DEPARTMENT OF HEALTH AND HUMAN SERVICES

PUBLIC HEALTH SERVICE

FOOD AND DRUG ADMINISTRATION

CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE

Twenty-eighth Meeting

Thursday, November 16, 2000

8:30 a.m.

Holiday Inn

Bethesda, Maryland

P A R T I C I P A N T S

Committee Members:

Daniel R. Salomon, M.D., Chairman

	Richard E. Champlin, M.D.
	Richard C. Mulligan, Ph.D.
	W. Michael O'Fallon, Ph.D.
	Edward A. Sausville, M.D., Ph.D.
	Esperanza B. Papadopoulous, M.D.
	W. French Anderson, M.D.
	Xandra O. Breakefield, Ph.D.
	Jeffrey S. Chamberlain, Ph.D.
	Jon W. Gordon, M.D., Ph.D.
	Carole B. Miller, M.D.
	Bruce E. Torbett, Ph.D.

Guests/Guest Speakers:

	Dale G. Ando, M.D.
	Katherine A. High, M.D.
	Donald Gay
	Deborah Hurst, M.D.
	John Levy
	Louis Zumstein, Ph.D.
	Richard J. Whitley, M.D.
	Amy Patterson, M.D.
	Estella Z. Jones, D.V.M.
	Mercedes Serabian, M.S.

FDA Participants:

	Kathryn C. Zoon, Ph.D.
	Philip D. Noguchi, M.D.
	Steven Bauer, Ph.D.
	Carolyn Wilson, Ph.D.
	Jay P. Siegel, M.D.
	Karen D. Weiss, M.D.
	Philippe Bishop, M.D.
	Anne Pilaro, Ph.D.

C O N T E N T S

Call to Order, Daniel Salomon, M.D., Chair	4

Conflict of Interest Statement, Gail Dapolito	7

Awards of Appreciation, Kathryn Zoon, Ph.D.	10
 and Jay Siegel, M.D.	11

Structural Characterization of Gene Transfer
 Vectors, Steven Bauer, Ph.D.	14

DNA Sequencing of Viral Gene Therapy Vectors
 During Preclinical and Clinical Development,
 Louis Zumstein, Ph.D.	27

Instability of Mini-Adenovirus Vectors, Jeffrey
 Chamberlain, Ph.D.	40

The CMV Promoter is Copied as "Extra DNA" from
 DNA Vaccine Plasmids, John Levy	65

Open Public Hearing, Dr. Janet Rose-Christianson
 and Dr. Russette Lyons	177

Preclinical Animal Models in Gene Transfer
 Research, Anne Pilaro, Ph.D.	183

Advantages and Disadvantages of the Use of
 Non-Human Primates, Estella Z. Jones, D.V.M.	202

Use of the Canine Model of Hemophilia,
 Katherine High, M.D.	218

Use of Aotus Monkey to Assess Neurovirulence of
 Replication-Selective Herpes Vectors,
 Richard Whitley, M.D.	238

