UNITED STATES OF AMERICA

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	FOOD AND DRUG ADMINISTRATION

	CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

	BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE

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	OPEN SESSION

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	THURSDAY,
	APRIL 5, 2001

The Committee met in the Second Floor Ballroom at 8120 Wisconsin Avenue, Bethesda, Maryland, at 9:00 a.m., Daniel Salomon, M.D., Chair, presiding.

Present:

DANIEL R. SALOMON, M.D., Chair
RICHARD E. CHAMPLIN, M.D., Member
RICHARD C. MULLIGAN, Ph.D., Member
EDWARD A. SAUSVILLE, M.D., Ph.D., Member
ABBEY MEYERS, Consultant
W. MICHAEL O'FALLON, Ph.D., Consultant
ALISON F. LAWTON, Non-Voting Guest
GAIL DAPOLITO, Executive Secretary
ROSANNA L. HARVEY, Committee Management Specialist

Also Present:

STEPHEN J. CHANOCK, M.D.
AMY PATTERSON, M.D.
BLAKE J. ROESSLER, M.D.
STEVEN R. BAUER, Ph.D.
THOMAS L. EGGERMAN, M.D., Ph.D.
SUZANNE EPSTEIN, Ph.D.
JOYCE L. FREY-VASCONCELLS, Ph.D.
PATRICIA KEEGAN, M.D.
MARY ANNE MALARKEY
PHILIP D. NOGUCHI, M.D.
JOSEPH P. SALEWSKI
MARJORIE A. SHAPIRO, Ph.D.

Also Present (cont.)

JAY P. SIEGEL, M.D.
KAREN D. WEISS, M.D.
CAROLYN A. WILSON, Ph.D.
SALLY SEAVER
JANET ROSE CHRISTENSEN
NEIL GOLDMAN
WILLIAM FREAS
KATHRYN C. ZOON, Ph.D.

	I N D E X

Call to Order, Daniel Salomon, M.D., Chair ...   6

Conflict of Interest Statement, Gail
Dapolito, Executive Secretary .........   7

Session 1

FDA Introduction .............................  13
Overview of March 6, 2000 FDA Gene Therapy
Letter
Product-Related Issues
Joyce Frey-Vasconcells, Ph.D.
Division of Cellular and Gene Therapies,
CBER

Responses to Gene Therapy Letter:
Multi-Use Facility, QA/QC Issues .............  39
Mary Malarkey
Division of Case Management, CBER

Questions from the Committee

Responses to FDA Gene Therapy Letter:
RCR and Different Packaging Cell Lines for
Retroviral Vector Manufacture .........  75
Carolyn Wilson, Ph.D.
Division of Cellular and Gene
Therapies, CBER

Committee Discussion

Responses to FDA Gene Therapy Letter:
Testing of Plasmids as Manufacturing
Intermediates in Gene Therapy Products ....... 103
Suzanne Epstein, Ph.D.
Division of Cellular and Gene Therapies,
CBER

Committee Discussion

Responses to FDA Gene Therapy Letter:
Adenovirus Vector Titer Measurements and RCA
Levels ....................................... 149
Steven Bauer, Ph.D.
Division of Cellular and Gene
Therapies, CBER

Clinical Issues of Adenovirus Infection in 
Marrow Transplant Recipients .......... 150 

Committee Discussion

Session II

Open Public Meeting .......................... 189

FDA Introduction ............................. 191
March 6, 2000 FDA Gene Therapy Letter
Preclinical and Clinical Issues
Karen Weiss, M.D.
Division of Clinical Trial Design and
Analysis, CBER

Responses to FDA Gene Therapy Letter:
Preclinical and Clinical Issues .............. 192
Patricia Keegan, M.D. 
Division of Clinical Trial Design and
Analysis, CBER

Results of Gene Therapy Clinical Site 
Inspections .................................. 217
Joseph Salewski
Division of Inspections and 
Surveillance, CBER

Committee Discussion

Update:  CBER Intramural Research Programs

Division of Monoclonal Antibodies ............ 284
Jay Siegel, M.D., Director
Office of Therapeutics Research and
Review

Marjorie Shapiro, Ph.D. ............... 286
Laboratory of Molecular and 
Developmental Immunology

Division of Cellular and Gene Therapies
Philip Noguchi, M.D., Director ........ 302

Thomas Eggerman, M.D., Ph.D.
Laboratory of Molecular and Tumor 
Biology ............................... 302

