in a minute.

  7             Prospectively, we are planning to test and analyze 
 
  8   the blood samples from patients treated with the 
 
  9   HepatoAssist system for evidence of the PERV, both of these 
 
 10   using the DNA PCR assay.  From a product standpoint, we are 
 
 11   looking at the cryopreserved hepatocytes.  We are looking at

 12   the animal analysis of these hepatocytes for evidence of 
 
 13   infectious PERV using both a standard RT assay and the RT‑ 
 
 14   PCR culture assay. 
 
 15             We are also looking at an interesting alternative 
 
 16   here which is the actual use of the system in the way it is

 17   being used and that is to run a bioreactor with both the 
 
 18   cryopreserved hepatocytes or the hepatocytes thawed out or 
 
 19   PK15 cells and being able to look at the ability for this 
 
 20   PERV to transmit from the bioreactor into the perfused 
 
 21   plasma in the system and then, again, looking at RT‑PCR

 22   culture for ability of this PERV to be transmitted. 
 
 23             [Slide.] 
 
 24             To date, we have treated 54 patients.  I include 
 
 25   one more patient that was treated under compassionate IND 

 
                                                               130 
 
  1   last month at Cedars Sinai and there have been no instances

  2   of any clinical manifestation of unknown viremia for periods 
 
  3   now ranging up to five years. 
 
  4             The peripheral blood lymphocytes of five patients 
 
  5   which we have now identified and, after duly‑informed 
 
  6   consent, have taken their blood and they have been tested. 

  7   Now, Allen Moore and his group, and recent results suggest 
 
  8   that they test negative for the PERV infection using the DNA 
 
  9   PCR. 
 
 10             These patients have been treated at various times 
 
 11   with one treatment up to three treatments and it should be

 12   noted that the oldest treatment is now 3‑94, which is about 
 
 13   three‑and‑a‑half years since the actual treatment date. 
 
 14             All but the 10‑95 patient received a transplant.  
 
 15   The 10‑95 patient is one that actually recovered and does 
 
 16   not have a transplant now.

 17             [Slide.] 
 
 18             In summary, we believe the HepatoAssist is 
 
 19   significantly different in risk profile compared to organs, 
 
 20   themselves.  Xenotransplantation, as well as whole organ, ex 
 
 21   vivo perfusion and freshly procured xeno tissue. One, he

 22   cells that we used were cryopreserved and allow for 
 
 23   extensive QC evaluation prior to human exposure. 
 
 24             Two, the cells are not in direct contact with 
 
 25   human tissue during or after the treatment.  Three, the 

 
                                                               131 
 
  1   cells are exposed for a short period of time via

  2   extracorporeally plasma‑perfused membrane bioreactor.  They 
 
  3   have no evidence to date of infectious PERV in cryopreserved 
 
  4   porcine hepatocytes.   
 
  5             We have sent samples for standard RT assay.  We 
 
  6   have retained hepatocytes from the hepatocytes used on our

  7   patients plus new patients and, right now, the data is not 
 
  8   suggestive of any PERV in that area.  We are still working 
 
  9   on some validation issues regarding that particular assay. 
 
 10             [Slide.] 
 
 11             Number three, we have no evidence of PERV

 12   infection in humans either via the clinical symptoms of the 
 
 13   unknown viremia for periods of one month to five years and 
 
 14   no evidence of PERV infections in the five patients tested.  
 
 15   Finally, the data from the phase I/II clinical study of the 
 
 16   hepatic system indicated a good safety profile and

 17   preliminary evidence of clinical efficacy in the treatment 
 
 18   of patients with acute liver failure. 
 
 19             Thank you. 
 
 20             DR. AUCHINCLOSS:  Thank you very much.  We heard 
 
 21   this morning, I believe, some information that suggests that

 22   the cryopreserved tissue might, in fact, not be infectious 
 
 23   for endogenous retrovirus.  Carolyn, did you want to make a 
 
 24   comment on that? 
 
 25             DR. WILSON:  I think the only thing maybe for 

 
                                                               132 
 
  1   consideration is just that we don't know what conditions are

  2   required for activation and whether or not it is reasonable 
 
  3   to try and explore some of those avenues. 
 
  4             DR. AUCHINCLOSS:  Other speakers from this 
 
  5   morning.  Is there reason to think the cryopreserved tissue 
 
  6   would be somehow safer?  No.  Shaking heads.  I am seeing

  7   "no" all around the table. 
 
  8             DR. COFFIN:  I have a different question.  What is 
 
  9   the nature of the barrier between the cells and the plasma.  
 
 10   I mean, what it pore size? 
 
 11             DR. SOLOMON:  It is a microporous fiber that has a

 12   pore size of about 100 nanometers, probably smaller after it 
 
 13   has been exposed to the plasma.  We are not making a claim 
 
 14   that it is a total barrier to the virus, but it is certainly 
 
 15   one that has a significant rejection of the virus, looking 
 
 16   at a virus that potentially could be as large as 80

 17   nanometers in size. 
 
 18             DR. COFFIN:  Have you actually looked at this 
 
 19   barrier with any viruses? 
 
 20             DR. SOLOMON:  We are doing those studies now.  We 
 
 21   are actually going to put PK15 cells into our bioreactor and

 22   look for that transmission right now.  The studies are 
 
 23   beginning right now.  We have just completed the study 
 
 24   design for that.  
 
 25             DR. HIRSCH:  I have two quick questions.  I am not 

 
                                                               133 
 
  1   sure what is meant by clinical manifestations of unknown

  2   viremia, if you clarify that.  The other one is getting back 
 
  3   to what Ron asked earlier.  Are there plans to look for any 
 
  4   kind of antibody in all of these people.  It seems to me 
 
  5   that might be a more sensitive way of looking at things 
 
  6   than‑‑

  7             DR. SOLOMON:  We can do that.  I am not aware of 
 
  8   any antibody test that has really been developed right now. 
 
  9             DR. HIRSCH:  Do you know if there are any 
 
 10   contaminating lymphocytes or macrophage monocytes into the 
 
 11   hepatocyte‑‑

 12             DR. SOLOMON:  We have done studies on that.  I 
 
 13   don't know if there are any‑‑at this point in time, we can't 
 
 14   say that they are absolutely free of any red cells or cells 
 
 15   in there.  We do see a purification of the hepatocytes from 
 
 16   a norm of 65 percent up to about 90, 95 percent.  But there

 17   are probably other issues in there. 
 
 18             They will be sequestered and kept on the outside 
 
 19   of the membrane.  Those will not move across the membrane. 
 
 20             DR. DESROSIER:  I didn't hear whether those were 
 
 21   fetal hepatocytes or adult‑‑

 22             DR. SOLOMON:  These are, I would say, juvenile pig 
 
 23   hepatocytes. 
 
 24             DR. DESROSIER:  I didn't hear anything said about 
 
 25   what infectious agents were specifically screened out in 

 
                                                               134 
 
  1   those pigs.  Are they reared in some of‑‑are they

  2   gnotobiotic pigs, for example? 
 
  3             DR. SOLOMON:  No.  But they are herds of pigs in 
 
  4   which we screen‑‑I think I showed at slide that showed all 
 
  5   of the different viruses. 
 
  6             DR. DESROSIER:  Were there any herpes viruses on

  7   that list? 
 
  8             DR. SOLOMON:  Pseudorabies. 
 
  9             DR. ONIONS:  Hepatitis C wasn't on your list which 
 
 10   is the virus with which we are all concerned now because of 
 
 11   the porcine hepatitis C which is very closely related to the

 12   human hepatitis C.  
 
 13             DR. SOLOMON:  We don't have that yet. 
 
 14             DR. PAUL:  My question also pertains to hepatitis 
 
 15   C because it has recently been reported it is very common 
 
 16   and we have some work going on in collaboration with NIH at

 17   our university.  I think this is a virus especially for this 
 
 18   particular application which should be looked at. 
 
 19             DR. WEISS:  With the type of perfusion you are 
 
 20   conducting, one might expect that the human complement of 
 
 21   human antibodies might get through into the liver cells and

 22   cause a sort of hyperacute injection of lysis of the liver 
 
 23   cells and that could, then, allow a lot of pig‑liver DNA to 
 
 24   come through. 
 
 25             I wonder if you are testing for this? 

 
                                                               135 
 
  1             DR. DEMETRIOU:  We have tested that and we don't

  2   see any evidence of cytolysis during the treatment.  
 
  3   Theoretically, if we treat patients more than ten or 15 
 
  4   times, it is possible that that would be an issue.  The 
 
  5   largest number of treatments we have done so far is five.  
 
  6   In those patients, we have not seen any evidence of cell

  7   damage, cell death, release of anything into the extrafiber 
 
  8   compartment. 
 
  9             We do measure‑‑all our patients, as I indicated 
 
 10   this morning, had baseline levels of xenoantibodies, both 
 
 11   IgG and IgM.  We see an increase and the increase is higher

 12   in patients who are treated more than two times and then, 
 
 13   about two or three months after the treatment, they come 
 
 14   down to the baseline, the antibody does. 
 
 15             DR. SALOMON:  I have a question.  My understanding 
 
 16   is, in other people's experience and I thought that you have

 17   also reported it, that after four hours of extracorporeal 
 
 18   circulation, you begin having a significant loss of these 
 
 19   cells and that starts at around two hours and continues and 
 
 20   you lose over 80 percent of their viability by six hours 
 
 21   after perfusion.

 22             People have talked about why, but the fact is 
 
 23   there is a lot of cell death going on on the other side of 
 
 24   the membrane; right? 
 
 25             DR. DEMETRIOU:  Yes.  Actually, we have not 

 
                                                               136 
 
  1   recorded these data.  But we do measure all possible‑‑during

  2   the treatment so we can monitor if there is a change in 
 
  3   viability.  Usually, we see about a 30 percent loss of 
 
  4   function at end of the treatment.  Where we do tripon blue 
 
  5   or something crude like that, we see approximately 30 
 
  6   percent cell death at the end of the treatment.

  7             It depends a lot on the underlying disease.  
 
  8   People with fulminant liver failure due to acetaminophen 
 
  9   ingestion with very high levels of plasma acetaminophen, you 
 
 10   see much higher levels of cell death.  
 
 11             DR. SALOMON:  It might be interesting, then, to

 12   look for antigen, for example, in the patients. 
 
 13             DR. DEMETRIOU:  We do measure porcine antigen in 
 
 14   the patients.  We measured more than seven or eight 
 
 15   patients.  It goes up and stays up for a couple of months.  
 
 16   It usually does not go up very high over the baseline in

 17   patients treated only once.  It certainly goes up in 
 
 18   patients treated over three times.  It stays up for about‑‑ 
 
 19   and also the anti‑albumin, anti‑porcine albumin, antibodies 
 
 20   stay up for several months. 
 
 21             DR. AUCHINCLOSS:  We will move on to our final

 22   speaker, Walid Heneine from the CDC. 
 
 23               CDC Collaborations and Surveillance 
 
 24             DR. HENEINE:  Good morning.   
 
 25             [Slide.] 

 
                                                               137 
 
  1             What I will be covering is the strategies we are

  2   using for the diagnosis of liver fractions.  I would like to 
 
  3   point out that to date they are limited to PCR analysis and 
 
  4   reverse‑transcriptase analysis.  We are working to develop a 
 
  5   serologic assay that will complement these tests. 
 
  6             The limitation with the developments serologic

  7   assays is the availability of antisera, so working on 
 
  8   producing these antisera as positive controls.  So you will 
 
  9   be hearing from me on the PCR analysis that now we are using 
 
 10   and the compartment we are tolerating because of its 
 
 11   accessibilities, the peripheral blood lymphocytes.  We will

 12   be discussing whether this is a good target or not. 
 
 13             But the assay, itself, we use amplification of two 
 
 14   different virus regions.  We have been careful in designing 
 
 15   the primers and probes for these two regions that are known 
 
 16   for the sequences.  For example, the generated sequences in

 17   our lab were generated from the Shimozuma.  This is the 
 
 18   secuba variant virus. 
 
 19             For the pol, we have compared our sequence with 
 
 20   the one published from Dr. Weiss' lab.  We have genetic 
 
 21   primers and an internal genetic probe based on the two

 22   sequences. 
 
 23             Remember, as Dr. Onions mentioned, that the 
 
 24   Shimozuma sequence is variant similar to the pol and the 
 
 25   MPK.  That may be different.  In the envelope, we know it is 

 
                                                               138 
 
  1   different from both A and B and maybe the C sequence.

  2             We define the positive PCR on the basis of two 
 
  3   different regions.  I will tell you why we do that, because 
 
  4   of region and so forth.  In addition, we see positive, we 
 
  5   need to look at the possibility of microchimerism.  A 
 
  6   positive PCR signal for the first sequence does not imply

  7   the presence of these sequences and infection of the human 
 
  8   cells but it may also be due to the presence of pig cells 
 
  9   and we need to have assays that can distinguish between 
 
 10   these two scenarios. 
 
 11             We use PCR analysis of a porcine cell‑marker

 12   sequence.  We have at least three different assays 
 
 13   developed, one on a low copy number, porcine‑specific 
 
 14   betaglobulin.  We now have mitochondrial pig‑specific 
 
 15   sequences which should present a high copy sequence and, 
 
 16   therefore, cannot be missed.

 17             All these assays, as I will show, you, the key 
 
 18   feature is that they should be highly specific to the pig 
 
 19   and should not be active to human sequences. 
 
 20             [Slide.] 
 
 21             This is the algorithm we are now using in our lab. 

 22   The PBL samples, after we obtain them, we lyse it and we put 
 
 23   it in different aliquots.  We test aliquot level 1 for both 
 
 24   sequences, the pol and the gag.  If it is negative, then we 
 
 25   stop there.  If it is positive, then we go back to a 

 
                                                               139 
 
  1   different aliquot, and we test it again for the positive

  2   sequence, either pol or gag or both, if that is the case. 
 
  3             If it is negative, then we report it negative for 
 
  4   third‑class sequences and this could be interpreted as 
 
  5   potential absence of infection.  It is possible to stop 
 
  6   there.  This is a confirmatory positive.  Then we proceed

  7   with the analysis for microchimerism. 
 
  8             We thought a little bit about a simple way on how 
 
  9   to differentiate between presence of infection or presence 
 
 10   of chimerism.  This is what we ended up doing. 
 
 11             Again, we wanted something simple but effective. 

 12   The way we are doing it right now is that we obtain 
 
 13   peripheral‑blood lymphocytes.  We dilute these before lysing 
 
 14   them for the analysis.  We dilute them and then we test 
 
 15   these different cell dilutions for the presence of the pig 
 
 16   mitochondrial sequence and the PERV sequence.

 17             This is a scenario which may reflect infection.  
 
 18   If you have PERV sequences in dilution, the titer of it is 
 
 19   much more significantly high than that of the pig 
 
 20   mitochondrial and that would reflect that you have the human 
 
 21   cells that are infected with PERV and maybe some residual

 22   chimerism with pig cells as well. 
 
 23             Now, if both PRC titers of the PERV and the pig 
 
 24   mitochondrial sequences are equivalent, then that may 
 
 25   reflect the presence of pig cells only in your sample and 

 
                                                               140 
 
  1   not the absence of human PBMCs that are infected with PERV.

  2             [Slide.] 
 
  3             Again, for the pig mitochondrial series, we used 
 
  4   the same algorithm.  If it is positive, we go to aliquot 2.  
 
  5   If that is confirmed, then we will go to what I have 
 
  6   mentioned before in microchimerism studies.

  7             [Slide.] 
 
  8             It is important that the assays are developed to 
 
  9   look at in the normal human population to see if those 
 
 10   sequences are floating out there or not.  It will give us 
 
 11   confidence about the basic viral activity in these samples.

 12             So we have obtained normal U.S. blood donor 
 
 13   samples and we have tested them for PERV, gag, we have the 
 
 14   pol now, and for also the pig mitochondrial.  All these 
 
 15   samples we have looked at so far, 54 here and 69, were 
 
 16   negative.

 17             [Slide.] 
 
 18             So what do we do when we identify a sample which 
 
 19   is positive for PERV sequences, meaning no evidence of 
 
 20   chimerism and this is a true infection of the human cell.  
 
 21   Of course, we are not interested in this only to stop there. 

 22   We would like to look at markers of expression, namely, do 
 
 23   we see any evidence of virus that has been released in the 
 
 24   plasma. 
 
 25             We can do that by our TPCR.  We have those assays 

 
                                                               141 
 
  1   available.  We can also do it by Amp‑RT which is an

  2   ultrasensitive PCR‑based assay that looks for reverse 
 
  3   transcriptase in the plasma.  We have included here serology 
 
  4   which was a very important marker, as I have mentioned 
 
  5   earlier. 
 
  6             We also do sequence analysis.  We are interested

  7   in looking at the divergence of the sequences that will be 
 
  8   infecting those human cells.  Of course, tropism, focussed 
 
  9   on the envelope to figure out what kind of envelope the 
 
 10   sequence is associated with these human infections. 
 
 11             [Slide.]

 12             My last part is what are the data we have 
 
 13   available right now from these studies.  Again, we have an 
 
 14   IRB‑approved study protocol at CDC which is open for 
 
 15   everybody and we don't charge for these testings. 
 
 16             We have looked so far at three humans that

 17   received liver perfusions extracorporeally.  They are from 
 
 18   three different institutions.  One is Johns Hopkins, Dr. 
 
 19   Klein.  Another one is from the University of Wisconsin, Dr. 
 
 20   Kirk.  The third one is from McGill University, Dr. 
 
 21   Trechenkow and Dr. Tector.  

 22             The McGill patient, the sample was obtained 
 
 23   approximately three months after the procedure.  The other 
 
 24   two were more than a year and a half, one year and a half to 
 
 25   two years.  All three patients were negative by DNA analysis 

 
                                                               142 
 
  1   from the gag and the pol PERV sequence.

  2             In addition, we realized the importance of animal 
 
  3   models to address the important issue of transmission.  We 
 
  4   are doing collaborations again to look at transmission of 
 
  5   these animal models. 
 
  6             This is an example of one collaboration with Dr.

  7   Allen Norin at New York State University.  There is a type 
 
  8   here.  It was six animals rather than eight.  I would 
 
  9   explore this for the second slide. 
 
 10             [Slide.] 
 
 11             This is the information we have on these six

 12   bonnet macaques that received two skin grafts.  One of them 
 
 13   is from a CD59 transgenic pig and another is a control from 
 
 14   a normal pig.  These are 2.5 cm by 2.5. 
 
 15             They are divided into three groups.  Group A 
 
 16   didn't receive any immunosuppression.  Group B received

 17   cyclosporine and steroid and Group C received, in addition 
 
 18   to these two, irrigation. 
 
 19             I have shown to you time to rejection of the CD59 
 
 20   skin graft which was higher than the normal graft.  So this 
 
 21   would give you an idea about the exposure time of these

 22   macaques.  These are the three time points we have tested on 
 
 23   these macaques.  Day 13, Day 40, Day 81 after the grafts 
 
 24   were put. 
 
 25             Again, all six animals were negative by DNA PCR 

 
                                                               143 
 
  1   analysis of all the gag sequences.

  2             [Slide.] 
 
  3             This is the example of the tests we did and it 
 
  4   will serve two purposes, to show you sensitivity and 
 
  5   specificity of the different assays.  This is pig cell PK15, 
 
  6   diluted in a constant background of DNA for 150,000 PBLs. 

  7   This is what we use as our PCR here for 150,000 PBLs. 
 
  8             Again, here, we can see reliably these two assays 
 
  9   detect the equivalent of 0.15 cell equivalent DNA from 0.15 
 
 10   PK15 cells.  We have now plasmas for these gag and pol, and 
 
 11   we will be determining the exact sensitivity of these two

 12   assays in terms of plasma copy numbers. 
 
 13             But, again, we go beyond the cell level because, 
 
 14   as you have heard, those sequences are high copy, more than 
 
 15   one copy per cell, and this is evidence of that as well.  
 
 16             This is the mitochondrial DNA sequence.  Again,

 17   this is high copy and here we can go beyond the 0.15 and we 
 
 18   see the strong signals associated with these dilutions. 
 
 19             This is a typical experiment.  These are different 
 
 20   animals, A1, A2, one type of experiment with other controls, 
 
 21   sensitivity controls.  They include every experiment, and if

 22   this does not show up, then the experiment will be repeated.  
 
 23   These are the negative results I have mentioned to you. 
 
 24             [Slide.] 
 
 25             I don't want to complicate your lives further.  We 

 
                                                               144 
 
  1   are focused right now on the porcine endogenous virus, but

  2   we should keep in mind that the virus in pigs have not been 
 
  3   very well studied as much as simians and humans. 
 
  4             One has to keep in mind the possibility of the 
 
  5   presence of another or a different retrovirus.  We plan also 
 
  6   to look genetically for expression of retroviruses and the

  7   presence of retroviruses in the plasma from these patients 
 
  8   by looking for reverse transcriptase activity using the Amp‑ 
 
  9   RT method. 
 
 10             If it is positive, of course, go to virus 
 
 11   isolation, cloning and so forth.  But also we will be using

 12   it on some of the PCR‑positive individuals as a marker of 
 
 13   expression of PERV and we may expand that to include it as a 
 
 14   basic component of our algorithm. 
 
 15             This is the team.   
 
 16             Let me add here that we have initiated additional

 17   collaborations here.  We will be starting looking at ten 
 
 18   Swedish patients that have received flutin bacteriatic cells 
 
 19   and, hopefully, we should have those data soon.   
 
 20             We also have additional collaborations of animal 
 
 21   models. One is with Dr. Colin Weber at Emory who is doing

 22   encapsulated bacteriatic eyelet cells in rhesus macaques. 
 
 23             DR. AUCHINCLOSS:  Any questions? 
 
 24             DR. ALLAN:  Your algorithm for PCR.  The question 
 
 25   I had is it looked to me like both PCR assays you used in 

 
                                                               145 
 
  1   the first case and then this second to confirm are both the

  2   same assays; is that right? 
 
  3             DR. HENEINE:  Right.  We will be running both 
 
  4   regions on every sample.  If you have a positive on one, or 
 
  5   on both sequences, then you will look at the second aliquot 
 
  6   and confirm that positive result.

  7             DR. ALLAN:  The reason I ask is because it seems 
 
  8   to me you may be biased for a negative result because, if 
 
  9   you have got a positive result and then you run it again and 
 
 10   you get a negative result, to me, that doesn't say that it 
 
 11   is negative.  It still could be positive.  You just got a

 12   false negative.  So I would want to go to a third one. 
 
 13             DR. HENEINE:  We have addressed this and this is 
 
 14   an important point.  We have determined the rate of false 
 
 15   negativity of these CRSs.  I don't have the data here.  It 
 
 16   is being compiled.  Usually, you do that by running a large

 17   number of reactions at your sensitivity levels. 
 
 18             We take the 0.15 dilution and we run about 90 or 
 
 19   100 seconds and you look for any times that have missed your 
 
 20   detection system.  So you can flag the negative by the rate 
 
 21   of your false negative results.  That data is available.

 22             DR. AUCHINCLOSS:  Any questions from our last 
 
 23   speaker? 
 
 24             If not, it is quarter of 1:00.  I am going to 
 
 25   suggest that the thing for us to do is go ahead and break 

 
                                                               146 
 
  1   for lunch and then we will begin to discuss these issues

  2   during the course of the afternoon. 
 
  3             But, in return for halting the discussion at this 
 
  4   point, I think we will try to reconvene here at 1:30. 
 
  5             [Whereupon, at 11:50 a.m., the proceedings were 
 
  6   recessed to be resumed at 1:30 p.m.]

  7             [Whereupon, at 1 o'clock p.m., the proceedings  
 
  8   were recessed to be resumed at 1:30 p.m.] 

 
                                                               147 
 
  1            A F T E R N O O N   P R O C E E D I N G S

  2                                                    [1:40 p.m.] 
 
  3             DR. AUCHINCLOSS:  We will resume the afternoon 
 
  4   proceedings.  We have another comment as part of the open 
 
  5   public hearing. 
 
  6                 Open Public Hearing (Continued)

  7             DR. LEVY:  Thank you very much.  My name is Marlin 
 
  8   Levy.  I am a transplant surgeon at Baylor in Dallas.  I 
 
  9   have some research sponsorship from Nextram. 
 
 10             [Slide.] 
 
 11             My comment really addresses the issue of where I

 12   live which is at the bedside.  I understand that the 
 
 13   committee and the FDA is charged with public‑policy issues 
 
 14   which are of tremendous importance.  
 
 15             This is a 17‑year‑old man who came to our 
 
 16   hospital, a 17‑year‑old youth, really, who came to our

 17   hospital in acute fulminary hepatic failure at the very 
 
 18   first of October and who was dying and had no liver 
 
 19   transplant available.  No human transplants were available. 
 
 20             We placed him, as you can see in this picture, in 
 
 21   an extracorporeal circuit with a transgenic liver.

 22             [Slide.] 
 
 23             This liver is transgenic for CD55 and CD59.  We 
 
 24   perfused him for six and a half hours during which the liver 
 
 25   worked very well.  The patient improved. 

 
                                                               148 
 
  1             [Slide.]

  2             He subsequently went on to receive a human liver 
 
  3   transplant and has done very well.  He is now 75 days both 
 
  4   from his perfusion and his transplant.  So, as I listen to 
 
  5   your proceedings and I understand the evidence that you are 
 
  6   weighing, I beseech you to keep in mind use like this one

  7   and others because I think they are very critical.  It is 
 
  8   very critical to think of them also. 
 
  9             Since this particular perfusion and transplant 
 
 10   were done, we had one more opportunity to have another 
 
 11   patient go this trial.  But, by then, of course, the trial

 12   had been placed on hold and so we weren't able to do that.  
 
 13   She was successfully transplanted.  It remains to be seen 
 
 14   what the outcome is going to be because she is 
 
 15   neurologically quite handicapped at this point. 
 
 16             Thank you very much.

 17             DR. AUCHINCLOSS:  Thank you. 
 
 18             Any other comments from the public as part of the 
 
 19   open public hearing? 
 
 20             We move on to the afternoon session.  We have the 
 
 21   data on the table from this morning.  In fact, however,

 22   those who have followed this field recently have been aware 
 
 23   of most of the data that we saw this morning for some time.  
 
 24   I don't know about everybody else, but it is far from clear 
 
 25   to me, despite knowing the data, exactly what we are 

 
                                                               149 
 
  1   supposed to do about it.

  2             That is really the topic for this afternoon, in 
 
  3   general.  As I mentioned earlier, the discussions of three 
 
  4   questions here will be initiated by presentation from the 
 
  5   focus groups.  In order to get the conversation going‑‑I 
 
  6   don't expect that to be very hard‑‑but, again, the emphasis

  7   is that this is open for discussion by all committee members 
 
  8   whether you are part of the individual focus group or not. 
 
  9             Again, our purpose here‑‑this is not a federal 
 
 10   regulatory body, this subcommittee.  We are here to provide 
 
 11   advice to people from the FDA and that won't necessarily be

 12   in the form of a consensus.  We really want to hear all 
 
 13   points of view and put those out in front of the people who 
 
 14   have some very difficult decisions to make in this area. 
 
 15             With that introduction, we will move on to the 
 
 16   presentation from the focus group.  John Coffin, I believe,

 17   is first at presenting the response and the general 
 
 18   introduction to Question No. 1. 
 
 19             Formatwise, what I understand we will do here is 
 
 20   the focus group leader will make a ten or fifteen 
 
 21   presentation and then you, Amy, will lead us through the

 22   questions part by part as a larger group; is that how we are 
 
 23   going to work this? 
 
 24             DR. PATTERSON:  We can do that or we can project 
 
 25   the questions; however the focus group wants to go through 

 
                                                               150 
 
  1   them.  But I think the strategy you have outlines probably

  2   makes the most sense. 
 
  3             DR. AUCHINCLOSS:  Is that all right with you, Joe? 
 
  4             DR. COFFIN:  It is okay with me. 
 
  5        Presentation of Focus Group Analysis of Question 1 
 
  6           Development of Strategies for the Detection

  7                 of Porcine Endogenous Retrovirus 
 
  8             DR. COFFIN:  We were fortunate enough, actually, 
 
  9   to be able to get together for a one‑hour teleconference.  
 
 10   So we were able to have some discussion and come to a 
 
 11   consensus.  I should say, starting off, we are taking a very

 12   stringent line on the consensus but I think it is a good 
 
 13   starting point for discussion of these issues. 
 
 14             The questions that we were asked to address can be 
 
 15   summarized as follows; first, should it be required‑‑I am 
 
 16   just giving a quick summary of them.  I am not reading them

 17   as they are written in the book‑‑it should be required that 
 
 18   assays for porcine endogenous viruses in graft material be 
 
 19   developed and applied and, if so, what assays, what are the  
 
 20   important considerations of sensitivity and specificity, 
 
 21   should there be induction regimes, how long should

 22   cocultures go on. 
 
 23             Second, we were asked to consider the issue of 
 
 24   biologic characterization of infectious agents; again, 
 
 25   discussion issues of sensitivity and specificity, 

 
                                                               151 
 
  1   infectivity for other cell substrates.  The big question is

  2   hidden in there and that is the impact of the detectable 
 
  3   virus on the suitability of xenotransplant material for use. 
 
  4             The final issue was how are these considerations 
 
  5   affected by different types of uses, for example, 
 
  6   consideration of transplant versus ex vivo applications or

  7   considerations of duration of use. 
 
  8             I should say, starting out, that the group was 
 
  9   somewhat concerned also that we should not forget 
 
 10   consideration of infectious agents other than endogenous 
 
 11   viruses.  But given the lack of information that we were

 12   provided concerning these other agents and the complexity of 
 
 13   this specific problem and the specificity of the charge, we 
 
 14   won't consider that issue any further.  But we certainly 
 
 15   shouldn't forget it. 
 
 16             Second, it is clear that our principle problem

 17   here is ignorance.  We know very little about the 
 
 18   distribution of infectious proviruses among pigs and 
 
 19   essentially nothing about their potential for replication 
 
 20   and pathogenesis in normal and immuosuppressed human hosts. 
 
 21             We are beginning to get that data, clearly, from

 22   what we have heard this morning but we have a long way to go 
 
 23   and we can certainly only guess about their potential to be 
 
 24   transmitted from one host to another. 
 
 25             Furthermore, there is no established animal to 

 
                                                               152 
 
  1   study these issues.  Clearly a major focus of

  2   clinical/preclinical studies should be to obtain this 
 
  3   information.  Again, we have heard a fair amount of research 
 
  4   in that direction.  That should certainly be continuing 
 
  5   until we feel more comfortable that we have some basic 
 
  6   understanding of what is going on.