P R O C E E D I N G S
	CHAIRMAN SALOMON:  Can we get started?  This morning, it is a meeting of the Biological Response Modifier Advisory Committee.  If you're here for another advisory committee, you're in the wrong room.  That is usually half the problem of being on the wrong airplane, I suppose.
	Just a couple of little minor things.  These are fancy new speaker microphones, but I think it is the same issue.  After you speak, you have to push the button so the light goes on and off.  Otherwise, what happens is two or three mics are on all at the same time and we drive this poor guy down there crazy, who seems to be on oxygen right now.  I have a number of pleasures this morning to get started.  One is to officially welcome Dr. Mulligan as a new member of the committee.
	I also have the pleasure of welcoming back three very distinguished past members, Dr. French Anderson, Abbey Meyers, and the former chair of the committee, Dr. Miller.  That was Julie.  Sorry.  Equally distinguished, who I fantasized was the chair.  Anyway, it is a pleasure to have them all back.  Thank you.  Lastly, I would like to welcome all our guest speakers, Dr. Dale Ando, Dr. Katharine High, Dr. John Levy, Dr. Louis Zumstein, Dr. Donald Gay, Dr. Deborah Hurst, and Dr. Richard Whitley, who I think really provide a critical input of important new information, and I appreciate that you could come here and join us today.
	What I wanted to do, we're trying to zoom along a little bit, because Kathy needs to get out of here.  But what I would like to do is just go around the table and introduce everybody, and just maybe give a sentence or two about what your area of interest is, because there are some new faces around the table.  If we could start at the end.
	DR. GORDON:  I shouldn't say testing, one, two, three, but recount, one, two, three.  I'm John Gordon from Mt. Sinai School of Medicine.  I'm interested in germ line gene insertion.
	DR. CHAMBERLAIN:  Jeff Chamberlain from the University of Michigan.  I work on gene therapy for muscular dystrophy.
	DR. TORBETT:  I'm Bruce Torbett from the Scripps Institute and we work on gene delivery to hemopoietic stem cells in animal models.
	DR. BREAKEFIELD:  I'm Xandra Breakefield.  I work at Massachusetts General Hospital and I use herpes vectors for gene delivery to the nervous system.
	DR. O'FALLON:  Mike O'Fallon, biostatistician at the Mayo Clinic, and I don't know anything about the election.
	DR. SAUSVILLE:  I'm Ed Sausville from the National Cancer Institute Developmental Therapeutics Program and we are interested in the preclinical valuation of drugs and biologics.
	DR. PAPADOPOULOS:  I'm Essie Papadopoulos.  I'm an attending on the allogeneic bone marrow transplantation service at Memorial Sloan-Kettering Cancer Center in New York, and my interest is in the area of allogeneic BMT for treatment of acute and chronic leukemias, as well as adoptive immunotherapy for relapse and viral infections.
	DR. CHAMPLIN:  Richard Champlin, blood marrow transplantation from the M.D. Anderson Cancer Center.
	CHAIRMAN SALOMON:  Dan Solomon, Scripps Research Institute, organ transplantation, xenotransplantation, tissue engineering.
	MS. DAPOLITO:  Gail Dapolito, CBER, executive secretary for the committee.  I would also like to introduce the committee management specialist, Ms. Roseanne Harvey, in the front row there.  Thank you.
	DR. MILLER:  Carole Miller, Johns Hopkins University School of Medicine, leukemia and bone marrow transplant, clinical.
	DR. ANDERSON:  I am French Anderson, interested in various aspects of gene therapy, specifically targeted retroviral gene therapy, in utero gene therapy and stem cell gene therapy.
	MS. MEYERS:  Abbey Meyers, president of the National Organization for Rare Disorders, and I was a former member of the RAC, and my interest is the informed consent document and patient protections.
	DR. MULLIGAN:  I'm Richard Mulligan from Harvard Medical School and I'm a gene transfer guy.
	DR. PATTERSON:  Amy Patterson, National Institutes of Health, Office of Biotechnology Activities, the office that supports the Recombinant DNA Advisory Committee.
	DR. ZOON:  Kathy Zoon.  I'm the director of the Center for Biologics.
	DR. WILSON:  Carolyn Wilson, member of Division of Cellular and Gene Therapies, FDA, CBER.
	DR. NOGUCHI:  Phil Noguchi, director of the Division of Cell and Gene Therapy.
	DR. SIEGEL:  Jay Siegel, director of Office of Therapeutics and Center for Biologics.
	CHAIRMAN SALOMON:  Thank you, everybody.  You got the button thing right, which is very good.  This bodes well for the rest of the day.  My next duty is to introduce Gail Dapolito to read the conflict of interest statement to initiate today's activities.
	MS. DAPOLITO:  Thank you, Dr. Salomon.  This announcement is made part of the public record at this meeting of the Biological Response Modifiers Advisory Committee on November 16th and 17th.  Pursuant to the authority granted under the committee charter, the director of FDA's Center for Biologics Evaluation and Research has appointed Doctors French Anderson, Xandra Breakefield, Jeffrey Chamberlain, John Gordon, Carole Miller, Bruce Torbett and Ms. Abbey Meyers as temporary voting members.  To determine if any conflicts of interest existed, the agency reviewed the submitted agenda and all financial interest reported by the meeting participants.  As a result of this review, the following disclosures are being made.  In accordance with 18 USC 208, Doctors Anderson, Breakefield, Chamberlain, Champlin, Mulligan Miller and Papadopoulos have been granted waivers which permit them to participate in the committee discussions.  Dr. Salomon, Sausville, Torbett and Ms. Meyers have associations with firms that could be affected by the committee discussions.
	However, in accordance with 18 USC 208 and 2635.502 of the standards of conduct, it has been determined that waivers or appearance determinations were not warranted for this meeting.  In regards to FDA's invited guests, the agency has determined that the services of these guests are essential.  The following interests are being made public to allow meeting participants to objectively evaluate any presentation and/or comments made by the guests.
	Dr. Dale Ando is employed by Cell Genesis, Inc.  Mr. Donald Gay is employed by Chiron Corp.  Dr. Katharine High has current financial interest in the topic.  Dr. Deborah Hurst is employed by Chiron.  Mr. John Levy is employed CTL Immunotherapies Corp.  Dr. Amy Patterson is employed by the National Institutes of Health Office of Biotechnology Activities.  Dr. Richard Whitley has current financial interest in the topic.  Dr. Louis Zumstein is employed by Introgen Therapeutics, Inc.  In the event that the discussions involve other products or firms not already on the agenda for which FDA's participants have a financial interest, the participants are aware of the need to exclude themselves from such involvement and their exclusion will be noted for the public record.
	Additionally, while some of today's meeting participants may belong to different professional societies and organizations, they were invited today to express their own individual ideas and opinions, not necessarily those of any organizations with which they may be affiliated.  With respect to all other meeting participants, we ask in the interest of fairness that you state your name, affiliation, and address any current financial involvement with any firm whose product you wish to comment upon.
	Copies of the waivers addressed in this announcement are available by written request under the Freedom of Information Act.  Just a couple of last administrative items.  As a courtesy to the committee discussions and to your neighbors in the audience, we ask that cell phones and pagers be set in the silent mode or turned off.  Please step into the foyer if you would like to use your cellphone.
	For the committee, there are slightly revised copies of the questions for committee discussion in the blue folders.  They are little bit revised from what has been posted on the Web and what you received earlier, and the hotel has asked for a little bit of patience in getting the temperature equilibrated in the room.
	Thanks.
	CHAIRMAN SALOMON:  The next item of business is actually both a pleasure and very important.  I just have to say that the FDA has a very strong sense of gratitude to retiring members who have really worked hard, and the people being honored today have--I just started on this committee when they were in full swing and all three of them have made just remarkable contributions to the committee and I really respect them.
	Kathy?
	DR. ZOON:  Thank you.  We're going to get our exercise running around the table today.  It really is a pleasure to be here this morning to honor two of our members who have been here and working with us so hard over the years, French Anderson and Carole Miller, and I just want to express the center's gratitude, plus my personal gratitude, for all your efforts and your contributions in public policy and just good public health contributions.
	I think the debt that the center owes you, not only for your participation in meetings such as this on very complex issues regarding gene therapies, cellular therapies, looking at bone marrow transplantation, whatever the topic, the contributions of these individuals have always been sound, have always been wise, and have always provided the balance and information for the agency to consider.
	They have also participated in helping with our science programs.  We have had our site visits and have done outstanding work in trying to help us improve the quality of our science at the FDA so we can do the very best job we can overseeing the products that we have.  We thank you for that.
	So, I would like to maybe ask Carole--if you could come up first--come on, Jay.  Jay wants to say thank you, too.  Carole, I just want to recognize you and thank you so much.  I hope you do not mind if we call on you periodically to help us out.  We would really appreciate it.  Thank you.
	DR. MILLER:  Thank you very much.
	DR. SIEGEL:  I just want to say, Carole, that I reviewed what we did over the last several years in this committee, and virtually every product we considered, Nupagen, Retexamab, intraleuken-11, the hematopoietic stem cell separators, you brought just some tremendous wisdom and help to us in dealing with them, and I want to express my great appreciation.
	DR. ZOON:  Next, French, can you come up?  French and I have known each other for probably longer than either of us ever want to admit, and we always appreciate--at the very beginning of gene therapy, French came and educated us on the science of gene therapy and he has been helping to educate us ever since.  Thank you, French, and I want to just thank you for everything, and be sure we will still call on you.
	DR. ANDERSON:  We should choreograph this better.  I also want to add our thanks, not just for what you've done on this committee, but for what you've done in the community, in helping them understand what this committee does, what the agency does, our role in this process.  It has been very important, and we will continue to hope that you continue in that role, as well as a consultant to us, and I know you have some regret, that we share, that your tenure here did not see gene therapy move into the area of approved medical products, but I think that great progress has been made and I think this is a fitting begin to symbolize, in fact, the importance of what happens here and of your contribution to it.  Thank you again.
	CHAIRMAN SALOMON:  I'm working on Whoopi Goldberg to emcee the next award ceremony.  I think you guys did great.  Billy Crystal, I kind of like Whoopi, but--then it is my pleasure to get on with the scientific and discussion part of this meeting, which is, of course, why we're all here.  The way we're going to begin session one is to basically deal with issues involved in the structural characterization of gene transfer vectors, and we will start with a series of discussions from the FDA and then from invited speakers, try and create some sort of scientific background for the discussions that will go on, and then, starting around 10:30, we will hope, we will begin the discussions with the committee--and again encourage active participation of the speakers, as well, who I consider really bringing a lot of expertise here.
	There is a little time for questions after each talk, but what I would like to do, and I hope no one will interpret this as being impolite, is that I do not want to start the discussion of the questions, so the questions after the individual talks, I would like to sort of keep toward maybe highlighting an issue or clarifying something that came up, and I encourage you to ask those kinds of questions, but if it looks like we're veering off into a discussion of what we want to do at 10:30, I might interrupt and just put it off.  I apologize in advance if I have to do that.  Anyway, I'm going to try and stay on time.
	The first speaker is Stephen Bauer, from CBER.  He spent part of yesterday trying to educate me on the construction of various different kinds of gene vectors, and I guess we'll find out how successful later today.
	DR. BAUER:  It is a pleasure to be here this morning.  My goal during this talk is to set the stage for our discussions and our subsequent presenters about how we go about structural characterization of gene transfer vectors and what kind of information is important to get out of those characterizations, where we are with our current recommendations, what kind of information has come in that is new and has stimulated us to think about changes in our recommendations about what kinds of gene therapy or vector characterizations ought to be done.
	The goal, of course, of structural characterization is to know that what you start with when you begin vector production is what you really think it is, and, of course, that at the end of the production process, that what you have is what you put into the system.  If I could have the next slide.
	To give you an idea of the complexity of this subject, this is a chart that shows our current active, as of a month ago, gene transfer INDs.  There is about 190 of them and the majority are with three different vector types, retrovirus, adenovirus and plasmid, but we're also getting into more, at least to us, exotic systems like poxvirus, herpesvirus, and, in terms of structure, these range on the order of three KB from plasmids up to as much as 300 KB for poxvirus.
	And the systems that are used to produce these viruses or these vectors are divergent and they each come with their own properties, but they are all biological systems and inherent in biological systems is variability, which is why at CBER we look at the process, as well as the end product, in characterization of these gene transfer vectors.  Some of these vector production systems are relatively straightforward, such as plasmid productions where you take a molecular clone of a plasmid, you put it into a bacteria, you let the bacteria propagate, you generate huge numbers of plasmids.
	Others are much more complex, such as those used for adenovirus, in which you use intermediate plasmids which contain parts of the vector that will end up being the final construct.  You transvect those into a cell line which mediates homologous recombination, which is a complex process, and then, using that which has been produced by the cell, you isolate a plaque and then propagate that.
	Using a cell system that has the capacity to mediate homologous recombination, you're also producing a product, so there is inherent variability in that sort of production system.  But common to all of these, as I said, is--the principles that we use are you want to know in great detail the structure of the vector seed that you start with, and I think we have the most control of that.  Then, at the end of the production process where you amplify to very high numbers of molecules, you want to know what you have produced at the end.  If I could have the next slide.
	The structural characterization, as I said, is to know that what you produced both at the construction phase of the vector and in the production phase, after you have made large numbers of molecules, that you actually get out the expected vector.  You also can use the structural characterization to show whether or not the particular vector and production cell pair that you have is capable of making a reliable product in terms of its fidelity and structure.
	Mostly the way that these kinds of characterizations are done are by restriction mapping, by PCR and by nucleotide sequencing, and each of these methods has advantages and disadvantages.  Restriction mapping is a good way to look at the overall structure, but it is low-resolution in terms of small changes.  Small changes can sneak by with that kind of analysis.  PCR, it really depends on how you developed your assay, what you're going to look for.  It is very sensitive, but again you're limited to knowing what you are looking for.  Sometimes you'll miss unexpected changes.  Your target sequences of the primers are well-known, but what's in between, you do not know, at least if the size of the product is the same.  Nucleotide sequencing we think of as the way to get the best answer for the complete structure of the vector.
	When you have a population of vector molecules that are homogeneous, of course you know exactly what the structure is after sequencing, but the inherent variability of some biological production systems means that if you sequence, what are you sequencing and how representative of the real population of molecules that you're looking at is that sequence?  If you sequence a clone, that is one clone.  If you do PCR sequencing, you are probably not going to pick up something that is there at 10 percent or lower, and that has to be a clone to see it.  If there is a variety of molecules in there, in the population you're looking at, you might miss things.  How important is that?  If I could have the next slide.
	The structural characterization has an impact on three very important areas, which are safety, efficacy and, again, production of your vector.  One of the primary issues related to structure of these viruses is whether or not replication-competent virus is produced.  That is something that we have very sensitive assays for some of our vector systems to detect, and I won't spend too much time on that subject today, but the other things that can change are vector backbone, the transgene itself can change, and our current recommendations have focused, at least early in product development, mostly on the transgene, the transcriptional control elements, and we have allowed product development to go forward with relatively less attention to the backbone.
	We will discuss later today some of the pitfalls that might come along with that approach.  But, if you alter the transgene, of course, you can easily imagine changing the safety of your product.  But if you alter the vector backbone, what kind of things can happen?  You can, for instance, introduce a new open reading frame that has a potential to make a protein that you didn't expect to be there.  We might see an example of that later today.
	Then, of course, you can also alter the efficacy of the product itself.  You can have transcriptional control that is a lot less than what you expected, so you have less product produced.  You can make a vector that just is not going to work as well as you had hoped.  Then, again, looking at structural characterization is a good way to look at the robustness of the production process.
	I am going to talk about a couple of examples of what we have seen under our current guidelines with the information coming in that is kind of stimulating us to think maybe we should change the way that we do some of this characterization.  If I could have the next slide.
	There are at least two sources of genetic alterations.  One is that what you start with isn't what you think it is, and this is surprisingly common in the world of molecular biology and vector construction.  People swap plasmids and you think you know what you are getting, but you don't really always know that what you have is what you think it is.  This illustrates the principle that, I think, for vector production, for what you're going to put into people, would like to have better characterization of the vector backbones that are being used.  The intermediates are not as well-characterized as we often assume.
	Then the other issue which is problematic and much more difficult to wrestle with is instability during vector manufacture, and, as I said, a lot of these systems are subject to inherent variability in which recombination rearrangements can occur during vector manufacture and also mutation as kind of a background rate of that, which we can't really do much about.  There can be improvements in this area by alterations in the vector and the packaging cell line that you use that will help minimize these, but I think this is just something that is inherent in the systems that we're using and it needs to be carefully thought about.
	Our job was always to encourage increased capability to detect these sorts of things.  Gene therapy seems to be--it's been around for 10 years but I think most of my colleagues and I look at it as a relatively new field still, and as we move towards treating patients who are younger, less seriously affected by disease, I think some of these things might become more important to look at.  If I could have the next slide.
	This is an illustration of this principle that you should know what you're starting with.  This represents a surprise that has cropped up during analysis, kind of creeped up on us there.  I just learned about how to do animation on PowerPoint, so you will have to forgive me.  But, at any rate, adenoviruses are constructed, at least some of them, using different intermediate plasmids which you transvect on the cell line and allow homologous recombination to occur.
	A relatively widely used vector system in which the E3 region has been thought to be deleted, it turns out that in the making of that particular shuttle plasmid, there was an insertion of unexpected sequence into that region of the shuttle plasmid.  If your characterization is focusing on this area of the gene, you can begin clinical trials without realizing that you have an altered sequence in the backbone of the vector.
	This gene does knock out the E3 region, but surprisingly has homologies to salmon, human and drosophila sequences, introduces an open reading frame which does not have homology to any known protein, and the question can you detect expression, you can see it by RT-PCR, and whether or not there is protein expression from that is still an open question, at least as far as I know right now.
	This illustrates the principle that, if you have not really completely characterized what you begin with, and then only later in the course of clinical development of a product you get around to complete characterization, you can be in for some surprises.
	The next topic I will talk about is, I think, the more difficult issue of alteration during production of a vector.  The idea is that if you have completely characterized a product, its intermediates, and you really have a good handle on the vector seed stock that you start with, you're going to put it into a production system and amplify tremendous amounts of molecules out of that.  If I could have the next slide.
	As I said, you can get during that process replication-competent viruses, and this has been observed with retrovirus and adenovirus production systems, but you can also get other kinds of mutations, and I think that is an area that we have relatively less of a handle on compared to our relatively sensitive assays for replication-competent virus.  If I could have the next slide.
	Here, if you start with a well-characterized product and you put it into your packaging or your expansion system, you get still the possibility that mutants will arise during this process.  For instance, in adenovirus, these are cell lines that will mediate homologous recombination and other kinds of recombination.  If this occurrence happens and generates a replication-competent virus, we have assays, at least for adenovirus, that detect on the order of one-in-10-to-the-ninth particles that are produced that are replication-competent.
	When we're talking about doses of 10-to-the-13th molecules, you could have 10,000 of these at that level of sensitivity administered to a patient.  Does that come along with any toxicities?  I think really we just don't know the answer to that, but I think it's important to think about.  But that also indicates that the system that we're using not only will generate replication-competent virus, but also other kinds of changes, and improve--what we would like to know from the committee and our discussions is what kinds of improvements of structural characterization could we make that would help us get a better handle on these other kinds of alterations during vector production?
	If this happens early in the production process, it is going to be relatively easy to detect, because it will be a higher proportion of the number of molecules.  If it happens late, it is going to be more difficult to detect and then how much should we really worry about that is an important issue.  If I could have the next slide, please.
	Our current recommendations that are in the guidance to industry are that during an early phase of development of product, you sequence appropriate portions of the vector or restriction-map and look at protein.  As I pointed out earlier, that has generally been taken to say let us look at the transgene and let's look at the transcription control regions and some of the flanking sequences, but it really hasn't meant that you can start in the clinic without knowing the entire--it means you can start in the clinic without knowing the entire structure.
	The kinds of information I talked about before, with relatively incompletely characterized starting materials and intermediates, has stimulated us to think about what would we like to do to improve this--the characterization of these products.  In discussions amongst our colleagues, we thought that perhaps the best approach would be to have, for vectors that are less than 40 KB, and this would include our three most commonly used virus classes or product classes, adenovirus, retrovirus, and plasmid, that there be a complete sequence of the vector and the intermediates before initiation of phase one.  This will be a point of discussion with the committee later.
	Then, for those vectors which are larger than 40 KB, which I discussed, poxvirus, herpesvirus, perhaps other types, that we ask for sequencing of the intermediates and focus on the introduced regions, so that would be anything that is changed during vector production, in the backbone or the transgene, and then flanking regions.  Exactly how much sequence is appropriate for a flanking region is also a question for discussion later this morning, and then that the sequence be completed before initiation of further trials in humans, expanded trials.  If I could have the next slide.
	I think the focus has mostly been on sequencing, and I think sequencing at the beginning of product characterization gives us the best confidence that we're starting with the material that we expect.  I just described this proposal, but then the other issue deals with this instability or the inherent variability in biological production systems, and should we do more to look at product lots?
	I have to point out that some of our sponsors sequence end-of-production vector, as well as at the beginning, so we have some focus just at the beginning.  In terms of looking at product lots, if you sequence, how representative is that sequence?  I think it is important to know what the structure is, but if you have very low-level impurities in your vector preparation, is sequencing going to give you that information?
	To my knowledge, nobody has sat down and sequenced 100 clones of a product lot to say well, this is the amount of variability that we have, and as I pointed out, it is a stochastic process, so one product lot is not necessarily going to be the same as the other, but we do have methods that will look at--to a lower resolution of product lot, such as PCR.  The limitation with that is that you're looking at what you're predicting, what you see is dependent on the target, the primers that you use, and then restriction mapping, that has its stimulations.
	But we would like to stimulate as much development in these areas as possible, so we know products we're developing are as characterized as possible.  Next slide, please.
	Another issue is what do you do with sequence that you have generated?  If you do sequence an entire vector, adenovirus or other vector, 40 KB or less, what should be done with that?  If you just submit to us a sequence without some analysis or sequence in a format that allows us to do analysis, that in itself is not informative.
	At a minimum, we would like some discussion of these proposals that there be comparison to expected sequence, that there be analysis for open reading frames.  That would be to look at this contingency that a mutation would introduce a new antigen into a vector backbone.  Finally, comparisons to nucleotide and protein databases that are easy access, and are publicly available.  This is an issue we would like some commentary this morning on.  If I could have the next slide.
	What do you do if you do find an unexpected sequence?  Say you're at the beginning of your process and one of your shuttle plasmids not the expected sequence.  You have invested nonetheless a lot of time and energy into producing that.  One could start over again or say what additional experiments should be done to examine the effects of those unexpected sequences, and you could do studies where you look further at characterizing the vector in its altered form or its unexpected form and, as I mentioned earlier, look for open reading frames, whether they express RNA or protein, for instance.
	You could decide preclinical studies to look at whether or not those particular changes really do have any effects, and you could add additional aspects or experiments in patient follow-up if there is sufficient rationale to go ahead with using that particular vector.  If I can have the next slide, please.
	To summarize, I have kind of gone over what our current recommendations are, and shown that, in that context, we have seen unexpected material crop up in some of our vectors.  And this has stimulated us to consider more rigorous structural characterization of products.  We have made some proposals that we would like some feedback from the committee today on, and I think that our next speakers will give you a greater level of detail and insight into this particular phenomenon.
	With that, I would like to thank the committee and the members of the audience and welcome any questions.
	CHAIRMAN SALOMON:  Thank you, Steve.  Are there any questions to enhance clarity of this very clear presentation?  Okay.  I think there is no doubt that that is going to be a guide for us later this morning.  We really will get back to those issues.  Thank you, Steve.
	The next speaker is Dr. Louis Zumstein, from Introgen Therapeutics, and he is going to talk about the identification and characterization of unexpected DNA found in an adenoviral vectors, and we really appreciate his willingness to come and share their experience in this issue with his vector construction.
	DR. ZUMSTEIN:  Thank you very much for the invitation.  I am Lou Zumstein.  I'm Director of Research at Introgen Therapeutics and I have been asked to give a case history, if you will, of our DNA sequencing efforts for our new gene therapy product.  In light of recent SEC regulations on fair disclosure, our lawyers insist that I show this slide and I would ask that you take note of the caution on forward-looking statements.
	The basic premise of this talk is very much in agreement with what Dr. Bauer just said, which is that by sequencing data, the predicted origins and sequence of a vector needs to be considered to be tentative.  The vector I am going to be talking about is RPRINGN201, less formally known as Ad5CMV-p53.  I will be calling it Ad-p53 in this presentation.
	We're developing this in cooperation with Aventis Pharmaceuticals for the treatment of cancer.  The current clinical status of this vector is that it is in Phase III trials for advanced head and neck cancer.  It is in Phase II trials for lung cancer.  The predicted and demonstrated mode of action is to over-express p-53 protein in cancer cells and to cause those cells to die by inducing apoptosis.
	A little bit more detailed background about how this vector is made and some general organization of the adenoviral vectors.  This part of the virus, the expression cassette encoding p-53 and some flanking sequences, originates from a shuttle vector.  The bulk of the rest of the virus originates from a plasmid called PJM-17.  The sequences in this plasmid trace back ultimately to an adenoviral variant called DL-309, and I will be going into that in a little more detail.
	This virus is E1-deleted.  E1 is essential for replication, so this is a replication and paired vector.  E2, E3, and E4 are other groups of early genes.  You should probably try to remember that the E3 region is not essential for viral replication.  The L. genes are late proteins, primarily structural proteins.  As is fairly common with gene therapeutics in development right now, Ad-p53 originated in academia and very shortly after the laboratories of Introgen were opened, Adp53 and the reagents used to make it came over from the laboratory of Dr. Jack Roth at the M.D. Anderson Cancer Center.  The documentation we got with these reagents were restriction maps, not sequence.
	I have shown you a map of the shuttle vector.  I have noted that PJM-17 was derived from the Ad5DL-309 adenovirus.  DL-309 was made in the laboratory of Tom Shank in the late 1970s.  They did some manipulations to get rid of several restriction sites in that virus, and clearly their intent at this time was not to make a reagents to go into the clinics.  This was a research reagent.  Also keep in mind that DL-309 was selected as a viable virus.  It grows as well as wild-type Ad5, so the changes that happened to get rid of these restriction sites do not seem to have been deleterious.
	At this stage, prior to clinical trials and before we started our sequencing program, we knew the historic building blocks of the virus.  We knew there had been changes to several restriction sites that did not seem to be deleterious.  And our program at this point was that, as the clinical program proceeded, as the number of patients treated got larger, as the trials got more important, our characterization of this virus was going to have to get more extensive and more sophisticated.
	At this point, we also had a predicted sequence of the virus, but that sequence was based on sequences in the Genbank databases, not actual sequencing.  The first sequencing we did was of a plasmid in which the P. 53 expression cassette had been cloned into.  We did this literally within two months of opening the laboratory, manual sequencing.  Again, this was prior to Phase I trials, and our conclusions from this was that the sequence of the expression cassette was as we had expected.
	The new information we got was that the polylinker sequences in between the large pieces were now defined, for instance, the pieces of DNA between the CMV promoter and the P. 53 open reading frame.  So, to give you a little more detail, this is the expression cassette with some flanking adenovirus sequences blown up down here.
	At this stage, we now knew the sequence of this region.  At this point in time, we also had a number of QC assays in place to identify this virus, to show that it made p53 both biologically active and it killed cancer cells.  The next piece of information we got on this virus was a paper from Bett, et al., in which they had sequenced a region in E3 where some of these restriction sites had been changed in the original DL-309 virus, and they noted that there was a two base pair deletion.  They got rid of an expocyte down here, six-base deletion right here, they got rid of a restriction site, and a slightly more complicated event here that was a deletion of about 700 bases of E3 sequence and the insertion of about 650 bases of DNA, and I will come back to that in more detail.
	The second piece of sequencing we did was GLP sequencing of the expression cassette and flanking sequences.  We took a lot of clinical material, isolated viral DNA from that, cloned the expression cassette out into a sequencing plasmid, had that sequence to GOP standards, and we also sequenced straight from the viral DNA from within the expression cassette out in the adenovirus flanks.
	Again, the expression cassette was as expected.  We now knew the sequence of the polylinkers between the expression cassette and adenoviral flanking sequences.  We also found out that about 300 bases of the left adenoviral flank, that is, the sequences just to the left of the expression cassette, had more similarity to Ad2 than to Ad5.  It is still very similar to Ad5 and in that region there are seven bases that agreed better with Ad2 than with Ad5.  That region of the adenovirus is not expected to code for any proteins.  The next piece of information we got on this virus was a paper by Gingras, et al., which sequenced the same region of DNA that I have been talking about in E3 and came up with a slightly different sequence than that of the Graham laboratory.  At this point, the exact sequence of that insertion deletion event was unclear.  We had restriction and PCR data of this region.  We had been working on it, but the data we had did not differentiate between the two sequences.
	The next step in our program to better characterize this virus as the clinical trials progress was to do full GLP sequencing of the entire virus prior to the initiation of pivotal trials.  Again, after we sequenced the whole virus, the expression cassette was as expected.  The E3 region sequence agreed with that of--from Cordova's laboratory, not from Frank Graham's lab, and aside from the E3 region, there were 23 discrepancies between the actual sequence of the virus and the sequences in the database.
	To go into these discrepancies in a little more detail, 10 of them are expected to be either in non-coding regions or silent, nine are predicted to change amino acids in either known or predicted open reading frames, and four are expected to change the size of open reading frames.  We believe that most, if not all, of these discrepancies are actually errors in the original Genbank sequence.  The Ad5 sequence in Genbank is not the result of a large, well-coordinated sequencing program.  It is a piecemeal compilation of sequence from a large number of laboratories.
	In addition, when we have gone in and looked, the more recent literature agrees with the actual sequence of Ad-p53, not with the old Genbank sequence.  Let me go into a little more detail about the changes that happened in E3.  The top part of this map is the E3 region from wild-type Ad5 and predicted open reading frames.  The bottom is the E3 region from Ad-p53 with predicted open reading frames.  There is a 6 base pair deletion and a 6.7K open reading frame.  It changes the expression level of this protein.  It changes the number of glycocylated forms that one finds.  As far as I know, the function of 6.7K is still unknown.
	This is the insertion deletion event down here.  646 bases are inserted, where about 700 bases are deleted.  The open reading frame for 14.5K is completely removed.  10.4K truncated.  18 amino acids are truncated off the end and you get a fusion into sequences within this insertion, and you get a novel open reading frame that starts within this insertion and reads out into Ad5 sequences.
	There's no protein similarity between this sequence and 14.7K because we're reading in a different frame here.  14.7K is not predicted to be made at all.  Let me give you a little more detail about these proteins and what they do.  6.7K, I have run you through.  10.4K, 14.5 and 14.7K are all involved in preventing TNF alpha mediacytolisis.  They are part of the mechanism Ad5 uses to avoid the host immune response.
	The other point to make is that there are 27 amino acids fused onto 10.4 K.  Neither those sequences, nor the novel open reading frame, have any significant similarity to known or predicted protein coding sequences in the databases.
	What I'm going to do now is blow up just this region, this insertion/deletion of that 646 basis.  That is down here.  This is the novel open reading frame, 10.4K region from the left here.  What do we actually know about this sequence?  If you do a homology search with the sequence against Genbank, the only statistically significant similarity I pull up is a 92 percent identity to some sequences in the salmon prolactin 2 gene.
	It is a 135 base pair region.  That is right here.  That region in the prolactin II gene is downstream of the open reading frame.  As far as I know, it is not predicted to encode any proteins.  From Gingras, et al., we know that this DNA insert hybridizes to salmon DNA and not to human DNA.  It is possible that the hybridization is just a result of this high homology region.  From studies at Introgen and from Gingras, et al., we know that RT-PCR detects RNA made from this region and northern blots do not.
	Northern blots are a fairly insensitive assay for RNA.  RT-PCR is a very sensitive assay. Our guess at this time is that the transcript that is detected is a low abundance one.  The only thing we don't know from RT-PCR is which strand is being transcribed here.
	To summarize, we have now sequenced the whole virus prior to the initiation of pivotal trials.  E1 deletion, p53 expression cassette--there was a two base pair deletion down in this part of the virus and a six base pair deletion here, but we also had a more complicated insertion/deletion of that, which resulted in some novel open reading frames.  I will stop there and take any questions.  Thank you.
	CHAIRMAN SALOMON:  Thank you very much, another very clear presentation.  For the non-gene vector experts in the audience, can you explain why all of a sudden there is salmon DNA and in a vector you're constructing?
	DR. ZUMSTEIN:  Sure.  First, I'm not entirely convinced it is salmon DNA.  There is a high degree of similarity between that DNA and salmon sequences.  It is not identical.  Beyond that, there is speculation in both Bett, et al. and Gingras, et al. that that DNA originated from the transvection process.
	When dl309 was made, they were trying to get rid of restriction sites, so they would cut the end of viral DNA, do a limited ligation and transvect that back in, basically looking for viruses that have lost some sites.  It was typical at the time that when you do transvection, you use salmon DNA as a carrier.  Whether they actually did that or not, I do not know.
	The hypothesis from Gingras, et al. and Bett, et al. is that that carrier DNA got incorporated in as a result of an illegitimate recombination of that, if you will.  Beyond that, I don't know where it came from.
	CHAIRMAN SALOMON:  Thank you.  I guess the point I wanted to make, and we'll get to it later, is that one of the things to consider is, sort of, guidelines during the construction of these sort of vectors, and too, what additional products are being added to them, much along the way that we have looked at it in cell-transplantation and tissue engineering and xeno-transplantation.
	All of a sudden, somebody adds fetal calf serum or a co-culture with the a xeno line or something like that.  The same thing, in a way, is happening in the construction of these vectors.  You decide you have extra salmon DNA in a carrier and it gets incorporated in a vector.
	DR. ZUMSTEIN:  Certainly, when these regions were originally made, there was not the anticipation that they would end up in the clinic.
	DR. MULLIGAN:  This is off the topic, but what do you do about the p53 sequencing?  One of the issues we will get into is, what amount of mutation of the coding sequence is actually going to be of issue?  That will, of course, depend upon on what the protein is.  I am kind of curious in the case of p53.  First, what is your thinking in terms of what is acceptable in terms of having mutant p53 in your virus?  And second, how did you go through the thinking of how to test for a one percent or a 10 percent mutation or something?
	DR. ZUMSTEIN:  Both of those questions are very good and very much out of my league.  I think I'm going to pass on those.  They seem to be topics, actually, for the committee.
	DR. MULLIGAN:  The answer is you have not, in your group, addressed this question?
	DR. ZUMSTEIN:  No, that is not the answer.  The answer is that it is personally out of my expertise.
	CHAIRMAN SALOMON:  I think one of the things that is--they agreed to come and present this as a case history, and what we definitely do not want to give anyone the impression of is that Introgen or any other product going into phase III is part of the discussion today.  I think it is very appropriate not to get into the specifics of that.  We will deal with everything in general.
	DR. ZUMSTEIN:  We have certainly been in detailed discussions with the FDA, and they know exactly what we're doing and what we have.
	DR. NOGUCHI:  The questions that you raised were also discussed with Dr. Roth's original constructs, and there was actually a fairly extensive discussion about mutations in p53 and how one would detect these low-level mutations.  As you correctly point out, p53 can mutate rather rapidly.  I am not sure that we really came to a total resolution of the issue.  I do know that that has been discussed both within FDA and publicly, at the rack, very extensively.  It was about six or seven years ago.
	CHAIRMAN SALOMON:  Dr. Breakefield and then Dr. Anderson.
	DR. BREAKEFIELD:  I just wondered if you have done any in vitro transcription translation just to see that in an in vitro system--if you can make a protein?
	DR. ZUMSTEIN:  No, we have not done that.  We have certainly been discussing whether and how to go looking, to see if a protein was made off of this, but we have not done in vitro transcription translations.  I think our first step would be to see which strand is being transcribed there, and those experiments are in progress.
	CHAIRMAN SALOMON:  That is a very good question. We will come back to that one, specifically, in question five of the discussion.  Dr. Anderson.
	DR. ANDERSON:  Just for purposes of historical accuracy, when this vector was built, it was planned to go into patients, not by Introgen, but in terms of the discussion of the next two days, this reflects the fact that academic investigators are not as versed in all of the issues having to do with clinical trials that a large pharma is.  But this vector, as built, was planned to go into patients.
	DR. ZUMSTEIN:  So, what I was saying was that ad5, dl309, when constructed in Tom Shank's laboratory--certainly was intended to go into the clinic?
	DR. ANDERSON:  You're absolutely right, yes.
	CHAIRMAN SALOMON:  Excellent.  Thank you very much.  The third speaker this morning is Jeffrey Chamberlain, University of Michigan Medical School.  And he's going to talk about the instability of mini-adenovirus vectors.
	DR. CHAMBERLAIN:  Well, I would like to give an overview of a slightly different type of adenoviral vector system, in order to raise some questions about how we may want to consider making recommendations in the characterization of the system.
	My laboratory, and a number of others, are interested in the development of so-called helper dependent adenoviral vectors, also known by various other names, such as gutted adenoviral vectors, or gutless adenoviral vectors.  This system is unique from a conventional adenoviral vector system in several ways, in that a so-called gutted or gutless adenoviral vector is the end product that is being sought.  This is a vector that does not contain any coding regions from the adenovirus itself.  However, these vectors, at least with current technology, cannot be grown except in the presence of a more conventional adenoviral vector that functions as a helper virus.  As a result, the system requires repetitive growth, where a particular packaging cell line is producing both the so-called gutted and the helper adenoviruses together.
	I think this raises some additional issues.  In particular, there is the potential in the system to encounter rearrangements, not so much by a conventional mechanism that you might see with a single adenoviral vector, but by homologous or non-homologous recombination between the two vectors that are present in replicating in the same cell line.  I would like to just give a little background on this system, and then point out some problems that we did encounter early on in the development of the system that may be instructive in terms of trying to figure out what types of requirements to impose on these types of vectors.
	So, just to give a little background on how this system is used--there are several different versions of it in use in a couple of different labs.  I think most laboratories today have a starting product of a plasmid-based vector, that contains small portions of the adenoviral genome, in particular, the left and the right inverted terminal repeat, as well as a packaging signal, and then a therapeutic gene expression cassette cloned into that plasmid vector.
	Certainly, in my laboratory, we do all of our experiments starting with super-coiled plasmid preps.  This is then co-transvected into a packaging cell line, together with one form of adenoviral DNA.  Again, different laboratories use different forms of the helper.  Some use co-infection, with a packaged adenovirus vector.  We prefer to use either cloned adenoviral helper DNA in the form of a plasmid, or a purified protease treated in fetal chloroform extracted helper viral genomic DNA sequence.
	In any case, once these are transvected into the packaging cell line, the helper virus produces all the adenoviral proteins needed in-trans for replication and packaging of your gutted vector, and these are both released from the cells and can be separated from each other in various methods.  There are genetic selection methods and there are also physical methods, usually involving cesium chloride gradient centrifugation.
	This system has some inherent inefficiencies, in that you're starting with a cloned version of these vectors, which typically do not replicate very well.  Laboratories that are using this type of a system generally have to do a number of serial passages in order to slowly increase the titre of their gutted adenoviral vector.  Here is an example from an early experiment we did, measuring titre by expression of a reporter gene.  You can see that we would typically have to go out five or six serial passages, where we're growing both the gutted and the helper virus together in the same cell lines, before we would achieve a high-titre stock of the gutted virus, which was the goal of these studies.
	Now, there has been a number of modifications to this system, where the number of serial passages can be reduced considerably, down to three or four even.  However, I am not aware of any system where a person has been able to do this by a simple co-transvection and immediately obtain a sufficient yield of a gutted virus to be useful without doing subsequent serial passages.
	There are a variety of tricks that different labs have introduced to try to speed up the rate of growth, and to select against the so-called helper virus in this system.  The most commonly used one is--takes advantage of Cre-lox mediated recombination.  In this case, this is a system originally developed in Frank Graham's lab--the packaging signal of the helper virus is flanked by inserted lox p-sites.  Then the adenovirus packaging cell that's used expresses the Cre recombinase. By co-transvecting your gutted virus DNA with your helper virus DNA, you can get Cre-mediated excision of the packaging signal of the helper virus.  This serves a couple of purposes.
	One, it tends to render the helper virus unpackageable by virtue of removal of that packaging sequence, yet the remainder of the helper virus sequence is still able to replicate and produce all the viral proteins you needed in-trans to replicate and package your gutted adenoviral vector.  As a result, when the system is working well, the lysates prepared from these cells are tremendously enriched for the so-called gutted adenoviral vector, although inevitably there are still small trace amounts of contamination by the helper virus.
	Typically, at the end of a preparation, one might want to do some further purification on cesium chloride.  I apologize, this was not a particularly good gradient, but it was a convenient one that I was able to throw into this presentation.
	Here is an example where one has a fair amount of residual contamination by the helper virus.  This is the gutted virus here, and they're different sizes, typically, so they're resolved by a difference in their buoyant density on cesium chloride gradients.  You can do multiple gradients, if you need to, to get fairly homogeneous preparations of your gutted virus.  One of the reasons I illustrate this is to point out a couple of things.
	One, even under the best of circumstances, there's generally at least .1 percent, if not higher, levels of the helper virus.  Secondly, particularly with better gradients than I have shown here, which are not that difficult to achieve, you can get a reasonable feel for what types of viral genomes may be present in your mixture by potentially observing the presence of additional or unexpected bands or bands that might be migrating at a buoyant density different than what one would predict from their particular vector.  That is a point I will come back to in a little bit.
	Let me talk a little bit to some of the issues that might be involved in analyzing a preparation of gutted adenoviral vectors.  As I mentioned, I think essentially all laboratories that are using this system start with a cloned plasmid vector, such that your entire gutted or helper dependent adenoviral vector has been cloned into an E. coli plasmid vector and, as a result, it is really a fairly straightforward--one has a straightforward ability to go in and restriction map and sequence completely the gutted vector genome.
	I think it is important that people also keep in mind the structure of their helper virus, since you're growing these two vectors together in the same cell and there is the potential for recombination or rearrangement, either between these two vectors or in one or the other of them.  It is important that you know the exact nature of your starting helper virus.  At least in the methods that we're using it my laboratory now--I mentioned that we use cloned helper virus DNAs where the entire helper virus is cloned into a plasmid.  And again, it is a fairly simple matter, although somewhat expensive, to go through and restriction map and completely sequence the helper virus, as well.
	I think an important issue in starting to characterize preparations of these viruses, is that you have very accurate titres of both the gutted and the helper adenovirus, so that you can make sure that what you are seeing is what you really have there.  I would suggest that before this technology is really going to be useful in the clinic, the efficiency of the growth of these vectors needs to be taken to the point where one can effectively generate a seed stock of the gutted adenoviral vector that will allow subsequent analysis of the initial preparation of the virus.  What I'm trying to do here is to contrast that with the starting material, which is a plasmid vector that is subsequently converted into a viral vector, and that viral seed stock should be subsequently verified, prior to doing the individual preps that would then go into some sort of clinical trial.
	The one question that we may want to address a little bit is at which individual stage is one going to require verification of the ultimate sequence?  I think it is a simple matter to make sure that the plasmid you're transvecting into your plasmid cell line are highly verified and sequenced.  When you get to the level of a seed stock, you're starting to deal with smaller and smaller quantities of these materials.  And one may ask is it important to sequence off of the seed stock also.
	I think it is a relatively simple matter to at least verify the density of these particles by doing cesium chloride gradients and to do some restriction analysis of the seed stock, but we may wish to consider whether it were the seed stock itself or, perhaps, a sample preparation from that seed stock should be further analyzed.  Probably, I would suggest by restriction analysis and biological evaluation.  I think it is an open question whether additional sequencing might be required at that time.
	Here's just an example of a generic gutted adenoviral vector, one of the ones we're interested in in my laboratory.  I just want to point out a couple of things.  Again, this is a conventional plasmid backbone.  We usually use blue scrip from Strategene.  It contains a CDNA cassette, a gene regulatory element.  Often, you need a stuffer fragment to bring this up to an efficient size for packaging.  It is important that one is aware of the nature of their stuffer fragment and what the sequences in there are.
	And then, the only part of this vector that is derived from adenovirus, again, is a packaging signal and the left and the right inverted terminal repeat.  Different versions of these vectors have a different size from these regions of adenovirus.  It works well with a region as small as 600 base pairs.  Some labs have taken that up to about two KB.
	What are the requirements that I think are necessary to get this to be a viable system?  One, you need a very stable vector backbone.  It would be nice to have something that is well sequenced and characterized, that enables cassette cloning capabilities, such that minimal modifications could be made to the vector.  And that everything can be done either by homologous recombination, E. coli, or direct cloning, without the necessary, somewhat dirtiness of recombination of mammalian cells.  You need to be able to derive a consistent, predictable structure from these vector backbones.  You need very efficient and well characterized packaging cell lines that are able to grow these vectors to high titres without generating a replication competent adenovirus.
	Again, you are going to need a well optimized helper virus that can support high titre growth of the gutted virus, preferably one that is self-disabling in order to minimize contamination of the preparations without generating RCA virus.  And something that preferably gives minimal, if no gene expression in vivo, to prevent side effects from the helper virus.  A problem I think of has plagued a lot of labs with this system is to be able to put all of this together and get very efficient, large scale growth of these vectors.
	Let me talk about some of the issues in terms of being able to characterize these things.  One is the issue of titreing of a gutted adenoviral vector.  I think it is important that one is able to clearly define exactly how much of this vector they have prepared, in order to start analyzing perhaps trace contaminants or impurities.  A problem that has plagued a lot of labs is these gutted vectors are incapable of forming plaques, which used to be sort of a conventional assay.
	You can determine particle counts, but you have to account for how much helper contamination is in there.  You can put reporter genes into these things to look at transducing units, but preferably, I think you really want to go right at how many vector genomes you have by using some sort of quantitative PCR method, and this is a method that can be used to quantify both the helper and gutted virus, and is very useful in guiding the growth, but also assessing the purity of your preparations.
	One question that is a little less clear, is what is the ratio of these different titreing units?  I will show you an example of that, with just a sample prep from my own laboratory, where we grew up some gutted adenoviral vector and we assayed the titre by using a Taqman assay to do quantitative realtime PCR and got a titre a little over two-times-ten-to-the-ninth. We had a reporter gene in this particular vector and when we determined titre by tranducing that into 293 cells, the titre was about tenfold lower--excuse me, this is the helper virus I'm quantifying here.  When we tried to quantify the amount of helper virus by doing a plaque assay, as you can see, it was about another tenfold lower than that.
	When people want to try to figure out exactly how much contamination they're dealing with, in terms of helper virus or perhaps other rearranged products, it is critical that you take into account exactly which assay you're using and what you expect to be able to detect with those various assays.