	P R O C E E D I N G S
	9:10 a.m.
DR. SALOMON:  Good morning, everyone.  I'd like to get everyone to sit down now and initiate this meeting today, April 5, 2001, Biological Response Modifiers Advisory Committee.
I decided coming here that one of the things that we need to do from here on in is title these meetings because I never really quite know what to say after this.  You know, this is the 18th Annual -- we'll have to work on that one.
            Okay, anyway, welcome everyone.  I know it's always something to make time in busy schedules to participate in these meetings and I say that both for our expert panel as well as visitors and the representatives of several government agencies that are here and I hope everyone will feel welcome and also feel like they had an opportunity to participate actively in the deliberations of the committee over the next two days.
Certainly, if anyone on any part of the table or in the audience feels they're not getting a chance, that they should definitely feel comfortable to come and talk to me at the break because that would not be my strategy.
I'd like to turn to Gail Dapolito to read into the orders the Conflict of Interest Statement.
MS. DAPOLITO:  Thank you, Dr. Solomon.  This announcement is made part of the public record of the April 5-6, 2001 meeting of the Biological Response Modifiers Advisory Committee pursuant to the authority granted under the Committee charter, the Director of FDA Center for Biologics Evaluation and Research has appointed Ms. Abbey Meyers and Dr. Michael O'Fallon as temporary voting members.  To determine if any conflicts of interested existed, the Agency reviewed the submitted agenda and all financial interests reported by the meeting participants.  As a result of this review, the following disclosures are being made:  in accordance with 18 U.S.C. 208, Dr. Richard Mulligan has been granted a waiver which permits him to participate in the Committee discussions.  Drs. Champlin, Kurtzberg, Salomon and Sausville and Ms. Meyers have associations with firms that could be affected by the Committee discussions.  However, in accordance with current statutes, it has been determined that none of these associations require the need for a waiver, a written appearance determination or an exclusion.  
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 presentations and/or comments made by the guests:  Dr. Steven Chanock is employed by the National Cancer Institute, National Institutes of Health; Ms. Alison Lawton will be serving as a 
non-voting industry representative for this meeting.  She is employed by Genzyme.  Genzyme has associations with various universities, investigators and research foundations that are involved in gene therapy.  Ms. Lawton also has interests in several firms that could be affected by the Committee discussions.  Dr. Amy Patterson is employed by the National Institutes of Health, Office of Biotechnology Activities.  Dr. Blake Roessler is employed by the University of Michigan and has interests in the field of plasmid vector production that could be affected by the Committee discussions.
In the event that the discussions involved 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.
With respect to all other meeting participants, we ask in the interest of fairness that you state your name, affiliation and address any current or previous financial involvement with any firm whose product you wish to comment upon.  A copy of the waiver addressed in this announcement is available by written request under the Freedom of Information Act.
And just a household item, housekeeping item, we would like to request, just as a courtesy during the Committee deliberations that you turn your cell phones and pagers off or put them on the silent mode and if you wish to speak on your cell phone please go into the foyer.  
Thank you.
DR. SALOMON:  Thank you, Gail.  Another little quick thing from the Chairman's perspective, just housekeeping is if you notice the red light, red light off, I think most everybody here has been on this Committee before, so this is not news, but just remember to turn it on and off during your -- after you've made your comments because otherwise you get feedback through the loop and they won't be able to get the kind of recording that's necessary to keep track of all of this.
Usually, what we've done at the very beginning is just gone around the table really briefly, again, not so much for our sake, but for the visitors' sake, to know who's sitting on the Panel.  If you can just give a sentence, a name and a sentence or two about why you're here, what your area of expertise is.
Amy, do you want to start?
DR. PATTERSON:  Yes.  I'm Amy Patterson.  I'm Director of the Office of Biotechnology Activities in the Office of the Director of NIH.  My office houses three federal advisory committees, one on genetic testing, the Secretary's Advisory Committee on Genetic Testing; one on xenotransplantation and the third and probably most relevant to today is the NIH Recombinant Advisory Committee.
DR. CHANOCK:  Yes, I'm Stephen Chanock, an Investigator in the Pediatric Oncology Branch and particularly the Immunocompromised Host Section with a strong interest in infectious disease and immunocompromised hosts.  I'm a consultant for infectious disease at the Clinical Center and I also serve on the Institution of Biosafety Committee for the NIH.
MS. LAWTON:  I'm Alison Lawton.  I'm Senior Vice President of Regulatory Affairs for Genzyme Corporation.  I'm the Industry Rep.  I'm also the Chair of Cell and Gene Therapy Committee for the PhRMA Industry Association.
MS. MEYERS:  I'm Abbey Meyers, President of the National Organization for Rare Disorders known as NORD.  I'm a former member of the RAC and I'm currently on the National Human Research Protection Advisory Committee.
DR. MULLIGAN:  I'm Rich Mulligan from Harvard Medical School and I'm involved in gene transfer research and stem cell research.
DR. CHAMPLIN:  Richard Champlin.  I'm from the M.D. Anderson Cancer Center.  I'm a hematologist and Chairman of the Blood and Marrow Transplant Department.
DR. O'FALLON:  Michael O'Fallon from the Mayo Clinic.  I'm a biostatistician.
DR. SALOMON:  Dan Salomon from the Scripps Research Institute in LaJolla, California.  My interests are in organ and cell transplantation and gene transfer.
MS. DAPOLITO:  Gail Dapolito, CBER, Committee Executive Secretary and the Committee Management Specialist, Rosanna Harvey.  Thank you.
DR. SAUSVILLE:  I'm Edward Sausville.  I'm the Associate Director for NCI's Developmental Therapeutics Program, involved in the discovery and development of drugs and biologics for early clinical trial and I'm a Medical Oncologist.
DR. WILSON:  Carolyn Wilson, a member of the Division of Cellular and Gene Therapies at CBER, FDA.
DR. FREY-VASCONCELLS:  Joyce Frey, Deputy Director for Cellular and Gene Therapies.
DR. NOGUCHI:  Phil Noguchi, Director of Cell and Gene Therapy in the Office of Therapeutics at CBER.
DR. SIEGEL:  Jay Siegel, Director of the Office of Therapeutics Research and Review at CBER.
DR. SALOMON:  Okay, thank you all very much.  Unless there's anything that needs to get read into the record at this point, I'd like to get started.
Dr. Joyce Frey is going to present to us an overview of the March 6, 2000 FDA Gene Therapy Letter which then leads into a discussion on the responses to the gene letter and some of its implications in terms of discussion of the Committee.
DR. FREY-VASCONCELLS:  Okay, today I'd like to give you an overview of the famous March 6th letter and kind of the process that we went through in issuing this letter and reviewing the responses.
Next slide.  There are several reasons that we issued this letter.  One of them was safety concerns related to recent events.  This included the death of a patient on a gene transfer protocol and the conduct of that trial.  There was also a report of potential risk of transmission of infectious agents by inadequately tested product.  And then finally, there were violations that the Agency noted on several directed inspections.
In addition, we realized that gene transfer was a rapidly developing field and over ten years a lot of things had changed.  So standing testing requirements that the Agency was looking when the field began, began 10 years ago, is clearly not adequate by today's standards.
In addition, based on our regulations for annual reporting, for product information, a sponsor is only required to submit a summary of significant manufacturing or biological changes.  So it's very difficult for the Agency to ensure over time whether sponsors were changing and testing their product by current standards.  Generally, what we would receive in annual reports is no deviations on sterility.  Well, I think you can all see where that's probably not sufficient information for a novel technology.
Next slide.  So we went through the process of getting experts, both from product and clinical and pre-clinical together, and trying to figure out what information does the Agency need to receive in order to address our concerns that were listed in the previous slide.  Once we had identified what issues, what information we wanted to received, we issued the famous March 6th letter.  In that letter, because of what had happened with the death of a patient and report of potential transmission of an infectious disease, we actually put a 3 month time line for sponsors to respond to this letter.  We realized that this was an enormous task, both for the Agency and for sponsors, but in talking to industry and people that were in this field, everyone felt that this was an effort that clearly needed to happen and needed to happen in a relatively short period of time.  So about March 7th we had received basically all of the responses from all the active files.  In those responses, we reviewed them and we analyzed the data for each vector system and as you can see by the agenda, we're going to be discussing specific issues related to three different vector systems.
We also wanted to identify whether there were common problems in regulatory compliance across the board that would be to all areas and I'm actually going to be talking about two of those at the end of my talk.
In addition, with this information it helped us to identify areas where we needed to increase our training and outreach.  Based on the information, we have proposed draft policy recommendations that will be discussed and finally to seek outside advice on these recommendations and that's part of the purpose of today's meeting.
Next slide.  I'm going to focus mainly on the product questions.  There were seven questions in the letter.  I'm going to talk about the first five.  And then this afternoon, Question 6 which related to the clinical trials and 7, the preclinical, will be discussed by Drs. Karen Weiss and Pat Keegan.
For product questions what we wanted to know was we wanted a list of all gene transfer products, cell banks and viral banks that were ever produced in your facility.  What we had noticed over the years was when gene transfer first started, most people were making one product in their facility using one cell bank or one viral bank.  What had happened over time is due to relationships with companies and things like that, people were making multiple gene transfer products and it was also a mechanism for us to find out what areas did we need to start thinking about in relation to facility-type information.  
The second question was a list of cross reference files.  There was a lot of people that were cross referencing and we weren't really sure that we had a good handle on who was cross referencing who.  So we wanted a list of both what files you -- the sponsor cross referenced and what files the sponsor had authorized to cross reference their file.  
Then Question 3 was probably the most intensive.  And that was a list of all lot release and characterization data for each lot and each cell bank and viral bank that had been produced to date.  
The fourth question was reasons for rejecting lots.  This was so that we could get a feel for were there particular areas, vectors systems that we needed to keep a close eye on, were there common reasons for rejecting lots, were there certain facilities that were having problems that we needed to work with?
The fifth question was the quality assurance program.  We wanted to ensure that there were appropriate checks and balances in manufacturing and releasing product in order to treat subjects.  
And then finally, we asked sponsors to commit to yearly updates of this information.  That doesn't mean that they have to submit all their lot release and characterization data on all lots ever produced each year.  It was an update on that information.
Next slide.  The goals that we had set for this letter was (1) to ensure that all gene transfer products met today's testing standards.  That was really the most critical thing we wanted to get out of this.  The second one was to evaluate the testing requirements.  Were there areas that we needed to make the testing requirements more stringent?  Were there areas that we had gained enough experience that we could potentially relax the testing requirements?  
Then we wanted to use this information to provide appropriate guidance and also to be able to look to areas on what we needed to focus in order to move these products towards licensure.  It was also a mechanism by which we could increase public confidence in our oversight ability and then it also provided a mechanism for ensuring annual reporting of information, adequate product information to the Agency so that we could have proper oversight.  And then finally, to increase, to identify the training and outreach needs and to develop appropriate policy recommendations.
Next slide.  Okay, there were two areas that we had identified, that there was -- that the Agency had not been real clear as to exactly what we wanted to see.  And one of them was the area of potency.  The CFR defines potency as a test shall consist of either in vitro or in vivo tests, or both, which have been specifically designed for each product so as to indicate its potency and then potency is actually defined as a specific ability or capacity of the product to affect a given result.
Next slide.  What we meant by potency is actually a measure of biological effect.  It's a functional activity of your product.  A lot of sponsors we noted wanted to use a measurement of viral titers, their potency.  The problem with doing this is that if something happens during your manufacturing process and you lose your gene insert, just measuring viral titer will not detect that you have lost your gene insert.  
Another common measurement that people wanted to use for potency was gene expression and we actually do allow gene expression for potency in their early phase of product development.  But as you move towards licensure, you need to move to a more functional assay.  The problem with gene expression is that protein may be expressed, but your gene may be mutated slightly to where the protein that's expressed is actually not active.  If all you're measuring is gene expression, you're not going to pick up that your gene actually, that the protein is actually not active.
Next slide.  The next area that I'd like to talk about that we have increased actually the testing is the testing for adventitious viral testing and that is what we're asking now is that on each production lot you do an in vitro viral testing and this is usually done either on the lysate or the end of production cells.
Next slide.  So this morning's session, the first talk following mine will be more of a training outreach on Question 5 and what constitutes a quality assurance program.  And then also to discuss issues related to multi-use facilities.  Then following that there will be three different discussions on policy recommendations that we're seeking advice on.  The first one will be on RCR and the appropriateness of different packaging cell lines.  The second policy recommendation will be for testing of plasmids when plasmids are used as intermediates to produce the gene transfer product.  And then the final discussion will be on adenovirus vector titer measurements and RCA levels.
So I think I'll turn the mike over to Ms. Mary Malarkey to talk about quality assurance.
DR. SALOMON:  Joyce, can I ask a question or two, briefly?
DR. FREY-VASCONCELLS:  Sure.
DR. SALOMON:  So just so that I have the right context for this, you looked at a minority of the total programs in the country.  You took a random sampling.
DR. FREY-VASCONCELLS:  You mean for the inspection part?
DR. SALOMON:  Uh-huh.
DR. FREY-VASCONCELLS:  Right.
DR. SALOMON:  And so can you -- if we're going to get to this later, then you can tell me wait for the next talk, but one key question I think is how did you do a random -- how is this random?  I mean if we're trying to reassure everybody that this was done right.
DR. FREY-VASCONCELLS:  Oh good, I don't have to answer this.  No, there's actually going to be a talk this afternoon by the compliance and discuss how the randomization was done.  So I think I'd rather let -- since I was not involved directly.
DR. SIEGEL:  Let me just put that into a framework.  The March 6th letter that's been discussed was sent to every sponsor doing gene therapy and every sponsor going gene therapy sent us a response regarding their viral testing, their validation, their test methods, their quality control for manufacturing and the talks you're going to hear this morning are based on those responses and interactions with the sponsors.  So that is 100 percent overview of what we regulate.  
Similarly, it's on the clinical practices, clinical oversight and clinical monitoring.  We got responses from everybody to the same letter and in terms of what they do, but we sent inspection teams out to a random sampling.  Those were good clinical practices inspections.  We did some good manufacturing practices inspections, but those were not part of the random process.  Those were for cause where we had specific concerns.  So that will be discussed this afternoon, but it's not terribly pertinent to this morning's topics.
DR. SALOMON:  Okay, thank you.  I actually didn't understand that as well as I should have.
The second question I had was is it reasonable to ask what would be then -- this was sort of the first fly at what kind of things was out there, what kind of information you get back and then -- but use some of that information to help guide the policy decisions.
DR. FREY-VASCONCELLS:  Right.
DR. SALOMON:  What do you see, in general, at this point, in terms of going forward in the future?  Would this be a yearly event?  Would this be a constant reporting requirement from these sort of production facilities?  Would it be individualized?  You need to show this, this and this before we'd allow you to have an IND.
DR. FREY-VASCONCELLS:  Well, what we ask is first of all for anybody submitting a new IND, yes, they have to answer all the questions in the March 6th letter.  In addition, so that we can maintain proper oversight, we are asking people to update the information requested in this March 6th letter on a yearly basis.  In the letter, there's language that we -- for convenience, you can submit it in your annual report, but we want to see this information on a yearly basis.  That way we can keep closer tabs on each vector system and what people are seeing and that's why we want updates on why people are rejecting lots.  And then that way -- right, we can develop appropriate policy recommendations and have further discussions as we see trends in vector productions.
DR. SALOMON:  One more question, and again, I personally think this is an extremely important thing that we're talking about here today.  This, to me, is about as important as anything we've had in front of the Committee for a long time in terms of its implications about and its impact on the way we'll be doing gene therapy in many different sites around the country.  
So one of the questions I have is right now, correct me if I'm wrong, but right now, there is no official certification for a gene therapy production facility.
DR. FREY-VASCONCELLS:  Right.  I guess I'm not quite sure how -- what you mean by quote certification.
DR. SALOMON:  Well, I mean for example, clinical laboratory has to be CLIA certified.
DR. FREY-VASCONCELLS:  Right.
DR. SALOMON:  And all the technicians in the laboratory have to have CLIA certification and that's necessary for any data that is reported to a physician that would impact on their management, therapy or decision making in any way, shape or form on the patient, so I was just questioning whether the situation I see as an investigator in the field looking back at Washington instead of being here in Washington today is the idea that we essentially can set up gene production facilities in many different sorts of venues without any very high level of local oversight except for perhaps approval by an institutional, by a safety committee.
DR. FREY-VASCONCELLS:  But I think if you -- you have to understand that even if you're doing investigational studies, you still have to follow the GMP regulations.  GMPs don't kick in at licensure.  They kick in when you're doing clinical trials.
DR. SALOMON:  But a single center gene therapy trial doesn't require production of the vector in a GMP facility.
DR. FREY-VASCONCELLS:  It's supposed to be in the spirit of GMPs.  I mean there are -- I know, what's the spirit of GMP.  
DR. SALOMON:  Wash your hands in the morning.
DR. FREY-VASCONCELLS:  Yeah.  I mean you do have to follow the appropriate record keeping.  You have to be able to say at any point in time what you did and how you made your product and how you tested it.  In testing, it may not be that you have a validated test method, but you clearly have to have run appropriate positive and negative controls to ensure that your assay was working.
DR. SALOMON:  So is there -- do we feel that that is -- right now, basically, we're policing ourselves then in the sense that we have our institutional review committees, our biosafety committees, our institution review boards and then, of course, if we have NIH grants or we have a RAC approval, etcetera, we have several different federal agencies and an IND, then the FDA is involved.  So that's quite a bit of regulation.  I agree.
DR. FREY-VASCONCELLS:  Right.
DR. SALOMON:  And the minute you become multi-center it probably gets kicked up a degree.  So are we ever going to the point where we need to be thinking about some sort of a qualification that goes beyond just saying this is a GMP gene vector facility, but this is a qualified gene vector production facility and that would be something then at academic centers would aspire to or perhaps would only be community resources?
DR. FREY-VASCONCELLS:  I think there are discussions on that.  The problem is the Agency doesn't necessarily have the resources to do that at this point in time and so I think that that may be -- I know there has been talk with ASGT to potentially do something like that.  In fact, a couple of weeks ago we just had a manufacturing meeting for -- to let people know what would be expected of them and I think part of the oversight is related to the quality assurance program that you set up.  There are clearly checks and balances that are built into the system and that's another reason that normally we don't ask for that information up front, but we have found that there's a lot of misunderstanding of what an appropriate quality assurance program is in the responses that we received.  And so it's clearly an area that we feel that we need to do more outreach on and we need to have that information up front in the IND and we have in many situations have told people that if you don't have appropriate checks and balances, we're not going to allow the trial to go forward at this point in time.  And that's one of the reasons we're asking for the information prior to even you starting a clinical trial.
DR. SAUSVILLE:  Dr. Salomon, one point that I'd like to make in response to your question are we setting up a certifying, if I heard the word.  One problem with the gene therapy field is that it's been a very evolving process, rapidly evolving and I think that's where the analogy with the CLIA laboratory issue that you raised sort of breaks down.  A serum sodium is a serum sodium is a serum sodium.  Where as a viral gene product circa 1992 was different in many respects than the type of things that I think the industry is contemplating today.  
So I would actually caution against making standards for facilities and rather focus on products.  In other words, each product needs to have elements that I guess are addressed by the GMP regulations and by what's brought to each product both by the sponsor and the Agency.
DR. SALOMON:  Yes, I wasn't selling any particular agenda.  The CLIA I was just using as an example of a certification of a lab.  You could argue still that we are taking these products and putting them into patients and I could, for example, create a plasmid, create or clone a viral producing cell line with high titer and show that I had a high infectious titer, show that I had biological activity and show that it would target the appropriate cells and do the safety, but in the end, if you went back through my production facilities, it could be that it started off in the hood in my regular room which was also doing human, rat and pig studies and on and on and on, and I didn't have full tracking of all the fetal calf serum and other additives that were in the mixes.  So we have to be careful then that when -- I don't think you can have alternatively your comment that we should set standards for the product, we should just be reasonable about the fact that the standards for the product don't necessarily become standards for its production.
DR. SIEGEL:  Let me put a little perspective here too regarding the analogies to CLIA and other issues.  If a gene therapy is being manufactured for commercial use as a licensed product, it will be regularly inspected and it will be licensed which is a process I'm sure as rigorous as certification of a clinical laboratory.  So what we're talking about here is experimental products and they are held to good manufacturing practices.  The concern and the issue that Joyce was talking about in terms of what she termed the spirit of GMPs is that the good manufacturing practices, regulations, recognize that they need to be phased in during certain, during clinical development and the reason is that some of them, some of the extensive validation and process controls are appropriate when you're making thousands or millions of doses, but are not appropriate for a Phase I clinical trial in significant part because they'd involve such an investment of time and effort that no drugs would ever be developed.  
So we require good manufacturing practices appropriate controls to ensure even at the small scale the quality, sterility, purity, potency of the product, but some of the specific regulations, particularly those involving validation, but others as well, don't have to be met in the same way or in as rigorous or detailed a manner as they do as one moves through production.  So it's a graded in -- it's a -- you know what I'm saying.
DR. SALOMON:  I do.
DR. PATTERSON:  I just have three brief questions for Joyce.  They're sort of overview questions.  One has to do with numbers, the second with the cross referencing of master files and the third with the process.
In your background material, you mention that you received 200 out of 270 responses to the letter.  Could you speak about the fate of the 76 INDs for which you didn't receive responses for or maybe someone else will cover that.  I just want to have an understanding what the denominator is here.
The second question I have and I'm confused and hopefully you'll be able to lead me into the light.  You mentioned that some sponsors cross reference other INDs and/or master files and in cross referencing these INDS or master files they are relying on data, pre-clinical data, in particular sometimes product manufacturing data.  And you mentioned also in the background materials that sometimes these files don't contain the data that was being cross referenced or relied upon.  And my question is how can that happen?  How can an IND  be authorized if, first of all, it would be the sponsor's responsibility to know if the data is truly there that they're relying on and secondly, it would be the review staff's responsibility to look at the INDs and master files and make sure that the proper data is there to support authorization of the IND that's relying on it.  So I'm -- I probably missed something very fundamental in here, but I'm perplexed. 
And then my third question has to do with process.  You're developing recommendations and training and outreach and coming to the Committee here for their very valuable insights and expertise.  But I'm wondering if at least some point today you can talk about what the process is for outreach to the broader scientific community and the investigators and industry to I guess have a relative consensus about how to achieve what are very, very laudable goals, apart from today's deliberations.
DR. FREY-VASCONCELLS:  Okay, to answer the first question in relation to the number of INDs, the responses we received were for active INDs.  We felt it was important at this point in time to bring INDs where patients were actively being treated and studied to today's standards.  So we got -- we did receive responses from a number of people who clearly indicated that their file is no longer active.  They just had never bothered to inactive the IND.  So that's why a lot of letters went out and it actually proved to be a very useful exercise for the Agency because a lot of sponsors didn't realize they had never inactivated the IND.  So that's the difference in those numbers.
As far as the master file, this is one of those areas that actually is quite troubling to the Agency.  Generally, when we get a letter of authorization for cross referencing, the letter says that the sponsor, that the holder of the IND is authorizing cross reference to a particular sponsor.  That's the limit of what we get.  We don't know exactly what they're authorizing.  Now our regulations clearly state that when you provide a letter of authorization, you need to include exactly what information you're authorizing that can be cross referenced, the page numbers, volume numbers, where it can be found.  So that has been an issue for us is when we get these global letters, what exactly is being cross referenced?
The thing is is that a sponsor, the purpose of a master file is so that we can use information in the cross reference file to support an IND and to be able to keep information proprietary.  So if you're a sponsor and you want to cross reference something, no, you're not going to necessarily know what is in that file. It may be proprietary information and this is a mechanism by which it can be used to support your IND.  But like I say, the problem is if you don't know -- if you don't clearly state what's being -- what you're authorizing, it makes it difficult for the Agency to make that assessment and so you're right, we have had situations and that's one of the things we found out in this is we had situations where a sponsor was cross referencing a file and they weren't on the same page as to what the information was and it wasn't clear to us that they understood.  So it's something that we've been clearing up through this process, outreach and other activities.
DR. SIEGEL:  Before you leave cross referencing, I want to say something though about -- you asked about how would the clinical reviewer not have picked this up.  What we've discovered, in part, is the IND comes in, it cross references the master file, the clinical reviewer reviews the master file and reviews the INDs.  And there's adequate manufacturing testing or preclinical testing.  Now it's three years later and we say have you done changes in manufacturing testing or new animal studies that you haven't told us about that you were supposed to and the sponsor says yes, we have, it's in the master file.  And we look at the master file and it's not there.  So that's what they're talking about.  It's not that there was a deficiency in what was sent, but that there was additional information that we're asking since the IND went into effect that in some cases sponsors thought were in the master file, but in fact, weren't and it's part of this bigger issue of sponsors not always knowing what's in the master file and what has been submitted.
DR. FREY-VASCONCELLS:  So in regards to that what we are now actually doing is if we get one of these global letters, we're not necessarily accepting the global letters.  We're going back and saying no, we need to know exactly what you're cross referencing.
MS. LAWTON:  I have a question with regards to the analysis of the responses.  I know in some of the specific questions responses to some of the specific questions you look at the responses as far as the different types of sponsors, for example, is it sponsor investigator, was it industry sponsored, etcetera.  And I particularly on the manufacturing side, I would like to know did you break down the analysis into sponsor investigator, small company, large company type situations to understand whether there are any particular trends with your concerns around the manufacturing facilities and the QA/QC controls and whether that's something we should indeed be looking at?