  7             It was noted that currently ongoing 
 
  8   xenotransplantion trials in primates could and should be 
 
  9   utilized to provide some of the necessary information‑‑I 
 
 10   think we have heard a little about this‑‑as well as, of 
 
 11   course, all the data from the human studies that have gone

 12   on. 
 
 13             Given these considerations, it was the consensus 
 
 14   of the committee that human graft recipients should not be 
 
 15   exposed to infectious virus either present and/or inducible 
 
 16   from xenograft tissue until such time as animal studies or

 17   human studies provide a measure of confidence that spreading 
 
 18   infection will not result in the recipient. 
 
 19             Stringent testing of the potential donor tissue 
 
 20   will be required.  Because the proviruses are inherited, 
 
 21   however, it may not be necessary‑‑it probably won't be

 22   necessary‑‑to test each donor organ or tissue prior to use.  
 
 23   If it can be established for a given strain that the 
 
 24   prospective donor animals have the same composition of 
 
 25   proviruses as the test samples, which is the genetic effect 

 
                                                               153 
 
  1   we are looking at, there needn't be a burden of extensive

  2   testing of each organ if you understand the genetic 
 
  3   composition of the host relative to this particular issue. 
 
  4             Conversely, if a given tissue or a specific donor 
 
  5   strain has tested positive for infectious virus, then that 
 
  6   tissue must be assumed to be infectious in all other animals

  7   of the same strain unless we demonstrated genetically the 
 
  8   responsible provirus is not present in the donor. 
 
  9             So, given those principles, our answers to the 
 
 10   specific questions as posed are as follows: yes, appropriate 
 
 11   tests for the release of PoEV infections, infections for

 12   humans should be developed and applied.  That clearly is 
 
 13   ongoing. 
 
 14             The development process should also include 
 
 15   studies on the genetics of the proviruses since, again, it 
 
 16   can be assumed that if the virus can be obtained from a

 17   specific organ from one individual, all individuals carrying 
 
 18   the same composition of proviruses will have the same 
 
 19   property. 
 
 20             Conversely, it can be established that, if a 
 
 21   tissue does not release virus from a sample of animals, then

 22   all donor animals with the same proviruses will also have a 
 
 23   same property. 
 
 24             Testing should include in vitro cocultivation 
 
 25   studies as well as follow‑up studies in appropriate animal 

 
                                                               154 
 
  1   models and long‑term clinical monitoring of human

  2   recipients. 
 
  3             Second, the best sort of in vitro test is 
 
  4   cocultivation with as sensitive an indicator line as can be 
 
  5   found.  It is not necessary for the cocultivation to be with 
 
  6   a human cell line.  The mink cells or the cat cells that Dr.

  7   Onions mentioned would seem to be reasonable‑‑this requires, 
 
  8   of course, a careful study to determine this‑‑under 
 
  9   conditions which will vary depending on the donor tissue. 
 
 10             I would think that, although we haven't heard much 
 
 11   of this used, the subtoxic levels of inducing agents such as

 12   demethylating agents, such as 5‑azatadine or 
 
 13   bromodeoxyuridine, also are good adjuncts to that because 
 
 14   that gives the best chance of seeing if anything in there 
 
 15   can be induced.  These are well known to be good inducers of 
 
 16   endogenous proviruses in other animal models.

 17             Filtered supernatant from coculture can be tested 
 
 18   for infectivity in appropriate indicator human cell line.  
 
 19   We have seen a lot of this this morning.  I hardly need to 
 
 20   belabor it‑‑using specific PCR assays and so on.  Certainly, 
 
 21   coculture is to be maintained as long as practical,

 22   particularly since we consider they are being used to 
 
 23   clarify the situation with a given strain of animals rather 
 
 24   than with a specific individual. 
 
 25             Recipients should then be tested for spreading, or 

 
                                                               155 
 
  1   localized transmission of infectious virus.  The next focus

  2   group will consider these issues.  Certainly, any recovered 
 
  3   infectious should be characterized by sequencing and to 
 
  4   identify it as a novel or known agent.  
 
  5             As an aside, an issue that came up this morning.  
 
  6   It will be important to get an idea of how many proviruses

  7   can release infectious virus.  One way to do that is to 
 
  8   sequence a lot of these and see if you get even slight 
 
  9   differences.  Even a single base difference, reproducibly in 
 
 10   sequence from what is otherwise the same virus would provide 
 
 11   fairly strong evidence that there are actually a couple of

 12   different proviruses that can give rise to essentially the 
 
 13   same thing.  It will be important to know that for future 
 
 14   genetic analysis. 
 
 15             Any new agent with a novel sequence should, of 
 
 16   course, be characterized by determination of host range for

 17   cells in a variety of species in part with the goal of 
 
 18   identifying potentially suitable animal models.  Also, 
 
 19   infectious for primary human cell types should be tested, 
 
 20   again to provide guidance as to how best to assess infection 
 
 21   in the human recipient.

 22             Guiding by these results, infectivity and 
 
 23   pathogenicity should be evaluated after infection at as 
 
 24   large a possible of virus in appropriate newborn animals.  
 
 25   Again, experience with retroviruses indicates most 

 
                                                               156 
 
  1   pathogenesis models, especially with viruses like these,

  2   require the use of newborn animals in order to really see 
 
  3   what is going on. 
 
  4             Injected animals should be monitored 
 
  5   appropriately.  They, obviously, should be thoroughly 
 
  6   analyzed by PCR and by antibody studies.  Until such studies

  7   provide some evidence that there is no spreading infection 
 
  8   or pathogenic consequences in the models used, the bottom 
 
  9   line sort of is the likely presence or inducibility of virus 
 
 10   infections for human cells and donor organs should preclude 
 
 11   its use in xenotransplant studies until there is good reason

 12   to expand and to go forward, given clean negative results in 
 
 13   appropriate models. 
 
 14             In answer to the last question, although it was 
 
 15   recognized that some applications such as short‑term 
 
 16   extracorporeal perfusion and introduction of small numbers

 17   of cells into sites such as the brain where virus spread is 
 
 18   likely to be inefficient, that such treatments would have a 
 
 19   lower probability of giving rise to spreading infection than 
 
 20   others. 
 
 21             It was a consensus view that all procedures

 22   carried some risk of transfer of infectious virus and should 
 
 23   not be performed if virus is present. 
 
 24             That is basically the bottom line of our 
 
 25   discussion. 

 
                                                               157 
 
  1             DR. AUCHINCLOSS:  Before we go to putting the

  2   individual components of the question up, at least I am 
 
  3   going to ask for a couple of clarifications on this and 
 
  4   maybe other members of the committee do as well, and then we 
 
  5   will go through the stages of your question one‑by‑one for 
 
  6   the committee at large.

  7             There are really two points that I wanted you to 
 
  8   clarify for me, John.  One is this issue of is there an 
 
  9   animal model and, in particular, does pig into non‑human 
 
 10   primate constitute an adequate test of the infectivity, 
 
 11   pathogenicity, of the pig endogenous retrovirus?

 12             During the course of the morning, we seem to hear 
 
 13   that infecting non‑human primates with this virus was harder 
 
 14   than infecting human cells, but we did see several examples 
 
 15   where it appeared to be possible.  But, on the other hand, 
 
 16   would the tropism that you might find or not find and the 

 17   pathogenicity you might find or not find in a non‑human 
 
 18   primate tell you anything about what would happen in the 
 
 19   human? 
 
 20             Is there an animal model for this or is the 
 
 21   information going to come from clinical studies?

 22             DR. COFFIN:  It is a very good question.  I think 
 
 23   attempts should be made to develop some kind of an animal 
 
 24   model, perhaps even in the mouse, just to get some handle on 
 
 25   how you work with this or what you might want to look for.  

 
                                                               158 
 
  1   I think the points that you raise are very good ones. 

  2             Starting off from scratch, there is no assurance 
 
  3   that what you are going to see is going to be relevant.  I 
 
  4   think one thing we didn't appreciate, at least I didn't 
 
  5   appreciate, when we had this discussion, was the size of the 
 
  6   human population that is available for study already.  Given

  7   that, I think I, at least, would modify somewhat and I see 
 
  8   some nods from other subcommittee members, on going more 
 
  9   toward detailed in‑depth studies of human recipients. 
 
 10             DR. AUCHINCLOSS:  A second point of clarification 
 
 11   for you, John, is that it sounded like what you were saying

 12   is that if you can demonstrate infectious virus in a donor 
 
 13   source that that source is out.  That is what I thought I 
 
 14   heard you say.  That, I though, translated to pigs are out. 
 
 15   Actually, I thought it translated to no further 
 
 16   xenotransplantation.

 17             DR. COFFIN:  That is a possible outcome of this.  
 
 18   That actually, I should say, in all honesty, is a more 
 
 19   stringent position than I, myself, personally take but it 
 
 20   was the consensus.  It was the consensus of our group that 
 
 21   we should at least start with this point, start with this

 22   position. 
 
 23             DR. AUCHINCLOSS:  Am I right in saying that this 
 
 24   is a Catch 22, the only way of testing the issue is clinical 
 
 25   trials.  That was the point of my first clarification.  But 

 
                                                               159 
 
  1   no clinical trials should go ahead, would be your focus

  2   group‑‑ 
 
  3             DR. COFFIN:  It was suggested that there be a 
 
  4   graded sort of series of things as you get more and more 
 
  5   comfortable.  That is what I would suggest. 
 
  6             DR. AUCHINCLOSS:  Is it not correct that we are

  7   all taking‑‑is it correct?  Are we working with the 
 
  8   assumption that all pigs have an infectious endogenous 
 
  9   retrovirus that can go into human cells? 
 
 10             DR. COFFIN:  Until shown otherwise, I think we 
 
 11   have to.  I don't think we have any choice.

 12             DR. HIRSCH:  I am surprised by the recommendation.  
 
 13   I think it takes an awfully draconian position. 
 
 14             DR. COFFIN:  It does that. 
 
 15             DR. HIRSCH:  That the virus is guilty until proven 
 
 16   innocent and that the tissues are proven guilty until‑‑

 17             DR. COFFIN:  You can't prove innocence here as in 
 
 18   a court of law. 
 
 19             DR. HIRSCH:  But the point being that the data I 
 
 20   have heard this morning is that most pigs have this or all 
 
 21   pigs have this and all pig tissues must be assumed to have

 22   it.  And yet it seems to have a low infectivity‑‑ 
 
 23             DR. COFFIN:  Can I just work through the 
 
 24   alternative because the alternative really is that you go 
 
 25   ahead, obviously, with the extensive monitoring and, 

 
                                                               160 
 
  1   perhaps, with some limitation on the kinds of things that

  2   are done initially limiting to more life‑threatening 
 
  3   conditions and so on until a better experience is obtained 
 
  4   and work sort of through in that way. 
 
  5             What that means is that basically you don't pay 
 
  6   any attention to whether the virus is in the tissue or not,

  7   because that is the alternative.  You actually don't test.  
 
  8   You don't bother because you assume it is there but you are 
 
  9   not going to care. 
 
 10             DR. AUCHINCLOSS:  There is a middle position.  I 
 
 11   would take that position that you don't care, because I

 12   assume it is there.  I think the tests are worthwhile 
 
 13   primarily to validate the test because what you really want 
 
 14   to be testing later on is the patient. 
 
 15             DR. COFFIN:  There is a second use for these, and 
 
 16   that is what we have heard today so far, is to get as much

 17   information as possible to work later on. 
 
 18             DR. AUCHINCLOSS:  So you can argue for testing 
 
 19   without saying that because the virus is assumed to be 
 
 20   there, you should never do xenotransplantation‑‑ 
 
 21             DR. COFFIN:  I am in a slightly uncomfortable

 22   position here. 
 
 23             DR. ALLAN:  I think part of the reason there is 
 
 24   this rigidity and, obviously, it is a starting point‑‑the 
 
 25   real question is whether we have done enough homework in 

 
                                                               161 
 
  1   terms of understanding what a pig endogenous retrovirus

  2   might do. 
 
  3             Obviously, you have got the non‑human primates 
 
  4   that have already been used in preclinical studies.  Those 
 
  5   really need to be evaluated and it sounds like people are 
 
  6   beginning to do that.  But you can do something very clearly

  7   and, even though Carolyn's studies would suggest that maybe 
 
  8   it doesn't go into primates, I would probably suggest that 
 
  9   three cell lines isn't enough to be able to tell. 
 
 10             I would think that probably you could find a non‑ 
 
 11   human primate species, whether it is a macaque or baboon and

 12   we should probably use several of the species, is that you 
 
 13   can do everything possible to set up an ideal situation to 
 
 14   transmit a virus and to get to express in a non‑human 
 
 15   primate. 
 
 16             You can use newborn baboons and macaques.  You can

 17   bolus them with large amounts of cell‑free virus, also virus 
 
 18   that has been put through a non‑human primate to set up an 
 
 19   ideal situation to get the virus to express itself in that 
 
 20   particular primate. 
 
 21             If, in fact, the virus doesn't infect a baboon, it

 22   doesn't infect a macaque or it is only at very low levels, 
 
 23   you may be more assured, going forward with these particular 
 
 24   studies, that you may have less of a risk than you initially 
 
 25   have thought.  You have some sense of where your starting 

 
                                                               162 
 
  1   point is.

  2             If you just start into humans going blind, then I 
 
  3   think you have got the problem.  The real issue for me is 
 
  4   that you are dealing with, and as John Coffin said, the 
 
  5   question of whether the virus is infectious.  He is starting 
 
  6   at the point that it is infectious.

  7             It may be infectious, but minimally infectious.  
 
  8   If you look at a newborn baboon and you see that you have 
 
  9   got a cell‑free virus, it is being expressed and it is in 
 
 10   high virus loads or something like that, you are going to be 
 
 11   more careful about going further in humans because what you

 12   need to understand is that we can talk about pathogenicity 
 
 13   and whether these viruses are pathogenic or not, but there 
 
 14   is no assay, there is no way to determine whether or not pig 
 
 15   endogenous retrovirus is going to be pathogenic in humans. 
 
 16             We have enough information from human retroviruses

 17   that suggests that their pathogenicities may only show 
 
 18   themselves after many years and after a lot of people are 
 
 19   infected. 
 
 20             So I think that if you can demonstrate that you 
 
 21   have got very little virus expression in that particular

 22   animal model system, it is going to give you a lot more 
 
 23   assuredness that going forward is not going to be‑‑or is 
 
 24   going to be, maybe, less of a problem. 
 
 25             So the issue is what do you need to do.  Can you 

 
                                                               163 
 
  1   do it in parallel at the same time you are doing human

  2   studies or do we need to do a few non‑human primate studies 
 
  3   very quickly.  We can argue that point, too. 
 
  4             DR. DESROSIER:  The committee didn't start with a 
 
  5   complete knowledge base but I think most members of the 
 
  6   committee were not comfortable with the notion of knowingly

  7   putting in replication‑competent pig retrovirus into people.  
 
  8   I think that was our basic starting assumption. 
 
  9             We were not aware of much of the data presented 
 
 10   this morning, particularly the numbers of humans who have 
 
 11   already received either the brain implants or the hepatocyte

 12   treatments.  So I think there is a wealth of information now 
 
 13   in these people who have already been exposed to pig 
 
 14   materials. 
 
 15             I will frankly say that I think it is entirely 
 
 16   possible that all of these humans that we heard about this

 17   morning have been infected with pig retrovirus, but 
 
 18   appropriate assays have not yet been used to demonstrate 
 
 19   that. 
 
 20             I think if all of these people had been Herpes 
 
 21   simplex virus negative and we had knowingly inoculated

 22   Herpes simplex virus into these people, then it would not 
 
 23   have been detected with the assay procedures that were used. 
 
 24             I think it is very important, very important, that 
 
 25   sensitive, reliable antibody tests be developed and that 

 
                                                               164 
 
  1   these people who have already been used in these early

  2   trials be monitored for antibody responses to the pig 
 
  3   retrovirus. 
 
  4             I think that information is going to be enormously 
 
  5   valuable in deciding how to go forward. 
 
  6             DR. AUCHINCLOSS:  I am going to continue with

  7   general comments here for a couple more minutes and then we 
 
  8   are going to tie ourselves down to the sort of particular 
 
  9   questions.  So let's go ahead. 
 
 10             DR. VANDERPOOL:  I am not a biologist or a 
 
 11   specialist in the areas in which most of the people this

 12   morning have spoken so far.  I am primarily interested in 
 
 13   ethics and history, the history of medicine.  But it seems 
 
 14   to me that the surprise we all had with the new information 
 
 15   about the porcine retroviruses certainly seems, on the 
 
 16   surface, to have entirely legitimated the FDA's putting

 17   studies on hold. 
 
 18             Yet, as I listened this morning, I kept saying, 
 
 19   "Okay; should I get more scared or less scared, more scared 
 
 20   and less scared?"  I went back and forth more or less for 
 
 21   most of the morning.  

 22             And then I began to reflect, okay; there are 
 
 23   xenotransplant trials and there are xenotransplant trials.  
 
 24   Some seem to be less risky than others and, certainly, more 
 
 25   efficacious than others.  I just want to put a word in for 

 
                                                               165 
 
  1   those trials that appear to be greatly efficacious for

  2   desperate patients and to say unless there are clear signs 
 
  3   that there is real danger out there, that we ought to give 
 
  4   those a chance to go forward. 
 
  5             It depends on how squeaky clean you want to be 
 
  6   about harm and risk, but the challenge, as was introduced

  7   this morning, is to balance harms, conceivable harms, and 
 
  8   certainly benefits.  So I think we need to restore the 
 
  9   benefit side of our deliberations as we go forward. 
 
 10             DR. WEISS:  Unfortunately, I wasn't able to join 
 
 11   in the long conference call that John Coffin conducted, but

 12   maybe that is an advantage.  It gives me an individual 
 
 13   opinion.  As one of the guys who sort of set the hare 
 
 14   running by showing that these retroviruses, that some of 
 
 15   them can replicate in human cells, I still feel that it 
 
 16   would be too draconian to say we have got to stop

 17   everything. 
 
 18             Just as we have heard from Dr. Vanderpool, there 
 
 19   is a risk in every medical procedure.  I think the 
 
 20   risk/benefit calculations for the indication recipient‑‑ 
 
 21   well, if I was one of them, I would take the risk.  So, to

 22   my mind, the big question before us is the most difficult to 
 
 23   answer; what is the risk of human to human transmission if a 
 
 24   recipient of a xenograft becomes infected with the virus. 
 
 25             I think C‑type viruses are different from 

 
                                                               166 
 
  1   lentiviruses.  They tend to be less pathogenic when

  2   introduced into adults, although severely immunosuppressed 
 
  3   adults is a very different matter from immunocompetent 
 
  4   adults. 
 
  5             I think the probability on the transmission, of  
 
  6   starting a human epidemic iatrogenically through

  7   xenotransplantation, is a remote one but it is a very 
 
  8   devastating one. 
 
  9             So my opinion wouldn't be that we absolutely must 
 
 10   not proceed in a single further patient but, rather, as we 
 
 11   have heard already, that we should extract the maximum

 12   information that we can from the people who have been 
 
 13   potentially exposed because they have been exposed to live 
 
 14   pig cells. 
 
 15             We should get more information on that.  We should 
 
 16   try and develop animal models, imperfect though they are. 

 17   We should proceed slowly.  But I don't think we should go 
 
 18   into reverse.  That is just a personal opinion. 
 
 19             DR. AUCHINCLOSS:  Amy, I am going to turn to you 
 
 20   to put up the questions and we will really go through 
 
 21   question 1 piece by piece.  But, before we do that, I am

 22   going to suggest two things that I think I have heard that 
 
 23   maybe there is a consensus on at the table here. 
 
 24             Let me start with the one that Robin Weiss just 
 
 25   expressed, that, at least in my view, the risk/benefit 

 
                                                               167 
 
  1   analysis for the individual recipient is well within reason

  2   and not the issue, certainly not the retrovirus issue or 
 
  3   even any infectious issue related to xenotransplantation.  
 
  4   Frankly, if you compare it to allotransplantation, it is 
 
  5   much safer. 
 
  6             So the issue is not the individual.  The issue is

  7   public health.  Is that something you would agree with at 
 
  8   this table? 
 
  9             [Affirmative responses.] 
 
 10             DR. AUCHINCLOSS:  The second thing I am going to 
 
 11   suggest that there might be agreement on is that one must

 12   assume, at this point, that all pigs have an endogenous 
 
 13   retrovirus capable of infecting human cells.  Is that 
 
 14   something we would all say? 
 
 15             DR. SIEGEL:  Let me get a clarification of that, 
 
 16   though, because there was a lot of discussion.  I am not

 17   sure all the people were addressing the same issue.  Maybe 
 
 18   Dr. Coffin, you can clarify what your subcommittee was 
 
 19   talking about.  On the one hand, this committee, everybody 
 
 20   sat around and said yes, all are infected. 
 
 21             But Dr. Coffin, I thought, said you shouldn't put

 22   in tissue in which you identify or can induce infectious 
 
 23   virus.  I am not a virologist, but it is not clear to me 
 
 24   that just because all pigs both have the genome and, in some 
 
 25   tissues, you can induce virus, that that will necessarily be 

 
                                                               168 
 
  1   true of all products.

  2             We have heard from a couple of sponsors who, to 
 
  3   date, have cocultivated‑‑they haven't induced with BRDU but 
 
  4   have cocultivated and have been negative.  Is, then, what 
 
  5   you are getting at is it doesn't matter what they test or 
 
  6   induce, whether it is negative or positive, we treat it as

  7   though there is infectious virus there? 
 
  8             DR. AUCHINCLOSS:  Jay, that would be my answer is 
 
  9   that I would treat all tissue as if it was potentially 
 
 10   infectious.  But let's put this as a question because it was 
 
 11   one that I was going to ask of the committee.

 12             There is, I think, the assumption, the way you 
 
 13   phrased the issues in question 1, and, indeed, in the way 
 
 14   you have presented your report on question 1, that some pig 
 
 15   tissues are better than others and maybe some pigs are 
 
 16   better than others and maybe some strains or lines of pigs

 17   are better than others. 
 
 18             Is that what you mean to imply and is that what 
 
 19   the FDA believes? 
 
 20             DR. COFFIN:  I don't know what the FDA believes, 
 
 21   but that is what we meant.  We meant to leave the door open

 22   on that particular point until we have more information.  It 
 
 23   certainly is possible that one could identify or breed 
 
 24   animals that, in fact, don't have infectious proviruses. 
 
 25             DR. AUCHINCLOSS:  Do other members of the 

 
                                                               169 
 
  1   committee want to comment on that?

  2             DR. ONIONS:  I really want to echo Robin Weiss' 
 
  3   comments because, normally, I am very conservative on these 
 
  4   matters.  But I think one of the problems is often we use 
 
  5   the HIV paradigm.  These viruses are, biologically, very 
 
  6   different.  

  7             To actually infect mice or cats with these 
 
  8   viruses, there is an age resistance.  Once you get beyond 
 
  9   the newborn, you have to use very high titers of virus to 
 
 10   establish a persistent lifelong infection, over 103, 104 
 
 11   infectious units per animal to actually establish an

 12   persistent viremia. 
 
 13             I think that the kinds of infection that we might 
 
 14   see here, I don't exclude the possibility that we would see 
 
 15   infection of human cells.  But, to my mind, the real danger 
 
 16   is the establishment of a persistent viremia which may also

 17   lead, then, to shedding of virus in saliva or other tissues.  
 
 18   I think it is very important that we distinguish those two 
 
 19   patterns of infection that might occur in patients. 
 
 20             Whilst I think the first might occur, I am not 
 
 21   convinced that the second will occur.  I think what we have

 22   at the moment is a very good pool of patients, these 100 or 
 
 23   so, or more than 100 patients, that have been exposed to 
 
 24   porcine tissue who may help us answer that question. 
 
 25             DR. AUCHINCLOSS:  Can you give us some words that 

 
                                                               170 
 
  1   we can use to refer to these different‑‑I have used

  2   infection in my own mind to mean viruses in human cells.  
 
  3   Then there is a second level at which the viruses in human 
 
  4   cells replicating and being shed; what do we call that? 
 
  5             DR. WEISS:  John has just whispered to me and said 
 
  6   productive.  I would sort of call them either persistent

  7   productive infection or persistent viremia. 
 
  8             DR. AUCHINCLOSS:  So there is infection, producing 
 
  9   infection and then, of course, the issue of pathogenicity is 
 
 10   still separate from that. 
 
 11             DR. WEISS:  Sure.

 12             DR. HIRSCH:  I also wanted to ask John, I am 
 
 13   willing to accept what you said that every pig has, 
 
 14   potentially, infectious virus.  But I don't think I am 
 
 15   willing to say that every cell in every pig has potentially 
 
 16   infectious virus.  Again, you are assuming the cell is

 17   guilty. 
 
 18             It may be that a thyroid cell or a neuronal cell 
 
 19   or a hepatic cell can't be induced to produce this virus 
 
 20   whereas a lymphoid cell could be.  I think we should accept 
 
 21   that possibility.

 22             DR. COFFIN:  In some cases, clearly, you can't put 
 
 23   in pure tissue.  You can't put in pure kidney cells if you 
 
 24   are transplanting a kidney.  In other cases, there is room, 
 
 25   clearly, for substantial purification of cells. 

 
                                                               171 
 
  1             DR. AUCHINCLOSS:  My point is that there is an

  2   implication to the fact of testing which you may or may not 
 
  3   mean to include, and that is that if you test and the pig 
 
  4   tissue shows that it has got the endogenous retrovirus and 
 
  5   you can induce it, then you shouldn't use it.  
 
  6             You should only use tissue that you can't

  7   demonstrate the virus because you believe that that is, in 
 
  8   fact, better tissue.  I don't believe that.  I would love to 
 
  9   know whether you people think that that is true.  The tissue 
 
 10   from pigs that you can't get the virus induced is better 
 
 11   than tissue from pigs in which you can?

 12             DR. SALOMON:  I would also like to point out, just 
 
 13   from the point of developing guidelines, until we formally 
 
 14   prove that there are novel agents that we are not currently 
 
 15   testing for, I would agree with our chairman's more 
 
 16   conservative view that we ought to act as we do in the lab

 17   for HIV that all tissues are potentially infected and then 
 
 18   use our best judgment and good scientific principles to move 
 
 19   forward. 
 
 20             So I am not excluding your argument, Dr. Hirsch, 
 
 21   that there would be cells that might not produce a known

 22   virus, but we would never be able to formally exclude it at 
 
 23   this early stage in the process. 
 
 24             DR. COFFIN:  In some sense, our approach to this 
 
 25   aspect of the answer was framed by the way the questions 

 
                                                               172 
 
  1   were phrased.

  2             DR. AUCHINCLOSS:  Let's go to the questions.  I 
 
  3   think it is time that we tie ourselves to some words on the 
 
  4   piece of paper and see what we really say in response. 
 
  5             Discussion of Question 1 by Subcommittee 
 
  6             DR. PATTERSON:  The Center for Biologics at the

  7   FDA currently requires that sponsors of porcine 
 
  8   xenotransplant clinical trials develop assays capable of 
 
  9   detecting infectious porcine endogenous virus potentially 
 
 10   present in their porcine xenograft products as part of the 
 
 11   preclinical screening evaluation process.

 12             The first question is a very fundamental one.  Is 
 
 13   it appropriate for FDA to require that sponsors of 
 
 14   xenotransplant clinical trials develop assays to detect 
 
 15   infectious porcine endogenous virus potentially present in 
 
 16   their product.

 17             DR. COFFIN:  There are actually two issues here 
 
 18   that Hugh separated.  One is the research aspect of coming 
 
 19   to understand what is there, what is out there to be 
 
 20   obtained.  And that needs to go on for a while, although 
 
 21   probably not forever.  Clearly, I don't think anybody would

 22   disagree that that has to be done and should be required to 
 
 23   get that information into the bag so that we can get an 
 
 24   understanding of what we are facing here. 
 
 25             DR. AUCHINCLOSS:  I think Amy thought this was a 

 
                                                               173 
 
  1   "gimme."  We are going to say yes, but we are not going to

  2   draw implications from it at this point.  We are just going 
 
  3   to say that good science says that you should be testing 
 
  4   your tissue. 
 
  5             DR. SIEGEL:  Let me clarify where we are on this 
 
  6   because, when I do that, it may or may not be a "gimme." 

  7   The position the agency has taken at this point in time is 
 
  8   not a position on the recommendation which was just made 
 
  9   which we will get to in a later question about what to do 
 
 10   when a test is positive but that, in fact, we are at a state 
 
 11   of technology now which calls for doing the assay, that

 12   there are sufficiently sensitive and informative assays that 
 
 13   can lead to risk control and risk and risk containment, that 
 
 14   those assays should be done on the tissues and on any 
 
 15   patients that have been treated to date for whom adequate 
 
 16   specimens are available prior to proceeding with further

 17   experimentation which is implicit in the word "preclinical." 
 
 18             That is why, at the present time, a number of 
 
 19   these investigations are not currently proceeding because 
 
 20   they are gathering the data that we have asked them to 
 
 21   gather.  And so part of what we are asking the committee is

 22   not simply should these data be gathered but is it 
 
 23   appropriate, while we have out there patients who have 
 
 24   already received treatments who have yet to be tested in any 
 
 25   way and tissues that have yet to be tested, is it 

 
                                                               174 
 
  1   appropriate to continue to treat patients or should we get

  2   that information and then proceed. 
 
  3             DR. COFFIN:  That is a very different question. 
 
  4             DR. SIEGEL:  That is that question.  It is just 
 
  5   worded that way. 
 
  6             DR. COFFIN:  We certainly read it differently.

  7             DR. SIEGEL:  I will take full responsibility for 
 
  8   any confusion in the wording, but that is the question we 
 
  9   need an answer to. 
 
 10             DR. COFFIN:  It is no longer a "gimme" then. 
 
 11             DR. AUCHINCLOSS:  No; it is not a "gimme."  It is

 12   distinctly not a "gimme" and, in fact, we have been trying 
 
 13   to get this distinction since we first began with this 
 
 14   question.  We are all going to say, all right; yes.  Testing 
 
 15   is a good thing.  Fine. 
 
 16             Now, my own personal view would be testing is a

 17   good thing because you want to gather information and you 
 
 18   want to prove that company is capable of doing testing that 
 
 19   is sufficiently sensitive because what you are really going 
 
 20   to want to test eventually is patients who received the 
 
 21   tissue.