	Some of the safety issues that will have to be dealt with are--some of these are similar to a conventional adenoviral vector, the toxicity of the vector.  Obviously, a lot of preclinical testing needs to be done.  Again, this issue of helper contamination, the structure and the stability of the helper virus, and then this question again of what type of quality control you want, in terms of when you assess it.  Do you assess only the plasmid, your seed stocks or the final vector preparation?
	I want to come back to this point of the serial passaging that is required to grow these vectors. I'm going to show you some problems that we ran into early on, before we started to get a better handle on this system.  We initially encountered a very serious problem in growing these vectors, that we have largely overcome, but I think is still worthwhile making this point, because similar events could happen at a much lower efficiency.  Then they might be more difficult to characterize.
	Here is an example of a helper virus and a gutted virus that we are growing at one time, that expressed the dystrophen CDNA, of interest to my laboratory, and we also had a reporter gene, the Lac Z gene that was driven under the control of its own promoter to help us titre this vector.  Unfortunately, we had several relatively small regions of similarity or sequence homology between the gutted and the helper virus.  In particular, the promoter that we had driving our reporter gene and the helper virus was the same promoter we were using to drive the Lac Z gene.  It was relatively small, about 500 base pairs.
	A second region of similarity was we had a 195 base pair polydelineation signal that was present in common between these vectors, and it was, in fact, located in two different places in the gutted adenoviral vector.  It turned out when we grew these vectors together, we consistently recovered very high levels of rearranged products that, upon characterization turned out to be homologous recombinations that occurred in three different places.  Either between these two polydelineation signals, these two polydelineation signals, or these promoter fragments.
	Here is an example of the two most common ones where this homologous recombination event here between this promoter resulted in the transfer of the left inverted terminal repeat and the packaging signal from the gutted adenoviral vector onto the right end of the helper virus, generating a helper virus that could no longer be selected against by Cre-lox mediated excision of the packaging signal.  And instead, we now had a very robust, unselectable, robust growth that we could not select against this helper virus, that started contaminating our preparations at a very high level.
	A second rearrangement did a similar thing, but resulted from homologous recombination between these polydelineation signals, that, again, brought the left inverted terminal repeat onto the right end of the virus and brought in a new packaging signal, such that this helper virus now started taking over the growth.  One of the problems with this, is that these resulted in different sized vectors that no longer migrated at the density we predicted on cesium chloride gradients.
	We were able to confirm these rearrangements by extracting DNA from these growths and doing southern blots with various sub-portions of the helper virus.  I won't go into details, but on this particular prep here, we're missing a large portion of the right end.  We have lost the right inverted terminal repeat, and we have a new fragment that hybridizes with the left inverted terminal repeat derived from the gutted adenoviral vector.
	Again, let me come back to the question of cesium chloride gradients.  In all of these cases, these rearranged products were immediately visible when we went to cesium chloride, because we started generating aberrantly migrating bands.  However, we've also encountered some rearranged products that were smaller in size than normal, and which co-migrated with the gutted adenoviral vector.  In which case, when we purified one of these particular viral bands to apparent homogeneity, and then we came back and started doing PCR assays, particularly the Taqman assay, together with southern blots and titreing of reporter genes, we clearly realized we had a mixture of co-migrating viral products in that band.
	Not to leave you with too pessimistic of an outlook, when we had gone back and removed all of the sequences of homology between our gutted and helper viral vector, we are no longer seeing these rearranged products, but that does not necessarily mean one might not occasionally encounter those at a very low level.  We feel that, interestingly, one of the things that is actually leading to the appearance and selection of these rearranged products is, in fact, our method that we used to try to select against the helper virus, which is Cre-recombinase, mediated disabling of the helper virus.
	The real advantage of Cre-recombinase is that if you do a gutted adenoviral preparation in the absence of Cre, such as we've shown here, you always have very high levels of the helper virus.  And it's very difficult to get your gutted virus, shown in the blue-green here, to catch up to that.  In the presence of Cre-recombinase, however, you have a constant selection against the helper virus, shown in yellow, and your gutted virus can accumulate to a level well over 100 times as concentrated as the helper virus.
	However, we believe it is the very nature of the selection that allows us to end up with contaminations of rearranged helper viruses that have picked up a competent packaging signal.  And, in all the cases we have characterized this, that competent packaging signal has always derived from the gutted adenoviral vector, although that is not to say that at some point one could not drive a competent packaging signal from the packaging cell line itself since a lot of those cell lines are E1-positive.
	The other point I would make--even with these vectors that we worked with early on that had a propensity to recombine on us--when we did not grow those in the presence of Cre-recombinase selection, we never saw those rearranged products.  This is something we were bringing out by the very nature of our attempt to select against helper virus.
	Let me just close out with some suggestions from my point of view.  I think is critically important to quantify a gutted and helper by quantitative, real-time PCR methods.  It is important to have a very firm grasp of the structure of both the gutted and the helper virus.  I would propose that the starting plasmids from both of those be completely sequenced.  At a minimum, there should be extensive restriction analysis and sequencing of ambiguous regions of the helper virus, if it's not possible to completely sequence your helper virus, although I would suggest that it should be possible to sequence a helper virus.
	The question is, I think it is critical to have some sort of seed stock that can be verified, and the question is, will extensive restriction analysis and checking these preps on cesium chloride gradients be good enough, or would one want to require that the seed stock itself be re-sequenced for verification?  I think I'll just stop there.  Thanks.
	CHAIRMAN SALOMON:  Thank you.  Again, another really nice talk and I appreciate everyone staying on time.  It makes my job easy.
	The question I have is, you talked about homologous recombination, if I understand right, and here, I'm getting out of my area of expertise, but I understand non-homologous recombination is also quite possible, and that can occur between the helper virus and the gutted vector, but also with the packaging cell line.  To the extent that that occurs, can you make some comment, again, with your expertise, just how often does non-homologous recombination occur?  If so, how do you see that as having any implication, because given the fact that you're growing these things together in a packaging line, it almost seems like you'd have to sequence the lot every time you made one.
	DR. CHAMBERLAIN:  The most common example of that is perhaps the ability to occasionally pick up E1 positive sequences from the cell line.  Although the rate of that occurring from a non-homologous event versus a homologous event is tremendously lower.  I do not know the exact frequency, but there are examples of that occurring in the literature.
	So, I do not know what to say in terms of how often that is going to happen.  I guess, all I can say, with the limited experience we have doing a number of these growths, we have not observed a non-homologous recombination event that has cropped up in one of our gutted adenoviral vector preparations.  That does not necessarily mean that it's never going to happen.
	It probably will happen at some low efficiency, but I think that is certainly an issue that we want to consider--and why it's going to be important to characterize these seed stocks as well as possible, and also, to try to develop methods that allow these vectors to be grown up with minimal rounds of serial passaging.  I think the more times that you serial passage your two vectors together in the same cell line, the odds of a non-homologous recombination event are going to begin going up higher and higher.
	CHAIRMAN SALOMON:  Dr. Breakefield and then Dr. Sausville.
	DR. BREAKEFIELD:  I just had a question about the viral particles.  You mentioned the possibility of titreing those, and I was wondering if, if they would really be resolved by the Cesium chloride, or whether they might give you a clue, for instance, if you had some background in that band.  If you have, like, many more particles than you could account for by transducing units, you might suspect there wasn't the contamination, with empty particles or particles containing the smaller pieces of DNA.
	DR. CHAMBERLAIN:  Right.  I agree.  I think cesium chloride gradients can be quite useful in a number of different ways.  I mean, we have typically been running these, just to help us purify the vector, although that is something that hopefully won't be required in the long-term.  But, nonetheless, it does give you a good visual feel for what you have, and that gives you something to compare with.
	I think you're right, if you simply take a viral preparation and start doing PCR assays on that, you don't really know how accurate those numbers are, but you do get a very visual feel for what you have on the gradient, in terms of the number of bands you have, but even if you have a pure band, if your PCR assays are not giving your numbers that jive with what you've see on those gradients, obviously, you would have a problem there.
	I think where that may become an issue, is if you get rearrangements that generate viral vectors that are essentially the same size, you are not going to resolve those on cesium chloride, but when you start doing quantitative PCR against select regions of the genome, the numbers may not match up all the way along the viral genome.  If you test for the quantity of your transgene and then you test for the quantity of an inverted terminal repeat, or a sequence that is only present in the helper virus, those ratios should come out with what you're predicting or what you saw on the cesium chloride gradient.  If you start to get rearrangements arising in the preparation, you're going to see altered ratios and they're not going to match up with what you might have predicted from looking at the cesium chloride.  I think it's important to do both.
	DR. BREAKEFIELD:  So, do you think it is necessary, then, to actually measure numbers of viral particles or not, I guess, is my question?
	DR. CHAMBERLAIN:  Yes, I do.
	DR. BREAKEFIELD:  How do you do that?
	DR. CHAMBERLAIN:  I think the best way to do that is by quantitative PCR, to actually quantify the amount of vector DNA that you have in your preparation.  I mean, if you're talking about taking a banded virus and also just measuring the optical density, to get a conventional viral particle count, yes, that should be done also.
	In our experience, we generally get very compatible numbers with those two methods, but the PCR assay, I think, is a little more accurate.  That is a good point.  In fact, we do do that.  We measure the viral particle count by conventional assay, and then we come in with several sets of quantitative PCR reactions, to pick up different regions of both the helper virus and the gutted virus, and make sure those numbers add up to what we saw on the particle count.  If you had a rearrangement, then you would get a situation where those would not add up to what you had seen.
	DR. SAUSVILLE:  To pursue this line of questioning, my concern is, is there facile technology to not only detect the gutted virus plus the expected helper virus, plus a number of other things that one could conceive, or even not conceive rearranging, in relation to your particle count?  Is there facile technology for that, and would the incidence of this problem change from gene to gene, so that potentially you would have to reinvent this methodology, each time you chose a different target?
	DR. CHAMBERLAIN:  Well, I think the best method to use is really southern analysis.  You can go in and take some of your viral preparation and digest it with a variety of different restriction enzymes, and hybridize it with different parts of your vectors, and make sure the restriction fragment pattern you see is identical to what you would predict from the sequenced material you're starting with.  That is not necessarily the fastest and easiest method to do, and unfortunately, it takes, actually, a fair amount of a viral preparation to do that, which is, in fact, it can use up a reasonable amount of a seed lot to do an extensive restriction analysis like that.
	Nonetheless, one could prepare a seed lot and then do subsequent growths from that, and use all of those subsequent growths for verification by southern analysis.  The reason I would favor southern analysis is that it gives you a visual representation of the entire viral genome.  You know, you can always go through it much more simply with less biological material, to do PCR assays across different regions of your different vectors.  But if you don't know the nature of potential rearrangement, you're not necessarily going to pick it up by PCR, where southern analysis should reveal all of the sequences present in your preparation, with perhaps the exception of a very low level contaminant. At the same time, I'm not sure there is any method that's going to pick up low level contaminants at a guaranteed--
	DR. SAUSVILLE:  Southern analysis--you really wouldn't expect to pick up things at less than a, say, 1.0, maybe .1 percent pressing it.  You're still talking about a large number of potential particles or genomes that you're going to miss.  Right?
	DR. CHAMBERLAIN:  Yes, that is true.  But that is true of any viral vector, I think, so I think that is an issue that is certainly not unique to this system.
	DR. O'FALLON:  That kind of gets to my question, some of us were born at a time when 99.44 percent pure ivory soap was the epitome of perfection.  I think I heard you say, and you just repeated it, that you had some of your gutted, which was 99 and--90 percent, 100 percent pure, and yet, we're talking about a lot of objects.  Did I misunderstand you?  You do have some that is that pure, but what is our target?  Maybe that is our primary question here.
	DR. CHAMBERLAIN:  The purity I was talking about there, 99.9 percent pure, that is a purity of gutted viral vector versus the helper virus.  I think the more critical issue is what percentage of a rearranged product may be further contaminating those preparations.  It is perhaps a separate issue, that we may not want to be dealing with here, is if one is going to do clinical trials with a gutted adenoviral vector, how much helper virus contamination would you allow to have in those mixtures?  That is an issue of contamination of a known sequence.
	DR. SAUSVILLE:  I actually wouldn't be too worried about the helper virus.  As you had described, there are robust methodologies to detect that. You know what you're going after.  I must say I'm much concerned about the pieces I don't know might arise, with all the lines and colors that you had on your chart.  I guess the bottom line is, in this technology, if there are 100 particles, can you say with certainty what fraction of them has the desired product, the helper virus that you think about?  Then, is there a way of assessing what you cannot basically foresee or predict?
	CHAIRMAN SALOMON:  .1 percent of the 100 particles are, you know--
	DR. SAUSVILLE:  Are helper--
	CHAIRMAN SALOMON:  That is correct, but that is one point.
	DR. SAUSVILLE:  I'm not worried about the helper virus.  We can look for the helper.  I'm worried about what we don't expect.
	DR. CHAMBERLAIN:  Well, maybe to bring it into context, we can detect the helper virus contamination without too much difficulty.  At that level of impurities, is detectable by southern analysis.  I mean, I guess maybe what you're asking is, if the vector carrying you're therapeutic gene were to rearrange on you at a level of 0.1 percent, would you pick that up each and every time?  With current technology, I would say probably not.
	CHAIRMAN SALOMON:  That is a good answer, and we will get into that issue in a minute.  I just have one quick question, again.  This may be a stupid question, but you're using 293, which has an E1 sequence, but your E1 sequence is in your helper virus.  Why are you doing that?  Why don't you just use a different packaging line?
	DR. CHAMBERLAIN:  I may have mis-spoken.  Our helper virus is E1 deleted, but it contains the packaging signal, which I believe is present 293 cells.  I may be wrong about that.  Certainly the 293 cells are E1 positive.  I assume they have the packaging signal, also.  The helper virus is E1 negative, but we're trying to remove the packaging signal from the helper virus, and it can be picked up, in theory, from the cell line.
	CHAIRMAN SALOMON:  So, the point, though, is still, that the packaging line you're using has an extra gene not needed in the system that you have.
	DR. CHAMBERLAIN:  Well, more or less.  293 cells have been used for many years to grow E1-deleted adenoviral vectors, but at a very low frequency, you can pick up E1-positive replication-competent adenovirus due to homologous recombination with the E1 sequence that is in the cell line.  There are some cell lines out there now that people have made different versions--inserted different versions of the E1 gene in there, to try to prevent that.  Most of the cell lines are essentially unavailable to academic laboratories, though.
	CHAIRMAN SALOMON:  The direction I was going with those questions is, one of the things that's happening here, I think, is this tension at the nexus between an academic laboratory and Pharma.  Everybody uses 293, so you're doing all these elegant, you know, Cre-lox changes and things in the helper virus, and yet we're all still using the same packaging line, which, at a certain point, probably is not the ideal packaging line for the system.
	DR. CHAMBERLAIN:  Right.  I think that gets into a little different issue.  There are alternate packaging cell lines that can be used, there are also other ways to select against RCA besides concerns of the E1 region; particularly, by using vectors that have additional modifications, such as deletions and the E2 region or the E4 region and things like that.  I think there are very effective ways to select against and to screen against the E1 positive viruses.  I think a more significant concern now, is how to deal, though, with less predictable rearrangements.
	CHAIRMAN SALOMON:  Are there any other questions?  Well, remarkably, we are at 10:10 a.m., exactly time for a break.  I will see everyone back here in 10 minutes, please, and we will keep on going.  Thank you.
	[Recess.]
	CHAIRMAN SALOMON:  I am always reluctant to cut short these first breaks, since I know for a lot of people it is a nice opportunity to get together with people and of course, you're all discussing the FDA questions during the break.
	MS. DAPOLITO:  They shouldn't be.
	CHAIRMAN SALOMON:  Is that right?  We're really not off time, because we actually started 10 minutes ago, is where I was going with that, but maybe not.
	As we get started again, there is one more speaker.  I would like to introduce Dr. John Levy from CTL ImmunoTherapies Corp. to talk about "The CMV Promoter is Copied as "Extra DNA" from DNA Vaccine Plasmids."  And after that, then we will go into our discussion.  Thank you.
	MR. LEVY:  Well, thank you.  I'm the Quality Control Manager at CTL ImmunoTherapies Corp., and I have been invited here, I take it, to talk about extra DNA sequences.  That certainly fits the description of what we found in our product.
	The identification of this, I will go into some of the issues related to that toward the end, but essentially, we're using plasmid DNA.  We are not using the traditional viral vector systems.  The DNA we're going to be using is going to be used as a polynucleotide vaccine.  From the standpoint of how we put the vectors together, I would just talk about a couple cloning constructs.  The backbone we chose to use as our starting point was a vector from invitrogen PVAX, which has apparently been engineered to become a very simplified, streamlined vector to contain reasonably desirable elements that could be used in these polynucleotide vaccines and, hopefully, not other elements that would be not particularly useful in vaccine research.
	It contains a PMB1 origin replication, a CMV enhancer promoter region, bovine growth hormone polydelineation sequence, and a kanamycin resistance chain.  We wanted, for the purposes of what we wanted to do for our vaccine, is express two genes simultaneously.  I understand there are a number ways to do that to promoters.  There are systems you can use that would involve differential splicing, but we chose use an IRES sequence as our way of simultaneously expressing a gene from the cap-dependent site upstream and the cap-independent site downstream of the IRES sequence.
	We obtained our IRES sequence from a commercially available vector called PIRES.  Essentially, we put these two constructs together, cloning the IRES gene sequence into the multi-cloning site of the PVAX gene to make a hybrid vector PVYY1.  You'll have to excuse the--I was learning to use animation the other night, too, and actually, me and John took this stuff off the presentation, but it persisted--the contamination of an animation sequence.
	I should point out, as I get a few slides into this, I'm going to describe a small region called a sequence terminator that is a sequence found within the uppermost part of the IRES that is available commercially.  And we did a little bit of analysis to determine what effect that has.  Essentially, the bottom line is, when we make these plasmid vaccine preparations and run them out on gels, where the vector contains the PVAX backbone with an IRES sequence in it, and independent of the transgenes, we see as in either lanes B or C, the super coil DNA high mobility migration in the gel, but we see these other bands and we see them very persistently.  They gave us pause when we first saw them, and the fact that they were 600 base pairs and a couple of sequences below that, led us to believe that we potentially had some double-stranded fractions of DNA in our preparations, but what they were, we weren't really sure.
	What this slide is meant to represent is an early attempt to see if there's any way to get rid of the 600 base pair, and the two sequences below it are approximately 300 base pairs and 360 base pairs.  In lane C, we're using a host strain that is fairly common in the field, DH5As.  What we found is if we, instead, put our polynucleotide vaccine into a host strain DH10Bs in lane B, that we could just by doing that, consistently reduce the relative content of these bands about threefold.
	The question is are these things related?  Is the 600 base pair piece of DNA that we're getting in these vaccine preparations related to the 300 base pair and 360 base pair piece?  Actually, when we run this out on a two percent augerose gel, run it quite a ways, it may not be visible from the slide, but the 300 base pair band actually is a doublet that migrates as a 280 base pair fraction and approximately a 310 base pair fraction.  We cut it with an enzyme--well, I should probably say that, in looking at the literature, we just formed the idea that the most likely possibility of this is that they are what are described as replication intermediates in the literature.
	There are a lot of papers out there where experimental replication intermediates are deliberately formed by putting two origin replications, basically facing right at each other, to form an active and silent origin replication producing these fragments.  There are some instances in the early literature that these were happening on PPR345 plasmids.  Essentially, we went from that direction--that this was probably an origin of replication phenomenon.
	If you look at a unique restriction site, about 130 base pairs down from the origin replication, and consider that a unique restriction site and digest these bands with this enzyme, ASC1, what it does is it actually cuts all of the bands and generates an accumulation of about 130 base pair common fragment, leading to the idea these are all a family of fragments, all originating from the same point.  And, based on this enzyme and, at least in our plasmid, it is only represented in a position, which you would expect this behavior if it was occurring at the origin of replication.
	We gel-isolated, and purified, cloned and sequenced the 600 base pair predominate fragment and aligned that to--our first best guess was it was something probably generated from our vector in the host strain, and the replication intermediate, or replication anomaly, has 100 percent homology match to a sequence within our vector.  And that sequence is a sequence that would imply it starts with the origin of replication and extends through the entire CMV enhancer region and is terminated at approximately the CMV enhancer/CMV promoter boundary.
	In lieu of actually doing some genetic manipulations to the plasmid to address the question of can we get rid of this thing within the context of the arrangement of the genetic elements in the vector, we did some cell culture techniques to see if there were just some straightforward things we could do in the fermentation or production process that would attenuate a level of these replication intermediates.  We noted that if you change the temperature of the fermentation production process, that lower temperatures--when you lower from 37, down to 35, down to 33, you get approximately a twofold reduction in the accumulation of these replication intermediates.  In this case, it is in the DH10B cell lines, which themselves have already been threefold attenuated, in comparison to the DH5A cell line.
	You can further affect the level of accumulation of these replication intermediates by choosing a time in your fermentation process that extends beyond a 12-hour time point.  It seems that, at 12 hours, when your host cell strains are first coming out of their mid-log phase growth, that these replication intermediates are at their peak accumulation.  They tend to, if you will, de-accumulate over time.  If you go to 16 hours or 20 hours--it seems to tail off if you go out to 24 or 48 hours, that you can effect about a two-to-threefold reduction on top of the reductions that you've seen already.
	So, cell culture conditions can actually lead to some attenuation of this phenomenon.  We wanted to study what this replication intermediate was, how it occurred and, if possible, how we could eliminate it within the confines of our construct.  We performed a deletion analysis, since it is that the sequences mapped--two sequences in the CMV promoter originating from the start side of the origin of replication--we first decided to do a deletion analysis on the--some sections of the CMV promoter.  And we have kind of an opportunistic deletion, one of those laboratory anomalies where you got, apparently, a star activity digestion of the enzyme that cut some portion of the IRES sequence out at this stage of the deletion analysis.
	And what you see is that in an undeleted vector preparation, you can compare the replication intermediates that you see to the deleted.  So if you delete out essentially two-thirds of the CMV enhancer and CMV promoter sequences, you effectively get rid of the predominate 600 base pair replication intermediate band, but retain the lower doublet, 280, 310 and 360 replication intermediate bands.  And it is not until you actually delete further into the E-CMV IRES sequence, that you're actually seeing a complete elimination of all of the replication intermediates.
	From the standpoint of is this a strategy that would actually make a better vector, the problem, of course, is that you're actually having to delete out sequences that you need for function of your vector in the vaccine activity of the vector.  We went back, and I know this seems a little bit backwards, but this is a deletion where we took the entire CMV promoter out.  There are a couple of ASE1 sites.  This ASE1 site was the one I used to map the upstream portion of the replication intermediates.
	What we found was that if you consider our deletion that we just performed here, taking the entire CMV promoter out, you produce, actually, a new replication intermediate that would map now within the IRES sequence.  If you take the original backbone vector, which PVAX represents a, essentially, IRES deletion, you do have still the approximately 600 base pair band that you see from the vector that has both the CMV and the IRES.  However, in both cases, there is a tremendous attenuation of the accumulation of these species.
	The opportunistic deletion that actually cut off a sequence within the IRES was something that we looked into a little further.  We knew that in the sequencing of this vector--we have sequenced this vector in its entirety multiple times at master cell bank and production lot phases to ensure its integrity throughout the process.  We noticed that there is a sequence within the five-prime (ph.) region of IRES sequence that we could never completely sequence beyond in either direction.
	Actually, this is a very dominant sequence termination site that actually became kind of a map site for alignment and context analysis, because you always knew where you were at by just watching where all of the context converged--was a sequence that is very C-rich, CT-rich.  It is actually--this portion of the IRES maps to a portion in the wild-type strain of the virus where that sequence is just coming out of the poly-C track.  So, this is a C-rich sequence within the traditionally-defined IRES domain.
	It is very difficult to sequence beyond it.  You can sequence through it, and then these big-dye terminator, cycle-sequencing reactions will fail after that in either direction.  I speculated, well, this is a polymerase, a bacterial polymerase.  I'm not sure if the wild-type polymerase is in--the host strains are having not necessarily the same problem, but at least the sequence is potentially giving them some biological response.
	We simply just deleted out that sequence, this sequence right here, and we can see by simply doing that--this is our vaccine vector with the CMV and the IRES sequences in it.  If you just take out the sequence terminator, you get a tremendous attenuation in the accumulation of these replication intermediates, implying that there is some contribution of the IRES to the termination that is occurring on CMV and the IRES sequence.
	If you just cut the sequence terminator, you get a tremendous attenuation in the accumulation of these replication intermediates, in ponderous some contribution of the IRES determination those occurring on CMV, considering what is known about termination interactions in host strains, considering that perhaps maybe the sequence is inciting something in the bacterial host strain that is leading to a biological response that maybe we would predict, and that potentially would be an interaction that is known as replication termination in the host strain, through proteins called tuss (ph.) and termination sites called terr (ph.), and that body of literature, that phenomenon of termination within the E. coli host genome is orientation dependant, so we figured by simply just turning the origin replication in the other direction, perhaps we would actually produce a plasmid vector, and these are meant to be circular, of course, produce a plasmid vector that would test the hypothesis of whether these are contrahelic cases that are binding the CMV promoter or whatever they may be, that there are operating in an orientation-dependant fashion.
	And what we see is that our plasmid vaccine, here in line A is the comparison, simply by turning the origin replication in the other direction within this vaccine, there are no replication intermediates seen anymore, at least at the level, unloading--there are probably 40 micrograms of DNA loaded on these, we do not see anything in these lanes.  Within the detection limits that augerose gel electrophoresis can give you, there is nothing that we can detect as far as existence of these species anymore.
	Just as a note, there is another vector used traditionally or has been used traditionally in the DNA vaccine, PCDNA-3, and I think I put the map for it on there.  I do not know if this necessarily has anything to do with it, but within this vector, there's an ampicillin resistance marker ribbon, kanamycin resistance, that does have a CMV promoter.  You can put an IRES sequence in there if you like, but the origin of replication in this construct is running in the opposite direction of the CMV promoter, similar to the construct that we modified from our own laboratories.
	On the last lane of that gel, I would just kind of reiterate what was in the last line of the last gel, is PCDNA-3 vector does not produce these replication intermediates, not even at low levels.  We can say that our plasmid vectors do contain these sequences that result in replication anomalies, replication intermediates, that the CMV enhancer promoter contains sequences that seem to synergize with demands within the IRES to produce more of these replication intermediates than you would see in plasmids that would just contain either one or the other of these sequences, and that replication anomalies--we have actually--I have not actually brought light to this--but we have actually seen replication anomalies in other plasmids with similar arrangements that do not necessarily have IRES sequences, but have sequences similar to this termination, the sequence termination or C-rich element within the IRES.
	Deletion, obviously, is not always going to be a practical means in these vectors, if deletion means actually getting rid of the promoter enhancer sequences that you're going to need further down in your clinical work to actually express the genes that you need for your DNA vaccine, but that inversion of the origin replication apparently eliminates these replication intermediate anomalies and may reflect the orientation-dependant nature of how these things are actually produced.
	Thank you very much, and I will be happy to answer any of your questions.
	CHAIRMAN SALOMON:  Thank you very much, another real interesting view of an alternative way of getting sequence anomalies in vector production.  Any questions?
	MS. MEYERS:  Just as an overall perspective on this morning's presentation, the public, I think, has more understanding of pregnant chads than anything that was said here today.  It is a whole new vocabulary.  Since there are these problems and there are--these replication anomalies occur in a lot of different experiments, there seems to be production of abnormal viruses that have never appeared on Earth before.  How do you throw them away?  Do you throw them in the dump?  Do you throw them down the drain?  What do you do with these very different viruses?
	MR. LEVY:  Oftentimes--well, in the case of a double strand of PCDNA, DNA is exquisitely sensitive to common chemicals like acid, so autoclaving is a high enough heat to actually remove, not only viruses, but DNA, so there are some common sterilization techniques that laboratories always produce within the normal confines of actually developing production protocols or manufacturing protocols that should address actually eliminating these sequences.
	MS. MEYERS:  You destroy these viruses before they are thrown away?
	MR. LEVY:  If there is a proven or perceived safety risk, certainly I think someone would want to evaluate, you know, what the expediency for actually removing these entities are.  In some cases, obviously, it is necessary to generate enough of these entities to study them, to understand what they are.  If one were to see something that was anomalous and immediately get rid of it, you might not actually ever know what it is that produced it.
	In some cases, you would have to assess what the risk of the entity was and find ways to either safely study it or expediently get rid of it.
	MS. MEYERS:  You do keep some of it alive in your laboratory, maybe forever?
	MR. LEVY:  Well, in our case, these sequences are DNA.  They are not viruses.  On their own, they will not replicate.  If you put them in a tube, frozen away in a freezer, presumably, if you came back in 100 years, in the cases from our studies, you would hope that even if you came back in 10 days, that they would still be there.  In the case of viruses, though, if you freeze viruses away, there are ways of actually containing viruses over long periods of time.
	I think it would be difficult to assess, you know, what the safety of each of the viruses is unless you had some idea of what it is you're actually working with.
	MS. MEYERS:  My understanding, though, is a lot of times these viruses that have been made up in these experiments, we really don't understand, because they've never existed before.  Am I right?
	CHAIRMAN SALOMON:  Well, yeah, I think in this particular case--it is a really good question you asked.  That is why I didn't say anything, but what he's talking about now are just pieces of DNA.  These are not viruses.
	MS. MEYERS:  There's no danger of them escaping?
	CHAIRMAN SALOMON:  Well, this particular instance, yes, there's no danger, but the whole point that we are here, however, is on point for your question, and that is we could generate viruses that, through recombinations of various sorts, did generate new sorts of viruses.  So, your question is excellent.  I think it threw the speaker off a little bit because his DNA fragments are not viruses.
	One of the things you would use this for, though, is a vaccine, so did you do any studies in which you actually used this preparation contaminated with these replication intermediates and injected in a mouse model, let's say, and see whether or not this protein was expressed in the target cells?
	MR. LEVY:  When we identified that this contained regulatory sequence and that there was a durable percentage of our vaccine preparation that contained this sequence, and, of course, then that would mean if you were to inject this into a recipient, that they would be receiving these regulatory sequences.  There are two thoughts, one, these are just mirror images of regulatory sequences that are already in the plasmid.
	However, we do have a very active and thorough toxicology program, where these vaccines are screened in advance through a number of animal studies, and we're looking at not only just the pharmokinetics and distribution and half-life of the DNA entities, but we are actually looking histopathologically at the response to these DNA vaccines--as well as all of these DNAs, of course, are going through the traditional identity testing and safety testing for endotoxin and sterility--sterility--
	CHAIRMAN SALOMON:  I guess I was just trying to get at--I mean, Abbey is making the point here that a lot of this is so arcane that people here who are not gene experts are not going to follow the implications here.  I was just trying to translate a little bit that, in a medical instance, in use, there are clinical problems here, is one might, through the generation of these sort of anomalous DNA species within a vaccine, now taking your product, you would be, let's say, exposing the individual eventually to expression of proteins that might represent new molecules or neoantigens, than if these were based on sequences, let's say captured from proteins that are normally present, one could generally create--potentially, rather, create an autoimmune disease, accidentally, or some other sort of immune complex disorder that might be totally unexpected, without knowing that you had done this.  I think that is the question that we were asking.
	MR. LEVY:  Okay.  I understand.  Do you want me to answer that just globally or in our particular case?
	CHAIRMAN SALOMON:  No, I don't think this is relevant exactly to your situation, so I'm not worrying about your 600 base pairs, but just in general, that is what Abbey was asking.
	MR. LEVY:  Well, it is something that--in more fully characterizing what it is that you are actually developing and finding that there are some things that are new or different or unexpected, particular in the case of producing new proteins, if your gene is producing a new open reading frame, certainly.  We had an instance where there was a question about open reading frames, and even if it means changing a single amino acid, certainly there are plenty of things out there in the literature that imply that changes of even that can be dramatically immunogenic or have a disease potential.
	So, I think you have to bring to bear the knowledge you have in the field, but, at the same time, be willing to actually act on even things that would be seemingly subtle, because the potential for them to actually have dramatic effects in humans is certainly there.
	CHAIRMAN SALOMON:  Thank you.
	DR. CHAMBERLAIN:  I would sort of like to make the general comment, though, that in some ways, these are not unique issues that apply only to genetic vaccines, that the possibility of rearrangements or not completely characterized products is an issue that has to be dealt with with any type of vaccine, whether it is an attenuated virus, a completely killed virus or a fusion protein that has been purified.  There is always a potential for uncharacterized biological material to be in there and enter the vaccination protocol, so this is an issue that pertains to any type of vaccination.  It is not unique to genetic vaccines.
	CHAIRMAN SALOMON:  Any other questions?  Thank you.  Then we will move to the discussion period.  The one thing I want to clarify during the discussion period here is the speakers have presented information at the request of the FDA that was specific, and it is something that, as chair, I have pushed, that we get specific information presented to the committee, so that we get away from just always dealing in generalizations, because I think this is very critical in sharpening the kind of discussions we have.
	However, I want everybody to realize that, in the discussions now that follow, the only thing that is off-limits is going back to these speakers and putting them on the defensive about their individual products, because that was never the intention of today's meeting.  If there are intellectual points that evolve directly from the talks, that is excellent, and go at them, but put them in general terms, because we certainly do not want to bushwhack any of these guys who have done us the courtesy to come and really present their data to us and suddenly mess up, you know, a clinical trial that they are in the middle of evolving from Phase II to Phase III pivotal trials.
	That is only thing that is off-limits today, but otherwise it is all there, and forward-thinking statements are perfectly encouraged in the following discussion.  I have been asked by Dr. Anderson that, before we get started on the individual questions, that he promises me he has a three-minute general perspective, and I have also told him that we're not going to spend a half-hour discussing it, but anyway, out of respect to Dr. Anderson, I think it is very reasonable and I look forward to his general comments.
	DR. ANDERSON:  I am here as a gadfly.  I will raise an objection or raise concern about every single issue the FDA has brought up, and I want to put that in perspective at the beginning.  I have had many, many discussions with the FDA over the years, all of them friendly, usually friendly.  This one really is part of what I think is an absolutely invaluable process of having public discussion of issues.
	If this were a real world--I take that back--if this were an ideal world, if we had infinite time, infinite money, that everything that could be done to help patient safety would absolutely be fine, but it is a real world.  We deal with real patients.  We deal with real budgets.
	Therefore, what is critical and what these two days are designed to do is to look at balance, is to balance priorities, and I know this is what the FDA has the meeting to do.  In fact, that is the reason I was asked to come, because I will provide a counterbalance perhaps to other views.  If one plots, and everybody in this room knows plots, patients safety versus cost, what everyone knows is initially there are many things that can be done at minimal cost that really benefits patient safety, and as one goes up in terms of cost, patient safety starts to fall over a bit, and then it becomes asymptotic.
	You can pour in tons of money and the increase in patient safety becomes really very minimal.  The issue is where are we in that curve for each individual recommendation, and many which I might object to, I actually agree with, but that--my role here is going to constantly look where are we on that curve.  I believe, as the FDA knows, that the increased monitoring requirements is right on that steep slope, will make better protocols, and that is well worth the extra money.
	Well, is there any reason why one should not say, let's not worry about the money, let's go all-out, because anything that improves patient safety is a good thing?  Well, no, everything has repercussions, and the repercussions in this case could be catastrophic, and that is what I want to point out.  First, let me tell you where I'm coming from.  I have been involved in 16 clinical protocols.  I'm involved in two now that are at the pre-IND/IND stage.  Of those 18 clinical protocols, the two present ones involve no commercial money at all.  It is all gift money or grant money.
	Of the other 16, most involved either a start-up, a small or a medium-size biotech company, all of it GTI, one of it was a large pivotal Phase III, done by big pharma, Novartis, and I was a consultant to that, the brain tumor trial.  So, I have firsthand experience in all aspects of how you do clinical trials and who pays for them, and the thinking behind what goes into doing them.
	Having said that, what are the repercussions of going up in this curve when you can superimpose another curve on this curve, and that is what happens to the initiation of clinical trials?  Increased cost is not going to affect big pharma much at all.  It is not going to affect major biotech much at all, but it is going to affect physician-sponsored INDs where the budget is really very minimal.  It is going to affect new, small biotech companies.  That is where the effect is going to be.
	Many of the most innovative protocols are going to come from physician-sponsored INDs and small companies.  So, the more you inhibit those trials from taking place, the more damage you are going to do to the field as a whole.  Cancer research isn't going to be particularly affected.  Cardiovascular disease is not going to be particularly affected, but rare genetic diseases, a cause that Abbey and I have been fighting for for decades, I guess, is going to be catastrophically affected.
	If one superimposes on this plot, here is your patient safety versus cost, in terms of physician-sponsored INDs, it is going to go high and then gradually drop and then precipitously drop as the cost goes up.  What I will argue on every point is where are we on the curve?  Is the amount of increased patient safety balanced by the loss of physician-sponsored INDs?  That is where I'm going to be coming from the entire time.  Do you feel comfortable now, Mr. Chairman?  You were really worried, turning the microphone over to me.
	CHAIRMAN SALOMON:  I am comfortable.  I think that the issue here that has been framed by Dr. Anderson is really pretty much the charge that I believe we have as a committee, and that is to see two sides, actually, if not three sides of this area.  One is that we have a responsibility to the public.  Henceforth, we have public meetings and invite the public to participate in these sorts of discussions.
	The second is we have a responsibility to the field in the sense that we are trying to deal with new areas.  Obviously, if these were well-established areas, they don't need advisory committees for it.  As we deliberate on these things, I also agree with Dr. Anderson that we need to be cautious, that we do not make recommendations in regulatory fervor that would damage the ability to progress in this field, because in a way that is also our agreement with the public, that science is going to move forward, but that science is going to move forward responsibly.
	I think the last thing that really is particularly important here, and I think it is going to be how we respond to Dr. Anderson's point, and that is this positive tension between the development of things in academic centers, by individual investigators and small pioneer companies, and the transition to large trials and big pharma, in which case you're dealing with many patients and probably the only thing I would probably take issue with, Dr. Anderson, is that we need to think of that as two separate issues.  I mean, there may be points at which we demand less in order to facilitate the first transitions of novel technologies, but then later crack down and accept the higher expense as the public is exposed more to these things.  Yes?
	DR. GORDON:  I just wanted to speak to something related to that, although you said you didn't want to belabor these points for half-an-hour, so I'll keep it under half-an-hour.  It would seem to me that what we're seeking here is the best possible testing paradigms for quality of preparations and characterizing preparations.
	What one wants then is to find those and then to implement them with uniformity for all preparations that are going to be administered to people.  Some of these testing paradigms may prove to be costly and involved, and because they are costly and involved, their uniformity is much less likely to be maintained, if it is done in 1,000 different places over the country by 1,000 different groups, who have 1,000 slightly different ways of doing this or that technique.
	Overall, when one looks at this problem, one has to think of methods of establishing uniformity and reproduceability for these tests, and deciding whether or not that is something which should be left to individual entities to do anymore, or whether or not we should consider referral centers for quality-control testing.
	CHAIRMAN SALOMON:  Good point.
	DR. MILLER:  I just have one clarification, when we discuss these questions for session one, is there a distinction between the in vivo and ex vivo use of vectors, and when and how can we determine if there is any distinction, from a safety standpoint and from what has already been--cell lines that have been set up years ago with vectors, and are we going to then subject them to the same scrutiny as what is going in now into patients?  I just wanted some clarification on that.
	DR. WILSON:  We don't believe that the discussion should be delineated along those lines, of ex vivo versus in vivo.  The question you raise about those trials that have established allogeneic cell lines that were transduced ex vivo some number of years ago, and how we would apply any new recommendations coming from today's discussion is an important one, but I think that for the purposes of these questions I would rather just lump them both together.
	CHAIRMAN SALOMON:  Last comment, Dr. O'Fallon.
	DR. O'FALLON:  This will be very brief.  I have a profession only because there is variability in systems, and we struggle in all of our contexts to separate out that variability that we can explain and hopefully then control from that variability which, until we understand it, we call randomness.  We are exposed to some questions here in the next few minutes we're going to discuss that is essentially going to be talking about controlling variability in a system which I maintain still is going to have massive amounts of random variability connected to it, so these issues have to be balanced as we are discussing this.
	We do not want to see how many things we can put on the head of a pin until we decide how many pins we have.
	CHAIRMAN SALOMON:  That is probably an excellent introduction to the next part.  Let's get started.  The one thing I might suggest to everybody, it is very useful to me, is in the material that was put together by the FDA, there is background information titled, "Structural Characterization of Gene Transfer Vectors," and at the very last page is a table that gives you essentially all the common vector classes, which, I think, for everyone who is not instantly conversant with the different sizes of the genomes of these different vectors and some of the replication properties, et cetera, you just might want to have that in front of you.  It's just a suggestion.
	Question one is for vectors up to 40 KB in size, which, referring to Table 1, would include plasmid, retroviral vectors, and adenoviral vectors and adeno-associated viral vectors, but would specifically exclude herpesvirus vectors, poxvirus vectors and some other EBV virus vectors, which is a type of herpes vector, et cetera, so there is a delineation here, is the point I'm making.
	For vectors up to 40 KB in size, the FDA proposes that the full sequence, coding and non-coding, should be determined prior to initiation of a Phase I clinical trial.  Do you agree with this proposal?
	DR. GORDON:  It sounds good, but let me just say that I think there are some problems with it, political mainly.  I mean, I think it will be very difficult to explain to somebody receiving a larger vector why theirs wasn't sequenced, and if you are establishing sequencing as the gold standard, you're going to put yourself in the position of deliberately applying less than the gold standard to some individuals and not to others.
	CHAIRMAN SALOMON:  Yes, I actually agree with that.  Let's divide that question into two, because we will get that in the next question.  It is absolutely on point, however.  The first question I'm trying to keep relatively simple, and that is, for a vector under 40 KB, would you agree that the full sequence should be provided to the FDA at the time of the initiation of a Phase I investigator-initiated or other trial?
	Dr. Anderson?
	DR. ANDERSON:  The issue here is Phase I, because you're certainly going to do it higher up, and anybody who would go into a $100 million clinical trial and not have that information, just it is not going to happen.  We are only talking about a Phase I, and the problem with doing this or anything else is that it adds an additional increment.  If the NIH is willing to fund it, well, that is fine.  I mean, every one of us would love to send all of our material and have it all sequenced for us.  That is great, but it costs money.  What are you going to do with that information?  If it turns out that you have a vector and it has got some salmon sperm DNA, and it's the salmon sperm DNA that cures cystic fibrosis, well, wonderful.  What difference does it make?  At a Phase I trial now, where you're only talking about a few patients and you're looking at safety and you're looking for a little bit of efficacy, as the FDA is prone to say, if it works, that's what is important, so long as it doesn't hurt the patient and it works.
	To require physician-sponsored INDs to sequence everything up front, before they ever go forward, might be a good idea.  Nowadays, it is pretty cheap to do.  But it is going to be the same fundamental issue every time you require an additional step and an additional requirement that really doesn't have a real need in a Phase I trial, you're going to reduce the number of trials.
	CHAIRMAN SALOMON:  Dr. Mulligan?
	DR. MULLIGAN:  I mean, I can't disagree more.  I think this is the most no-brainer of a question.  This is the standard way people do science at this point, and the availability is such that the cost is negligible.  When we get to point two, I'm going to say we ought to do that, too, but with regard to this question, I think it would be tremendously irresponsible to conduct any clinical experiment without having a DNA sequence.
	Our own personal experience at a major Ivy League university, in receiving DNA samples from people to help them make vectors, is that close to 50 percent of the things you get in-house by very reputable people are not as advertised.  I hope we don't spend more than about five minutes on this question.
	CHAIRMAN SALOMON:  Well, this is public discussion.
	DR. SAUSVILLE:  Yes, I would heartily agree with Dr. Mulligan on this point, and where I respectively disagree a little bit with Dr. Anderson is that Phase I is safety and that is the linchpin of what you are going to build subsequent decisions on, and you could potentially make a bad decision by not knowing exactly what you have.  I would agree with the characterization of Dr. Mulligan and strongly call for that.
	DR. TORBETT:  I will have to agree with Dr. Mulligan; when you start off these kinds of procedures, I think it is first knowing what you have in hand and going forward is the critical first step.  This is the beginning of the whole process, and I think knowing the sequence and having the same experience as Dr. Mulligan, we do sequence most of our vectors.
	We get them from others, because there are number of errors.  However, that being said, when unknown sequences are found, I think going to a standard database is useful as long as that sequence is present in the standard database.  That is, I think, something we have to consider, that is, unknown sequences, I think, need to be documented in different kinds of vectors.
	CHAIRMAN SALOMON:  I guess the point I would like to add is the research we're doing should be hypothesis-driven.  I don't need to tell Dr. Anderson that.  He helped teach me that.  One of the things that would be a true event is that if salmon sperm DNA cures cystic fibrosis.  I mean, if it does, I would be very happy for cystic fibrosis, but I think we have to have sequence, too, so I happen to agree with that.
	Dr. Anderson?
	DR. ANDERSON:  Having made my gadfly point, every vector we have, we sequence totally.  We sequence it repeatedly through the entire process.  It is absolutely required, and Richard, the only thing I disagree with, and on our West Coast, we don't get 50 percent of them right.  Most of the things we get in are about 80 percent are wrong.  Our rule of thumb is if you get it from your best friend in the next lab, you sequence it.
	CHAIRMAN SALOMON:  One more comment.
	DR. GORDON:  I just want to say there may be a way of harmonizing these two very disparate points of view, and that is the difference between having sequence in hand or making sure that sequence is becoming available as one moves forward.  The reason I say that is because I think it would be very unusual indeed--suppose salmon DNA was found in a vector, would that then cause you to discard the vector or not?  What are we going to do with the information, is basically what I'm saying.
	I think most of the time we're not likely to do much with it except shrug our shoulders and say, "Gee, that is interesting," and then go forward.  I think one way to harmonize this issue with issues of dauntingly difficult sequencing is to say that samples of this material have to be made available for sequence ongoing and introduction into some sort of meaningful database.
	CHAIRMAN SALOMON:  The point that I am not so comfortable with is that if you get sequence and if there is anomalous sequence present, that you just say, "Well, you know, it is present.  I'm going on to the clinical trial," I'm not sure I'd buy that actually.  One of the things that we will get to in question five is what to do when you find anomalous sequence, you know, what would be a limit--I think right now I would like to just deal with the question should we have sequence, and I think we're getting pretty close to agreeing that we should have sequence, and you raise now the additional and important point of what you do when the sequence doesn't match, and I think we can't leave today without having addressed that issue.