DR. FREY-VASCONCELLS:  We didn't do a real in-depth.  It was more just an assessment as we were going through.  And to be honest with you, we didn't find that there were really any issues related to one that quote big manufacturers were doing things any better than academic manufacturers.
The issues were the same across the board.  There didn't seem to be trends in that area.
Outreach.  You're right.  That's probably one of the most difficult areas for us, but clearly any recommendation that we put forward, it will be to get -- to also get public input into those recommendations.
DR. NOGUCHI:  Although I have to say what we're presenting here are our evaluation of what we view as current safety issues and in terms of implementing them I think there's ample room for discussion, but in terms of discussing whether they're important or not at this point in time, a large part of what we're discussing are these are things that we do think need to be implemented in terms of safety at this early stage in gene therapy.
DR. SALOMON:  Yes, I would go back to my comment.  When the staff showed me the questions and the things that we were going to be talking about in the next two days, again, my response was this is probably one of the most important meetings we'd had in a long time and given the potential impact to some of these discussions we're going to have, I was surprised that it wasn't standing room only of sponsors, concern with how we were going to develop things.  That doesn't, by the way, mean that the audience isn't still -- every person is important to me.  It's just surprising because of the global nature of these things.
But I do feel, Amy, that you're bringing up a point that everybody is sensitive to and I think these are things that ought to go on to discussion at big groups like PhRMA, the American Society of Gene Therapy meeting in Seattle later in the year and I think we need to, many of us involved in those organizations should make an effort to bring them forward so they are discussed there.
Joyce?
DR. FREY-VASCONCELLS:  Actually, we do every year have heavy participation at the ASGT and in fact, there's going to be a two-day training session on clinical trials and I don't know exactly -- I haven't seen the latest agenda on that.
DR. SALOMON:  It's sort of -- it's basically a workshop on doing for clinical investigators in how to take a clinical gene therapy trial from beginning to the end with active involvement with several FDA staff.
DR. FREY-VASCONCELLS:  And I know other years we've done training sessions, workshops on manufacturing.  I think this year we're going to try and have a booth at the ASGT and so we're constantly, and I know we have taken issues to the RAC for public discussion.  And so as much as we can, we try and get out there to get our message and to get input from the public.
DR. SALOMON:  Abbey, did you have a question and then we need to move on?
MS. MEYERS:  In terms of something that's been in the news lately which is upsetting the public about these people who are claiming that they're just going to go out and clone a human being and is there anything to stop me from manufacturing gene therapy vectors in my garage, since you don't have any requirement for certification?
(Laughter.)
DR. SALOMON:  In terms of giving them to people are you suggesting or just making them?
MS. MEYERS:  I'm talking about opening up a gene therapy clinic in my basement which is what the cloning people are claiming they can do.  What is there to stop me.  I don't have to have a factory approved by the FDA in order to make these things.
DR. SIEGEL:  Yes, you do.  You have to have an approved IND with the FDA and you have to submit the responses to all these questions and extensive data about manufacturing and about your clinical study plan before you get authorization to proceed and to do otherwise would be a violation of law.
DR. SALOMON:  Abbey, let me clarify.  It's important that -- my questions were specifically about the production facility.  You took it another step further and we're talking about actually giving it to a human being.  Once you want to cross that line then all the existing regulations are fine.  There's no issue.
MS. MEYERS:  It's the certification question that I'm concerned about is you know, for example, scientists who are developing genetic tests for people with rare hereditary diseases, academic laboratories don't have CLIA certification and FDA could walk in and say we want you to stop developing this test because it's not a CLIA lab.  Several government agencies can do that.  But now you're saying that gene therapy manufacturing facilities, vector manufacturing facilities don't have to be certified.
DR. NOGUCHI:  But when we're talking about certified from the FDA viewpoint, we license both manufacturing and products at the same time, once they're approved and have been shown to be safe and effective.  So that's our level of certification.  That means you can legally sell this and administer it by a physician in the United States.  Prior to that, all our regulations for the pre-IND do pertain.  If we learn of deviations or of labs starting up in the night, we will take appropriate action which in the case if there is no IND that's associated with it, we can shut them down, we can seize, we can move for injunctions.  There's a whole variety of things and we would do that as a matter of fact.  So certification is not a necessary component for the FDA to take action and to prevent illegal activities from happening in this area.
DR. SALOMON:  Well, themes of this can come up later, but I'd like to  move on here to Mary Malarkey is going to talk about the QC/QA analysis.
MS. MALARKEY:  Good morning.  That was a lively discussion on that topic.  As Dr. Frey said, Item 5 of the Dear Gene Therapy Sponsor letter caused, we believe a lot of confusion, that is, what is the expectation for Phase I in relation to quality.
In addition, I'm going to speak very briefly on multi-use contract facilities because that's another area that causes a bit of confusion.  That is if a sponsor contracts out the manufacture or testing and/or testing of their product, what are their responsibilities and what are the responsibilities of that contract manufacturer?
Next slide, please.  Quality is a GMP expectation.  That is under Title 21 of the Code of Federal Regulations, Parts 210 and 211.  It is expected that a quality unit will be in place.  Once you prepare product for administration into humans, then technically speaking, the GMPs fully apply.  However, as has been mentioned by Dr. Siegel and Dr. Frey, we have looked at this as a step-wise approach.  Certainly, there are certain GMPs that are expected right from Phase I, but things such as validation and end process controls develop along with the product.
Another point of confusion, good laboratory practices are not GMPs.  The GLPs are specific to pre-clinical studies.  The GMPs cover all phases of manufacture, controls and documentation as well as testing.  One thing the regulations do not make the distinction of is the difference between quality control and quality assurance.
Next slide.  Under 211.22, the quality control unit is defined and the first three bullet points here really are more of what we look at today as quality assurance, keeping in mind that the GMP regs were published in 1978, so expectations have changed over time.  The quality assurance function is to approve and reject all components, intermediates or products, to approve and/or reject all of the procedures that are used and the specifications, to review all the records for a given lot of product to ensure that it meets the specifications and if there are deviations that investigations are performed to try to find where the problem lied and to correct that problem so it doesn't recur.
The fourth bullet here is more what we think of as QC today and that is the laboratory function, the actual testing function.  And all the responsibilities, regardless of whether it's QC or QA are expected to be in writing.  
Now the last bullet here is not in the regulations, but it's become an industry standard and an Agency expectation over time.  And that is that the quality unit needs to be separate from production and this is the system of checks and balances, so there isn't a conflict of interest between the people manufacturing the product and actually releasing the product to the public.
Next slide, please.  In 1996, there was a proposed revision to the 211s and industry asked that the Agency define quality assurance and quality control.  At that time the Commissioner, Commissioner Kessler said that we don't really care what you call your unit as long as you have the functions that are needed.  So as I said earlier, quality control has generally evolved to mean the testing activities to ensure that the specifications are adhered to whereas quality assurance is really the oversight responsibility, really the QC of QC, if you will.
This unit is responsible for auditing all the methods, the results, the systems and the processes and trending of data to show where things are starting to get out of a state of control.
Next slide, please.  The next couple of slides go into some other regulations that give quality definitions.  In the GLPs, we have a quality assurance unit definition and at the very end of that you can see that it talks about being entirely separate from and independent of the person engaged in the conduct of the study.
Next slide.  The proposed rule for the Good Tissue Practice Regulations which is a fairly recent publication defines a quality program and this is where we see the terms preventing, detecting and correcting deficiencies and this is, of course, the language that was in item 5 of the Dear Gene Therapy Sponsor letter.
We understand that there are some unique considerations for these products, particularly in Phase I and Phase II.  That is, the QC unit and the QA unit may be one person as opposed to in a manufacturing facility where you would see a whole unit of people devoted to these tasks.  Most QC, that is the testing function may, in fact, be contracted out, so the sponsor may not have a QC unit per se.  Validation and qualification activities may also be contracted out and many vendors are involved, that is, rather than manufacturing media or putting in a pharmaceutical water system, these may be purchased already pre-made.
And in the case of the National Gene Vector Labs we have a situation where we have multiple sponsors that are using the same facility or having products manufactured in the same facility.
Next slide.  The most general consideration with quality is documentation.  Everything needs to be documented and this is an expectation right from the very beginning.  It is understood that these procedures will evolve over time as the process evolves.  However, batch production records are a requirement.  This is every step in the process is documented along the way.  The equipment, the cleaning and use of the equipment, what lot of product was in the particular piece of equipment on a given day, laboratory records, standard operating procedures are basically procedures that go to everything that is done within a given facility.  Distribution records, which I have here in quotes as the distribution may, in fact, be just right down the hall in the hospital setting to a patient if it's a direct vector or cell product.  
And finally, complaint files are something to start thinking about, if in fact, you are a facility that is multi-use and is actually distributing product to other people.
And the main point here is that adequate documentation allows traceability, so if there is a problem, you're able to find where the problem lies and hopefully to correct it.
So going through the letter or that item in the letter, the first bullet was preventing deficiencies.  And of course, this is the most important thing.  If you can prevent deficiencies from occurring in the first place, then you're far along the way.  These are some examples of things that would be preventive measures.  Of course, testing of all cell and viral banks.  If you aren't doing that testing yourself as a sponsor, it's expected that you will review all the SOPs that are used, any validation protocols for the assay methodology, and of course, all the results that are obtained from the test lab.
Testing or certification of components, I just give one example here, of course, of our concern with bovine-derived materials and certifying that they are from BSE-free countries.  And screening of patients or if you don't choose to screen patients, if you're using cells of multiple patients in your facilities, then one would expect that you would use universal precautions, that is, just assuming that there is everyone is potentially infectious or every cell line is potentially infectious.
The facility, there's been a lot of discussion about the facility itself.  We do expect that it would be adequately designed and validated for its intended use.  The equipment needs to be calibrated, qualified and certified.  There should be maintenance and monitoring procedures to ensure that the facility maintains the state of control and requalification, recertification, recalibration activities should be in place.
Cleaning becomes extremely important, particularly with multi-use facilities and we recommend a variety of cleaning agents be used because no one agent is effective against all potential organisms that one may encounter.  And segregation is extremely important as well and this is a 
cross-contamination prevention issue.  
Finally, the manufacturing process itself and this could be, of course, the vector or when I say product here I mean if it's cells or the actual product, controls need to be developing and again, Phase I and II, we don't expect full controls to be in place but towards Phase III and then into licensure it's expected that in-process testing will be performed and specifications set.  
Validation of aseptic processes, on the other hand, is an expectation right from Phase I.  Sterility is extremely important and if you're doing aseptic processing, that is, after filtration or not able to filter a particular product, then it's expected that you will validate, that you can maintain aseptic conditions during its manufacture.  Operators, of course, need to be adequately trained and qualified for their intended tasks and you need to have procedures to look at deviations when they do occur.  		And finally, of course, the testing of the product and review of all records associated with the lot need to be done prior to release of any given batch.  
Some detection considerations, monitoring, of course, of the facility as well as the personnel.  This is environmental monitoring as well as monitoring of temperature, humidity, pressure differentials, whatever is important to maintain that state of control.  Testing, not just of the final product, as I said, but components, everything that's going into the product as well as starting to set up some 
in-process tests.  And finally, I mentioned trending before.  It's not specifically a requirement, but it's a good idea in order to demonstrate that you're maintaining control over time.
When problems do occur and even in the best of circumstances they do, you need to think about what to do to correct them and this is the importance of traceability and all the documentation that I mentioned earlier.  You need to have procedures in place for performing an investigation.  What are you going to do?  What are you going to look into?  What data are you going to review?  You should have an idea of what corrective actions you may think of performing if you do find the problem and of course, procedures for handling of complaints or any adverse events that are tied to manufacturing.  And finally, procedures for notification of physicians, patients, FDA, all of these components.
The letter also asks for an identification of authority and this is really the important checks and balances issue.  Again, the quality unit should be separate from production and of course, production is sometimes the sponsor themselves.  This quality unit has to have the ultimate authority to release or reject so they can't both be producing and testing and reviewing and releasing.  Again, we have a conflict of interest there.  The ideal situation is a separate unit with ultimate reporting to the sponsor, but the authority to basically override the sponsor and this is a difficult concept and we understand that, but even in a licensed manufacturing facility, we don't expect that the CEO would be able to override the quality unit decisions.
There was also a request for the date of the last audits that were performed.  Of course, this suggests that there needs to be a plan in place for audits, what you need to audit, how you're going to perform an audit and the frequency of your audits.
Under the regulations, it's required that an annual review of your manufacturing operations for each given product be performed.  This would be a representative number of batches.  All associated records of those batches and after that review is complete and compiled, it needs to be reported to the responsible individual.  So if the quality unit was doing this, it would then report those results to the sponsor.
Vendors, we understand there could be a lot of vendors involved and I think at Phase I-II, the expectation is that you'll get a certificate of analysis, but over time you need to start putting some testing into place, not just relying on the C of As.
The contract validation activities, again, the validation of a facility is a difficult task and not often can be done in an academic setting.  So once you -- if you have people come in to help you validate a facility, for example, you need to be involved and you need to pick up the ball so that you can maintain that facility or that validated state.
And finally, contract manufacturers.  Years ago this was generally testing, so the cell and viral bank testing is contracted out.  Even most final product testing is contracted out, but again the quality assurance function of the sponsor in this case would be reviewing and approving all the SOPs that are used, validation protocols that are used and reviewing, of course, the test results.  But we're seeing more and more where the entire manufacturing process is being contracted out.  And often, it's being -- the products are being manufactured in 
multi-use facilities and this brings up some questions as to who is responsible for what.  
The bottom line is the sponsor is ultimately responsible for the quality of the product.  So again, review and approval of all relevant procedures, including product testing, all the data generated during production and testing would apply.  And this is again the QA oversight function.  Even if you're not the manufacturer.  Now we also recognize that many sponsors may perform some specific testing and not contracted out, such as potency.  If this is the case, then you have the QC function.
And finally, if you are contracting out, it's expected that you will have enough information on other products that are being manufactured to evaluate all the cross contamination procedures that are in place.  So there may be proprietary information.  That is the exact products that are being manufactured, but you need to know enough about them to know that the cleaning procedures, etcetera are appropriate and that your product will not become contaminated.
Now the contract facility also has responsibilities and of course, the main one is they need to operate under appropriate GMPs.  They're also usually the ones responsible for validating the cross contamination prevention procedures and this would be such as cleaning procedures and we don't have any current expectation on how this will be performed.  We are certainly open to review data and make suggestions.  This is a very interesting topic and how you demonstrate that you're not contaminating one lot of product with another or one vector with another.
And finally, the contract manufacturer may submit a Type V Drug Master File and in this file they may put all the proprietary information that they do not want to share with the sponsors that are using their facility or having their product manufactured in their facility.  We did away with Type I Drug Master Files last year.  This was the historic -- historically, that's where this information would be, but now we're saying you can submit a Type V without prior permission from the Agency.
So in conclusion, sponsors should be in compliance with GCMPs with respect to these quality functions and we do have these special considerations that we're getting more and more concerned about for multi-use facilities.  But keep in mind that the sponsor does have the ultimate responsibility for product quality, but that the contractor also has responsibilities which is adherence to GCMPs and validation of cross-contamination procedures.
Thank you.
DR. SALOMON:  Mary, can you clarify, I just don't understand the difference between a Type I and a Type V Master File?
MS. MALARKEY:  Okay, yes.  Master Files are defined in 314, 420.  There are five types or there were five types.  The Type I was specifically for facility information.  And this was done away with.  Generally, mostly CBER using the Type Is.  The Type V is kind of the catch all for everything that doesn't apply or doesn't fall into the II, III, IV category.  And the regulation does say that you need to get prior permission from the Agency to submit such a file.  But we are saying for certain circumstances, we will accept one without that prior permission.
I hope that helps.
DR. SALOMON:  So I'm just not sure what would be in the Type V.  In the Master File, for example, let's say I had a proprietary viral producer cell line or a helper system or something like that.  Is that what you're talking about?
MS. MALARKEY:  No, what I'm talking about --
DR. SALOMON:  Or verification or viral concentration?
MS. MALARKEY:  I'm talking specifically the facility.  So if I'm a contract manufacturer and I manufacture multiple sponsors' products, then I could submit a Type V Master File with my facility design, my diagrams, the flows, the SOPs, the general SOPs that are in place, as well as a list of those products, specifically that I manufacture, because again, that information would not necessarily all be shared with the sponsor.
DR. SALOMON:  So just to follow up on that, I mean in a number of different kinds of gene delivery vector systems, there are standard quote unquote cell lines or helper cell lines or various things depending on whether you're talking about the adenos or plasmids or retroviral vectors.  That you could do multiple kinds of studies by inserting in the gene of choice is plasmid and then you deliver it with these proprietary vector-producing lines.  Where would they be?  The production facility could control these GMP level producer cell line systems.
MS. MALARKEY:  Yes, as I mentioned, there were Types I, II, III and IV and I believe that this would fall into a Type II Master File.
DR. SALOMON:  Any other questions?  Richard?
DR. MULLIGAN:  I'm interested in the cross-contamination issue.  When you looked at the contract facilities, I would find it hard to believe there's almost any contract facility that actually would do the direct sorts of cross-contamination tests, so were there cases where, for instance, people making an adeno vector and retrovirus vector actually looking for retrovirus vector, not just a generic retrovirus, but retrovirus in their adeno, perhaps, and other than doing those direct sorts of tests, it's not clear how you'd really ensure that there's not cross-contamination.
MS. MALARKEY:  That's a very good point.  And that's something we are all struggling with and I think what I'm talking about here is more demonstration, not product testing, but actual cleaning validation, really, equipment and facility validation of the cleaning processes as opposed to testing one lot of product for another type of product, so to show that your cleaning processes are effective in removal.  There are other things such as using different pipetters or other controls that can be put into place to ensure that cross-contamination won't occur.
DR. MULLIGAN:  Is it fair to say that, in fact, there hasn't been any case where people have done these direct tests as far as you're aware?
MS. MALARKEY:  No, I don't believe that that is the case.  Dr. Epstein?
DR. EPSTEIN:  There are some cases, for example, it's not the one that you're talking about, but we're asking for PCR looking for the wrong plasmid, the previous one.  And we're asking a lot of questions about dedicated equipment, that changing of tubing and so on.  So sometimes we directly as-test for the product before yours.
DR. MULLIGAN:  A more general question is this issue of what it means for the sponsor to be responsible for production, if it's a contract facility.  What is the -- can you give a better sense of what you can possibly mean as being responsible if, in fact, you don't have a lot of proprietary information about things that are going on in the facility that are likely to cause contamination.
MS. MALARKEY:  Well, the 
cross-contamination issue is certainly a separate one and it does involve proprietary issues of its own.  However, if you are contracting out your product to be manufactured, then our expectation would be that you would review all the batch reviews, that is the blank records up front, you would approve -- you would ensure that, in fact, the facility was doing the production as they should, in addition to all their standard operating procedures and those types of things would have to be reviewed.  I mean you would want to know how your product is being produced, what testing is being done, what procedures are in place to prevent not just cross contamination, but contamination of the product.
DR. MULLIGAN:  I think that there's many cases where there's a very non-industrial investigator who is getting product from a company because, in fact, they don't have the expertise to know what a batch record is from whatever is.  And the question is whether or not you actually have the expectation that an investigator would have enough expertise in these specific areas to actually be capable of reviewing the manufacturing process.
MS. MALARKEY:  Well, it may not be the investigator themselves, but someone that they have on their staff that would be that quality person that we're talking about.  I understand exactly what you're saying, but you do need to be concerned as an investigation and if you aren't, if you don't feel able to do that, then you need to have a quality person in place to do those types of functions and that's where we're seeing problems.  There isn't that.  There isn't the responsibility being taken and there are problems in that area.
DR. SALOMON:  Michael and then Dr. Sausville.
DR. O'FALLON:  The presentation was actually overwhelming as far as my sense of the complexity of the entire thing and you've just hit on one of the areas where that's almost certain to fail.  I mean one of the basic tenets of modern quality assurance is the more complicated you make things, the closer you are to having the probability that one, that something will go wrong.  I was really awed by your presentation and by the fact that there are so many different things that we are trying to control here.  This is an observation, not a question, nor do I have a solution to it, but I can imagine the guru of modern quality, Deming, is probably turning over in his grave as he tries to imagine how we could handle this and it is so critical. I agree completely with our Chairman's earlier comments.  It is so critical.  Just setting up more rules and regulations is not a solution to that problem.
DR. SAUSVILLE:   Yes, and picking up on that and also on Dr. Mulligan's comment, I think it illustrates the point that was made previously that trading and outreach as this field evolves is really going to be an absolutely critical function because on the one hand, the innovation that gives rise to many of these products does clearly originate in academic settings.  And we have encouraged often as part of the illusion of that innovation that the academic investigator take the lead in actually developing a product to clinical trial.
On the other hand, though, I think what we've heard today and I commend Mary Malarkey for really going very lucidly through a complex area is the -- something in our experience, academic investigators, they just don't get it.  When you start talking to them about issues of quality control and quality assurance, they fall asleep, they're not interested.  It's not something they've been trained to do and I think that this is absolutely key.  And I actually believe that it also impinges on the doing of science because ultimately, the scientific experiment which is the early phase clinical trial, you need to know what you have that has given you the result that you're going to interpret and move on.  And that's really what quality control and quality assurance gives rise to.  So without, and again, this is more in the spirit of an observation.  I think this underscores the training in outreach.  I think that if we're going to have and we should actually encourage academic investigators to be active and viable in this area, we -- the greater we, that is, NIH, FDA, the people who are entrusted by the public with promoting this enterprise, need to put in place the support for investigators so that they feel they're empowered to make these types of decisions and whether they want to get involved.  Because I think as the recent tragic events have proven, we want to create a scenario where recognizing there are going to be errors, there are going to be problems.  When you say a probability of one, yes, that's right.  Errors do happen.  We have to have in place an orderly and systematic way to understand where the errors come from and have the academic investigators buy into a ready participation in that process.  I'm sermonizing, but I think that's what we have to do.
DR. SALOMON:  I'm kind of enjoying this because that was exactly what I was thinking and of course, I have the advantage of having seen this stuff a little bit ahead of you and that was where my comments were coming from.  I mean the way I'm thinking about it and trying to take what I've heard just in the last few minutes and make it, think about it in a constructive way, is that we have issues, of course, where we have a lot of different companies that I think are much -- that have vector and viral production facilities and they know what they're doing.  They come from an industrial culture.  They understand what a GMP facility is.  Oftentimes, they already have existing GMP facilities and they've just taken over a part of them, so that they have very sophisticated QC and QA and they know how to deal with the FDA, etcetera.  But then we have a whole other world.  I think that's where Dr. Mulligan and Dr. Sausville and myself are coming from when we're saying there's a lot of stuff going on outside our labs.  We're talking about setting up an GMP facility at Scripps for islet isolation and for gene therapy to do our own trials and there it gets really very complicated because even in a grant, I know my first version of my NIH grant for gene therapy they cut out two of the technicians because they were well, you know, Dr. Salomon doesn't need that technician to do this trial and that's where -- there would be your quality control, they were supposed to be data monitoring technicians and they cut them out of two of the centers in the trial.
(Laughter.)
DR. SAUSVILLE:  So that illustrates the lesion, okay.  Because you create a situation where it's really impossible for the academic investigator, even if you, in your particular case you plan for it.  So one interpretation which some have given is that's a reason why academic investigators in a sense shouldn't play in this game.  I don't hold that, but you could take that as the limit case.
On the other hand, as I stated emphatically, innovation in this field comes from academia in the main.  It is brought to fruition certainly by the industrial sector, but I think we have to define a set of rules of engagement that allow facile participation by academia.
DR. SALOMON:  I totally agree with that and so I think that therefore some of where we could start would be creating a couple focused places where you could go if you were in an academic institution and get first just some real education in it.  I know when I did it, I was very fortunate, I happened to be able to call Phil and Joyce and Amy and they were kind enough to spend some time taking my ignorant self and educating me about what I needed to do.  Oh yes, you might have to go in front of the RAC, thank you, Amy, that kind of stuff.
(Laughter.)
But that's not really very efficient.  So perhaps the first thing that we ought to be doing is setting up sort of a website area that might be a collaboration between the FDA and the RAC, the NIH, where you could go and there might be then if you have further discussions you could call these people to get sort of specific answers by e-mail.  I'm not saying that everyone has to be running around with cell phones to know that this is an emergency.