 22             But my own personal view would be that just 
 
 23   because you got a positive on the test doesn't mean that 
 
 24   that is tissue that you shouldn't use.  Now, that is far 
 
 25   from a "gimme." 

 
                                                               175 
 
  1             DR. SIEGEL:  The question now is is it okay for

  2   companies to continue to use untested tissue and not to have 
 
  3   any way to test the recipients to see if they are viremic, 
 
  4   whether they need to have those tests done before they 
 
  5   proceed. 
 
  6             MS. MEYERS:  Could I address that?  During the

  7   last decade, all the blood‑products companies didn't 
 
  8   properly test their blood products for HIV and look what 
 
  9   happened.  So any company, knowing about this virus, who is 
 
 10   dealing in xenotransplant products, who goes ahead and 
 
 11   transplants tissues without testing for this virus will be

 12   liable. 
 
 13             So even if FDA doesn't stop it now, if the tissue 
 
 14   is positive, then the courts will have to stop it later 
 
 15   because that is what is going to happen when the first 
 
 16   person gets sick.

 17             DR. NOGUCHI:  Abbey, you mentioned companies.  A 
 
 18   lot of these studies are done by companies but many are also 
 
 19   done by transplant surgeons as part of the medical 
 
 20   community.  Do you see any distinction, because that is the 
 
 21   other thing that we have done.  We have made no distinction

 22   in our clinical hold. 
 
 23             MS. MEYERS:  There really shouldn't be a 
 
 24   distinction, the way I see it, because if you don't test to 
 
 25   see if the virus is there, then you are guilty, whether you 

 
                                                               176 
 
  1   are a university or a company.  If you do test and you find

  2   that it is positive and you go ahead an transplant, you are 
 
  3   double guilty. 
 
  4             DR. COFFIN:  The question is you stipulate the 
 
  5   virus is there, which is what we are trying to do, and then 
 
  6   move on.  

  7             DR. AUCHINCLOSS:  That is the issue. 
 
  8             DR. COFFIN:  You stipulate.  You don't need to 
 
  9   test it in that sense. 
 
 10             DR. MICKELSON:  I would agree with you about the 
 
 11   testing, in particular, not testing.  But I think what that

 12   testing then does is allow you to inform the patient or 
 
 13   recipient of the status of the tissue.  An alternative might 
 
 14   be, then, to allow the patient to try to make some sort of 
 
 15   informed decision. 
 
 16             Presumably, the recipients at the moment are in an

 17   extremely life‑threatening situation that these would tend 
 
 18   to be bridging.  Most of them are bridging transplants.  
 
 19   They are not considered long‑term therapeutics but possibly, 
 
 20   at the moment, the way to look at the testing is a way to 
 
 21   sharpen in informed consent document with the proviso that,

 22   of course, efforts are being made to develop donor tissues 
 
 23   that are getting freer and freer of these PERVs. 
 
 24             DR. MICHAELS:  I think it is very dangerous to 
 
 25   actually make the connection with HIV in testing because, in 

 
                                                               177 
 
  1   fact, when the HIV testing was being done, HIV was

  2   recognized as a pathogen.  This organism, even if the 
 
  3   testing is positive, at this point in time, we don't know it 
 
  4   to be a pathogen at all. 
 
  5             Now, I do believe the testing should be done.  I 
 
  6   do believe that follow up, and I certainly concur with

  7   Claudia's comment that it really leads to the informed‑ 
 
  8   consent part.  But I think that it is very dangerous.  I 
 
  9   think what we have heard from the retrovirologists today is 
 
 10   that it is a very different virus. 
 
 11             MS. MEYERS:  So you think the tissue should be

 12   transplanted even if there is a positive result and it does 
 
 13   carry the virus. 
 
 14             DR. MICHAELS:  I think that is something we are 
 
 15   going to discuss.  We are talking about the choice. 
 
 16             DR. AUCHINCLOSS:  We are going to come to that

 17   question in a few minutes. 
 
 18             DR. ONIONS:  I think there is confusion.  What do 
 
 19   you mean by positive.  I would just like a clarification of 
 
 20   what you mean by positive.  All of these pigs, at least to 
 
 21   date until we really characterize the loci and determine

 22   whether it is possible or not possible to remove some of 
 
 23   these loci, all the pigs that I have looked at and, I think, 
 
 24   all the pigs that Robin has looked at, all contain the two 
 
 25   viruses we know infect human cells. 

 
                                                               178 
 
  1             So they all contain the provirus, the genetic

  2   information.  I would strongly suspect that, at least in 
 
  3   solid‑organ transplants where you are going to be 
 
  4   transplanting lymphoid cells, I would suspect, but do not 
 
  5   formally know, that you can induce those viruses from those 
 
  6   cells.

  7             So I am not quite sure what you mean by positive.  
 
  8   Do you mean that they are genome positive?  Do you mean that 
 
  9   they are expressing the virus?  My third point would be, if 
 
 10   you really mean that, how are you going to do that in the 
 
 11   context of the xenotransplantation of solid organ.   You

 12   don't have the time to take the organ, test the organ for 
 
 13   production of virus‑‑not in the presence of the genome, 
 
 14   production of virus‑‑and put it into a patient. 
 
 15             So I think we have to be very clear about the 
 
 16   terminology here.

 17             DR. COFFIN:  Built into our response to the 
 
 18   question was a comment that if it is true for one, you 
 
 19   assume it is true for all, leaving open the possibility it 
 
 20   might be possible to get some tissues that you could never 
 
 21   get virus from.  But if you get it once, then you have to

 22   assume that you get it every time. 
 
 23             DR. AUCHINCLOSS:  Back to the question, now, for a 
 
 24   second because I need some help from you, Jay.  We are 
 
 25   saying yes, it is appropriate to require that assays be 

 
                                                               179 
 
  1   developed.  We are saying that we are going to deal a little

  2   bit later on in the questions with what to do when you get a 
 
  3   positive. 
 
  4             What is the distinction, now, in this question 
 
  5   that you want us to‑‑ 
 
  6             DR. SIEGEL:  I guess one could argue that even if

  7   you know it is going to be positive, that having an assay in 
 
  8   place, more than informing, informed consent, provides 
 
  9   certain safety features. 
 
 10             DR. AUCHINCLOSS:  I believe that. 
 
 11             DR. SIEGEL:  You really know what sequences to

 12   look for in monitoring the patient.  You may know which 
 
 13   organs to look at because you might know what organs‑‑ 
 
 14             DR. AUCHINCLOSS:  Precisely.  It is good to have 
 
 15   the assays in place. 
 
 16             DR. SIEGEL:  So part of question is should the

 17   assays be in place and done before the‑‑not necessarily in 
 
 18   the specific heart, but in the product, in the type of 
 
 19   tissue that is going to be transplanted. 
 
 20             DR. AUCHINCLOSS:  Again, I would say yes.  But I 
 
 21   think there is some narrowing to the yes.

 22             If you have proven that your assay works, if you 
 
 23   have proven that your source of pigs is positive or negative 
 
 24   or a particular source of tissue from your source of pigs is 
 
 25   positive or negative, I wouldn't imagine that you need to 

 
        sgg                                                                180 
 
  1   prove that "every batch" be tested because the purpose is,

  2   again, not to define the virus.  The purpose is to prove 
 
  3   that you have the assay. 
 
  4             DR. VANDERPOOL:  I agree with Marian that we are 
 
  5   not assuming that this is an AIDS‑type virus.  We are just 
 
  6   saying we need the data right now, and if later studies show

  7   that these are not as problematic as they might will become 
 
  8   or appear, then the requirement could be eased. 
 
  9             I want to register one more objection to this 
 
 10   question as worded and that is that the sponsors be required 
 
 11   to develop assays.  I think that the sponsors should be

 12   required to use assays or utilize assays and, in that way‑‑ 
 
 13   as it reads, it looks like every single sponsor needs to go 
 
 14   out there and develop a particular series of assay. 
 
 15             DR. AUCHINCLOSS:  Jay, how do you feel about that?  
 
 16   Do you have to access two assays?

 17             DR. SIEGEL:  The sponsor is responsible for 
 
 18   performing them.  It shouldn't say developing. 
 
 19             DR. HIRSCH:  Jay, I am still confused as to 
 
 20   whether you are saying that company X who is doing this 
 
 21   procedure, there has to be a moratorium on any further

 22   patients getting this procedure until they have shown that 
 
 23   using every conceivable assay that the previous recipients 
 
 24   don't have infection.  Is that what you are saying? 
 
 25             DR. SIEGEL:  What we have told companies now, and 

 
        sgg                                                                181 
 
  1   there is a letter in your book under Tab 9, is that before

  2   proceeding, they should have in place assays for both the 
 
  3   product and for the patient and informed consent for the 
 
  4   patient. 
 
  5             What we are asking you now is, is that an 
 
  6   appropriate approach and then what you just asked me, what

  7   assays?  Is it every conceivable one, what should they have 
 
  8   to induce?   
 
  9             DR. AUCHINCLOSS:  We are going to come to (b) 
 
 10   right now.  What tests, and what do we do with the results?  
 
 11   We have made no decision that just because it is positive‑‑

 12   we understand that that would be a hold.  I think, rather, 
 
 13   our current situation is based not so much, as I view it, on 
 
 14   a risk‑benefit analysis but a risk‑control analysis.  We 
 
 15   don't always accept a risk simply because it is overwhelmed 
 
 16   by benefits.  There are different types of risks.  There are

 17   some that are intrinsic to a product.  You are going to get 
 
 18   them; if you are going to get the benefits you are going to 
 
 19   get the risks.  There are others that are controllable.  To 
 
 20   use what is probably a poor analogy, we, in blood, say if we 
 
 21   were to use less sensitive blood screening mechanisms for

 22   HIV we could probably still prove, and they were cheaper‑‑ 
 
 23   some company could probably come and say, you know, for 
 
 24   every 1000 units we give we are going to save a lot more 
 
 25   lives than are going to transmit HIV.  But we would say that 

 
        sgg                                                                182 
 
  1   is not adequate risk control because you can do better.  

  2             So, where we are now is saying, well, you can do 
 
  3   these assays and these assays are going to be informative 
 
  4   and potentially important in terms of assessing the arm and  
 
  5   controlling arm in looking how to follow the patient, 
 
  6   knowing what the potential are and knowing what to tell

  7   future patients, and since that can be done, and it appears 
 
  8   at this point reasonable to do that, and since it is 
 
  9   evolving so rapidly we need expert input as to whether, in 
 
 10   fact, it is reasonable and how much is reasonable.  I can 
 
 11   assure you that if we ask them to do every conceivable assay

 12   by the time they are done there will be a better assay.  
 
 13   Somebody will have a more sensitive one.  You know that‑‑ 
 
 14   more sensitive strains.   
 
 15             We recognize it is an evolving thing and we are 
 
 16   just trying to get at that.  So, let's go to (b) because for

 17   (a) I think the answer is yes and (b) is fundamentally, all 
 
 18   right, what assay?  So let's go from there.   
 
 19             DR. PATERSON:  Actually, if I may take the 
 
 20   liberty, Carolyn had a quick question to clarify one of Dr. 
 
 21   Coffin's comments. 

 22             DR. WILSON:  I just wanted to follow up on a 
 
 23   comment you made before.  You said if you did testing on a 
 
 24   particular organ and it was positive you would then be able 
 
 25   to assume that in all cases it would be positive.  What if 

 
        sgg                                                                183 
 
  1   it was the opposite, where the first few tests you did were

  2   negative? 
 
  3             DR. COFFIN:  Clearly then you would have to come 
 
  4   to some consensus or decision as to what you would consider 
 
  5   an adequate sort of negative representation and the 
 
  6   sampling, and that is something that would have to be

  7   considered separately.  
 
  8             DR. AUCHINCLOSS:  Let's see what we feel about 
 
  9   (b). 
 
 10             DR: PATERSON:  Yes, question (b):  Please discuss 
 
 11   the types of screening assays most appropriate for accurate

 12   detection and identification of infectious porcine 
 
 13   retrovirus  in porcine xenotransplant products. 
 
 14             DR. AUCHINCLOSS:  You have this broken down into 
 
 15   (i), (ii), (iii).  Do you want to go directly to (i)? 
 
 16             DR. PATERSON:  Right.  

 17             DR. COFFIN:  I think we have seen good examples of 
 
 18   this today in some of these things being done.  
 
 19             DR. AUCHINCLOSS:  Maybe that is one way of asking 
 
 20   it.  
 
 21             DR. COFFIN:  With the exception that I would like

 22   to see some induction studies included as well to see if we 
 
 23   can get other things.   
 
 24             DR. AUCHINCLOSS:  We have heard one statement that 
 
 25   glaringly missing is antibody.  This is tissue now at this 

 
        sgg                                                                184 
 
  1   point, so we will come back to antibody when we come to the

  2   patients.  I am jumping the gun.  That is question 2.  Sorry 
 
  3   about that.  So, stick with the tissue.  So I guess we can 
 
  4   turn to Carolyn and say, Carolyn, you have heard two 
 
  5   companies tell you what they now do with their tissue.  Is 
 
  6   it okay?  

  7             [Laughter] 
 
  8             DR. WILSON:  Well, I was going to turn it around.  
 
  9   I think one of the areas that is being explored, and we 
 
 10   heard data from Robin Weiss and David Onions about 
 
 11   alternative cell substrates that may be more sensitive for

 12   detecting certain strains and whether or not we need to have 
 
 13   more exploration in that area.  Right now, we are currently 
 
 14   recommending that 293 and ST cells be used because we have 
 
 15   seen the most data on that and have the most experience.  
 
 16   But if there are recommendations for other substrates, or

 17   whether or not we need to even explore that more fully, I 
 
 18   think that might be useful to discuss.   
 
 19             DR. AUCHINCLOSS:  So, ST is the positive control; 
 
 20   293 is the test cell.  Is that what you are recommending at 
 
 21   this point?  

 22             DR. WEISS:  But in our experience, which is 
 
 23   limited, you know, the mink cells are clearly more sensitive 
 
 24   to the 293 cells for detecting viruses then have the 
 
 25   potential to grow in human cells, and from what David Onions 

 
        sgg                                                                185 
 
  1   said, his in‑house cat cells line is even more sensitive

  2   than that.  So, I think we have to test human cells but if 
 
  3   we know that there are other indicator cell lines that are 
 
  4   sort of surrogate markers for infection and are more 
 
  5   sensitive, they should be included.   
 
  6             DR. ONIONS:  Yes, but I would be cautious because,

  7   again, our experience is limited because we see fantastic 
 
  8   patterns, certainly on 293 cells, I wouldn't like to stand 
 
  9   up and say it is more sensitive but, certainly, our 
 
 10   experience echoes Robin's.  That is, these two cell lines at 
 
 11   least are more permissive than 293 or, indeed, Raji.  In our

 12   hands, actually Raji is slightly better than 293 but, again, 
 
 13   it is limited experience.   
 
 14             DR. AUCHINCLOSS:  But Carolyn said Raji didn't 
 
 15   work for her.  
 
 16             DR. WILSON:  I didn't do Raji. 

 17             DR. AUCHINCLOSS:  It was somebody else who didn't 
 
 18   have a positive on that.   
 
 19             DR. COFFIN:  It is important to keep in mind that 
 
 20   the co‑culture cell is different from the indicator cell you 
 
 21   eventually use in testing.  You want to give yourself your

 22   very best shot at getting something out on the first round 
 
 23   and then see what you get.   
 
 24             DR. AUCHINCLOSS:  So, does somebody from the 
 
 25   Committee want to tell the FDA what a basic set of assays 

 
        sgg                                                                186 
 
  1   should be?

  2             DR. ALLAN:  I have just one comment on that, and 
 
  3   that is with the RT‑PCR.  There were some studies that have 
 
  4   been implemented.  One of the things I noticed was that none 
 
  5   of them have been standardized.  I saw 10‑3 and 5 
 
  6   microliters of concentrate and soup, and based on what we

  7   know about working with HIV and some of these other viruses, 
 
  8   you really need to have better standardization.  You need to 
 
  9   know how many virus particles or what the equivalents are, 
 
 10   RNA copies/ml, something like that, something that is more 
 
 11   standardized rather than we just have this culture soup and

 12   we diluted it out and this is the sensitivity.  So, that is 
 
 13   very specific but may be helpful.  
 
 14             DR. COFFIN:  Let me say as well that there is 
 
 15   probably room here for development of some rather more 
 
 16   efficient assays where one could co‑cultivate with an VM1 or

 17   a cell line. 
 
 18             DR. ONIONS:  I think we need to distinguish, 
 
 19   again, between what one might test on the donor organ or the 
 
 20   donor pig and what one might test on the patient.  In the 
 
 21   donor pigs I think it is going to be very important,

 22   experimentally at least, to demonstrate variance produced in 
 
 23   the organs in the whole pig and are they produced in organs 
 
 24   that are being transplanted into immunosuppressed hosts, 
 
 25   that is, find out whether you are actually producing 

 
        sgg                                                                187 
 
  1   virulence in either of those two situations, and I mean

  2   virulence not RNA transcripts because we know we can 
 
  3   actually detect RNA transcripts.  But we do know situations 
 
  4   in other species where we can find transcripts and no virus, 
 
  5   clearly no virus.  So, we need to define that, it seems to 
 
  6   me.  

  7             In the patients, I think really, as I tried to 
 
  8   allude to at the end of my talk, we need a battery of tests, 
 
  9   at least in the first instance to try and identify ‑‑ 
 
 10             DR. AUCHINCLOSS:  I have already slipped on the 
 
 11   patients once.  Questions 2 is the patients, and I have

 12   drawn us in there and I am sorry ‑‑ 
 
 13             DR. ONIONS:  Right.  
 
 14             DR. AUCHINCLOSS:  ‑‑ otherwise we will just get 
 
 15   confused.   
 
 16             DR. SOLOMON:  The issue that we had before, and we

 17   got into it with the cross species is where infectivity 
 
 18   comes in.  Again, I realize we want to be pragmatic, so you 
 
 19   want to go to the incubator every day and pull out your mink 
 
 20   cell line or your mutant 93 but these are not real and you 
 
 21   are really concocting it for a specific set of known

 22   viruses, which is okay but rather limited, I would think, at 
 
 23   this early stage.  I would think it is very important that 
 
 24   we agree ‑‑ and here I defer to my infectious disease 
 
 25   colleagues ‑‑ I would think it would be very important to 

 
        sgg                                                                188 
 
  1   agree that certain primary cell lines, human cell lines,

  2   transformed, activated T‑cell macrophages as well as 
 
  3   possibly primary cell epithelial cell cultures be tested as 
 
  4   well.  
 
  5             DR. ONIONS:  Could I just make a comment?  I think 
 
  6   certain co‑cultivation should be in when you look at these

  7   and you take them from the transplanted cymologous or baboon 
 
  8   or whatever your species is.  But I actually thought that 
 
  9   once you put those infected cells in, there are other ways 
 
 10   at looking for virulence, getting virulence not 
 
 11   transference, and I think those should be introduced.  We

 12   have done this in the past in animals.  Robin's group have 
 
 13   done very similar studies in another context where you then 
 
 14   put these on gradients and then look for viruses at the 
 
 15   right density.  These can be very sensitive assay systems 
 
 16   and I think it is worth considering those in these two

 17   contexts. 
 
 18             DR. AUCHINCLOSS:  Well, let me ask all the members 
 
 19   of the Committee.  You could make your dream wish list of 
 
 20   assays and they can be impossible for any reasonable company 
 
 21   to survive with.  Have you just told me about the sort of

 22   assays that no ordinary company in this world could perform? 
 
 23             DR. ONIONS:  I don't think you have to do this on 
 
 24   every pig.  Don't get me wrong, what I was trying to say is 
 
 25   to try and find out what the parameters are in a number of 

 
        sgg                                                                189 
 
  1   experimental situations.  I am not saying you should do this

  2   on every xenotransplant at all.  That would be impossible.  
 
  3   In fact, my point earlier was that I don't think that is 
 
  4   going to be possible.  What we need to know is are virulents 
 
  5   produced in these pigs normally in the line of pigs that you 
 
  6   are using?  And when you put them into, say, twenty

  7   transplants is there any evidence of virulence produced in 
 
  8   these?  That at least gives you some kind of idea of what is 
 
  9   going to go into a patient with a whole organ transplant.   
 
 10             But I would also go back to the early comments 
 
 11   that I think different transplants have very different risk

 12   factors.  That is, bridging transplants carry a much lower 
 
 13   risk than, say, a solid organ transplant.  I think we also 
 
 14   need to distinguish between these transplants.   
 
 15             DR. AUCHINCLOSS:  Indeed, because at least one of 
 
 16   the groups hopes to go ahead soon and put pig cells into the

 17   brain and hopes they will stay there very long term.  So, it 
 
 18   is not just bridge transplants that we are talking about at 
 
 19   this point.  
 
 20             DR. ONIONS:  Right.   
 
 21             DR. AUCHINCLOSS:  What I am hearing from the

 22   Committee is that they can make you a list of some assay to 
 
 23   do, and I am not sure that we want to sit down and write out 
 
 24   all the details, but you and some of the experts here can do 
 
 25   a reasonable set of assays that are doable and, you know, I 

 
        sgg                                                                190 
 
  1   think from a practical point of view you would like to have

  2   that set of assays be doable, affordable and not going to 
 
  3   change every two and a half weeks, or that the company be 
 
  4   told that the rules changed last week and next week we are 
 
  5   asking for something different.   
 
  6             DR. SIEGEL:  One thing that Dr. Coffin and I guess

  7   the subcommittee mentioned is that there is some further 
 
  8   discussion on induction.  The comments were limited to BRDU.  
 
  9   We have considered other types of induction.  Should human 
 
 10   cytokines ‑‑ is there any other dialogue? 
 
 11             DR. PATTERSON:  We asked the Committee to discuss

 12   whether agents should be used to induce viral activation to 
 
 13   enhance detection of virus.  
 
 14             DR. HIRSCH:  I think that is going a bit too far, 
 
 15   frankly, because I think from what we have heard this 
 
 16   morning you probably will be able to induce virus from most

 17   of the tissues if you use enough chemical or mitogens or 
 
 18   whatever.  The same could be said if you took human blood, 
 
 19   most of them carry EB virus latently; many of them carry CMV 
 
 20   latently; many of them carry HHV8 latently and you can 
 
 21   activate them too with the appropriate chemicals.  Does that

 22   mean we shouldn't be using these known human pathogens?  I 
 
 23   think that is going a little bit too far.  I think it is 
 
 24   important to know whether these pigs or these particular 
 
 25   tissues have virus, but to take every one of them and induce 

 
        sgg                                                                191 
 
  1   them, I think, doesn't tell us anything very useful. 

  2             DR. COFFIN:  You are arguing against yourself, 
 
  3   Martin, in a sense because EBV in fact is a real problem in 
 
  4   transplant settings.  
 
  5             DR. HIRSCH:  But it is not being tested ‑‑ 
 
  6             DR. SOLOMON:  EBV is tested on all transplant

  7   situations.  
 
  8             DR. HIRSCH:  No, you don't.  You don't try to 
 
  9   induce EBV in every transfusion ‑‑ 
 
 10             DR. COFFIN:  But you know you can.  The issue here 
 
 11   is partly the research issue, the searching for things, and

 12   there, clearly, you want to give yourself the best chance of 
 
 13   finding whatever is there and all induction does is increase 
 
 14   in frequency events which will happen without it given 
 
 15   enough cells and enough time.  
 
 16             DR. SOLOMON:  So what are you going to do with the

 17   data when you have it? 
 
 18             DR. COFFIN:  Presumably you will obtain a virus 
 
 19   that you either know or don't know.  If you know what the 
 
 20   virus is, then fine; if you know what the virus is you 
 
 21   already know how to deal with it or you know what has been

 22   done before.  If you don't know what the virus is, then you 
 
 23   work it up.   
 
 24             DR. SOLOMON:  How do you deal with the issue that 
 
 25   really at this point there is no transplant, except maybe 

 
        sgg                                                                192 
 
  1   the identical twin transplants, that doesn't engender an

  2   acute immune response or a chronic immune response?  So 
 
  3   there is going to be local infiltration of cells, release of 
 
  4   cytokines and some inflammation, period, no matter how you 
 
  5   concoct this system ‑‑ at least in the conceivable future.  
 
  6   I mean, things could change five years from now.  So, the

  7   idea of induction is something that is going to occur, at 
 
  8   least with current technology, every time we do one of these 
 
  9   transplants.  Don't you think it is reasonable to come up 
 
 10   with some sort of induction protocol?  I defer to you if you 
 
 11   say certain kinds of induction protocols get ridiculous,

 12   like ionizing irradiation maybe, but an appropriate set of 
 
 13   inflammatory cytokines might be more acceptable.  
 
 14             DR. HIRSCH:  I personally don't think you are 
 
 15   going to learn anything that you don't know already.   
 
 16             DR. ALLEN:  I would just say that since you are

 17   dealing with an unknown ‑‑ these are viruses that may or may 
 
 18   not cause any disease so if you can actually characterize 
 
 19   them ‑‑ we may be talking about unknown viruses; maybe not 
 
 20   pig endogenous viruses but maybe something else you activate 
 
 21   that you get out of pig cells that you may be transplanting

 22   to a recipient that you didn't know about, and this is one 
 
 23   of the ways you could be able to study that virus if you 
 
 24   already have transmitted it.  So these are just, like, new 
 
 25   viruses we are talking about.   

 
        sgg                                                                193 
 
  1             DR. MICHAELS:  I think from a research interest

  2   that is reasonable to do, the inducible studies and look for 
 
  3   other viruses.  I am not sure that it, in itself, should be 
 
  4   the issue that allows the FDA to say to move forward or not 
 
  5   move forward, but I certainly think from a research area to 
 
  6   try and push the system as much as the system can be pushed

  7   is reasonable.  
 
  8             DR. AUCHINCLOSS:  Key point; absolutely key point. 
 
  9 
 
 10   We all agree, or at least I would agree that it would be 
 
 11   nice to have induction studies; that the FDA should require

 12   induction studies in order for a company to proceed with the 
 
 13   use of that tissue ‑‑ no, I don't understand why that would 
 
 14   be the case.  I can't imagine that we would be learning 
 
 15   something that would make a go/no go decision, or that 
 
 16   knowing how to do the induction studies makes you better at

 17   detecting the virus later on in the patient.   
 
 18             DR. SOLOMON:  Well, I would agree with that only 
 
 19   to the point where it was not long ago that Dr. Hirsch was 
 
 20   making the argument that some cells that we transplant might 
 
 21   not be showing virus and, therefore, would be kind of less

 22   infectious risk.  My point there is if you don't induce 
 
 23   those, then your statement ‑‑  
 
 24             DR. HIRSCH:  If you have a research basis, then 
 
 25   you should certainly try to do that but for the FDA to 

 
        sgg                                                                194 
 
  1   require that mitogens and cytokines be used on every single

  2   cell that you are thinking of transplanting ‑‑ 
 
  3             DR. ONIONS:  If I could just make a comment, what 
 
  4   this discussion I think reflects is in fact our ignorance, 
 
  5   and our ignorance is really that we don't know which of the 
 
  6   many lines that we can identify are productive.  If they all

  7   are, the likelihood is, and as Robin pointed out most of 
 
  8   them will not be productive, we really do need to get to the 
 
  9   stage where we can know these loci and whether these are 
 
 10   spontaneous producers or inducible and, actually, those are 
 
 11   the data we will eventually need to get so I am very clear,

 12   as Robin is, that that is where the research emphasis should 
 
 13   go.   
 
 14             My only comment about induction, and I defer to 
 
 15   John Coffin who is certainly expert in the area, that the 
 
 16   experience tends to be that those that are at least

 17   inducible by 5‑azacytine or RDU tend to be those viruses 
 
 18   that also on occasion come up after prolonged ‑‑ 
 
 19             DR. COFFIN:  That is correct.  
 
 20             DR. ONIONS:  ‑‑ passage in the cell so their 
 
 21   inducibility ‑‑

 22             DR. COFFIN:  That is the point I was trying to 
 
 23   make.  It really increases your efficiency of detection.  
 
 24   You are unlikely to see anything that you wouldn't see given 
 
 25   a long enough assay otherwise. 

 
        sgg                                                                195 
 
  1             DR. ONIONS:  Sure. 

  2             DR. COFFIN:  But, for example, in mice where you 
 
  3   have these endogenous proviruses that are very highly 
 
  4   repressed, in cell cultures you can grow the cell cultures 
 
  5   for quite some time before the viruses spontaneously appear, 
 
  6   and they spontaneously appear in animals right around the

  7   time of birth but until that time the development of these 
 
  8   animals has kept this virus ‑‑ this virus has been kept out 
 
  9   but if you have an inducing agent they come up right away.  
 
 10   So, it is really a matter of enhancing your sensitivity in 
 
 11   detection.

 12             The other aspect of induction ‑‑ there are two 
 
 13   things that get mixed up here and the other thing is the use 
 
 14   of cytokines as stimulating agents for cells ‑‑ I think that 
 
 15   is actually fairly important, to get cells in cycle as much 
 
 16   as possible in order to get your best chance of getting

 17   things going.   
 
 18             DR. AUCHINCLOSS:  I am going to move us on to 
 
 19   subgroup (iii) which is a relatively technical point, how 
 
 20   long should your co‑culture be for before you feel like you 
 
 21   have done a good job?

 22             DR. PATTERSON:  Right, the committee was asked to 
 
 23   discuss the appropriate time periods for maintaining co‑ 
 
 24   culture assays for treating more sensitive detection of 
 
 25   porcine endogenous retrovirus.  I think we saw this morning  

 
        sgg                                                                196 
 
  1   data out to 35 days presented by Diacrin.  One of the

  2   questions is how long. 
 
  3             DR. AUCHINCLOSS:  Again, I am going to give you my 
 
  4   perspective on this, which is I think consistent all the way 
 
  5   through here.  Since I believe that you can always get virus 
 
  6   out if you do the assay correctly with the right induction

  7   and long enough and into human cells, this is not the issue 
 
  8   as to whether it is a go/no go.  So, learning how to do good 
 
  9   assays and the type of virus is important but co‑culturing 
 
 10   for 52 days instead of 35 is not, to me, important.  
 
 11             DR. ONIONS:  Can I make a very quick point?  In

 12   our hands it behaves ‑‑ in those cultures where it is 
 
 13   productive it behaves just like MELV or FELV in that we can 
 
 14   do a nice time course and by three passages it has 
 
 15   plateau'd; it has gone through the culture.  So, we always 
 
 16   do it routinely for five cultures and at three cultures it

 17   tends to plateau.   
 