	DR. SAUSVILLE:  You may want to discuss this later, but I would certainly say it depends on the stage in which you discover it.  If it is the beginning of the process, I mean, this is technology, this can be fixed.
	DR. O'FALLON:  So why is nobody asking whether it should be 40 or 35 kilobytes or 45 kilobytes or--I mean, how did we arrive at that?  And that is where I assumed we would see some of this discussion.  I don't really think Dr. Anderson was voting against this.
	DR. ANDERSON:  No, the reason it is 40 is that encompasses the adenovirus, the retrovirus and the plasmids.  That is why nobody has objected to 40.
	DR. WILSON:  I think also the other reason we cut it off at 40 was to take into account some the comments Dr. Anderson was reflecting earlier, in terms of the cost versus the benefit, so when you go into the larger classes, which is question two, where you're looking at a couple of hundred kilobases, the cost becomes much more of an issue.
	CHAIRMAN SALOMON:  Carolyn, can you, just for the committee's sake and the public, tell us what you guys found?  You did some research on what it would cost at a contract lab to do 40 KB of sequence.
	DR. WILSON:  At a contract lab, which is probably, for most people, the most expensive way to do sequencing, most people would have access to in-house automated DNA sequencers, it would cost around  $20,000 to $22,000 to do a typical adenovirus vector.
	CHAIRMAN SALOMON:  Would that be both strands of a double-strand DNA?  Total?  Not $44,000?  About $22,000, and we could probably do that for half at an academic center or at the NIH.
	DR. O'FALLON:  It seems to me then, let's assume it's past and I said this to Jay at break.  I hope that wasn't illegal, but this will become obsolete, because that $22,000 will be going down to $10,000, will be going down to $200, in which case we can move this up to 100.  I like the other answer better, which is saying that it encompasses most of the current viruses we're interested in.  It will certainly cost less, even before any recommendations can be published.
	CHAIRMAN SALOMON:  Phil, did you want to make a comment?
	DR. NOGUCHI:  That's a valid point.  I think some of the further discussion will also illustrate that this is what can be done with an actual virus without further manipulation.  To get to the larger viruses, you will probably have to re-manipulate the virus itself or the vector.  That introduces then again another error of randomness to it or lack of scientific understanding that makes that problematic.  So, 40 KB is what is practical, and what can give us accurate information, as well, I think, at the present time.  Maybe next week, it will change.
	CHAIRMAN SALOMON:  I have been asked to call for a vote on this particular question, specifically then that we would agree to recommend to the FDA--
	DR. SIEGEL:  Given concerns about hand-counts, we've prepared some punch cards.
	CHAIRMAN SALOMON:  Only if representatives from industry, pharma, university and the NIH are all present.
	DR. WILSON:  Right.  We're going to distribute some punch cards.  Is that not it?
	CHAIRMAN SALOMON:  Very funny.
	DR. GORDON:  Just a quick procedural question.  I'm sort of an ad hoc or a visiting member and I was under the impression, when recruited, that I was a nonvoting member, but in the preamble this morning, the implication was that I would be voting.  Please instruct me.
	MS. DAPOLITO:  Yes, you have been appointed as a voting member.  I apologize if that was not made clear.  You do have the option of abstaining.
	CHAIRMAN SALOMON:  Again, for those of you have not been on the committee before, I just want to clarify  that these votes are not going to determine the next President or the next FDA policy, for that matter.  These are recommendations of the committee and they're meant to just give a very clear message on specific points.  We won't vote on everything in the next two days.
	With that said, I think everybody is clear about what the motion is, and a set of hands would be okay, Jay?  Those who say aye, if they could please raise their hand.
	[Show of hands.]
	MS. DAPOLITO:  11, yes.
	CHAIRMAN SALOMON:  And nays?
	MS. DAPOLITO:  One nay, so we are one shy.
	CHAIRMAN SALOMON:  Okay.  Actually, in the interests of making sure we have discussed this whole thing, I'm not sure we came up with a reason why you would vote nay, so could you explain that, just because again my job, I think, is to make sure that all the sides of this is on the table.
	DR. GORDON:  If this is causing some sort of grinding halt to the proceedings, I can abstain or something.  I felt the discussion did not result in an absolute consensus that, prior to initiating a Phase I trial, all of it should be on the table, and I still think there are many aspects of that will be addressed in some of the other questions put forward.
	If one says, well, should we not accept sequences that are anomalous or unexpected, one could say that, but of course that discussion will evolve to what level of anomaly or unexpectedness is acceptable.  This can go on and on, so that is why I voted nay, to reflect that there was this--more complexity of opinion expressed.
	CHAIRMAN SALOMON:  Again, it was gray, I just wanted to make sure everyone understood why you would vote nay.  One thing I did not say, and I apologize, is that I do always encourage the audience to participate.  This is a public meeting.  There is a microphone there, and if you choose to come to the microphone, which I see a gentleman doing, then just identify yourself for the transcription.
	I would like to make the same point to our invited speakers, and they should specifically use the microphone there on the left by the podium.  Again, I would encourage them to participate, and the only thing is I'm going to try to stay on time.
	DR. BYRD:  I'm Paris Byrd, from Maxogen in Redwood City, California, and I want to address the issue of sequencing AAV vectors at the production lot level.
	To actually do that requires at least one entire production lot.  The amount of DNA that is in one of these production lots for AAV would then go for sequencing and you would not have enough DNA to actually do an entire sequence by the standard sequencing methods that are now in use.
	The question is would you accept then using it in its plasmid form, which is one step removed from the actual production lot?  The size is not really captured for adenovirus, as it would be for AAV, in terms of what is feasible.
	CHAIRMAN SALOMON:  That is a really good point.  I think we should have some discussion of what we are exactly talking about sequencing there, but I think the general intent right now, with this first, question, was just that you had the complete plasmid that you started your experimentation with sequenced, and in a few moments we will get back to talking about should you also have sequenced the master producer line and/or clinical lot.  That is a good point.  Thank you for bringing that up.
	FLOOR QUESTION:  In order to answer this question, then, what are we actually sequencing?  Will it be the production lot?  Will it be intermediates?  I'm not quite sure that we have an answer that we would want to live with now, based on this, and one thing that's not captured is are we talking about GLP-validated sequencing or research-grade sequencing?  They are two very different things, and for AAV, we're talking not about $20,000, we're talking in the range of $200,000 to $250,000 to actually dedicate an entire or multiple production lots to actually getting a sequence on the final production lot under GLP.
	CHAIRMAN SALOMON:  Excellent.  Excellent point.  Yes?
	DR. CHAMBERLAIN:  From my own personal point of view, I mean, I think these are good issues and they apply not just to AAV, but to other viruses.  What I was voting on, at least in my own opinion, was that the starting material, which in most cases is going to be a plasmid, should be sequenced.  I think the question of whether one would also want to sequence a production lot or viral growth, we should come back to on point three.
	CHAIRMAN SALOMON:  I agree.  That is what I assumed we were voting on, and Carolyn, that follows the discussion we had yesterday, that that is what we would be voting on, so this would be the plasmid initially.  The idea is that we don't know there is like a gene stuck in the middle that some fellow put in trying to help us along, and got past.
	DR. SAUSVILLE:  I think that is a key point.  I think the spirit of this should be that latitude needs to be potentially available to the FDA, to make the situation reasonable to each biological situation and to be driven by the science of each vector.  The key point is exactly that there is a definable starting point as to where you then evolve the sequence for clinical use.
	CHAIRMAN SALOMON:  There actually was a question raised there that, I think, might be worth a few minutes of discussion, and that is what are we talking about when we're talking about sequencing.  Before we go on to question two, there's a question I had a little bit later in my notes for discussion, but I think it actually is not inappropriate to kick it around for a minute here.  I think, as we just heard, there are different kinds of sequencing that we could do, and you could sequence once or sequence 100 clones.
	What exactly does the committee think about that?  What should we define in general terms to the FDA, sequencing?
	DR. MULLIGAN:  I would say simply just getting the right sequence, that at this point, as opposed to one of the other questions, it's just that what you bring in to begin whatever process is what you thought it was, and so I think people know how to sequence, and I think the criteria is getting the right sequence, not making any errors.
	CHAIRMAN SALOMON:  That would be saying that this initial 40 KB sequencing is academic-quality sequencing, where we trust each other.  It is reasonable.  There isn't a whole open reading frame that got stuck there and we know if there are some deletions in the starting material.
	DR. MULLIGAN:  I guess one more point, and it's that I think we keep getting off on what if there are other sequences, and I think we should look at it very philosophically, just knowing what we have in our hands, okay, and we'll address the issues at another point, in terms of if it looks like there are other things we didn't think.
	So, it is really having something where you know exactly what it is, and then there is a separate interpretation of whether that is okay.
	CHAIRMAN SALOMON:  Very good.  Agreed.  Dr. Anderson?
	DR. ANDERSON:  Let me just ask our FDA colleagues, is the FDA position that an academic sequencing, assuming that all the data is there and so on, is adequate, or are you going to require GLP-level sequencing?
	DR. NOGUCHI:  The really correct answer is at all levels, regardless of who does it.  We want to make sure the information you supply us is accurate and is controlled and is reproducible.  That can be done in academic center or it cannot; it can be done in the pharmaceutical industry or it cannot.  It depends on exactly what you submit.
	CHAIRMAN SALOMON:  I have a series of questions that follow, sort of what does it practically mean to sequence, I left for later, but I think we will just follow them now.
	The second question then is, I think everyone would agree, but if you disagree, that this could be manual or automated sequencing, core lab or contract lab, and if there's any discussion there--it doesn't seem like it.  The next question is that you should submit the sequence, and the question now is what does it mean to submit sequence?  How should it be submitted?
	One of the things that I feel fairly strongly is that sequence should not be submitted as just a run of A's, G's and C's, but rather should be submitted electronically and submitted with comparison to relevant published data banks, and that should be specifically stated.  Right now, there is no requirement or even request for electronic submission of sequences.
	It would be, I think, valuable if the rest of the committee agrees to make a recommendation to the FDA that at least there be electronic submission of the sequence, not of the IND.  It is there any discussion of that?  This is just on how to submit sequence to the FDA.
	DR. BREAKEFIELD:  We were discussing what if you had some contaminated sequence and it was being kind of generated in multiple vectors, kind of the example we saw with the salmon sperm.  It would be nice of these sequences that would then be available to other investigators that are sequencing, you know, that this database would be accessible to some extent.
	CHAIRMAN SALOMON:  Well, that, I think, we should defer to our FDA colleagues, and a lot of that I don't think could be publicly available, because if a company submits a sequence on their vector, I don't think there is any law that would allow that to be publicly available.  Again, I would defer.  I don't know if a lot of academic investigators developing new things, intending to sell them to pharmaceutical companies, are going to share their sequences publicly, either.  Any comment from the FDA?  That is pretty clear, though.  Right?
	DR. NOGUCHI:  That really gets back to the accuracy of the data.  We would expect that people know they need to control their own systems internally, and if you have a problem, that you would address that.  You can always ask us for help or advice on that, but if you have contamination of your whole system, you need to clean it up.  That is independent of FDA evaluation.
	CHAIRMAN SALOMON:  The next question would be does everyone agree you should sequence both strands of a double-stranded DNA vector or plasmid?
	DR. CHAMBERLAIN:  I think that gets back a little bit to what we mean by an accurate sequence.  You said what we submit should be accurate and reproducible, but I think a typical academic sequencing lab that is just going to do one-pass, one-strand sequencing is going to have an error rate of about one percent.  If you do both strands, you're going to drop that error rate, but even the professional labs doing the genome sequencing that are doing 6X, 10X redundancy are getting error rates of one-in-10,000.  I don't know how pedantical we want to get here about error rates, but I think the difference between one strand and two strands is mostly a matter of what your error rate is acceptable at.
	CHAIRMAN SALOMON:  That's actually what it intended to do, was to reduce your error rate.  That was question--that was my last question, is what would be the error rate you would tolerate?  As we all know, there's an error rate of about one-in-1,000 bases with automated sequencing.
	DR. NOGUCHI:  Dan, I think what we're asking here are really advice on the major points.  The technical details is one reason why the specific guidance FDA will have will need further discussion and interaction with reality as to what can be done.  We really unfortunately need to move and get some very fundamental questions on the table, which we have already alluded to in our previous discussions.
	CHAIRMAN SALOMON:  Good enough.  If everybody is comfortable, I realize it takes time, but the issue here is we keep talking about sequencing without any discussion yet about what sequencing means.  I think I was detecting some people were uncomfortable with it.  Let's move on to question two.  Vectors greater than 40 KB in size, it's proposed that coding sequence of the gene of interest, transcriptional control regions, and regions including and flanking any sequences that are altered during production of the vector should be determined prior to initiation of a Phase I clinical trial.  This is very early.
	For this group of vectors, which now are the herpesvirus vectors, the poxvirus, EBV and--should the full sequence be determined prior to initiation of a Phase II clinical trial or later?  That is the next thing we need to discuss.  I guess the specific proposals are the extent of flanking sequence determination that would be adequate.
	DR. MULLIGAN:  Just to get the ball rolling, I think how you actually do the sequencing would be a tough issue, but I think I would very strongly push for having a complete sequence of the herpes vector, EBV vector, and I think the issue of do you sequence every new vector is perhaps something I would not have great concerns about.
	I think that every vector or the parent vector, ought to be sequenced, and some are, some are not, and I think this is the genome generation, and I think having information, when you can get that information, is something that is very important.  I'm interested to hear what Xandra thinks, but I think with these big DNA viruses, I think there are still questions about whether isolates may have certain sequences.
	They could have unusual pathogenic characteristics, and sometimes the biology of the test systems may not identify those characteristics.  If we were to see patches of human sequence in a herpes isolate, one particular researcher's herpes isolate, that could be very, very important.  It could point us to other pre-clinical tests to see whether there was something important or not important about that sequence.
	I just think that this is a generation where, when you can get the information, you must get that information.  I think the cost-benefit is a benefit.  There is a real benefit to having a sequence, but whether, again--to reiterate, I don't think that you necessarily want to make everyone, every time they put a gene in a herpes vector, do a sequence.  I think there ought to be strains that are used by people, and when they use it, if they use to choose to use a big virus, it's not that they're penalized for using a big virus, but if they're using it, it's more complex and provides more risk.
	CHAIRMAN SALOMON:  What we have to focus on now is do we accept the basic principle that you need sequence, which means then it is really why do we not hold that principle to a larger vector.  That is basically the question.
	DR. MULLIGAN:  Yes, one more point that might be helpful to have the FDA talk upon, is the proposal here I found is interesting, because it says when you go to a Phase II, then you ought to have it, and I think it would be helpful to have their perspective.  My feeling is it is just information.  You must have that information before you initiate a Phase I, and I'm curious as to the reasoning about why in the Phase II do you all of a sudden have to have that information.
	CHAIRMAN SALOMON:  Good point.  Okay.  Dr. Sausville?
	DR. SAUSVILLE:  So the question allied to some of those considerations is--one thing about the size cutoff is clearly the really new creations would likely be most frequently, particularly plasmid-derived or things of that nature.  I think the question I put out on the table for people who actually work with these viruses, is does the community of herpesvirus workers or poxvirus workers have a sort of repertoire of common strains that could potentially addressed this, that as long as you start from someplace that you know where everybody came from and there is sequence or information, and then what you are going to be doing or what the individual investigator is going to be doing is going to be editing that in relation to their local and regional context.
	That might be, to me, a more defensible compromise that wouldn't call for making this simple distinction on the basis of size.
	CHAIRMAN SALOMON:  Dr. Whitley has gotten up, because I was going to call on him in a second anyway.
	DR. WHITLEY:  I will let Dr. Breakefield join this discussion, as well.  I think it goes back to what Dr. Anderson said earlier, and that is there are potential risks versus benefits in terms of sequencing the entire genomes of the "herpesvirus family".  I don't think it's wise to say herpes simplex is the same as cytomegalovirus or Epstein Barr virus is the same as HHVA.  I can well-understand Rich's argument about KHSV and EBV.  I think the argument is a little bit less cogent when we talk about herpes simplex and varicella.
	Cytomegalovirus is somewhere in between, but it is certainly going to be a risk-benefit analysis in terms of whether you sequence these viruses.  It is a tremendous amount of work even in the era in which we live, I wouldn't deny that, and it will be very costly, more costly than the smaller genomes that we talk about sequencing.
	To address the other question that was put on the table, are there prototype herpes simplex virus strains that have been used in the engineering of these viruses?  For the two that are currently in clinical trials now, one that Bob Martuz has developed that is known as G207, and the other, which is known as NV1020, which is a variant of a candidate vaccine strain that was studied 10 years ago, they came from different parental lineages.
	Whether other herpes simplex viruses arise from wild-type viruses remains to be seen, but for EBV, the folks who are working with that, there at least four strains of EBV that are being used for vectors.  So, it is going to be very complex and has to be very carefully thought out by this committee.
	CHAIRMAN SALOMON:  Rich, before you sit down, for those of you don't know, Dr. Whitley's doing herpesvirus vector and clinical research with them.  First of all, when you say it is difficult to do, how long would it take you to fully sequence a clone?  How hard is it to do in real terms?
	DR. WHITLEY:  I don't know.  What would you say, Xandra, from what you have been sequencing at MGH?
	DR. BREAKEFIELD:  We just sequenced the amphlicons, because we tend to use that system.  I have to agree with you, taking on sequencing of the herpes genome, there are so many repeat elements in it and it's not just the size.  I do think that sequencing is getting better and better, but right now, that would definitely be prohibitive to an individual investigator trying to move forward, just because of the difficulty right at this point in time.
	If you are going to decide on some strain backgrounds and then sequence what they've manipulated, at least initially maybe, when sequencing methodologies improve, you know, make that a requirement.  But, right now, it would definitely slow things down.
	CHAIRMAN SALOMON:  To both of you, one of the things that I think Dr. Sausville was commenting on would be let's say I want to start--I decided I'm excited.  I'm going to do a herpesvirus trial tomorrow.  I call one of you up and I get what?  Do I get a plasmid that has already been sequenced, even though I know everybody has messed around with it for umpteen weeks or months or years?  But are there sort of parental strains?  I think that was one of the questions.
	DR. WHITLEY:  I can tell you what we're doing, and I am going to speak for Bob Martuz, as well, because we sort of have been doing this together, and Xandra can correct me if I'm wrong, but for G207, which is his virus, the inserts into ribonucleotide reductives have been sequenced, the deletions have been sequenced, and about 1,000 base pairs on either side of the deletion have been sequenced.  That is a standard virus that is being given out either by Bob or by the sponsor, who is developing that particular virus for therapeutic indications.
	For the viruses that we have made, we have sequenced the deletions.  We have sequenced the inserts, so we know basically where we stand with that, and we're giving out the same parental stock.  It is one parental stock that has been aliquotted into 1,000 lots, and that's the virus that goes out.
	CHAIRMAN SALOMON:  The point is that what I would get from you as I started my trial is basically not a fully-sequenced vector.
	DR. MULLIGAN:  That is correct.
	DR. BREAKEFIELD:  They kind of come in two forms.  They have been cloned into the F-plasmids, also, and typically we digest with like 10 different restriction enzymes, because there is instability and there is a lot of change that goes on in herpes vectors just as their passage.  I think if you don't do sequencing, you definitely have to do a very extensive restriction analysis of the genome to make sure you don't have major rearrangements.
	DR. GORDON:  I just want to say there is a little bit of reverse logic expressed in the question, I believe, here, too, in the sense of implying that you would want to be more careful with a vector after it cleared its safety trial than before it cleared its safety trial.  Here you are saying let's not worry about the full sequence until after we know it is safe in a Phase I trial, and once we know that, we should sequence the whole thing before doing a Phase II trial.
	I think there is a reverse logic there, which, if corrected, could allow you to escape some of these problems.
	DR. SIEGEL:  Let me address that question, not to at all suggest what the right answer to the question on the table should be.  The first thing to point out is that virtually all clinical trials are safety trials, certainly Phase II and Phase III trials are products in development, which extend exposure to much broader populations than--you know, if you do a Phase I trial in one dozen people, you could events occurring at a 30 percent or 40 percent frequency that you just don't happen to see.
	So, they are all safety trials.  Now, the question was raised before about the logic of phasing in a requirement, and I have to say a lot of different types of requirements are phased in during clinical development.  The whole notion of good manufacturing practices and of process validation is phased in.  There was a question about--Abbey had a question about getting rid of contaminating virus that was talked about, autoclaves and acid treatment.
	If you were making something for production, you would probably have to test every corner of your autoclave to ensure that it did, in fact, function as anticipated to get rid of that contaminating virus, but we don't impose that at any early phase of development for those sort of pragmatic reasons French was mentioning.  We might impose some practical standard that you have an autoclave that is known to reach the right temperatures and is appropriately tested, but with a simpler form of validation, and then we phase in requirements with size.
	Many, especially in biotechnology, many, many, many products that go into Phase I do not go into Phase II or Phase III.  Requirements that come a little bit later, but as you get into Phase II or Phase III development, you both get increased risks because of increased exposure, but decreased costs overall, because you may be screening 10 molecules in a few patients in Phase I for that one you're going to advance, or 10 viruses in this case.  The actual screening costs decrease tenfold.
	There is a tradition and a logic here, which is that more testing later on can provide a higher increment to level of safety.  There is, as presaged by French's earlier comments, plenty of precedent for taking one level of safety concerns in early studies and advancing them as you move on, one level of controls to address safety concerns early and advancing as you move forward.
	CHAIRMAN SALOMON:  The answer that Dr. Siegel is giving you is it is not unreasonable to have less of a safety profile from the point of view of not knowing the whole sequence as you go into a Phase I, but as you go to the Phase II or later.  One of the issues we ought to deal with in a second is when we think we should have more sequence, and I will get to that in the next point.
	DR. SIEGEL:  And since you recharacterized what I said, I do want to make clear that I was not making a statement specifically about whether it's reasonable not to have that in Phase I.  I was simply making the statement that, in general, it has been seen to be reasonable to increase certain types of controls and requirements through development.  I have no expertise on which to base whether or not sequencing of herpesvirus should be done before Phase I and prefer not to comment in that regard.
	CHAIRMAN SALOMON:  Okay.
	DR. MULLIGAN:  All that being said, who in the FDA did suggest that in Phase II there should be complete sequencing?  I would turn then the question in the opposite direction.  Why is there a reason at any point then to know the sequence?  I know I have my opinion, but I'm curious, if it's necessary at some point, why does it become necessary at any point?
	DR. NOGUCHI:  We are making practical decisions and I think part of the answer is what French said, in an ideal world, all sequence before you do an experiment, but that is not technically feasible in the case of herpes.  I think that point is quite cogent that even within a passage, you get multiple rearrangements and we have not even yet gotten to the question of what do we do with sequence that we do have in hand.
	Part of this is a balance between what we can, as a group, actually do in a timely enough manner to make product production move forward, as well as the consideration, well, if you took the information being needed absolutely, then herpes and some of the poxviruses wouldn't move until the technology to sequence those accurately come into being.  We are going through a series of both risk-benefit evaluations, as well as practicality, trying to move things forward in as safe a manner as possible.
	We are transferring some of this discussion to the public domain, so that you can actually understand why we are on some occasions being somewhat arbitrary.  Everyone has picked up, in a way, you're sort of reversing your stance on the small versus the large, but the fact of the matter is the large, you cannot get as much accurate information at this moment in time, and it may take awhile to get that.
	The corresponding question is, as a society, which is what this is going toward, should we then prohibit anything moving forward that is larger than 40 KB until we have the methodology to get the basic information, such as sequencing?  That does not address it all, but I think that is our basis of thinking.
	CHAIRMAN SALOMON:  I think that is well-said, Phil.
	DR. SAUSVILLE:  I just wanted to pick up and potentially ask Dr. Breakefield to amplify on this line of, shall we say herpes biology, because to follow on what Phil was alluding to, one basis for what was called reverse logic is exactly that, the sequence that you get with respect to herpes, with respect to present technology, would not ultimately be as meaningful as the sequence with the smaller.
	That might actually be a reason, since I personally don't want to see herpes use prohibited, I think we just need to make this balance, again basing it on the biology of the system.
	DR. WHITLEY:  I think what Dr. Breakefield said before is obvious, and that is serial passage of these viruses leads to genetic change.  We're going to have to be very careful about that, and certainly the critical trials that we do, that has been built in as one of the standards.  The one point I want to make is that I don't think sequence data in and of itself should be taken in the absence of relevant biologic models, and we're going to talk about that this afternoon.
	It is one thing to talk about sequence, but do not isolate that discussion from the relevant biology of this virus.  We know how it behaves in certain systems and hopefully we can spend some time with those issues later today.
	DR. BREAKEFIELD:  Really, in my heart of hearts, I think we should sequence everything that goes even into Phase I before it goes into patients.  I think the government should provide us the means to do that.  This is an important thing to do, and it is just the logistics of it right now that if you have people kind of developing these vectors in academic institutions, they don't really have the resources to carry that out.
	I find the situation of arguing for something just based on the practicality of it--that I think is actually a good idea, and I feel uncomfortable about it, and I certainly would abstain from this vote just because of that, but I think it is a very hard call.
	CHAIRMAN SALOMON:  I think that is really well-put, and that, I think, is the whole reason we're here this morning, and this has got to be one of the major questions I think we will deal with in this morning's session.  What are we really telling the FDA?  I think we're really going to have to face this as a hard thing.  At one point, we accept the overall concept that we do not want to stop the development of new programs and new technologies.
	At the same time, we have a responsibility to the public to do it safely.  It is okay if the message to the FDA is that you feel as experts that it should be sequenced, and that then the recommendation to the NIH and the FDA would be to provide alternative ways to do it.  I think we have to be ready to make that sort of recommendation.
	MS. MEYERS:  A few years ago, in the early development of gene therapy, we quickly saw that it was going too far into the commercial sector.  By that, I mean the science was so basic at that point, it was much too early to go into to the commercial sector, but it was going there anyway, a lot of it because of the cost, and then the government set up some vector manufacturing facilities for academic scientists.  I think there are three of them.  Do they still exist?  Okay.  Which was not enough, but it was something, at least.
	The government really should do something to set up a facility that could do this type of sequencing and cater to the academic scientist, because, if not, then the field will continuously be in the commercial sector before it is ready.
	CHAIRMAN SALOMON:  I would just point out for the record that the national gene vector laboratories, which do produce vectors for clinical trials and is, of course, supported by the NIH, does not do sequencing of vectors.  That is all done by the sponsors or the investigators.
	DR. MULLIGAN:  I think one piece of all this we're missing is expertise from the real high-tech sequencing people.  I think it would have been helpful to have some of those people, because I think the cost of sending it to a contract laboratory is very different than sending it to the Whitehead Institute or someone who really knows how to do large-scale sequencing.  I think this is high-tech gene therapy.
	We ought to use the highest tech that we have in the country and I guarantee you we will find people who will be able to do this, and I think we would be shocked by the cost.  That is, there are companies, there is a whole range of people who really look at sequencing in a very different fashion than any of us, and I don't think they would look at a herpes sequencing project as anything more than trivial.
	MS. MEYERS:  I think that the public is generally very uncomfortable with the idea of poxvirus as things being played with in laboratories.  If you do not assure the public that it is being done in the safest manner possible, then you're making a big mistake, because one accident will be just a dramatic effect on this whole field.  We have already seen, for example, what one bad example has done to this field.
	I cannot vote for that the way that is written, because I feel strongly that the first person who takes the first dose should be assured that it is a safe as possible.
	CHAIRMAN SALOMON:  We get back again to the idea is the sense of the committee at this point that there should be full sequencing of any vector and that this arbitrator division by vector size is just arbitrary?
	DR. SAUSVILLE:  I remain troubled by the issue that, with current technology, and maybe again there is better people to do this, the meaning of the sequence that is going to be generated for the larger viruses, because they have to be taken apart in order to get the sequence.  There is going to be sort of an uncertainty, sort of, factor always operating, and that is in contrast to the smaller pieces.
	Again, I just ask the question, and again following on Dr. Anderson's concern about incremental gain for required activities, by just saying we require a certain level of sequence or a sequence above a certain size, are we ignoring the practicalities of what that information means at this point?
	CHAIRMAN SALOMON:  I think, I mean, that is a question open to discussion, but the point is, I think what Abbey is trying to make us cognizant of is what we're saying to the public then is we're going to go forward with a Phase I clinical trial with a vector that has been around for years, been passed through several different laboratories and has never been fully sequenced.  I'm not going there.
	DR. MILLER:  Can you just update me on where we are with these viruses and the INDs that are already filed?  Are they ongoing?  How much of the water is over the dam already?  If we have clinical experience already in the INDs that have been filed and have been started, on the herpesvirus and the poxvirus, it's not like the first patient getting the poxvirus is going to be prevented from getting it.  The first patient, I assume, has probably already gotten it, but I may be wrong.
	DR. WILSON:  That was in the graph that was presented this morning, but may have gone too quickly.  Herpesvirus, we have two clinical trials ongoing, and poxvirus, we have about 12, although there are, I don't know exactly how many, but poxviruses are also being used in Office of Vaccine as vaccines, as well.  This number only reflects those that are being used for the purpose of gene transfer type of clinical protocol.
	DR. MILLER:  Not being a gene therapist, there is more heterogeneity in the smaller, in your potential for the smaller, less than 40 KB, than--is that not true?
	DR. WILSON:  No, I didn't think that it is necessarily true.
	DR. ANDERSON:  Just out of curiosity, what is the status, what are the requirements in the DNA vaccine field?  I mean, there are lots and lots of trials with vaccines, with pox and the rest of it.  If those are not being sequenced, then Abbey, what we're talking about is there are already 10,000 patients who have gotten it, and to say, "Oh, we have got to sequence it before our first patient."
	DR. WILSON:  I don't know exactly what our Office of Vaccine sequencing policy is, but I can be certain that they aren't requesting full sequence analysis of a poxvirus being used for a vaccine.
	CHAIRMAN SALOMON:  I am a little bit stuck here, because I want to try and give the FDA the answers to the questions.  That is about the only thing I'm supposed to get done.  At this point, I'm a little stuck because I'm not sure what I should push as the next question.  If we're kind of agreeing--Carolyn, if you want to comment, I mean, at this point, I'm getting the sense from the committee, and don't worry, I don't think we're calling this to a vote, right, we agreed on that.  This is just discussion, so everyone should relax.  We're not going to try and come to a vote on it, but what I'm hearing from the group is no one really feels comfortable in this idea of a principle applied to smaller vectors versus larger vectors, simply on the size.
	Yet, Dr. Sausville several times made the point of sequence is sequence, what is the significance of sequence, and I think that's a fair point that I can't respond, but I think Dr. Mulligan's response to you might be, well, yes, I don't know what every sequence means, but at least it's a damn good starting point for genetic manipulations of materials intended to going into human patients.  I mean, if there's a big open reading frame for a transcribed protein, then I think we ought to know about it.
	Carolyn, do you have a comment that you want to--
	DR. GORDON:  I just wanted to comment that I sense, as a quasi-outsider here, that people are looking for information that will mollify them and make them feel more comfortable with using these vectors, and for psychological reasons, perhaps as much as scientific, they have latched upon sequencing as the type of information that they want.  I am not all certain that sequence information is, perforce, enlightening, and I just wanted to say that I'm not so sure that it always is enlightening.
	CHAIRMAN SALOMON:  I think Dr. Whitley has made the point to us, and I totally agree with that, that no one is trying to say that sequencing is the only demonstration of safety or responsibility before going on.  He is saying you have to do the animal models and we all agree with that.  What we are dealing with, though, is sequence information, a piece of information that should be required, and that is the key.
	DR. CHAMPLIN:  You know, the issue that Dr. Anderson had raised is what is the relative balance in the small vectors that was a small cost for potentially a large benefit.  Here, it is a big cost for potentially a small benefit, in terms of really understanding what is going on.  I'm not sure, I think it may be more of an impediment to progress than a facilitator of progress, to make a rigorous sequencing requirement for the larger vectors when it is both impractical and hard to interpret.
	CHAIRMAN SALOMON:  Well, I guess that is what is sticking me right now.  I mean, what is it you guys are saying?  I have not got it straight.  Is it a large cost for a small benefit or is knowing sequence important?
	DR. CHAMBERLAIN:  Well, I agree with the previous speaker, that I personally would not be in favor of requiring sequencing determination of an entire large vector, because I think the information you gain is not worth it.  In the case of the smaller vectors--well, let me back up a minute.  I think what we really need to get it, as Richard said earlier, is that what you're producing is what you think it is, and the amount of sequencing that you need to do to confirm that with the small vector, it doesn't take that much more to complete the vector and that can be done rather inexpensively.
	I don't think that argument really holds up for vectors that are approaching 200 KB, even though our large genome sequencing centers are routinely sequencing tens of hundreds of, you know, bacs (ph.) a day.  There are difficulties in giving them new clones.  They generally agree to take on larger-scale sequencing projects only as a subcontract, where you pay them, or if they have a collaborative relationship where they are going to get co-authorships out of that.
	I don't think they are going to be that receptive to suddenly having these large vectors dumped on them, without them getting anything in return for it, and the important issue, I think, is to make sure that when you have constructed a new herpes vector or whatever, that it is essentially what you think it is, and I believe that can be done by sequencing the relevant regions you have modified and then following that up with extensive restriction mapping.
	Until there's some mechanism to make it affordable to do this large-scale sequencing, either through improvements in technology or a huge infusion of new funds from NIH, that we should stop short of making that a requirement.
	MS. MEYERS:  It seems to me that this whole discussion, the determination of whether it should or should not be sequenced, is based on finance, and that doesn't make any sense.  It's just a matter of how much is it going to cost, and I don't know whether I would be willing to risk one of my kids going through a Phase I trial in gene therapy when the investigator has to admit that he does not really know whether the construct of the thing is what he thinks it is, and it should not be a financial cutoff.  It should be a scientific cutoff.
	DR. SAUSVILLE:  I guess I would respectfully disagree with the contention that it's strictly financial, because if it were strictly financial, I would be in your camp.  I remained troubled with the biology--not troubled--I am struck by the fact that the biology of these viruses is intrinsically different than the smaller-sized ones, and this gets back to the question of the meaning of the sequence, because if in order to determine the sequence you're breaking apart and there's no certainty that when you put it all back together again, that that is what is going to go into your theoretical patient, what is the meaning of it?
	I think that is where somebody remarked earlier that the behavior of the construct in a relevant biological model and an additional biology needs to be factored into the decision.
	CHAIRMAN SALOMON:  Just in trying to get just a little bit of agreement on this, would you agree that even though you should not--perhaps it is not reasonable to require sequencing of everybody's vector before the clinical trial.
	I know that there's disagreement on that, but is it reasonable that the parent vector has been completely sequenced?  I think what I was trying to get at was the parent vectors, in many of these instances, have not been sequenced anytime in the recent past, which means then that you really are not knowing what you're putting in.
	DR. SAUSVILLE:  But this gets to what I had raised a number of minutes ago, that is there consensus among workers in the field as to what those parent vectors are?  I guess we have heard that for herpes, there are two that people use, but for Epstein Barr virus, there are four, and so I think--
	CHAIRMAN SALOMON:  None of them have been sequenced.  That's the problem I'm having.  If you would tell me that they were sequenced, and then it was 1.8 years since they were sequenced and it passed through three different labs, I could deal with that.  You're saying, if they're never sequenced, then I really have no idea.
	DR. SAUSVILLE:  That is a relevant question for our FDA colleagues.  I mean, the FDA has a historical, long-standing interest in defining standards for biological reagents.  Are we talking about really then, as an outcome of this discussion, asking the FDA to call for, perhaps themselves to establish, these standard strains and then they would be sequenced and that's what everybody would build their biology on?
	DR. BREAKEFIELD:  I think that sounds like a great idea.  I actually think, practically speaking, if you said there are two strains and we want you to get it like from the ATCC and put your thing in this, we would all do it.  That would make life very much easier and it would be a very practical solution to it.
	DR. NOGUCHI:  What I would like to do, Dan, is to try to wrap up this question, and I hope we can get a little discussion on question five.  Again, we're running into practicalities.  FDA is committed to a very large program for standardization of adenoviral vectors.  That is not being done at all simply by FDA.  We are in the middle of creating an MOU.  We're talking about at least four reference laboratories who will be doing the characterization, which now will include sequencing.
	The production of the vector itself may come in through competitive bidding.  We're talking about a multi-component, both academic, private industry and federal government working collaboratively to do this.  We cannot put this on any one entity, none of this, because there simply isn't enough wherewithal at any one entity to complete all the tests, all the production, all the quality control that needs to be done.
	I think if the outcome is a recommendation that consideration be made for really addressing the issue of these larger vectors, we're comfortable with that, but we are really not comfortable with being given mandates for which we really have no current funding or would ever appreciate getting that.  We're committed to many things, but we also need a whole lot of help in that.
	DR. GORDON:  I just want to say quickly how very good it sounds to try to introduce the principle of sequencing the parent vectors.  It has been asserted here that these vectors are biologically distinct from the smaller vectors and therefore it may be more relevant to sequence them that way.  That may not always be true for the smaller vectors.
	There may be smaller vectors that have different biology in the future where parent vector sequencing makes more sense or whatever.  I think it is sort of a way of jumping that bridge between the below-40 and above-40 KB, and it is a way of introducing some practicability into all of it.  I really like that notion.
	CHAIRMAN SALOMON:  Phil, when we discussed this yesterday, Steve and Carolyn and I looked at each other and said there is no way we are going to get this done before noon, and that doesn't surprise me, but yet I think these are really important questions, and that's why I haven't jumped ahead out of this discussion, because I think this is a really important question.
	What I would like to do is take a chairman's prerogative and add another 15 minutes now and about a half-hour after lunch to try and get at some of this, because I think we should go on.  We can try going on to question five right now, but I think question three has got some important issues there.
	One of the issues, just before we leave this one, what is still bothering me a little bit now is one of the issues was if we accept the fact that you do not sequence the 40 KB, greater than 40 KB vector, and I'm not even going to try and do a consensus this time, because I don't think there is a consensus around the table here on this, but what is begging an issue then is do you ever sequence it?  If you do not sequence it before the Phase I, does the committee have a comment or discussion on do you sequence it before Phase II?  Do you sequence it before Phase III?  Do you sequence it before licensing?
	DR. BREAKEFIELD:  I guess I feel that most of the innovative work is kind of done in Phase I, and if it looks promising, hopefully some company or something will move forward with it, and then there would be the funds to sequence it.  I think it is more that first step of trying to get into a Phase I that I would certainly like to encourage the academic investigators to be able to do.
	I would also like to restate that they should be sequenced even at that level, but right now it is impractical.  I agree with Richard that you can sequence big pieces, but these have a lot of repeat elements and you would have to have somebody committed to doing it.
	CHAIRMAN SALOMON:  But you like the idea that you would have to sequence before going to Phase II.
	DR. BREAKEFIELD:  I--
	CHAIRMAN SALOMON:  I mean, I'm not trying to put words in your mouth.
	DR. BREAKEFIELD:  I--that's what I would--
	CHAIRMAN SALOMON:  Okay.
	DR. SAUSVILLE:  I would certainly take the position that before Phase III, which is looking at marketing that's going to go on for some large number of years, you would want sequence even if there are ambiguities in meaning, because what you're basically defined are patterns of behavior that you're going to expect this construct to exhibit over perhaps decades of use.
	The issue of whether to require it before Phase II, I think that's going to depend on the nature of the disease, nature of the indication, the level of--whether a Phase II would be possible if we did require it.  I guess what I'm saying is I would still be ambiguous or desire the sequence, but live without it going into Phase II, but would really want it before Phase III.
	DR. MULLIGAN:  I don't see the consistency of that point.  I think you're trying to raise the question that I have been troubled with, that it either is or isn't important to have the sequence, and what is it other than the cash, essentially?  We should address it, either it is or it isn't, and if it is at Phase III, I don't understand your reasoning why it is all of a sudden at that point.
	I think, as Xandra was saying, it is the cash, that maybe you've got someone to sequence it, but I think that's maybe now the reasonable question on the table, is is it ever important to have that sequenced and maybe divorce it from the cash issue?
	CHAIRMAN SALOMON:  I think one of the things that we can maybe just not come to consensus on is I think what Phil is trying to say is if we get too specific here, we're going to also reduce the flexibility the FDA has.  Someone will come back and say, well, your advisory committee said you had to sequence it, which might be a dangerous position to take.
	Again, I'm making this all public.  I may be a little concerned, as chair, to go there, that we'd come out with an official statement here.  I think there is enough disagreement and lack of consensus here that I don't think we can give the FDA that sort of recommendation, but I think I've been very straight in the record that I see--I'm a little confused.
	The only thing is this whole thing recently, with the gene therapy scandal or whatever, if that happened, if somebody died in one of these trials and, you know, the Washington Post and New York Times published that we had no idea what the sequence was of the vector that went in, I just don't know how that would play the next time Jay and Phil confront Dr. Frist, but I think that's their problem right now--thank God--if the sequence was fully known and something about it had not been noticed, especially if the vector was 200 KB.
	DR. MILLER:  I think that's the best point, I mean, if you believe doing the sequencing would prevent the deaths or severe untoward outcomes in these studies, such that you would not want to expose patients, then yes, you should sequence.  If we're saying that we don't think, with these large viruses, that there's going to be enough benefit gained to potentially make both Phase I and Phase II trials at least able to be done in a reasonably or safe and sort of broad-reaching--not broad--innovative patterns, I mean, Phase I and Phase II are still looking for evidence, you know, safety plus some preliminary evidence of efficacy before you take it to a large Phase III trial.
	They're more likely to be investigator-sponsored or small-group-sponsored, as compared to a Phase III trial, where you really need a big support.  So, I mean, I'm saying that if you feel that it is truly that sequencing is going to save it, then I think we would all say vote, but I don't think the feeling of the people--that that is going to be the major vector on whether this is a safe or not safe thing to do.
	DR. SAUSVILLE:  I would agree, and that is the  basis for the potential perceived lack of consistency that you pointed out, in that when you get to the level of Phase III and what is entailed by Phase III, by definition implying marketing, then at that point I think it is reasonable to demand the sequence, but to not stymie investigation and to allow this broad reach is where I, at least, was trying to make the balance.
	DR. MULLIGAN:  I think there's just a lot of opinions upon the usefulness of sequencing information.  I think, if you talk to the genome people, there's one point of view.  There are certainly people like John that do not think the same way.  But I think this is exactly the issue, what is the value of having sequence information?  And I think there are just two camps.  There are some people, and I am in that camp, that thinking that having that information is better than not having it, and I think there's a good chance that it can be helpful in the future.
	Other people will say, though, it is just too complicated.  What if you miss the thing?  But you'll never catch the thing if you do not have the sequence.  I'm going to stop talking, but I think the reason I'm so really positive about pushing this is that I think this does have a very important implication for the evolution of gene therapy in the future.
	I think the whole discipline ought to think about having some perceived rigor that it may not have had.  I think that although you could even say this is a symbolic type of thing, it is just having information.  I think Abbey appreciates that, that having the field say, look, we may not be able to understand information, which is always said about the genome project, but we have the information.
	I think your point is very well-taken.  If there's a death due to a herpesvirus vector and no one can figure out what happened, and, in fact, people said this never occurred before in the preps that we had before, you might be concerned that there was some variant herpes strain.  What would you do?  I would almost guarantee you that someone would ask the people to sequence that herpesvirus vector.
	DR. TORBETT:  I have somewhat of a philosophical question.  It would be really useful to have this information, but if there is no way to access that data and compare it to your own parental strain, then the data remains meaningless.  There has to be a way of taking that information, comparing it to what you have, and know that your particular strain is within a certain realm or not.
	I think that is something that has conflicted me during this whole discussion, not so much with the smaller viruses, but with the larger viruses, and perhaps the committee would like to comment on that point.
	DR. MULLIGAN:  I think that we've been drifting from a compromise.  You know, the first thing was sequence your herpes vector.  The second thing was that is too tough, let's use something that has been sequenced.  I think you're getting to the point of maybe that, in practical terms, does not make much sense, you know, once you're slightly off the mark, you're completely off the mark, and I think that is a good point.
	DR. TORBETT:  I am in favor of sequencing, but what are you going to do with the information once you have it, and I think that's what I think some of the committee, at least I feel, is somewhat conflicted about, what can you do with that information if there is no way of figuring out what it means or even having access to other pieces of information other investigators have?
	I know it's not the FDA's point to comment on these, because these are proprietary pieces of information, but that somewhat conflicts me here.
	CHAIRMAN SALOMON:  I think that one thing that you could say is if you find sequence information that is, let's say, base pair changes that are conservative or not, change an amino acid here or there, at least you can now begin to see whether or not, for example, there is immune response in your animal models reflecting these changes.  You could look to see whether or not you might be--I think more critical is whether there are whole sequences that have been adopted.
	This viruses, as you well know, have survived in evolution by grabbing host cell genes and incorporating them and then using them functionally.  The idea there could be a chemokine that was in there or a fake Class I molecule, all of which are examples in CMV and EB virus and HHVA, and I think those are the things that Dr. Mulligan and I are concerned about.
	I have to say I continue to be more on the bullish side of sequencing, but--
	DR. SIEGEL:  As a nonexpert, let me ask, though, particularly about that last risk.  Those larger changes, such as insertion of a major open reading frame that might be a Class I or a chemokine, to what extent would restriction mapping or other techniques less fine than sequencing likely detect those or exclude them?
	CHAIRMAN SALOMON:  That is question three.  I thought we would get at that at lunch--after lunch--not at lunch.  Excuse me.
	DR. SAUSVILLE:  Right.  It bears, though, on how compelling it is to do sequencing.
	CHAIRMAN SALOMON:  I agree, Jay.  That is really a good point.  I'd like to kind of wrap up this morning with the idea that we will spend a half-hour after lunch trying to get at question five and then three.  Is that okay, Carolyn, as an order?  We sort of did four, but the question here would be if you are in the camp that was advising the FDA not to do the sequence, and there is considerable sympathy for that as a recommendation, are we then suggesting there is no sequence, or are we going to go back to that we should do 40 KB, just not--
	DR. SAUSVILLE:  No, I would say--I mean, the question is written here, you know, the parts that are relevant, I would categorically agree we should have that sequencing.
	CHAIRMAN SALOMON:  I just wanted to get this part on the record.
	DR. SAUSVILLE:  We're talking about what is not in those parts.
	CHAIRMAN SALOMON:  Obviously, I'm just trying to end here by making sure that we do not miss getting on the record a critical point here, and that is does everyone agree, regardless of whether you want to sequence the whole vector or not, does everyone agree those areas of the vector that have been specifically modified by the investigator, particularly the insert and/or the promoter regions and the linker sequences, they should be--are we at least agreed on that?
	Do we agree that it should be 40 KB, that you take the middle and go 20 KB in either direction?  That was my suggestion, just that I didn't like the idea that someone working on adenovirus got punished by having to do 40 KB sequencing, but someone working on herpes did not.  I am being partially facetious, but at some point here you should decide how much flanking region you're going to include in this, and I suppose we ought to leave it at that.
	Well, again, I think we've pretty much summarized this morning's discussion.  Do you want any more summary, Steve, Carolyn?  Are you okay with that, and are we okay with the idea of a half-hour after lunch to deal with question five and three?  I have been told no more, though.  Please.  Okay.
	It's 12:30.  I would like to start again at 1:15.  Thank you all very much.
	[Whereupon, the committee recessed for lunch at 12:32 p.m., to reconvene at 1:15 p.m.]