I think that there ought to be some sort of --
DR. SIEGEL:  Those -- we have those websites.  There are extensively and recently updated websites at CBER and FDA with pages for clinical investigators, pages for sponsors, phone numbers, 
e-mails, whatever.
DR. SALOMON:  Specific for gene therapy?  I guess that's what I was kind of saying, Jay.
DR. SIEGEL:  Single site.
DR. NOGUCHI:  It's evolving for gene therapy.  We have a site, but these types of information you're talking about are precisely the feedback that we are already getting and we're going to be implementing.
DR. SALOMON:  Jay, what Ed and I are saying is that yes, I know, again because I've just been educated by you guys that I can go to the FDA websites and you can go through there and find, for example, what's a good laboratory practice, what's good manufacturing practice, etcetera, etcetera.  But if you're not in that culture, if you're not thinking that way, I wont' necessarily know where to look and what's relevant if I want to set up a gene therapy development -- that's all I'm thinking about and I don't have that sort of culture in academia.  Maybe I should if I want to get into this area.
DR. NOGUCHI:  I'd like to just comment on the comments to say that in a way, yes, this is a critical moment for the field of gene therapy, but to also offer the other side of it is yes, it is very complicated, but many things are complicated.  We didn't put a man on the moon without a lot of complications and science was the beginning, but hardly the mechanism by which we get there.  And that's what we're talking about here.  
The idea, the demonstration that an idea, that a vector may have an approach is the easy part.  We're talking here about the very, very hard part, hard because it's hard to get a hold on, hard because it is not -- it is not rocket science, but it's very much in the course of how do you assure to the best of your ability that every trial being done is of the highest ethical, highest scientific quality and has the best chance for success.  It can be done.  It does need a commitment and an understanding by everyone here and throughout the academic and industrial community that yes, we can do this.  We just have to commit to it.
If you were a cook, a gourmet cook, you would be assured that you would know everything that came into your kitchen and if anybody got sick, you would be devastated.  You might even close your restaurant for just that.  This is no less the same.  We're talking about quality.  It is achievable.  It is do-able.  It is work.  But I am positive and FDA is positive and the reason we're holding these kind of conferences is to just say, yes, it's complicated, but you can do it.  We can all do this.
MS. LAWTON:  Can I just follow up on that, Phil, with a question?  Obviously, the education piece is a critical component here, but given the presentations and what we're hearing is that this is being an issue identified from the responses.  
What is the FDA's perspective at the moment around the compliance side and how you are going to monitor?  You said, for example, in new INDs, you're going to be asking questions about the QA/QC.  Will you put INDs on clinical hold unless they have those appropriate answers and then also, a second part of that is are you expecting to up the number of audits, the compliance side of things to make sure that you identify and things are corrected in areas where there are issues?
DR. NOGUCHI:  In regards to the first part, yes, we are considering that the answer to these questions are a part of your IND submission and are part of the information that we need to ensure safety.  If you don't provide the information or we feel that it's inadequate, we will put the trial on clinical hold until they're addressed.
In terms of the specific audits, this last year, doing the -- what you will hear later on as roughly 15 percent of active INDs, actually was an enormous strain not just for our CBER compliance people per se, but for the entire FDA inspection team.  We were able to do it in a relatively short amount of time.  We don't expect to be able to do that continually, however, we will have through -- in the future we will have a smaller number of audits of gene therapy trials, very likely not nearly as many as we've had, but yes, we will continue to have some spot checking to make sure that things are going.  But a large part of it is going to be in terms of being up front.  This is the information required.  Part of it is also trying to expand the infrastructure of people who are qualified to do these QA and QC types of roles.  There are not very many of those types of people and yet obviously they are critical to the whole enterprise.  
We know of several cases in academic institutions where literally a one QA/QC person is being bid for and has his choice of going to Harvard, St. Jude's, Baylor, any of the major institutions.  So a large part of where we think industry and academia could help is programs to actually train people who understand this and who live and breathe this and make it sure that it becomes a viable career for people.
Right now, most of these people, other than in the areas of high demand like in gene therapy, they're sort of looked down on, well, you know, you do QA/QC, yet, they're the heart and soul of getting these products to the patient.
DR. CHAMPLIN:  It's obvious, I think, to everybody doing it that there's two major areas.  There's the gene vector production which is very different than the center that is administering the gene therapy therapy, so often the vector is produced by a company and then shipped to the hospital where the self-processing laboratory will actually do the transduction of cells ex vivo, for example, and then
administer those to the patient.  So the QA/QC issues are obviously very different for the manufacturing of the vector and the clinical approach to individual patients.  And I would view it that one would probably not need quite the same level of QA/QC rigor in dealing with the individual patient on the treatment end, perhaps, than producing a vector that's going to be given to thousands of patients by the manufacturer.  At least, it's a very different type of process that needs to be considered.
DR. NOGUCHI:  That actually is one reason why if you look at the current good manufacturing practices, they are not proscriptive in the sense of you must have a person in QA/QC who has four years of college and has been certified by X number of people.  We allow for local approaches to how you actually address the issues that are there.  It's true that the complexity may be somewhat different for a single patient versus the breadth of the field, however, we do expect that at the very least, if you listen very carefully, documentation, documentation and then again, documentation is where you start.  
Again, as Mary has pointed out, we know something will go wrong at some point.  This is experimentation.  We're talking about experimental products.  That happens.  But you want to make sure that if it happens, it happens once and not twice.  The way you do that is to have the documentation regardless if it's for a single patient or for thousands or hundreds of thousands of patients.  How do you prevent an accident that you know about from every happening again?  How do you learn how to do it better?  You have to document that.
DR. O'FALLON:  A comment -- in the academic environment we commonly refer to QA, whatever terms we want to use frequently falls in the hands of technicians as we've just heard you losing your data clerks and your concept of independence is absolutely ludicrous in that setting.  Those people have no independence whatsoever.  Indeed, if they report something they may get shot as the messenger who has reported the bad news.  And of course, one final observation, this is much more complicated than rocket science, we haven't sent anybody to the moon for a quarter of a century and if we have a catastrophe such as the Challenger in this arena, it will set this whole business back I don't know how long, but a long, long way.  So this is extraordinarily important stuff.
DR. SIEGEL:  Let me just say with the use of terminology being a little bit confusing here, there's a couple of issues I want to address.  Dr. Champlin, your comment about the importance of QA and QC for the single patient, I assume is directed to the issue of QA and QC over manufacturing of a product for a single patient.  The concept of QA and QC over the treatment of the quality of the clinical trial and the treatment of an individual patient is a critically important and under appreciated concept that we'll be discussing this afternoon.  And indeed, the issue of the independence of those processes and how you monitor a clinical trial independently from the investigator is an issue that's every bit as complicated and I would hope not to use the word impossible, but let us say complex and difficult as the issue of how you QA and QC manufacturing independent of the actual people doing the manufacturing.  I think we use the word independent as a gray scale term rather than a black and white term when we talk about these things.
DR. SALOMON:  Comment from Amy and I'd like to quickly summarize this.  I'm going to try to make an executive decision.  We're supposed to have a break at 10:30 and Carolyn hasn't had her -- introduced the idea of the Replication Competent Retrovirus.  
So what I'd like to do is have a comment, summarize this really briefly and then ask Carolyn to come up and talk, so we'll delay the break just a little bit, if that would be okay with everybody.  
Is that okay, Carolyn?  Does that work for you?
Okay, Army?
DR. PATTERSON:  Three very quick things.  I thought that Dan, our Chairman, raised a very important point about having something up on the web that would essentially really walk people through in addition to the various, somewhat complex, but very critical guidances that FDA has up on their website.  And a suggestion I'd put forward is that the workshop at ASGT and any further workshops not evaporate after the workshop is over, but rather a set of facts, frequently asked questions that come out of that workshop could be put up on the web.
The second point, and I think Jay started to address this, I want to make sure it's clear for the public record the point raised by Dr. Champlin.  It is just as critical for single patient as it is for large studies that involve multiple patients to make sure that the product that is administered to the patient is appropriately screened and tested.  I think that there are larger issues of complexity when one moves from small scale to large scale production, but I don't want anyone to leave this room thinking that there's a lesser standard for small single patient studies as compared to larger scale studies.
DR. CHAMPLIN:  My point isn't that it's less important, but it's clearly different.  The problems related to manufacturing a vector is very different to the problems related to running a 
self-processing facility where you're treating a series of patients with transplants of various types, some of which may be genetically modified and how basically to regulate your practice environment of the cell processing laboratories is totally different issues than in the manufacturing of any sort of product.
DR. SALOMON:  Well, just by virtue of just a quick summary here to make sure that we sort of give everyone is on the same page on this, what I've heard pretty consistently here is that there's generally and I hear it also from the FDA staff that one of the things that came out of the letter and reporting is that things aren't so bad out there, that the quality of the understanding in most of these vector production facilities is very high and that reflects I think a tradition in this country for GMP facilities that is just being used now for gene therapy production, but has been long out there and validated and every -- a lot of expertise out there.  
What I also hear us all saying is that in gene therapy just as in any brand new cutting edge technology, the contributions and the ability to contribute actively by academic centers is critical and there, things get more difficult because the culture in an academic center is very, very different, obviously, than that in industry.  And the problem then is that sponsors, including those at the table, are not -- and we're probably a lot more sophisticated through our interaction with you, are not, in general, going to understand and/or appreciate these critical details of QA and QC and GMP and GLP and cross contamination and validation and that more education needs to be there, a higher level of appreciation at the level of the NIH study section needs to be there, education of the faculty needs to be there and I think two key points here came out.  The one key point from Drs. Mulligan and Champlin was just if you don't -- you've got to understand that what happens now is if a sponsor is going to send out something and get back their gene therapy product to deliver, that they're going to do and I think that's what Dick and Rich Mulligan were talking about, when that happens, they're not going to really understand the intricacies of going back on all the lot release forms that they get in some big packet because their face is going forward to the bedside or to the cell processing laboratory.  That's a real issue, I think, from the point of view of the simple statement the sponsor is responsible.  So I mean I think if we want to hold investigators in a system in which we're going to be sending a lot of this stuff, then there really has to be some serious education in the academic centers in order for that to be fair because if a disaster happens, I can just tell you right now, that these guys, in general, are not going to go through all this by just innocence.  They're not going to realize it. 
The other thing, I think, is what Ed said and -- Ed Sausville -- and that is if you do a trial and you don't really know the quality of what you did, and the trial is negative and so an academician on a cutting edge of a new technology sort of closes that door off, that's a tragedy.  And I think a lot of that is even more of a tragedy in a field that up until now has been struggling for its big successes in the last several years.  So I think again, there's just this educational process is going to be critical for the field in academia.
Any comments?  I mean does anyone disagree with that summary?  Did I miss something important?
Okay, Carolyn, you're on.  Talk about retroviral vector production.
DR. WILSON:  Good morning.  I want to first just begin by clarifying that what I'm talking about today are retroviral vectors that are currently used in clinical trials and these are vectors that are derived from a class or group of retroviruses known as, known now as gamma retroviruses.  These vectors have been engineered so that when they are produced they are defective.  They can no longer replicate in their target cells and this is an important safety feature.
However, there are occasions when there can be what are called recombinational events that occur during manufacture of these vectors where  replication properties are regained by these vectors.  And those are termed replication competent retroviruses or RCR.  And we consider these contaminants and on the next slide the Agency's point of view is that these are not only contaminants, but also pose a safety concern and a risk to subjects in these types of clinical trials.
 		To underscore this, I just wanted to briefly remind people of a study that was done early on by Robert Donohue and his co-workers at Art Neidenai's lab where -- this actually wasn't a serendipitous finding where they were doing some preclinical studies for an ex vivo gene therapy using bone marrow transduction and it turns out that their preparation of retroviral vector was heavily contaminated with RCR.  And when these immune suppressed monkeys received the bone marrow transplant, within 200 days, three out of 10 developed lymphomas and died.  
Subsequent molecular analyses of tumor tissue from these animals demonstrated that there were sequences present in that tissue that were recombinants between the vector and helper sequences from the vector producer cells or vector and cellular sequences.
Next slide, please.  Because of the recognized concern of presence of RCR actually over a number of years, the Agency has been developing guidance in this area and as early as 1993, developed more stringent guidance about how to test these types of products for presence of RCR during manufacture.  The most recent guidance was issued in October 2000.  I'm not going to go into that in any detail.  It's available on the web and the title is shown here, but I just wanted to briefly say we do give detailed recommendations about how to do RCR testing at  multiple points during manufacture, but we also still ask for a follow-up of patients in these clinical trials.
Next slide, please.  In the March 6th letter, as Joyce mentioned this morning, the fourth question asked for information about lots that were rejected for clinical use and the reasons for why it was rejected.  We viewed this as an opportunity to gain some information about what types of vector producer cells had reported incidents of RCR detection during manufacture.  Again, as Joyce also mentioned, I wanted to just point out this represents only those currently active files.  So the files that are no longer treating patients did not provide a response to the March 6th letter.  So it's meant to really represent trends and only in the currently active files.
Before I go on with that data, what I want to briefly do just so you have an appreciation of what these different vector producer cells are about is to just quickly go through the technology and how some of the vector producer cells have been designed to try to reduce incidents of RCR. 
Next slide, please.  Essentially, the gamma retrovirus, these are simple retroviruses compared to the virus that you probably most know about, Human Immunodeficiency Virus.  It has only has three open reading frames called Gag, Pol and Envelope and then it has LTR or Long Terminal Repeats at either end and this size sequence is a packaging sequence which allows for a viral RNA to be packaged in the particle.
In the design of retroviral vectors, you can typically think of this genome or actually this is a provirus structure being divided into what are called retroviral helper sequences which encode the transacting elements for production which have the coding sequences, Gag, Pol and Env and the vector sequences which contain the cyst acting elements that are required for packaging, reverse transcription integration and transcription.  
On the next slide this a sort of cartoon of what a typical vector producer cell might look like.  The helper sequences and the vector sequences have been introduced on plasmids and then become integrated into the genome of a mammalian cell and the RNA is expressed.  In the case of the helper sequences, these are translated into viral proteins.  Because this RNA does not contain the packaging element, that RNA isn't packaged into the vector particle, but rather the helper, I mean the vector sequence that does contain the psi element will be in the vector. 
So these elements come together at the surface of the plasma membrane, but through and you get a vector particle.  These particles are structurally identical to a retrovirus, a wild type retrovirus, but they no longer contain the coding sequences to make progeny variants. 
In a stoicastic manner, there are occasions where you get recombinational events between these sequences or in the case of, for example, murine cell lines that have their own endogenous retrovirus sequences that have homology to these elements, those can also participate in recombination and generation of a wild type viral RNA which can then be packaged and then we have an RCR.
Next slide, please.  Over the course of really the last 15 years, scientists have been working on designing vector producer cells which have reduced incidences of RCE for obvious reasons and some of the strategies that have been applied are to eliminate sequences of homology so that there's less opportunity for recombination, so overlapping sequences between vector and helper, using cells that don't have homologous endogenous retroviruses, splitting up the helper sequences into more than cassette, for example, Gag-Pol is typically separated from the envelope.  And introduction of stop codons in any of the open reading frames that might still be present on the vector sequences, for example, the Gag overlaps with the packaging element, so you usually have a little bit of Gag on the vector sequence.
Next slide, please.  So now I just wanted to very quickly go through this summary table and I know this is going to be hard for people in the back to see and I apologize, but I wanted to be able to capture for each vector producer cell that's being used in clinical trials, that critical information as it may correlate with the detection of RCR during manufacture.  So I've listed whether or not it's a murine or human cell line, how the helper sequences are designed, the envelope, and what we observed in response to Question 4 in terms of reports of RCR detection.
PA317, notably, is the only vector producer cell still being used for clinical trials that has only a single expression cassette  for the helper sequences.  And we've observed that it was fairly common that manufacturers who were using this packaging cell line have lots that are positive for RCR.
Two -- what you might consider second generation cell lines that have two expression cassettes, but are still in a murine cell background also had reported lots that were positive for RCR, but at a lower frequency.
PG13 which is still a murine, two expression cassettes, but now has a more heterologous envelope given a leukemia virus with reduced homology to the endogenous murine retroviruses so far has not had any reports of RCR and this other category actually represents several different producer systems that are used in a human cell line, also two expression cassettes and the amphotropic envelope in this case and so far, again, no reports of RCR positive lots.
I just wanted to mention these last two, in particular, have been used -- their implementation for production of vectors in clinical trials is more recent and so our cumulative experience with these is somewhat less.  So this may not be an absolute no as time goes on.
Next slide, please.  I wanted to just give you also a snapshot from one production laboratories.  This is actually a National Gene Vector Lab at Indiana University.  This data was kindly provided by Ken Cornette and Lilith Reeves.  The provided to the FDA their total summary of all their production lots for clinical trials with the different vector producer cells.  And consistent with what we saw in the response to the March 6th letter in general, they see really a fairly high incidence of RCR positive lots when they used PA317.  This is reduced with AM-12 and although this is only an N of 2 for Psi-CRIP, so far they haven't had any RCR positive lots.  PG13 for 14 lots produced is still, so far have not produced any RCE positive lots.
Next slide.  What I just want to finish with is first of all, I think we need to recognize that there are some assurances here that, in fact, in the later generation of vector producer cells, what we're seeing is that design elements that were meant to reduce incidents of RCR during actual manufacture for clinical lots is resulting in a reduced incidence of RCR and I think that's the good news.  And I also just wanted to sort of put a plug in, not only is training and outreach from our perspective important, but it's equally important for us to be able to continue to have these kind of dialogues and the public and for investigators to continue to try to get as much of their data out into the public domain on these issues as well.
And so with that, I'll just turn to the next slide which has the question for discussion for the Committee.  Thank you for your attention.
Do you want me to read the question or do you want to read it?
DR. SALOMON:  I can probably read it.  Thank you, Carolyn.  The -- I'm trying to think of the best way to do this.  I think what I'd like to do is take a break now.  I think we've been sitting here for a while and come back and deal with these questions and the subsequent presentations, if that's okay with everybody.
So ten minutes, and be back here at ll.  Thank you.
(Off the record.)
DR. SALOMON:  If we can get everybody back to their seats so we can start again after the break.  As everybody knows whenever Jay sits down, that's my official signal.  Jay is good.  I might have to pick someone else like Phil, because you've gotten real good about it.
Okay, if we can get the audience to sit down as well.  I need a gavel.  Can we have like maybe a sudden loud feedback?
(Laughter.)
Okay, thank you, everybody.  I'd like to first introduce on new Member to the Panel.
Dr. Roessler, can you just give us a quick brief on who you are and what your expertise is?
DR. ROESSLER:  I'm Blake Roessler at the University of Michigan and at the National Gene Vector Laboratory and our center has been manufacturing plasma DNA for use in clinical trials.
DR. SALOMON:  Thank you.  Okay, well.  So the point where we're at now is after the presentation of Carolyn Wilson on replication competent retrovirus and retroviral packaging lines, she posed for us a question that based on currently available data regarding RCR detection during vector manufacture, and she's referring now specifically to the table showing the different vector packaging cells, the VPCs, is it reasonable for CBER to disallow in the future in INDs, the use of VPCs with a single expression cassette for the helper sequences such as PA-317? 
Any comments from the Committee?  Richard?
DR. MULLIGAN:  I think it's not exactly a burning question is my opinion, but my answer would be no.  I think one thing that was listed on the overhead, but not really appreciated is how key the vector is to a packaging cell, that is, pairing the right vector to the packaging cell is really the key and you can take a lousy packing cell and use a good vector in the packaging cell and tend to get no difficulties, or you can use a very good packaging cell that is constructed, designed in a proper fashion with a lousy vector and have difficulties. 
And so I think that you cannot in a blanket fashion take a particular packaging cell and say no.  I think I would rough up anyone who would suggest using PA-317 and really ask them why would you possibly do that, but I think it's not actually worthy of a lot of our time and effort.  I would focus on issues that have to do with the details of vector design and how they influence things and also the issue of how you introduce the vector to the packaging cell.  One thing that we've never really published, but we've always had a sense of, is that doing 
so-called ping-pong infection or cross infection to make the packaging cells is not a good way to make clinical grade packaging cells and there's some technical reasons we don't really need to go into at this point on why that may not be a good idea.  But I think there's some real reasons where I would -- I think that's a far more significant question of whether there's mutations associated with cross infection.  that means you have to test the product in a different fashion.  There's issues of whether or not the use of cross infection simply improves, increases the frequency of transmission of other endogenous sequences.  So I think those are more the kinds of issues.
The last thing I'd mention is people over the years have really not appreciated other things, other transmission issues other than replication competent and I think although it may not be that much of an issue at this point with the Lentivectors, the whole issue of whether or not these packaging systems are predisposed to transmission of a portion of the packaging sequences I think is very, very important and I think that if you look at all the literature, people really have not addressed.  I think very recently there's some people that have begun to look at this, but I think that's going to be key, that is the fact that even without any packaging sequences of nonretroviral genomes can be packaged with some frequency and depending with what else is co-packaged, you can transmit those sequences.  And so, I think that those are the kinds of issues that are key.  But here, I would just leave it to the sense of the FDA which I think is a consensus of the scientific community that these single genome packaging cells are just not the latest and the greatest.  So why would you use them?
DR. SALOMON:  Carolyn, do you have a comment on that?
DR. WILSON:  I just wanted to make one point for clarification which is that in production of clinical grade retroviral vectors, we don't approve INDs that use the ping-pong method of manufacture.  That's been the status for quite some time.
DR. SALOMON:  So -- what I see here as an interesting issue --
DR. MULLIGAN:  Excuse me, just to clarify.  Even a single pass of virus?  Because I think the same holds to the single pass.
DR. WILSON:  Okay, yes, we do allow for the single pass.  I thought you meant when they go back and forth.  And when they do, we do ask for additional testing, for example, for the ecotropic RCR that could be introduced from that method.
DR. MULLIGAN:  Because even the single pass predisposes you to things that couldn't happen by the transvection.  So you know, if you're unlucky, very unlucky and some people are, you can actually get a cross infection that will give you a point mutation, even though it may be an error and if you happen to pick that as your producing cell, then your product has mutation in it and you're probably not or you haven't in the past asked people to actually sequence the proviral DNA.  But that is a difficulty that you wouldn't have, at least not the same extent by just the transvection of the sequences.
DR. WILSON:  That's correct, but as you know, that's actually a topic that we discussed in November and we're trying to evolve our policy in that area as well, so that a master cell bank that you would derive then would have sequencing of -- we were thinking of the viral genomic RNA as a one-time basis to qualify and make sure that just the type of thing that you're suggesting wouldn't have occurred.
DR. SALOMON:  Yes, Richard, you missed the last one.  I know you had to leave, but the final -- the second day.  The final was any vector up to 40KB had to be sequenced.
DR. MULLIGAN:  Yes, well, I did miss it, so I'm not sure what you said, but I thought when I left that you had to sequence the parental DNA.  But the question is, what we're talking about here is the actual if you were to do a viral infection and generate the packaging cells, would you have to actually sequence what had undergone reverse transcription and become DNA from the input of RNA?
DR. WILSON:  And that's where we're in the process of evolving our policy recommendations on to address that exact point.
DR. SALOMON:  That was certainly the spirit of it, at least my understanding of the spirit of it.  We knew what we were producing, not what we thought we went into at some point.
DR. MULLIGAN:  Right.  The one last issue with the helper is that although there's obviously other events that are important for a retrovirus, a non-oncogenic or a non-oncogene containing retrovirus to cause a tumor, it's generally thought the simple version is that it's the number of hits, the number of integrations that are important and I used to make this joke in the past, at these BRMAC meetings 10 years ago that all people have proven so far in gene therapy is that if you don't have any gene transfer occur, it's a perfectly safe approach.
(Laughter.)
And it's very important here because the results that we have showing the tumors are presumably the results of many more integrations of sequences and people ought to be aware of the fact that as the vectors get higher titer by thousand fold or so, and we finally figure out how to transduce stem cells, that you're going to have a risk not from replication competent virus, but you're going to go back to the risk that everyone never really wanted to talk about which was if you load enough proviral copies, you're going to hit a location that's not a good location.  And therefore, going back and trying to analyze those earlier Neinheis results might be quite important from the point of view of do we really have the sense of just in the simple number of book integrations that have occurred that led to this event, how far away are we from an in vivo gene therapy with a vector that now integrates into resting cells at high efficiency.  Could we actually get the same number of events and would we then be concerned about that?
DR. WILSON:  To address that point, actually, I believe it's the Purcell paper that I cited, does show, if I recall correctly, around 20 to 30 copy numbers in the tumor tissue, so your point is well taken, that it certainly does take multiple hits.  And I also just to focus on that point, that's the reason why even in our patient follow-up, when we look for evidence of RCR infection and it's even negative at one year, we continue to recommend follow-up of patients past that point for the very reason you're suggesting, that the vector per se can also have the potential to integrate into a cite that could have potential tumorigenic consequences.  And that will also be a topic we'll be discussing more tomorrow.