 18             My only caveat to that is, and it is quite 
 
 19   interesting, in some of the cell lines where we have only 
 
 20   got the virus in as a provirus it appears to be 
 
 21   transcription silent.  If you are not careful you can

 22   actually show extinction of that virus by passing the cells.  
 
 23   For instance, I think I showed that we can get it into 
 
 24   baboon cells, and we are confident of that but, as we pass 
 
 25   those cells, because it doesn't replicate, you can actually 

 
        sgg                                                                197 
 
  1   lose it because it is so weak and if you keep passing the

  2   cells, if that clone doesn't replicate as efficiently as the 
 
  3   others, you can lose it by PCR. 
 
  4             DR. AUCHINCLOSS:  Do you want to give any days? 
 
  5             DR. ONIONS:  Well, I just say five passages and 
 
  6   you have to keep the cells mitotically active. 

  7             DR. ALLAN:  It seems to me that it is more 
 
  8   important in isolating out from the patient and then the 
 
  9   days become important because if you are trying to isolate 
 
 10   out from the patient those days ‑‑ it may be a while before 
 
 11   you see virus.

 12             DR. AUCHINCLOSS:  Fair enough, all right.  What I 
 
 13   took away from (b) in general was that, yes, there are some 
 
 14   reasonable assays that a company should be able to do, and  
 
 15   I can't phrase them for you but there are people here who 
 
 16   probably can, and they ought to somehow be doable and

 17   affordable.  That is a sort of general summary of part (b). 
 
 18             DR. PATTERSON:  All right.  In the event that 
 
 19   infectious PoEV is identified during the screening 
 
 20   evaluation of the porcine xenograft, please describe 
 
 21   biologic assays (in vitro and in vivo) most appropriate to

 22   further characterize the infectivity of porcine endogenous 
 
 23   retrovirus present in xenotransplant products.   
 
 24             DR. AUCHINCLOSS:  Well, obviously we are now about 
 
 25   to come into sort of the heart of the matter.  Do you want 

 
        sgg                                                                198 
 
  1   to go directly to (i) because you have broken this question

  2   into sort of stages of discovering infectivity etc? 
 
  3             DR. PATTERSON:  Right.  The first part of this 
 
  4   question is to discuss what sensitivity and specificity 
 
  5   might be reasonably achievable and appropriately required ‑‑ 
 
  6   two separate concepts ‑‑ of assays to further characterize

  7   the infectivity of porcine endogenous retrovirus present in 
 
  8   xenotransplant products.  Of course, the answer to this is 
 
  9   dependent upon your answer to the first part of that 
 
 10   question, what assays would be suitable to characterize the 
 
 11   infectivity of porcine endogenous virus. 

 12             DR. AUCHINCLOSS:  Let's be clear where we are.  
 
 13   You have the pig cells co‑cultures with the human cells and 
 
 14   from the human cells got an endogenous retrovirus, and now 
 
 15   you want to know what should be done to determine the 
 
 16   subsequent infectivity.  

 17             DR. COFFIN:  The first thing, actually, is to do a 
 
 18   sequence analysis and see if that virus has been identified 
 
 19   before.  I think it is clear from what we have seen before 
 
 20   that if you sequence a reasonable section of the GP70 
 
 21   portion of the envelope gene that you can have some

 22   confidence in identifying the virus with a previously known 
 
 23   agent of interest with whatever is known in the literature 
 
 24   about its infectivity.  For new viruses, I mean clearly I 
 
 25   would argue again, I think for the introduction of attempts 

 
        sgg                                                                199 
 
  1   to get some newborn animal model infection with this virus,

  2   starting perhaps with some cell culture assays for various 
 
  3   primate or even non‑primate species.  I mean, if you can see 
 
  4   spreading infection in the mouse, if you injected a newborn 
 
  5   mouse, I think you would be a long way toward being much 
 
  6   better biological characterization of these viruses.  So, I

  7   would argue for some studies that go toward the idea of 
 
  8   developing some kind of animal model where you get viremia, 
 
  9   even if transient viremia, and being able to see what the 
 
 10   consequences are.   
 
 11             DR. ALLAN:  I think what John is answering is

 12   research questions, and I think the question directed 
 
 13   towards us is clinical.  Is that right? 
 
 14             DR. AUCHINCLOSS:  That is the way I see it, is 
 
 15   this a go/no go finding and is there something you should do 
 
 16   to determine whether it is a go/no go.  

 17             DR. LERCHE:  Maybe this gets back to the question 
 
 18   Dr. Solomon was raising and perhaps what you were getting at 
 
 19   was at this stage to look for the ability of these activated 
 
 20   viruses to infect primary cell lines, and what relevance 
 
 21   that would have or more relevance to the clinical setting. 

 22   For example, is the virus not only infectious but will it 
 
 23   transform human cells?  That would certainly be ‑‑ so, 
 
 24   perhaps that would be included here to see what the range of 
 
 25   infectivity is in human primary cell lines.  That may be 

 
        sgg                                                                200 
 
  1   something that should be discussed or considered.  

  2             DR. COFFIN:  Regarding the research versus 
 
  3   clinical, I mean, they run together.  You don't have the 
 
  4   opportunity to take a kidney that you are going to use for 
 
  5   transplant, if you get a virus out, to do this workup.  It 
 
  6   is just not possible.  You have to have done it before in a

  7   research setting and then apply what you have to what you 
 
  8   see.  So, the two questions are really not different; they 
 
  9   run together.   
 
 10             DR. AUCHINCLOSS:  Well, the sponsor has done the 
 
 11   co‑culture experiment; the human cell indicator line has the

 12   virus in it; and now you tell the sponsor you can't use that 
 
 13   tissue until you have done X to determine its human‑to‑human 
 
 14   infectivity? 
 
 15             DR. HIRSCH:  No, I don't think we should say that 
 
 16   but, John, perhaps one of the more important things we could

 17   do is to say very strongly that the NIH, or whoever, set up 
 
 18   systems where we can evaluate the infectivity and the 
 
 19   pathogenicity of porcine or other endogenous retroviruses in 
 
 20   suitable animal model systems, whether they be primates, 
 
 21   whether they be nude mice, suppressed mice or newborn mice,

 22   or whatever.  I think that is what is critically needed in 
 
 23   this field.  If you can take a porcine, a rabbit or whatever 
 
 24   endogenous virus and show that it causes lymphomas in some 
 
 25   other species I would be much more concerned about these 

 
        sgg                                                                201 
 
  1   than I am today.  

  2             DR. AUCHINCLOSS:  But it is not what the sponsor 
 
  3   needs to do when they tell the FDA that they have a tissue 
 
  4   as their potential tissue source for clinical trials.  So, 
 
  5   it is a clinical versus a research issue.  
 
  6             DR. SOLOMON:  Right.  So, to take that guidance

  7   from the Chairman, to be perfectly practical then one of the 
 
  8   ways we could frame this is if you have a specific protocol 
 
  9   ‑‑ all protocols are going to be different so let's say it 
 
 10   is pig islet cells and you want to put them into the 
 
 11   intraportal circulation in a given protocol, which is going

 12   to be then exposed to the portal elements, which are the 
 
 13   Kupffer cells, the hepatic epithelium and stromal elements, 
 
 14   you should be able to provide the FDA as a go/no go, to use 
 
 15   your terms, with some evidence for primary hepatic cell 
 
 16   lines, and those are perfectly achievable.  There are a

 17   number of different ways of growing primary cell lines in a 
 
 18   lab.  As well as leukocytes that would then infiltrate the 
 
 19   graft that was rejected.  Those would be the two elements ‑‑ 
 
 20   I made that point earlier ‑‑ that would contact those cells 
 
 21   in an in vivo biological setting. 

 22             DR. AUCHINCLOSS:  So you are saying every sponsor 
 
 23   who gets a transferred human co‑cultured cell now has to 
 
 24   test for infection of human ‑‑ 
 
 25             DR. SOLOMON:  No, I said you have to take the 

 
        sgg                                                                202 
 
  1   protocol that is being proposed.  I am trying to be

  2   practical.  So, I was giving you an example ‑‑ 
 
  3             DR. AUCHINCLOSS:  No, no, I understand.   
 
  4             DR. ONIONS:  I am a little bit concerned about 
 
  5   this conversation, or at least with one misconception 
 
  6   already.  That is, first of all if you put these viruses

  7   into primary cells, in general they won't transform as cells 
 
  8   because to do that, as John explained, they have to sit next 
 
  9   to an oncogene or they have to transform an oncogene.  This 
 
 10   does not occur ‑‑ well, I am not going to go into arguments 
 
 11   about the probability of that but essentially if you put

 12   these viruses onto primary cultures in vitro you do not get 
 
 13   transformation, with some exceptions.  That is the first 
 
 14   comment.  
 
 15             The second comment is that you have to define what 
 
 16   you are trying to do here.  I am still a little bit puzzled

 17   about what the aim is here.  If you look at a clinical 
 
 18   situation you are going to have a pig and it is going to be 
 
 19   in quarantine and at some point you are going to use an 
 
 20   organ from it.  I can see that if you are going to use 
 
 21   hepatocytes or islet cells it is possible to test those

 22   because you can bank those cells frequently.  You could test 
 
 23   them for infectious virus and you could determine which of 
 
 24   the viruses that we have talked about today are being 
 
 25   released from those cells.  That won't be the situation if 

 
        sgg                                                                203 
 
  1   you move on to solid organ transplants.  You are not going

  2   to have the luxury of being able to do that.  The only 
 
  3   situation would be to look at, say, peripheral blood cells 
 
  4   and assume ‑‑ and it would be an assumption ‑‑ that the 
 
  5   pattern of expression is the same in those cells as it is in 
 
  6   the particular target organ, and I am not convinced of that. 

  7 
 
  8             I am also concerned about the idea that you should 
 
  9   look at a representative cell like a hepatocyte.  What you 
 
 10   want to use is a cell line that is the most permissive for 
 
 11   those viruses, and it might not be.  It might be a bat lung

 12   cell or it might be, you know, a clawed toe cell.  It 
 
 13   doesn't matter.  What you want is a cell line that is 
 
 14   permissive for the range of viruses that we have been 
 
 15   talking about today.  
 
 16             DR. SIEGEL:  Actually, I am confused by your

 17   comment which seems inconsistent with others we have heard.  
 
 18   You said if we use a solid organ, since we can't test that 
 
 19   we would have to test the plasma or serum.  But several 
 
 20   other speakers said that for a strain of pigs, since it is 
 
 21   going to be the same why not just use the same solid organ ‑

 22   ‑ 
 
 23             DR. ONIONS:  No, that is a different argument 
 
 24   because, as I think we have discussed in previous meetings, 
 
 25   they will be sentinel animals and it is possible to test 

 
        sgg                                                                204 
 
  1   sentinel animals that you would take out.  

  2             Again, I would slightly caution that there is a 
 
  3   quick assumption that all these animals are the same.  
 
  4   Actually, I think there are some interesting data from 
 
  5   Gibian Langford where she has looked at expression of these 
 
  6   viruses in different tissues and, in fact, even within a

  7   closely defined line of pigs there is actually a very 
 
  8   different pattern of expression in different animals.  Those 
 
  9   have not yet been mapped to different loci but these pigs 
 
 10   are not inbred mice; these are still outbred.  Indeed, it 
 
 11   will be very necessary for the health of these pigs to keep

 12   them to a degree outbred.  We do not want completely inbred 
 
 13   animals for all sorts of other reasons.  So, I don't think 
 
 14   it is going to be feasible to say that because a few animals 
 
 15   in a line behave this way that every individual animal will 
 
 16   behave that way.  I don't think that is the case at all. 

 17             DR. COFFIN:  Throughout our report it was very 
 
 18   clear that we need to get the genetics of this under some 
 
 19   control so that one knows.  It is possible to identify the 
 
 20   loci now that give rise to the viruses ‑‑  
 
 21             DR. ONIONS:  I think that is the key end result ‑‑

 22             DR. COFFIN:  Another thing that is important to 
 
 23   keep in mind in these discussions is that there are not ‑‑ 
 
 24   this discussion is being approached a little bit as, well, 
 
 25   there is a chance that every time you turn around you will 

 
        sgg                                                                205 
 
  1   see a new virus that you haven't seen before.  That is not

  2   going to happen.  We may well have already seen all the 
 
  3   infectious proviruses that there are.  It is unlikely that 
 
  4   there is a great deal more.  So, the workload here of 
 
  5   working these up is not infinite.  This is not completely 
 
  6   open‑ended.  It is merely a matter of getting hold of as

  7   many as possible in a research setting, figuring out what 
 
  8   they are, what their properties are and then, as new ones 
 
  9   come along, simply having probes and tools for telling which 
 
 10   ones you are looking at.  
 
 11             DR. AUCHINCLOSS:  I just want to translate for a

 12   brief second some nodding heads and shaking heads.  What I 
 
 13   thought I saw there was that it is unlikely we have yet 
 
 14   identified all PoEV's; it is quite possible we have 
 
 15   identified all infectious PoEV's. 
 
 16             DR. COFFIN:  I didn't say that.  I just said it is

 17   possible.   
 
 18             DR. WEISS:  We have already identified extra sets 
 
 19   of PoEV's that are less related to C type, more related to D 
 
 20   type but none of the sequences so far have open reading 
 
 21   frames.  There are certainly other endogenous retroviruses,

 22   and we have lots of them in pigs, that haven't yet been 
 
 23   described.  So, the key thing that John Coffin said is are 
 
 24   any of them going to be infectious and, yes, it is likely 
 
 25   that there is a small  set there and we are most of the way 

 
        sgg                                                                206 
 
  1   there.  I think there will always be surprises.  

  2             DR. COFFIN:  But even in outbreeding, once you 
 
  3   have identified the loci that are involved, that are 
 
  4   segregating, you can still use the presence or absence of 
 
  5   loci to inform your breeding program that you have been 
 
  6   maintaining outbred animals.  

  7             DR. ONIONS:  It might be that the long‑term goal 
 
  8   is loci identification.  So you fingerprint a pig and you 
 
  9   will know what those loci relate to in terms of your 
 
 10   patients.  So, it might be that that is definitely the way 
 
 11   to go for an intermediate, long‑term goal.  

 12             DR. AUCHINCLOSS:  But now let me go back to the 
 
 13   sponsor who says he has his tissue here and, yes, I admit I 
 
 14   have infectious endogenous retrovirus here.  What assay 
 
 15   should we require the sponsor to perform? 
 
 16             DR. ONIONS:  I think, again, it depends on the

 17   type of tissue that we use.  I think at the moment, if it 
 
 18   would b feasible ‑‑ correct me if I am wrong ‑‑ the people 
 
 19   who are doing work with isolated hepatocytes, and I think 
 
 20   that is a very interesting system, and also the people who 
 
 21   are doing islet cell transplants ‑‑ it would seem to me

 22   feasible that they could actually look at the particular 
 
 23   lots where you could look for expression of viruses in those 
 
 24   systems.  But I think the problem comes when we get to solid 
 
 25   organ transplants where you can't do that, or you can rely 

 
        sgg                                                                207 
 
  1   perhaps on at least demonstrating what is expressed in some

  2   other tissue in that pig before you get to slaughter.  
 
  3             DR. NOGUCHI:  One of the things I think Dr. 
 
  4   Solomon was trying to get at is as we are moving forward in 
 
  5   this particular area, above and beyond being able to detect 
 
  6   a virus, are there specific assays, such as using primary

  7   hepatocytes, that might allow us some comfort in saying this 
 
  8   may be infectious, a virus that can be activated under 
 
  9   certain conditions but when put into primary cells of a 
 
 10   particular target organ or target embedded tissue, will that 
 
 11   help us judge whether or not to go forward, or if you

 12   develop a sensitive assay at some point it will be positive.  
 
 13   We all realize that that is necessary for research 
 
 14   development to get the best possible assays, but in a 
 
 15   practical sense how will we judge what to do with this 
 
 16   infectious virus? 

 17             DR. AUCHINCLOSS:  I think this is the point, and 
 
 18   you have heard my point of view over and over again.  There 
 
 19   is no comfort you can take from any negative, at least in my 
 
 20   view on this because I start with the assumption that the 
 
 21   infectious virus is going to be there and it is going to get

 22   into human cells one day when you have done 
 
 23   xenotransplantation, ten years or twenty years from now.  I 
 
 24   don't know when it is going to be.  It is going to happen.  
 
 25   And just because you have done a hundred assay that say that 

 
        sgg                                                                208 
 
  1   tissue didn't have it happen in vitro, it is still going to

  2   happen.  So, all of these tests that you are requiring of a 
 
  3   sponsor, are not in fact worthwhile, except to the extent 
 
  4   that they demonstrate their capacity to mono patients later 
 
  5   on.  There are lots of things we will learn but for 
 
  6   validating tissue for transplantation they are

  7   inappropriate.   
 
  8             DR. SIEGEL:  We are asking whether we should 
 
  9   characterize what sorts of tissues they might infect and 
 
 10   what sorts of viruses they are, and we are saying they are 
 
 11   irrelevant except ‑‑

 12             DR. AUCHINCLOSS:  No, I am saying you should go 
 
 13   ahead and characterize and our experts should characterize 
 
 14   but the sponsor, for use of that tissue, does not have to 
 
 15   characterize.  
 
 16             DR. SOLOMON:  I think  part of the problem is the

 17   way you are putting it.  You are making it that if you 
 
 18   demonstrate a positive transmission of an endogenous PoEV, 
 
 19   that means that the sponsor can't go on and do a clinical 
 
 20   trial.  I certainly never meant to imply that by any of my 
 
 21   comments and didn't say anything like that.  So, that I

 22   think  is creating a problem here.  I mean, nobody wants to 
 
 23   see any regulations or guidelines that are so rigid come out 
 
 24   of these discussions, at least I certainly don't want to be 
 
 25   associated with them, that kill xenotransplantation.  I 

 
        sgg                                                                209 
 
  1   don't think the FDA wants that.  That is what we are doing

  2   here.  So I think you are putting it into wrong terms. 
 
  3             What we are saying is are there certain tests 
 
  4   that, by their biological relevance, appropriately blur the 
 
  5   line between basic research and clinical practice that are 
 
  6   relevant?  I would say, as a transplant clinician, that

  7   after I have to take care of my patient who just had islet 
 
  8   cells put in the liver I would like to know ahead of time if 
 
  9   there is a high likelihood that PoEV is going to be 
 
 10   transmitted to primary hepatic cells in culture.  Now, that 
 
 11   doesn't mean that I won't go ahead with the transplant. 

 12             DR. AUCHINCLOSS:  Do you want to try (iii) under 
 
 13   (c)?  It is important that (ii) came before (iii) because it 
 
 14   reflects what Dan was just saying, the fact that we might 
 
 15   ask a sponsor, and consider it part of their responsibility 
 
 16   in terms of safety assessments to do studies of infectivity

 17   of different tissues does not mean to imply that a specific 
 
 18   result would influence whether they could go or not, which 
 
 19   is what we are now asking. 
 
 20             DR. PATTERSON:  Right.  This is the major 
 
 21   question, the heart of question 1, in the event that

 22   infectious PoEV is identified during the screening 
 
 23   evaluation of porcine xenografts, please discuss whether 
 
 24   these xenografts would then be suitable for use in human 
 
 25   clinical trials.   

 
        sgg                                                                210 
 
  1             DR. COFFIN:  Let me just say something first.  Let

  2   me state that it was the consensus of the committee that 
 
  3   such materials should not be used.   
 
  4             DR. AUCHINCLOSS:  The focus group.  
 
  5             DR. COFFIN:  The focus group, right.  That doesn't 
 
  6   necessarily mean it was the opinion of everybody in the

  7   group but that was the consensus in the discussion that we 
 
  8   had ten days ago.  
 
  9             DR. DESROSIER:  I would say in the absence of 
 
 10   additional information on the potential infectivity of that 
 
 11   virus for humans or experimental animals, the committee was

 12   not comfortable, in the absence of any other information, 
 
 13   with putting in live replication competent virus into 
 
 14   people, not knowing whether it would take or not.  I think 
 
 15   we heard this morning that it has been put in ‑‑ there have 
 
 16   been multiple exposures of people and of monkeys in a

 17   variety of settings, and I think the answer to the question 
 
 18   is dependent upon further analysis of those individuals.   
 
 19             As I stated before and I will say it again, I 
 
 20   think it is possible that all 24 of those people who got the 
 
 21   brain implants could be infected with pig endogenous

 22   retrovirus but we don't know because we haven't done the 
 
 23   right tests yet, and we need to know those results.  If they 
 
 24   are antibody positive, persistently antibody positive to the 
 
 25   pig endogenous retrovirus and the population at large is 

 
        sgg                                                                211 
 
  1   negative, that is going to tremendously influence our views. 

  2   I think even Robin then might take a step back ‑‑ 
 
  3             [Laughter] 
 
  4             ‑‑ and reevaluate these comments about slowing 
 
  5   down a little bit.  If they are negative even by that 
 
  6   sensitive assay, then that changes the picture too.  So, I

  7   think we need to know.  If the information is there, and it 
 
  8   is gatherable, but in the absence of any further information 
 
  9   I, personally, would not be comfortable with the notion of 
 
 10   putting live replication competent pig retrovirus into 
 
 11   people, not knowing what was going to happen. 

 12             DR. AUCHINCLOSS:  Let me be clear.  Would you be 
 
 13   comfortable putting apparently replication not competent pig 
 
 14   tissue in based on our current knowledge?  I have heard you.  
 
 15   It was no. 
 
 16             DR. DESROSIER:  I mean, if there is no replication

 17   competent virus, I mean it is being done in gene therapy all 
 
 18   the time now with replication defective murine leukemia and 
 
 19   I can live with that. 
 
 20             DR. AUCHINCLOSS:  But would you believe any assay 
 
 21   that said that this was pig tissue that doesn't have

 22   replication competent virus? 
 
 23             DR. DESROSIER:  Well, if I were to assume that it 
 
 24   is  highly unlikely that all those tissues will, so yes.  
 
 25             DR. KASLOW:  One of the problems, it seems to me, 

 
        sgg                                                                212 
 
  1   with the way it is phrased is that it is almost as if on one

  2   day you have no information and the next day you have all 
 
  3   the information you need to decide when, in fact, it is 
 
  4   going to be weeks, months before you get it.  So, it really 
 
  5   perhaps ought to be rephrased at what point you have enough 
 
  6   information to make this decision rather than when it is

  7   identified as such.  What will tip you over?  At what point 
 
  8   will you have enough information after all the studies are 
 
  9   done to say one way or the other? 
 
 10             DR. DESROSIER:  I am not saying that that 
 
 11   information is.  I can't answer the question.  I know that

 12   based on what I have seen today on PCR analysis of blood 
 
 13   cells and all results being negative, it is not overly 
 
 14   convincing to me that those individuals are not infected.   
 
 15             DR. WEISS:  PCR data of blood is of limited value 
 
 16   really.  You know, most of us in this room are infected with

 17   EVV, but if each of us today donates 10 ml of blood and we 
 
 18   turn the buffy coat into DNA, only about 15% of those 
 
 19   samples will show up positive TPV.  You know, we have lots 
 
 20   of viruses on board at levels of detection in the blood that 
 
 21   are just below that, and even have tropism for white blood

 22   cells.  I think the antibody tests are going to be very 
 
 23   important.  They have lagged behind the PCR test so far 
 
 24   because, as we have heard this morning, you know, there are 
 
 25   tests coming along.  So, one thing we can take away is to 

 
        sgg                                                                213 
 
  1   perhaps put more effort into developing them.  I know the

  2   CDC is.   
 
  3             DR. ONIONS:  We are having a go in a rather more 
 
  4   desultory way.  We do need those tests.  We can study the 
 
  5   proteins; we can make some study peptides.  We can progress, 
 
  6   make positive reference and test those against human sera,

  7   against pig sera and do it.  It will take time but we are 
 
  8   probably talking about months, not years, in that regard.  
 
  9   So, I think there is something positive to do there that 
 
 10   will be useful.  
 
 11             DR. ALLAN:  I would just comment on the fact that

 12   you could do the animal models, as we discussed before.  One 
 
 13   of the things that strikes me about this type of a setup is 
 
 14   that you may not be looking for this but one of the things 
 
 15   that came to mind to me was the fact that it may be that you 
 
 16   design a standard assay to detect infectious pig endogenous

 17   retrovirus.  You may be able to design your assay system 
 
 18   such that you can detect low levels and high levels so that 
 
 19   you may find certainly in animals that when you isolate 
 
 20   viruses you get a notion that that particular animal may be 
 
 21   more likely to transmit to a human than, say, another pig or

 22   another virus.  So, all viruses are not equal.  Some may be 
 
 23   more likely to induce an infection or produce a lot of virus 
 
 24   that could be infectious.   
 
 25             These are questions that we can't answer yet, but 

 
        sgg                                                                214 
 
  1   you could design assay systems to be able to begin to

  2   determine those kinds of things, depending on what your 
 
  3   indicator cell line is.  You can do that in conjunction with 
 
  4   the animal model system so that you can take the virus that 
 
  5   comes out that is a high level, inject that into a primate 
 
  6   and see if that is more likely to be productive in a primate

  7   versus a virus that you inject that is at a low level in 
 
  8   human cells or monkey cells.  So, I think we can do those 
 
  9   things. 
 
 10             DR. AUCHINCLOSS:  I don't have a sense from this 
 
 11   committee as to whether or not when they hit (iii)(c) here

 12   they have said is not tissue you can transplant or this is 
 
 13   tissue that can be transplanted.  Any answer to that? 
 
 14             DR. COFFIN:  Or the middle ground where there are 
 
 15   levels of risky procedures in tissues, which actually comes 
 
 16   up in the next question. 

 17             MS. MEYERS:  Can I address that?  As a layman, it 
 
 18   just seems this is a science fiction and how it is going to 
 
 19   hit the home newspaper when we are talking about this should 
 
 20   be of concern to scientists and everybody else.  So, 
 
 21   transplanting tissue from any animal that contains a virus

 22   that is live and can replicate into a living human being 
 
 23   just seems unethical.  I can't see how anybody would do 
 
 24   that, purposely expose them to a known disease.   
 
 25             DR. AUCHINCLOSS:  But, Abbie, we do that all the 

 
        sgg                                                                215 
 
  1   time in humans, with CMV for example, ones that we actually

  2   know are pathogenic as opposed to this one.  
 
  3             MS. MEYERS:  But you know what those diseases will 
 
  4   do.  
 
  5             DR. AUCHINCLOSS:  No question of that.   
 
  6             DR. COFFIN:  But would you feel better if we knew

  7   that these were dangerous viruses? 
 
  8             [Laughter] 
 
  9             It is actually a serious issue that comes up over 
 
 10   and over again in other kind of FDA things as well.  It is 
 
 11   much easier to deal with the dangerous agents than the ones

 12   we don't know what they do. 
 
 13             MS. MEYERS:  But shouldn't somebody be working 
 
 14   with those viruses to see how dangerous they are before you 
 
 15   expose the patient plus their family and maybe the people 
 
 16   they get on the bus with, the people on the airplane with? 

 17   I mean, we have no idea what these viruses can do and we 
 
 18   shouldn't be doing it knowingly.  
 
 19             DR. SOLOMON:  The issue that you raise is very 
 
 20   important.  What the transplant community has come together 
 
 21   and said is that the organ donor shortage is such that we

 22   are losing literally thousands of patients every year, 
 
 23   thousands in the United States alone because we are not able 
 
 24   to transplant.   
 
 25             So we are looking at individual risks and public 

 
        sgg                                                                216 
 
  1   health risks, and I think that was well articulated earlier. 

  2   We know that regular human‑to‑human organ transplantation 
 
  3   regularly transmits viruses that create serious illnesses in 
 
  4   our patients and sometimes illnesses that kill them, but we 
 
  5   accept that as not a zero risk procedure because it is life‑ 
 
  6   saving in the great majority.  

  7             What we are trying very hard to do is to 
 
  8   responsibly not raise the bar too much higher for xeno.  It 
 
  9   is never going to be a zero risk procedure.  I think the 
 
 10   challenge for this group of experts and for the FDA and the 
 
 11   public health service in general is to come to grips with

 12   these obvious issues and positively allow 
 
 13   xenotransplantation to go forward and save thousands of 
 
 14   lives in the future, realizing as best we can what the risks 
 
 15   are so appropriate informed consent can go forward to my 
 
 16   patients and to the public, and that I think is the only

 17   thing that we have to do, to be responsible.  
 
 18             DR. AUCHINCLOSS:  Yes? 
 
 19             MR. BENEDI:  Miss Meyers alluded to it, I am a 
 
 20   transplant recipient; I am a liver recipient, and the risk 
 
 21   to the recipient from human‑to‑human transplant is one that

 22   is known and the recipient takes that risk.  What I think is 
 
 23   being expressed is the risk not just to that recipient of 
 
 24   death or infection or whatever, but a risk to the general 
 
 25   population.  That is what one recipient can't be held 

 
        sgg                                                                217 
 
  1   accountable for when they sign that consent form when they

  2   may infect a number of people in their families and outside.  
 
  3   So, that is a whole different issue. 
 
  4             MS. MEYERS:  That is right.  
 
  5             DR. AUCHINCLOSS:  I completely agree that that is 
 
  6   what distinguishes this from all other forms ‑‑ not all

  7   other forms but the standard risk‑benefit calculation that 
 
  8   an IRB evaluates.  We agree with that, we really do.   
 
  9             Now, people in the FDA, help me out.  We have not 
 
 10   answered what you consider to be, and what I consider to be 
 
 11   the central question in group 1.  Do you want me to kind of

 12   have a show of hands here?  Do you want to get some more 
 
 13   information?  I don't feel like I have satisfied you with 
 
 14   advice from your experts, recognizing that we have very 
 
 15   different levels of expertise here and lots of opinions.  
 
 16             DR. SIEGEL:  Right.  Let me state where I think we

 17   are and what I think I have heard relative to this question 
 
 18   just for feedback.  Obviously there is not full agreement 
 
 19   but I think most people say that if next week somebody 
 
 20   reports to us that they have grown this virus out of their 
 
 21   product and, indeed, most of the committee thinks that that

 22   is likely to happen, if not next week, sooner or later 
 
 23   because it is going to be there, I have yet to hear that 
 
 24   they should go ahead because I think I heard some 
 
 25   reservations from Dr. Desrosier and from others that, in 

 
        sgg                                                                218 
 
  1   fact, perhaps there is more information that we need on the

  2   patients who have already been treated, and we will get to 
 
  3   that in question 2.  But, as a point of information itself, 
 
  4   what I think I am hearing is that that really makes 
 
  5   relatively little difference; that we already knew that that 
 
  6   is going to happen.  We know that these tissues have virus. 