[1:29 p.m.]
AFTERNOON SESSION
	CHAIRMAN SALOMON:  I would like to begin now with the afternoon session.  However, as I said before the break, I am going to put in 30 minutes of discussion on topic one now, before, and then everything will just be pushed back a little bit, and I apologize to the speakers and to the audience if they had timed their afternoon and I'm messing it up.  I think that I feel comfortable, after having a chance to just think about what we did this morning, that no more needs to be said about this morning's discussion.
	I, in discussing it with each of the FDA people--people got the sense that they heard what we were saying and I think we've done a responsible job representing the diversity in the community and there are no complete answers, and I think I am content with that.  So, let's go on and deal with question five, and I am going to try and, so it is 1:30 right now, just see how we go on five, and if we're not getting there, I'd like to sort of skip on just so we can briefly discussion question three, so we will see how we do here.
	So, question five is--I have to go back and forth, because I have my notes on one sheet, but I have Jay's version on another.  Hold on.  Okay.  Should unexpected sequence and/or open reading frames be identified during analysis of the vector sequence?  What additional steps should be recommended?
	This is now picking up, I think, where we were at, at the end of the morning session, specifically what do you do with sequence?  I think this was asked by several members, Dr. Sausville, Dr. Torbett, so what do you do with the sequence if you have it?
	If unexpected sequence is identified during analysis of a vector sequence at any point now.  Let's not get hung up whether it's before Phase I, Phase II or Phase III.  What should you do?  For example, A, expression analysis of the reading frame for RNA and protein; B, if sequences identify open reading frames, analysis should include retrospective studies of the subjects, for example, an antibody response or, I would imagine, a T-cell helper response or a cytotoxic T-cell response to the potential protein.
	If sequences identified include transcriptional control elements, should we look at expression pattern of the vector sequences?  Is there any discussion of that?  What should we do if unexpected vector sequences are found?  Again, a resounding silence here.  That is fine, then we go on to the afternoon session.  They'll be happy with me.
	The point here is that I'm not going to let you out of it this easy, though.  We all agree that there are going to be sequence differences, because even if there's nothing else going on, most of these vectors have spontaneous mutations.  There are errors in the sequencing procedure.  There are homologous and non-homologous recombination.  So, I think we have to face the facts that whenever the sequencing is done prior to licensure, there is going to be sequence differences, deletions or insertions, and we have to say something about this.  We can't just be silent.
	DR. SAUSVILLE:  I think the answer to the question is it is a resounding "it depends" really.  I mean, you know, clearly, it also depends when in the sequence it is detected and also the nature of the event, because we all would agree that if it were in a production lot for something that was going to go into clinical trial and the change affected the actual gene of interest, that clearly would be a basis for rejecting the lot or trying to fix it or something along those lines.
	If, on the other hand, it is in a portion, particularly for those under-40 KB entities that we likely will have lots of information, if it is in a somewhat less well-defined place where it is not obvious that it is going to affect anything, I don't see anything needs to be done about it.  I think the point about open reading frames is germane, and that even if it is a vector-related open reading frame, one could imagine some type of immune response, et cetera.  I would not necessarily even say that that should be changed.
	I guess the more detailed answer is that then the sponsor should seriously consider whether the change detected would impact the nature of the clinical question being asked or considered.
	CHAIRMAN SALOMON:  Okay.  That is one way.  French?
	DR. ANDERSON:  First off, the reason I came in late, as you know, is the big issue that we were discussing earlier was sequencing, and the point was made, Rich made, that there is all kinds of expertise in the genomics program, and 200 KB, come on.  That is sort of an afternoon's work, but it is a big deal for us.  So, Phil Noguchi and I, sitting together at lunch, and Phil and I, as we often do, say, well, how can we get around this problem, and one issue would be perhaps if the Human Genome Project could pick up these big viruses, both in terms of gene therapy, as well as vaccine development and so on.
	So, I called Francis Collins, and that is who I was on the phone to when you got started.  Thank you for delaying, by the way.  I saw you talking up here, but it took a little while to get through.  There is, of course, a procedure to go through, so we will get together and go through the procedure.  There is a reasonable possibility that this would go onto their program, and therefore there would be a sequencing of the significant vaccine gene therapy vectors, viruses.
	Okay.  Now, in terms of answering this issue, we are all going to agree on this if it is important, you have got to do something.  If it is not important, you don't do anything.  I will now argue against what I initially said.  When I first was talking, it was as a gadfly, to try to open up thinking.  I will now respond in this context to what rather horrified you in saying, well, salmon sperm DNA is curing cystic fibrosis.
	The way that could take place, in fact, is that that piece of salmon sperm DNA happens to be homologous to an enhancer region or locus control region or MAR, and SANR, an insulator or something else that then allowed the cystic fibrosis gene to be able to function better in that particular context.  If you do not have the sequence, you will never know that, and then when somebody else makes exactly the same vector, they think, it will not work.
	Having the sequence really is vital, but then the issue comes down to when do you do it?  Your starting material very well might be exactly what you think, but it changes as you go through all the production and all the rest of it.  The fact is, one needs to have as much sequence information as possible, and then what you do with it is just what Dr. Gordon said.  You have got to figure out what you're going to do with it, but if you don't have it, you can't do anything.
	It is a balance, but the general principle, the FDA has to be left flexible to be able to use its own decision on a case-by-case basis of what policies they are going to set forth in terms of what is going to be required and not required, but balancing between cost and benefit as much sequence information as possible, as late in the production process as possible, is a good thing.
	DR. GORDON:  I think sequence discrepancies fall into two categories, and the resolution at which you find these discrepancies would be irrelevant if they were categorized in this way.  One is in which you expect them to have some functional significance, and the other is when they don't.  When they don't, I think it would be a mistake to advise that anything be done because I think we could end up chasing our tails with errors and other minor anomalies.
	When things are functional or potentially functional, I think the appropriate action to take is to determine if they are, in fact, functional, and use the appropriate test mechanism, depending on the situation, to determine if they are actually functional.  Whether or not you should insist that they then be removed if their function is apparently extraneous to the purpose of the vector, is a far more complicated issue.
	I would say that dividing these sequences up into ones that examine--when examined, determined to be potentially functional and non-functional, are in two categories, and I think we can deal with the potentially non-functional ones by saying we are going to log the information for the moment, but not take action.
	CHAIRMAN SALOMON:  I think that could be a beginning of trying to come up with some kind of consensus.  You have to the sequence at some point, remember we are not getting into exactly when this is, and if you've got that sequence now, you analyze it, and you analyze it for mutations that have no clear biological or other functional significance.  Those are catalogued.  Then there could be two other kinds.
	There could be insertions that create new open reading frames or there could be new sequences that might be promoter-enhanced or other transcriptional elements.  Is that fair?
	The question I have for the experts is how good is genetic analysis of sequence for the identification of those kinds of differences?  I mean, how good are we at guaranteeing that we catch all the open reading frames, and particularly I am interested in how good are we at identifying transcriptional enhancer and other promoter elements from sequence.
	DR. BREAKEFIELD:  I would say that it seems like when people do sequence analysis for transcriptional control elements, they always find them.  I think it's hard to know when they're active or not, and that's a more difficult issue.  On the other hand, if you come across an open reading frame of any substantial size, I think even the people who find them, they're going to see if they make RNA just like--since we can't refer back, I mean, I think they would naturally want to make--whether it was expressing a protein and whether the protein was antigenic.  It is hard to believe somebody would not want to do that.
	CHAIRMAN SALOMON:  I think if you find an open reading frame and it that is clearly identified, I don't think we need knew to waste any time discussing what to do there.  I don't think there's anyone around this table who can't figure that out.  The FDA can, unless you guys disagree.
	I'm more concerned with, number one, how good are we at identifying these sequences that would be for promoter enhancer and other transcriptional elements, and if so, then what do we do, because your point is very well-taken, you know, there are all these different short sequences that could be potential interaction sites.  What do you do?  Do you have to make reporter gene constructs of each one of these now and look to see whether or not they're active in transduced cells or not?
	DR. TORBETT:  Given that, as Dr. Breakefield noted, promoters and enhancers seem to be everywhere and most of them are context-dependent.  That means they are defined as what cell they're in and if they function, and I think that kind of opens up a large Pandora's box, but I think noting that they are present and have a potential function is worthwhile.
	The next point of getting at the function could very well be somewhat problematic, I think.  Again, not all promoters, not all enhancers, function equally well, and again it is many times cell-type specific.  I would like to have the comments from the rest of the committee on that.
	DR. BREAKEFIELD:  I would say that, just like Richard Whitley talked about with the herpesvirus, there are certain things you expect of certain viral vectors in terms of what cells they are going to, in fact, replicate in, and presumably those types of biologic assays would be done and might be looked at a little more carefully if there were some potential sequences in there.
	You might say, well, gee, does it really show the same spectrum of infectivity that we saw before and I don't think that's that complicated to do and, again, I think should be probably done.
	CHAIRMAN SALOMON:  One of the ways we could say is that if you find sequences that may be representing these sort of control elements, that some work be done in the model systems for each of the vectors appropriate to each of the studies to look at genomic expression, right, transcript numbers, et cetera, to see whether or not they are significantly different than what might have been predicted from native or wild-type species.
	DR. BREAKEFIELD:  I think, since we are really thinking about issues of toxicity, that could be done, too, but I think you need to see that the infectivity spectrum of this virus, the types of cells that it infects and if it is replication-competent, what it replicates in, is like what all your toxicity studies before showed you for that virus completely--have the same ballpark.
	DR. O'FALLON:  From the simplistic standpoint of a statistician, if we were talking about Phase III studies where we have randomized these subjects into different therapeutic arms, what we are assuming we have done is give the same therapy to everybody that is in the same arm.  You're describing a situation where apparently we will discover that that is not the case later on, and you are saying, well, it doesn't make any difference when this is discovered.  Of course it makes a difference when this is discovered.
	I would assume if it was discovered before treatment was administered, it would not be administered, that something was not what we thought it was.  We're faced with some extraordinarily complicated analysis problems, I would gather, after the fact.  If we discover in a randomized clinical trial, that what we thought we had administered is not what we administered, how on Earth are we going to make sense out of that?
	We're certainly going to have to study as much of it as we possibly can in order to make sense out of it.  So, to not follow up on every lead that we discover, I would think, would make us in an intolerable situation, from the analytic standpoint.  We would not see them being able to bring to us anything that they could explain if they did not try to follow up on that.
	CHAIRMAN SALOMON:  What we're going to going into in the afternoon session, of course, is preclinical models, which is very important to keep that in context, so if there were some tumor-inducing promoter region that got stuck into this, one would hope we might find it, but that is a discussion for later.
	DR. MILLER:  The more basic question from the regulatory standpoint is not so much what are we going to do with the information, but have you already or is there already in place the responsibilities of how you report this and when you report it, because I think that people who are looking at this scientifically, when they find something, an abnormality, what I'm sort of wanting to know as a non-gene therapy person, is at what point are they going to report it and how much--how soon into the studies, as soon as they find it out, do they report it first and then do all the analysis, something like an adverse drug reaction, something you have 24 hours to report if it's life-threatening, something not like that, but is that the idea from this or is this something that you're going to say to the sponsor of the IND that they figure it all out and then they include it in their annual report, or is this something you feel should be put in more--earlier on--because I think that helps us shape how much you require, because if after they show that there is a difference and if there is an abnormality and then it is reported and then an analysis plan is then proactively worked on, that gives more comfort than if you say, okay, you can, you know, to the sponsors, figure it out and then we will hear about it down the road.  So, I wanted to get that clarified, how that works.
	DR. NOGUCHI:  In fact, the reason we're bringing these discussions out here is we're literately learning all about these things in almost real-time here and bringing them out for discussion.  Our basic concern is exactly what Dr. O'Fallon referred to, is if there something there you didn't know, then that is not the product that you thought it was, and that should be reported as soon as possible.
	Typically, what we do is when we get the information, we work directly with the sponsor to create a plan to either study it further or to change the protocol, any of a number of things.  What we're looking for here is to try to get some more generalizable principles so that we can, in fact, just as has been stated, if it's not an open reading frame, our concern and our need to have a plan in place might be less or it might not, depending on how the discussion goes, but if it's an open reading frame, I think what we're hearing is that there is an expectation that you have the open reading frame, what protein is it, and do some more analyses.
	That should be done, I would think, as soon as possible.  It's not something that you leave for an annual report.  Gene therapy is a very embryonic field, despite having been around for over 10 years.  Every new piece of information literately can help shape how future studies are done or even how the current study is being done.  All the questions you're asking are kind of we are trying to help frame our own staff for how we should proceed and we're asking advice on that.
	DR. MILLER:  I think that is more specific to each different vector in product, just like as we saw this morning, where we saw that road map of how--one of the presentations this morning, they actually did this and they showed what they found as they went through their analysis, which seemed like a very reasonable approach to the detection.
	I assume that was done based on some discussion with the agency back and forth.  I think it's more important to sort of set a threshold for when you report and how quickly, so it is very clearly stated.
	DR. SIEGEL:  We do have a regulation, it's at 312.32 in the Code of Federal Regulations, that does require 15-day reporting of information which extends beyond adverse events if, in the judgment of the sponsor, it can be animal data and it can be product data if, in the judgment of the sponsor, it has significant potential implications for safety in humans.
	Beyond that, there's much less specificity because the type of information is so much broader.  We're much more specific in our regulations about reporting adverse events than reporting each new piece of information that develops regarding a product as it develops.  However, we work closely with sponsors, and as Phil indicated, there is a fairly high level of expectation that important information is reported, reported promptly.
	That doesn't mean, though, that if it is not, it is a violation.  I do know that we know that we hear everything that happens when it happens.
	CHAIRMAN SALOMON:  I think that my comment is on regulatory sequences that might be identified.  Today, I think we have some really very good technology in DNA arrays to look at transcriptional regulation of genes, and I think that is one of the things that would be relatively straightforward, I think, for a company or even for an individual investigator to do, to do some simple DNA arrays right now, most of which are commercial.
	Yes, they can get expensive.  No one needs to tell me that, but the technology is there and I don't want to get hung up on the expense issue, but I think that if transcriptional elements were found that were not expected in the original sequence, I think it would be a minimal thing to ask for some sort of experimental data looking at transcript regulation in those transduced cells or tissues.
	DR. CHAMBERLAIN:  I think it is still a real tough issue because it depends on what you mean by a transcriptional element.  If you're characterizing a vector and you find that the CMV enhancer has somehow integrated into it, then that is going to be pretty obvious, but as Xandra was saying earlier, I think with current DNA sequence technology, for most insertions, you're really not going to know whether you have a regulatory sequence or not and it really gets to be a very tricky issue.
	The other problem is again, as we heard, the context.  If you're designing a vector for muscle gene therapy, let's say, you're never really going to know if some of that vector leaks into the spleen, that you may have an enhancer that only works in spleen and not in muscle and things like that.  I don't know what the test system would be and how you would go about doing that.
	What I might suggest is that a lot of this depends on when you find these sequence alterations.  Obviously, if it is early on, before you have started your clinical trials and the simple matter is simply to go back and remake the vector and make sure it's correct, if for some reason you find an alteration and it's very difficult to remake the vector, then I would think one would be expected to go through the typical pharm-tox preclinical studies that are required of any vector where you have made a corrective manipulation and proved that this alteration is not causing problems.
	Maybe where the critical issue comes in is if you've already been doing a lot of clinical trials and something arises in your stocks that wasn't there originally, what do you do then?  I think at that point then the responsible thing to do would be again make this decision, can you go back and clean up your stocks and go back to using the correct thing or, if you feel you do not want to do that, then you should be expected to go back to the animal studies and repeat your pharm tox studies the same way that any vector would be expected to go through the approval process and show that this new change is not having adverse consequences.
	CHAIRMAN SALOMON:  We essentially agree.  I was dealing with when you find a transcriptional element that you recognize, then you should be forced to show some genetic transcriptional regulatory data.  I realize that it can be complicated because it could be cell-type specific in a tissue and then do you go in with a laser dissection microscope and all that or did it leak to the spleen.  It can't be perfect, but at least what you're saying I agree with, that you should go back to whatever you established as the models that were approved on the entry direction for the initiation of your trial and go back and show that what you expect is what you expect and not that there is now suddenly a tenfold difference in expression of a gene of interest.
	DR. NOGUCHI:  Dan, I think we're getting the feedback that we need on that and while I would personally like to continue, we could probably spend a day here, but we do have another whole session.  If we could move to three, we would appreciate it.
	CHAIRMAN SALOMON:  Let's spend the last 10 minutes here and go to question three.  This is an issue of changes in vectors during the manufacture process leading up to the trial.  You have sort of gotten in through your original trial.  You've done your proof of concept.  You've demonstrated a potential clinical utility.  You're gearing up to do your Phase I or Phase II.  Again, we promised not to get hung up on that, and as you roll into it, you now make a master production cell bank, and the questions in three now are meant to briefly suggest what should be done at that level.
	And this now gets to the issue that this gentleman raised in the morning, of, well, wait a minute now, it is one thing for me to sequence my plasmids, but for an AAV vector, for example, if I now have to do a whole production run and sequence it, that is a serious issue.  We don't have a whole lot of time to discuss this, but is there like any key points that the group wants to make on this issue in a few minutes?
	DR. NOGUCHI:  Dan, just a clarification, I think we have to some extent beaten the sequence and--
	CHAIRMAN SALOMON:  I'm not talking about sequence now.
	DR. NOGUCHI:  Okay.  I mean, here we're talking about as you're producing lot after lot, some of the lots may not be exactly the same.  What are the most sensitive ways that we can detect that?
	CHAIRMAN SALOMON:  Phil, given that our time is short, what would be the one question you want us to comment on then in this time, you or Jay?
	DR. SIEGEL:  Just to frame the broader question, then I'll let the others frame it more specifically, but the issue we're talking about here, I believe, is not so much a change in the masters cell bank, which hopefully you establish later on, but from production run to production run, each time you expand a virus many logs to produce a lot of clinical material, you run a risk that, at some early stage in that production run, there could be a mutation, and so that lot of clinical materials in substantial portion are different from other lots, and then the gist of these questions is about how best to assess that sort of genetic stability.  Is that not right?  How can we focus best in a five-or-10-minute discussion in terms of what we could use?
	DR. BAUER:  I think the best focus would be if we could have a discussion of what kind of techniques could be applied.  I think that Jeff Chamberlain talked about some of those issues earlier in his talk, but to expand on that would probably be the best.
	CHAIRMAN SALOMON:  Let's pick that up.  If you have to do an analysis of a vector lot during production, can we do restriction mapping and, if so, how should that be done?  Is there what we're talking about?  I don't want to use the "S" word, the sequencing work.
	DR. SIEGEL:  You've sequenced and done whatever else you want to on your starting cells.  Now you've produced something to give to humans.  What do you need to do in process and at the end?
	CHAIRMAN SALOMON:  Southern blotting, PCR, quantitative.
	DR. BAUER:  The bottom line is with the techniques that are available, such as restriction mapping, Southern blotting, PCR-sequencing one lot, whatever it is, are we confident that those catch the events we want to see and how concerned are we with that, and what would be the techniques, first of all, to apply to that?
	CHAIRMAN SALOMON:  We have a lot of virus at ten-to-the-ninth viral particles, but what do you guys want to know about that?
	DR. ANDERSON:  I think what is clear is we don't know any more than you know and that is what you wanted to find out.  That is the reason for having an advisory committee.  If the advisory committee can't advise you, then you know as much as there is to know.  Certainly as much information as you can get--and you already know if you do a restriction map you will find big things, and as you be more refined, you will find smaller things.
	I think what I am comfortable with is that the FDA recognizes that there is a balance here, that you can't just say you have got to tell us everything, and I am comfortable that you are as aware of the issues as we are.
	CHAIRMAN SALOMON:  French, I think the issue that they want us to address is also is there a limit--I mean, we have to recognize the fact that any of these viral products used in a clinical trial, in vivo or ex vivo, is going to have a percentage of recombinant or deletional mutations or whatever in the mix in these ten-to-the-ninth particles.  Is there some sort of a limit to that that we want to advise them for allowing--we're talking now production runs?
	DR. ANDERSON:  I am very aware of what the issue is.  There is no question that that happens.  Those of us who have been, in part, responsible from looking at data from production runs are very aware of it, and my feeling is the FDA knows as much as we know.  In fact, you know more than we know, when you get right down to it.
	If we had lots of time, we could talk about it all day, but I don't think we have anything useful to tell you that you don't already know.
	CHAIRMAN SALOMON:  That's going to make for a short day.
	DR. SAUSVILLE:  I would just want to know if one is comparing, for example, a master cell bank with what is produced ultimately to be used in a clinical trial, why would we want to have any different criteria than what went into setting up the master cell bank?  If sequencing is as easy as it is thought to be in certain size ranges, why not sequence the product lot, as well, at some frequency?
	DR. NOGUCHI:  Not to prolong that, but I think that was one of the things that Dr. Bauer was bringing up.  If we really wanted to detect a low level of genetic variation, sequencing 100 clones would probably not even begin to be adequate, and so from a practical point of view, even if you could sequence 100 clones, could you do it in a several-week period and then analyze it throughout all the areas, do the back-crossing?  While it is an ideal situation, what we're really asking here is other than sequencing, what are the genetic tests that can give us an indication of how good that lot is or how reproducible that a lot is?
	DR. GORDON:  I think certainly sequencing 100 clones would not be very helpful.  That would only give you a one-percent resolution.  I think the test applied would have to be appropriate to the situation.  If you are looking for infectious particles, for example, that can be an extremely sensitive test.  Where that is not relevant, there are PCR, quantitative PCR methodologies--there a lot of methodologies that would be more than sequencing.
	I think one of the other issues, though, is what level of detection would you find to be unacceptable?  If you found some mutants, at what level does that mean you have to do something or not?  Every lot is going to have something in it, so I think the more relevant question is how to intervene in the context of getting that information or if to intervene.
	DR. BREAKEFIELD:  Well, I was just trying to look through this list and see what I thought would be reasonable.  I think certainly a restriction digest with a lot of enzymes, you want to make sure there's no big change, like French said.  With these vectors, you can't really PCR across all of them, but PCR is very sensitive and it could pick up if there were some changes, some more subtle rearrangements, maybe one in 1,000, like a restriction digest might be one-in-100, if you were really lucky--this might get you down to another level.
	It wouldn't be comprehensive, but it would just give you some idea if there had been some major rearrangements.  You would have to do like PCR sets across the region, and also I was struck by what Jeff said, that in some of these like gutless antibodies, you might get rearrangements and if you actually kind of did quantitative PCR and compared the relative ratios across the genome, you might get an idea whether there had been a loss of something in a major fraction, you know, and could be fairly sensitive, and then some kind of relatively straightforward gene expression profile, like you were talking about, seems like something that could be done fairly straightforward, and at least we'd be covering a little bit of the gross changes, you know, some minor changes and then some gene expression changes which we certainly want to know about.
	You have to make them doable, you know, and sequencing isn't going to help you in this case.
	DR. CHAMBERLAIN:  Yes, I agree with those points.  I would just follow up a little bit to keep in mind that when we're talking about production lots, you're going to be somewhat limited with the material that you can deal with, and so there are so many assays that you can do, and the restriction mapping, southern analysis, PCR methods, are ways, are probably the most sensitive methods that we have that will go in and pick up major abnormalities.
	I don't think any of these methods are going to pick up more than about a one-percent variation in your sample.  I don't really see any way around that.  The ultrasensitive assays that we have referred to a little bit, for example, with adenovirus, there are replication-competent adenovirus assays that will pick up one-in-ten-to-the-ninth and all that, but those are extremely unusual assays that require a specific and selectable biological event, and there's not going to be any assay comparable to that for random genetic variation, and I think we're just going to have to live with the idea of testing your vector as extensively as you can before you get into the clinic, and then just live with the limitations of the assays, that as long as it is 99 percent the way it is supposed to be, that you will have to live with that.
	CHAIRMAN SALOMON:  I think, for interest of the time, that is as well-put as I can do it.  I think we have to realize the fact that these are biologics and I think that is the spirit of the committee, if no one disagrees, that if you look at an erythropoietin production run or recombinant human growth hormone or any of these production runs, you know, then you would say, well, yeah, we need then sequencing of ten-to-the-ninth molecules of EPO before we will accept it, and that is kind of what we're doing here.
	I think we have to accept the fact as physicians and the public needs to realize that these are biologics and there's going to be variations there and it is going to have to be part of the consent process, I would suggest.
	DR. MULLIGAN:  I think, though, even out of one percent, there are certain contexts where one percent would not be detectible, say placqued assays or PCR, subtle differences, point mutations that make mutant proteins.  I think those are contexts where you would want to perhaps, in preclinical work, at least, say what are the chances that, at about one percent, I will get a mutant hemophilia sequence, so I think these are all context-dependent, but those sort of issues are very key.
	Also, I think that for the question of how heterogeneous is your virus prep, those things, I think, should be under the preclinical stage.  So, if you want to look at adenovirus vectors, say gutless vectors, I do not think it is unreasonable to sequence 100 or 200, do some sort of find restriction or restriction mapping with four cutters or something just so you can present, you know, we don't think this is a likely thing that is going to happen because we looked at 100 of these, so we know it's not going to happen with frequency.
	I don't think you would actually do that from lot-to-lot, you wouldn't test it that way, but I think we should, as Xandra said, make sure that the test we do, however gross the tests are, would pick up real rearrangements.  I know in the Wilson trial, there are gross rearrangements that were trackable back to an earlier point, in some of the virus seeds, and those things we have easily the methods to make sure that does not happen, and so those tests should definitely be on the final product that goes into the patients.
	The other thing I would mention is like with retrovirus vectors, is that some of these vector systems have a literal inherent variability, and that will never change.  If you look at your retroviral particles, you will, if you sequence enough things, see that there are differences.  I think the FDA and everyone has to be comfortable when you're using these complicated things, there are just biological processes that are not perfect and we may see more and more, with other vector systems, that the same thing does happen.
	It is possible we may have to cope with accepting a certain level of natural biological difference that we have.
	CHAIRMAN SALOMON:  Obviously, I agree with that.  I think we have come to some consensus, in that for each of the vector systems that you approve for clinical trials, we're going to have to look initially at--in the preclinical phase, to get some sort of indication from the investigator or sponsor whether or not, if you have 16 passages of a producer cell line, that suddenly 20 percent of the viral particles are recombined and mutated, and if there is that much drift, then it is probably a very reasonable question for the FDA to either say go back to the drawing board and come back with a more stable system, or impose a more strict post-manufacture monitoring, as opposed to let's say a system that came along around the same time that had a much higher degree of fidelity over the long-term, but in the end, it is a biological product.
	Okay.  Now we get to go on to what we're supposed to be doing this afternoon, and I again am grateful for the patience of the speakers who are to start the open public hearing.  Without any ado, I believe I'm okay in introducing Dr. Janet Rose-Christianson, of Targeted Genetics, who has been invited to speak.  Please.
	DR. ROSE-CHRISTIANSON:  I believe all of you should have a copy of what I'm going to be reading, but I would like to just go over some comments.  Targeted Genetics Corporation is a gene transfer product manufacturer, as well as a clinical trial sponsor.  Our products encompass both synthetic and viral vectors, delivering a variety of genes for therapeutic and prophylactic use.
	Therefore, we have a very keen interest in the topics being discussed at this advisory committee, product characterization, preclinical animal models and long-term patient follow-up.  I would like to address these topics individually.
	Regarding product characterization, from that perspective, we believe that the currently available CBER documents provide sufficient guidance at this point and in this area.  We believe that gene transfer products can be handled in a manner analogous to the approach previously used for other formerly-novel biologics, such as recombinant DNA and monoclonal antibody products.
	We also believe that a gene-transfer product has the potential for meeting the definitions of a well-specified or characterized biologic.  Regarding preclinical animal models, we do support the judicious, but not gratuitous, use of animals.  Toward that end, we support the use of appropriate animal models, using rodents whenever possible.
	The need to use non-human primates should be carefully evaluated before any study is initiated.  Each animal must be necessary to achieve the study objectives.  As more information continues to be garnered pertaining to specific vectors, we would propose that a consideration be given towards platform preclinical studies.  For example, adeno-associated virus, or AAV, is the vector that we're using to deliver genes targeted to treat several different diseases, such as cystic fibrosis and hemophilia.
	Clearly, preclinical studies must mimic the anticipated route of delivery in the clinical trials.  However, we believe that there should be some consideration given to the biologic behavior of the vector class itself.  This would reduce the need for costly and redundant studies, yet provide sufficient information to initiate clinical trials.
	We would ask the committee to consider the use of smaller confirmatory studies to evaluate biodistrubtion, for example, of the same class of vectors produced by similar methods and delivered by the same administration route, despite a different transgene.
	Long-term patient follow-up--we have amassed significant experience with long-term patient follow-up and really thank you for the opportunity to share these experiences.  This includes the lifetime follow-up for retrovirus vector trials performed in the early 1990s, as well as adeno-associated virus vector and non-viral trials, which is required by the RAC.
	To summarize, long-term patient follow-up is challenging at best and impossible at worst.  Although our experiences have been shared with the FDA, I would like to tell you about some of the problems we have incurred in performing lifetime follow-up.  Number one, once a patient has completed the study, there is often little motivation for the investigator to keep track of each subject.  The value of performing this follow-up is difficult to ascertain, because to the best of our knowledge there has yet to be a report issued from the data collected by the FDA to date.
	Number two, autopsy specimens, in general, have not been available because the request for an autopsy was denied or the investigator was not promptly advised of the death, therefore the patients had been interned, cremated, et cetera.
	Number three, we have encountered several barriers to patient follow-up, the most problematic include, A, a lack of investigator motivation to perform follow-up once the site is closed to active follow-up; B, from a scientific perspective, many of the gene transfer systems do not result in the long-term persistence of the transgene and transduced cells.  It is, therefore, difficult to justify performing expensive and complex testing on autopsy samples in situations were there is little, if any, likelihood of finding vector sequences.  Further, the false positive rate of these type of PCR assays at high sensitivity levels makes it probable that positive signals will be seen at some frequency, further confounding the issue of true long-term persistence.
	C, patients are often lost to follow-up; D, the costs associated with long-term follow-up, especially when it is a lifetime requirement, these costs are attributable to sponsor and investigator time, the need for continuing regulatory filings and FDA interactions, and long-term database management; E, to date, we're not aware that there is a compelling scientific data indicating that lifetime follow-up is really useful.
	Although the data are presumably submitted by other sponsors and investigators, there is yet to be any data or analysis released to those supplying that data.  Going forward, we offer the following suggestions for consideration in addressing long-term patient follow-up.  Number one, define patient follow-up requirements considering the following:  the vector class, the level of the effective dose; the disease being treated; the maximum number of patients needed to be follow-up, not just everyone; route of administration and the treatment being investigated.
	Additionally, we would like to suggest that we need to ensure there is some sort of sunset and/or review provision to reassess the need to continue long-term patient follow-up.  Would a maximum of five years be appropriate?  For example, we performed trials involving the delivery of the aerosolized AAV vector for treating cystic fibrosis.
	As our patients have mild-to-moderate disease, it is likely that many will survive for 20 more years.  How long should we follow these patients, especially in Phase I studies were a no-effect dose was administered?  Is this really cost effective?
	Number two, delineate the relationship of long-term patient follow-up and product registration.  Will trial completion and closure be a requirement before a product can be registered?  If so, this could be a very huge issue.  Three, issue an epidemiological report of the long-term patient follow-up data each year.  To date, we're not aware of this information being made available.  It would truly be useful to all those involved in this research to have access to this data.
	Four, assess the cost-effectiveness of this reporting with respect to patient safety.  What has been learned so far?
	Five, specifically define the expectation of sponsors pertaining to long-term follow-up and what is considered acceptable.
	Six, harmonize any long-term follow-up with the RAC.  Otherwise, there will be additional costs associated with providing different reports with differing monitoring strategies to two HHS agencies.
	Seven, use vector system and route of administrations as two major criteria for follow-up.  How many patients must we treat before we can stop performing long-term follow-up?  Can given classes of vectors be excluded from long-term follow-up at some point?  It would be also useful for the committee to provide the rationale for long-term patient follow-up.
	As you may know, long-term follow-up is not required for patients who participate in clinical trials of other non-gene-transfer drugs that are known to modify DNA or vaccines that are remarkably similar to current gene transfer vectors, yet have escaped similar regulatory scrutiny.  Is there a scientific basis for this type of follow-up being performed exclusively on gene transfer patients?
	To conclude, we believe the length of time for following a patient who has received gene transfer agents in a clinical trial should vary and be based on defined parameters.  We believe that these parameters should take into account the phase of the clinical trial and the disease being treated.  As an increasing number patients are not imminently facing death due to their disease are enrolled in these trials, the length of follow-up should be modified or appropriate to the disease.
	I would like to thank you for your time and your attention.
	CHAIRMAN SALOMON:  Thank you.  I should point out for, unless just so there's no confusion, the first part of your comments will be this afternoon.  The long-term follow-up issues will be dealt with tomorrow morning.
	I would like to invite the second speaker now, Dr. Russ Lyons from Genetic Therapies, Inc., Novartis.
	DR. LYONS:  I want to thank the advisory committee for giving me a few minutes to talk about some of our preclinical data.  My comments will be limited to comparison work that we have done in mice and monkeys with adenoviral vectors, specifically an AB3 vector that is deleted in E1, E2A and E3.  We do have plans to extend these studies to gutless vectors based on the findings that we have achieved with an AB3 vector.
	To give you background on what the study designs look like, the route of administration is IV.  The does used in both species was three-times-ten-to-the-twelfth particles per kilo, and we included four monkeys in the study and 30 mice.  The time line is shown here.  The important time points that I want to point out are the one-week time point where we took a liver biopsy on all four monkeys in necropsied 10 mice.
	We did the same type of analysis at one month, again liver biopsies on all monkeys, necropsy on 10 mice, and then all animals were terminated at the two-month time point.  The dots along the time line indicate other observations that are made.  We do every-other-day observations during the first week, including hematology and serum chemistry, and then weekly observations throughout the study.
	To show you what the clinical pathology looks like in both species, one representative enzyme, AST, both species peak at about two days after administration and resolve within one or two weeks and continue to be normal throughout the study.  The platelet drop that has been seen by other investigators, we see as well, with nadir at four days after administration and resolution by one-to-two weeks, and they remain normal throughout the study.
	What I do want to focus on are the microscopic changes that were observed in the liver, and I have summarized these here at each of the biopsy or necropsy time points.  At the one-week time point, the most prominent feature is extramedullary hematopoiesis in both mice and monkeys, although we do see a few changes with increased mitotic figures and increased actin immunoreactivity in inappropriate cells, and the mouse and the monkey look very similar in terms of the number of animals presenting these findings.
	By the one-month biopsy and necropsy, the more prominent features are prominent sinusoidal lining cells, which we believe, based on immunostaining to be stellate cells or EPO cells.  We also see now apoptosis in about half of the animals, a cellular infiltrate, more animals presenting with increased mitotic figures, and now many of the animals with actin immunoreactivity and the beginning of a laminin immunoreactivity.  We also begin to see the onset of hepatic fibrosis.
	By two months, the majority of animals show hepatocellular apoptosis, as well as these indications of an ongoing process of hepatic fibrosis.  What I would like to summarize in terms of our comparison of mouse and monkeys is that we do see very significant species similarities, both in the acute effects, the time and durations of either an elevation of liver enzymes or the transient thrombocytopenia mimic each other quite well in both species.
	The sub-chronic liver changes that were seen in both species are extramedullary hematopoiesis, activation of the stellate cells, and this is the inappropriate smooth muscle actin standing, activation of the stellate cells progresses to a morphological transformation to myofibroblasts, and we believe this is the mechanism that is initiated, leading to hepatic fibrosis.
	What I didn't mention where nuclear inclusion bodies.  By the two-month time point, we see nuclear inclusion bodies that are fibrillar in nature, that have not been described in monkeys or mice by anyone else with any other compounds.  So, these are still under investigation.  The differences that we see in mice all pertain to the sub-chronic liver changes.  The hepatocellular damage appears to be more severe in mice than in monkeys.
	There is a more prominent cellular infiltrate.  The distribution of the findings and effects in liver seem to be more patchy.  We have also detected nuclear inclusions in non-parenchymal cells.  The nuclear inclusions that I referred to up here all occur in hepatocytes and in mice we also detect nuclear inclusions in stellate cells.
	Another finding in mice that we have not at this point tried to duplicate in monkeys is the activation of oval cells.  Oval cells are liver stem cells that are activated in response to severe hepatocellular injury in instances where the hepatocytes can no longer or no longer have a regenerative capacity.
	I would like to conclude with this summary of findings.  We believe the acute and sub-chronic changes are similar in mouse and monkey.  The acute changes in clinical pathology are dose-dependent in both species.  The sub-chronic changes are dose and time-dependent.  The effects progress over time and the microscopic changes cannot be identified with standard H&E.
	Many of the changes we found required immunostaining and analysis at the ultrastructural level with TEM.  The liver fibrosis does not correlate with any clinical pathology parameter.  All the clinical pathology parameters are normal by the one-and two-month time points, yet there is a chronic process of active hepatic fibrosis.
	So, our conclusions with regard to AB3 vectors for systemic use are that mouse is an appropriate species for assessment of adenoviral vectors.  We did not detect anything in the monkey that was not present in the mouse.  The long-term microscopic evaluations are needed, regardless of vector persistence.  In the monkey, we have not been able to detect vector in the one-month and two-month biopsy, yet the process of hepatic fibrosis is continuing.
	The surrogate markers of liver fibrosis will be needed to monitor patients treated with adenoviral vectors, since we do not have any clinical pathology correlate at this point.
	Thank you.
	CHAIRMAN SALOMON:  Thank you, Dr. Lyons.  That was extremely on point to this afternoon's discussions about models.  I think these two speakers have made us a nice transition into the afternoon session, which is entitled Preclinical Safety and Efficacy Testing of Gene Transfer Vectors.  I think that really the big challenge here is to--I think of us accept, particularly as we have evolved the discussion in the morning, that safety issues are critical for allowing regulatory decisions on gene therapy trials.
	We also were reminded that these kind of preclinical models are critical measures of safety.  In other words, just doing sequence analysis, which I think was clear to all of us, is not going to guarantee safety.  It doesn't mean it's not important, but is not going to guarantee safety, and so these have become very important.  At the same time, insisting that every gene therapy trial have a primate model study is something this committee would have to consider very carefully, as the effect on trying to develop trials in academia would be devastating, if that was required.