DR. SALOMON:  Yes.  I want to say that these are really important discussions, that's why I didn't cut them off, but we want to talk right now about production issue and the questions we're not segueing into are critical, but they're more what's going to happen after you institute the trial and we should get back to those.
I guess the comment that I had, just to make sure that we have a little bit of discussion before -- I always like the basic principle of not just making a policy that then creates a rule that might later reduce some flexibility that a reasonable scientist, based on very rational sets of thinking suddenly wants to use this vector, for example, 
PA-317.  On the other hand, I think there is a safety issue that's overarching this.  When you have, I guess, data where 75 percent of the production lots had RCR contaminating them, and you concerned yourself with the idea that any sort of testing  strategy might miss that once in a while, is it really safe?  Should we not just ban single help, single package, single cassette, expression cassette lines like this when they have such a bad record?
DR. MULLIGAN:  I haven't really looked at the data enough to look at vectors that are used with the packaging cells, but I think you'd have to do a very careful analysis, a very detailed analysis of what vector was used in a case where this did happen, what were the circumstances in terms of how they did the transvection, how they picked the things.  The other issue that probably is not pertinent here is how often are they actually false positives?  How often can you repeat the positivity?  Again, I could go either way on this, but I don't think it's a real big enough issue to set a precedent because that means that then as other things come down the pike, I mean this is such a no brainer in a way that this is okay if you want to set the policy on this, but things are not going to be no brainers in the future.  There's going to be something that's a little better than 
PA-317, but still not very good and then you're going to have to say now where do I make the cut and who's going to make that cut?  I think everyone probably agrees that this is a nefarious cell relative to other things and I don't know of any single biological property of those cells that would make someone say well, we got to use those because they do something.
DR. SALOMON:  Exactly.  That was my point.  On the other hand, I hear what you're saying, that -- I guess that's always the thing that we're monitoring, right?  At what point are we in development of a field where we don't need necessarily to make a complicated decision, yes, you can't have a single expression.  I guess that's probably not correct and maybe what we ought to do is leave it as I think there's general agreement here that you don't want to see replication competent retrovirus.  I think the other thing that the data shows is that this kind of data actually will continue to grow and be available in the sense that the percentage of replication competent retrovirus contaminating lots will come out and that should be a gauge.  You should have to show that kind of data.
Moreover, I guess it would be something that would raise an appropriate alarm if I came up to you and said I've got a new trial and I've got a new packaging line that has no history at all and then questions like what kind of vectors do you put in, maybe there ought to be some data where you put in four or five different kinds of vectors and show that you're not getting RCRs.  I mean maybe the general principles are on the ground and we don't need to do anything more.  I think that's what I'm hearing here.
DR. MULLIGAN:  I just thought of one case in point that someone might make for the PA-317 which is that a company might say look, optimize the large scale production of these cells for five years and this is unique and every cell is different and that's why we want to use these and I think that's somewhat of an argument, so I think there are going to be compelling reasons, but I think it's perfectly reasonable to discourage the use of it and try to probe why it's necessary and you know, -- that's all.
DR. NOGUCHI:  If I could comment, I think we appreciate the need to be flexible as much as possible, but the other way to look at it is we're asking for a scientific evaluation of what data that we have and in the context of the larger picture that all these things are complicated.  We have not had any true demonstrated, long-term success as yet.  Maybe some encouraging results.  
Would you, as part of this Committee advising us, do you really think that it's, as you correctly point out, there are many larger issues, does that mean then that we can't put this one to rest so we always have to have open the ability for somebody to come in and say well, look, I want to use this for the following reasons.  We have to evaluate that.  We have to figure out what kind of designs or tests we're going to say to make sure that, in fact, the reasons for using this outweigh the risks that it might occur?  
Partly, the question we're asking is a simple one, but it is an important one.  Are there some things that just aren't worth pursuing?  That's the question.
DR. MULLIGAN:  I think that I just don't personally have enough of the scientific information to fully evaluate the properties, the total properties of packaging cells and vector, so I'm just saying you may have that, but I wouldn't be convinced until I went and looked at that information.  For instance, it may be that there are certain kinds of vectors that are generally used because someone develops the vector and someone develops the packaging cell, so it might be that typically you use a certain type of vector with PA-317, just because you get sent a vector and you get the packing cells in the same person.  And it may be, it may tell you something.  It may tell you that using this vector leads to this difficulty, but another vector doesn't lead to it.  So I don't think there's enough, but before I as an expert could actually commit to saying you ought to derive a stake in the heart of this packaging cell, I would want to see much more detailed scientific information about the properties and what happened.
DR. NOGUCHI:  At the cost of more clinical trials?
DR. MULLIGAN:  Oh no.
DR. NOGUCHI:  No, I mean with -- using this vector or this packaging cell line with at least some potential for being less efficient in producing a clinically acceptable vector.  That's part of the equation here. 
If we could be assured that in fact, scientists would be looking and stressing the system in experiments not designed for clinical trials, that would be one way to approach it, but that's not what's being done.  What is being done is these are being used at very high titers, or relatively high titers, specifically because that's the amount that is needed for a clinical trial.
We're pushing you and asking, do you think it's worth doing any more clinical trials or some more or how many more clinical trials with a product where we know a goodly proportion of the vectors that are produced will not be acceptable, that's an additional cost that does leave the potential risk because the limits of detection may not be appropriate or always the same?
That's the real question.  We're asking a very hard question between the starkness of scientific discovery and pushing the envelope and finding out as much as we can versus a very real concern that to progress, we need better vectors.  Is this the best way to do that?  And is it worth the human experimentation that is going to drive the production of these vectors?
DR. MULLIGAN:  I remember this argument over a decade ago with the evolution of the first 
Psi-2 cells and so forth and I remember distinctly the consensus point of view being this is all theoretical.  Because I remember when we developed the first of these split packaging cells and we were mentioning the importance of the theoretical safety advantages, I remember many people said well, gee, isn't this really theoretical?  The test is the test.  If you do the test and it comes out clean, then you use the stuff.  You could argue from, I think, the line that you're taking, if you ever saw with any packaging cell, a batch that had replication competent virus, you might think that there's something deficient with that cell and you might then be concerned about using that in the future.  I mean the argument is not that different, if you ever find something happening.  I suppose it's the case that if you had something that you never saw any helper virus, you might think that that meets the absolute test, but as you know, as the tests get more sensitive, you begin to pick up things you didn't pick up before.  
DR. NOGUCHI:  Yes, but this is not a single case.  This is multiple cases.  This is 10 years of experience.  
Are you saying that we cannot use our experience to exclude things?
DR. MULLIGAN:  Well, I would feel more comfortable, I cannot say here that  I would want to get rid of it without seeing what the data is.  I think you owe it to everyone to -- well, I mean you have to tell us how much investigation you had of the actual vector and the method of generating the packaging cells, because I think that that's important.  Because what you might be doing is revealing something that's not PA-317 specific and if that's the case you want to know that.  That is, your assumption is that if something because it's a single gene, but it may not be, and you would hate to miss that if turned out it was the way you did -- the way people generally did those transvections, the way they cultured the cells or some other property.  So I think there's incomplete information.  
I'd be happy, if you want, to review that information, but I would not be comfortable saying that you shouldn't use this. 
All that being said, I'm sorry, we spent so much time on it.  I mean I actually think it's -- I wouldn't feel awful if you rammed it, I just think it's something -- clearly there's better things.  There's no question about that.  But in this field, as you know, there's always better things.  And I think it begins to get complicated once you try to figure out what's better and how much better does the next thing have to be before you can the first thing.
DR. SALOMON:  Dr. O'Fallon?
DR. O'FALLON:  Yes, I was just going to point out that the data that you brought to us is summarized in Tables 1 and 2 of our previous presentation and it says yes, fairly common.  That's not a very good quantifiable concept and in the next table there's three out of four, also not very impressive from a statistical standpoint.  
We might start on a slippery slope, but if this Committee makes judgments on such small lot of data that we certainly wouldn't make the same judgment and approve a product if somebody said we would have 3 out of 4 successes on a clinical trial.
DR. SIEGEL:  If you had 3 out of 4 fatalities though you might make a judgment that it was unsafe.
DR. SALOMON:  Right.
DR. O'FALLON:  I agree.  
DR. SALOMON:  Dr. Chanock.
DR. CHANOCK:  I was going to say on the last point, I agree fully about the question of the statistical nature, but the other issue to come back to the question is are we sure that we can blame it on the PA-137s per se and not some methodologic question?  And I think that with this amount of data I wouldn't want to throw the baby out with the bath water, so to speak if there are opportunities for people who are still trying to develop and improve the vector technology to be able to use that and I think that you are going to find that if there's a set of restrictions or a set of guidelines set down for RCRs, then those are going to help to drive the choice on the part of the individual investigators into commercial outfits, so I think it will partly drive itself, so I would be worried about cutting it off at the pass right now, unless there was more information that would be more compelling, at least that is forthcoming.
DR. SALOMON:  I think to just in the interest of moving on, I think what I hear from everyone and I certainly agree as well, is that the Committee is willing to consider the possibility that a cell line with a bad enough track record might be taken off the market unless someone -- it wouldn't stop anyone from coming back later and saying look, if I do this and this, it's wonderful.  You should take that for merit.  I think that what you're hearing is is that we ought to set a series of guidelines for the use and selection of these vectors.  To pick up something that was emphasized in the last meeting in November, right, was again the data base is still really incomplete.  We get data, we've shown data today from just the NGVL, from Ken Cornetta's group which is great because it gives us data, but I think that Dr. Mulligan's and Dr. Chanock's points are well taken.  It's not every case.  We don't really see the whole universe and I think in the absence of really getting all the data the way scientists want to see it, it's not reasonable to ask the Committee to kill a vector or to even make those kind of decisions.  And I think that you're trying.  I mean that was the message in November as frustrating as it was sometimes, that this whole area is suffering in one major way from a data search crisis.  We need all the data in one place.  And I know that you guys got that message last time.
DR. MULLIGAN:  My message was that there is no social redeeming value to that cell line.
(Laughter.)
I don't think -- there's no special properties of that that I can think of that would make me support it the way it appears that I am, but nevertheless, I wouldn't do it right now.
DR. SALOMON:  Okay, are we comfortable with that?  All right.
The next presentation is from Suzanne Epstein.  Dr. Epstein is going to talk about responses to the FDA letter on testing of plasmids.  If you haven't already, obviously, looked ahead a little bit here, it's -- we talked about replication competent retrovirus.  Now we're going to talk about plasmids.  Then we're going to talk about adenoviral vectors and we're also then going to hear some more about adenoviral infection.
Somewhere in the line here I've got to juggle this with lunch and stay reasonably on time so someone doesn't strangle me by late this afternoon.  I'll worry about that.
DR. EPSTEIN:  Can you hear me?  How's that?  I'll try and make up some time.  I'm going to be talking about testing of plasmid DNA when it is used as an intermediate in manufacturing other gene transfer gene therapy products.
Plasmid DAN is used in a variety of ways in the manufacture of biologicals.  In one extreme it is the actual product administered directly to the patient.  In the case I'll be discussing today, plasmid is used as an intermediate during the 
lot-by-lot production process of other products.  And then finally at the other extreme, plasmid DNA can be used early in deriving a construct that's used for any of a wide variety of purposes, but then it's a reagent.  It's not something used every time and its status is really not very different from any other reagent.
The goals in this area were as follows:  testing of plasmids used as intermediates would help achieve consistency of manufacturing of the gene transfer product and would prevent contamination of cell cultures that are used as the product or in making another vector.
What we decided from the March 6th exercise was that we needed to clarify CBER's expectations for testing of plasmids when used as intermediates and also seek advice from the Committee about the reasonableness of our set of recommended tests.
So first to give you some examples of what we mean by this, when plasmids are used as intermediates in production, they're used during production of each lot of a gene transfer product, not just during deriving some kind of a construction of a cell line.  Some examples include ex vivo transvected cells, AAV vectors and retroviral vectors in certain cases, certain production schemes by transient transvection.
One thing we noted in the responses to the March 6th letter were that sponsors were confused.  There was tremendous in whether or not they reported on plasmids and in some cases they reported only on the final product.  In other cases they reported surprisingly only on the intermediate and didn't, for example, provide data in answer to questions 1 and 3 about cellular populations which in those cases may have been the actual product.  So anyway, there was confusion as to what our expectations were.
So here's one of the examples.  If cells from a patient, this is often a patient-specific population, but could be a cell line, are transvected with a plasmid that contains a transgene, you then end up with a cellular population expressing the transgene product and this is your final material given to the patient, but this vector is quite important and is used every time and will have tremendous impact on the consistency and quality of this production scheme.
Here's another example, production of AAV by a certain method.  This is from a paper by Grimm, et al., and in case two plasmids are used, one containing the vector, the intended AAV, and one containing the rep and cap functions and the adenovirus helper functions.  Both are used to transvect a cell line and the cell line then produces the AAV.  So again, the intermediate is used every time and its identity and quality will have an impact on the consistency of this manufacturing and whether these cell cultures become contaminated.
There are lots of precedents for regulatory scrutiny of an intermediate.  First of all, reagants and intermediates in general, when used to produce biologicals are subject to quality control testing, some of this is in the GMPs in qualifying source materials and so on, and this is quite general.  Then specifically, the uses of plasmids I've illustrated for you are analogous to use of retroviral vectors when they are used to transduce cells for ex vivo gene therapy and in that case the transduced cells, not the retroviral vector are administered directly to the patients.  There are some other cases where retroviral vectors are given directly to patients, but the analogy here is with the ex vivo case.  In those cases, even though the vector is an intermediate, retroviral vector preparations are subject to extensive quality control testing.  So what I'll be talking about is nothing new.
What I'll do now is just throw out there a proposed list of quality control tests, but this is for discussion purposes and is certainly open to change.  These would be plasmid intermediates being used, as I've discussed and what I mean here is that each lot of plasmid DNA that was prepared for use would be tested in these ways.  So this lot-by-lot testing and we'll come to some one time testing.
Sterility is pretty obvious to avoid contaminating the cell culture.  Residual toxic reagents such as organic solvents also could have a negative impact on the cell culture.  Endotoxin can interfere with transvection.  Then identity is particularly important because of the number of 
multi-use facilities and we've heard before the discussion of mixups.  This could be a variety of types of tests.  We're thinking of, for example, restriction mapping, but I'm hoping Dr. Roessler will comment on this because sequencing is a possibility here also.  And in a multi-use facility this might include excluding contamination with particular other products.  Purity -- these interact -- purity could include ruling out a variety of contaminants and in this case might be an agarose gel electrophoresis.  Concentration might be absorbance.  We're certainly not specifying particular assays for these things.  
And then activity or gene expression can be very important to know that the protein expressed from the transgene is active if you're not doing a full sequence here.  So these types of tests are proposed and the sponsor would have to establish acceptance criteria which would depend partly on the amounts being used and so on, what levels of endotoxin might be acceptable, for example.
Note that an activity assay is not necessarily a fully quantitated, validated assay like a potency assay.
Next.  We're proposing as a one-time test, full plasmid DNA sequence and homology search for open reading frames.  As you probably know from the earlier meeting, this type of analysis has to do with finding extraneous material in the construction that shouldn't be there, looking for rearrangements and so on, fairly gross features.  
This would be performed once, not necessarily on every lot although we'll return to that in the discussion because there are different points of view there and it would be performed prior to Phase I because these are small vectors and there should be no problem conducting that analysis, so you should know what your construct is.
Then we'd like to raise the question of whether certain other tests are necessary or not for plasmid intermediates.  Residual E. coli DNA, RNA and protein are contaminants that can matter in some situations.  They can indicate inconsistent manufacture or a sloppiness, but they may not matter if the product isn't going into patients and it's just being added to a cell culture.  Then a potency assay is a more quantitative, formally validated assay of activity and we're proposing instead only an activity or expression assay.  These additional tests, these purity tests and potency tests are expected for plasmids that are being given directly to patients, so we're proposing a less stringent standard for intermediates.
And that brings us to the questions for the Committee.
DR. SALOMON:  Thank you very much.  Can you do one level of clarification while everyone sort of gathers their thoughts and that is you used these words very clearly, potency and activity.  Can you maybe just --
DR. EPSTEIN:  Give examples?
DR. SALOMON:  Yes.
DR. EPSTEIN:  A true potency assay would be a measure of what the product is supposed to do to have its biological effect in the therapeutic setting.  		An activity assay can just demonstrate something the product does.  To try and give an example, suppose the plasmid is being used to transvect lymphocytes which are then infused in the patient and lymphocytes are supposed to go and kill something.  An activity assay might be that the plasmid causes the appearance on the cell surface of that protein.  A potency assay might be some correlative killing or an animal model in which the tumor regresses, something like that.  So it's much more difficult to provide a potency assay and for a final product for patient use by Phase III you have to at least do your best.  For an intermediate, we feel if you are getting, for example, the proper enzymatic activity of insert, that's good enough or whatever.
DR. SALOMON:  Good.  That's great.  Okay, specific questions then?  
Are the quality control tests listed appropriate tests to be performed on each plasmid lot?  Sterility.  Yes?  Obviously, right.
Residual toxic reagents, for example, solvents.  Now my response there is I'm not quite so clear, so maybe this is where we need some discussion.  Number one, it's not always so easy to assay a preparation for the presence of solvents unless we're talking about gas chromatography or thin layer chromatography.
DR. EPSTEIN:  There hasn't been any resistance to that.  Organic solvents, phenol or ethanol or whatever that are used in plasmid purification can be detected by means like gas chromatography, very sensitively and it doesn't seem to trouble people to do that.
DR. SALOMON:  Well, okay.  I'm just bringing it out.  Here, again, we make this segue, to have a gas chromatograph I think minimum would be $75,000 to $150,000 investment.  
DR. EPSTEIN:  They just have a firm do it for you.
DR. SALOMON:  Okay, fine.  I guess the other question would be if there's phenol or something contaminating it, what's the concern?  If I now add it to my T-cells in order to deliver this potency product, I'll affect the T-cells and I won't know it, right?
DR. EPSTEIN:  Right.  Say there's chloroform in there.  You may kill your T-cells or even if you didn't, you don't want to give chloroform to your patients.
(Laughter.)
DR. SALOMON:  I certainly don't want to give chloroform to the patients.  I'm just trying to be reasonable here.  At some point, you're going to wash the cells I would hope.
DR. EPSTEIN:  Yes, but basically, the reason to be concerned in the case of intermediate, would be the health of the culture.  It's more of a consistency.  In fact, all of this is more of a consistency.  In fact, all of this is more of a consistency issue than safety.  It's very unlikely you'll put enough of something in to create a safety hazard to the patient.  It's more likely that you'll kill the cells and/or mess up, contaminate your production.
DR. SALOMON:  Good.  I'm fine with that if everyone else is fine with it.
Endotoxin, I think we pretty much all assume that lot testing should include endotoxin testing and that is a danger and I don't want to give my patients endotoxin and it's a real issue in any manufacture.  Is there any disagreement there?
Identity.  Now that covers a lot of ground.  I hope that there's some discussion from the group here.  I'll play the stupid guy for a while here, but in some way identity is important.
(Laughter.)
I'm telling you identity is important and I want discussion on this one.
(Laughter.)
DR. ROESSLER:  I'll take the bait there.  I think that there probably is a role to consider the value of sequencing of lots and I think that's largely on the basis of a specific transgene in the effect that a specific transgene might have during the production process in terms of an adverse selection pressure it might exert on your E. coli producing strain that would allow for mutations, rearrangements or deletions to occur at a high level that might affect the fidelity of your product and might affect how much of your viral reagent was manufactured 
post-transvection or how much of your transfusion of interest was produced post-transvection.
DR. SALOMON:  So the last time we met, we all agreed that you had to supply sequence identity for plasmids under 40KB, in like 40KB or less.  And the interesting thing that came out today, I think partly in Richard's comments was where is that sequencing validation done and how often do you have to do it in order to stay within the Committee's spirit that you know what's in, right?  We all agreed that on the way in you need sequencing because you just don't know what someone helped you with and it's just really hard when people are giving you little vials to know exactly what's in there, right?  And your point is well taken that some of it's even selected again by differential growth of the production bacteria.  
So we all agree on that, but how about -- how about at the end of production?  How often does one have to determine the identity?  Is it only on the first day when I give you the vial, you grow it up?  So what is an appropriate recommendation from the Committee on tracking identity over time?
DR. ROESSLER:  Well, we wouldn't expect that there would be changes in the actual sequence over time and is your question related to product storage?  For example, that you're going to make a large batch of plasmids that express rep or cap and that you'll need some stability assay to be performed, but that doesn't necessarily have to involve sequencing.
DR. SALOMON:  I agree.
DR. EPSTEIN:  I'm not sure you realize that Dr. Roessler was proposing lot-by-lot full sequence.
DR. ROESSLER:  No, not full sequence.  I think we just need to consider the role for sequencing of lot-to-lot production material.  
DR. SALOMON:  That's the question that was asked.
DR. ROESSLER:  I don't think that I would -- I would just say that for every gene vector that's being produced that restriction analysis alone is going to be absolutely adequate.  I think for the vast majority it probably would be adequate, but I wouldn't discount the added value of doing some limited sequencing, primarily of the transgene insert or flanking regions.  I think once again it echoes Dr. Mulligan's point that you have to take advantage of the available technology and changes in technology.  Clearly, sequencing is evolved and it's become more rapid and less expensive.  And so whenever you have a technology that is moving in that direction, it represents from my perspective added value to the manufacturer and to the sponsor.
I think the one other issue is that in the academic sector, specifically, if you have a sponsor who is going to be responsible for performing the potency assay, and that assay fails, then they're going to come back to the manufacturer and say the potency assay suggests that there was a problem with the plasmid and as the manufacturer what data or evidence can you provide to me that it wasn't a problem with the plasmid.  So I think it's just another series of checks and balances and it may be that my perspective from the academic sector is trying to think through that scenario where we might send a plasmid intermediate to a sponsor for a potency assay or a functional assay and then it may not meet specifications and then we have to problem solve that unexpected result.
DR. SALOMON:  Well, at the risk of oversimplifying, right now when we think about what kind of things we'd do with plasmids in gene therapy, one is that we would do ex vivo exposure to effector cells, right?  They could be stem cells or it could be T-cells or macrophages, something like that, that would give them a property to target or to kill or to home to some sort of area and maybe produce a growth factor.
The second thing is actual injection of the plasmid directly into a site, in vivo, right, such as the VEGF trials being injected into areas of ischemia, in the heart or into peripheral, ischemic peripheral vascular tissues.  So that would be two major things.
Now injection of the plasmid, I would be comfortable that you had made a large lot of plasmid, sequenced a aliquot of it and so I know what I was injecting.  That would be easy.  The question I have now is in the experiments where I'm putting plasmid into 10^10 purified T-cells for a study that I want to do, just for example, do I need to - -and I know that the input plasmid is sequenced because it came from the same lot you would have given me had I wanted to do a direct in vivo injection of plasmid.  Do I also need to grab a couple of the T-cells and sequence the plasmid in the T-cells?  Is that something that we're suggesting?
DR. ROESSLER:  I think that's obviously technically much more difficult and presents much more costly scenario.  So I don't think that we have enough information.  It's kind of a theoretical anecdote that you raise, but I see where the point is, but I think that from a manufacturing product perspective, once again, it seems reasonable to do the complete sequencing and then restriction analysis and then on a case by case basis to consider added value for limited sequencing of a specific lot.
DR. MULLIGAN:  I think Blake's point about how you can take a perfectly good plasmid and grow it up to a large quantity and have something happen to it is very, very key.  So I think the issue we dealt with at the issue meeting was essentially just knowing that you're beginning with the right thing and everyone agrees, I think, at this point we ought to have a sequence.  I think I would still go for my interest in seeing lot-to-lot sequencing for the very reasons that I'm not sure you really know that the coding sequence is going to necessarily be the relevant place that would affect gene expression.  It could be the upstream sequences or something.
The issue of the post-transvection, I think, is an easy no, except for coming back to that black hole of single genome retrovirus things.  There is a context where you have to consider a plasmid intermediate within the cells and that's when you're doing transient transvection that make retrovirus vectors.  And one of the things that I think most people don't appreciate is even if you have separate, separated viral functions if you in a transient transvection introduce those separate functions, there's a remarkably high rate of recombination among the transvected sequences so that I don't know if anyone has really looked at this, I don't know whether you've assayed, want to look at this, but I bet you see there's a high rate of those dangerous single genome packaging sequences.  
So you might want to ask people to at least assess whether or not that happens because what you're going to see, I think is that everything is going to link to everything, so you're going to have a little bit of retrovirus vector linked to a little bit of lung packaging sequence.  It's not exactly what I would say is the best way to go about making this.
Now AAV is a slightly different case.  It's probably less of an issue, but it may nevertheless be worth looking at.  
Okay, well, I wanted to say one thing.  I certainly don't think it's an anecdote to be doing this.  At every transplant meeting you go to now, you have somebody, at least one person, getting up and singing the praises of ex vivo gene transfer to target cells and then infusion back into patients.