  7   The fact that we are screening and we are going to continue 
 
  8   to screen for virus is not based upon the fact that we think 
 
  9   it is going to make a difference in how we act whether they 
 
 10   recover or not because we think it is going to be there 
 
 11   anyhow, and if they don't recover we think it is probably

 12   simply that they haven't been tweaked hard enough and, yet, 
 
 13   we are not going to ask them to try to induce it in any 
 
 14   case.  So, we are just going to get that information.  We 
 
 15   are not going to ask them to take that virus and see what 
 
 16   sorts of tissues it infects, but we would like maybe to find

 17   somebody to do that or sequence it ‑‑ 
 
 18             DR. AUCHINCLOSS:  If you heard all of this, you 
 
 19   heard me, but I would be very surprised if this committee 
 
 20   agreed with all of that.  
 
 21             DR. SIEGEL:  When this happens, when this culture

 22   is positive, you know, Dr. Zoon and the Commissioner and 
 
 23   other people are going to ask me, well, you know, what are 
 
 24   you doing about that?  And I am going to say, well, we 
 
 25   consulted with the experts and they didn't seem to think 

 
        sgg                                                                219 
 
  1   that this made much of a difference ‑‑

  2             DR. AUCHINCLOSS:  All right, you have heard my 
 
  3   point of view now let's hear from the rest of them and see 
 
  4   what they really think.  
 
  5             DR. VANDERPOOL:  Do you want me to answer number 
 
  6   (iii)(c)?  It totally depends on the nature of "infectious"

  7   PoEV.  I might be able to assume that that is not serious.  
 
  8   So, proceed, by all means.  But if I think, hey, this is 
 
  9   going to form a mutant virus; it is going to be equivalent 
 
 10   to AIDS I would say no way.  So, I think it is going to be a 
 
 11   judgment all the way.  Just the fact that infectious PoEV is

 12   present doesn't mean to me that one can't proceed with human 
 
 13   trials.  It totally depends on the extent of harm and the 
 
 14   degree to which it might be spread.  And that is a judgment 
 
 15   call and we can't guess the call.   
 
 16             DR. SIEGEL:  I don't understand why you can't

 17   guess the call.  Largely, some of  you have told us not to 
 
 18   require the companies to generate information to address 
 
 19   that question so we are going to have to make that call on 
 
 20   the basis of current information ‑‑ 
 
 21             DR. COFFIN:  No, no, no.  

 22             DR. SIEGEL:  Well, you said it would be an 
 
 23   interesting research question ‑‑ 
 
 24             DR. COFFIN:  No.  I think everybody has said that 
 
 25   you absolutely have to have follow‑up on animals that have 

 
        sgg                                                                220 
 
  1   received these and on humans that have received these.  That

  2   I think is a complete given.   
 
  3             DR. SIEGEL:  Right.  
 
  4             DR. COFFIN:  That is the information that we 
 
  5   really need.  
 
  6             DR. MICKELSON:  I think we also mentioned that

  7   there should be a drive for eliminating these from the donor 
 
  8   sources as well.  I think one of the issues here is, again, 
 
  9   the risk‑benefit ratio.  I think it is really hard.  There 
 
 10   are no data.  Even though there may be a hundred or so 
 
 11   people who have these transplants, that is not really a big

 12   group.  The infectivity and whatever else might happen, 
 
 13   those should be relatively rare events and a hundred people 
 
 14   is not statistically robust by anyone's consideration.  But 
 
 15   to assume that the companies can test and say it is 
 
 16   positive, no.  It should be a burden placed upon sponsors,

 17   whether they are universities or companies or whatever, to 
 
 18   the whole group working together to remove and reduce 
 
 19   whatever they can detect that is potentially a danger.  But 
 
 20   the word is "potential."  I don't understand how we can 
 
 21   honestly deal with a lot of this because we are not data

 22   driven here.  We have in vitro tests.  I don't know what it 
 
 23   means to have these viruses, after being induced with PHA or 
 
 24   something, being able to infect primary human cells, blood 
 
 25   cells, in culture.  I mean, there is no immune system; I 

 
        sgg                                                                221 
 
  1   mean, even if the recipients are immunosuppressed ‑‑ I don't

  2   know what to do with this data.  It is fearful but it is not 
 
  3   a reflection, and I do not want us to deny something that is 
 
  4   potentially helpful to people who are in dire need as well 
 
  5   until we would have better data that this is a real issue.  
 
  6             DR. AUCHINCLOSS:  At the beginning of the day Jay

  7   said to me we know we have asked you impossible questions 
 
  8   and we feel bad about that, but they are, in fact, the 
 
  9   questions that we are being asked to answer every day.  
 
 10             DR. MICKELSON:  But there are answers here and 
 
 11   they are all personal as to what you consider risk or

 12   benefit, but I think one of the things that we must always 
 
 13   fall back on is exactly what is your data telling you is 
 
 14   going on, and if there isn't any data then we have moved 
 
 15   into the realm of value judgments as to what these 
 
 16   experiments mean.  

 17             DR. ZOON:  I think it is a little bit of the 
 
 18   chicken and the egg syndrome because, you know, we would 
 
 19   like data as well but the reality is the science is evolving 
 
 20   and the question is at what stage do you let things go 
 
 21   forward, knowing that you have to scientifically monitor and

 22   study as you proceed, deal with informed consent with 
 
 23   individuals that potentially may receive these life‑saving 
 
 24   therapies.  I think, again, the public health balance if it 
 
 25   were only affecting the individual would be fairly 

 
        sgg                                                                222 
 
  1   straightforward to assess, and part of the complexity in

  2   this, and I think the committee rightly so is saying that 
 
  3   any data that you do have or can explore with respect to 
 
  4   those individuals already treated, their family members or 
 
  5   whatever, to raise issues that are, are there additional 
 
  6   data that may raise concern or not.  But part of this is do

  7   you stop things while you are waiting to get that data or do 
 
  8   you allow it to proceed?  I think part of what we are trying 
 
  9   to ask you is to help us balance that so we can move 
 
 10   forward.  
 
 11             DR. AUCHINCLOSS:  Here is what I would like to do. 

 12   I would like to basically just run around the table and let 
 
 13   any person who is in the mood to make a comment to this 
 
 14   question do so, and then I think we can actually relatively 
 
 15   quickly address (d), and then we should have a coffee break. 
 
 16

 17             [Laughter] 
 
 18             So, I am just going to go around the table and 
 
 19   anybody who wants to make sort of their opinion known on 
 
 20   this central issue of question 1.   
 
 21             DR. PAUL:  Having worked with a number of

 22   different infectious agents in swine, and also I have worked 
 
 23   with bovine leukemia virus, if with this retrovirus there 
 
 24   was any experimental evidence that it caused leukemia in 
 
 25   swine I would be very concerned.  I am personally more 

 
        sgg                                                                223 
 
  1   concerned about some of the agents in swine more than the

  2   endogenous retrovirus.   
 
  3             DR. DESROSIER:  I would just restate what I have 
 
  4   already said ‑‑ 
 
  5             DR. AUCHINCLOSS:  Your feeling is get the antibody 
 
  6   test up and running before you go forward.  Is that fair?

  7             DR. DESROSIER:  My feeling is that I am not in 
 
  8   favor of putting replication competent endogenous virus into 
 
  9   people unless we have some sort of evidence of whether it is 
 
 10   infectious for people and might potentially cause disease.  
 
 11   I think in the absence of that information, we should seek

 12   information from animal models and the people who have 
 
 13   already been exposed to rigorously as possible examine 
 
 14   whether there have been infectious events. 
 
 15             DR. KASLOW:  I think I would proceed on the 
 
 16   current course.  We do not have any evidence of human

 17   transmission.  We don't have any evidence of disease as a 
 
 18   result of this agent in humans, and it seems to me that it 
 
 19   is appropriate to collect the information that would change 
 
 20   that view as expeditiously as we can, and then proceed to 
 
 21   meet again and to think again about it, and this is going to

 22   be a continuous process.   
 
 23             DR. LERCHE:  I agree with that last statement.  It 
 
 24   is a constant feedback process that is going to be going on.  
 
 25   I think in the framework that we, as the focus group, 

 
        sgg                                                                224 
 
  1   discussed this, essentially the question was posed to us

  2   would we be comfortable giving replication competent virus 
 
  3   to human patients, and that is sort of the basis for a 
 
  4   consensus or, I guess, a non‑consensus.   
 
  5             I think that my own opinion is that I think that 
 
  6   there needs to be a little stepping back and looking at

  7   where we actually are.  I think we have two sources of this 
 
  8   information.  One is on animal studies, and I think the 
 
  9   importance of this has been made very clear.  The other is 
 
 10   to fully evaluate the patients who have already received 
 
 11   that.  This morning we have seen a very good start on that. 

 12   I do agree that some of the assays that need to be done have 
 
 13   not yet been done but could be done.   
 
 14             So, I think my own opinion is that if this is 
 
 15   going to proceed it should be done very cautiously and the 
 
 16   protocols and the tissues that are being used have to be

 17   evaluated independently.  All these procedures are not 
 
 18   really the same in terms of risk and should be evaluated 
 
 19   that way.  
 
 20             MR. LAWRENCE:  With Deputy Commissioner Pendergast 
 
 21   gone, I am the only lawyer left at this table.  We will talk

 22   about some of the legal stuff a little bit later.  I am also 
 
 23   a liver recipient, along with Tony.  I would like for a 
 
 24   moment to put a human face on some of this because I hear 
 
 25   these outstanding scientists talk about accepting risk, and 

 
        sgg                                                                225 
 
  1   I would like to suggest that perhaps the people who are

  2   accepting the risk are people like myself.  So, we have a 
 
  3   voice here, and I would like to thank the FDA for inviting 
 
  4   us and asking us to participate in this.  I resisted when 
 
  5   Dr. Patterson called me.  She called me several times to get 
 
  6   me to agree to come here but I think this is good. 

  7             I would like to say that we have been accepting 
 
  8   risk for a long time.  In the '70s we accepted the risk that 
 
  9   we were probably going to die because the problem then was 
 
 10   immune response.  I mean, we are talking about a porcine 
 
 11   virus now and that is threatening but it was more

 12   threatening when the probability was that your immune system 
 
 13   was going to kill you if we did this procedure now, and 
 
 14   would you like to proceed?  And some of us ‑‑ I say us, I 
 
 15   mean recipients ‑‑ proceeded anyway, and science progressed 
 
 16   and now we are in much better shape probably for that.  

 17             It seems to me we are on the cusp again of a major 
 
 18   stride.  When I was transplanted ten years ago there were 
 
 19   15,000 people on the waiting list waiting for organs, and 
 
 20   today, as we sit there, there are almost 60,000.  So, the 
 
 21   problem is getting worse.  As we deliberate today 10 people

 22   will die in this country.  Every 24 hours 10 people die.  
 
 23   So, to make this short, we need to proceed.  Recipients 
 
 24   would say to you we will accept the risk.  We want to be 
 
 25   informed; we want information.  We want to participate with 

 
        sgg                                                                226 
 
  1   you here but let's go.  Caution is fine but let's not stop. 

  2 
 
  3             DR. MICHAELS:  I think a lot of the comments 
 
  4   actually that I would make have already been voiced and I 
 
  5   should probably just try to say that I think different 
 
  6   procedures do have somewhat different risks and I think that

  7   we can proceed cautiously with small numbers as we go 
 
  8   forward.  I actually didn't realize how many numbers of 
 
  9   people had already received tissue, and I believe it was 
 
 10   Immunotron that have over 100 patients that have had some of 
 
 11   the human tissues, and it would be nice to hear at some

 12   point how many numbers of those patients they actually have 
 
 13   already been able to contact and get samples from to at 
 
 14   least by the best ways possible that we have right now, 
 
 15   while the assays are being developed, and I certainly know 
 
 16   that a lot of effort is going into developing the other

 17   assays.  
 
 18             DR. HIRSCH:  We have no knowledge about 
 
 19   pathogenicity and we have no knowledge about risk, but 
 
 20   extrapolating from other viruses and trying to get back to 
 
 21   Jay's question, I certainly would be more worried about a

 22   culture that was producing five logs of infectious virus 
 
 23   than I would be about one that you had to induce with IUDR 
 
 24   to get anything out of, but where you draw the line I 
 
 25   haven't the vaguest idea.   

 
        sgg                                                                227 
 
  1             DR. ALLAN:  I think that in a vacuum you would

  2   like to see the animal studies done before you go forward.  
 
  3   That may not be reasonable.  Each of these procedures has 
 
  4   different types of risks and you are looking at fetal 
 
  5   neuronal cells; you are looking at certain types of 
 
  6   procedures that probably have less of a risk than if you are

  7   transplanting an organ with a whole ecosystem of cell types 
 
  8   and microbes that are associated with it.  So there are 
 
  9   differences in risk.   
 
 10             But I think you need to do animal model systems, 
 
 11   and I think just looking at the animal models

 12   retrospectively is not enough.  I think you have to do the 
 
 13   hard core science.  You have to blast the monkeys or 
 
 14   whatever animal species it is with boluses of viruses, 
 
 15   immunosuppress them and you can do that within six months.  
 
 16   I think it needs to be done.  Who is going to do it I don't

 17   know.   
 
 18             DR. ONIONS:  I think we have two sources of data 
 
 19   that are coming through now that are going to be very 
 
 20   important.  One is the study that is going on the patients 
 
 21   that are being exposed either through liver transplants,

 22   skin grafts or a variety of other procedures.  I think 
 
 23   evaluating those patients for the presence of virus, and I 
 
 24   think also crucially for the presence of antibody ‑‑ one of 
 
 25   the reasons we developed recombinant P30 is because I think 

 
        sgg                                                                228 
 
  1   that is a critical part of the evaluation of these patients. 

  2   I think those data are going to be very important, and I 
 
  3   think some of the primate data is going to be useful.  
 
  4   Unfortunately, I think not all of it is.  I think we need to 
 
  5   know more about the infectivity of these viruses for 
 
  6   different environments and some of that data is beginning to

  7   come through.  
 
  8             I would like to see some of those data before I 
 
  9   would be happy about putting in any tissue that I knew was 
 
 10   expressing a virus that could infect human cells, whether 
 
 11   that be pancreatic islet cells or a whole organ transplant. 

 12   Having said that, once those data are on the table, I think 
 
 13   you can begin to evaluate a little bit more sensibly what 
 
 14   the risk factors are.  I said I would like to pause at that 
 
 15   state because I would like to reevaluate the data because, 
 
 16   in my own view, I think it is unlikely from what we know of

 17   the virus already ‑‑ and I can only use the word unlikely ‑‑ 
 
 18   unlikely that the virus would establish a persistent 
 
 19   productive viremic infection in a human patient, unless 
 
 20   immunosuppression modifies that significantly.  It seems to 
 
 21   me that that is the danger because that is the public health

 22   danger when you get high titer infection and you probably 
 
 23   get onward transmission.  
 
 24             So, I think we need to do it step by step, and to 
 
 25   come out with the question straightforwardly, I think it 

 
        sgg                                                                229 
 
  1   depends on the data coming through at the moment and then I

  2   think we can make a sensible decision about going on with 
 
  3   the data that is going to be available in the next few 
 
  4   months.   
 
  5             DR. VANDERPOOL:  To give a bit of very recent 
 
  6   history, the full reports released on xenotransplants

  7   recently, two of them were the Nufield Council of the U.K. 
 
  8   and the Institute of Medicine Committee in the U.S.  One of 
 
  9   the significant differences between the Nufield Council in 
 
 10   Britain and the U.S. was on this very issue.  The Nufield 
 
 11   Council appealed to the "principle of precaution," saying we

 12   should not proceed until we are assured that the procedures 
 
 13   are safe.  That is one way to go.  The Ireland Committee is 
 
 14   different.  Our stand in the Ireland Committee is proceed 
 
 15   with caution; proceed as long as you think the risks are 
 
 16   controllable.  So, I said a moment ago that we can't make

 17   that decision for you.  What I meant is I can't make that 
 
 18   decision for you now.  I will be with you and helping you 
 
 19   sort through that decision in the future if I am so called 
 
 20   to help do that.  But I think we should proceed as long as 
 
 21   the risks are manageable and controllable, and as long as

 22   they are not serious.  It is interesting how it would be so 
 
 23   nice to appeal to mathematics for comfort, and we do that 
 
 24   all the time, but ultimately, in my judgment, we fall back 
 
 25   on just straight word adjectives ‑‑ serious, controllable.  

 
        sgg                                                                230 
 
  1   As long as the risks seem manageable and controllable I

  2   think we should proceed.  When the red flags come up you 
 
  3   will know it and we will know it, and that is when we take a 
 
  4   different course in my judgment.   
 
  5             DR. MICKELSON:  I must say I agree whole‑heartedly 
 
  6   with what you have just said.  I do believe that

  7   xenotransplantation trials should go ahead cautiously but in 
 
  8   parallel with the animal research.  I think the data that we 
 
  9   have seen and what is known now, and the types of viruses 
 
 10   that have been recovered ‑‑ the risks seem low and they seem 
 
 11   manageable, and placed in context with the need and the

 12   other adjectives that accompany descriptions of the diseases 
 
 13   that could be treated by this, I do think they should 
 
 14   proceed cautiously.  I think the FDA can encourage the types 
 
 15   of research to answer the questions that are needed so that 
 
 16   if and when things happen they can be detected.  I am not

 17   sure PoEVs are ever going to be the issue, but if that is 
 
 18   what we are dealing with today, you know ‑‑. 
 
 19             DR. SIEGEL:  We encourage a lot of things that are 
 
 20   happening ‑‑ 
 
 21             [Laughter]

 22             ‑‑ some of the same people who, in fact, said that 
 
 23   animal research was critical but it shouldn't be go/no go, 
 
 24   but then subsequently said until we got the human and animal 
 
 25   data we shouldn't be going forward. 

 
        sgg                                                                231 
 
  1             DR. MICKELSON:  Can I ask one question?  When you

  2   go back to the patients who have already received 
 
  3   transplants what do you tell them as to why you are asking 
 
  4   for new samples?   
 
  5             DR. AUCHINCLOSS:  Good question! 
 
  6             DR. SIEGEL:  Per the guidelines that we put out a

  7   while back, substantial samples have been banked and 
 
  8   archived from these patients and are being done on a regular 
 
  9   basis.  So, we are not specifically asking, in most cases ‑‑ 
 
 10   I am sure in some of the projects you have heard about there 
 
 11   will be a need for specifically asking for additional

 12   samples but by advice of many of the people on this panel a 
 
 13   lot of sample collection has been going on routinely.  
 
 14             DR. MICKELSON:  But you don't have to go back to 
 
 15   ask for permission to test.  That is what I am saying.  They 
 
 16   were collected with the understanding that they were

 17   blinded, or what? 
 
 18             DR. NOGUCHI:  In the larger issue those samples 
 
 19   for which specific authorization had not been, which is 
 
 20   really the case of a lot of patients who had received it 
 
 21   under IND, if you are asking what do you tell them, you tell

 22   them the truth as far as we know it.   
 
 23             DR. AUCHINCLOSS:  I am going to move on with our 
 
 24   answers pretty quickly as a matter of fact because I think 
 
 25   we are all going to drop before we get to number 3.   

 
        sgg                                                                232 
 
  1             DR. WALKER:  I just would like to ask a question

  2   of clarification of the people from the FDA.  Am I right in 
 
  3   thinking that the clinical hold applies to all 
 
  4   xenotransplantation studies at this point?  
 
  5             DR. SIEGEL:  For this specific porcine xeno‑ 
 
  6   transplantation.

  7             DR. AUCHINCLOSS:  The effect of the answer is yes. 
 
  8 
 
  9             DR. SIEGEL:  The hold that is pending having an 
 
 10   assay for porcine endogenous retrovirus is for all porcine.  
 
 11             DR. AUCHINCLOSS:  I think you have sort of stated

 12   my point of view so I am going to pass.   
 
 13             DR. WALKER:  So essentially we are being asked 
 
 14   whether we think the clinical hold should be taken off and t 
 
 15   he research proceed now while data are being gathered.  I 
 
 16   think that is one way to operationalize the question that is

 17   being asked here.   
 
 18             DR. SIEGEL:  Well, that is one of the ways.  The 
 
 19   question I thought was being asked now is when the data are 
 
 20   gathered and the virus culture comes back positive, should 
 
 21   that ‑‑ if otherwise you were going to life the hold, should

 22   we go ahead and lift the hold?  However, we are getting a 
 
 23   lot of advice on a lot of different questions and I don't 
 
 24   want to discourage you from giving it because it is needed. 
 
 25             DR. WALKER:  Well, it is a slightly broader 

 
        sgg                                                                233 
 
  1   question than this one that is on the screening, but I guess

  2   generally speaking I favor seeing research go forward and I 
 
  3   think moratoria are a bad idea.  In this case, I guess I 
 
  4   would argue that another two months should be taken with the 
 
  5   clinical hold and that during that time a risk assessment 
 
  6   and risk control program should be developed.  

  7             DR. COFFIN:  I am not of the opinion that it 
 
  8   matters very much whether a given tissue is producing virus 
 
  9   or not.  It only takes one infection event ‑‑ if an 
 
 10   infection is going to be initiated in a patient, it only 
 
 11   takes one event to do that.  The real issue is whether you

 12   get a spreading infection in the patient.  To my mind, that 
 
 13   is the key and that is what the research has to be directed 
 
 14   at.  And that is two things.  One is that newborn animal 
 
 15   studies should go forward as fast as possible, with negative 
 
 16   results being fairly useless but to try to find a positive,

 17   try to find an animal in which things do happen and then one 
 
 18   can ask focused questions.  That is one thing.  
 
 19             Secondly, I think it is not unreasonable to 
 
 20   consider that certain kinds of transplant studies could go 
 
 21   forward with the idea that they are being used to accumulate

 22   this data but that these studies should be assessed on the 
 
 23   basis of the risk that if there is a spreading infection 
 
 24   will transmission occur?  Because it is really transmission 
 
 25   that we are talking about.  We are not talking about 

 
        sgg                                                                234 
 
  1   individual risk to the patient despite what some of the

  2   people have said.  So, I think that should take into account 
 
  3   something we haven't talked about before, and that is what 
 
  4   the patient population is.  Clearly, some of the patient 
 
  5   populations are going to be much more at risk for 
 
  6   transmission, for example vertical transmission.  If you

  7   excluded women who were within childbearing age you would 
 
  8   essentially eliminate that risk from the patient population.  
 
  9   So, one could grade up the risk by also considering what the 
 
 10   patient population being treated is, as well as what the 
 
 11   various types of procedures are.  

 12             DR. SOLOMON:  I have already made the one comment 
 
 13   that I don't think any longer the issue here is should we go 
 
 14   forward in a safe way with xenotransplant trials.  I think 
 
 15   under the right circumstances we should go forward.  You 
 
 16   have to remember that there are a number of barriers after

 17   the infectious disease issue is put into context that are 
 
 18   standing in the way of us deciding whether there is any 
 
 19   benefit, and we can't have a risk‑benefit ratio when we know 
 
 20   nothing about benefit, and I think Dr. Hirsch made that 
 
 21   comment as well as others.  We need to get into clinical

 22   trials in order to define the benefit, and the sooner we do 
 
 23   that the sooner we benefit the patients and that is a major 
 
 24   responsibility in transplantation.  
 
 25             So, what is the responsibility of a group like 

 
        sgg                                                                235 
 
  1   this?  I would leave you with the idea that it is to provide

  2   leadership.  We need to provide leadership in where the 
 
  3   research should be going, how the research is moved to the 
 
  4   clinical side and, lastly, how we protect patients and 
 
  5   public safety.  With that in mind, I think it is very 
 
  6   critical to acknowledge the important role the FDA, the NIH

  7   and the CDC has had.  They have provided leadership that has 
 
  8   moved this field forward in a practical way in the last two 
 
  9   years in a way that I personally think is remarkable.  I 
 
 10   mean, there is data now when two years ago at this very same 
 
 11   sort of thing we were talking about "what if?"  Don't, in my

 12   opinion, give up the leadership role you have taken so far.  
 
 13   It is going to be a very profitable industry.  There are 
 
 14   very fine scientists out there in the biotechnology world.  
 
 15   They are doing very fine science.  Don't give up.  Don't 
 
 16   take the pressure off them.  Don't wave your hand and say

 17   oh, the NIH is going to support this because we all know 
 
 18   that is baloney.  They are going to try and do their part, 
 
 19   and they are doing their part, but a tremendous amount of 
 
 20   this work is being driven right now by the leadership you 
 
 21   guys have provided.  Don't let up on that in my opinion. 

 22             DR. WEISS:  I am also still in favor of proceeding 
 
 23   with caution.  Caution tells me that there is more 
 
 24   information to gain in the short term from people who have 
 
 25   already been exposed or transplanted with porcine cells.  I 

 
        sgg                                                                236 
 
  1   hear what our colleagues on the panel who are individual

  2   recipients of transplants, of allograft transplants, have 
 
  3   said, and my mind remains focused on what is a new issue in 
 
  4   this field of medicine, the possibility of transmission.  We 
 
  5   are going beyond the risk and benefit of the individual and 
 
  6   we have to address the rest of the population at large,

  7   which I think is a lower risk but a potentially more 
 
  8   devastating one, and one that we are not going to get a 
 
  9   sensible answer from today with the blind leading the blind 
 
 10   at the moment.   
 
 11             DR. AUCHINCLOSS:  Abbie, do you want to make a

 12   comment? 
 
 13             MS. MEYERS:  Well, of course I am going to get in 
 
 14   trouble with what I say, which I always do ‑‑ 
 
 15             [Laughter] 
 
 16             ‑‑ but that is probably because, you know, among

 17   the members of my organization is the Huntington's Disease 
 
 18   Society and two of the Parkinson's groups and a lot of the 
 
 19   diseases that are involved in this area.  Besides which, for 
 
 20   ten years my organization gave away millions of dollars 
 
 21   every year for cyclosporine to transplant patients.  We

 22   handle 8000 patients a year.  So, we certainly know the pain 
 
 23   and the suffering and anxiety of these people, and how they 
 
 24   are counting on this technology to move forward.  However, a 
 
 25   hemophilia patient is about 30 years old, and he was HIV 

 
        sgg                                                                237 
 
  1   positive, and that is not really what destroyed his life;

  2   the hemophilia didn't destroy his life and the HIV didn't 
 
  3   destroy his life.  What destroyed his life is that he had 
 
  4   given HIV to his wife before he knew it.  He found out he 
 
  5   had HIV when his wife died of HIV, and that was a public 
 
  6   health tragedy and that is what we must avoid at all costs. 

  7 
 
  8             So, we must have a complete understanding of what 
 
  9   this virus does; what its potential is.  Maybe it is benign.  
 
 10   Maybe it just gives you thick finger nails or toe nails for 
 
 11   one week of your life ‑‑ who cares?  But get the answer

 12   before you start using this technology in large numbers of 
 
 13   people because then you are transplanting patients who are 
 
 14   going to come to you and saying, "my wife died and I killed 
 
 15   her and I didn't know it. "  So, that is where I stand.  
 
 16             DR. AUCHINCLOSS:  Okay.  

 17             MR. BENEDI:  Being a recipient, it is a question 
 
 18   that is really about our lives and everyone in this room 
 
 19   obviously is here because they are concerned with saving 
 
 20   lives, and 60,000 people, as Bill has mentioned, are 
 
 21   waiting; ten will die every single day.  The science

 22   community needs to look at ways to save those lives.  
 
 23   Obviously, the donor pool is not meeting the demand.  So, to 
 
 24   proceed cautiously is something that is responsible to do, 
 
 25   but also, and this is a good start, to create a partnership 

 
        sgg                                                                238 
 
  1   because there needs to be a partnership between the

  2   scientific community and the general public out here about 
 
  3   this issue and about the understanding about the 
 
  4   ramifications of this issue.   
 
  5             If it were up to a recipient alone to decide 
 
  6   whether they were going to be infected or not, I think that

  7   that is an individual choice but to think that a recipient, 
 
  8   if they were posed with a question whether they could live 
 
  9   or die but if they lived there is a potential, just a 
 
 10   potential that they will infect their loved ones when they 
 
 11   are gone or in their lifetimes or others in the community, I

 12   think that is a bigger question, and I think posed to a 
 
 13   recipient like myself, and I can't speak for Bill, if that 
 
 14   question were posed to me before my transplant I would have 
 
 15   probably said no.  Actually, I know I would have said no.   
 
 16             So, I think we need to really look at it as a

 17   community to come together and to see what the risks are.  
 
 18   Obviously we need to proceed.  There are people who die but 
 
 19   we need to proceed with caution.  Thank you.  
 
 20             DR. AUCHINCLOSS:  Well, thank you all very much ‑‑ 
 
 21             MR. BENEDI:  And if I could say one more thing,

 22   with 60,000 people waiting for organ transplants and 10 
 
 23   dying every day, I would like to challenge everyone in this 
 
 24   room to sign a donor card before Christmas.  Thank you.  
 
 25             [Laughter] 

 
        sgg                                                                239 
 
  1             DR. AUCHINCLOSS:  Thank you.  It is time for a

  2   coffee break so let's just put this last question out, (d), 
 
  3   very briefly.   Does anyone wish to make a statement about 
 
  4   the issue of whether or not, if you were to put xenografts 
 
  5   so they are not in contact with patient cells, which I 
 
  6   assume basically means barrier devices and isolation

  7   devices, and/or do bridge transplants specifically, alter 
 
  8   the way you feel about anything you have heard? 
 
  9             DR. ONIONS:  Yes, it matters because bridge 
 
 10   transplants are so much safer.  I don't know that they are 
 
 11   but I would just guess that they are because the period of

 12   exposure is shorter.  You might be the unlucky thousandth 
 
 13   person who gets the hit straightaway but, yes, I am sure 
 
 14   bridge transplants are safer and I am pretty sure that the 
 
 15   kinds of barrier devices that some of the islet people are 
 
 16   talking about, and we have heard today from groups who are

 17   using hepatocytes in various systems that potentially those 
 
 18   are significantly safer.   
 
 19             DR. WEISS:  The microchimerism that can occur over 
 
 20   a short term of a fixed transplant could actually result in 
 
 21   long‑term presence of the tissue even after the solid organ

 22   is removed.   
 