	I think it is really important now to consider these sort of issues, is when is the mouse model equivalent or even better than a monkey model?  When is a monkey model required, and if a monkey model is equivalent, what kind of additional information we can get from it?  With that, I would like to introduce Dr. Anne Pilaro, who is going to do the FDA introduction on preclinical models.
	Dr. Pilaro?
	DR. PILARO:  Thank you, Dr. Salomon.  I'm going to speak you today about some of the uses of animal models in development of drugs or biologics or gene transfer vectors and focusing today on vectors for gene therapy.  The initial steps in the development of a new gene transfer vector can really be broken down into three areas, and that is the characterization of the product, looking at the manufacturing and the quality control issues, some of which we touched on this morning, the demonstration of biologic activity is proof of concept that the vector is doing what you think it does, and safety, and that includes toxicology testing in animals, as well as biodistribution.
	The purpose of doing the bioactivity studies in preclinical models, especially models of the disease, are really to establish the rationale for conducting the clinical trial in humans.  What we really hope to get out of these models is an understanding of the feasibility of the gene transfer, the duration and levels of gene expression and the degree of functional correction that is needed to give an important clinical benefit to the patients.
	We also hope from the animal studies to be able to optimize the dosing and the regiment for the clinic.  What is the optimal schedule for administration of these vectors?  What is the optimal route of administration?
	We also, from doing these preclinical activity studies, frequently get additional information about what species is more appropriate for conducting further testing, including toxicity and biodistribution studies.  The goals of the preclinical toxicology testing are really no different from the goals of testing for other biological therapies or drugs.
	What we want to be able to get out of these studies is to be able to recommend the initial safe starting dose for the clinical trial and a safe dose escalation scheme for humans.  We want to be able to identify potential target organs for toxicity, which leads us to be able to identify appropriate parameters to monitor for the clinical trial.  For example, if you see a liver toxicity in the animals, you would want to be monitoring patients for serum levels of transaminases.  You also want to be able to identify any patient populations that may be at risk from the intervention.
	Using the hepatic example again, you would want to exclude patients with any previous evidence of hepatic damage.  The biodistribution studies, we will hear a little bit more about tomorrow morning, but they're really designed to address two issues, and the first is the dissemination of the vector to the germ line and the potential for any transfer to future progeny.
	To date, total gonadal tissue has been identified and assayed.  It is not been germ cells themselves.  The other purpose of doing biodistribution studies is to look at non-target tissues.  Does the vector stay where you put it or does it go to a non-target organ and what are the consequences if the gene actually gets there?  This gives you information that lets you plan a better toxicology or preclinical safety trial.
	Both of these issues may be addressed in the same preclinical study.  What you want to get when all your preclinical work is done is an idea of what is the relationship of the dose of the vector to the biological activity and what is the relationship of the does to the toxicity and how far apart are those two, what do the curves look like, do they overlap or is there a good separation that gives you a good margin of safety?
	You want to be able to understand whether the dose or the route of administration or the schedule of administration can affect the toxicity or the bioactivity, and you want to be able to identify the risks for the clinical trial.  The bottom line is that animal data are important because prior to availability of human data, the toxicology and the bioactivity or pharmacology data provide the sole source of data on which safety assessments can be made.
	I want to touch for a minute on some of the models of animal diseases that model human disease that have been very useful and been evaluated in some of the gene transfer studies.  These include the Wobbler mice for ALS or Lou Gehrig's disease, the W/WV mice in Fanconi's anemia, the sparse-fur mouse model for the ornithine transcarbamalase (ph.) deficiency, the MDX mice in muscular dystrophy, the cystic fibrosis knockout mouse for CF and several non-gene-modified or non-knockout models that are actually spontaneously occurring, including the Watanabe rabbit for hypercholesterolemia and hemophiliac dogs and various mucopolysaccharide defects in dogs, cats and rats.
	These models have all been utilized for preclinical efficacy studies and, in some cases, safety studies and have really proved very useful in gene transfer research and forwarding the field.  I want to present a couple of case studies now on how preclinical data have allowed clinical trials to go forward and what the different areas have been.
	The first case study will focus on using an animal efficacy model to support the study of a gene therapy for cancer.  The disease is metastatic breast cancer.  The gene defect is not really known.  It may include several.  It may include alterations in BRCA-1 or in c-fos expression or P53 down-modulation or others.  The clinical outcome is usually very aggressive disease.  It is spread through the lymphatics.  It mets to the lung and to the chest wall, peritoneal cavity.
	There is poor response to chemotherapeutics and the medium survival is less than 40 percent over a five-year period.  The gene therapy approach taken by this particular group is they used a retroviral vector, expressing the multi-drug-resistance-1 or MDR-1 gene.  They took CD-34 stem cells, transduced them ex vivo with the retroviral vector, and planned to reinfuse them into patients after serial high-dose chemotherapy.
	The hypothesis behind this approach is that MDR-1 expression by the stem cells will confer resistance to taxol and other marrow-toxic agents, allowing the doctors to give higher doses of these agents and potentially increasing the anti-tumor affect.  The preclinical studies in support of this came both from in vitro and in vivo data.
	The in vitro data were done as proof of concept studies showing that they could get effective gene transfer and resistance to taxol in both murine and human stem cells.
	What they found in these studies is that there was a higher level of gene transfer observed in the murine than the human stem cells by about 30-to-50-percent higher.  They did see no inhibition of cell proliferation or altered cell phenotypes after flow cytometric analysis.  They also saw that the gene was functionally active.  They used rotamine-123 eflux and flowcytometry as a measure of MDR-1 function and they actually saw that following transduction, they had an increase.  Although it is somewhat modest in the human cells, it did support their hypothesis.
	In vivo studies were then planned, serial bone marrow transplants in lethally irradiated mice with MDR-1 gene transduced stem cells and escalating taxol doses were done to select for the MDR-1-positive cells.  The safety issue behind this trial was would transfer of the gene or expression of the MDR-1 gene actually affect engraftment or reconstitution or the function of these marrow cells.
	For this particular study, there was no formal toxicity testing conducted.  However, what this group did was they built the toxicology endpoints into the efficacy study and followed it out over the duration of their study.  What they saw was there was no adverse effect of the gene transfer on engraftment or on reconstitution of marrow populations.
	They had a beneficial effect on survival in these mice.  They had resistance to escalating doses of taxol.  They were actually able to treat these animals at LD-100 values for taxol and all the animals survived.  There was also support from the literature that this group used that showed that transgenic MDR-1 mice had no adverse effect on engraftment.
	In this first case study, what we have seen is an animal model of efficacy was also used at the same time to gather safety information, and this gave us a better understanding of what was actually occurring in something close to the clinical setting.
	In the second case study, I'm going to compare toxicity of vector in monkeys and mice, such as we just heard from Dr. Lyons.  The diseases is hemophilia-A.  The gene defect is a mutation in the Factor VIII gene.  This results in either defective or absent Factor VIII production.
	Clinical outcome, this is an X-linked disease that affects males only.  There about 15,000-to-20,000 males in the United States affected.  The severe form of the deficiency results in factor levels of less than one percent of normal, and this is associated with increased morbidity and mortality in these patients.
	Uncontrolled bleeding events can require exogenous Factor VIII to stop them.  Patients can be treated prophylactically with IV Factor VIII, but it is very expensive and very difficult to do.  They have to be given frequent injections.  The repeated bleeds into joints can lead to significant joint disease?  Bleeds into the--intracranial bleeding, rather, is fatal, and so there was a need to develop a therapy to treat these patients.
	The gene therapy approach was to take a mini-adenovirus or gutless adenovirus that we heard from Dr. Chamberlain this morning carrying the Factor VIII CDNA.  It is given by IV infusion to target the liver.  Preclinical studies showed that about 90 percent of the vector uptake was in the liver after IV dosing.  The vector itself contains a liver-specific promoter region, so it is only expressed in hepatocyte tissue.
	Patients can be monitored for gene presence and for expression by measuring the levels of Factor VIII in the plasma, and this is done by an ELISA assay.  The goal of this study was really to see if correction of Factor VIII levels could be achieved in a range that might be therapeutically beneficial, and the hypothesis is that correction to as low as one-to-five-percent of normal could actually lead to an improvement in phenotype.
	The efficacy data were obtained in hemophiliac mice, the Factor VIII knockout mouse models on a black six background.  Doses from 2.4-times-ten-to-the-eleventh to eight-times-ten-to-the-twelfth vector particles per kilo were given IV.  Detectable Factor VIII plasma levels were present in the plasma out over six months and the longest mouse actually went out to 247 days.  Peak Factor VIII levels were actually present in levels that are therapeutic.  They were 164-to-892 nanograms per ml, while the normal level in humans is approximately 200 nanograms per ml.
	In the hemophilic mice, they had restoration of clotting time to within normal limits.  Similar results in terms of the factor levels of production and the duration of expression were also seen in normal C-57 mice or normal bulb-C (ph.) mouse, so on a different background, you got the same effect.  The toxicity studies for this particular product were conducted in normal mice.  Animals were dosed with four-by-ten-to-the-tenth to four-by-ten-to-the-twelfth vector particles per mouse by IV injection.
	This is a single injection on day one and they were followed out in this study for 30 days.  Toxicities included a transient dose-related decrease in platelets at day four and a dose-related increase in liver enzymes, ALT, in particular, also on day four.  This is again similar to what you just saw from Dr. Lyons.
	On histologic evaluation of the different sacrifice time points, there was minimal microscopic pathology in the liver.  We were able to determine that the no-effect level dose for toxicity in this group of mice was four-times-ten-to-the-eleventh vector particles per mouse.  This group also did preclinical toxicology studies in cinemologous (ph.) monkeys using two monkeys per group, three dose levels, separated by half-log.  They also, I should point out, used the dose of 4.3-times-ten-to-the-eleventh, which is the maximal dose plan for their clinical trial.
	Again, they saw transient dose-related increase in platelets and an increase in ALT on study day three.  Histology in these animals was not done until study termination at day 30.  At that point in time, there was no distinctive toxicity observed in the liver.  These livers were no different from the control animals.  The no-effect level dose in this group of monkeys was 1.4-times-ten-to-the-twelfth vector particles per kilo.
	What we actually did was go back and compare the data from the different studies in mouse and in monkey and look across them to see what are the similarities or the differences.  This is looking at data from individual animals in terms of monkeys and mean values for different groups across a couple of different studies for the mice.  Factor VIII levels were pretty comparable in the mouse studies between the balb-C(ph.), the C-57 and the Factor VIII knockout mouse.
	The range of Factor VIII levels seen in the GLP tox study was 202-to-490 nanogram per ml.  In the two monkeys that actually were at the highest dose group, peak Factor VIII levels were seen of 88 and 24 nanograms per ml, so they did not quite reach the same levels as the mice did.  ALT was also elevated in both groups, but this is still within normal limits.
	Pointing out to the monkeys, the elevation was barely one-and-one-half times over background.  The elevation of the mice was a little bit different.  There was one outlier that actually had to be discarded because the sample was hemolyzed.  The other two animals in that group had elevations that were just outside normal limits or within normal limits.
	The platelet value is a mean value for three animals in the group, for the mouse, and it was 44,000 at day four and that was the nadir that was reached.  At day three in the monkey, the nadir that was reached was 15,000 or 34,000, so similar to what was seen in the mouse.  When we actually went back and recalculated the no-effect level dose for the mouse, it was previously presented as vector particles per animal.  When it was gone back and calculated on vector particles per kilo, it was determined that it was virtually identical to that which was observed in the monkey, 1.6 versus 1.4-times-ten-to-the-twelfth vector particles per kilo.
	I do want to point out that these data have been publicly presented before at the RAC meeting and we did request permission from Genstar Therapeutics, their representative is in the audience today, and they were gracious enough to allow us to present these data today.  The last case study that I want to present to you is how safety data in animal models compare to actual safety data in humans.
	This is a somewhat old set of information here, but it is still one of the best representative sets of information we've seen with different species, compared to the human.  The disease is cystic fibrosis.  The gene defect is the mutation in the CFTR gene resulting in defective chloride secretion and defective mucus and bacterial clearance in the lung.
	The clinical outcome is chronic respiratory infections and digestive disorders, sterility in males and death by the age of 35-to-40, although this is beginning to creep up a little bit.  The gene therapy approach for these initial trials in CF was to use an adenoviral vector carrying the CFTR CDNA.  The plan was to instill this through a bronchoscope into one lobe of the lung so that if any toxicity was seen, other lobes would be spared.
	You could also monitor for the presence of the gene in airway epithelial cells by doing brush biopsies and analyzing them by PCR and RT-PCR.  In looking at the safety profile for this, animal models of the disease were not appropriate for obtaining information about the pharmacology or the efficacy.  The CFTR knockout mouse does not have the same lung pathology as the human does.
	It has a secondary backup chloride channel in the lung that functions in place of the missing CFTR.  As a result, it has the digestive disease, but no long pathology, so it is not a really useful model to get activity information.  Basically, what was done here was in vitro studies were used for efficacy.  Safety studies were done in different animal species.
	The preclinical safety or toxicology studies showed a dose-limiting inflammation in the lungs.  This was observed in all species tested, be they rodent or non-human primate.  It was dose-related and there was a very sharp threshold.  There was only a two-to-five-fold difference between the no-effect level dose and the toxic dose.  The original clinical trial was conducted starting off at a dose of two-times-ten-to-the-seventh platforming units, or PFU, of the virus instilled into the lobe of the lung.
	A dose-limiting inflammatory reaction similar to what was seen in the animals was observed at the next-highest dose level, which was two-times-ten-to-the-ninth PFU.  This trial was stopped because of the toxicity and re-evaluation of the data was done at this point in time by both us and by the groups involved.
	What we basically did was went back and conducted an analysis of all the different species that were used, and what the no-effect level doses were.  When we actually looked across the species, the no-effect level dose was between 1.7 and 2.4-times-ten-to-the-ninth platforming units per meter squared.  The doses here are normalized to total body surface area, since lung surface area, which would be what we would expect the toxicity to be based on, scales to body surface area, and this is a more appropriate way of comparing the data across the different species.
	What is important to note is that at this point in time when these data were obtained, the no-effect level doses in the animals were approximately equivalent to the toxic dose that was observed in the human.  The toxic dose in the human was 1.2-times-ten-to-the-ninth infectious units, in this case, per meter squared, as compared to the PFUs per meter squared up here.
	We're right about in the same range and, as I mentioned, there was a very sharp threshold here.  What this wound up doing was the clinical trial was reevaluated and then redesigned.  A smaller volume was used to treat patients, since that was one of the contributing factors.  A second generation E1E3-deleted vector with some small changes in the gene sequence to make it safer was also used.
	A spray-gun approach, by which the director rather than being instilled into the lung, was actually applied locally in a tiny droplet formation.  Single and repeat administration dose cohorts were included in this trial design.  The clinical data from this trial showed that a single administration was well-tolerated up to 3 million PFU.  It was a dose-related, positive gene transfer.
	Actually, that should be three-times-ten-to-the-eighth PFU.  Repeat administrations were tolerated up to two-times-ten-to-the-ninth PFU per dose.  They actually did more higher dose levels in this trial since they had shown in the single-dose levels that they were safe.
	Gene transfer was positive after the second administration, but not after the third repeat dose in the highest dose group, and this was due to development of antibodies.  These data have been published and have been used by permission, as well.
	In summary, what we can say from the studies is that safety data can be obtained in a well-designed efficacy model where you can answer two questions at once.  Mouse studies can provide similar information as studies conducted in monkeys.  We have seen that now in two separate instances, and no one species may be predictive of toxicities in humans.  Not all toxicities may be seen in all species and even humans may not be predictive of toxicity seen in other humans.
	Our next three speakers today are going to give us a little more information about preclinical models for gene transfer.  Our first speaker, Dr. Estella Jones from the Division of Veterinary Services at CBER, will give us an education on the use of nonhuman primates in biomedical research.  Our next speaker will be Dr. Katherine High from the Children's Hospital, Philadelphia, who will talk about answering questions in an animal model of the disease that could not be answered in a normal animal model.  Our last speaker will be Dr. Richard Whitley, from the University of Alabama at Birmingham, who will speak to us about the use of aotus monkeys to address the latency issues involved with herpesvirus.
	I would like to thank you and introduce Dr. Estella Jones.
	CHAIRMAN SALOMON:  Dr. Pilaro, before you sit down, just to make sure that, if we're going to use a jargon, that we are all familiar with it.  Can you just be a little more specific as to this no-effect level dose?  There were lot of numbers thrown around and you were using it to compare the different models, so please explain that a little better.
	DR. PILARO:  In pharmacology and toxicology jargon, there are actually two no-effect level doses.  The NOAEL is no observable effect level dose.  This is the dose at which you have no pharmacologic or biologic activity.  What I was discussing today, calling the NOAEL, is the NOAEL, or no observable adverse effect level dose.  This is the highest dose a vector or drug or biologic, whatever it is that you're studying, that can be given with no discernible toxicity or no difference from control animals.
	CHAIRMAN SALOMON:  You compared them, then, in trying to come up with a way of using this to objectively compare the value of different animal models, you compared the NOAEL--
	DR. PILARO:  We compared the NOAEL, the no observable adverse effect level dose.
	CHAIRMAN SALOMON:  To another parameter that you didn't show on the slides, which was the therapeutic dose.
	DR. PILARO:  The therapeutic dose, I actually only touched on in the hemophilia study, and the therapeutic dose was actually tenfold lower than the no observable effect level dose.  You had therapeutic levels of the gene being expressed at lower doses.  That's what gives you your margin of safety.
	CHAIRMAN SALOMON:  So, that is a good--
	DR. PILARO:  Yeah, that is a good thing.  That's a good thing.
	CHAIRMAN SALOMON:  So, if your effective dose is lower than your no adverse effect--
	DR. PILARO:  If you're tenfold lower than your toxic dose, then yes, you're in great shape.
	CHAIRMAN SALOMON:  The last question is when you compared all those species, it was in the cystic fibrosis trial where you had the NOAEL's for mouse, hamster, cotton rat, rhesus monkey, baboon and human, how significant are these comparisons then?  I mean, the rhesus monkey, for example, gave you 8.2-times-ten-to-the-seventh and the baboon, 1.8-times-ten-to-the-ninth, and the human, 1.2-times-ten-to-the-seventh.  Does that mean that the baboon is not a good model for the human?
	DR. PILARO:  Actually, the data out of that study are somewhat old now.  The rhesus monkey no observable effect level dose is probably at least two logs higher now.  Those studies were conducted with early generation of vector, and it is probably not fair to have that one in there because that was actually done very early on.
	The baboon, the monkey and the mouse actually showed the same pathology in the lung.  They all had perivascular infiltrates.  They all had peribronchial or cuffing of leukocytes.  There was inflammation present.  There was edema present in the lungs.  When you actually looked at the one patient that developed the toxicity, she had a white-out of her lungs on X-ray.  She had basically infiltrates all over the place, too.  It was very predictive when you looked at it, but the mouse was just as predictive as the baboon in that particular group of studies.
	CHAIRMAN SALOMON:  Not to belabor it, but the data for the human, though, is 1.2-times-ten-to-the-seventh, which is two logs lower.
	DR. PILARO:  Well, and that was the only dose that was studied lower, so at this point in time we actually have additional data that shows that the NOAEL, the later study that I showed you, the NOAEL is three-times-ten-to-the-ninth PFU after repeated administration.  It actually is higher.  It is up in the same range as what was seen in the animal studies now.
	CHAIRMAN SALOMON:  Thank you.  That clarifies things.
	DR. JONES:  Hi.  My name is Estella Jones.  I am a primate veterinarian and I'm the chief of the primatology lab for CBER, Center for Biologics Evaluation and Research.  Today, I am going to discuss the advantages and disadvantages of using primates in biomedical research.  I will frequently try to stay away from acronyms, but the animal care world has its own life of acronyms, as you will see.  You will see me using NHPs, and that means nonhuman primates.
	I try to stay away from the use of the word monkey, because it means a lot of things and a lot of times every nonhuman primate is not a monkey to us.  Now, we know--the things that come to our head when we say advantages.  We know that nonhuman primates closely resemble us, genetically and biologically, so we think that they are good animal models for a lot of diseases and we have obtained a lot of relevant information, and they're valuable research subjects for a lot of diseases, certainly.
	But, to date, we have really only used about 30 of these species out of 200 that are available.  There are some disadvantages that come to mind, especially when you talk to primate veterinarians.  There are increasing animal welfare regulations and legislations, cost and supply limitations.  They are comparatively lower with reproduction rates.  They have very long gestation periods, if you compare that to a mouse, every 20 days.  They have a lot of special regulations, as you're about to see.
	We will start with federal agencies.  You can decide for yourself whether that is a or a con.  Department of Interior, some of these will be surprises.  Fish and Wildlife Service, actually, Fish and Wildlife regulate the Endangered Species Act.  They also enforce the Lacy Act.  Some of these things don't seem like they need enforcement, but, for instance, the Lacy Act came up with the idea that animals do need ventilation when they travel overseas.  That one should be a no-brainer, but actually that one needed an act.
	There's something called CITES, which you will see if you have ever worked with chimpanzees.  By the way, chimpanzees are not monkeys.  Most veterinarians get very offended if you call chimpanzees monkeys.  They are great apes.  CITES stands for Convention on International Trade in Endangered Species of Wild Fauna and Flora.  For that one, I will use an acronym.  Department of Health and Human Services, under that one falls the NIH.
	NIH is responsible for developing PHS-funded organizations that are ALAC-accredited.  That one, I will also use an acronym.  ALAC stands for--it used to stand for American Association for the Accreditation of Laboratory Animal Care, but now they're calling themselves ALAC International, and that one is too long to say.  There was OPRR, which is now OLAW.  We like to make up acronyms and then change the name, too.  That keeps everybody confused.
	There is the National Chimpanzee Management Plan that also falls under NIH.  The Interagency Animal Model Committee for Review of Research Protocols Utilizing Chimpanzees--there is the NIH Intramural Nonhuman Primate Management Plan, and then there is the SPF Rhesus and Cinemologous Breeding Program that NIH is responsible for, and that started in 1988.
	Then, of course, there is the Centers for Disease Control, and they are responsible for overseeing the importation of nonhuman primates into the United States and preventing the introduction of new diseases; one of those is--some of you may remember the Ebola-like scare that we had back in 1989.  I was around for that one--yellow fever, monkey pox, there are quite a few regulations with CDC, and they were kind enough to share some statistics with us, and I will get to those later.
	FDA, we have GLP regulations and criteria for viral vaccines that a lot of you are familiar with.  Department of Transportation--we're facing some problems now with importation because airlines are deregulated and they can pretty much choose what they're going to ship now.  A lot of them are refusing to ship nonhuman primates because of the disease risk.
	The Justice Department is involved with the controlled substances that we have to use to transport a lot of these species.  And a lot of states have developed laws to try to protect the cities that the primates come into with some of the communicable diseases.  If you can get past the federal agency involvement, then we go to national laws, regulations and policies.
	Really, I am only touching on these subjects, because we have a limited amount of time today.  The document that you see on your right is a guidebook for everyone who uses laboratory animals.  It is pretty extensive and it has been revised many times.  It started off being called the Guide for Laboratory Animal Facilities and Care in 1963, and it was revised in 1972, and now it's called the Guide for the Care and Use of Laboratory Animals, and this is pretty much our Bible.
	This is a new revision and it is revised about every five years.  In 1966, we had the Animal Welfare Act, and there are so many revisions on that that I cannot list them all on this document, but you can find out a lot about the Animal Welfare Act, if you're interested, on the Internet.  Every revision is listed.  There is some current legislation on pain management that is in a comment period, and if you like to comment, now is the time.  There is IPSC, and that was renamed to IRAC, and that is currently a group that meets quarterly, I believe.
	Then there is the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals.  That is a published document that is amended currently, as well.  So, you should be familiar with all of these documents if you plan on using laboratory animals, including nonhuman primates.
	The Animal Welfare Act is a very important document in using especially nonhuman primates, because as you know they are a very visible animal with the animal rights activists.  I chose to pull out Policy 12, because when you write an animal study proposal, or some people call it an ASP, you have to fill out a lot of sections and justify why you're using--why you have chosen a nonhuman primate, particularly.
	If you look at number four, the written assurance that the activities do not unnecessarily duplicate previous experiments, this particular wording means that you have to categorize your pain classification into column C, D or E.  C means minimal pain or distress, to the USDA.  D means pain or distress that you will alleviate.  In other words, if you're going to classify your animals under column D, that means that you're going to use an analgesic or have some means of relieving pain.  E means that you do not plan on intervening at all, and if the animal dies while that animal is on your experiment, then it has to be justified and you have to provide a narrative with your protocol.
	The trick is if you have any nonhuman primates or any animals at all that go under column E, these are the protocols that are targeted by groups like PETA, and there are lots of them.  It is not just PETA, but animal rights groups frequently pull all large animal protocols that are classified as column E under the Freedom of Information Act, and you may be a targeted scientist, i.e., they may follow you home and protest on your lawn and threaten your children and all of that stuff.
	That is an important thing to know, should you choose a column E protocol--that is a very sensitive category.  Importer requirements, this is a CDC requirement and you have to be registered with the CDC to important nonhuman primates into this country.  Their use has to be certified.  You have to implement disease control measures that the CDC approves of.  They have to be isolated for a minimum of 31 days.  That is if they are healthy, if they are not showing signs of clinical disease.  You have to report suspected zoonotic illness and maintain records regarding distribution.
	Also, it is important to know that if you have animals that you have arranged to come into this country, and there is an outbreak, a lot of times these animals are caught in nature and there's an outbreak of a stress diarrhea, and that might look like some type of zoonotic illness, it is your responsibility and your expense to have these animals in containment until you can prove that they are healthy.  The CDC does not take on that expense.  That is up to the shipper--I mean, up to the person that is importing.
	This was the total number of primate imports over the last six years.  CDC was kind enough to provide these statistics for me.  As you can see, we're going into a decline, because there is a fear, a concern, that we are eventually going to get some kind of zoonotic disease outbreak from a nonhuman primate.  Like I said, we had our Marberg scare about 11 years ago.
	Right now, we only have 26 registered importers.  The slide on the right was from a colony in Africa.  This species is now endangered, so you would not see this species used in research in the United States.  That is the black and white collibus monkey.  Out of all the imports last year, fiscal year 2000, ending in September, there was only a .4 percent mortality, but 2.9 percent morbidity.
	So, carriers are decreasing the numbers that they're willing to carry and this is creating a problem of availability, unless we breed our own animals at breeding institutions.  Also, this slide is just designed to show how many shipments these were spread across and also how many species came into the country.
	There were 10 carriers in the last 12 months across the continent that were willing to carry nonhuman primates.  It is not just across the continent.  There was one airline that carried nonhuman primates within the U.S. 24 times within the last 12 months.  That statistic was provided by the CDC.  As a matter of fact, my facility had chimpanzees come in last month and the researcher had to go and pick up the chimpanzees himself.  He drove to New Mexico to pick up five chimps.  From what I understand, it was not a pleasant trip.  That is dedication.
	By the way, chimpanzees are now costing $120,000 each.  Macaques, these are rhesus macaques on the right.  These are imports by country, and this is just giving you an idea of the majority of the cynos and macaques that are coming in and what countries are importing these.  This, of course, will change based on any trade restrictions that go into place in the coming years.
	If you have been reading the Post, then these numbers may definitely change.  This is also by country for African Greens and baboons.  These numbers are not nearly as high as the cynos and rhesus numbers.  I was fortunate enough to spend some time with World Health at the Institute for Primate Research in Kenya, and there they have a vast availability of baboons.  They are considered pests, kind of like roaches are here.  They bargain with the pineapple farmers not to kill them and they use them for research there.
	Another import-by-country slide, and you see the owl monkey is almost not in importation at all.  So, now we're on to quarantine, and there is a need for quarantine.  If you're lucky enough to get them here, okay, now we're into quarantine, and this is pretty self-explanatory.  You have to have a staff that is separated from your healthy staff, because if you go through all of the requirements to get them here, and you bring a sick colony in and your whole healthy colony dies, it does you no good.  So, you want a healthy colony to come in, but there is a requirement to keep them quarantined.
	Standard at NIH is 90 days and our facility is on NIH's campus, so we do abide by their standard quarantine rules.  If you get a positive TB test and they do have to pass three negative TB tests every 30 days at NIH, you have to start your quarantine all over again, this is at the investigator's expense.  This can get pretty expensive if this were to happen.
	If you get an illness in quarantine and it's nondescript, that starts your quarantine period all over again.  Also, you cannot have cross-contamination of personnel, equipment and clothing, because that just defeats the whole purpose of having a quarantine, obviously.
	Cost factors, and this really is what is driving the cost of primate research up so high, and that is really what the animal rights activists would like to see, is to make it so expensive that we just cannot do it at all, and then they have reached their goal.  Animal procurement is expensive.  The average rhesus monkey, you can expect to pay anywhere from $4,000-to-$6,000 per animal these days.
	You also have to expect to pay--these are actually NIH numbers--46 percent of your cost in caretakers' salary and benefits.  Now there's discussion about the hazards in this job, because just normal macaque work is a very hazardous job.  I'm sure most of you know that macaques carry diseases that are not threatening to them, but are very deadly for us.  So, a very innocent exposure can cost a human its life.  I lost a classmate to a monkey disease.
	Caging is very expensive and we will discuss that in a minute.  Of course, you have to provide veterinary care and all of these expenses have to be taken into account.  Housing is something else that a lot of people do not realize can be very, very expensive.  There are lots of different types of housing.  We're trying to go back to the more natural habitat.
	Again, a lot of the language in the Animal Welfare Act and the guide is to provide a more natural environment.  We're finding that you get more natural behaviors if you can provide a natural environment.  Some of these are outdoor group caging that is designed to create a more natural environment for the animal.  They seem to thrive, to be able to breed and do better, perform on the trials better, if you can do that.
	Some of these cages are designed to provide enrichment where they can be pair-housed.  Actually, pair-housing is now a requirement of the guide.  If you do not pair-house your animals, you have to provide a scientific justification of why that is not occurring.  This cage on the right is averaging about $6,000 to $7,000 right now.
	Environmental enrichment is a requirement now under the law.  It has to be natural.  You have to be able to assess their well-being.  That includes keeping records of what you're doing to enrich these animals, also what you're doing to assess their psychological well-being.  You have to give consideration to species differences and what is natural for them and be able to record their cognitive and motor enrichment, because now we're finding that psychological distress can give you an adverse research results.  That is actually not surprising.  That is something that has been shown in humans, as well.
	These are just some of your options for what we consider to be enriching.  That is a puzzle feeder.  Most nonhuman primates can figure that out in under two minutes flat.  Humans, maybe a couple of days.  These are toys.  You do have to watch the size of your spaces, because you do not want them to swallow this thing whole and then have to take them to surgery to remove it.
	We're going to touch a little bit on taxonomy.  Like I said, monkeys are not really all monkeys.  There are prosimians.  These species, I just wanted to show you, they vary so much in size, some of them are really just not much bigger than a rat.  If you're looking for vascular access, that might be hard.  All of these are considered endangered now, so you're not going to find these guys in research; same thing for the Tarsiers.  You're not going to find these in research.
	There are few places that are running breeding programs, but they don't breed very readily in captivity.
	CHAIRMAN SALOMON:  Dr. Jones, as long as there is a natural break here, how much long do you have?  I just am trying to stay on time here.
	DR. JONES:  I think I'm about four slides from the end here.
	CHAIRMAN SALOMON:  Okay.  Thank you.
	DR. JONES:  You can read these for yourself.  I won't read to you, but we split monkeys into two groups, platyrrhines and catarrhines.  These are new world versus old world.  It's just a fancy name for a new world monkey from the left, versus old world, and they are characteristically different.  If you're going to select a group, you have to know these differences, because they may impact your research results.
	As you can see, some require vitamin D3 in their diet, the others don't.  You see the ischial callosities, just pads for them to sit on, and the others don't have them.  Some have them, some don't.  The opposable thumbs just help them peel bananas and things like that.
	This is a marmoset.  Also, you have to know you that you cannot house squirrel monkeys with marmosets because squirrel monkeys carry very generic diseases that are fatal to marmosets.  Owl monkeys are used in research, especially for antimalarial drug development.
	That is a squirrel monkey.  Also, I wanted to mention that there are so many species and subspecies within each group, if they have a lot of subspecies like the African Green, we recommend that you do karyotyping because you can get different results based on just having different subspecies within the same research group.
	This into the old world monkey species here, African Green, or Cercopithecus, rhesus monkey, which is very commonly used in the U.S.; 80 percent of these macaques carry the herpes B virus, which is deadly for humans.  If you're scratched or bitten or even exposed with urine or feces through a mucous membrane, you are at risk for herpes B, which can be deadly in humans.  It causes a fatal encephalopathy.  Apes and humans are not monkeys, and the chimpanzee is used for hepatitis research.
	This is a first-response posting that we're required to have in all of our facilities at the NIH.  You should enroll your employees in what we call an animal exposure surveillance program, because this is a big risk if you have employees working with nonhuman primates, and these are the zoonotic diseases that come to mind when you have nonhuman primates in your facility.
	I also listed the various uses of nonhuman primates in biomedical research, and that list is there for you to read on your own.  And I believe we are at the end here.
	CHAIRMAN SALOMON:  Abbey, point of clarification?
	MS. MEYERS:  Before you sit down, I really want to know, the Animal Welfare Act and all of the regulations, do they apply to privately-funded research, as well as publicly-funded?
	DR. JONES:  Yes.
	MS. MEYERS:  If you have monkeys, you have a pharmaceutical company and you have these things at your headquarters, you still have to follow them through?
	DR. JONES:  Yes, you do.  That applies to industry, as well as government agencies.
	MS. MEYERS:  I would also just like to comment that there's no equivalent to the Animal Research Act for human beings.
	DR. JONES:  No, there are actually more regulations for animal study protocols than human protocols.  I used to work at Baylor College of Medicine, and we frequently got that complaint from the people who reviewed the human--the animal protocols versus the human studies.
	MS. MEYERS:  Yes, we do not have any enrichment.
	DR. JONES:  Right, no human enrichment.
	CHAIRMAN SALOMON:  Thank you very much.  I think everyone has been sitting for quite awhile.  There are two more talks before we get to the discussion, so I would like to call a 10-minute break and ask people to really be back in 10 minutes this time.  Thank you.
	[Recess.]
	CHAIRMAN SALOMON:  I would like to introduce one addition to the panel at the table.  It is Dr. Joe Tomaczewski, who is the chief of Toxicology and Pharmacology Branch, the Developmental Therapeutics Program of the National Cancer Institute, National Institutes of Health.  Ed Sausville had an internal site visit going on and asked to--that he had to leave for a few hours, but gave us his super expert, and so I think we're well-served and we appreciate your joining us.  You have to answer one brilliant question sometime in the next hour-and-a-half.
	Before we start with the next speaker, Dr. Anderson, I pointed out, had stolen my chance to make a big dramatic introduction to his concept that we could get the Human Genome Project and Dr. Collins to begin the sequencing of these larger vectors.  He says he has a follow-up, so Dr. Anderson?
	DR. ANDERSON:  Yes, Phil Noguchi and I had additional phone calls over the break, and we have now gone from a very possible to a very probable for getting these herpes simplex, pox and so on that are directly involved, not only in disease, but also vaccine development and gene therapy.
	Phil now has the ball firmly in his own hands to make direct contact tomorrow, to go about the process of determining which viruses will be done.  That's it.
	CHAIRMAN SALOMON:  I think this is the government at its best.  I appreciate that very much.  Okay, Dr. High, to talk to us about the use of the canine model of hemophilia, and then that will be followed by Dr. Whitley, and then we will go on to the discussion.
	DR. HIGH:  I'm a hematologist on the staff at the Children's Hospital of Philadelphia and a member of the faculty at Penn and our laboratory has had a long-standing interest in the molecular basis of blood disorders and on the use of gene transfer techniques to treat hemophilia.  As Dr. Pilaro mentioned at the outset, we have use the hemophilia B dog model to do safety, efficacy and toxicity studies of AAV vectors used to treat Factor IX deficiency, and the short version of what I am going to talk to you about over the next several minutes is that the major complication of our current protein-based method of treating hemophilia is the formation of inhibitory antibodies, that it is likely that this complication will not be avoided by gene-based approaches to treating the disease, and that it is, in my opinion, an obligation of investigators to assess the likelihood of inhibitor formation using their gene therapy technique in an animal model of the disease.
	As I mentioned, the major complication of the current protein-based method of treating hemophilia is the formation of antibodies that inhibit the activity of the clotting factors.  Those occur 20 percent of the time in individuals with Factor VIII deficiency and in three percent of individuals with Factor IX deficiency.
	The reason that there are serious complications, this is a picture of a boy with a compartment syndrome as he has bled into the soft tissue in the thigh, and you can see also he is unable to straighten his knees because of repeated bleeds into these joints.  The concern over inhibitory antibodies is that normal therapy fails.  It is much more difficult to control hemostasis.
	There are products that can do it, but they are much more expensive than they already very expensive clotting factor concentrates.  Individuals who develop inhibitors suffer increased morbidity and mortality.  They suffer from much reduced ability for us to maintain hemostasis during either necessary or elective surgery, and individuals who develop antibodies to Factor IX can develop anaphylactoid reactions to the infusion of concentrates.  It is a serious problem, if it develops.
	Clinically, inhibitors are measured in a unit called the Bethesda unit, which measures--one Bethesda unit is defined as the quantity of inhibitory antibody that can result in the loss of 50 percent of factor activity when the test sample is incubated with a sample of normal plasma.  Clinically, these are divided into high-responding, which is over 10 Bethesda units, or low-responding, and that is important because they are managed differently.
	They can also occur either as long-lasting persistent inhibitors or more transient inhibitors, which tend to be lower titre.  It is known from 30 years of experience with protein concentrates for the treatment of hemophilia that there are certain risk factors that predispose toward them, and first and foremost among those is the underlying mutation causing hemophilia in the individual, and so, for example, in Factor IX deficiency, many individuals who develop inhibitory antibodies have large gene deletions or early stop codons in the Factor IX gene, and if one views the underlying mutations on a gradient moving from large gene deletions through stop codons, missense mutations associated with no circulating protein or missense mutations associated with a circulating protein, there is a gradient with many inhibitors occurring here and much less frequent as you move to the right.
	The induction of inhibitors can be understood in terms of moderate concepts of tolerance.  Inhibitors are promoted by T-helper cells.  In a normal individual, during fetal development, self-reactive T-cells are deleted or energized, but depending on the underlying mutation, a hemophilic individual may not express the epitopes recognized by those T-cells and thus they persist, and on encounter with the antigen during initial treatment with Factor VIII or Factor IX concentrate, these T-cells can promote induction of a neutralizing antibody response.
	Clearly, the nature of the underlying mutation and the amount of coding sequence that is lost is a risk factor.  In protein based therapy, certain inherited characteristics of the immune response are also risk factors.  We know this because there are many individuals who have this same underlying mutation, but not all of them will form inhibitory antibodies when exposed to factor concentrate.
	The clotting factor itself can be a risk factor for antibody formation.  There was a so-called outbreak of inhibitory antibodies in the Dutch population some years ago, following the introduction of a new method for viral inactivation of the plasma used to produce the product.  Finally, there is a great deal of information in the clinical literature for hemophilia that suggests that individuals exposed to clotting factor for the first time, under situations where there is extensive inflammation or tissue injury, may be more likely to form inhibitory antibodies.
	I believe that it is likely inhibitory antibodies will also be a problem for gene-based approaches, as well as those that we currently use that rely on the intravenous infusion of Factor VIII or Factor IX protein, and one can list many ways in which antigen presentation may differ between a donated gene approach versus intravenous infusion of a protein, and this slide illustrates one of those.
	For protein-based treatment of hemophilia, the protein is exogenously synthesized and infused intravenously and antigen presentation occurs almost exclusively in the context of MHC Class II, which has as its job the display of peptides derived from proteins that are taken up from the environment and a gene-based approach where now, for the first time, the individual will begin to synthesize the protein endogenously.
	Antigen presentation will surely occur in the context of Class II determinants, just as it does for intravenous infusion of the protein, but may now also occur in the context of Class I determinants which have as their function the display of the peptide fragments that are derived from proteins synthesized inside the cell that displays them.  That is just one example of the differences between antigen presentation and these two different approaches.