DR. ROESSLER:  I just meant as an anecdote a single case as opposed to a specific protocol.
DR. SALOMON:  So what I'm hearing now is we all have agreed before we even came here today that we would have to know the complete sequence of the plasmid when it arrived at the facility.  Then we talked today about the fact that there should be a lot-to-lot control because in expansion of the plasmid DNA in bacterial systems, there can be changes, right?  Although Dr. Mulligan, Dr. Roessler, you guys have given two slightly different statements.  You started to sound like you were going to do every sequence should be, every lot rather should be sequenced and then you sort of backed away from it and you were saying every lot should be sequenced.
DR. ROESSLER:  I'm trying to make it broader in terms of giving the Committee the perspective that you need to consider that every plasmid is a little bit different and there may be plasmids that are used over and over again that are known to be quite stable in terms of their genetic identity.
So from that perspective, it doesn't seem to make as much sense that you would need to do complete sequencing of that particular component, that particular intermediate, whereas you might have a transgene that has a particularly negative selection pressure on your E. coli strain so that you believe there would be a higher incidence of either recombinational events, mutations, deletions and in that instance, that particular intermediate component, there may be significant value to doing sequencing.  Now whether that's complete sequencing or sequencing only of the promoter transgene or whatever, I think you have to deal with that on a case by case basis and that you can't just assume that complete sequencing is going to be the best way to go at this stage.
DR. CHANOCK:  Can I ask a question at this point?  Just in terms of the utility of sequencing, I understand that theoretically the question is on a practical level.  Is the sequencing tied to the release of any plasmid or any material as it goes forward?  In other words, you do the sequencing, but is that tied to when that material is then made available only when someone has actually done the sequencing, looked at it and verified it and moved on or is this more protective in the sense of being able to look back and say all right, we have that information, we now need to go look at that because something has gone awry.
It seems to me those two different tracks have two different implications.
DR. SALOMON:  Well, that's important to clarify.  Certainly my thinking and the Committee can modify it, is that we're actually saying that for lot sequencing or pre-manufacturing sequencing that these are events that have to be done, checked off before you go on to go forward.  It's not a parallel sort of testing that might occur under other circumstances.  So trying to take this and make a practical statement out of it, if you have a plasmid that you're going to make large lots and go forward into trial, then basically the manufacturing facility and the sponsor has to answer the question that you posed, is this a plasmid that maintains its integrity and is not an issue, which means there has to be some data on at least several lots sequenced that would satisfy FDA staff, that this was correct and then if you did, perhaps after that there would be a more limited obligation for quality control of the lot.  That might make a lot of sense, vice versa if it turned out that either (a) you didn't have data, but you still wanted to push forward in the trial, you might accept the onus until you do have data to sequence all lots.  Or, if you had data that actually showed you had a difficult plasmid that you would have to sequence a portion of lots, if not every lot.  Is that -- again, I'm just trying to be practical in terms of a recommendation.
DR. EPSTEIN:  I just want to explain why we took a different position.  If you have both a good restriction map and an activity assay, so I'm accepting what he's saying and we'll take that back for consideration, but if you have an activity assay that's well controlled and highly specific, then you know the insert both is expressed and has not either mutated or been rearranged to such an extent that it loses its biological function.  Minor mutations elsewhere that don't lead to wrecking the promoting and so on, we would simply accept.  So if the restriction map shows it's what you think it is, you know the sequence when you went in and now some degree of mutation has occurred, but without losing transgene expression and function, that's where we were starting from.
DR. SALOMON:  I would just point out though that there's a problem potentially there because when you infuse it back into the patient or you inject the plasmid into the patient or into a tissue, if a mutation occurs, let's say and most of these are natural biological products.  Some of them might generate antigens or other unwanted effectors on other portions of the molecule that wouldn't regularly be anticipated and might really be devastating.  So I think you have to be a little cautious at this early in the field. 
Again, I'm not saying that I could take the sequence and go aha, I've got an antigen created here, that's not my point.
DR. CHAMPLIN:  Just to clarify, what you had said and I believe our consensus was was that you would want to fully identify the lot, but if that lot is going to be used to treat a series of patients, you wouldn't then need to take the T-cells from each patient and redo the sequence at that point which I believe would be onerous.
DR. SALOMON:  Okay, no, I was just trying to be logical and walk it through and I -- we haven't gotten to that one yet.
DR. MULLIGAN:  I think the issue with the sequencing is -- my own philosophical view that we're in the age where you can easily do that, like wash your hands after you go in the bathroom or something.  		(Laughter.)
So I would still push for the complete sequencing, but on the other hand, it's very obvious that's a very low resolution determination of how pure the thing is because you're only going to get the major sequence.  So I mean it's of course -- it's a dicey type of thing, that is if you had 20 percent of some different thing, when you sequenced it, you might detect and you might not detect it by a good high resolution, a gel assay either.  So but I just think it's so simple, if I were a company that was making DNA, I would be shocked if we wouldn't be sequencing a sample from every lot.
DR. CHANOCK:  Just on that end, I think the technology is clearly there for -- I mean, I think your point is very well taken.  It may be 15 or 20 percent representation of a variant, but the whole SNIP world of SNIP detection has exploded and the technologies are there and the software is there, so I think it may very well be possible if you know, if you do the right aliquotting so to speak, to be able to identify at a certain place that you may have 20 percent sequence that goes off when you look at your standard phred phrap scores off of your ABI sequencer or whatever.  So I think that that is pretty much at hand already and it's something that we may want to think about.
DR. NOGUCHI:  Just some clarification, Dan, on where you're taking this.  It just seems like there's still one part of it is that nobody is routinely, as far as I know, looking for how stable a plasmid is through the production process by sequencing, so wouldn't you have to say that at this point in time everyone would have to do that first and then we would back off on a case by case basis?  Is that what you're kind of suggesting?
DR. SALOMON:  That's what I said.  I mean in other words, either you have data, you have a stable plasmid which I was picking up on what Dr. Roessler was saying, or you don't.  If you don't, then you have to show that it's stable.  When you've satisfied that it's stable then you can -- you don't have to maybe do it as often.
I think that the technological issues are well taken.  We all have core laboratories now.  It really is like washing your hands to send something for sequencing.
I like the idea of incorporating even the newer technologies that more bio-informatics than another technology in the sense of looking for SNIPs and satellites and other groups that might rapidly give you information on subspecies that would address the question Dr. Mulligan came up with.  That's a good idea.
All right, again, just kind of plodding forward is the question Dick Champlin had said and the next step would be do you have, what do you have to do with the -- this will be cells that have been transduced.  They put the plasmid in and you get the transduction and you want to go forward into the clinical trial.  Do you have to do anything with those?  I'm not saying that I think you do, but I just think that's something the Committee ought to make a comment on.
DR. MULLIGAN:  I can't understand why you'd want to do that.
DR. SALOMON:  Fine.  I'm just being the devil's advocate right now.  I don't want to do that, no.
DR. SIEGEL:  You're specifically speaking of when you say do anything, you mean do sequencing?
DR. SALOMON:  Yes.  I'm talking about right now, I give you 10^10 T-cells from a pheresis because I want to put in a granzyme and stimulate it with a dendritic cell antigen for my tumor and then inject it into the patient with melanoma.  That's a very scenario.  That's the kind of things that people want to do with plasmids right at this second.
DR. SAUSVILLE:  Yes, but it seems that that's going to vary in a case by case basis and where you're going to efficiencies of detection, how you detect it, etcetera, that could be viewed as I think very onerous to actually require that type of thing.
DR. SALOMON:  I wasn't, again, I know that that's the way this is going and I think we should comment on it as part of this discussion.  I don't think that that's what we should be doing right now. I agree with that.
Okay.  Any other comments on identity then?
Purity.  So I guess here we're referring particularly to things like E. coli DNA and RNA as well, I guess, you could add that to things like solvents, etcetera, that would go forward?
DR. EPSTEIN:  Actually, the second question deals with that.  I think it's the second one.  We're talking here about general purity.  Should you have to do something like an agarose gel to show what you've got, then looking for specific contaminants is where we'd like to distinguish it from plasmid for patient administration.
DR. SALOMON:  Okay.  Any comments on that?
DR. SAUSVILLE:  Agarose is cheap.
DR. SALOMON:  Agarose is cheap.  Okay.  Would that be okay?  Is that enough these days?  I mean an agarose gel, you sustain with ethidium bromide, you turn up the integration on most image processing packages.  You can always find an extra band here and there.  What exactly is it that I'm supposed to show to reassure everyone that I have a pure plasmid?
DR. EPSTEIN:  Consistency.  You can have contaminants at some level.  The cells would be washed and so on, but you're just going to look at the purity of what you're using.  And have some reasonable standard.
DR. MULLIGAN:  We had a talk, I think, last time or several times ago about someone getting into the details of dimers circles, and that type of thing.  What is your opinion on -- I mean that can vary from batch to batch.  You have basically the same identical construct, but it may be as a dimer, trimer or some complicated multimer.  Have you thought about whether or not you want to have a consistency in that?
DR. EPSTEIN:  Well, for plasmids at the later stage, say for patient administration, you certainly want to note the percent that is supercoiled and the percent that's in various forms.  I don't know if it's consistent which form is the active one.  Do you know whether transvection of cells is always the same species?
DR. MULLIGAN:  Well, I think it makes a big difference how big it is and it would make a difference.  So if you have different species, it would make some difference.  I'm not actually personally all that concerned with it, but I think that it is, it will be different.  You'll have a giant piece and that will probably behave differently, depending on how the multimerization occurs.  You may have different gene expression potential.
DR. EPSTEIN:  So you could simply report all the forms observed and if you are way out of line with your experience of a reference, say you have a reference standard, that would be useful.
DR. MULLIGAN:  Again, on these things I would think the investigator and the manufacturer would really want to have a product that's as good as possible, as homogenous as possible.
DR. SALOMON:  When we went over these questions with the staff before this, my comment at this part was that we're dealing with things that are production quality issues that I think certainly are beyond my expertise.  I mean we agarose gels all the time with plasmid DNA, but I'm just excited when I see a big band around the right molecular weight and then we cut it out and go do our blunt, clone it into something and go on.  And that's not what we're talking about today, so I feel a little bit -- one of the comments that I made back to the staff on that was that did we have enough expertise on the Committee at the production level.  That's one of the reasons we asked Dr. Roessler to join us.  
Does anyone in the audience want to comment on this?  I mean if you see two or three different bands on it, you can't just cut it out and purify it.  I mean when you're talking about commercial lots, right?  So what do you do when you see these?  It's just you mark it down and it's part of the record which is what Dr. Epstein suggests and is that okay with everybody?  These are the things that concern me is that the Committee is making some comments on things that I feel, I certainly feel is not in my area of expertise any more.
DR. MULLIGAN:  I mean I think a key message that's probably the most important message is that as we march down more biologicals, there's going to be more and more of the issue that these entities are not homogenous like a drug would be and the viruses are clearly the case and it's most important to have the FDA get a sense of how they're going to deal with that philosophical issue and the plasmid DNA is one of those, but clearly virus preps, when we get into the different ways to purify and the inability to completely characterize the composition of a virus prep and so I think we have to begin to think about the fact that you can't be that specific.  And I think plasmid DNA is that guy who talked to us, some expert in making plasmid DNA and I think he had some ideas of how to reduce the amount of the multimer forms and so forth and you want to encourage that sort of thing, but I think the state of the art is that you're going to get this sort of thing and I agree that just at least having a good, accurate description of what you have is probably the best you can hope for.
DR. SIEGEL:  This is not an issue for gene therapy.  It's present for our protein products, for vaccines and so forth.  It's not a -- the question, as worded up there and I'm not sure exactly whether our group wants a different question answered, but the question that's asked up there is what testing should be done, not what specifications should be set for those tests.  It's very common for development of complex products that we require a test be done and that the initial specification is that the results are to be reported and reviewed and that over time that tells you, among other things, not just how homogenous the product is, but how consistent it is and whether your Phase II studies are being done with the same stuff as your Phase I studies and ultimately it collects the data base that as you move into more advanced studies and licensing, you can set specifications based on not only what is considered safe and effective, but also what is considered achievable levels of consistency so that if something unusual happens, you have an indicator and you -- so I'm not sure we really at this point are asking or need to be discussing whether where to set the limits or what to allow or whatever.  The question really was I think the one that Dr. Sausville answered pretty succinctly, that agarose is cheap and we should be testing it and accumulating the data, if that's, in fact, the sense of the Committee.
DR. SALOMON:  I think that's fine.  Again, I was just making sure that we were comfortable.  To request tests that make no sense also is onerous and part of my feeling, the job of this Committee is not to support that sort of thing either.  So that's kind of why I was questioning is this something you are going to make a decision on or are we just archiving it.  I'm okay with that.
Please identify yourself.
MS. CHRISTENSEN.  Yes, Janet Rose Christensen with Targeted Genetics Corporation.  I think in response to the question that you asked about what are people doing, I think this really dovetails back to what we heard from Mary Malarkey earlier.  Specifically, that yeah, you can get a band, you can do the ethidium bromide staining, whatever, but again the controls and the amount of characterization, the understanding of what your assay is telling you and as Dr. Siegel just pointed out about as you move through production and developing those controls I think is a very common type of approach we take in the industry and I think that that's a very reasonable thing as we gain more experience, that I would expect that we should be able to quantify and characterize what we should be seeing and as part of that you're going to have to understand the sensitivities of your assays and it gets back to assay qualification which again, I think, is an important attribute of these types of assays, even very early on.  So when we get a question from the Agency specifically well, that's a nice looking band, what is it, and does it have any relevance, we can answer those questions.
I think what you're hearing, certainly from my perspective is taking the approach of Dr. Siegel and what the FDA has advised us is a very use and I think it really supports the ultimate issues of product safety, product consistency and patient safety.
DR. SALOMON:  That's exactly the kind of feedback that I think I'd like to hear more of from the audience as again, as you see appropriate to back up some of the manufacturing experience that we don't have sitting up at the table.
Yes?
MS. SEAVER:  Sally Seaver, Seaver Associates and I consult on CMC issues and I would like to back something up and really ask the Panel if they want to do this and that is if you wanted to complete sequencing, is your sequencing -- I know you all do it in academia, but can you validate that method and are you doing it under full GMPs?  I think that goes back to Mary Malarkey's talk and I would like to remind you that we do not do full sequencing of the amino assays on every protein lot, even in Phase -- even in clinical trials of our recombinant proteins and clearly a change in amino acid could affect amino genecity.
DR. SALOMON:  Thank you.  Good comment.  Do you want to comment on that, Dr. Roessler?
DR. ROESSLER:  Well, once again, a main reason that I said I saw a role for sequencing and I'm not sure that I would advocate complete sequencing along the lines that Dr. Mulligan articulated, was for the quality control aspect that's a necessary part of our role in the NGVL programs, specifically when you sent plasmid material to a sponsor and they do a functional assay and that functional assay fails, then you have to problem solve and having that sequence data allows you the opportunity to get some insights into what the problems may have been.  And once again, I think that there's always value in doing whatever you can at some level within the cost-effective constraints to try and verify the identity of the material that you're using to manufacture your final product.
So I think that my perspective may be a little bit different than your perspective.
DR. SALOMON:  I think appropriately so, which is good.  
The next question would be on concentration.  I mean that's kind of a no brainer unless I'm missing something.
Activity in gene expression.  And I see this one as sort of now segueing with the next question and that is should tests be added for potency.  Can we have some consideration for a second about activity versus potency assays?
DR. SAUSVILLE:  It gets back to this question, do you mean this in the case of ultimate use such as Dr. Champlin brought up, or do you mean in the expected performance in a model system as a description of the product?  I clearly would be in favor of the latter.  I would not be in favor of the former.
DR. SALOMON:  So you're saying that if there was an animal model that was used in the process of your pre-clinical and now you were a -- a year or so later you were doing your clinical study, if you demonstrated potency, it should be demonstrated in the animal model?
DR. SAUSVILLE:  Animals, in vitro cells, whatever.  I think that would be part of the complete description of the product package and in that sense be in the spirit of potency.
DR. EPSTEIN:  There wouldn't necessarily be an animal model if here, the intended function of this plasmid is to transvect 293 cells, for example.  So I think we have to back off yet another layer beyond what you're talking about.  This is not for patient use and the intended function is in vitro and is simply for transvection.  So I think we should simply the whole thing greatly and look for activity ability to transvect those cells and express what it's supposed to.  That can be by flow cytometry or enzymatic activity and it should be a very simple test.
DR. SAUSVILLE:  I didn't mean to imply that an animal should be used.  I mean one could imagine situations where they might be, but as you say, you want to imagine situations also where that's not necessary.
DR. CHAMPLIN:  It seems as if this is a product-specific issue.  Certainly, some things where there's a readily detectable functional assay you should probably do it.  If you're going to give 
T-cells that are going to kill a liver tumor that's unique in that patient, there's no readily apparent ex vivo assay that could show that you're going to kill that patient's tumor.
DR. EPSTEIN:  That's probably the wrong topic.  We're talking about the plasmid intermediate being qualified for use.  We're not talking about taking the T-cells -- the potency assay for those 
T-cells might be an animal tumor model.  I want to back off that we're only talking about showing that this plasmid is good enough to use and --
DR. CHAMPLIN:  Let's go back.  When I asked you to clarify this, the idea was that activity would be, let's say expression of a gene in the targeted cell and that potency was some sort of cellular assay.
DR. EPSTEIN:  Oh, I'm very sorry then.  I was trying to define potency and get you to understand the concept.  For a cellular preparation, potency is the kind of thing I alluded to.  But here, we're talking about an intermediate and even the ultimate potency assay, if there were one, would be a very quantitative, very validated version of in vitro transvection because that's all this material is intended to do.  I'm sorry, I thought that the definition of potency was unclear.  And the easiest cases to give a definition where the product has to serve its biological function for therapy, but here, the real question before us is whether to do an assay with controls showing that this transvection works, or whether to really do a quantitative and validated version of that, a fancy assay requiring showing that the T-cells now have acquired a receptor that mediates lyses in vitro, for examples.  Or, can you just show that the plasmid went in and the T-cells now have a marker on their surface?
DR. CHAMPLIN:  Presumably we have shown this for the parent gene and your question is do you have to do this lot-to-lot reassessment, can you kill the tumor in nude mice or what have you and I would think that would not necessarily need to be done if you've shown that you've got expression of the protein of interest and that that -- you had met all of the other criteria that we have discussed.
DR. SALOMON:  I guess my concern here, just trying to keep the conversation going to all the different levels it could go, I'm not certain of that, in the sense that we keep repeating the obvious fact that this is a very new field and that there are a lot of rules that begin with manufacture and go all the way to administration that are far from clear.  And if that's true, I'm not so certain that I'm comfortable not having early on maybe more than later in terms of potency, so if there are -- if I'm giving it to 
T-cells and I'm going to take those T-cells and put them into the patient and put the patient through all the different things that I might do including radiation therapy and all that, based on these great plasmid transduced T-cells I'm giving them, then yeah, dammit, I think they probably ought to have some sort of assay lot-to-lot before I go to the bedside, particularly when everything is so new that we really don't have all the rules in place.  So it's -- I actually disagree with you with on that one.
Amy, I know you had a comment and then Richard.
DR. PATTERSON:  I think it might be helpful to parse these concepts out because in the dialogue they seem to be intermingled.
Suzanne, you were first asking about or one of the things you were asking about is efficiency of transvection and then someone asked about duration of gene expression, which genes are being expressed and I think those are fundamentally different concepts and they're lumped together here.  I think it might be helpful for the Committee to consider them separately.  Transvection efficiency, gene expression, is the gene expressed, to what extent and what's the duration?
DR. EPSTEIN:  And then the one that people are raising now is you're trying to show that the product of the expressed gene functions as it should and how close does that functional assay have to be to what you're concerned about or can it be a marker assay?
DR. SALOMON:  Well, I think -- yes, and I think there that's got -- I don't think we can give you guidelines for that.  That, I think is clearly product specific, but if I have a product that well, taking Dick's example, it's going to kill liver tumors, I mean there's got to have been a human model, I mean a mouse model probably for it.  I'm hoping, obviously, I think we ought to stop short of insisting on nonhuman primate model for the testing.  And so there would be let's say a skid mouse model.  I'm just making that up, but there would probably be a model like that and if you did have a model like that, then it probably wouldn't be unreasonable to test that until we become more confident that these sorts of things work.
DR. SAUSVILLE:  Yes, but I guess my position would be that although in the development of this product, such a model would have likely been used to gather confidence that things were -- this was a good thing to do.  I would be wary about establishing the response of a mouse model as a qualifying issue in the manufacturer's subsequent lots because at least our experience with mouse models is if they range from the health of the mice, hepatitis, I mean, so this gets into very problematic sorts of issues that could make things very expensive.
Certainly if there was the type of information collected and the expected duration of expression of the gene, the normative properties of the gene product, again, as we emphasized earlier in the morning, much of this preclinical testing and quality sorts of issues are to help explain the behavior ultimately in the clinic and as long as it's available so that there were a problem or lack of expected performance, you could go back and maybe address these issues.  It's fine.  Bottom line is I don't think requiring animal model behavior is a good thing.
DR. SALOMON:  And as I pointed out, what I'm trying to do is push the conversation as far as we can go to try and make sure we get a clear idea of the Committee, of what you want to do.  I would also agree that every lot having to be done in an animal model would be onerous and that would be fine.  However, I would say though in terms of the pendulum that if it was a simple cellular assay that that would probably be readily doable and so when those kinds of assays were available, a couple day assay of killing or something in a model, then I think it wouldn't be unreasonable to do that at this early point.
DR. SIEGEL:  I would note that there's not just an issue of being onerous here, but I think as Dr. Sausville correctly pointed out, potency assays are not dichotomous.  They're quantitative assays and a quantitative in vivo assay, you know, you're lucky if you can get within a log or so, base 10 of estimate of accurate estimate.  They are just not very useful and I think it is to be urged of all manufacturers and sponsors to be looking for relevant potency assays that can be done in a more reproducible way such as cellular assays.
I'm a bit concerned about time.  I wonder if we might want to move off this question.
DR. SALOMON:  We're almost done with this one.  
 		Dr. Mulligan, do you have a --
DR. MULLIGAN:  I was just going to say I think Amy's point about the transvection piece of this is that any assay of biological potency where you're trying to look at, I thought the DNA's potency is going to be totally variable based on DNA transvection efficiency.  It's like an in vivo assay.  So I would go very easy on biological assessments.  If what we're talking about which is what I think is is the DNA that's been now sequenced a number of times shown to be the right thing, does it have inherent biological activity it ought to have?  I think there's almost nothing you really have to do on that front.  Because if you try to transvect it and look for how well it performs, if you did five transvections, you'll get a variation of probably 30 percent and will that convince you that you'll have less potent DNA?
DR. SALOMON:  I don't think that would convince me I had less potent DNA, no.  But if I was going to do a clinical trial with this reagent in any shape or form, I would like to know if that would be fine to know that there was going to be a 30 or 40 percent difference in the product that I eventually put in the patient.
DR. MULLIGAN:  I'm just trying to, maybe in the spirit of moving along, say that's another issue.  That's not the DNA issue.  That's the transvection issue.  You know, you have to show that the procedure that you're going to use with the DNA is reversible, but that's different than the DNA issue.
DR. SALOMON:  Yes, I understand that though.  I have to say to this particular issue every time we've tried to clarify it, I get less clear where we're stopping it, because we start talking about potency assays and then we are talking about -- yeah, if you just want to talk about the plasmid DNA, then we're done.
DR. EPSTEIN:  We're not talking about the final ex vivo cells or the AAV or retrovirus.  They have their own potency assays.  They will fail their lot release criteria if this plasmid doesn't do its job.  What we're trying to do is avoid tremendous rates of failure.
DR. SALOMON:  All I'm saying is in the lot of clinical uses of the plasmid, it's the plasmid into a cell and that's your product.
DR. EPSTEIN:  Right, and we're talking about qualifying the plasmid, but the cell is subject to assay before it goes into the patient, including potency assay when there's a correlative of what it does.
DR. SALOMON:  Fine.  So you got some free discussion on that. 
DR. EPSTEIN:  Right, that was not the question being raised.  But thank you.
DR. SALOMON:  Then I think we're done.  The last one is full plasmid sequencing and homology analysis appropriate as a one time characterization test and such analysis could use plasmid from the master cell.  I think we've covered that adequately.
The other thing we did when we presented this kind of as a discussion before, we all agreed that time would be -- that this would be a real killer to keep all of this on time and we're seeing that this is very difficult to keep this all on time.
So the question here would be that I'd like some feedback right now.  Obviously, lunch -- we'd be right on time for lunch.
(Laughter.)
We haven't done the presentations on adenovirus.  So Phil, do you want to comment?
DR. NOGUCHI:  Yes.  What we would like to do is we certainly would like to have Dr. Chanock present his extensive knowledge about the adverse affects of replication competent adenovirus and Dr. Bauer has agreed that I could condense his to a very short, just statement that his talk would lead into it.  Dr. Bauer will actually update us at our next Advisory Committee meeting because it will be at that time even more information on this collaborative effort between academia, industry and the government in terms of producing a standard for vectors.  But Dr. Chanock's talk is actually could be done in the afternoon as part of the clinical and compliance issues because it's very relevant to the clinical situation.
DR. SALOMON:  So would a good plan be to break for lunch now and begin the afternoon session with Dr. Chanock's talk?
DR. NOGUCHI:  Yes.
DR. SALOMON:  Okay.  Then I want to see us all back at 1:15.  Thank you.
(Whereupon, at 12:39 p.m., the meeting was recessed, to reconvene at 1:15 p.m., Thursday, April 5, 2001.)