 23             DR. ONIONS:  Well, I would be very interested 
 
 24   because I am certainly not an expert in this area and I 
 
 25   would be very interested later on to hear from people.  My 

 
        sgg                                                                240 
 
  1   understanding from talking to a couple of people who have

  2   done bridge transplants is that actually microchimerism does 
 
  3   not occur in bridging of transplants.  You get DNA in the 
 
  4   circulation for the first few hours ‑‑ somebody might be 
 
  5   able to comment on this ‑‑ but detection of microchimerism 
 
  6   is actually almost unknown after the first few days, true

  7   microchimerism, by that I mean getting porcine cells in the 
 
  8   circulation.  But there may be somebody here who can 
 
  9   contradict or add to that, I don't know.  
 
 10             DR. AUCHINCLOSS:  I think I am going to suggest 
 
 11   that we take a break.  It is now 3:55 ‑‑ oh Lord, cancel my

 12   flight tonight and can we make some reservations in the 
 
 13   hotel?  Let's suggest that we come back in here and be ready 
 
 14   to start at 4:15.  
 
 15             [Brief break] 
 
 16      Presentations of Focus Group Analysis of Question #1:

 17        Development of Programs for Patient Monitoring and 
 
 18                        Clinical Follow‑up 
 
 19             DR. AUCHINCLOSS:  We will go right ahead and move 
 
 20   on.  First a comment, when you are speaking, members of the 
 
 21   panel, people in the back are having difficulty hearing

 22   sometimes so please bring the microphone closer.   
 
 23             The bad news is that that was the easy question.  
 
 24   I have heard from a number of members of the committee that 
 
 25   there are flights, including trans‑Atlantic flights, that 

 
        sgg                                                                241 
 
  1   simply can't be missed and we are going to begin to lose

  2   members.  What that functionally means is that we are not 
 
  3   going to answer all of the questions that we have in front 
 
  4   of us today but it is clear we didn't answer even the first 
 
  5   question in a fundamental way.   
 
  6             The important information I think for everybody to

  7   have is that this is not the last time that this 
 
  8   subcommittee will gather, partly because the information 
 
  9   will change and partly because the issues are, in fact, so 
 
 10   complicated that they deserve still additional attention.   
 
 11             With that introduction and the notion that there

 12   will be a time probably 45 minutes from now where we wind 
 
 13   down and complete, let's move right into question #2 because 
 
 14   there really are some very fundamental issues that come up 
 
 15   in this question as well.  We are going to have a 
 
 16   presentation from our focus group on question #2.  Claudia?

 17             DR. MICKELSON:  Well, just as a general summary 
 
 18   and just as before, we will go on a question by question 
 
 19   basis, our question in focus group #2 tried to deal mainly 
 
 20   with development of programs for patient monitoring in 
 
 21   clinical follow‑up issues.  

 22             Basically, the first four or five questions dealt 
 
 23   with actual tests that might be used to monitor recipients.  
 
 24   The questions had to do with appropriate tests, how to 
 
 25   identify PERVs and sensitivity and specificity.  Basically, 

 
        sgg                                                                242 
 
  1   I am trying to be quick so that we can get into the

  2   questions specifically, but those types of questions.  We 
 
  3   all reiterated words that you have all heard this morning, 
 
  4   such as co‑cultivation with stimulation of the pig cells on 
 
  5   PCR and reverse transcriptase, with the idea that we need to 
 
  6   have some sort of quantitative estimate of the viral loads

  7   that might be present in the patient. 
 
  8             I think we should note too that just as we all 
 
  9   agreed that it is necessary that CBER require sponsors of 
 
 10   xenograft recipients to monitor for PERVs, I think most of 
 
 11   us would agree that we would also ask that the sponsors of

 12   any of these trials be required to test the tissues and 
 
 13   cells and,  of course, you would want to test the recipients 
 
 14   as well.  
 
 15             We felt that in terms of what types of tests these 
 
 16   would change with time as the state‑of‑the‑art shifted and

 17   things got more specific and more sensitive and those, of 
 
 18   course, would be the tests that you would have to ask for.   
 
 19             The other question is at what frequency, what 
 
 20   times would you ask to obtain clinical specimens for post‑ 
 
 21   transplantation monitoring.  There is some flexibility in

 22   that, of course, and it would change depending on the 
 
 23   transplant type.  With things like bone marrow transplant 
 
 24   you want very early time points, mid‑ and long‑term follow‑ 
 
 25   up.  I think the point was also made that there would have 

 
        sgg                                                                243 
 
  1   to be baseline pre‑transplant testing and monitoring to be

  2   sure that there were true xenos., not only with PCR but with 
 
  3   any antibodies, and we are hoping that there would be 
 
  4   antibody tests available.  That is really necessary for 
 
  5   appropriate monitoring.  
 
  6             We looked at frequent intervals in terms of at

  7   least weekly, sometimes multiple times during the week, for 
 
  8   at least the first two to three months and then follow‑up 
 
  9   every six to twelve months for a very long time frame.  At 
 
 10   least the minimum requirement would be that seen for 
 
 11   allotransplant recipients.

 12             As to what types of tissue would be relevant and 
 
 13   informative in monitoring for PERVs, again it depends on the 
 
 14   type of transplant.  I think one of the points that was made 
 
 15   was that detection in peripheral blood was going to be 
 
 16   informative.  I think one of the points that was made was

 17   that by the time you see infection in peripheral blood you 
 
 18   probably have a reasonable infection elsewhere in the body.  
 
 19   If you have enough infection in cells to detect it by PCR 
 
 20   you have somewhere else a reasonable source of virus 
 
 21   shedding into the blood.  Also, the issue would be how

 22   invasive a monitoring you want to do.  Is it warranted?  If 
 
 23   these are non‑accessible sites, how far do you go in terms 
 
 24   of invasive biopsies on transplant patients who will also be 
 
 25   immunosuppressed?   

 
        sgg                                                                244 
 
  1             In the event of a positive test result, what

  2   strategies would be included in the diagnostic plan to 
 
  3   identify the source of the positive signal?  Now, we have 
 
  4   heard talk of species‑specific probes, but the key question 
 
  5   there is to be sure that the positive signal arises from the 
 
  6   human cells and not trace contaminants of pig cells.  So, it

  7   looks like you want not just the full complement of species‑ 
 
  8   specific probes and, so far, we are concentrating on porcine 
 
  9   ones but I assume there would be other species that would 
 
 10   eventually be donors.  So, it would be important to also 
 
 11   have very strong, clear human‑specific probes to use as a

 12   counterpart in all of your tests.  I have seen no one really 
 
 13   mention those.  I suppose those are easier to obtain, but 
 
 14   for each species‑specific one you should have a human 
 
 15   positive.  
 
 16             Then, you also want to be sure that in terms of

 17   the positive test results whatever result is obtained, 
 
 18   whether by PCR, you would also look at other host responses 
 
 19   to see the extent of the positive result.  If it is positive 
 
 20   by PCR, are you also getting humoral or cytotoxic cell 
 
 21   response; is there any kind of NK cell response?  Then, if

 22   you can isolate the virus from the patient, look at issues 
 
 23   of tropism, host range and then tissues capable of being 
 
 24   infected in the gene sequence.   
 
 25             If a patient were to test positive, what changes 

 
        sgg                                                                245 
 
  1   might be made to the clinical evaluation program in terms of

  2   safety and possible public health risks?  If PERV is 
 
  3   detected in human cells, all of us agreed that at least the 
 
  4   clinical trial should be suspended immediately until 
 
  5   extensive analysis of the signal or the positive assay can 
 
  6   be confirmed, and see if it can be supported by other tests.

  7 
 
  8             We left those other tests vague because I think it 
 
  9   was clear from the first discussion that a lot of the tests 
 
 10   are still under development and, as more tests become 
 
 11   available, we are just going to get much more information to

 12   assess exactly what is going on.   
 
 13             What happens in case one of the clinical 
 
 14   participants turns positive?  What do you do in terms of 
 
 15   patient counseling?  We asked that there be extensive free 
 
 16   transplant counseling for the recipient, the expected

 17   recipient and family and close contacts to deal with issues 
 
 18   that are usually addressed in HIV infection counseling, such 
 
 19   as not donating blood or tissues, using your own toothbrush.  
 
 20   The same kinds of items and issues that have been raised and 
 
 21   shown to be methods for HIV transmission should be pertinent

 22   in these instances. 
 
 23             In terms of immediate clinical care of the 
 
 24   patient, the counseling of partners and family pre‑ 
 
 25   transplant, as well as training and information for all of 

 
        sgg                                                                246 
 
  1   the patient health care personnel and treatment, the minimum

  2   starting point would be the same types of support given for 
 
  3   allogenic transplant recipients.  
 
  4             In terms of transmissibility, you would check, 
 
  5   just to be sure that what was being shed or detected, that 
 
  6   you hadn't developed any new types of phenotype.  C‑type

  7   retroviruses are not shed by respiratory methods.  You 
 
  8   rarely find them in certain type of secretion but they would 
 
  9   all be checked and you would try to screen all contacts on 
 
 10   an ongoing basis with PCR and serological tests, if 
 
 11   possible. 

 12             I think one of the issues, and one of the 
 
 13   questions that we sort of threw in is how might you change 
 
 14   the immune suppression regimen if the patient is 
 
 15   productively infected?  What can you do?  Are there possible 
 
 16   treatments for the infection?  I think one of the issues is

 17   if you do have a positive test, what are you going to do 
 
 18   about it?  Do you just sit there with it or are you going to 
 
 19   try and do something to suppress that signal?  Even if the 
 
 20   signal goes away, it doesn't mean, honestly, that the 
 
 21   patient is not infected.  But if the suppression can be

 22   safely reduced and possibly treatment instituted it might be 
 
 23   advisable.  But there was disagreement on that, that there 
 
 24   would be no changes made in any kind of immune suppression 
 
 25   regimen until there was evidence of pathogenicity or 

 
        sgg                                                                247 
 
  1   infectivity ‑‑ again, the potential treatment with

  2   antiretrovirals. 
 
  3             In terms of long‑term follow‑up for the patient, 
 
  4   we raised issues that these were different than 
 
  5   allotransplants and that there would be much longer‑term 
 
  6   follow‑up needed if survivability allowed that, and that it

  7   would constitute as many types of samples as we could get 
 
  8   without being overly invasive to the patient.  I think one 
 
  9   of the things that was very hard to set up is an idea of 
 
 10   what you would do for monitoring unless we knew what kind of 
 
 11   tests would work.  I think one of the things that came out

 12   of the discussions ‑‑ we conversed by e‑mail; we actually 
 
 13   never met altogether until today ‑‑ but I think one of the 
 
 14   things is how do you set up a monitoring program when we are 
 
 15   not sure how good the tests are and until they are well 
 
 16   standardized?  I am not sure what you do with the data, and

 17   then how do you use the data?  What is it going to mean if a 
 
 18   patient is positive if we don't know the outcome or have an 
 
 19   idea of what a reasonable outcome of the infection is?   
 
 20             Then, if clinical trials do proceed and we 
 
 21   accumulate all this screening information, how will it be

 22   used?  What will be important in terms of making clinical 
 
 23   decisions to treat one of the patients and then, also, how 
 
 24   are you going to safeguard all the information that you 
 
 25   would get from patients? 

 
        sgg                                                                248 
 
  1             We can go through the questions point by point.

  2             DR. AUCHINCLOSS:  Thank you very much.  Before we 
 
  3   do that, reading the handwriting on the wall, I guess, Leroy 
 
  4   Walters has suggested that you receive the summary of the 
 
  5   focus group #3 before we go into detail on question 2.  We 
 
  6   though that maybe we will sort of run out of energy before

  7   we got to 3 in detail. 
 
  8             DR. MICKELSON  I think there is so much overlap 
 
  9   that that is good.  
 
 10             DR. WALTERS:  Our group met for about 45 minutes 
 
 11   over lunch, and our group was actually growing over the

 12   weeks leading up to this meeting.  So, this will largely be 
 
 13   one person's reaction to the discussion of the group, and 
 
 14   some group members may want to disown this summary.  
 
 15             I will give you four generalizations, three 
 
 16   recommendations and two concluding comments.  Our first

 17   generalization was that this is clinical research, not 
 
 18   standard and accepted therapy, and it should be presented to 
 
 19   patients as clinical research.  
 
 20             The second generalization is that there are many 
 
 21   unknowns in the current situation, and this fact too should

 22   be disclosed very clearly to the subjects.  
 
 23             The third generalization ‑‑ there are two very 
 
 24   different contexts in which xenotransplantation trials are 
 
 25   being conducted or proposed.  One of them is the emergency 

 
        sgg                                                                249 
 
  1   situation, for example, with fulminant liver failure.  The

  2   other is a non‑emergency situation, for example, progressive 
 
  3   Parkinson's disease.  In terms of the consent process, these 
 
  4   two types of situations present very different scenarios and 
 
  5   very different problems.  In the emergency situation we are 
 
  6   really talking about consent by a family member or a close

  7   friend on behalf of someone who can't give consent for him 
 
  8   or herself.   
 
  9             A final generalization ‑‑ there are at least three 
 
 10   groups of people who are potentially affected by these 
 
 11   clinical trials.  First, the subjects themselves and there

 12   can be a consent process for them if there is no emergency.  
 
 13   Secondly, the close household contacts of the subjects and 
 
 14   there can be a consent process for them if there is not an 
 
 15   emergency.  Third, and I don't think we have really 
 
 16   addressed this today, the health providers who are involved

 17   in the subjects' care.  Here, we think there may need to be 
 
 18   some kind of notification procedure and, very likely, 
 
 19   universal precaution until we know more information about 
 
 20   actual risk.  
 
 21             Now, our recommendations fall into three areas. 

 22   First, on the description of risks, this was a suggestion by 
 
 23   one member of the group in particular, that there should be 
 
 24   a standardized and regularly updated and accurate and 
 
 25   balanced description of the major risks for each major type 

 
        sgg                                                                250 
 
  1   of clinical trial.  We even speculated about developing a

  2   web site and an 800 number to which IRBs or prospective 
 
  3   subjects or clinicians and investigators could turn.  Very 
 
  4   likely, this web site and 800 number would have to be 
 
  5   managed by a federal agency.   
 
  6             A second recommendation regarded selection

  7   criteria for subjects.  In our view, inclusion criteria 
 
  8   should be related to the clinical research and to nothing 
 
  9   else.  So, the subjects have to be willing to be monitored 
 
 10   for many years.  Their household contacts will also probably 
 
 11   need to be willing to be monitored at regular intervals. 

 12   Clearly, they have to have the capacity to understand the 
 
 13   research protocol and to give consent to participate.  But 
 
 14   we hope that the trials will avoid selection based on social 
 
 15   criteria.  That is, they shouldn't be based on social class 
 
 16   or gender, or ethnicity, or even a history of alcohol use

 17   early in life.   
 
 18             A third recommendation has to do with the consent 
 
 19   process.  Again, we go back to the web site and an 800 
 
 20   number.  There should be a relatively standardized 
 
 21   description of risks that is updated regularly on the basis

 22   of the newest information.  We also think that the consent 
 
 23   forms should be simplified; should be in lay language.  We 
 
 24   observed that the two consent forms that we read were fairly 
 
 25   complex at some points and a master's degree in immunology 

 
        sgg                                                                251 
 
  1   would have been very helpful in understanding those forms. 

  2   Again, household members may need to be involved in the 
 
  3   consent process. 
 
  4             Now, two concluding comments ‑‑ there is one 
 
  5   additional kind of subject monitoring that we think should 
 
  6   probably be included that is a little different from the

  7   types of medical monitoring that we have just heard about.  
 
  8   That is, gathering information on the effect of receiving a 
 
  9   xenotransplant on one's health insurance coverage.  That 
 
 10   wouldn't occur to our colleagues from the U.K. because you 
 
 11   couldn't easily fall out of that system, but it is at least

 12   conceivable that insurers will be a little worried about the 
 
 13   unknowns in xenotransplantation and be reluctant to cover 
 
 14   people who have had this kind of transplant.   
 
 15             A final concluding comment is that I think we need 
 
 16   to be prepared for the moment when and if the first infected

 17   subject is detected, and have to be resolved to protect that 
 
 18   individual's civil liberties.  It will be easy to overreact 
 
 19   and words like "isolation" and "quarantine" come readily to 
 
 20   some people's minds.  I would just say that there are many 
 
 21   people walking around with influenza.  You have sat beside

 22   some of them in airplanes when you have flown.  There are 
 
 23   many people walking around with tuberculosis or hepatitis B 
 
 24   or HIV infection and questions of isolation and quarantine 
 
 25   do not come up.  So, we need to be resolved not to overreact 

 
        sgg                                                                252 
 
  1   when and if a human patient becomes infected.  

  2             DR. AUCHINCLOSS:  Thank you very much.  I am going 
 
  3   to handle the individual questions slightly differently as 
 
  4   we move through question 2, and suggest, for example, that 
 
  5   question 2(a) which basically asks, as before, is it 
 
  6   appropriate for CBER to require that assays be done to

  7   detect virus, I think we are going to simply suggest that 
 
  8   the answer from this committee would be yes.  Is there 
 
  9   anybody who wants to disagree with that? 
 
 10             [No response] 
 
 11             Then, in moving to question 2(b), essentially it

 12   is a question similar to what we had with question 1, what 
 
 13   types of assays, how sensitive, and in many respects I 
 
 14   assume the answer is the same as what we talked about 
 
 15   before.   
 
 16             So, what I want to ask the committee is what in

 17   addition do they feel are tests that should be required for 
 
 18   patient monitoring.  We have already heard one statement 
 
 19   that we need to be looking for antibody responses.  But we 
 
 20   need to hear a little bit of amplification on this because 
 
 21   the antibody assay doesn't exist at this point.  Can we

 22   generate one, and is it realistic or is it necessary to wait 
 
 23   for an antibody test before any further progression of 
 
 24   xenotransplants? 
 
 25             DR. ONIONS:  Actually, Chairman, I think this 

 
        sgg                                                                253 
 
  1   series of questions is actually a lot easier to answer than

  2   the previous ones.  My own view is that if you are going to 
 
  3   test ‑‑ certainly you should test patients but my own view 
 
  4   though is those tests should be done perhaps 2 weeks, 1 
 
  5   month, 3 months at 3‑monthly for the first year.  What I 
 
  6   would do on the first test up to the 3‑month period would be

  7   to look for plasma viremia, and the way we would normally 
 
  8   look for plasma viremia, the way, in fact, it is done 
 
  9   commercially is 10,000 cats a year and we do 25,000 a year 
 
 10   in my lab to look for virus antigen to detect viremia.  But 
 
 11   what we always do is confirm the positives by virus

 12   isolation.  That, ideally, would be the way to do it.   
 
 13             However, I am concerned that the sensitivity of 
 
 14   the tests are not good enough to do it that way at the 
 
 15   moment, and the way I would actually do it at the moment is 
 
 16   to actually look for virus by PCR in the plasma.  You could

 17   do that by generic tests in the first place, and if it was 
 
 18   positive I would then confirm which of the viruses of at 
 
 19   least the four groups we now know of are present in the 
 
 20   plasma.  So, the first thing I would look for plasma 
 
 21   viremia.  

 22             Secondly, I certainly would look for virus in the 
 
 23   presence of peripheral blood mononuclear cells.  I think one 
 
 24   of the questions we will have to ask is how many cells you 
 
 25   should look at because you can look at 106 cells and you do 

 
        sgg                                                                254 
 
  1   this in 105 aliquots.  That only gives you a 96% certainty,

  2   if your assays are sensitive of course, that you are 
 
  3   detecting it in that 105 aliquot.  So, I would certainly do 
 
  4   peripheral blood mononuclear cells.  As Robin pointed out, a 
 
  5   lot of the viruses we know about that are latent are at 
 
  6   levels below 1 in 105; they are 1 in a million or so in

  7   circulation.  
 
  8             Thirdly, I certainly would have an antibody test, 
 
  9   and the reason for that was perhaps badly described this 
 
 10   morning.  There are at least four patterns of infection we 
 
 11   see in animals and, in fact, to understand which of the

 12   patterns of infection we are seeing in patients we must have 
 
 13   an antibody result to actually define those.  I think we are 
 
 14   very near to having an antibody test.  We are just 
 
 15   validating one for P30.  We are going through a lot of human 
 
 16   sera as well as going through defined polyclonal sera.  

 17             So, I think those four tests over those time 
 
 18   periods are the way I would approach it.  
 
 19             DR. AUCHINCLOSS:  Comments from other members of 
 
 20   the committee?   
 
 21             DR. KASLOW:  I guess the question would be do you

 22   simultaneously or sequentially add such tests as would 
 
 23   indicate transmissibility, other fluids at that point or do 
 
 24   you wait until ‑‑ 
 
 25             DR. ONIONS:  I didn't bother to show it this 

 
        sgg                                                                255 
 
  1   morning but if any of the tests were positive by any

  2   criteria, what I would then do, I would want to know if this 
 
  3   patient is secreting virus in the saliva and possibly other 
 
  4   fluids, yes, absolutely.  I think if PBMCs are positive, we 
 
  5   would really like to know if that is a true latent infection 
 
  6   or is there, in fact, transcription of virus going on in

  7   those cells.  That would be the second question I would want 
 
  8   to ask.  
 
  9             DR. AUCHINCLOSS:  Well, before you get to the 
 
 10   positive what tissues do you screen and how invasive are you 
 
 11   being?

 12             DR. ONIONS:  I wouldn't be invasive.  The 
 
 13   clinicians would be in a much better position to answer this 
 
 14   question but I would have thought it is not really ethical 
 
 15   to use invasive procedures on these patients.  I don't 
 
 16   actually see the necessity for doing that.  We are concerned

 17   about a public health risk here, and the public health risk 
 
 18   is when we get sufficient replication of virus for that to 
 
 19   be of concern, either through blood spillage or through 
 
 20   excretion in various body secretions.  So, I don't see a 
 
 21   justification for invasive procedures.  But there will,

 22   unfortunately, be patients coming through for postmortem to 
 
 23   provide those tissues to do those other analyses.   
 
 24             DR. AUCHINCLOSS:  As a clinician, I guess I 
 
 25   started from a slightly different point of view.  I can 

 
        sgg                                                                256 
 
  1   imagine actually getting tissue with invasive assays through

  2   a number of different sources, but I came back to the same 
 
  3   point that you just made.  I am not sure that it helps me 
 
  4   because the public health issue is the one at stake, and I 
 
  5   am assuming that the tissues that represent a public health 
 
  6   risk are the ones that we can easily sample.  Is that a fair

  7   assumption? 
 
  8             DR. ONIONS:  I think it is, yes. 
 
  9             DR. KASLOW:  That may depend on how easy you think 
 
 10   it is to sample cervical or seminal secretions I suppose.  I 
 
 11   mean, that would sort of be at the interface between easy

 12   and difficult.  
 
 13             DR. AUCHINCLOSS:  You are right.  
 
 14             DR. MICHAELS:  I think they can be requested ‑‑ 
 
 15             DR. AUCHINCLOSS:  Of course.  
 
 16             DR. MICHAELS:  ‑‑ and I would also second David's

 17   comment that if patients were to die that up front in the 
 
 18   consent process, which we will talk about in question 3, I 
 
 19   would discuss autopsies with families if that should occur.  
 
 20 
 
 21             DR. AUCHINCLOSS:  I don't know if that actually

 22   emerges in 3 but let me just make a comment.  I guess it has 
 
 23   been determined that you cannot require, and it doesn't do 
 
 24   any good even if you did because it is not the person who 
 
 25   gives permission for an autopsy, but you could potentially 

 
        sgg                                                                257 
 
  1   put in a statement in the informed consent that "I have

  2   discussed with my family my desire that a postmortem occur." 
 
  3             DR. MICHAELS:  I am not even sure that you have to 
 
  4   make the person agree to an autopsy but I think it behooves 
 
  5   the investigator to discuss those issues up front.  
 
  6             DR. AUCHINCLOSS:  Okay.  Under (b), I think your

  7   comments have addressed most of the specific questions.  Amy 
 
  8   and other members of the FDA, have you heard what you wanted 
 
  9   to hear about (b), and other members of the committee, do 
 
 10   you want to contribute further under (b) about the nature, 
 
 11   sensitivity, timing etc. of assays on patients?

 12             DR. MICHAELS:  I have just one other quick 
 
 13   comment.  If the patient were to have febrile illnesses or 
 
 14   unwellness that was not able to be ascribed easily to 
 
 15   another cause, that at that time, even if it wasn't in your 
 
 16   time point of surveillance, you would bring the patient in.

 17             DR. AUCHINCLOSS:  That sounds reasonable.   
 
 18             DR. SIEGEL:  Let me ask for clarification on one 
 
 19   point, which is that there has been a widespread, I think, 
 
 20   agreement that antibody tests would be nice and should be 
 
 21   developed rapidly.  Should we take home from that that they

 22   should be required as soon as they are developed, or I 
 
 23   should take home the alternative, that they need to be 
 
 24   developed, or they should be developed before we consider 
 
 25   moving forward?  We have said under 2(a) that before 

 
        sgg                                                                258 
 
  1   somebody starts a protocol they should have a patient

  2   monitoring scheme in place, and we take that to mean that 
 
  3   they should be looking with PCR in the blood, in the 
 
  4   lymphocytes, whatever.  Does that mean they should be 
 
  5   looking at antibodies or they should be looking at 
 
  6   antibodies at such point, as soon as that becomes a

  7   reasonable, controlled and validated thing to do? 
 
  8             DR. HIRSCH:  I would think you would want to 
 
  9   collect the sera. 
 
 10             DR. SIEGEL:  Collect it now? 
 
 11             DR. HIRSCH:  You know, we don't know how long it

 12   is going to take to develop a satisfactory assay.   
 
 13             DR. SIEGEL:  I just wanted to clarify that.  
 
 14             DR. MICKELSON:  We just felt it is very important 
 
 15   that one be in the works somewhere. 
 
 16             DR. MICHAELS:  Also, looking at a patient that is

 17   a year out from having had porcine tissue in them for a 
 
 18   finite period of time, it is true that antibody testing in 
 
 19   that person, if it is a sensitive test, might be better than 
 
 20   PCR or PBL because you might not be having a latent organism 
 
 21   viremic.  But looking for a primary infection, I think the

 22   time points that David Onions brought up would be very 
 
 23   sensitive, and I would concur with the others that serum 
 
 24   should be collected.  
 
 25             DR. AUCHINCLOSS:  Which, I guess, Jay, indicates 

 
        sgg                                                                259 
 
  1   that the committee is not suggesting to you that not only

  2   should the antibody test be in place but that it should be 
 
  3   applied to all existing patients who have had porcine 
 
  4   transplants in the past before proceeding with new trials, 
 
  5   which is yet a further extension of what might have been 
 
  6   suggested to you but I am not hearing that either.  Is that

  7   a fair statement from the committee?  
 
  8             One suggestion is, yes, but don't go forward with 
 
  9   any pig trials until you have the antibody assay and until 
 
 10   you have used it on all those 100 or whatever patients, and 
 
 11   this subcommittee is not making that recommendation to the

 12   FDA is what I am hearing.   
 
 13             Again, within this issue of testing patients there 
 
 14   is another specific angle which is slightly different from 
 
 15   testing tissue ahead of time, and that is how do you know 
 
 16   whether the virus is in the pig cell or the human cell?  Is

 17   there a technical answer to that?  What do they need to do? 
 
 18             DR. ONIONS:  I thought we heard a very nice 
 
 19   presentation from Walid this morning about his approach 
 
 20   which I think is a valid approach.  I don't know whether I 
 
 21   am allowed to say it but I am going to say it, Ed Otto, from

 22   GTI has developed a very interesting system using the tapman 
 
 23   quantitative PCR system, and I think it is a very elegant 
 
 24   system and I think that also provides the tools for 
 
 25   answering this question.  It allows you to get a really 

 
        sgg                                                                260 
 
  1   accurate ratio between the proviral DNA and the cellular

  2   DNA.  So, I think there are systems to do that.   
 
  3             I am also going to be interested, actually, in 
 
  4   what really is the true problem of microchimerism.  I wonder 
 
  5   if it is going to be as big a problem as we think it is.  I 
 
  6   know there are different opinions but I think it is a

  7   solvable problem.  
 
  8             DR. AUCHINCLOSS:  Any other comments on this?  Any 
 
  9   of the experts want to add anything?  So, you get the virus 
 
 10   and you suggest that it is infective in a human cell, in a 
 
 11   patient, and you do some things to characterize its

 12   infectivity and to further characterize the agent.  I think 
 
 13   we have indicated before that you sequence it, you look at 
 
 14   tropism, and I think that list is already complete in your 
 
 15   summary.  Have I missed an important aspect of this because 
 
 16   we are going to move into what it all means, sort of the

 17   heart of it? 
 
 18             Let's go there, which is (f), which is if the 
 
 19   virus is in a human cell, in a human patient, what do you 
 
 20   do?  Let's go to the bottom line here, (iv), what do you do 
 
 21   with the clinical trial?  You suggested that we all agreed

 22   that that is the stop point. 
 
 23             DR. MICKELSON:  Yes, we all ‑‑ 
 
 24             DR. AUCHINCLOSS:  Well, I didn't agree with that 
 
 25   and I was on that focus group.  Remember, I am assuming that 

 
        sgg                                                                261 
 
  1   that event is ‑‑

  2             DR. MICKELSON:  You didn't answer that question ‑‑ 
 
  3             DR. AUCHINCLOSS:  Oh, yes, I did.  I am assuming 
 
  4   that event is going to happen.  What do other people think? 
 
  5             DR. MICKELSON:  Well, not everybody that I e‑ 
 
  6   mailed all the questions to answered me back on that one. 

  7   The one answer that was given as to what you would do in 
 
  8   case you did eventually have a patient that came in positive 
 
  9   on a test was that the program be suspended immediately.  I 
 
 10   assume that that was done in context of until you have 
 
 11   further analysis of what the positive signal was and if it

 12   is a true positive.  He left but that was Dr. Solomon.  
 
 13             DR. HIRSCH:  I can't remember whether I answered 
 
 14   that question or not but I think it makes good sense to 
 
 15   suspend the trial at least temporarily so that the FDA can 
 
 16   gather the data.  They may want to reconvene a committee and

 17   say, "well, what do we do now?"  I don't think that means 
 
 18   necessarily that the trial is permanently discontinued but 
 
 19   that it is temporarily suspended seems to me the prudent 
 
 20   thing to do.  
 
 21             DR. MICKELSON:  Yes, until you figure out what it

 22   actually means in terms of the patient.  
 