	Work by a number of investigators over the past 10 years has demonstrated that a number of these factors listed here may be risk vectors for inhibitory antibody formation to clotting factors in a gene-transfer approach for the treatment of hemophilia.  I should stop and say that I am talking about hemophilia, but these remarks in general apply to genetic deficiency states characterized by the absence of some circulating secreted protein.
	The vector itself can be a risk vector for inhibitor formation.  The target tissue that is chosen, the dose of vector, the presence or absence of tissue-specific promoter elements and immunomodulatory maneuvers at the time of antigen presentation.  I will not have time to go through all of these factors, but I will show you one example of experiments that we published earlier this year, looking at the role of the vector itself and immune response to the transgene product Factor IX when a vector is introduced into skeletal muscle of mice.
	For these experiments, we made either an AAV Factor IX vector or an adenoviral Factor IX vector and injected it into the hind limbs of mice and at time points later sacrificed the mice and examined them for the presence of CD8-positive cells, and you can see these were present in the context of adenoviral vector, but not in the context of the AAV vector.  In additional experiments, we harvested lymphocytes from the draining lymph nodes and the splinocytes of these animals and carried out experiments looking for cytotoxic T-lymphocyte response  specifically to Factor IX, so use target cells expressing Factor IX and used the harvested lymphocytes as the effector cells and were able to show that there was Factor IX-specific CTLs in the lymphocytes derived from the Ad-injected animals, but not from the AAV-injected animals, and in additional experiments we looked at CD4 profiles, again from lymphocytes isolated from animals that had been injected either with Ad Factor IX intramuscular or with AAV Factor IX intramuscular, and you can see that when these lymphocytes were stimulated with Factor IX, lymphocytes from the Ad Factor IX injected animals produced interleuken-2, interferon gamma and IL-10, whereas the animals that had been injected with the AAV Factor IX had at most a limited IL-10 response to Factor IX antigen.
	Clearly, the vector itself can be a determinant of the immune response to the transgene product.  As Dr. Pilaro mentioned, we have done a number of studies in hemophilic dogs looking at other determinants of inhibitor formation in the setting of gene transfer in this approach to treating hemophilia.  I'm going to talk a little bit now on some of our studies that analyzed dose as a determinant.
	For these studies, we were using an AAV Factor IX vector, and you talked about AAV earlier in the day, so I'm not going to spend a lot of time, but the strategy that we are currently using involves the introduction of an AAV Factor IX vector into skeletal muscle.  In earlier experiments, we had demonstrated in immunodeficient mice that the introduction of the vector into skeletal muscle could result in long-term expression, and actually this went out for more than a year, long-term expression at levels that would be therapeutic in humans.  These correspond to levels of five-to-seven percent of normal circulating Factor IX and would convert an individual with severe hemophilia into one with mild hemophilia.
	Based on those studies, we wanted to extend our studies into the hemophilic dog model, and as I said before, one of our major goals in using the hemophilic large animal model was to determine the likelihood of an inhibitory antibody response using this particular strategy of introducing AAV Factor IX into intramuscular sites.
	For these experiments, we collaborated with the canine hemophilia B colony, with a group that runs the colony at UNC Chapel Hill.  The defect in these dogs is known and was determined some 10 years ago.  They have a missense mutation at a highly conservative glycine residue in the catalytic domain of the protein.  They have normal Factor IX transcript levels, but no circulating Factor IX antigen, and they have severe hemophilia B, less than one percent activity.
	The experiments that we did made use of the vector that is diagrammed here.  It directs the expression of canine Factor IX--this is a very important point--that without the use of a species-specific transgene, these animals will rapidly develop antibodies, and it is not possible to continue to follow the work.  Using a CMV enhancer promoter and a synthetic intron to derive canine Factor IX CD in an expression, we carried out the experiments outlined here.
	Our initial experiments in dogs were actually done as a dose-escalation approach, starting with about ten-to-the-eleventh vector genomes per kilo and moving up to nearly ten-to-the-thirteenth vector genomes per kilo.  The dogs ranged in size from six-to-20 kilos, so the larger ones were as big as a nine-year-old child, and then they underwent intramuscular injection of vector at day one of the protocol and have been followed since using a number of coagulation, hematologic and chemistry clinical pathology measurements, as well as serial biopsies of the injected muscle tissue.
	One point that I would make, which is an advantage of the dogs over mouse models, is that, of course, dogs are very long-lived compared to mice, and we've been able to follow these out now for a period of over three years.  What we've seen in the dose escalation study is that the more vector injected into intramuscular sites, the higher the circulating levels of Factor IX.  The blue line across the middle here denotes 50 nanograms per ml, which would be a level of one percent in humans, and levels above this, we know from experience with clotting factor concentrates, should improve the clinical phenotype in an individual with hemophilia.
	So, we have been able to follow these animals for long periods, over three years, with the study continuing as long as we continue to be funded.  We have also done serial muscle biopsies in the animals and these are muscle biopsies done six weeks after injection and again two years later, and you can see there is no evidence of inflammation or degeneration in the muscle, and the dog vector was co-injected with carbon particles, which shows up here as a sort of reddish material and allows us to go in and biopsy exactly the right location, and you can see again the carbon particles are evident here two years later, and although there is freeze artifact in this muscle, again, there is no evidence of any inflammatory infiltrate or evidence of deterioration of the tissue.
	Then, we also did immunofluorescent staining for Factor IX and injected muscle tissue, and you can see cells that demonstrate the fluorescent stain here.  We did serial chemistry studies in these animals, again over a period of years.  Except for transient elevation of the creatinine kinase immediately after injection, which is also seen when saline is injected in the animals, there was no evidence of any changes following vector injection, no changes in hematologic parameters.
	We did viral shedding studies in the animals, including collection of semen from the male dogs that were injected and saw no evidence of transmission of vector in the semen and other serial studies have all been negative for any toxicity.  In addition to the toxicity studies in dogs, we obviously did toxicity studies in other species, as well, including mice, rats and rabbits.  Dogs were important for these studies, and also another important issue we were able to address in the muscle biopsies was to look at gene transfer on southern blot from the biopsied muscle, and without going through it in detail, I will just mention that using undigested DNA, vector sequences are detected as a high molecular weight smear, and the genomic DNA is cut with either a single-cutter or a double-cutter within the mini-gene cassette, it releases fragments of the appropriate size and allows us to estimate gene copy number, which in a dog is generally about one-to-six copies of vector per diploid genome in the biopsied muscle.
	Finally, this other issue, the issue of the formation of inhibitory antibodies, in this first sequence of dogs, we measured both on western blot and using that Bethesda assay that I referred to earlier, the presence of an inhibitory antibody in an animal that received the highest dose.  As you can see on the western blot, two weeks after injection, we first detected an antibody on western blot, and it peaked at about five-to-six weeks and then slowly receded.
	The data on the Bethesda assay tracked very well in terms of the temporal cores with what is seen on the western blot, and it peaks at about seven Bethesda units and then slowly recedes.  During the time the inhibitory antibody is present, it's not possible to measure Factor IX, but as the antibody recedes, now you can see Factor IX appearing in the circulation.
	We viewed this as an important cautionary note and, as you will see, we did additional studies on it later on, but this looked to us as something that would be important to follow up on and probably a dose to stay below in human studies.  So, a human study has been initiated based on this, and this is a study done in collaboration with Avigen and with investigators at Stanford University and is an open labeled dose escalation study with three subjects in each of three dose cohorts.
	The study is essentially an outline of what you had seen in the dogs.  The individuals undergo intramuscular injection of vector into the vastus lateralis and then we do a battery of hematologic, chemistry and collaboration studies, and periodically these individuals undergo muscle biopsies to look for evidence of gene transfer and expression.
	One thing you will see here is that in the initial studies, we did make the decision to use individuals with missense mutations, and the reason for that was because the dog studies we had done had been in animals with these relatively less severe mutations, missense mutations, and we felt it was important not to get out too far in front of the animal data that we had on this important safety issue.
	Fortunately, missense mutations account for a large percentage of individuals with hemophilia B, so we were able to do that.  First of all, in terms of inhibitory antibodies, these have been measured weekly, initially, and then monthly since the first vector injection.  Fortunately, none of the individuals on the trial have developed any evidence of inhibitory antibodies at the doses tested so far, which are still about fourfold lower than the dose where we first saw a transient inhibitory antibody in the dog study. and then southern blot on muscle DNA harvested at the muscle biopsies has demonstrate findings similar to what was seen in the dogs; that is, if you look at uncut DNA harvested from these, one sees again a high molecular weight smear, and if you cut with a single-cutter, echo R1, within the mini-gene cassette, you release the unit length 4.5 KB fragment and again copy number can be computed from this and has run about one-to-four copies per diploid genome.
	Note that these were actually subjects that were in two different dose cohorts, but because the dose per site is held constant, there's not really a substantial difference in the copy number per diploid genome.  Again, I will say little bit more about the dose per site in just a minute.
	If you look at immunofluorescent studies for Factor IX in the biopsied muscle, this may be a little difficult to appreciate, but I want to make the point here that immunofluorescent stains for Factor IX, and by the way, these look quite similar to what we've seen in mice and dogs, the expression appears to be primarily--these are two adjacent sections, one stained for Factor IX and one for slow-twice miocene, and you can see that it appears to be the slow-twitch fibers that take up and express the AAV vector.
	I won't say more about that at the minute.  I will say that, for the muscle biopsies, both immunofluorescent stains and immunoperoxidase stains are done and the immunoperoxidase stains show similar information; that is there is a typical checkerboard pattern where there are positive fibers directly adjacent to negative fibers, probably reflecting the mix of slow and fast fibers in the injected muscle.
	The reason immunoperoxidase stains are done, of course, is that they are very long-lasting, compared to immunofluorescent stains.  The importance of the studies that have been done so far is simply that they do suggest that the dogs accurately predicted what we saw in terms of gene transfer and expression in the human data that are available so far.  We did want to follow up on the evidence of dose as a possible risk factor for inhibitory antibody formation, and in additional studies done in the Chapel Hill dogs, which are demonstrated here, and this is a busy slide and it is difficult to sort through, but this is gradually increasing doses per kilo in the series of dogs, and we assessed both anti-canine Factor IX antibodies on western blot, which may or may not be inhibitory, and inhibitory antibodies, and you can see that in this series of dogs, the three animals that got the highest dose per kilogram, diagrammed here, one had no evidence of inhibitory antibody formation, one had a transient inhibitory antibody--that is the one I had shown you earlier--and one had a longer-lasting inhibitory antibody.
	Without showing you all of the data, I will indicate to you that it appears the best predictor of inhibitory antibody formation may indeed be the dose per site.  These two animals got two-times-ten-to-the-twelfth vector genomes per site, and it had either no inhibitor or a transient one.  This animal got a five-times-higher dose per site and had an antibody that lasted for a period of nearly a year and had a much higher titre, and these are diagrammed together here.
	This is the animal that I had shown you earlier in red, and then this other animal had a much higher titre and longer-lasting antibody, following a higher dose per site, and based on these data, we do recommend to the clinical investigators that they confine the dose per site to well below the dose given to this animal here.
	Hypotheses as to why the dose per site may matter are listed here, and we're trying to, in the laboratory, sort through these.  Is there a contaminant in the prep that can act as an adjuvant?  Does a higher dose per site lead to better transduction of antigen-presenting cells?  Could a higher number of viral particles at a local site change the cytokine milieu and lead to increased danger signals as the Factor IX is being produced or, finally, could the higher levels of local Factor IX expression, combined with the high number of viral particles, trigger an immune response against Factor IX; that is, could the antigen be essentially now presented in the context of a viral infection?
	We're working to sort through this, but it was actually through the dogs that we saw this as a potential problem.  Let's see.  I would just maybe mention one more set of experiments.  With intramuscular delivery of AAV vector, the likelihood of inhibitor formation appears to be dose-dependent and dose-per-site-dependent.
	To follow up on our observation that we're concerned about using individuals or admitting to the trial individuals who had mutations more severe than missense mutations, we did a series of studies with Clint Lottrop and Auburn University in Alabama, and they have dogs that have a very severe mutation, essentially an early-stop codon, and they have essentially no Factor IX transcript.  These are normal Factor IX transcript levels.  These are from the Auburn dogs.
	What we saw in those studies is that even at this dose per site, but at a much lower total dose, inhibitory antibodies developed in these dogs with a more severe underlying mutation, and even if the dose per site was lowered, this still occurred.  This is again the Bethesda titre in these animals and you can see following injection the inhibitory antibody appears, and it does seem that this can be altered by dosing the animals with cytoxan at the onset of the injection, so they got four doses of cytoxan, one dose every two weeks immediately after vector injection, and following that, they had sustained correction of the whole blood clotting time and no evidence of inhibitory antibodies.
	We have additional mouse data that suggest--that actually was the basis of performing this experiment--but the important aspect is that when we went into dogs with a more severe mutation, we did indeed see this problem of inhibitor formation, I think giving support to the idea that it was probably not wise to admit individuals with severe mutations to this trial.
	I'm going to conclude summarizing that inhibitory antibody formation is the most common complication of the current method of treating hemophilia, that is, intravenous infusion of protein.  Neither theoretical considerations nor experimental data suggest that gene transfer approaches would avoid this complication.
	One can attempt to assess these things in murine models of hemophilia, but these models are essentially very limited, because all of them are murine models, hemophilic models due to gene deletions, which are found in only a small percentage of the human population and which do not mimic the gene defect in most individuals with hemophilia, and moreover, strain differences in these mice may confound interpretation of data, and there is some very nice published work by Sheila Connolly and her colleagues at GTI Novartis, that demonstrates using a third-generation adenoviral vector, no inhibitory antibody formation in hemophilic mice, but the presence of inhibitory antibodies in hemophilia dogs using an adenoviral vector with a tissue-specific promoter.
	The dog model of hemophilia does allow assessment of inhibitor risk, which I think is really the most likely complication.  It is the most likely complication of our current method of treatment.  It is likely the most common complication of a gene-based treatment.  As I said before, I think that investigators have an obligation to design and carry out experiments that will let them assess the risk of inhibitory antibody formation, using their gene transfer technique, that requires the use of a species-specific transgene, and those are cloned and available.
	The vector, the promoter used in the mini-gene cassette, the dose, the route of administration and the underlying mutation in the recipient all will influence the likelihood of inhibitor formation, and it is probably that each of those will need to be evaluated independently.  With that, I'm going to stop.
	CHAIRMAN SALOMON:  I had one question before you step down.  I don't understand, how did you explain getting a transient antibody response in the first set of dogs that effectively cleared circulating antigen, and then it spontaneously resolved and antigen was detectable.  That's not an immune response I'm familiar with.
	DR. HIGH:  I can't tell you how many immunologists we have talked to about this, but I will tell you that using protein-based therapy for the intravenous infusion of protein, this is a very commonly observed phenomenon, and some substantial proportion of individuals who develop inhibitors have these transient inhibitory antibodies, and despite 30 years of using these protein concentrates, the immunologic mechanism of that phenomenon is not worked out and people fight about it.
	One explanation I have heard is that it is a B-cell response with no T-cell help.  If you have any other ideas, I'm interested to hear them.
	CHAIRMAN SALOMON:  We will fight about it later.  It's very interesting.  It's very interesting, because it suggests that you have some sort of tolerance induction or an antibody getting turned off, but that's not the purpose of today's meeting.  I had to ask in the context of trying to use these models to model what might happen in human patients.  It is atypical.
	DR. MILLER:  Very nice, but I have a question about whether or not it is clear that the dog model is valid until you reach your first immunogenicity in humans, because right now you have it in dogs, but you don't know it is modeled in humans because you never see it in humans, and not that I want the humans to get antibody responses, but building on that, and it's a beautiful model, but without having some positives, it's hard to say that it models.
	DR. HIGH:  That brings up a very important question, because I didn't have time to show you, but again Sheila Connolly's data from GTI Novartis would suggest that using an adenoviral vector, there's also a dose-dependent increase in likelihood of inhibitor formation, and it does appear from many studies that I did not have time to show you, that as you escalate the dose in animals, first you see, if you're going to get inhibitors, first you see transient antibodies and then you see more persistent higher-titre antibodies.
	There are two ways to look at that.  One is that you will just keep dose escalating until you begin to see that in humans, and I have real concerns about that because of the risk of inhibitory antibody formation and the lack of certainty that the first inhibitor you encounter would, in fact, be transient.  The other way you can use the data is to try to define situations that either enhance or reduce the risk of inhibitors and use that information to construct the clinical trial, and we have opted for the second strategy, not the first.
	CHAIRMAN SALOMON:  Excellent.  Thank you.  Very good presentation.  Then it is my pleasure to introduce Dr. Richard Whitley from the University of Alabama, who is going to talk about the use of aotus monkeys to assess neurovirulence of a replication-selective herpes vector.
	DR. WHITLEY:  [Off microphone.]  --vaccines for the whole family of herpesviruses, I think it is essential to weight the risks and benefits of what has been learned over the last 20 years using a variety of animal model systems to take engineered viruses from animals into humans, and this has been done both in the vaccine arena, as well as in the gene therapy arena at the present time.
	For those of you who are not aficionados of the herpesvirus family, please remember that there are eight viruses that are herpesviruses.  Three are in the alpha herpesvirus family, three are in the beta herpesvirus family, two are in the gamma herpesvirus family.  These viruses have been sub-classified according to their predisposition infect and establish latency in target issues.
	For example, for the alpha herpesviruses, we know they have a neuronal predilection and therefore establish latency in neuronal tissue and can be reactivated in that site.  That is very different from the beta herpesviruses who tend to establish latency in endothelial cells, lymphocytes and macrophages from whence they are reactivated.  Gamma herpesvirus is EBV and HHVA--excuse me--establish latency, for the most part, in lymphocytes.
	I want to make four points about this slide.  The first and the most important one is please remember that animal models for all of these viruses have been developed for the purpose of studying pathogenesis and antiviral therapy, historically.  It has only been recently that animal models of herpesviruses have been used to study gene therapy.  That is very important to remember, because if you stop and ask the fundamental question, which is really what you're being asked to address this afternoon, and that is do animal models correlate or predict benefit or harm in human disease?  The answer is only in the context of antiviral therapy.
	Whatever I say about the aotus is relevant only to the studies that are ongoing now, that are being done at three or four hospitals in the United States.  The second point that I want to make is even though there are members of a subfamily of viruses, namely the alpha herpesviruses, they behave very differently in animal model systems.
	Herpes simplex Type I is less likely to establish latency than herpes simplex Type II, although we can drive it to latency and we can reactivate it in that animal model system.  The last point about this slide is just to pursue the discussion of this morning and the ongoing phone calls that have been taking place.  There was a program project grant that has just been funded to go to Lyn Enquest, Tom Shank, Bernard Roizman and Elliott Keith, relating to the sequencing of several of these herpesviruses, and I just point out that that grant was funded for five years, not six months.
	Okay, with that in mind, I think you really need to began in looking at the relevance of animal models, whether it is in the mouse, the guinea pig, the rabbit or in the aotus, with what is the biology of human disease?  What are we trying to prove here?  Why are we doing this in the first place?  What are the risks and benefits when we think about the predisposition of herpes simplex, which is the common vector used in these studies to establish disease?
	Remember, herpes simplex viruses live on mucosal surfaces.  They cause oropharyngeal disease and they cause genital disease, for the most part.  However, they can cause herpes simplex encephalitis in adults.  That will occur in approximately one-in-150 or one-in-200,000 individuals annually, and that is what we have to prevent when we are talking about using herpes simplex as a vector for gene therapy.
	We also know that if it causes disease in the genital tract, newborns can become infected, and if they do, disease can be life-threatening.  With that in mind, let's go one step further and let's ask ourselves about the natural history of this disease, and when we think about the natural history of disease, infection enters the body from a mucosal surface.  Virus replicates as a function of intimate contact, either kissing or sexual contact, in the oropharynx or at a genital site, with initial penetration of nerves and then accession of virus to a sensory dorsal root ganglia.
	It is at this site that virus will replicate and either be transported back down to skin sites to cause lesions or, ultimately, become episomal and resident in the ganglia until it is subsequently reactivated.  Why do I worry about this?  I worry about because I have spent the last 30 years of my life trying to treat this disease, which is herpes simplex encephalitis, and it is the one disease that if it occurs in an individual who is on one of our gene therapy studies, we all have to be held accountable for understanding why this disease occurred.
	With that background in mind, I want to look at how attenuated herpes simplex viruses can be used for direct gene therapy, for vectors for foreign gene expression or as attenuated vaccines.  I will just begin by saying that for 20 years Bernard Roizman, a colleague of mine at the University of Chicago, and I have been working on the latter area, namely attenuated vaccines utilizing principles from his laboratory on the engineering of herpes simplex, and then taking them into animal models that we have tried to establish at the University of Alabama at Birmingham.
	Here is a listing of some of the animal models we used.  I don't intend to bore you with it, but I do want to point out that you can adequately and, in fact, in detail study these animals to get a better understanding of the safety, efficacy and ability of these viruses to establish latency and be reactivated.  For example, in the mouse, we clearly know that we define safety following intracerebral inoculation, either in an immune-competent or in an immunocompromised mouse, particularly a skid mouse.
	We can study efficacy and challenge experiments.  We can study latency by harvesting ganglia and reactivating them in vitro, and the systems we can use then are those of immune-competent, immune-compromised mice.  We can study genetic stability, and I'll illustrate that for you in a minute, and we certainly can study neurovirulence to get an assessment of that before progressing into subhuman primates.
	The guinea pig is a good model for latency and recurrences.  The rabbit is a great model for using the eye and the issue of establishment of latency at the trigeminal ganglia, and in my opinion, when we use the aotus trivirgatus or a nancymae, we're really looking at safety more than efficacy or the establishment of latency.  These are difficult systems to use.  This is not a system that I would recommend for the casual investigator, but we can use immune-competent, immune-compromised animals in both systems and assess neurovirulence as well as pathogenesis.
	I want to illustrate this for you with a couple of examples.  Here at two viruses that have been taken from bench into humans.  The top is the example of a virus that has deletions in the inverted repeats of a gene identified his gamma 134.5, and I'll show you how that virus behaves upon inoculation into the central nervous system in a minute.
	This virus was developed in the laboratory of Bernard Roizman about a decade ago.  Remember, the problems that we were talking about sequencing other, this is 150 KB of DNA.  There are inverted repeats that bound the unique long segment, inverted repeats that bound the unique short region, and it is going to be very confusing to sort this out, although I'm sure sequencers can do that with facility.
	The second construct is a virus that was initially known as R7020, that was a candidate vaccine that consisted of both HSVI and HSV Type II.  It currently has been reconstituted, reformulated as a virus known as NV1020, and it's entering into a clinical trial for metastases of colorectal carcinoma.  I would point out that both of these viruses have herpes simplex virus thymidine kinase, and as such they are susceptible to acylovere, and that is a fundamental principle when considering herpes simplex viruses.
	TK-negative viruses, in my opinion, should not be used in human experimentation because you cannot treat them with the drugs that we currently have available, unless you want to go to potentially toxic medications.  Why use these viruses and what is the safety data that allows us to advance them forward into human investigations?  The purpose of this study was to define the platforming LD 50 ratio, in other words, the number of viral particles required to kill 50 percent of the animals.
	This is an assessment of neurovirulence and neuroattenuation.  Those data are shown in the column on the far right.  These are viruses that are deleted in gamma 134.5, and that is shown here, compared to the parent virus, which is known as HSV1F, it is wild-type virus.  For wild-type virus, 200 viral particles inoculated intracerebrally will kill 50 percent of the mice.
	For a virus deleted in both copies, because remember this gene maps in the inverted repeats of the unique long segment, identified R3616, you cannot kill the mice with over one million particles of virus inoculated into the brain.  If we put a stop codon into gamma 134.5, it remains its avirulent phenotype, as we have to do with these viruses, restoring 34.5 restores neurovirulence to these viruses.
	With that as background then, we have a candidate virus then that potentially could be used either as a backbone for gene therapy or as a vector for foreign gene expression.  Here are the experiments that we do to establish genetic stability, and these are very straightforward experiments that utilize the mouse to determine whether or not we can address one of the two fundamental issues that herpes virologists always worry about; one is a revertant to a wild-type phenotype and the second is a second site mutation which would lead to virulence in the animal system.
	We will take a mouse, we will inoculate herpes simplex intracerebrally, on day three of our avirulent, our aneurovirulent virus will harvest brain tissue, isolate virus in cell culture, re-raise it to a stock titre, inoculate it intracerebrally and repeat this process eight times.  We will then, as we do this, continue to calculate PFLUD50 ratios.  If we see no change in PFULD50 ratios, it indicates the virus has not regained neurovirulence.
	Our ability to detect genetic variance of reversion or second-site mutations is one-out-of-ten-to-the tenth viral particles.  This is a sensitive way to screen genetic stability in the candidate herpes simplex virus that have been used.  So, where are we then with the viruses that have been developed for human administration?  R7020 was the first virus, and I'll illustrate data for you from this candidate vaccine strain from aotus.  It was administered to 33 volunteers in Lyon, France, in studies that were done with Institute Merieux.  G207 is a study that was just finished and reported in Gene Therapy; 21 volunteers who had glioblastoma multiformi were inoculated directly into the tumor stereotactically with this construct.
	This study was done by Bob Martuz of Mass General and Jim Markert of my own institution.  NV1020 is a virus that I showed you at the bottom of the viral construct slide, entering Phase I studies in volunteers with liver cancer.  The first volunteer went on that study this week.  Then the last candidate strain is a virus known as AB9395, which is a herpes simplex Type II deleted in gamma 134.5, which is undergoing evaluation for potential vaccination.
	What do these animals look like that we use?  Here is the aotus nancymae.  This is an HSV hypersensitive animal.  I want to emphasize that for you, because this represents both the pro and the con of using an animal like the aotus in evaluating potential herpes simplex vectors; 10 platforming units of wild-type virus given intracerebrally will cause fatal encephalitis.  These animals will die in a period of 10 days.
	At the present time, whether it should be or not, it is the standard for preclinical evaluation of genetically engineered herpes simplex viruses.  We can inoculate virus intracerebrally, intraocularly, intravaginally or intramuscularly and then we can perform a variety of studies that are relatively routine for other animal models in assessing outcome.
	For survival, we can look at dose dependence, site of administration versus long-term survival, clinical signs of disease, radiographic evidence of encephalitis that would occur in these animals.  For those animals that are sacrificed, we can look at histopathology, in situ hybridization, immunohistochemistry and certainly we can look at cellular pathogenesis and differential jean expression within the central nervous system.
	For virology, it's not just viral quantitation and viral isolation, but it is also PCR evaluation of where viral DNA and what viral DNA is distributed in the brain itself, and certainly we can look at foreign gene expression within the model, and I will illustrate each of these principles for human studies that we have done.  Over a period of 14 years, there have probably been four different types of studies that have evolved.  The first, Bernard Miniet, Bernard Roizman and myself evaluated R7020 in the aotus, and I think it was probably the first time the aotus, both the immune-competent and the immune-compromised, was used to evaluated herpes simplex and get some understanding of the pathogenesis of the viruses under those circumstances, and I'll illustrate those data for you in a minute.
	Four years ago, we began evaluating 9395, which is a deletion in gamma 134.5 and HSV2.  Sam Rabkin and Bob Martuz evaluated G207 and published their data in Journal of Virology last year, and then currently we're working with a herpes simplex virus that expresses IL12, and I will show you how that particular virus behaves in animal model systems.
	The first is deleted in the joint region.  The second is an HSV2 deletion with a deletion in genes responsible for recognition of the virus by the host cell.  The third construct is a 34.5 deletion, as well as a second site segmentation, and that second site mutation is a ribonucleotide reductase and it was done intentionally to avoid second site mutations that would occur naturally when administered to the host.
	The last viruses is one that I just mentioned and that is a virus that expresses IL12.  What do you learn from these experiments?  This is the aotus trivirgatus.  It was R7020, so it's HSV1 in the long segment, HSV2 in the short segment.  Virus was given by one of a variety of different routes, intradermal, intravaginal, HSV1, HSV2, the period of viral shedding was relatively brief.  These animals all died in a very short period of time with very low exposure to replicating virus, ten-to-the-one, ten-to-the-two.
	With R7020, we could administer up to ten-to-the-sixth to ten-to-the-seventh viral particles by one of several different routes, with all mice under these circumstances surviving.  This should be S, not five.  All those animals survived following administration of virus.  If we look at multi-inoculation with a TK-negative or a TK-positive virus, we can actually quantitate duration of shedding by site.
	We know the longest shedding will occur about 22 days in all animals, but we also have the opportunity to determine whether or not virus is picked up from ganglia when animals are sacrificed and dorsal root ganglia harvested, and you can see here that approximately one percent of ganglia expressed latent virus.  That latent virus is R7020 or the TK-negative virus, R7017.
	There are other things that we can do.  We have standard procedures for intracerebral inoculation of virus, and I won't go into the details of what we've done, but I just illustrate some of the work that was done first with G207 and then with virus constructs that we've made in Birmingham.  Here's the virus and the does that was employed.  You can see up to ten-to-the-ninth viral particles were put in.
	The animals were followed until they either died naturally or were sacrificed.  One animal died, for example, from an aneurysm; the other was sacrificed at 20 months to determine whether or not virus could be detected at latent sites, whether it could be reactivated from the central nervous system and whether there was PCR evidence of virus.  This is in contrast to wild-type virus, HSV1F, ten-to-the-three viral particles inoculated intracerebrally led to death at five days from encephalitis.
	You can also see that with mock infection, these patients lived their natural life course.  For those animals sacrificed  here and in other experiments to detect evidence of latent virus, one can find latent virus, but it only in approximately one-to-five percent of all ganglia which are assessed.
	We can also do repeat intracerebral inoculation.  Here, ten-to-the-seventh viral particles was put into the central nervous system to determine whether or not there was an additive effect if virus was given a second time, and the animals surviving the first series of experiments were reinoculated, followed for ten months, to determine whether or not there was any additive effect and these animals are perfectly fine and well.
	What we've done is taken this one step further, and that is pose the question what happens when you use herpes simplex as a vector for foreign gene expression?  Here, we have used IL12 to determine whether or not it had adverse effects when expressed in herpes simplex upon inoculation into the central nervous system.  No changes in behavioral or feeding patterns.  The monkeys remained--excuse me, the aotus remained normothermic throughout the period of time they were evaluated.
	There was a period of temperature to 104, which was associated with increased activity.  If we look at MRI scans on the monkeys that we evaluated in our studies, this was 10 days post-inoculation of ten-to-the-seventh, both sagittal sections, as well as sections that were coronal sections, looking for evidence of encephalitis.  There's no evidence of hemorrhage.  There's no evidence of edema.  There's no evidence in shift in midline structures, all characteristics of herpes simplex encephalitis.
	The conclusion that we reached from the encephalitis component of this study was there was no evidence of central nervous system disease.  We did follow up this monkey.  We found that the monkey developed an infection that was treated with antibiotics, and this makes an extremely important point.  It was treated with presumptive antibiotics, developed a diarrheal illness, ultimately went into renal failure with nephritis, secondary to the antibiotics that were administered.
	The reason I bring this up is the aotus is a difficult monkey to deal with.  They are fragile little creatures and one has to manage them very carefully.  If we looked at the brain upon evaluation of this monkey, there was no evidence of encephalitis or necrosis.  There was an inflammatory response in the choroid plexus, but no ventriculitis.  We did not find any evidence that led us to believe that herpes simplex expressing IL12 led to disease in the central nervous system of this animal.
	We looked for additional evidence of encephalitis at multiple other sites and we found absolutely nothing that was indicative of disease.  Studies that we will do in the future will expand the histopathology in specimens from this one monkey, to try and get a better understanding of what the nature of the initial site was, particularly as it relates to PCR and in situ hybridization on brain tissue, and we will try and push this does probably in one or two other monkeys before going on.
	I just want to make one point, and that is as a clinician who has to take care of patients with herpes encephalitis, primum no nocere.  We have to remember that herpes simplex can cause disease in the brain and we have to be exceedingly careful.  I just want to end with a couple thoughts, and that is what is okay with herpes simplex using a mouse, guinea pig, rabbit and aotus certainly is not going to be okay if you consider cytomegalovirus, human herpesvirus-6, human herpesvirus-7, EBV or HHVA.
	Each one of the members of the herpesvirus family has a very, very different spectrum of tissue trophism and susceptibility in animal model systems, and unless that is well-understood by all parties involved, you should not embark upon these studies casually.  The last slide is there are both pros and cons of using the aotus.  The pros are that this is an exquisitely sensitive animal model for preclinical toxicology, but the con is the biggest one, and that is it is too sensitive, and therefore ultimately we're going to have to find a happy medium that we can use.
	Thank you.
	CHAIRMAN SALOMON:  Rich, before you sit down, I had two quick questions.  One is, with retroviruses, frequently many of the viral particles are not infectious.  In comparing your titres between wild-type and your vector, the question is obvious, so I have a follow-up to that.
	DR. WHITLEY:  That is the standard problem that we have with all the herpesvirus, and that was one of the rate-limiting steps, I think, in my opinion, and the folks CBER don't have to comment on it, in the licensure of the Occa vaccine strain, was because the vector particles account for a significant volume of that virus, and it is a problem with herpes simplex, but to a lesser extent, and that is an issue that we deal with all the time, very difficult to quantitate.  The only thing you can do is look at infectious particles that are put into the tissue.
	CHAIRMAN SALOMON:  When you did your titres, those were infectious titres, so when you say you put in ten-to-the-sixth or up to ten-to-the-ninth, got no infection, that was of actual infectious titres.
	DR. WHITLEY:  That is known virus.  It titred before it went in.  It was titred from the syringe after the inoculation was done.
	CHAIRMAN SALOMON:  That is perfect.  The other question I have is you have a mouse model and you have this nonhuman primate model.  One of the things you and I talked about before is, is this now an example in which we have added to the preclinical development by having a nonhuman primate model, or are we find this is a nonhuman primate model that is excellent, but we could have actually found out everything in the mouse?
	DR. WHITLEY:  I think, at this point in time, the aotus adds very little to what we learned in a mouse system, particularly a skid mouse system, which is also exquisitely sensitive to HSV.  It was developed because of the concern of both Bernard Roizman and myself, that we didn't want to take herpes simplex into people and have something bad happen, but we're having to re-question that at this point in time, just to be honest.
	DR. GORDON:  Have you ever tried interrupting either wild-type virus or looking at latency rates for the recombinants--for the vector--with acyclovere in the monkeys?
	DR. WHITLEY:  I haven't done it in the monkey, but I've done it in the mouse and I could tell you what we've learned in the mouse.  We did it in our tumor model in the mouse and we've done it with both acyclovere and gancyclovere, and just to tell everyone what the experiment is, we take a skid mouse, we put in human tumor cells, establish the tumor for five days, actually MRI the mouse brain to make sure we have a tumor, then put virus directly into the tumor itself, and then we can follow survival as a function of dose and as a function of other manipulations that we have tried.
	If you administer gancyclovere within a day or two days of putting virus into the brain, you lose any potential anti-tumor effect of the virus itself, and, in vitro, all of the viruses I have described to you are as sensitive as wild-type herpes simplex to acyclovere and gancyclovere.
	DR. BREAKEFIELD:  What is the frequency of mutation of the TK locus?
	DR. WHITLEY:  It is very, very, very low in the normal host.  In fact, there's a nice study that was just submitted to NEJM, looking at the development of resistance in the normal individual after exposure to acylclovere, both episodically and chronically, and these are people who were on drug for five or six years, and it is less than .03 percent, and that is--don't forget, these are acyclovere-exposed patients.  It is not the normal population.  It is even lower in the normal population.
	FLOOR QUESTION:  If the difference between the mouse and the aotus is not that great, what is the advantage of using the aotus at this point over the mouse?
	DR. WHITLEY:  To make those of us who are taking this into people feel more comfortable.
	CHAIRMAN SALOMON:  Excellent.  Thank you.  I think this was really a series of superb talks that focus on the issue in front of us.  To finish the day, our job is to get through two questions, and the key one is to discuss the appropriate basis for determining whether safety studies of a gene therapy product should be in small animals, for example, rodents and/or nonhuman primates, which was clearly what I was getting at with my question to Rich and to the others along the way.
	I think it is important, one of the agreements that I had with the FDA in discussing sort of these kind of questions was that we deal specifically with the idea that we don't get into defaulting to nonhuman primate models, for reasons that I think are obvious to everyone.  At the same time, I think we're all grappling with the same issue that Rich responded to, and that was, yes, he now knows that the nonhuman primate model told him the same things that the mouse model did, but how would he have made that kind of conclusion confidently prior to doing these nonhuman primate studies?
	Even when you have a good mouse model, one could make an argument, and obviously I'm putting this out here for discussion, could make an argument for going forward to a nonhuman primate study, even with the unknowns, and then the question to the group is what do you think of that kind of a statement and how could we do that intelligently and reduce the use of nonhuman primates, if possible?
	That is kind of the issue.  Does someone want to pick up on that?  Let's start with how do you tell when to request a nonhuman primate model, versus a mouse model, at all?  Let's just start with the basic decision of I have got a great product and I've got proof of concept.
	DR. GORDON:  I just want to make some comments on this.  I think selection of an animal model--I think it was very interesting, Dr. Whitley's last comment, why did you select a primate, and the answer was because it made us feel more comfortable before going into humans.  I think that this is a very historically common reason for choosing primates, but if I were to make a recommendation to the FDA, it would be to discourage doing it for that reason and to look for other reasons.
	These reasons may be what about the organ system you're actually studying, and we don't need to think only about primates, but about other larger animals, does the organ system physiologically resemble the human more in a dog or in a pig than it does in a mouse?  What about, in the case of gene therapy vectors, the distribution of receptor for the vector?  You don't really want to test gene therapy vectors in a dog for liver gene therapy if the receptor is not on the liver of a dog, and it is on a mouse and it is on a human.
	I think the physiologic, biologic criteria are the ways to select a model, not because the animal is closer in size to a human, not because the animal's eyes are both in the front of its head, as they are in a human, and not, because it just makes you feel better in some sort of nondescript way, which I have a lot of sympathy for that feeling, may I say, but I think it is costly and potentially more controversial and also much less efficient.
	Let's remember that if we're looking for a rare effect, something that would occur one-in-every-50-animals, it is very difficult to do that in a primate and ever see the result.  We saw a bunch of studies this afternoon where there were fewer than 10 primates used, and that is only natural.  I think one should discourage going to animals like that unless there is a demonstrable reason for doing it, and that would relate to the biology of the disease, the biology of the organ system and the biology of the vector.
	DR. JONES:  If I can speak on Dr. Whitley's behalf, too, what he mentioned at the beginning of his presentation that we have not considered is when he chose the aotus, it was not just to make them feel better.  It was also because they're very susceptible to herpes diseases, as well, and I think you have to take that into account when you are selecting an animal model.  If the dog were a model that were susceptible to that, then maybe the dog would be your first choice in a large animal model, so I don't think that that was the entirety.  Would you agree?
	DR. CHAMPLIN:  There may be some unique features about--you know, herpes, obviously, is a dangerous virus that can cause human disease and to mimic that in an animal species makes sense, whereas some other viruses that are not toxic on their own, the AAV, for example, would not make sense in the same rationale.
	DR. WHITLEY:  No, I think that's exactly the point.  I think you have to understand the pathophysiology of disease and that was the point that I made with the third slide.  If you don't understand the pathophysiology of the disease, you should not be doing these experiments in the first place.  I think the key issue is that we didn't know when we began these studies is what we learned in the mouse and what we learned the rabbit, the same is what we learned in aotus.  It wasn't until we got the experience of three different groups now that have used this animal and have basically reached the same conclusion, that we feel like we're on firmer ground.