	A F T E R N O O N    S E S S I O N
	1:35 P.M.
DR. SALOMON:  Sit down and we can get start with the afternoon session.
(Pause.)
Can we have everybody sit down, please, so we can get started?  We've got a pretty busy afternoon here and I appreciate the Panel already sitting down and all that.  That's great.
Okay, so this afternoon we're going to pick up what we interrupted for lunch with some slight modifications that I will explain in a second, but essentially going on to some, the clinical issues in adenoviral infection and to start that I'd like to introduce Steve Bauer who is going to make some comments to put this into context.
DR. BAUER:  I just had a few short quick comments about adenovirus and what we learned from the March 6th letter response and some of our changes and the first one is we are going to recommend from this point forward that the ratio of virus particles to infectious unit be less than 30 virus particles per infectious unit for adenovirus lots and I know there's been a lot of discussion of this at various forums in recent times so I wanted to make that announcement.  
The other is that our recommendation for RCA which had been based on a radio with infectious units, we're now going to change to a specification of less than one RCA in 3 times 10^10 viral particles and that's in response to a lot of discussions about assays and their precision and accuracy.
And then finally, I wanted to just set the stage for our next speaker, Dr. Stephen Chanock who has agreed to come and talk to us.  The specification that I just mentioned, less than 1 RCA and 3 times 10^10 virus particles is going to be used for clinical lots, at least currently, regardless of the clinical indication.  And the appropriateness of that stance is what, I think, the next speaker will address.
The background issue is are there clinical indications for which this recommendation might be too stringent and/or are there clinical indications for which that might not be stringent enough.  So without further ado, I'll turn the floor over.
DR. CHANOCK:  Thank you.  I thank Stephen and Phil for inviting me to speak this afternoon.  When I was first invited I wasn't quite sure what I was going to do and coming this morning I wondered whether I was supposed to be the comic relief before lunch, but then they moved to after lunch, so now hopefully while everyone is digesting I'll be able to provide a little bit of comic relief, but more importantly, the clinical questions and particularly the clinical issues that come up with an issue that we were discussing earlier about lots, what is the consequence about what we would consider in a very practical terms of lot failure, in other words, too much of an infectious load being challenged or being infused into an individual who is clearly at risk.
So in order to do that, what I'd like to do is talk about several issues.  I'll take the first slide, please.  And we'll start with this slide.  And there are a couple of very broad, important points that I think are very important to the comments that Steve just made in terms of really trying to set a bar, per se, and having it be more specifically addressing the actual host who is going to be receiving the gene therapy product and that really comes to the critical point is the host immune function is really what's crucial about adenoviral infection and I'll take us through adenoviral infections in normal individuals as well as individuals with immuno-compromised systems such as bone marrow transplant recipients, patients with HIV infection and then primary immunodeficiencies.  So there's a very important issue that I'd want to jump right into as just a primary point and that relates to primary infection and we know that there are many different serotypes of adenovirus and immunity appears to be specific to the different serotypes of which they're well into the 40s now.  And we know this is very important because this really provides for recurrent risk for exposure to different adenoviral infections.  Now the question is what specific serotypes are being used as the backbone for the generation of the specific gene therapy vehicles.  I think that's a very important question and I know that there's been a big debate in the literature about going beyond using serotype 2 and 5 for reasons related to immunogenicity.
The clinical side, particularly, the immuno compromised hosts really in adults it's much more important to think about reactivation because in adults it's much rarer that a primary infection is taking place, whereas in a child, particularly a younger child, primary infection is taking place, so I think there's a pediatric/adult dichotomy that we need to factor into this as well in thinking about who are the hosts and who is potentially at risk per se.  		And then that reactivation is really determined by the underlying events, in other words, alterations are changes in immune function, not only from the point at which you start, but then what happens during the course of that such as in a bone marrow transplantation, particularly an Allogen A  transplantation with HLA mismatch, T-cell depleted cells.  That's a very high risk for having reactivation of adenoviral infection as opposed to someone who may have an autologous transplant with no manipulation whatsoever of their marrow.  Those risks have very significant implications with respect to the likelihood of developing infection, plus the question of co-infection and the ever present of which I think there is very strong data to really argue against, but I at least want to bring it up, the question of the oncogenic potential based upon the animal models and the information that's seen in other systems, but not in humans, per se, for oncogenic adenoviral infection. 		I might just add at this point that I really have not been convinced or seen anything in the literature to suggest that chronic or persistent adenoviral infection has clearly been linked to any known human cancers at this time and if anyone can come forward in making that point I would very much like to see that data.  I think this question has been studied for an extended period of time.
Next slide, please.  So really the adenoviral serology is really based on a number of standard references of sera that have been generated with the primary epitope being the capsid protein, one that's not necessarily going to be eliminated in the standard approaches right now towards generating adenoviral vectors and particularly it's a different question when we talk about adeno associated virus, reflects the heterogeneity of the adenovirus genome which we know has the ability to evolve and when we look at the sequencing now that a number of the different serotypes have been sequenced, have been classified into these so-called DNA homology groups.  And this is very important when you think about the question of recombination and there are these anecdotal cases of recombination between serotypes that are of the same VNA homology group.  For instance, there's a well known reported case in an HIV individual that had serotype 7 in 37, apparently have a recombination in vivo and I think that's a very important question to consider.
And then also, we know that the serotypes are associated with pretty specific clinical manifestations and we'll review some of those.
Next slide, please.  So just taking the large DNA groupings, we think of the A subgroup as really being associated with common upper respiratory infections that affect us all, that we certainly see in military recruits or in prisons or adult populations that are in very close proximity as well as in the pediatric setting.  We know that a large percentage of children who come in with apparent strep throat turn out not to have strep throat and adenovirus certainly is an important contributor and plays and important part in that.
Now the B subgroup is very important, particularly in the immunocompromised population.  We know that in, for instance, particularly the allogen A transplant recipients, hemorrhagic cystitis can be an extremely disabling and actually a very dangerous long-term complication because of the propensity to have continual bleeding and hemorrhaging and an inability to really stop that.
Similarly, the respiratory tract with pneumonia.  We know that there are endemic URIs in the tonsillopharyngitis, certainly with the subgroup C and then the very epidemic keratoconjunctivitis and I want to just pause there for a second and talk about what we know in terms of the transmission of adenovirus.  We usually think of as a relatively stable virus that can be transmitted from individual to individual by droplets, by cough or by touch and certainly we know with the D group that we certainly see the keratoconjunctivitis or so called, associated with swimming pool outbreaks where one person can go in a pool and everyone else who gets in that pool for the next or two is certainly at risk for developing that infection and certainly those in the audience who have children and have gone to a pool or you've had that and then three or four days later are beset with that, certainly understand that.
The conjunctivitis and the pharyngoconjuctival fevers are also very important, particularly the pediatric setting.  And the gastroenteritis is really a much more complicated story.  When the adenovirus was first described people thought that it was an important cause of diarrheal events, particularly in young children and I think that that's waned as we've gotten better at identifying other pathogens.  It appears to be sort of dropping on the list of pathogens associated with clinically significant diarrhea, particularly in younger children.  But when you look at the immunocompromised hosts, i.e., the transplant recipients, or HIV population, that's where the gastroenteritis, the F pops back up and we certainly know that in HIV there are a number of studies of individuals with HIV infection who have chronic gastroenteritis in which the Fs are isolated and are presumed to be responsible, or at least partly responsible.
Next slide, please.  So how do we detect that in a virus clinically?  We take material and we inoculate into cell lines and we look by a number of different effects and what many laboratories use now are fluorescent antibody staining per se, but we also use direct tissue detection and this is particularly important in the immunocompromised host where you're addressing questions of either pneumonia of hepatitis in an individual who is getting sick very quickly with what we would describe as disseminated disease.  And we either do an in situ hybridization, Southern blot analysis or PCR.  It's not that easy, necessarily, to make the diagnosis of an adenoviral infection per se.  You have to look for it and you have to have a diagnostic virology laboratory that's thinking about it and not every laboratory clearly is and I would say that that's a point we may want to come back to in terms of linking where and who is going to be following up and looking specifically at specimens and individuals who may be receiving these base products.
Next slide, please.  We know in the healthy child, roughly 80 to 90 percent of children have antibodies to 1 or more serotypes between ages 1 and 5.  Serotypes 2 and 5 which are the backbones that have been used for a number of the vectors that have been commercially and/or academically advanced at this point, 2 and 5 fall into that 80 to 85 percent, so we know it's a very common adenovirus out in the general community, causing up respiratory infections and many, many children develop antibodies to it.
We know that mild illnesses generally last less than 10 days, usually on the order of 3 or 4 days.  There's a latency in lymphoid tissue as well as adrenal tissue and an interesting thing that's been published, sort of buried in the review about two years ago was when they looked at a number of individuals and saw this serologic profile and then went back and looked at the actual lymphocytes isolated from a small subset of individuals.  About 75 percent of individuals who were serologically positive were positive by PCR and their lymphocytes, but not symptomatic at the time.  So in other words, there's this idea that adenovirus once infected can certainly be maintained in the lymphocyte population as well as we know that important targets are respiratory epithelial, particularly for regeneration, for making new viruses as well as for infection.  And then certainly, the renal and particularly the bladder epithelial are other very interesting targets.
Next slide, please.  So we know that adenoviral infection, particularly in the normal host, particularly in children, sort of here we look at the decreasing frequency of pharyngitis, a very, very common cause of pharyngitis and similarly conjunctivitis.  Gastroenteritis should probably be down about the equivalent of pneumonia and from some of the early 1960s and 1970s studies of these large sort of sweeping prevalence studies of children with pneumonia through the United States, pneumonia represented between 5 and 10 percent causes associated with adenoviral infection.
Next slide, please.  So really, we know that there are sort of calculable attack rates in the general population and we now have these numbers that have been published and have been verified and confirmed by other groups, not only in the United States, but certainly in Western Europe this has been looked at and it's roughly about 40 per 100 person years below the year age 1 where you know that somebody is infected and that there is an ascribable clinical manifestation.  Now whether there's actually a culture that links that is a whole other question and as individuals get older you see a decrease in the infection rate, but we still see that it's substantial, roughly 14 in 100 per years for people above age 10.  And we know that acute diseases are the most important thing, particularly in the upper respiratory tract.  Roughly 5 percent of URIs across the population, 8 percent of childhood pneumonias and adult pneumonias probably a little bit less than that.  		Why don't we go on to the next slide?  So now I want to, having taken that sort of background, I wanted to take a step back and really think about the immunocompromise population where we know that many, many people have been exposed to adenovirus, the question is is there a difference between adenovirus infection and adenovirus disease when we now launch into looking at, for instance, the reported experience in bone marrow transplant, in HIV-infected individuals and primary immunodeficiencies.  And indeed, there is.  I think it's important to just pause and use this definition as we look at the literature so that we understand two questions.  One is who actually has reactivation or at least the ability to be able to see and isolate that they have adenovirus from a sterile site which is a palpable risk in a palpable number that can be generated in any number of studies and then out of that a subset, generally on the order of anywhere from 20 to 40 percent of individuals who will actually what we consider to be clinically significant disease, in other words, there are clinical symptoms that are linked to the isolation of the particular pathogen at that time.  So if you want to turn this around, in other words, there's a good percentage of circumstances where we may find adenovirus is isolated from urine or from sputum or from the gastrointestinal tract, but yet it doesn't link very closely with a clinical event that's taking place.  So there is this disjointed nature that I think we have to take into account when we look at these numbers and are trying to calculate what would be estimable risk that we would use or apply in trying to come up with particular guidelines at this time.
Next slide, please.  So what are the clinical syndromes that are associated with adenoviral infection, I just want to give them sort of generically and then we'll start to look at what the literature really tells us at this time.  Well, the disseminating disease is really defined as having two or more of the following and this is one that we worry about.  This has mortality rates of anywhere from 30 to 80 percent, depending upon the host.  In other words, what's going on in the patient, what's the status of their immune system, are they in that terrible ablated stage, 15, 20 days post-allogenic transplant?  Do they have a primary immunodeficiency?  That's very different from an individual who may be just minimally immuno suppressed.  In other words, they may have just a perturbation of one part of their immune system, but not a complete loss.  Pneumonia certainly is a clinical syndrome that we worry a lot about as is the fulminant hepatitis and pancreatitis.  Colitis and gastroenteritis certainly in the transplant population.  As I mentioned before, the hemorrhagic cystitis and I just put up here for the sake of completeness, the encephalitis, but this is exceedingly rare in this population and I don't think this is something we should really concentrate on.  These are rare case reports and I think that there are some issues about whether that should really be applied in any given model.
Next slide, please.  We know that the distinct serotypes have been associated or presumably cause disease in the immunocompromised hosts.  Serotypes 5, 11, 34 and 35 and I just underscore the importance of 5 showing up there have clearly been associated with infection and immunocompromised adults and if we just look at, for instance, a series of 46 patients with Adeno 35, I mean this is sort of looking the other direction.  Clearly, a number of HIV infected individuals have problems with 35 and 35 is strongly linked to the hemorrhagic cystitis problem.  Bone marrow transplant and renal transplant as well as severe combined immuno deficiency and then a few individuals who are otherwise healthy, although that's always a very difficult question of what's going to happen, what diseases are they evolving and at that time we have not characterized per se.
Next slide, please.  What lessons have we learned from the patients with primary or secondary immunodeficiencies?  We know that there are sporadic neonatal adenoviral pneumonia which can be very severe and they are very localized outbreaks with a fairly high case fatality rate in newborn nurseries.  We see less and less of that now that we're better and better at being able to recognize and cohort neonates in the NQ.  SCID population, in other words, patients with severe combined immunodeficiencies and the absence of B and T cells, the patients who are at significant risk in that circumstance.  There's very high morbidity, mortality.  Small sets of case reports of either hepatitis and pneumonia with extremely high fatality rates, 80 percent or greater.  The DiGeorge syndrome-case reports of fatal hepatic necrosis and then certainly now we move into the solid organ transplant where we know both the infection of the transplanted organ as well as reactivation in the donor and these kinds of cases and reports have clearly increased over the last 5 to 6 years in the literature and if you just try and look at those very carefully, part of it, I think is the reporting bias of people beginning to catch on and look for this, but there's no question that there is a clear morbidity and some mortality associated with particularly in solid organ transplant, individuals who are receiving particularly severe immuno suppression.
And then the HIV population has been a very interesting population for a number of reasons.  We've been able to identify new serotypes from the HIV population and then the ever-present co-infection with other pathogens which is a very important question in adenoviral, particularly with pneumonia that there may be other pathogens that may have kicked off a pneumonitis or a type of infection in the lung that then has the adenovirus reactivation.
Next slide, please.  So let's just concentrate on bone marrow transplant.  If you look at the different published studies, the mortality is anywhere from roughly 20 to 60 percent with risk factors being the very young and the older patients who are at greater risk for poorer outcome.  Graft versus host disease is a very important risk factor for reactivation of adenoviral infection, particularly the acute GVH, but it certainly can be seen in the chronic and then the conditioning with T-cell depletion and in a particular, HLA mismatching.  I think they're very important things.
Now the risk for adverse outcomes, we know that individuals who have multiple sites, those patients who have disseminated infection, as I put that list 3 or 4 slides ago of two or more sites.  Those individuals are the highest risk for a poor outcome.  We know that serotypes, for instance, 2, 5, 7 and 9 are particularly important for pulmonary disease in the younger patients and 11, 34 and 35 in the hemorrhagic cystitis.  And then this 
ever-recurring question of co-infection with opportunistic infections.
Next slide, please.  Now when we actually look at some of the studies, for instance, we go to the Flomenberg study, adenoviral infection occurred in 21 percent of 200 patients who were undergoing bone marrow transplant.  And of that, 6.5 percent overall, or in other words, one third developed clinically significant disease, so just taking that paradigm that I was trying to convey in that slide a little bit earlier, we know that from that study, particularly, that the isolation of the virus for multiple sites and the presence of GVH were very important risk factors, as well as infection appeared more common in children and this comes back to a point that I made with the very first slide and that is with children who may be immunologically naive, this very important question of primary infection I think is all the more pressing because of the clinical implications in an immunocompromised child are quite literal, quite significant.  The time of onset in children, we knew that these things come on much sooner, whereas in adults you see them over a period of time.  Again, this may have something to do with reactivation versus primary infection.
Next slide, please.  From the big Mirza study of 1300 adults, in that situation they looked at specifically adenoviral disease; 6 percent versus 1 percent in the autologous setting.  They  did not find that GVH was a risk factor.  They had a lower case fatality rate than in some of the other studies, but again, I think it was very important.  If you look at the Shields study going back even further, about 5 percent and what I think the message here is, even though it may have been lower numbers earlier on, we're getting more aggressive as a community with respect to bone marrow transplant, longer, more intensive therapies that put patients at greater risk and probably more commonly used in the last 10 years than they were the 10 years before or the 10 years before that.  So I would use that as one way of understanding that and not to say that we've gotten worse, per se in treating, just that we are better at creating the circumstances where somebody is at greater risk for developing that infection as we've pushed the envelope of immunosuppression per se.
And then certainly the Blanke study of 13.5 percent among T-cell-depleted allogenic bone marrow transplants.  There again, a mortality of roughly 50 percent GVH and co-infection were not contributory in that study.  So there are certain current, such as GVH, which show up in some studies and don't show up in other studies and I think that we have to sort of factors those into what specifically is being done in those particular transplant populations.
Next slide.  So when we look specifically at children, I think this is very important that the adenoviral disease is about 18 percent in children which is a higher number than we've really seen in adults, particularly high in individuals who have significant GVH, but also individuals without GVH so I think in the pediatric setting it's still more of an open question of the importance of GVH.  We saw a lot of adeno-12 in this particular study which is uncommon in the normal host and the most important thing that the authors of this study really suggested was preconditioning, but I again point to the fact that this was done 12 or 13 years ago.
Next slide.  And then now we look at a retrospective study looking in the last decade and we see that -- I'm sorry, that's a mistake.  That should be 6 percent adenoviral disease, not infection, in the pediatric population.  I just noticed that.  In that setting, it was really restricted to mainly patients with hematologic malignancies which raises this other question, what's the underlying condition that the child or the adult is receiving the transplant for?  Is that a contributing factor.  And certainly having an underlying suppression or loss of a immune function is very, very important.  And as we looked here, down at the bottom, the type of graphs seen at the mismatched or matched unrelated donors, appeared to have a higher likelihood than the HLA-match and the Autograft.  Fits with the model that we'd seen before.  But as you can see, the numbers are floating about 3, 5, 10 percent at most of individuals in a cohort of transplant recipients which develop significant adenoviral disease and I think that's a number that continues to be relatively consistent.
Next slide, please.  And then the question is where is this disease?  Well, when we look at this Hale study, the hemorrhagic cystitis was really a very significant problem.  We know that 7 of the 13 individuals died, but only one of them clearly died as a result of the adenoviral infection that was associated with significant hemorrhage and other complications.   
  		Can we really implicate other risk factors such as total body irradiation or type of graft?  Certainly by different kinds of statistical manipulations these things are brought up, but again, I think that's not the purpose of the discussion today, but clearly these are underscoring the importance of other events that are taking place in these populations.	
Next slide, please.  So the hemorrhagic cystitis is certainly something, I think, that we all have to pause and think about because we know that there a small cadre of otherwise healthy children who develop hemorrhagic cystitis with adenoviral infection and it can be a chronic debilitating problem.  But when you then put in the circumstance of a bone marrow transplant and an underlying disease, it is a particularly difficult disease and entity to treat and this is one that I think we really have to watch very closely.  At the same time, we're able to actually monitor by looking at urine samples and specifically culturing urine samples for adenovirus.
Next slide, please.  So really what do we take from this transplant literature?  Well, for the older individuals we really look at this question of reactivation and I think most people would agree that it's reactivation in those populations who have a defect or a set of defects that have been introduced in the adult population.
In children, there clearly is this risk for primary infection and you still have the possibility of reactivation in children and I think that that's something that still has to be underscored, but the younger the child, the greater the risk for a primary infection per se.  We know that case reports of primary infection can be devastating in particularly young infants and we may really, as we begin to bring bone marrow transplant into the infant population, children under two years of age expect to see increases and talking with some of the pediatric transplant centers and being at some of their meetings in the last year, there clearly are sort of anecdotal references to this, but no one has put this together in a large enough series, anyone who would have the gumption to get up and show in front of an advisory panel per se, but that's something that we need to be concerned about and over the next year the Panel should particularly have a close eye on that.
Next slide, please.  So in the adenoviral setting and particularly in the bone narrow transplant population, we know that the primary infections are things that we worry about, particularly in the younger children.  Reinfection is clearly another issue that I've just barely touched upon, but nosocomial transmission is clearly a very important question and the question is who is susceptible to that reinfection or exposure to a serotype that they may have had, immunologic response to and have lost it due to their underlying either disease or therapy and then certainly reactivation which we know is very important.
Next slide.  Treatment, unfortunately, at this time is still relatively limited.  We don't have good antivirals that are clinically in hand that have progressed at least to a Phase III or to licensure in the United States at this time.  Ribaviris and Ganciclovir have each been used.  They are rare anecdotal cases of successes, but I think the overwhelming experience is that these are not primarily successful therapies for adenoviral infection and in particular adenoviral disease.  Intravirus IGG has certainly been used and there are again anecdotal cases, but I would -- it's safe to say that our treatment options at this time are extremely limited, so an immunocompromised host who develops this significant infection with adenovirus is really in a very perilous state and much of their, I think the reason for survival or success of getting someone through really has to do with supportive care and really most importantly the reactivation and the -- and really, the reimplementation of their immunologic system being reconstituted.
Next slide, please.  Now in HIV, we see enough people in the 1980s predicted that adenovirus would be a very significant pathogen in the HIV population and it really has not been.  Other than the chronic diarrhea, it really has not been a significant problem in the HIV population which is something that I think most of us would not necessarily have predicted 15 years ago per se, in just thinking about the transplant and/or cancer paradigms being applicable to HIV.  We know that there are a number of individuals who can excrete adenovirus, particularly in their urine and there's this famous case of the question of recombination between 7 and 34 which I think is a very important point that we have to at least be aware that this has been shown in vivo or at least suggested in vivo.
Next slide, please.  So really the issues again come back to this same slide for the sake of time.  I think I've emphasized them very strongly.  The state of the host immune function of the individual and I think when we're thinking about gene therapy protocols, this is very important.  It's probably a very different risk in an individual who is undergoing cancer therapy or in conjunction with chemotherapy as opposed to an individual who may have an underlying congenital disease or primary monogenic disorder in which there is no known defect in the immune system per se, but this is a vehicle to be able to approach neurodegenerative disorders.  I think those are two very different poles.  The exposure to primary infection, certainly as we get younger becomes more of an issue as well as the reactivation in terms of what other concomitant therapies or changes are potentially taking place as a result of the natural history of the disease or therapies or supportive therapies that are being offered to the individual at that time.
Steroid, corticosteroids is always an issue that's brought up and there's very, very little data to really link corticosteroid usage and the development of adenoviral disease and the reactivation.  That's a topic that has not really been addressed and I have never really found anything satisfactory in the literature to be able to address that.  But that's certainly something that I think is important for this population.
Next slide.  So really the future issues we want to look towards the development of new antiviral therapies and I know of several different approaches that are going forward, as well as the use of cytotoxic lymphocytes is certainly being addressed in several academic centers right now.  
Early detection is very important and then personally, my own laboratory is very interested in this host susceptibility factors, looking at things at the genomic level and asking the question are there certain SNIPs that are going to predispose or protect an individual who is at high risk for developing adenoviral infection, but I think those are still some way away.  So I don't think that these, the host susceptibility factors are really available in any meaningful way to apply to any of the things that need to be addressed by this Committee at this time.
Next slide, please.  So really, in my mind, there are some very important things I'd like to end on, sort of as points for discussion and thinking about and that has to do with the use of adenoviral vectors and the question of adenoviral infection, whether it's Iatrogenic or whether it's a natural 
co-infection per se in the gene transfer protocols, really has to do with the response and site of the inoculation because there are certain places we know that the adenovirus replicates particularly in the respiratory epithelium and we know that pulmonary disease can be a significant problem.  And similarly in hepatic cells so the inoculation into each of those places, I think, raise very important questions.  The state of the host immune function is very important, both with respect to the changes that are being undergone at that time that are either iatragencic or disease related, but then also this very, very rare case, I think, at least gives us pause to at least consider this question of recombination events and thinking about really what kinds of things could potentially go wrong and there's no real data in the animal literature to really validate this per se with respect to gene therapy reported studies per se, but again, it's a theoretical question that I feel morally obligated to at least toss that out for discussion.
Next slide.  So why don't I stop there and see if there are any specific questions and I'm sure we'll have discussion.
DR. NOGUCHI:  Dr. Chanock, what is the experience in terms of adeno infection in patients with chronic hepatitis?  Does it add to any risk?  Is there any literature on that?
DR. CHANOCK:  There are a couple of very small studies that suggest that reactivation of adenoviral infection in the course of chronic hepatitis, whether it's with a known B or C or whether it's chronic active hepatitis without a known pathogen, it may contribute to that, but I would say that that literature is extremely rudimentary and I'm not sure there's a lot we can do to generalize on that, simply because what we're looking at are case reports, basically, where someone sees something and says yes, this may mechanistically make sense, but I don't -- I'm not aware of an extensive literature on that.