 23             DR. COFFIN:  It seems to me one should make a 
 
 24   quantitative distinction here between localized infection 
 
 25   and spreading infection.  I think in the case where it is 

 
        sgg                                                                262 
 
  1   clear that there is a spreading infection, viremia,

  2   appearance of virus at sites other than in the blood or at 
 
  3   sites at other than where the transplant was, unless in the 
 
  4   meantime the collective wisdom has been brought to bear on 
 
  5   this problem better than we can do right now, I think that 
 
  6   definitely calls for an assessment of what is going on and

  7   assessing what is going on.  
 
  8             DR. AUCHINCLOSS:  Actually, that is what my answer 
 
  9   said, no, not necessarily, but I want to hear from the 
 
 10   experts what the features are that are positive that would 
 
 11   lead you to say no, no, no.  

 12             DR. ONIONS:  I can't cast myself in that role but 
 
 13   I agree with John.  If you get viremia, I would certainly 
 
 14   want to hold things until we know a lot more about that 
 
 15   situation because the viremia might increase ‑‑ if you get 
 
 16   over viremia, I think that is five years ‑‑

 17             DR. AUCHINCLOSS:  I see a lot of nodding heads.  
 
 18   Viremia is a bad thing.   
 
 19             DR. ONIONS:  If you get just a few cells, maybe 
 
 20   one in a million cells in the circulation infected, I would 
 
 21   still be concerned but I don't think that is the same degree

 22   of concern, particularly if you can demonstrate that is true 
 
 23   latency ‑‑ 
 
 24             DR. KASLOW:  By viremia you mean free virus? 
 
 25             DR. ONIONS:  Free virus, yes.  

 
        sgg                                                                263 
 
  1             DR. ALLAN:  I would go a little farther.  I would

  2   think that if you had infected cells, and they are human and 
 
  3   they are circulating the peripheral blood I think that is a 
 
  4   red flag, and I think if you do have that you will probably 
 
  5   also get antibody responses.  If you are going to start 
 
  6   seeing infected human cells floating in the peripheral blood

  7   you may have antibody responses, and is that a red flag?  In 
 
  8   other words, you start to see antibody responses to the 
 
  9   viruses probably at least temporarily and maybe longer.  
 
 10             DR. HIRSCH:  I think with the different answers 
 
 11   you are getting it means that when you get any evidence of

 12   human infection you pause and you reevaluate.  I mean, I 
 
 13   think we are extrapolating a lot from HIV where you get 
 
 14   viremia.  With HTLV, let's remember, you really don't get a 
 
 15   plasma viremia and it is pathogenic, albeit a human pathogen 
 
 16   of low virulence, and it can cause serious disease and you

 17   don't get plasma viremia. 
 
 18             DR. ONIONS:  But they are very different and I 
 
 19   really must reemphasize again that this is not like HTLV.  
 
 20   HTLV carries transactivated genes.  Its method of 
 
 21   pathogenesis is quite different from these viruses.  I think

 22   it is always risky dragging back to the systems we know 
 
 23   behave in the same way.  In the systems that we know of this 
 
 24   representative group, in general, in over 90% of the cases 
 
 25   it is the plasma viremia that is the bad news.  You do find 

 
        sgg                                                                264 
 
  1   tumors that are associated with recovering latency because

  2   transduction or insertion of oncogenes occur but generally 
 
  3   it is when you get plasma viremia that you get into trouble 
 
  4   and, honestly, I think HTLV‑1 is a bad model in this case. 
 
  5             DR. HENEINE:  If I could add to the criteria for 
 
  6   positivity, a persistent ability to persistently detect

  7   viremia, antibody and all that ‑‑ it is not like a one time 
 
  8   point when you detect the evidence of infection but if you 
 
  9   are able to detect it over time and persistently, that is 
 
 10   what would give you evidence of a persistent infection.  
 
 11             Regarding the viremia, we know very little about

 12   the biology of this virus and how it is going to behave in 
 
 13   humans.  You can argue both ways.  I was about to raise Dr. 
 
 14   Hirsch's point as well, like in HTLV‑1 and 2 we don't see 
 
 15   cell free virus in infected people.  We see persistently 
 
 16   infected PBLs.  Also in fomi viruses we don't see cell free

 17   virus in the plasma, but we see infected PBLs all the time.  
 
 18   So, there are different biologies but we are not sure how 
 
 19   this PERV would behave in a human.  
 
 20             DR. ONIONS:  But I think we have very good 
 
 21   circumstantial evidence.  If you look at the same group of

 22   viruses and we look in the context where they have been put 
 
 23   into a foreign host, into a primate host, if we take alpha 
 
 24   tropic murine leukemia virus that has been put in by 
 
 25   Cornetta and by Donahue, if you look at the Donahue study, 

 
        sgg                                                                265 
 
  1   the tumors occurred in those animals developed fulminant

  2   viremia, high titer plasma viremia.  Those were the animals 
 
  3   that developed disease.  In Cornetta's study and also in 
 
  4   Donahue's study the animals that didn't develop a viremia, 
 
  5   or they still might have had PCR evidence of virus around, 
 
  6   they did not develop disease.  So, I think actually it is

  7   confirmed.  This is the likely way that this group of 
 
  8   viruses will behave in a primate. 
 
  9             DR. HENEINE:  This is another point, pathogenesis 
 
 10   associated with or without viremia.  But with the other 
 
 11   viruses there is or there is no viremia irrespective of

 12   disease development in HTLV.  
 
 13             DR. AUCHINCLOSS:  What I think I have heard as a 
 
 14   general statement is that detection of virus in a human cell 
 
 15   is a break point.  Lots of information should be gathered, 
 
 16   including especially viremia; that you should call the

 17   subcommittee back for another conversation with you and 
 
 18   maybe we will be smarter then. 
 
 19             DR. HENEINE:  Let me add again about the serology, 
 
 20   the limitations and the usefulness and utilities of the 
 
 21   different tests or different assays we have, we look at this

 22   as different markers of the infection.  Marker number 1 is 
 
 23   proviral sequences and peripheral lymphocytes.  Marker 
 
 24   number 2 is antibody production.  Marker number 3 is 
 
 25   viremia, which is cell free virus in the plasma.  And the 

 
        sgg                                                                266 
 
  1   more markers we can gather for an infection, the better it

  2   is.  But to say that we select only one marker, antibodies, 
 
  3   and forge the rest is not optimal.  We need to gather as 
 
  4   much evidence as possible on all these different markers of 
 
  5   the infection, and we will be facing a situation where we 
 
  6   will have patients that are antibody positive, PCR positive,

  7   RT‑PCR positive and RT positive, and others will have only 
 
  8   one marker which is persistency of positivity but negative 
 
  9   for all the rest, and we will have to figure out what is 
 
 10   going on.  
 
 11             DR. KASLOW:  Is it so unlikely that we would see

 12   shedding of cells in some other secretory pathway instead of 
 
 13   or sooner than you would see it in peripheral blood, that 
 
 14   the order in which all of that is done is kind of irrelevant 
 
 15   and we should start with and rely on peripheral blood first? 
 
 16             DR. ONIONS:  That is a difficult question to

 17   answer because I think the point was made this morning that 
 
 18   maybe peripheral blood cells are not easy to infect.  What 
 
 19   we know of the pathogenesis of these viruses as exogenous 
 
 20   viruses, not as endogenous viruses being reactivated, is 
 
 21   that they tend to get carried to bone marrow.  The events

 22   that go on in the bone marrow are critical.  So, in fact, 
 
 23   you find a high viral load  suddenly coming into the blood 
 
 24   stream and actually the infected cells are in the bone 
 
 25   marrow.  So, in fact, you even find cells like platelets 

 
        sgg                                                                267 
 
  1   which, of course, are non‑dividing are actually releasing

  2   virus because the megakaryocyte is being infected.  So, you 
 
  3   suddenly get a burst and then you get a plasma viremia 
 
  4   following very quickly.   
 
  5             So, I would still think that is the likely cell I 
 
  6   would still go for.  It is possible that you could get a

  7   sequestered pattern of infection which we see.  That always 
 
  8   comes after transient infection in the plasma and then the 
 
  9   virus is just hidden somewhere in an epithelial surface.  
 
 10   But if you do, your timing is correct.  So, you are looking 
 
 11   for this early phase of a transient viremia, two weeks and a

 12   month.  Then I think you would pick that up.  
 
 13             DR. KASLOW:  So we might miss it at two weeks but 
 
 14   we are not going to miss it altogether.  
 
 15             DR. ONIONS:  I don't think so, yes.  
 
 16             DR. HIRSCH:  The exception to that might be the

 17   central nervous system ‑‑ 
 
 18             DR. ONIONS:  Sure.  
 
 19             DR. HIRSCH:  ‑‑ you may never get a viremia.  
 
 20             DR. ONIONS:  That is a very good point and that is 
 
 21   really quite distinct.  

 22             DR. KASLOW:  The distinction there though is that 
 
 23   that is probably not transmissible ‑‑ 
 
 24             DR. HIRSCH:  Right, but you still want to know ‑‑ 
 
 25             DR. KASLOW:  You want to know but it is not urgent 

 
        sgg                                                                268 
 
  1   in terms of needing to make a different decision as a result

  2   of that.  
 
  3             DR. AUCHINCLOSS:  Question number 2(f) has some 
 
  4   other points here as well.  What would you do about patient 
 
  5   counseling?  I am just going to summarize what I sort of 
 
  6   felt was the response here.  Patient counseling, where you

  7   have been counseling the patient appropriately ahead of time 
 
  8   and so this is more patient counseling with honest 
 
  9   information that it has occurred but we don't know what it 
 
 10   means, and long‑term follow‑up and evaluation of patients 
 
 11   really remains where it was before because we have things

 12   set in place for exactly this contingency.   
 
 13             The question, however, that I don't know the 
 
 14   answer to is what do you do about treatment for this 
 
 15   particular individual.  
 
 16             DR. HIRSCH:  This is one point I feel very

 17   strongly about.  I think you do not treat this patient.  You 
 
 18   don't know if it causes any disease at all.  You know that 
 
 19   all of the antiretroviral agents we have are terribly toxic.  
 
 20   We don't know whether they are active against porcine 
 
 21   endogenous retrovirus so there is zero indication to treat

 22   these patients.   
 
 23             DR. ONIONS:  Can I make a statement ‑‑ 
 
 24             MS. MEYERS:  I can't accept that.   
 
 25             DR. ONIONS:  I think we need to go backwards.  

 
        sgg                                                                269 
 
  1   First of all, I think we do need to know the effectiveness

  2   of the known antivirals on the porcine endogenous virus and 
 
  3   John, I think, made a point at the last meeting that he 
 
  4   treated MELV with some of the nucleoside analogs and not 
 
  5   found them effective.  But the critical point is actually is 
 
  6   it is often so dependent so there is phosphorylation between

  7   nucleoside analogs and we need to know that.  Of course, 
 
  8   those will be operative in human cells, which are the cells 
 
  9   we are concerned about and so we then need to know in human 
 
 10   cells if the porcine virus inhibited by the nucleoside 
 
 11   analogs.  I think there will be some evidence that it will

 12   be actually.  Sow e need to have those data.  We must have 
 
 13   those data.  We would certainly not treat a patient who has 
 
 14   just had cells that were provirus positive.  If there was a 
 
 15   viremia beginning, I have been persuaded by others that it 
 
 16   probably is effective, and I can only defer to people who

 17   treat HIV and they would suggest that, in fact, the toxicity 
 
 18   as I also thought.  I would have to defer to other people.  
 
 19             DR. HIRSCH:  We don't treat HTLV infections.  We 
 
 20   don't know that this virus causes any disease, and having 
 
 21   treated HIV patients now for 15 years, I can tell you they

 22   are toxic drugs.  You don't know what your endpoint is.  You 
 
 23   can't eradicate virus anyway.  So, it is something I feel 
 
 24   very strongly about.  
 
 25             DR. COFFIN:  Yes, I agree with that general 

 
        sgg                                                                270 
 
  1   conclusion.  There are other problems as well.  Models where

  2   antivirals were used to treat murine leukemia virus 
 
  3   infections really require pretreatment of the animal.  Any 
 
  4   treatment after infection is established in the animal ‑‑ my 
 
  5   memory of these experiments is that it doesn't work very 
 
  6   well.  Also, only a very few agents are likely to be

  7   effective.  Only a few of the nucleoside inhibitors are 
 
  8   likely to be effective.  
 
  9             DR. ONIONS:  John, if I remember correctly, those 
 
 10   experiments when you actually looked at MELV in mice, the 
 
 11   problem there is that actually mice do not phosphorylate the

 12   nucleoside analogs very efficiently.  
 
 13             DR. COFFIN:  But you can protect a mouse against 
 
 14   infection.  That is part of the problem, but the other 
 
 15   problem is as far as malignant consequences by the time you 
 
 16   see it the damage may already be done.  As far as

 17   transmission is concerned, that is another matter because 
 
 18   you probably would block transmission that way if that is 
 
 19   your concern, but then you are not treating the patient, you 
 
 20   are treating the public and the ethical issues get very 
 
 21   serious then.  

 22             DR. ONIONS:  This may be a question we can't 
 
 23   resolve today and I think it probably has a lot of ethics in 
 
 24   it as well, and I honestly can't answer it but, again, I 
 
 25   would not treat a person who was just provirus positive but 

 
        sgg                                                                271 
 
  1   I really, honestly, have to defer to people who know about

  2   this area more than I do.  I would think there is evidence 
 
  3   that you would treat a viremic person. 
 
  4             I think the other thing just to remember is that 
 
  5   there is evidence from studies mentioned earlier of cats 
 
  6   treated at a very early viremic phase, just going into

  7   viremia and viremia has been able to be reversed just by 
 
  8   using passive neutralizing antibody, which is very 
 
  9   surprising.  It is very good work and it looks real.  So, I 
 
 10   think there are other strategies that if you detect 
 
 11   infection really early, really early, you might be able to

 12   reverse infection. 
 
 13             DR. COFFIN:  That begs the question how frequently 
 
 14   you are willing to monitor because monthly monitoring is 
 
 15   probably too infrequent.   
 
 16             DR. AUCHINCLOSS:  Abbie, we have heard you gasp.

 17             MS. MEYERS:  I did because in my mind I am writing 
 
 18   the informed consent for this project: "Please come and 
 
 19   volunteer to go into this research program.  We would love 
 
 20   for you to be a guinea pig.  But once we do this, it has a 
 
 21   virus in it that we know nothing about and so we want you to

 22   know that if you get sick we are not going to treat you." 
 
 23             DR. HIRSCH:  That is not what we said, Abbie.  We 
 
 24   will treat you with the best medical knowledge we have and 
 
 25   the best possible way we can, but we don't treat you with 

 
        sgg                                                                272 
 
  1   unknown medications that we have no idea will work but we do

  2   know will be very toxic.  We treat you the best we can with 
 
  3   the best medical knowledge we have.   
 
  4             DR. KASLOW:  We don't do that when we have no 
 
  5   knowledge of the occurrence of clinical illness as a result 
 
  6   of this infection.  It would be very different if we knew

  7   that somebody was going to get sick as a result.   
 
  8             DR. AUCHINCLOSS:  The key word is "sick."  There 
 
  9   is no presumption here of sickness; there is only the 
 
 10   presence of the virus.   
 
 11             MS. MEYERS:  But that is not what you said.  You

 12   said that if they get sick I would not treat them.  I feel 
 
 13   very strongly that I would not treat them.  
 
 14             DR. HIRSCH:  No, that is not what I said.  I said 
 
 15   if you found evidence of infection in the human with a 
 
 16   porcine retrovirus I would not treat at that juncture

 17   because we don't know that it causes disease, and we don't 
 
 18   know that the drugs work, and we do know that the drugs are 
 
 19   toxic.  If, in fact, we find that porcine retrovirus caused 
 
 20   any disease and that they are susceptible to antiretroviral 
 
 21   agents I certainly would treat at that point. 

 22             DR. KASLOW:  Just briefly, would the committee 
 
 23   want to entertain a consideration or even a recommendation 
 
 24   about whether or not, if treatment were even considered, it 
 
 25   should be done under a protocol of some sort and not just ad 

 
        sgg                                                                273 
 
  1   hoc?

  2             DR. SIEGEL:  It would certainly be an off‑label 
 
  3   use.  
 
  4             [Laughter] 
 
  5             DR. HIRSCH:  And not necessarily compassionate. 
 
  6             DR. AUCHINCLOSS:  The one point that I wanted to

  7   stress, I think it is really implicit in the way these 
 
  8   trials would be set up that this is the point at which you 
 
  9   want to redouble your monitoring of contacts, which is 
 
 10   really the issue here, transmissibility, when now you know 
 
 11   you have a patient who has the virus.  But I guess that is

 12   sort of obvious. 
 
 13             Now, back to the FDA, have you heard in question 2 
 
 14   what you wanted to hear?  
 
 15             DR. SIEGEL:  Having discussed question 2, there is  
 
 16   something carried over from the earlier question I would

 17   want to get to.  There was a general sentiment expressed by 
 
 18   several panel members that having heard that there have been 
 
 19   many individuals who have received xenotransplantation of 
 
 20   cellular products, that it would be advisable to obtain some 
 
 21   amount of clinical screening, presumably the types of PCR we

 22   have been talking about in particular, as a database prior 
 
 23   to moving on.  Where the Agency is at the present time is 
 
 24   that we have asked sponsors to develop or access 
 
 25   appropriately sensitive assays of that form and consistent, 

 
        sgg                                                                274 
 
  1   I think, with your consensus advice, and to the extent that

  2   they have already treated patients and have available test 
 
  3   specimens stored, to test those specimens and report results 
 
  4   before proceeding.  We have not specifically asked or 
 
  5   decided that a sponsor who perhaps hasn't treated anyone or 
 
  6   has treated maybe 5 or 10 patients with a liver device, for

  7   example, we have not suggested that they would have to wait 
 
  8   until some other sponsor who maybe has treated 100 patients 
 
  9   with neuronal cells would have data from those 100 patients.  
 
 10   We could ask for that, but that would obviously be a very 
 
 11   different step in the sense that it would be entirely out of

 12   the control of that investigator to wait for results that 
 
 13   only another investigator, who might in fact be a 
 
 14   competitor, would have the ability to generate.  So, I guess 
 
 15   I would like some feeling, after having said that, yes, a 
 
 16   sponsor should have a test and do the testing before

 17   proceeding, is there some need for the whole field to wait 
 
 18   for a certain amount of information?  And I don't mean to be 
 
 19   putting that down idea.  Or, if tomorrow somebody has 
 
 20   submitted to us a really good PCR they are doing and they 
 
 21   have tested their cells, you know, in co‑culture and they

 22   have protocol for how they are going to test the patients, 
 
 23   is it okay to say tomorrow go ahead and do some transplants? 
 
 24             DR. AUCHINCLOSS:  And here we are back at our 
 
 25   science.  Why are we back at our science?  I will tell you 

 
        sgg                                                                275 
 
  1   why I think as I listen to this.  Question 2 turned out to

  2   be easier than I thought it would be, and the reason is that 
 
  3   I think there is more sense of comfort as to where the stop 
 
  4   point is on xenotransplant trials than where the sense of 
 
  5   comfort is with where the safe start point is, and you are 
 
  6   back to asking us where the start point is.  

  7             DR. SIEGEL:  There is a difference between saying 
 
  8   that there is no evidence for something and there is 
 
  9   evidence that something isn't there.  So, it is one thing to 
 
 10   say, yes, it is okay as long as we don't know that it is 
 
 11   there but there is a difference between not looking and not

 12   having looked ‑‑ 
 
 13             DR. AUCHINCLOSS:  I understand.  Let me try my own 
 
 14   personal response.  We are not talking about 1000 people 
 
 15   going out and getting pig transplants over the course of the 
 
 16   next two months.  We are talking, in fact, about very small

 17   trials and I personally view it as okay to proceed, given 
 
 18   the small numbers, because basically I don't think we are 
 
 19   going to really feel comfortable about this answer until we 
 
 20   have done 1000 patients or maybe even 10,000 patients, all 
 
 21   the animal studies aside.  We really just need to do

 22   patients and follow them extremely closely.  So, I think 
 
 23   that we should be pushing ahead under the kind of guidance, 
 
 24   controls, supervision, careful testing, monitoring, informed 
 
 25   consent etc. that we have been talking about all day.  So, 

 
        sgg                                                                276 
 
  1   there is one response. 

  2             DR. ONIONS:  I mentioned earlier that I would like 
 
  3   to see the data that has just been collected now.  There is 
 
  4   going to be very little in the next few months, the data on 
 
  5   the patients who have been exposed, before going on, but I 
 
  6   would say that after that, providing that there is

  7   reassurance from those data, that I honestly don't believe 
 
  8   we are going to create a public health hazard from going 
 
  9   into patients on a limited step‑by‑step basis.  It is just 
 
 10   almost inconceivable, I think, that you would create a new 
 
 11   world pandemic, which is what we are all concerned about,

 12   from a limited number of trials that are closely monitored.  
 
 13   That is not going to happen.   
 
 14             So, I think we just take it in this step‑by‑step 
 
 15   process.  Let's get the data from these people who have been 
 
 16   exposed.  If that is comforting, let's go to the next stage

 17   on a limited basis.  I think it is a fairly logical 
 
 18   progression.  We may be trying to define our terms a little 
 
 19   bit too rigidly and getting caught up in it.  I think there 
 
 20   is a fairly obvious step‑by‑step progression ahead of us.   
 
 21             MS. MEYERS:  Do you think that the public is going

 22   to feel the way you do having watched TV over the last few 
 
 23   days, worried about the Hong Kong chicken flu? 
 
 24             DR. ONIONS:  I don't know, but the thing to 
 
 25   remember about the Hong Kong chicken flu is the fact that I 

 
        sgg                                                                277 
 
  1   think the biggest world pandemic that has killed more people

  2   is the 1918‑1919 flu.  You are not going to stop these 
 
  3   events.  What we can do here uniquely is that we can 
 
  4   actually monitor the patients.  We can actually advise the 
 
  5   patients about their behavior.  You can't control it, of 
 
  6   course, but you can have a high degree of moral pressure on

  7   people and you can select them.  I actually believe that you 
 
  8   are unlikely to create the conditions for disaster by 
 
  9   limited clinical trials.  I think the problem, funnily 
 
 10   enough, will emerge a little bit later on, that is, when the 
 
 11   numbers start to go up because some of these problems may be

 12   quantitative.   
 
 13             MS. MEYERS:  But can you promise the public that 
 
 14   it will not start a major public health problem.  
 
 15             DR. ONIONS:  I personally could promise no such 
 
 16   thing. 

 17             MS. MEYERS:  No.  
 
 18             DR. ONIONS:  And never will be able to.  
 
 19             DR. SIEGEL:  I would like to note that while you 
 
 20   stated that this is the obvious thing to do, what you said 
 
 21   was very different from what Dr. Auchincloss said was a very

 22   reasonable and obvious thing to do in the sense that I 
 
 23   believe you said we should wait a few months until we have 
 
 24   the data from the current patients, and that sounds very 
 
 25   reasonable although there are folks out here who have told 

 
        sgg                                                                278 
 
  1   you that in those few months people will die from various

  2   diseases if they do not have access to treatment.  So, it is 
 
  3   a different decision from saying we will move ahead as we 
 
  4   accumulate the data.  
 
  5             DR. ONIONS:  The comment I should also have added 
 
  6   is that it does depend on the process.  I think, for

  7   instance, if we are using cells that are isolated cells or 
 
  8   in some form of containment, I think those issues are very 
 
  9   different from a whole organ transplant.  My comments were 
 
 10   actually directed at whole organ transplants.  I think there 
 
 11   are considerable differences in bridging transplants and I

 12   think there are considerable differences in isolated cell 
 
 13   systems that are in some form of containment. 
 
 14             DR. AUCHINCLOSS:  Is it fair to say, because I 
 
 15   think there are two kinds of clinical trials that are 
 
 16   probably closest to clinical application, that one involves

 17   barrier devices and the other involves central nervous 
 
 18   system placement, that those are the two kinds of 
 
 19   circumstances that you are perhaps most comfortable with? 
 
 20             DR. ONIONS:  Yes, I think so. 
 
 21             DR. AUCHINCLOSS:  Which is an important point

 22   which I wasn't personally willing to acknowledge in the 
 
 23   beginning.  I was sort of saying they are all the same and 
 
 24   this event is going to happen, but the experts are saying, 
 
 25   no, there are some that are safer than others.  The ones 

 
        sgg                                                                279 
 
  1   that you are looking at most practically in the near term

  2   are the safest ones.   
 
  3             DR. COFFIN:  I would also point out again that I 
 
  4   think attention should be paid to how one proceeds to the 
 
  5   nature of the patient population that is being treated.  I 
 
  6   think elderly patients, like Parkinson's disease patients,

  7   are at a much lower risk for spreading virus around than are 
 
  8   young patients from other groups, and I think that can be 
 
  9   taken into consideration as to how one proceeds and what 
 
 10   sorts of levels of concerns one has.   
 
 11             DR. AUCHINCLOSS:  Other comments from the

 12   committee on this issue?  They are sort of asking what they 
 
 13   should do tomorrow ‑‑ not sort of, they are asking.   
 
 14             DR. SIEGEL:  Those aren't the only protocols ‑‑ 
 
 15             DR. AUCHINCLOSS:  And I hesitated to make that 
 
 16   statement because I don't know what protocols you have and I

 17   don't mean to imply those are the only ones.   
 
 18             DR. SIEGEL:  And there are, for example, cellular 
 
 19   therapies that are implanted intravenously or otherwise in 
 
 20   ways ‑‑ some encapsulated and others not.  So there is a 
 
 21   spectrum of things out there.  But, you know, you have

 22   spoken to guidances and considerations that lead to higher 
 
 23   or lower risk and one of our questions was should we take 
 
 24   those things into account.  You know, if that represents 
 
 25   advice that is helpful.  

 
        sgg                                                                280 
 
  1             DR. ALLAN:  I think when you talk about bridging

  2   you have to be careful too.  If you are talking about doing 
 
  3   ex vivo or transgenic livers or something like that, where 
 
  4   you can actually get seeded with lymphocytes or porcine 
 
  5   lymphocytes or some other species lymphocytes, then that has 
 
  6   a higher risk than a barrier filter device or even the fetal

  7   cells in the brain.  It is different than if you are taking 
 
  8   an adult organ and you can get lymphocytes from those pigs 
 
  9   into the human body.  I think that is a much higher risk.  
 
 10   So, there are some differences.  
 
 11             DR. AUCHINCLOSS:  Question 2, any other comments

 12   from the committee?  Clarifications or questions from the 
 
 13   FDA?  
 
 14             [No response] 
 
 15             Basically what Jay Siegel said to me was keep 
 
 16   going until they drop ‑‑

 17             [Laughter] 
 
 18             DR. SIEGEL:  Until they have to leave.  
 
 19             DR. AUCHINCLOSS:  So I am going to push on to 
 
 20   question 3, but I do understand that if there are some 
 
 21   people who have things they have to do, like catch flights

 22   that they just have to catch, that some members of the 
 
 23   committee may not be able to stay around.  But his comment 
 
 24   was as long as you have a group here that can keep on making 
 
 25   intelligent comments or semi‑intelligent comments keep them 

 
        sgg                                                                281 
 
  1   talking to us.  

  2             DR. COFFIN:  Please let us know when the comments 
 
  3   stop being semi‑intelligent! 
 
  4             DR. VANDERPOOL:  Could I get us started on the 
 
  5   discussion of question 3?  I think the FDA should very 
 
  6   seriously consider setting forth a set of national

  7   guidelines for whole organ xenotransplant research.  I truly 
 
  8   appreciate the appeal of the Public Health Service in 
 
  9   section 2.5 of the guidelines that the standard procedures 
 
 10   involving the ethical principles in the federal guidelines 
 
 11   and the Belmont report ought to be honored.  Then, in

 12   addition to those, there were explicit guidelines regarding 
 
 13   the risk of infectious disease.  Question 3, as posed, deals 
 
 14   primarily with risk of infectious disease. 
 
 15             But before we focus on that, I want to make a plea 
 
 16   for the inadequacy of present day understanding of adequate

 17   review for whole organ xenotransplants.  Start with harms 
 
 18   and benefits.  The research community is very unclear as to 
 
 19   when whole organ trials are go or not.  Is this one month 
 
 20   survival?  Is this six months survival?  Is this a year 
 
 21   survival?  With what types of quality of life and so on? 

 22   These are unarticulated determinations, different ones being 
 
 23   voiced by different authors at different times.   
 
 24             Secondly, on questions of informed consent, I do 
 
 25   think there are critical questions dealing with informed 

 
        sgg                                                                282 
 
  1   consent regarding infectious disease.  But I think if there

  2   is ever an example of clinical trials that will exceed the 
 
  3   complexity of Phase I cancer research, which has been 
 
  4   discussed and aired year after year, it is these trials.  
 
  5   Informed consent will involve a host of things from the 
 
  6   effect of immunosuppressive drugs to how one is supposed to

  7   educate one's close contacts, to whether or not I understand 
 
  8   what it will mean to go through a life‑long surveillance, to 
 
  9   what I understand about infectious disease. 
 
 10             So, to me, these issues of harm‑benefit analysis 
 
 11   and informed consent are complex, and I don't think it is

 12   adequate to say, well, let's give the standard IRB review 
 
 13   but have the Public Health Service guidelines in your 
 
 14   pocket.  I think more is involved than that, and I appeal to 
 
 15   you to think about developing national guidelines.  Whether 
 
 16   they are required or not is a question for probably vigorous

 17   debate, but certainly recommended guidelines.   
 
 18             The third issue that is not here but I think is 
 
 19   very important was one that Dr. Walters mentioned from our 
 
 20   committee, and that is questions of informed consent for 
 
 21   close contact.  I think there are a lot of arguments to say

 22   there should be informed consent for close contact because 
 
 23   they will, in fact, be involved with many of the risks and 
 
 24   also be involved from time to time in the recipients' 
 
 25   process of coping with the trial, the transplant and so on.  

 
        sgg                                                                283 
 
  1   Where informed consent should also be required of health

  2   care workers is, as our committee discovered today at noon, 
 
  3   debatable.  Is this an ordinary risk that health care 
 
  4   workers will endure, or is this something much more?   
 
  5             I just want to mention one other thing, and this 
 
  6   is where the transplant survivors, Tony and Bill, can speak

  7   with far greater eloquence than I, but I think the question 
 
  8   of the process of informed consent needs to be very 
 
  9   seriously considered, and I would hope that protocols would 
 
 10   not just give an informed consent document, two 
 
 11   unfortunately poor examples of which we have in our

 12   readings, but would also see themselves the need to describe 
 
 13   the process of consent.  
 