	DR. ANDERSON:  Just historically, this same thought process as you went through is what happened with the original gene therapy trials with retrovirus.  We did a lot of studies in monkeys, took a lot of criticism from our colleagues, because I was at NIH, we could afford it.  People on the outside couldn't afford it, but the issue was, was something going to happen when we put retroviruses into nonhuman primates that didn't happen when we put them into mice and rats and so one, and the answer was no, there wasn't.
	There is not a need now to continue doing nonhuman primates, unless, as the example here, unless there is something unique that could only be answered in a nonhuman primate.  Short of that, we have answered the question, so we don't have to keep reinventing the wheel.
	CHAIRMAN SALOMON:  One thing that has come out in these conversations is that there are different study outcome objectives.  One would be safety of a vector, which I think loud and clear was what Rich was telling us.  He was concerned about safety of the herpesvirus.  Then, there are efficacy issues, which, for example, the dog model was one way of very clearly addressing the efficacy issue without using a primate.
	I think the early retroviral gene therapy data, French, you would agree, was safety again.  You were concerned about replication-competent retrovirus, for example.  Can we start to maybe become up with some things that we would say then would be principles upon which one would suggest the appropriateness of a nonhuman primate model?
	You started with saying that you certainly would have to document reasonable expectation of a similar distribution of receptors or permissivity for the vector that is being chosen in the primate.
	DR. GORDON:  That is right, I would say that.  That would relate both to efficacy and safety.  Efficacy would also relate to whether or not the organ system models physiologically that which you are treating in the human.  I just want to say parenthetically that I completely sympathize with the choice of the aotus monkeys here, and I would have been the first person to do the same thing after I heard they were that sensitive, so it's not like I'm criticizing.  But, yes, again it is the physiology of the system.
	Perhaps cardiovascular disease is better study in a pig than in a monkey, because the system is more similar to a human.
	DR. BREAKEFIELD:  I had a few things to bring up for discussion.  First of all, I think we don't know a lot about the receptors that are present for different viruses in different species, and if we had all that information, it would be very much easier to decide which model--just concerning safety, would be the best model.  I think sometimes it's hard, though, and it especially gets even harder to know now when people start changing the surface properties of the virus to try to target them to different tissues and start putting essential viral genes under different types of tissue-specific promoters, because some animals that weren't infected before, I mean, that is a trophism issue that has to do with entry of the virus and also whether the virus replicates or not.
	If you change those properties, you really don't know what to anticipate.  I would say I felt, when they showed the studies with a replication-defective adenovirus and they compared it in--I guess we don't want to use the word monkey, but you know, but monkeys and mice, and it was the same, I thought that was great.  It gives us a baseline.  We feel confident now that this type of vector is the same.  Somehow, you know, we talk about it, it is, in some ways, a little unrealistic or something to think we don't feel comfortable.
	After all, that is part of the public domain.  If we don't feel comfortable, we have to ask ourselves does the public domain feel comfortable?  Why do we feel comfortable testing monkeys?  Since we think they're closest to us?  Especially, in my opinion, if there is a disease where there is medical treatment available that is pretty good, hemophilia would be one of those or cystic fibrosis, that people are not in a life-threatening situation, those would not be the first people I would try a new route of administration or a new vector type or something very different without having a little more confidence in maybe multiple animal models.
	I'm not saying you have to use nonhuman primates, but using different models as kind of a first line of is this or isn't this safe, so I have other comments, too, but I'll--
	CHAIRMAN SALOMON:  Again, I'm trying to pick up on just trying to come up with principles then that the committee would agree on.  That principle would suggest that as a new vector is developed or a known vector is modified in a way that would significantly affect its range of trophism for different cell types, for example, or its expression in different organs, those would be points at which a trial might consider preclinical work in a nonhuman primate, providing that the first principle was correct, that there was a reasonable expectation that the vector was still permissive in the nonhuman primates.
	DR. JONES:  And making sure that we choose the correct species of the nonhuman primate, making sure that that nonhuman primate species is appropriate for what we have chosen.
	CHAIRMAN SALOMON:  So, another principle then would be, with 200-plus nonhuman primates, though, maybe only about half-a-dozen really available to any kind of research regularly, do you have a suggestion as to how we should do that?  How would you choose a species?
	DR. JONES:  I would say lots of consultation with your colleagues and people who are in the field, lots of information sharing and literature searches.  There is a lot of available information out there on what is being used for different areas of research.
	CHAIRMAN SALOMON:  Joe, did you have a comment?
	FLOOR QUESTION:  I would tend to agree with all that is being said in terms of trying to use some type of scientific rationale for picking the species.  In drugs, it is a somewhat different scenario, but we have actually gotten away from simply doing rats and dogs with drugs, as well, and we look for a rational scientific basis for using an animal model, rather than just arbitrarily saying we are going to do rats and dogs and that's it.
	The same thing with the biologicals, we had looked at a protein last year in which we knew the sequence of the human protein, and so we looked at the sequence of the mouse and of the cyno monkey, as well, and decided that the cyno much more closely approximated the human situation.  We were able to utilize the monkey then, we felt, as a reasonably good prediction of what would happen in man.
	You are talking about trophism.  We are actually working with another group on a trophism-modified virus at this point in time.  The majority of the studies we're probably going to be doing are going to be directed in mice, but there is also an imaging component with this, as well, so we're going to wind up doing some additional primate work, to a much more limited extent, in which we're going to be doing some imaging in addition to that to see whether or not the distribution of the trophism-modified virus is different than of the normal virus.
	CHAIRMAN SALOMON:  So there, the rationale or the principle you're going to use is you know that the virus is tropic in the mouse and in the nonhuman primates, but you now want to use the nonhuman primate to demonstrate if there is a distinct difference in the trophism, in terms of tissue specificity.
	FLOOR QUESTION:  Yes, to decide whether or not we need to target particular tissues in our evaluation of toxicity that we may have overlooked previously.
	CHAIRMAN SALOMON:  As a principle, that would get back to your talking about if you know the receptor or have some sort of measure of the receptor, even if you have not cloned and sequenced it, that one should argue that there is a reasonable expectation of a similar distribution of the receptor, and if there are second receptors, such as we know for certain, like, lentaviruses (ph.), that that would be important, as well.  Right?  Okay.
	DR. GORDON:  I would say I think primates should be chosen the same way other animal models are chosen.  There is a scientific basis for choosing them.  They are much more cumbersome to work with in a variety of ways that we have not yet discussed, however.  For example, at my institution everybody is screaming that every mouse has to be behind a barrier so they can be uniform with regard to what pathogens they have been exposed to in the past.  You start importing monkeys from Samoa and let me tell you, you don't know what they've been exposed to.  You don't know how old they are.  You don't know anything about your genetic background.  You're in a much less well-controlled system.
	CHAIRMAN SALOMON:  To what extent, if we think about the universe of vectors that are currently in front of us--to what extent do we know, based on what we know about these vectors, are they different between, let's say, mouse and dog and let's say nonhuman primates, in terms of the questions we have been asking?  Is there some obvious background knowledge?  Are certain classes of vectors more or less likely to require nonhuman primate work because of a species-specific distribution of receptors for the vector?
	DR. PILARO:  I wasn't going to hit on that issue specifically, I was just going to speak to the two different classes--well, two of the different classes we heard about today, the adeno and the gutless adenos--are lumped together because basically the outside of those viruses is what causes the toxicity, and it is the same for the two of those and the AAV, and they have been studied across a various variety of species, for lack of a better word, and really, with the adenovirus, what we have learned from the data we have looked at in the past seven or eight years is they are inflammatory no matter what route of administration you give them, no matter what species you give them in, and the doses are always very close, the dose at which you see no toxicity and the dose at which you see frank inflammation and pathology.
	There is a very sharp threshold.  They are very close when you scale between the species.  We have learned that adenotoxicity is basically comparable, no matter what species you're looking in.  There, you see the justification for doing the studies in the smaller animal models.  You can actually get higher numbers of animals treated.  You can do more things with them.  You can sacrifice them at interim time points and look at histopathology, whereas within a nonhuman primates you would really be limited if you wanted to do an interim time point to just doing either a biopsy or blood work.
	With AAV, we have learned across mouse, dog, nonhuman primates, I believe it is both rhesus and cinemologous monkeys now, and even human, that there is no known pathology with this vector.  It can be given into the lung.  It can be given into the muscle.  There is no inflammation seen.  That is another one we feel comfortable with.  You don't necessarily need a nonhuman primate study for that.  I believe rabbits have also been tested for that one, too.  That's in the literature, as well, there's no pathology with it.
	We are comfortable with those classes of vectors, saying that we understand what is happening based on the biology of the response to the virus.  We know what's going on here.  We do not feel that nonhuman primates would be added value to these two particular classes of vector, unless there is a specific question you're trying to ask.
	DR. WHITLEY:  I think there just needs to be a sidebar that is added just for the herpesviruses, because herpesviruses behave differently according to the strain of mice selected and the rodent species utilized.  I guess what that does is lead me to the conclusion that, when you're developing both safety as well as efficacy systems, they have to be individualized according to the virus and optimized for the information that can be retrieved, and to pursue what Xandra was saying, is we try and target viruses for different tissues, that is not to say you won't use an aotus at one point or another in time, but you better have a reason to use it and understand what the added value is going to be in that system.
	CHAIRMAN SALOMON:  Again, that is what I'm trying to do, is come up with a series of principles that would generally be--that everyone would agree with.
	DR. BREAKEFIELD:  Just, I mean, in general, I agree with what Anne Pilaro says.  I think sometimes, though, you come up with something that is not quite the same, like in mice, they always tell you well, we can't really do those hepatic arterial injections, you know, we do the portal vein, so your route of administration isn't quite the same and you wonder can that be a factor.
	I think the other thing, with replication-defective vectors, like adenovirus, once you know where it goes, it's going to go to the same place.  But I think by the time you start changing promoter elements that control critical genes and targeting elements on the surface, I think those are open questions again, and the whole distribution in different species may vary.  You just don't know.
	CHAIRMAN SALOMON:  Are you saying, again, species and tissue-specific differences in vector interactions--I mean, in enhancer and promoter interaction.
	DR. BREAKEFIELD:  If new issues arise in a protocol, then it has to be reevaluated, if the data we have speaks to it or doesn't speak to it.
	CHAIRMAN SALOMON:  It seems like we've come up with another principle, and that would be if a clinical trial that you have now specifically designed requires a type of administration that it would reasonably, on the part of physicians or the public or any regulatory agency, constitute something that was a specific risk for the administration route, and you could not do that in a mouse, that that would be another potential rationale for a large animal, not necessarily for a nonhuman primate, but at least for--
	DR. MULLIGAN:  I think another way to classify vectors is simply replication competence, that if you have a replication-incompetent adeno AAV retro, the issues are very different than if you're looking for something that you want to have a, you know, Pac Man function to chew up tumor or something.  Those are issues that are tough, because we know so little about the normal determinance of tropism, in the herpes case, in particular.  I think I would look to any replication-competent vector as very different in terms of the animal model system, and really look not very hard at the replication-incompetent systems.
	I think we've talked about the necessity to have receptors, the route of administration, but in the replication competence, I think that even for efficacy sake, which, you know, we're take a back burner to in this discussion, but for tumor approaches, where there's an effort to have actual replication just in tumor tissue, I think those systems that are now being looked at are very unsophisticated, and while from a safety point of view, things may be okay, I think looking from the point of view of efficacy, it may be very important to really look more carefully at the different systems.
	CHAIRMAN SALOMON:  Picking that up, what Anne told us was that there is such a body of information suggesting that the data between the nonhuman primates and the mouse, let say, for the adeno and the gutless adenovectors, is similar, that she is basically taking the position that she would be comfortable with that sort of data, yet in the context of the gutless adenoviral vectors, where you're going to be putting in helper virus at some percentage, I mean, do you want to comment on that, Dr. Chamberlain?
	I mean, then you've got replication-competent virus and the principle, I think, Rich was saying was that that might be necessary to do some limited amount of nonhuman primate data.
	DR. MULLIGAN:  Just on that point, I think the amount of replication-competent virus he is talking about is not important.  I'm talking about a gene therapy approach where you're attempting to have replication competence.
	DR. CHAMBERLAIN:  Yeah, it's a different issue, because the helper virus we're using is essentially a conventional adenovirus, so that would not really change anything.
	DR. GORDON:  Can I just say one little more thing about that, that even in regard to Richard's comments, I think when you look at a nonhuman primate, the key question is, is the response of the animal, on the basis of its toxicity response or its efficacy response, going to tell you something about what the human will do better than or for the first time, as opposed to another species of animal model.  If it tells you that, you should use it.  If it doesn't tell you that, you should not necessarily use it.
	DR. MULLIGAN:  I did not mean to suggest that that was a rationale for going to a nonhuman primate.  In fact, I think the discussion, if there is a consensus, as Ed said before, it all depends.  I was just saying that for replication-competent viruses or vectors, you definitely want to look at the system, any system, and make sure that you're as close to looking at the characteristics that are important as you can be.
	CHAIRMAN SALOMON:  Rich and Xandra, what do we know about the other herpesvirus vectors, CMV, EBV?  I guess you knew I was going to ask that.
	DR. WHITLEY:  Let me take a shot at it first and then you can jump in.  CMV is being developed as a vector, but we have a really, really fundamental problem, and that is we don't have a model to begin with to look at that virus, and we had a meeting at CDC about three weeks ago that was really quite a productive meeting, but at that meeting, it was very clear that we do not have an adequate model to begin to probe develop CMV as a vector.
	For EBV, we have tumor genicity models in nonhuman primates.  The question is, will they ultimately be used for the evaluation of gene therapy approaches, and it's just not that far along yet.  It is the next step, because you do not have a rodent system to evaluate those viruses, and that is true for HHV-6, HHV-7 and KSHV.  I think there you are going to be stuck using some form of nonhuman primate.
	CHAIRMAN SALOMON:  There, I guess, the principal would be one the one that we already discussed, and that is if it is not--if the tissue itself in the mouse, let's say, is not permissive for the virus, then you can't use it.
	DR. BREAKEFIELD:  But isn't that the only way an adenovirus--that it can infect mouse cells, but it can't propagate in them, so if you try to look with the replication conditional, you know, replication-competent vector, are you really evaluating toxicity in the mouse?  There may be, I think we've gotten to this before, the cotton rat is the only one where it does replicate and that is very hard to get, but, you know, these are the issues we get into, they are difficult to--
	CHAIRMAN SALOMON:  I didn't know that.  I didn't realize that, after everything we said about the mouse, that you were not getting replication in it.  Maybe Rich was thinking that when he made his comments.
	DR. PILARO:  Can I make a clarification on that?  With a cotton rat, we know we get a limited amount of replication of wild-type adenovirus in the lung.  However, no one has systematically looked to whether or not we get replication of the other tissues in any of the species, including nonhuman primates.  So, we're really at ground zero with that.  We know if you put a replicating adenovirus into a mouse liver or into a monkey liver, that you would get the same results.  We don't have that data.
	CHAIRMAN SALOMON:  But if I understand right then, really what you have said then, and I think Dr. Lyons, GTI Novartis, said something very much along the same lines with adenoviral work that they did, that from an adverse event point of you, it looks like the mouse and the nonhuman primates and even the human is fairly predictable, and that is a very good thing in terms of not having to do nonhuman primates for those studies, but it doesn't seem anything like that is true if the question one wanted to ask is what would be the risk of injecting a replication-competent adenovirus?  Can we agree on that?
	DR. PILARO:  You've got it.  That is the point.
	CHAIRMAN SALOMON:  Well, that was, I think, what Rich was trying to say and I missed it, so that was just me not understanding it.
	DR. PILARO:  We just don't know what an appropriate model to look at replication-competent adenovirus would be right now.
	CHAIRMAN SALOMON:  Do we know that a nonhuman primates is a model for it?
	DR. PILARO:  No, we don't, we don't have that information.  No one has really systematically looked across the different species, going into the different tissues, to see if you put a replication-competent virus in here, does it replicate in a mouse liver, does it replicate in a human liver, does it replicate in a monkey liver?  We just don't know.
	DR. CHAMPLIN:  Now you have human trials with adenoviruses.
	DR. PILARO:  What we know from those trials is the data that have been discussed before the RAC, is it appears that replication-competent viruses replicate in the tumor, but not in normal tissue, and that is the best data we have got out of those studies.
	DR. ANDERSON:  I'm not telling you, you know, but that is a correct statement, but that is a non-data statement, because those replicating adenovirus were designed not to replicate in normal cells.
	CHAIRMAN SALOMON:  Right.  These are the P53 mutant.  French, would you agree, with respect to retroviral vectors, I think you kind of said that, but just as long as we were going through each of the vector classes, to try and come to some sort of consensus, would you agree with the retroviral vectors that, at the moment, as long as one didn't do something again along the principal that Xandra came up with, that you didn't profoundly change the vector in some way that would raise concern, that the retroviral vectors do not need to be tested in nonhuman primates at this point?
	DR. ANDERSON:  They had considerable testing, including testing with RCR, with replication-competent virus, to the point they inadvertently did the definitive experiment of seeing what level of replication-competent virus would produce lymphoma in the animal.  All of that data was a report that I wrote, along with Gary McGarrity, to the RAC and the FDA, analyzing all of the data of retroviruses into monkeys.  We went into over 30 animals and many of those animals are still alive and still being followed.
	The total data now that represents well over 100 monkeys, or well over 100 monkey years, suggest that we understand sufficiently about what the risk factor is that probably no additional monkey studies need to be done, or nonhuman primate studies need to be done.
	CHAIRMAN SALOMON:  I have quoted some of those studies in grants and gotten mixed results.  Some people do not think that it is absolutely established that injection of replication-competent retrovirus produces lymphoma in these animals.  There are a relatively small number of animals in the end and there isn't spontaneous incidents of lymphoma in the animals, as well, particularly in captivity.
	DR. ANDERSON:  Let me refer you to the report that I am first author of that went to the RAC and FDA that analyzed all that data, as well as additional data out of the lab notebooks that was never published anywhere else, and the conclusions are sufficiency solid that it would be hard for me to believe that anybody who has analyzed the data would still question what the situation is.
	You could always say you could do 100 more animals and what if you did this and what if you did that, but there is no question that it was the replication-competent virus that caused the lymphomas.  There was no question that the retroviral vector did not cause them, and all the conditions required, including severe immunocompromised, the lack of antibody response, the necessary (sic.) for a long-term retroviremia lasting over 100 days, it was a clonal event in each animal.  The virus was actually isolated out of sequence.
	I mean, thorough studies were done.
	DR. BREAKEFIELD:  But if you just subtract out the lymphoma component and say that is established, let's say you had a retrovirus vector that had like a different targeting moiety on its surface and now you are going to inject very large amounts, IV, do you think it needs to be retested in primates or nonhuman primates or do you think the data is there that supports that would not be toxic?
	DR. ANDERSON:  Well, that is actually a question that we are directly facing, because we have now developed a targeted vector that can be injected directly into the bloodstream, and that will be discussed at the RAC in December, and is at the FDA, and our feeling is that we would not obtain additional information.  This is replication incompetence, to go along with Richard, that it is not necessary to go into nonhuman primates.
	If, however, we or someone else were to develop--well, I shouldn't say we or someone else--Dori Kasahara, in our program, has developed a replication-competent retrovirus specifically for treatment of cancer, and that is an interesting question.  I don't want to bias either us or the FDA by speculating at this point, but we're going to have to face that issue over the next several months.
	DR. NOGUCHI:  Not to prolong the discussion too much, but regardless about what people think about the lymphoma, what is clear and has been demonstrated is if you have replication-competent murine retrovirus that you give to a nonhuman primate, you will get chronic infection at the very least.  Some precipitating events like severe immunosuppression may then lead to further replication and activation out of that persistent infection.
	I think in at least a general sense we know what will happen with any contamination of replication-incompetent retroviruses.  I think French has also identified, though, just as adeno went from replication-deficient to replication-selective, they may an approach--approaching that with a retrovirus, then I think the question is open again.  But just talking about adenovirus, as an example, again it may or may not be the nonhuman primate that is the appropriate model.  That is still open for much discussion.
	DR. TORBETT:  I was just going to as Dr. Anderson if he felt comfortable with the many different approaches in his discussion, that many different types of targeting vectors now coming online, whether it's VSVG, whatever, which can probably hit a cell with much higher frequency than others, that simply using a mouse model is adequate?  That is to get back to your point that mouse models would be adequate.
	DR. ANDERSON:  Yes, yes, right.  Well, now that I'm sitting on this side of the table instead of out there, that is really sort of tough, because that is the other issue we have dealt with, because our targeted vector is much more efficient in our mouse models by having a mixture of VSVG with our chimeric targeted 4078.  I would tell you my gut feeling.  I would be a lot more comfortable going into primates for just the reason that Dr. Whitley said, is before we going into a patient that we get into a monkey, but I don't know if I could justify that on scientific grounds.
	DR. TORBETT:  We're back where we started.  I was just curious.
	CHAIRMAN SALOMON:  I'm glad to say this day will not end with consensus, any more than the morning did.  At this point, we have been talking about safety, and I think that we have come up with at least a series of principles that might at least be used to guide an FDA decision along the lines of whether a nonhuman primate or a large animal model should be used.
	Principles would include that you have permissivity, that you have a reasonable expression level of the receptors for the vector in different tissues, that if there is extensive experience with the vector choice in nonhuman primates to the point that there seems to be no evidence upon which to argue that the nonhuman primate adds information for safety, that you could not get in a mouse or a dog or another model, then there would not be a reason for doing it.
	But that changes--significant changes in a known vector class that could be argued scientifically would generate additional safety concerns, replication-competent virus whose behavior could be very different in a nonhuman primate than in a mouse, or where a clinical protocol would specifically require an administration route that would reasonably generate concern on the part of anybody looking at the study.  All those would be reasons--yeah, please, add one.
	DR. BREAKEFIELD:  The only thing I would add to that is if you had a transgene product that was active in humans and not in mice and was active in nonhuman primates, that might be another.
	CHAIRMAN SALOMON:  Right.  I was doing something specific here.  I was kind of ending with the safety and then I wanted to finish maybe a couple minutes talking about efficacy issues that I think have a whole other set of principles.  Does anybody have anything to add to what I sort of reviewed quickly?  Basically, it wasn't my idea.  I was trying to review what came out of the discussions here that would be then, as I said, some set of principles upon which to consider this, and I think we did answer most of the questions then about class of vector and when do it.
	At the end, like I said, I would like to talk about efficacy for a minute.
	DR. NOGUCHI:  I think the reasoning and the discussion has really been excellent here.  I'm reminded a little bit about sometimes we discuss questions about surrogate endpoints, and it seems to me the principle is the same, for our conclusion that you have come to today about where we can safely use a model other than a nonhuman primate in this particular case or any other model, someone had to do the studies in the first place or else you can't really make that evaluation.
	I think that overall it is a very good lesson for a lot of different things, not just for this, but things like surrogate endpoints and everything else.
	CHAIRMAN SALOMON:  Yes, I agree, and I think again, really I think an important principle here is whenever you would introduce a new viral class of vectors or non-viral vectors or really a significant change in the vector, then those would be another place where one would have to consider again the model.  Let's then segue at the end to are there any additional principles that should be added to the discussion for efficacy parameters?
	It is one thing, then, to have a model in a mouse in which we're comfortable that the adverse event profile is similar, but now how do we think about other issues with respect to expression and gene delivery, antibody responses, for example, really came up and I challenged Dr. High about the idea that, well, you know, this is a novel antibody response and then you're trying to reassure us this is a model for humans.  My response is, well, it might be, but then you have to see that happen in a human, which I think was Carol's point.  Can we discuss for just a minute what do you guys think about what does efficacy issues add to model choices?
	DR. TORBETT:  I think it depends on the system, if it is going into a human and you were using a cytokine, for example, or whatever that doesn't cross-react with a mouse, that mouse is going to see as foreign, you're already biasing the study and getting information that might not be relevant.  However, I think in all these situations, you have to judiciously pick the system that you're using, again if it something that is very toxic to mouse again, you're going to bias your study, so in these kind of situations, if it's a cytokine, IL-12, whatever, going into a nonhuman primate system, it might be the only thing that will work.
	DR. CHAMPLIN:  Even there, it may not work, I mean, immunogenicity isn't really, at least in my experience, it's not very predictive from animal models to humans.  Sometimes you have problems on one end or another and you just have to do the study to see.  The other issue is the model system for--you know, there are some nice diseases, hemophilia would be a classic example, where the model system could be very helpful, whereas there are many other diseases, cancer, where the animal tumors don't predict necessarily for human responses, particularly for immunotherapies or cytotoxics.
	I think, depending on the disease system, animal models may or may not be predictive and you really want to look for safety and for feasibility of expression of the transgene.
	DR. WHITLEY:  I was going to try and deal with one principle, and I think that when animal models exist for disease, they should exploited and they should be studied in the context of the natural immune response.  Having said that, if I just take a different hat and put on a vaccine hat, rather than a gene therapy hat, I can take a mouse and immunize a mouse with water and that will protect the mouse from challenge with wild-type herpes simplex infection.
	What we've learned from the mouse to humans with sub-unit vaccines has not been applicable.  Sub-unit vaccines don't work in people, they work in mice, so we have to be little bit careful about the analogies in terms of the meaning of the immune response in rodent systems compared to man.
	CHAIRMAN SALOMON:  That would underline the principle I started with, and that is that when we start talking about efficacy, the game is again different.  There may be very clear situations in which we would need to do some nonhuman primate studies to demonstrate efficacy or reassure ourselves that an immune response was similar.  Dr. High, I thought you might want to make a point.
	DR. HIGH:  I just wanted to make one point, and that is that in data that I didn't have time to present, using those two different dog models of hemophilia, we actually purified dog Factor IX protein that showed that, if you infused purified canine Factor IX protein, similar to the product currently used to treat humans, except that it is species-specific, if you infuse that into the Chapel Hill dogs, the ones with the missense mutation, they don't make inhibitory antibodies, but if you put it into the dogs with the early stop codon, they do make inhibitory antibodies.
	This correlates with what we have observed treating humans, that is a dog with a more severe mutation has a greater likelihood--so that speaks to the point raised by Dr. Miller, because actually the immune response to the transgene product in hemophilia is really a safety issue, so it is a safety issue related to the transgene product instead of the vector.
	CHAIRMAN SALOMON:  It is an efficacy issue, as well, right, there's no point in doing gene therapy if you're going to get an antibody and inhibit it.  Right?
	DR. HIGH:  Right, so it is both, but the point is this is a safety issue related to expression of the protein, and we do have information, from using purified protein, that what happens in the dogs is similar to what we have already observed in humans.  I think there is a rational basis for believing that the data generated by gene-donated approach may predict what happens in humans.
	CHAIRMAN SALOMON:  That would raise then a principle that I would put out for comment, that if in the roll up to a clinical trial you could convincingly argue, with data, of course, that the model you had chosen, that was a nonhuman primate, where it is a mouse model or a dog model or a guinea pig model, I don't care, reflects a clinical experience at a molecular, cellular, protein level in such a way that it would really give a convincing argument to people that this was a model, that you would not necessarily need then to go on to a nonhuman primate model to make your point.
	DR. HIGH:  I might turn that around a little bit, just to say that that fact alone would not exclude the necessity for nonhuman primates, but I would argue that it would support the use of an animal that was deficient in the protein.
	CHAIRMAN SALOMON:  That is the principle.
	DR. BREAKEFIELD:  I mean, I want to stress that point, too.  I think several people have said it, but if there is a non-primate model, a mouse model, of a disease available, I think it is very important to do the safety studies in those animals because sometimes, due to their illness or whatever else, they become especially susceptible to the virus.  If the nervous system kind of degenerates and the virus infects those cells, they may be much more affected, and it is a very important model to include if it is available.
	On the other hand, I don't think you can hold the investigators accountable if the model doesn't exist because it takes a long time to developed these models and sometimes you develop them and they don't even look like the human disease model.
	CHAIRMAN SALOMON:  I think that the issue I was trying to come up with was not if you don't have a model what do you do.  I can't help that.  I mean, that is not for this group to discuss.  But what I was trying to argue was if we agree that if you had a model and if you could demonstrate that that model paralleled what one saw in clinical experience, which is what Dr. High was saying, could we agree that you could then use that reasonably to argue for the use of that model as a surrogate for nonhuman primate data prior to going on, let's say, to a Phase I or clinical trial.
	I feel comfortable with that, if it would, of course, be an individual thing.  I would have to be convinced by the results and I would be skeptical, but I'd have to be convinced.
	DR. MILLER:  I mean, my point was, from the animal studies, is that you can use that data to suggest you don't need to go to a maximal tolerated dose.  I think you can say as long as you are getting efficacy, you can look for the minimal effective biological or pharmacological dosing, but using that model system, if you didn't get efficacy at the level which you felt was, based on your model system, not immunogenic, you would be forced to go up, so I think you can use the model system to try and help you as long as you're getting a biologic efficacy effect.
	While you are saying it is safety, it is also giving you--as long as you have efficacy, it can model only safety, but it has to be evaluated through both safety and efficacy.
	CHAIRMAN SALOMON:  I think it's a good point, too.  I think part of what I didn't say, but I agree with, is that if you are going to make this argument not to go to nonhuman primates for a new vector system, it shouldn't be just based on safety alone.  Picking up on what you said, there should be an animal model of the disease and you should be able to demonstrate efficacy, because I think at this point, in general, I think the public is getting shy of the idea to going on to risky trials when efficacy has not been demonstrated, even reasonably, in a model of the disease.
	DR. NOGUCHI:  I would just like to clarify what I think I hear Dr. High saying, and it is actually just slightly different than what we have been discussing.  What we were really discussing is in the absence--how best to put this?  There is ample evidence that inhibitors is one of the worst things that could happen with treatment of hemophiliac patients, because that can literally destroy any potential or real benefit that current technology can provide.
	What Dr. High is actually addressing is do we need to have the experience with gene therapy of developing inhibitors in a human first to say that then the animal model is valid, or can we had instead use the wisdom of previous experiments, and what Kathy High is saying is that we know that under certain conditions, with a big deletion that gives you no protein, puts a person at more risk, and she now has evidence that in the animal model with a gene therapy vector, that you can, under those conditions, induce inhibitors.
	The question of whether it is valid or not is a genuine one, but I think it's going a little bit beyond the issue here, is the question of do we need to have the human experience before we say let's not go there?
	CHAIRMAN SALOMON:  Actually, I don't agree with this at all.  What I'm saying is specifically that if you want to argue that you do not have to go on, that you have got a model that predicts what is going to happen in the human, you have got to tell me on what basis you made that prediction.  Look, we can cure all kinds of diseases in mice.  I've had enough of that in 20 years of curing mice.  There should not be a sick mouse in the United States, and yet my patients are not doing so well.
	I think that the point here is I would buy the principle that if you had a model in the humans--I'm not saying do it in the humans until you get a side effect and then congratulate yourself as part of a workup to a clinical trial, but I'm saying if there is a clinical experience in humans through the administration of exogenous Factor VIII in hemophilics, that now your model parallels and now you want to tell me now I've got a valid model and I don't have to do a nonhuman primate, that is what I'm saying I accept.
	If there's no human data, then all the arcane, beautiful, molecular baloney that you come up with for your animal model is just that.
	DR. CHAMPLIN:  Why would you need to do a nonhuman primate for the Factor IX approach?
	CHAIRMAN SALOMON:  I actually was saying I don't think you do.  I think that was a perfect example.  That would be a model in which I'd say you've got human correlation, you don't have to do nonhuman primates.  That was the whole point.  I agree with that.
	FLOOR QUESTION:  What I do not understand is, in the event that there was no canine model of Factor IX, what would you gain by a nonhuman primate study of a gene for Factor IX with regard to inhibitors?  I mean, I don't understand if there is no model, then a nonhuman primate does not help.
	DR. CHAMPLIN:  That's what we said.  We said we would not recommend it.
	FLOOR QUESTION:  And if there is a model, how does it help?
	CHAIRMAN SALOMON:  I could answer, but Xandra, do you--
	DR. BREAKEFIELD:  Well, from probably most of the primitive viewpoints, but just that if you been working with a vector and different species have different kinds of immune responses, I would imagine the nonhuman primate has an immune response more similar to humans with some subset.  Maybe that is wrong.
	FLOOR QUESTION:  But that is a safety issue.
	DR. BREAKEFIELD:  Well, it is efficacy, too.  If you make inhibitor, then your vector isn't going to work in those people that have the deficiency.  Right?  I see.  You're trying to just argue for efficacy.
	CHAIRMAN SALOMON:  Can you spell out what you're saying?  If you don't have a model--we were talking about if you had a model and how you ratify that model without having to do nonhuman primates.  That is not what you are getting into now.  You're suggesting now that what is the  situation where you don't have an animal model for the disease, and I'm not going to demand--
	FLOOR QUESTION:  Dr. High has made the case that we have this experience with the canine Factor IX, et cetera, and you have said there is a syllogism you can follow with regard to the canine Factor IX that might persuade you that maybe in that case you do not need a nonhuman primate model for Factor IX.  What I do not understand is in the absence of having a primate hemophilic model, what a nonhuman primate model for Factor IX would do.
	CHAIRMAN SALOMON:  Nothing.
	FLOOR QUESTION:  Okay.
	DR. GORDON:  I just wanted to make a little comment on efficacy and that is the one that was brought up briefly with the cystic fibrosis mouse, and here is an animal that does not have CFTR, but when it comes to efficacy at the level of alleviating disease, it is not a useful model.  I think if a primate presents a form of disease which can be examined for efficacy at the level of alleviating symptomatology, then it clearly is a model that should be sought.  I want to make one other political point, if I could.
	I think it is great to look for non-nonhuman primate animal models, but I don't think that it should be implied that one would hesitate for a moment to use a nonhuman primate if it was the best model.  I don't want to get into that dangerous ground of suggesting that we would not do adequate animal testing just because somebody does not want us to use a primate or something like that, not that anyone in this room agrees with that, but I just want you to know there are people who do think like that.
	CHAIRMAN SALOMON:  I agree.  I think that the principle that everybody is trying to grapple with is what would be the circumstances in which just sort of a knee-jerk response would be, we have to do a nonhuman primate because we're uncomfortable, versus we're going to do a subset of nonhuman primates to answer a specific question necessary for the safe introduction of this agent, to a question, as this gentleman posed, from Avigen, is that if you don't have a disease in it and in this particular case, the issue is you can't look for an inhibitor without an absence of the native protein, then it doesn't make any sense and I agree with that, as well.
	DR. GORDON:  Well, let me just make a brief amendment to the way you responded, although I do agree with it.  I think if you're in a situation where you are looking at vectors and looking for a suitable animal model and you don't have one yet, they shouldn't be left off the list, that is if you don't have a suitable animal model for looking at response, looking at toxicity, looking at supportive gene expression, then they must be included in the list and it would be a rationale for at least examining those species.
	CHAIRMAN SALOMON:  Right, and then I think again you would turn to the principles that we've kind of articulated this afternoon to ask critically whether if you fulfilled a number of those principles, whether a nonhuman primate model was reasonable, a new vector, a change in the vector, something specific that is species-specific.  One thing I wanted to comment on, and I think we're getting pretty close to the end here, but one thing that always bothers me is when we look at these animals models and when you look at these nonhuman primates, when you do these data, am I supposed to go into the nonhuman primate model with human Factor VIII or Factor IX or we have kind of beaten these factors to death, let's say IL-12 or any of these different things, if you're going in with the human protein into the nonhuman primate, I'm not getting that quite straight.
	At the same time, however, is then do I have to stop and remake my vector using the primate equivalent of the protein, which I can see a couple of the sponsors in the back fainted already, but anyway this is all Rich Whitley's idea, by the way.  I didn't have anything to do with it.
	DR. PILARO:  Can I address at least part of that?  It's been our long-standing policy that if you have a homologous gene available and you have done some preliminary efficacy work with that, that it is permissible and actually encouraged to do toxicity studies with that gene, so that you actually understand in the system you're looking at what the toxicity of that protein would be.
	There are differences between human and nonhuman primate IL-12 or interferon or any of these biologic proteins.  The monkeys usually all develop antibody against the protein within several weeks of administration.  So, it really--
	CHAIRMAN SALOMON:  Are you saying when you use human IL-12?
	DR. PILARO:  I'm saying when you use human IL-12.
	CHAIRMAN SALOMON:  But not if you use monkey IL-12?
	DR. PILARO:  If I had monkey IL-12 available, I'd give you an answer there.  I don't.  That is something that we have not got data available on yet.
	DR. CHAMPLIN:  Antibodies to cytokines in humans--and, you know, that may or may not be biologically relevant to their therapeutic use, and clearly things can be very different in primates and non-primates, in terms of their antibody response.  So I personally don't see that--any animal system really predicts for the human immunogenicity-related problems.
	DR. PILARO:  Okay, I did want to give the other half of that, though--that if you did not have a homologous gene available and the only gene you had was the human gene and the transgene product is species-restricted and only active in human and nonhuman primates--that would be a call where you would have to use a nonhuman primate.  We certainly wouldn't make you go clone the monkey gene and insert it into a vector and then do studies.  It is sort of six of one, half dozen of another.
	DR. HIGH:  Actually, since she brought up clotting factors, maybe I'll just mention it because it harkens back to another point that I had tried to make.  Monkey Factor IX, I know I said the wrong word, but Monkey Factor IX is about 97 percent identical to Human Factor IX at the sequence level, and it turns out that if you give an AAV vector expressing Human Factor IX into the liver of monkeys, they don't make antibodies to Human Factor IX.  If you use some other vectors, they do.
	CHAIRMAN SALOMON:  Well, that may be a reason we could talk about it, that we can get back to later as a resin-immune site, but that is another story.  I guess, just to pursue this just for another second and then we're done--if I, as a sponsor, did a study, then, with monkey IL-12--because these days, it is not that hard to insist on cloning a monkey homolog, to be honest.  But let's just say I did that study with homologous protein and I got this and this result.  And now, of course, I want to go to a human study, but I'm not going to use monkey IL-12.  I have got a fresh construct of human IL-12 in the same vector.  Is that okay?  I mean, is the FDA going to roll with that one?
	DR. PILARO:  Are you asking me to design your talks program for you?  If you are, what I would tell you is, if you had available that vector or that protein, the recommendation that I would make to you is, if you're going to go for the nonhuman primate studies, you build your talk study and you do your dose response with the monkey study, but you add in one dose group with the human protein, preferably close to the maximal dose that your giving, so that you can see what the differences are, what the similarities are.
	The human protein is what you're going to go into the clinic with, so it's always useful to have that data.  You want that information.  When you're dealing with a species restriction or when you're dealing with significant differences between a nonhuman primate and a human, you would want the information with the homologous molecule, if it's available--big caveat.
	CHAIRMAN SALOMON:  Good.  Okay.  Did we answer the questions that the FDA wanted?  Is there anything hanging out there that you want us to deal with?
	DR. PILARO:  You have basically given us some, what I call, red flags for when you think nonhuman primate studies are appropriate.  And, I want to actually commend the group because you go along pretty much with what the guidance in the ICHS-6 document for biotechnology-derived products is.  That document does not address gene therapy products, but it is basically what we use in biologic as our Bible.  So, I'm happy to see that we've been kind of going along the same lines with what you all are thinking.  We are very pleased to have that answer, yes.
	I think we basically had a very good discussion this afternoon and I hope that the committee has all learned something.  I know I have, especially about nonhuman primates.  I'm not calling them monkeys anymore.
	CHAIRMAN SALOMON:  Well, if we have answered the questions, I think it has been a long day.  We're actually finishing within 10 minutes of the time we were supposed to adjourn.  I thank everybody, including the audience.  See you tomorrow.
	[Whereupon, at 5:40 p.m., the Committee adjourned.]