 14             Now, that raises a final issue ‑‑ and I am just 
 
 15   giving the grid, and that is should you also consider who 
 
 16   else should be on an IRB besides infectious disease persons

 17   for xenotransplants?  Should a psychological counselor be a 
 
 18   required member of an IRB?  Should former transplant 
 
 19   patients be either regular consults or perhaps on the IRB?  
 
 20             Those are some of the questions I raise.  I don't 
 
 21   want to derail us from dealing with our specific infectious

 22   disease questions but I want to give that grid so we will 
 
 23   think in broader categories also.   
 
 24             MR. LAWRENCE:  If I could add, I agree with what 
 
 25   Dr. Vanderpool has stated.  I give a talk about liability 

 
        sgg                                                                284 
 
  1   issues in transplantation and one of the examples that I

  2   give is a comment an old law professor of mine used, that in 
 
  3   the medical setting determined informed consent reminds him 
 
  4   of the Holy Roman Empire which was neither holy nor Roman 
 
  5   nor an empire.  Informed consent is very often neither 
 
  6   informed nor consent.  What he stressed was that informed

  7   consent is not manifested by a signed document that says "I 
 
  8   consent."  It is a process of communication and 
 
  9   understanding.  If the consenting individual does not 
 
 10   clearly understand what has been said to him, the fact that 
 
 11   it was said to him is irrelevant.  And that is going to be a

 12   challenge to everybody involved here, I assure you.  My wife 
 
 13   is a registered nurse and she scarcely understood what was 
 
 14   being presented when I was in a hepatic coma and she was 
 
 15   having to consent ‑‑ she was consenting to everything, you 
 
 16   know, just "where do I sign?"  So we will need to be very

 17   careful with the consent process here, and I can't add much 
 
 18   more than that except that it is going to be quite 
 
 19   important.  
 
 20             DR. AUCHINCLOSS:  You bring up a point which is 
 
 21   going to be a peculiar problem in these trials, which we

 22   didn't kind of tackle head on, that you have this enormously 
 
 23   complicated consent process that we envision, and relatives 
 
 24   and offspring etc., and one of the treatments is for 
 
 25   patients in hepatic coma.  What are we going to do? 

 
        sgg                                                                285 
 
  1             MR. LAWRENCE:  Well, this is much easier legally

  2   than it is perhaps from the clinical standpoint.  Legally it 
 
  3   is well established what you do.  The persons who are 
 
  4   eligible to consent for the disabled person are well 
 
  5   understood ‑‑ spouses, parents and so forth, and there is a 
 
  6   hierarchy of those.  Obviously, those continue to apply

  7   here.  I am not sure that that is as troubling as it is to 
 
  8   be sure that you have communicated with this person, or the 
 
  9   patient himself if he is able to do that, exactly what is 
 
 10   involved and it is going to be very challenging.   
 
 11             DR. AUCHINCLOSS:  But you have indicated that your

 12   wife, a sophisticated, knowledgeable person ‑‑ you were in a 
 
 13   hepatic coma ‑‑ really fundamentally didn't understand or 
 
 14   didn't hear probably 90% of the issues involved in informed 
 
 15   consent for liver transplantation, and now we are suggesting 
 
 16   that she is instead going to hear not only about that but

 17   about something ten‑fold more complicated that we have spent 
 
 18   our whole life doing and not being sure we understand it 
 
 19   ourselves.   
 
 20             I am suggesting the stark way of asking the 
 
 21   question is are such people, in fact, not candidates for

 22   xenotransplantation trials because you just simply cant get 
 
 23   the kind of informed consent that you think is important? 
 
 24             MR. LAWRENCE:  No, I would reject that.  I 
 
 25   understand your concern but I have to reject that because 

 
        sgg                                                                286 
 
  1   the alternative is that you let them die because you don't

  2   want to get involved in a complex kind of a consent.  I 
 
  3   think all the law requires is that you do the best  you can, 
 
  4   but you must do the best you can.  It must be a good faith 
 
  5   effort and "good faith" is the legal operative word there.  
 
  6   But go ahead and do it; I mean, act in good faith but do it.

  7 
 
  8             DR. AUCHINCLOSS:  I am not saying all of this 
 
  9   because I believe it; I am saying this because I think it is 
 
 10   the question the FDA wants to hear the answer to.  But it 
 
 11   does put you in the position of saying this informed consent

 12   for xenotransplantation is so important and here is what you 
 
 13   are going to do, and you are going to have these educational 
 
 14   meetings, and then in the end you really say but, of course, 
 
 15   if the patient's life is at stake we can still dispense with 
 
 16   all of that and just move ahead and do the best we can. 

 17             DR. VANDERPOOL:  I think that is an excellent 
 
 18   question because we know what proxy consent is, or at least 
 
 19   we understand it in the clinical situations, but in terms of 
 
 20   medical research on human subjects, do we have a handle on 
 
 21   what proxy consent would involve?  And that is another issue

 22   to address.  
 
 23             MS. MEYERS:  Well, the federal government just 
 
 24   came out with regulations on emergency room types of consent 
 
 25   where a patient is unconscious.   

 
        sgg                                                                287 
 
  1             DR. SIEGEL:  That is different.  That is not

  2   pertinent.  That is when the legally acceptable 
 
  3   representative is not there and not available to consent.  
 
  4   So, this is a different circumstance.  
 
  5             I would like to explore the issue of consent of 
 
  6   the close contacts because you didn't say informing the

  7   close contacts; you said informed consent.  Now, consent 
 
  8   usually means in this setting that if you don't have consent 
 
  9   you don't proceed.  So, are you, in fact, saying, when you 
 
 10   say you should have informed consent of the contacts, that 
 
 11   family contacts of a prospective subject should have veto

 12   power over enrollment of that subject into a trial?  
 
 13             DR. VANDERPOOL:  No, what we are talking about in 
 
 14   informed consent for close contacts is informed consent 
 
 15   about what they will be expected to do and what they may be 
 
 16   required to do should X, Y and Z happen.  It is not informed

 17   consent in a proxy type situation; it is what is my role?  
 
 18   Surely, you know, all transplant trials will not have the 
 
 19   close contacts playing active roles that they need to 
 
 20   understand and consent to.  See, I have a problem on the 
 
 21   surface, although I could be convinced otherwise, that an

 22   educational requirement for the close contacts will do 
 
 23   because let's assume that some untoward event would happen 
 
 24   to a close contact and the health care worker would say, 
 
 25   well, we educated you; we told you about that.  Well, you 

 
        sgg                                                                288 
 
  1   have no indication that you talked with the person about it

  2   and that he or she actually gave consent.  So, I am not 
 
  3   saying we ought to move to that but I think we need to think 
 
  4   seriously about why we should or should not have informed 
 
  5   consent for close contacts.  
 
  6             DR. SIEGEL:  And you mean consent in the sense

  7   that they would have the right to withhold it and then you 
 
  8   wouldn't treat? 
 
  9             DR. VANDERPOOL:  They would have a right to say I 
 
 10   want to be involved in those ways or I won't take that role; 
 
 11   I will not support him or her.  But we can't just assume a

 12   Norman Rockwell via the family.  
 
 13             DR. SIEGEL:  So, blood testing ‑‑ 
 
 14             DR. VANDERPOOL:  Right, figure out what you have 
 
 15   to do to take the support roles that otherwise this person 
 
 16   would want to play. 

 17             MR. BENEDI:  Right now, in the regular transplant 
 
 18   scenario, there are questions that are asked the family, and 
 
 19   there are considerations by the surgeons as to what kind of 
 
 20   support mechanism exists for a patient to continue on a 
 
 21   long‑term basis or taking medications for life.  Those

 22   decisions are being made already in the regular transplant 
 
 23   community.  I think this extends it a little bit more 
 
 24   because it deals with that extended family person actually 
 
 25   participating in the testing.  So it is an extension of it 

 
        sgg                                                                289 
 
  1   but it does happen, and there are programs that have decided

  2   in the past that a particular potential recipient might 
 
  3   receive an organ because they don't have the support 
 
  4   mechanism, and that has been done and those people have 
 
  5   died.   
 
  6             DR. KASLOW:  I think this is a little different

  7   though.  If we are concentrating on the whole issue of 
 
  8   infectiousness and transmissibility there is more than just 
 
  9   the sort of passive involvement of the family and other 
 
 10   close contacts.  There is the potential risk not only to 
 
 11   that contact with actual disease if we think it is a

 12   pathogen, and also to the spread beyond the family of this 
 
 13   agent if it is, indeed, transmissible.  So, I think it is 
 
 14   qualitatively a little different from just more of the same 
 
 15   numbers of people and support.  And that is the issue I 
 
 16   think we have to deal with.  

 17             MR. BENEDI:  I guess my point is that there are 
 
 18   things that are different in this situation, and to take the 
 
 19   entire scenario of the individual, the family, those that 
 
 20   come in close contact and to have them also be informed and 
 
 21   give consent to the process in these early stages, when a

 22   lot of things aren't known about it, is absolutely 
 
 23   essential.  I mean, to say that there are four family 
 
 24   members in the house and three of them aren't going to 
 
 25   participate is irresponsible to take that recipient and 

 
        sgg                                                                290 
 
  1   introduce them when we don't know long‑term what is going to

  2   happen to that person.  Until we do know we are going to 
 
  3   have to be a little more cautious in what that support 
 
  4   mechanism and that family consent situation is.  
 
  5             DR. KASLOW:  Take the example that Abbie Meyers 
 
  6   gave, the powerful example, "the greatest guilt I feel is

  7   will I pass this infection onto my loved one."  I think 
 
  8   informed consent for the close contacts would alleviate some 
 
  9   of that.  That is, they would have been told whatever 
 
 10   worries, infections, risks and would have said "I'm willing 
 
 11   to take the chance" or they would not have been involved in

 12   that way.   
 
 13             Now, I am just thinking now from the top of my 
 
 14   head about some of the reasons ‑‑ not entirely from the top 
 
 15   of my head but on that point as to why informed consent 
 
 16   might be necessary for close contacts.  There might well be

 17   arguments that Tony has given from historic precedent to 
 
 18   show that it is not needed.  My point would be that we need 
 
 19   to think this one through beyond where we have gone at the 
 
 20   present time.  The IOM committee had one line on that.  We 
 
 21   need to think about this issue some, but I have never seen

 22   it really aired and discussed.  
 
 23             DR. MICHAELS:  I certainly agree fully with 
 
 24   everything that the others have been saying so far, and just 
 
 25   wanted to go a little bit further with it.  Certainly, with 

 
        sgg                                                                291 
 
  1   allotransplantation we do look at the family structure and

  2   support systems, and even the individual's ability to comply 
 
  3   and be able to take their immunosuppressive medication and 
 
  4   be able to come to follow‑up visits.  Now, it is easier to 
 
  5   do when you have the patient coming for evaluation as 
 
  6   opposed to the critical point when they are in hepatic coma. 

  7   But taking that type of patient who is able to come for 
 
  8   evaluation ‑‑ I guess I hadn't thought of it in terms of the 
 
  9   contacts actually having informed consent but that is 
 
 10   certainly very intriguing and worth considering.  I 
 
 11   certainly feel that education of the family members is

 12   critical, and certainly just taking some examples of what 
 
 13   has happened in the past, the patient who received the 
 
 14   baboon bone marrow, we did go through many educational 
 
 15   cycles with the person to let them know the unknowns, the 
 
 16   concerns for transmission if the unknown became a real true

 17   event of transmission to himself and, therefore, could it be 
 
 18   transmitted to a partner, then had the partner come in as 
 
 19   well and have more education with the partner, and 
 
 20   discussed, you know, things which I think Claudia has 
 
 21   already mentioned about using barriers with intimate

 22   relationships, avoiding transmission of secretions by 
 
 23   certainly not ever donating blood.  That patient, obviously, 
 
 24   had HIV and so those things were sort of a moot point to 
 
 25   start with.  But I think that that is reasonable even for 

 
        sgg                                                                292 
 
  1   patients who are not infected with a known virus to start

  2   with.  At this point of experimental research it is 
 
  3   reasonable for the partners and for the individual to use 
 
  4   barriers and to say that they will not choose to get 
 
  5   pregnant during that time or to conceive.  I think this is a 
 
  6   very experimental stage and I think that those are

  7   reasonable things to request and to discuss.  
 
  8             DR. NOGUCHI:  If I could just also say from the 
 
  9   FDA's point of view, having patients here, right here 
 
 10   talking about consent and the informed process is precisely 
 
 11   what we encourage very much.  There is this other part that

 12   you need to consider from our point of view, the one thing 
 
 13   about animal organs that is missing for human organs is that 
 
 14   they are not going to be scarce.  They are not going to be a 
 
 15   national treasure.  The whole process of being able to 
 
 16   actually take this on will need family support and community

 17   support, but the preciousness of the actual commodity, in a 
 
 18   way, takes away the ability of a physician surgeon to really 
 
 19   say, well, I know this family is not going to do it so I am 
 
 20   not going to give that person a liver.  You will not have 
 
 21   that option.  So, I think to the extent that this sounds

 22   important, it sounds even more important than the obvious 
 
 23   infectious disease question.  
 
 24             DR. AUCHINCLOSS:  I must say I don't quite buy 
 
 25   into that.  I think doctors in lots of ways continue to 

 
        sgg                                                                293 
 
  1   exercise those judgments about major procedures whether the

  2   resources are scarce or not, and I suspect they will 
 
  3   continue to do that in the future.  
 
  4             Let's bring this to a head here.  Is there, in 
 
  5   fact, a strong feeling in this group that informed consent 
 
  6   of close contacts of the recipient is a reasonable

  7   requirement in xenotransplantation trials? 
 
  8             [Several members respond "yes."] 
 
  9             DR. MICHAELS:  Yes, but I would be very surprised 
 
 10   if you were to develop a fully‑blown informed consent 
 
 11   document.  I certainly believe in the counseling and cycles

 12   of education where it is sort of documented what you are 
 
 13   trying to communicate to people but I am not sure ‑‑ that is 
 
 14   a new form of document.  
 
 15             DR. AUCHINCLOSS:  I want to know whether I am 
 
 16   hearing informed consent, signing the document.  Is that

 17   what this group is really suggesting they think is an 
 
 18   important part in xenotransplantation? 
 
 19             DR. VANDERPOOL:  I have been talking a lot about 
 
 20   it but I am not ready to move to that yet.  I am asking for 
 
 21   deliberation of the issues to get a good readout of the

 22   situation first.  
 
 23             MR. LAWRENCE:  I have one more comment about this.  
 
 24   Wearing my hat now as an official of UNOS, I am continually 
 
 25   surprised.  It is a daily event in this country that people 

 
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  1   are offered a human organ, after waiting on the transplant

  2   organ list sometimes for years, and they refuse it.  They 
 
  3   turn it down; they don't want it.  Transplantation is not 
 
  4   for everyone; xenotransplantation is not for everyone 
 
  5   either, and there will be times when the consent process 
 
  6   becomes so convoluted that you say this is not an

  7   appropriate therapy for you, and that is okay.  
 
  8             MR. BENEDI:  Could I just say something?  This 
 
  9   informed consent by family members as well is going to be 
 
 10   important in the long‑run when this new science takes hold.  
 
 11   In this information age there is a lot of information that

 12   travels throughout all kinds of different ways about each 
 
 13   and every one of us ‑‑ our medical histories and such.  I am 
 
 14   concerned about the insurance companies even looking at 
 
 15   someone who had lived with someone who had a xenotransplant 
 
 16   in the future, and whether they are at risk or too high of a

 17   risk to insure.  The portability insurance for us, 
 
 18   recipients, has been an incredible challenge that we now 
 
 19   have some laws to back us up.  But to go from job to job, we 
 
 20   couldn't do that without insurance companies closing the 
 
 21   door and saying that they are not going to insure us.  This

 22   is going to extend that window and that concern to family 
 
 23   members and to others that we come in contact with.  It is 
 
 24   just something to think about.  
 
 25             DR. WALTERS:  When Dr. Siegel uses the word 

 
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  1   "veto," that really challenges everyone's notion of autonomy

  2   and something very important that we stand for.  On the 
 
  3   other hand, this is clinical research.  Clinical equipoise 
 
  4   still exists, and these are both patients and subjects 
 
  5   simultaneously, and I do think that we have to remember that 
 
  6   this is qualitatively different from the context of an

  7   emergency situation with liver transplants or kidney 
 
  8   transplants.  This is still research.   
 
  9             MS. MEYERS:  I would like to say that for the 
 
 10   spouse or for the significant other of the patient there is 
 
 11   a risk involved, and they really have the right to know what

 12   that risk is, and that is why they should get the informed 
 
 13   consent document.  
 
 14             DR. AUCHINCLOSS:  So, what I think I have heard 
 
 15   from the committee is that there is a strong sense of lots 
 
 16   of information to the close contact, and it is almost up to

 17   a document signed but then there is sort of a gap on the 
 
 18   committee that they feel pretty uncomfortable with that. 
 
 19             DR. SIEGEL:  Well, I heard Dr. Vanderpool suggest 
 
 20   that there probably needs to be some further discussion and 
 
 21   exploration of the historical precedents, legal and other

 22   aspects of this.  
 
 23             DR. AUCHINCLOSS:  That is exactly what I heard 
 
 24   also.  Again, FDA staff, tell me if I am wrong.  I think we 
 
 25   have covered most of what is in 3, but there is an issue 

 
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  1   that I haven't seen us address yet which is whether you

  2   could take into account your judgment about the suitability 
 
  3   of the recipient from an emotional, psychiatric, clinical 
 
  4   view.  To me, that is sort of a no‑brainer.  We do that all 
 
  5   the time, as I think Antonio has already indicated.  It is 
 
  6   part of the evaluation process for liver transplantation and

  7   it would be even more so for a trial of this sort.  I open 
 
  8   it for further comment.   
 
  9             DR. SIEGEL:  Let me clarify a specific question.  
 
 10   In the report from one of the subgroups there was a comment 
 
 11   about not discriminating socially, such as based on past

 12   history of substance abuse, but there are, especially as we 
 
 13   are talking about liver support, potential patient 
 
 14   populations who are actively involved in intravenous or 
 
 15   alcoholic substance abuse.  You could have a chronic 
 
 16   cirrhotic who is still drinking and goes into acute

 17   alcoholic hepatitis deterioration.  In particular, since 
 
 18   those people are not generally considered suitable 
 
 19   recipients of human organs, they are potentially a very 
 
 20   desirable population.  Nevertheless, this committee and 
 
 21   others experts have told us these transplants should go into

 22   people that you can count on for the rest of their lives, 
 
 23   who will tell all of their contacts, will come in for their 
 
 24   annual checkups and screening, give all the specimens, and 
 
 25   should we be making the determinations whether or not there 

 
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  1   are populations that we really can't count on to do the

  2   things necessary to protect the public health? 
 
  3             MR. LAWRENCE:  I think we have to make a 
 
  4   distinction between the clinical trial phase where it is 
 
  5   very important to be able to follow these people and, should 
 
  6   this ever evolve to where it becomes common therapy are two

  7   different issues, to me.   
 
  8             DR. AUCHINCLOSS:  Jay, tell us about some of your 
 
  9   other concerns within the context of question 3 that we 
 
 10   might not have touched on.  
 
 11             DR. SIEGEL:  Yes.  One thing that we started

 12   touching on but didn't is an issue, and a very real issue.  
 
 13   If we say that before we proceed we should have in place 
 
 14   certain types of testing, being ready to test patients and 
 
 15   so forth, and we have done that and you have seen the letter 
 
 16   that was sent out, then what we will hear, and what we have

 17   heard, is, yes, but this particular patient is going to die 
 
 18   if we don't treat him now.   
 
 19             Now, in many protocols we certainly have the 
 
 20   authority and the general practice to really apply our 
 
 21   ability to grant single patient protocols, sometimes called

 22   compassionate use although that is not an official term.  
 
 23   Congress has supported that in recent legislation.  We will 
 
 24   do that assuming that basically the patient is dying and 
 
 25   signs the consent and understands the risk, obviously, you 

 
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  1   know, has a different benefit‑risk strata than just

  2   conducting general research.  However, here we are balancing 
 
  3   the potential benefit not just as a risk to the patient but 
 
  4   a risk to the general population, which has led us to this 
 
  5   quandary.  Is the risk to the general population, which is 
 
  6   not acceptable for certain patients, more acceptable if an

  7   individual patient is in more dire straights? 
 
  8             DR. AUCHINCLOSS:  As we have heard, something like 
 
  9   ten people are dying a day.  I see no reason why there would 
 
 10   ever be a reason for compassionate use for xenotrans‑ 
 
 11   plantation. 

 12             DR. SIEGEL:  You wee no reason why there ever 
 
 13   would be?  
 
 14             DR. AUCHINCLOSS:  Would ever be, no.  This goes 
 
 15   ahead because you decided it goes ahead or it doesn't go 
 
 16   ahead but it doesn't not go ahead except for the one patient

 17   that you hear about who is in somebody's ICU.  There are 
 
 18   patients in everybody's ICU.  
 
 19             DR. KASLOW:  We have made a public health 
 
 20   announcement here I think, and I think we have said that the 
 
 21   process that we have recommended proceeds on that basis, and

 22   I don't think we are going to make an exception to that.  
 
 23             DR. AUCHINCLOSS:  Anybody else? 
 
 24             DR. VANDERPOOL:  Well, that is a tough one when 
 
 25   you are saying, you know, the patient is going to die unless 

 
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  1   we do a xenotransplant.  They will probably truly die with

  2   one. 
 
  3             [Laughter] 
 
  4             But that is all the more reason why I think one 
 
  5   needs a basic set of general guidelines, not particulars, 
 
  6   about health benefit thresholds before xenotransplants

  7   should proceed.  I mean, you can read about xenotransplants 
 
  8   in Hungary and they do it just to see if it will work more 
 
  9   than a few days.  So, if you have some kind of a judgment 
 
 10   about what a success entails, then I think you would have a 
 
 11   better reason to say no to the truly desperate patient who

 12   perhaps would want to try the longest of long shots.   
 
 13             You know, it is interesting ‑‑ and this is a tough 
 
 14   question because our federal guidelines and the Belmont 
 
 15   report could have gone in a different way than they did.  
 
 16   The federal guidelines and the Belmont report are fairly

 17   paternalistic.  We don't allow you to volunteer for research 
 
 18   unless certain harms and benefits are in balance.  We could 
 
 19   have gone the other way and said anybody who really wants to 
 
 20   go for it can go for it, with a wild and crazy chance; that 
 
 21   is your prerogative.  But I think we realize that this could

 22   sully all kinds of reputations and would lead to probably 
 
 23   more consternation than success.  But I think we need to 
 
 24   stay with the spirit of the guidelines and the Belmont 
 
 25   report that certain harms and benefits need to be in place 

 
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  1   before the long shots can enter in. 

  2             DR. WALTERS:  I just think that the clinical trial 
 
  3   ought to proceed.  There is a lot that is unknown.  There is 
 
  4   a lot of information that needs to be gained.  The time and 
 
  5   effort of the investigators is a very important and scarce 
 
  6   resource, and in some cases the non‑human organs may be a

  7   fairly scarce resource at this point.  
 
  8             MS. MEYERS:  I think that for a compassionate 
 
  9   program you are really talking about a drug or a biologic 
 
 10   where you have something in a bottle that you can ship off 
 
 11   to a doctor out in Iowa or the patient who doesn't live near

 12   a big institution, but here you have to have a surgeon; you 
 
 13   have to have the transplant team.  You know, you just can't 
 
 14   have it done wherever.  The local GP isn't going to give you 
 
 15   a transplant.   
 
 16             DR. SIEGEL:  But it is not a question of that. 

 17   The question is largely whether while companies are, as we 
 
 18   have asked and as I think you have supported, getting 
 
 19   together and developing these porcine endogenous retrovirus 
 
 20   assays to ‑‑ 
 
 21             DR. AUCHINCLOSS:  The answer is no.  

 22             DR. SIEGEL:  ‑‑ so they are presumably planning to 
 
 23   start in a month, three months, whatever, when they have the 
 
 24   assays ready so that they can gather the data as to whether 
 
 25   the patients are getting viremic.  Should they be treating 

 
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  1   patients who are in dire straights in the interim but not

  2   other patients ‑‑ 
 
  3             MS. MEYERS:  No.  
 
  4             DR. AUCHINCLOSS:  Anybody want to say yes?   
 
  5             [No response] 
 
  6             Are there points in (iii)?

  7             DR. SIEGEL:  Well, did we skip over (d)(ii)?   
 
  8             DR. AUCHINCLOSS:  I said we do this all the time 
 
  9   in transplantation and, of course, we would do it here.  We 
 
 10   would assess the likelihood of compliance.  We do it for 
 
 11   will they take their medicines; will they cooperate with the

 12   program.  So, of course, we would do it here.  There, in 
 
 13   fact, would be a higher bar that you would need to go over 
 
 14   to be part of a xenotransplant trial.  When a patient is in 
 
 15   encephalopathy, well, that is very hard and we don't do it 
 
 16   well.  We do have lots of surrogate markers that we look at,

 17   however, and they are not perfect; they are terrible but we 
 
 18   do the best we can.  So, there is a process for dealing with 
 
 19   this and it seems to me you would just say, yes, the process 
 
 20   exists and we will do the best we can and it is appropriate.  
 
 21

 22             DR. WALTERS:  There is a history on this topic, 
 
 23   however, that goes all the way back to 1962 and the Seattle 
 
 24   committee selecting who would get access to dialysis when 
 
 25   access was very scarce.  One critic of the Seattle committee 

 
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  1   and the way it smuggled criteria of social worth into the

  2   selection of patients commented that the Pacific Northwest 
 
  3   would have been a very tough place for Henry David Thoreau 
 
  4   to get access to renal dialysis.  So, I guess just think of 
 
  5   Henry David Thoreau and whether he would be considered a 
 
  6   suitable candidate. 

  7             [Laughter] 
 
  8             DR. AUCHINCLOSS:  Jay, I have the feeling that my 
 
  9   committee is shrinking around me; that it is time to wrap 
 
 10   up.  One of the advantages of lasting longer than anybody 
 
 11   else is that you asked me to summarize and, believe me, this

 12   summary will be very brief.  But there is getting to be a 
 
 13   smaller and smaller number to disagree with me ‑‑ 
 
 14             [Laughter] 
 
 15             DR. SIEGEL:  Before you summarize, if we could 
 
 16   possibly take five or ten minutes perhaps to address the

 17   question that is before us now as it has been asked, and we 
 
 18   need to answer, and that is (e), which takes a whole page 
 
 19   but boils down to whether close contacts of xenograft 
 
 20   recipients, and this is closely related to something we were 
 
 21   just discussing regarding their informed consent, should be

 22   counseled not to donate body fluids such as blood, and 
 
 23   whether, in fact, they should be deferred from donation ‑‑ 
 
 24             DR. AUCHINCLOSS:  Again, I thought that was 
 
 25   obvious.  They should be counseled and deferred.   

 
        sgg                                                                303 
 
  1             DR. SIEGEL:  Counseled and deferred?  Okay. 

  2             DR. AUCHINCLOSS:  I assume that that was really 
 
  3   part of this information ‑‑ 
 
  4             DR. SIEGEL:  But it is another thing to tell all 
 
  5   the recipients that for the foreseeable future you need to 
 
  6   tell all your intimate contacts that they can no longer

  7   donate blood, for example.   
 
  8             DR. AUCHINCLOSS:  To put it in the overall context 
 
  9   here, the likelihood that we are going to create a public 
 
 10   health disaster with xenotransplantation I would take to be 
 
 11   one in a million, or something like that.  But this is the

 12   event that contact donating ‑‑ it is the transmission of 
 
 13   that one in a million event that you are trying to prevent.  
 
 14   I mean, this is the one thing that for sure you ought to do, 
 
 15   keep the contacts from spreading the new pathogen that we 
 
 16   create in that incredible unlikely event that we do. 

 17             DR. KASLOW:  The more difficult question is what 
 
 18   else should we tell them perhaps they shouldn't be doing 
 
 19   besides that.  
 
 20             [Laughter] 
 
 21             DR. VANDERPOOL:  You have to be monogamous from

 22   now on.  Until we give you the green light you have to be 
 
 23   monogamous.  I think that is the epidemic that we know about 
 
 24   has really spread, blood transfusion aside.   
 
 25             DR. AUCHINCLOSS:  So do you have your answer? 

 
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  1             DR. SIEGEL:  Actually, in the Center for Biologics 

  2   we regulate blood but, to date, we do not regulate sexual 
 
  3   intercourse! 
 
  4             [Laughter] 
 
  5             Summary Points by the Subcommittee Chair 
 
  6             DR. AUCHINCLOSS:  Now, under the prerogative of it

  7   is not over until you hear the Chairman singing, or 
 
  8   something, here is the Chairman's summary. 
 
  9             What I think I know is that there are replication 
 
 10   competent viruses in pig cells that are possibly, or even 
 
 11   very likely, going to infect human cells in the course of

 12   xenotransplantation; that we have absolutely no idea what 
 
 13   the consequences of that event will be in terms of 
 
 14   subsequent infectivity or pathogenicity; that somehow around 
 
 15   this table there is a sense that we ought to proceed with 
 
 16   clinical trials of xenotransplantation; that we would like

 17   to do so with the greatest degree of comfort that we can 
 
 18   muster.  We are not quite sure how really comfortable we can 
 
 19   every be; that the suggestion from the experts is that there 
 
 20   are things you can do to find safer forms of clinical trials 
 
 21   than other less safe forms, and you have heard the specific

 22   suggestions; and that there are assays and information that 
 
 23   still may, in fact, need to be accumulated to make us feel 
 
 24   as safe as we are likely to ever feel before these trials 
 
 25   proceed ahead.   

 
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  1             Is something like that the consensus of this

  2   group? 
 
  3             MS. MEYERS:  Very good.   
 
  4             DR. KASLOW:  For so late in the day you are 
 
  5   wonderfully articulate.  
 
  6             DR. AUCHINCLOSS:  Is there any other question that

  7   the FDA wants to ask?  I think I am supposed to ask for any 
 
  8   other public comment.  We are done on that?   
 
  9             DR. SIEGEL:  I want to thank you all.  I think we 
 
 10   have a lot to digest but a lot of very useful information 
 
 11   and advice, and we will look forward to further discussions. 

 12   As Dr. Auchincloss said, we intend this, whether it be a 
 
 13   subcommittee, committee or whatever, to be ongoing task of 
 
 14   public consultation and we really do appreciate your help.  
 
 15             DR. AUCHINCLOSS:  Those who have stayed to the 
 
 16   end, thank you very much. 

 17             [Whereupon, at 5:56 p.m. the Committee adjourned] 
 
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