DR. SALOMON:  One of the things that I'm trying to now put this back into the context of the way it was, we were going to try and present this in the -- so we're not going to talk about the replication competent adenovirus issue because that's going to come up later, not during this session but in a subsequent session, but I think everybody should see first just so that the record is clean for later that this thinking about what the implications of adenoviral infection in different patient groups that you so expertly presented today is very relevant to our thinking about the quantity of replication competent adenovirus that might be contained in a gene therapy trial.  But I'd like to take a moment, you do bring up a couple other issues about adenoviral therapy that might be worth mentioning and the question I had with regards to this reactivation.  So I'm used to it as a transplanter with CMV infection and CMV disease and reactivation, so this is just all -- I'm used to this one.  This is easy.
But what we're saying here is there are two things that we're interested in in this session.  Tomorrow, we're going to talk about long-term 
follow-up.  One of the ways that we tried to think about long-term follow-up an dhow that impacted on regulation was the idea if you had a non-integrating virus, that long-term follow-up was maybe less of an issue than with an integrating virus.  However, if this non-integrating DNA, double strength DNA virus, the adenovirus is actually capable of reactivation later, then I have two questions.  One is maybe that's not true.  Clearly, just integration is not the measure of long-term follow-up and then the question is is the production of adenoviral vectors being done in such a way that they are not taking on latency, that this is unique to the wild type virus.  That's one question I had for you.  And then the second is closely related and that is, if that's not true and if you have long-term persistence of adeno-DNA and adenoviral vector exposure, how much possibility is it that every year you get an adenoviral infection and you get recombination finally?
DR. CHANOCK:  Let me start with the second one.  I think the second is -- they're both excellent questions.  The second one I don't think anyone has data at this point to really answer that sufficiently and that's the kind of thing that I would think that as we go forward with these trials that we would need to continue to monitor because I think that is a real concern and a real question.
The issue of using a very immunogenic adenoviral serotype such as 2 or 5 is the backbone which we know generates a very good immune response that presumably is for life in most in vitros unless there's some kind of insult or diminution of their immune function, but that doesn't mean that closely associated by DNA homology groups, adenoviruses may not undergo a recombination event and that HIV case is one that just points that out in my mind as something to think about.  Again, I want to emphasize that's occurring in a very particular individual who's at very high risk, who the presumption is at least in our current understanding of adenoviruses is that that person probably has very high titers and was continually infected per se with adenovirus.  And that's a very important question because I'm going to slide into your first question because then it comes back this issue of how do we make sense of this paradigm that's been put forth of adenovirus not being an integrating double stranded DNA, but yet that Columbia data suggesting that IPCR in the lymphocytes of individuals who have been infected, that they may be asymptomatic, but a good percentage of them have adenoviral sequences.  That kind of study is, I think, provocative.  It hasn't been carried to the point that you would say that all the controls are done to be sure that some portion of the adenoviral genome hasn't been necessarily integrated or hasn't been picked up by some other pathogen, whether it's EBV, any number of other things that we know can go underground, so to speak, in lymphocytes.  But I think that's the kind of question that we need to go back to the community and look at much more closely and that people who are interested in this have to at least address that and think about that because the answer really is not available at this time.  I think that's the kind of thing that although I recognize that this body is trying to help guide and set guidelines, but at the same time we can also identify questions that we hope experts who are looking at this in the community would say yes, we can apply our system and analyze the appropriate data or animal system, whatever to be able to address these kinds of questions.
DR. SALOMON:  I think my comment to that and we can get some more comment is just exactly that.  I think one of the responsibilities, at least that I take on as chair is that I don't want us to be making advice when there isn't the information and the community to make it, so it's exactly what you said.
DR. MULLIGAN:  I'm interested in the question of how many virus particles would it take to initiate an infection and I love the swimming pool --
DR. CHANOCK:  I thought I was going to get away for voting before that. 
DR. MULLIGAN:  I love the swimming pool issue and that is what I'd really like to know is every time someone jumps in the swimming pool when you have these outbreaks, what's the probability that they will get a disease from this because the amount of virus particles, I was trying to calculate how many liters a swimming pool was to what the concentration of virus would be to give you a small number.
The suggestion is it's a very small number that's necessary, right?
DR. CHANOCK:  Correct.  And you know, in the swimming pool the question is how much of it is the water and how much is it the lack of hygiene in the dressing rooms and the close proximity of people who are barely clothed, touching and bumping into each other.  I mean you can imagine the hospital epidemiologists are very interested in this kind of thing and it continues, it's a classic board question and things put before people in training.  But what I think is also very important is not all adenoviruses are the same in terms of their infectivity, as well as their tropism for both tissue and subsequent development of disease.  Two and 5, we know, certainly have been certainly seen in sort of small, endemic respiratory outbreaks in military recruits, prisons, centers where a number of children are kept, for instance.  And so there is some information on that.  The question is how many actual particles, I don't think anyone really knows that answer.  The animal models and particularly the cottontail rabbits that are used for infection, those kinds of systems are helpful, but in terms of being able to actually calculate what's the viral titer of actual replication competent adenoviruses that it takes to engender an infection, that's still very much of an open question.
DR. MULLIGAN:  What about a single cough?  Is there any experimental work that says how many virus particles are put out from a single cough of an infected person?
DR. CHANOCK:  Actually, going back a number of years there were studies of that nature and that's the infectivity of a cough?  I would have to go back and look.  I know there's very strong information for respiratory, stentitial virus and parainfluenza 1 and 3 which are big problems in the pediatric setting.  I'm not sure that those studies have been carried on in adenovirus per se, but I think that information may be available.  I just don't have it in my fingertips.
DR. MULLIGAN:  I think that the question on this is just if you want to set a certain limit, does the limit always have to be zero, that is, how many -- if you thought that one or a couple of virus particles had a certain measurable frequency of giving -- you should be very, very serious about setting those limits far off.
DR. SALOMON:  I was actually laughing when you said that because I like the swimming pool concept myself.  I think that the agreement that we sort of had going into this is that there's no question that this is really an important thing to talk about, but that we didn't think that we had, we really were setting it up for this discussion yet.  
So I think I'm going -- unless my -- the FDA tells me no, go for this -- no.
DR. MULLIGAN:  I'd just like to ask them what is the topic here?
DR. SALOMON:  What we were -- there's two things.  That's what I was trying to explain before.  I didn't do a good job apparently.  Initially, what we wanted to do was get into this sort of new setting of how many replication combinant adeno could contaminate a clinical lot.  And it was decided just because of the interest of time and the important of that discussion that we wouldn't get into that right now, that we would make it a separate committee discussion later and I said that, but I guess I didn't make it clear enough.	
Nonetheless, I think that we felt very strongly to have -- to go on with the presentation, changing its focus a little bit, in that it's really a beautiful introduction into the afternoon's topic about clinical issues in gene therapy, because it's the way of saying that really, our understanding of the behavior of the wild type pathogens that we've made into vectors and their behavior in different situations and their biology, like this issue of are there episomal, double-stranded DNA or is it integrated in some.  I don't think any of those -- is really the kinds of directions this committee and the whole field need to go into.  So I think we'll take it as an introduction and realize we won't get to discuss all of the -- because I think there's a lot of interesting things to talk about here.
DR. CHAMPLIN:  Quick comment.  I'm impressed that this is really a safe virus.  You really have to have a pretty profound immune deficiency to get sick here and even more than half of the bone marrow transplant patients do just fine with this virus and in the absence of profound (The document referred to was marked for immune deficiency, it doesn't cause serious disease.  So not to say we shouldn't be concerned about it, but of the spectrum of viruses that one can think about this would seem to be on the safer end of the spectrum because the immune system seems very effective to deal with this particular virus.
DR. SALOMON:  Well, remember though again, this is kind of segueing into the discussion, we have the sense that it's a safe virus because we're not seeing a whole lot of sick patients say in our bone marrow transplant patients, but there's a whole lot of protection between them and people with adenovirus.  So we don't really know how many particles are floating by.  So if 6.7 percent is of the patients are getting -- is the incidence of getting infected and having disease is actually because one viral particle got sneezed out on the parking lot, floated through the ventilation system and into your transplant patient's bedroom, then it's a damn serious virus in an adenoviral gene therapy trial.  I don't think we can answer that question.
DR. CHANOCK:  I think there is some information that, in fact, this is not as infectious as for instance measles or chickenpox which, in fact, can have that where someone can be on a ward one floor away and be highly at risk and develop it because somebody coughed and it went through the ventilation system.  I think the point is very well take that it's remarkable that this is such a ubiquitous virus and so many people see it and in many ways we're lucky and it's important to recognize that that many immunoviral compromised patients are not coming down with it, the majority are not.  But again, what I would want to leave as a very important point is really age, I think is an important thing that we really have to think about and I realize that's always a difficult issue when you're trying to set up programs, but for the clinical implications, you know, of adenovirus in a very young child, they're probably very different than an adult because someone at age 20 who may be undergoing a gene therapy protocol has seen most or all of the adenoviruses that are going to be used as the background multiple times, over and over and they're probably going to be able to handle those whereas the very young child is a very different question.
DR. SALOMON:  I'm ready to go on, except Abbey, I didn't mean to cut you off.  Is it okay?
MS. MEYERS:  Maybe somebody can answer this later on this afternoon, but they've been using the adenovirus all these years in gene therapy.  I remember there was one experiment with cystic fibrosis where there was a very severe reaction.  I'm wondering if somebody can tell us what the results are, what were the adverse events in adenovirus experiments and was there any pattern?
DR. SALOMON:  Again, I don't think that's exactly where we want to go this afternoon, but I think Dr. Chanock is the world's expert on this, but he can certainly your question briefly, I think.
DR. CHANOCK:  I know there have been a couple of instances where individuals have received an adenovector and had an acute pulmonary type of infection where it looked like they had pneumonia or indeed actually had pneumonia and of course that's a risk that you have to face, for instance, in a patient with cystic fibrosis, but I would take the more philosophical step back that I think that for many of the reasons we talked about before, this is a remarkably hardy and very useful vector and I know, you know, we have to be very valued in pushing the envelope and if we don't we're not going to make the next steps because as you know, gene therapy still has a ways to go before it really is a defined and truly successful therapy and I think that those kinds of risk benefit analyses, again, I would fall more on the side of using an adenovirus knowing that we have that risk in certain patients, but as long as they understand and everyone else understands, those are questions we can talk about alter.
DR. SALOMON:  Yes, I think we'll have to stop there and realize there's a lot to talk about with adenovirus.  That was a good introduction.
I'd like to introduce -- no.  I've been reminded by my better two-thirds that I now have by my obligation to open this up to anyone in the public who would request to speak.  This is the open public portion and no one had asked to speak, but I'm still requesting if anyone would like to.
Yes?  Just identify yourself.
MS. CHRISTENSEN:  I'm Janet Christensen with Targeted Genetics, please excuse my voice.  I'm not trying to imitate Lauren Bacall.  It's just coming out that way.
I wanted to take a couple of minutes just to address some of the issues that were raised this morning about quality assurance and quality control.  I realize it's kind of wedged in here at kind of an awkward time.  But I think there were some good questions raised by the committee about complexities of quality control and quality assurance and the issues on investigators and sponsors as they're trying to develop these new technologies.
I've had the pleasure and sometimes I reflect on that, yes, it's been a pleasure, in the last 22 years of being direct, very involved in quality assurance and quality control and I got involved in the entire recombinant DNA process back in the early to mid-1980s.  At that time, I would say that the recombinant DNA issues and activities back then were probably pretty analogous to where a lot of the gene therapy and gene transfer issues are today and even though there's a lot of different types of concerns about documentation and what appear to be very, very onerous issues, it's not reinventing the wheel here.  
Back in the 1980s it was well, gee, biotech is different, we don't need to follow GMPs because we're different.  Well, at the end of the day the answer is guess what, it's not different.  The issues are the same.  The documentation systems, the way that companies and investigators can structure their quality program can be an added value to not only the study, but the patient and the product as well.  They don't have to be highly complex.   My view on this sliding scale for GMPs is that you have GMPs in Phase 1, but they may not be as complex.  They may not be as detailed.  The compliance issues for quality control and quality assurance in my view help to validate the clinical trial.  They help to ensure that the product and the result that you're seeing from the patient, albeit safety or efficacy or whatever, are really founded in science by reducing variables.  So I think in viewing, excuse me, in viewing the whole issues about quality control and quality assurance, I thought Mary did a great job in kind of giving a good framework for that today.  But I'd like to assure the committee that there are a tremendous amount of resources available for the industry.  I think it's been raised and I think it's a very good point that we somehow need to bring those two together, either through ASGT, meetings like this, the outreach program the FDA is doing to ensure that we can get information to these groups earlier and investigators earlier rather than later, so we can keep maintaining some momentum with the industry.
Thank you.
DR. SALOMON:  Well said.  Okay, anyone else?  
All right, then the two photographers jumped up.  I thought my God, they're going to address the audience on --
(Laughter.)
You're more than welcome, too.  
(Laughter.)
I'd like to introduce two more people to the table, old friends, Karen and Weiss and Patricia Keegan for the afternoon, and Dr. Salewski, I'm sorry.
DR. WEISS:  I was just going to say as Dr. Keegan is walking up to the podium that as you know we're shifting gears a little bit this afternoon to talk specifically on clinical issues and more specifically to issues on clinical trial conduct, issues that deal with monitoring of the clinical trial, oversight functions of the sponsor of the whole clinical program.  The presentations this afternoon will be two.  First, Dr. Keegan will continue on with the responses to the March 6, 2000 letter that specifically asked our sponsors to address their monitoring and oversight functions and some specific issues related to the pre-clinical program.  Then Mr. Salewski will follow to talk about inspections that were done at various clinical trial sites and after that we can open it up then for some discussion and we have some focus questions for the committee regarding trial conduct.  So with that let me introduce Dr. Patricia Keegan who is the Deputy Director of the Clinical Trial Division to discuss the additional responses to the March 6th letter.
DR. KEEGAN:  Okay, thank you, Karen.  What I'll do is review a little bit of the background and the process and then our review of the responses to the letter and the process that has continued beyond the initial set of responses.
Go to the next slide.  In way of background which is obviously redundant to this committee, but part of the issue with regard to the March 6th letter was the death of the patient participating in a gene therapy protocol which was a highly unexpected event and in reviewing the circumstances surrounding that adverse event there was an inspection conducted of the clinical study site which revealed deficiencies in the conduct of the clinical trial, including failure to adhere to the clinical protocol, failure to report on modifications to that protocol to the appropriate bodies and failure to provide all relevant animal safety data.
Next slide.  Based upon the concerns raised by that inspection and the events surrounding that event, FDA determined that there were certain actions which should be undertaken to further assess the scope of this problem and those actions were really two fold with regards to clinical protocols and clinical trial conduct.  The first was a series of unannounced inspections of a limited number of randomly selected sites participating in gene therapy studies and Dr. Salewski will review that process.
I will discuss the March 6th letter one of the aspects of which requested information on the clinical trial monitoring program from all IND sponsors as well as requested confirmation of adherence to reporting of all relevant animal safety data.
Next slide.  I'm going to review what was in that letter, in part, because I actually needed to review it several times in looking through the responses and in talking to my reviewers because it's clear that very few people carefully read the contents of the letter and availed themselves of the references cited in the text and therefore the responses really didn't address the question, but the question as originally asked was as follows:  that the sponsors provide a two to three page summary of their procedures in place that ensured that the clinical trial conduct was appropriate.  In particular, it asks that the summary of procedures that were in place to ensure that there was adequate monitoring of the clinical investigations and to demonstrate that the trials were being conducted in accordance with both the regulatory requirements for the IND regulations, good clinical practices and the written protocol.  
Next slide.  It further stated that these procedures would be those that would ensure that the monitoring was adequate to demonstrate that the rights and well-being of the human subjects were protected and that data reporting, including safety data reporting was being made in accordance with those regulations to the IND sponsor to the Investigational Review Board and to the NIH Office of Biotechnology Assessment and that it was complete and accurate.
Further, that the procedures demonstrate that the IND sponsor had adequate oversight over the clinical investigation and in order to address that question we specifically asked for an organization chart which identified the individuals responsible for the oversight of the clinical study and a summary of his or her duties.
And in those instances, where the IND sponsor had transferred some or all of his regulations to another organization, we asked that -- we have a summary of the procedures that demonstrated that there was adequate oversight and for the CRO that there be verification or for the monitoring body that had overtaken or some of these obligations from the IND sponsor, we asked the sponsor themselves to verify that they were aware that the obligations for oversight were being appropriately met and that they were to provide a summary of the CRO's oversight procedures.
A separate item in that letter requested confirmation that all required animal safety data have been submitted to the IND or if there was some areas of clinical studies which had been of -- of the animal safety studies which had not been appropriately submitted, that they be submitted at this time in response to the letter.
That the animal studies in regard were those which suggested the clinical -- significant clinical -- I'm sorry, that the results from the animal studies that we were requesting confirmation had been submitted were those animal studies that suggested that significant clinical risk might exist and that those studies were required to be reported in writing to the FDA and that all investigators should be aware of their obligations to report such studies within 15 calendar days after initial receipt of such animal studies and that IND annual reports are intended to include a summary of major preclinical findings.
The March 6th letter was sent to 156 individuals who were holders of 276 total IND or master files.  The number of letters were less than the number of files because certain individuals held more than one file.  The responses to date as of March 8th of 2001, we have had, we have received responses to 200 INDs.  The total number of INDs are smaller than the total number of sponsors and master file holders for certain reasons.  In those instances, as I've explained in the briefing document where active studies were on-going and it was no response to receive those INDs have been placed on clinical hold.  In other instances, INDs had been previously withdrawn or there had been an error in terms of the relevance of a certain master file to the March 6th letter and so there are some differences in terms of the number of responses.
The response to the letter has been reviewed and comments communicated to 165 IND holders regarding the adequacy of the clinical monitoring program.  
Next.  For those 165 INDs, we noted that there were really sort of two categories of initial response.  There were a number of studies, approximately 30 or 15 percent of the total active INDs at the time that the March 6th letter was sent out where the sponsors replied that they had completed all studies, no further development was planned, no further studies were planned, no patients were in active follow-up and those sponsors chose as their response to the March 6th letter to indicate that they would either inactivate or withdraw their IND.  And generally, I think there are only two exceptions, didn't provide further information on the clinical monitoring program.
I'm sorry, go back one.  In the remainder of the INDs where the studies were active and we have reviewed the initial responses, it's clear to us that there was some confusion about what we were asking for because most of the responses really did not contain adequate descriptions.  They were deficient in their description of the program.  I'm not saying that the programs themselves were deficient, just that they didn't contain enough information to describe the programs.
With regards to those 165 INDs where we've completed all review and made communications, there are 26 INDs where the description of the program has been reviewed and an in some cases has involved review of multiple submissions and resubmissions to the IND and we've determined that the program as described is adequate to fulfill good clinical practices.
There are 139 INDs under which there are 212 protocols which have or are being conducted where the description of the clinical monitoring program is not full or complete.  For six of those, the INDs were actually withdrawn prior to or inactivated prior to that March 6th letter and we don't anticipate receiving additional information on those.  
There are 27 INDs where the sponsors asserted that all the clinical studies have been completed, no additional patients continued under follow-up and they have chosen to withdraw or inactivate their INDs and again, we don't really have any information on their programs at this time, but should they choose to reactive the INDs that will be a condition of their reactivation, that they provide complete and full detail on their monitoring programs at that time.
There are 106 INDs which remain active, where there is not complete information, sufficient information to assess the adequacy of the monitoring program and for those 106 INDs all the sponsors have been contacted and provided with a description of the deficiencies.  And that gave an example letter in the background materials as to the kinds of information and the level of detail so that it would alleviate the confusion of the initial, more summarized letter.
There were, as of March 1st, 35 INDs that remained under review.  Those INDs have been cursorily analyzed, but either the information has not yet been collated and entered into our database and/or we are in the process of requesting that the sponsor send additional information.  So we haven't completely closed them out in terms of the first cycle.
I would also summarize the experience since the March 6th letter in terms of new gene therapy INDs and we have held that the INDs, all gene therapy INDs should contain this information, including those submitted since March 6th.  There have been a total of 32 new INDs submitted since March 6th through March 8th of this year.  Five were withdrawn prior to initiation, there were 16 active INDs which provided at least some of the information regarding the clinical monitoring program and a few of those we've requested additional information to tie up some areas which need further detail.  There were 11 INDs which were placed on clinical hold for failure to -- generally, for multiple reasons, among them failure to provide information about their clinical monitoring program as described in the March 6, 2000 letter and again, those sponsors have received a more detailed letter regarding exactly the type of information we would like to see.
Next.  In terms of the initial response to the March 6th letter, the major issue really seems to be that we -- although we thought we were being clear, we obviously weren't and most people essentially missed the boat on what we were asking for and simply failed to provide an answer to the question being asked.  In particular, most individuals failed to describe the procedure for monitoring adherence to the protocol and to GCPs.  Most of them failed to describe their auditing procedures, for auditing the primary study information and verifying the accuracy.  And interestingly, many -- virtually all of the sponsors, with the exception of some of the industry sponsors, really failed to understand that we did indeed mean that we wanted to see an organizational chart of the individuals who were responsible for this program.
Next slide.  What did we get?  We did generally get a description of the procedures that investigators use at the time of implementation of the protocol that they hoped would ensure that the protocol ran smoothly.  For example, the type of things that we would receive would be the investigator would generate an eligibility checklist and would agree to fill out the checklist prior to entering or registering a patient on to study.  That is different from the type of information we expected to see with regards to monitoring which documented that, in fact, those checklists were indeed filled out for every patient registered.  So we considered that perhaps there seems to be confusion about what we mean by monitoring the conduct of the protocol versus implementing the protocol and getting the procedures in place before the first patient is enrolled.
And that's what I mean about frequent confusion regarding the distinction between those procedures to start the protocol versus adhering to the protocol as written which is something that goes on as patients are being enrolled in regular review.
Next.  After we clarified what we were looking for in much more explicit detail, the subsequent series of letters and communications we've received to the INDs indicate that, in fact, there are very few deficiencies in terms of the programs which are described in their ability to actually meet all elements of good clinical practices.  The deficiencies that did exist were few, but they included both issues of procedures and description of organizational structure or staffing so that what I will describe to you in the second and sometimes third rounds of communication between the FDA and the IND sponsors, the kinds of things that people still seem to have trouble making sure that their monitoring program has in place.
Next slide.
DR. SALOMON:  Patricia, may I interrupt for just a --
DR. KEEGAN:  Sure.
DR. SALOMON:  I guess what's really bugging me right now is the -- maybe I don't have this right.  But what I'm looking at here is that were 20 
-- you sent out this letter.
DR. KEEGAN:  Yes.
DR. SALOMON:  And 26 INDs covering 64 protocols were reviewed.
DR. KEEGAN:  No.
DR. SALOMON:  And then you sent out a subsequent thing.  This is a whole year.
DR. KEEGAN:  Yes.
DR. SALOMON:  And after a whole year there are still 106 INDs that are active with insufficient information to assess the monitoring program.
DR. KEEGAN:  Uh-huh.
DR. SALOMON:  And 32 new INDs have been submitted and 16 of them are active with some attempt to address the March 6th letter.  I guess when you go back a slide and you say there were very few deficiencies, are we talking about then this small subset of 26 that you can evaluate because you've still got four times that many that you haven't got information back yet.
DR. KEEGAN:  What I'm talking about is yes, on the 26 and in addition some of the 106 were still going through it, but on the second review of the responses which again we haven't collated in full detail, so I couldn't give you the numbers on that.  On the second time around, people usually get it, but I can't give you the exact number where we've gone through and ascertained that everything is absolute and complete, other than for the first round, but on the second round we generally have.
DR. SALOMON:  Okay, I hated to interrupt you, but just for me to be processing what you're presenting, we're talking about a study that's not complete yet, that you have maybe 25 percent or 30 percent maybe by now, I'm just guessing, close to that and based on that 30 percent, you're giving us some feedback.
DR. KEEGAN:  Right.
DR. SALOMON:  So all these statements about there aren't that many deficiencies, etcetera is based on this subset of total -- then I can sit back and --
MS. LAWTON:  Can I just comment on that though because I understand that you're also providing us feedback on the additional INDs that you've had answers on the second round which -- so it brings it higher than percentage.  It's not just the 26 INDs.  It's the others in addition to that.
DR. KEEGAN:  It's the others, but I can't give you a firm number for that.  This is basically in discussions with the staff.  Like I said, when we sort of closed it out and put it officially in our database as where the review stands, then I'll have better numbers, but it's in terms of trying to do that.  Again, as regards to process, you should recall that the March 6 letter gave sponsors up to three months to respond.  The number of responses that we got prior to June was a handful.  I'm estimating less than 10.  So most people waited until the last second.  Many of those people, I should say that there were a number of people who didn't even respond to that, so we had to send out a second letter, basically putting people on notice that if they didn't do something, we would put their INDs on hold.  So by the time we had information in to begin our review, it was really the summer of 2000.  So it's taken a while to get through the number of INDs and protocols.  So I think it's just the process of getting through this and giving you numbers is the issue and I'm supplementing it by the flavor of the responses on the second round for which I don't have solid numbers.
DR. SALOMON:  Just for the record I in no way mean to criticize presenting preliminary data.  We do that every week in my lab.  I wanted to make sure that I was sitting here listening with the appropriate context.
DR. SIEGEL:  Let me put this in context because it's, I think, a little less preliminary than you may think.  I hope so because we're talking about thousands of hours of reviewer time to generate it.
The Agency and I'm not talking about just gene therapy or just biologics, but the Agency as a whole has always required that clinical trials be monitored and that there be QA and QC, that there be assurance that there's good clinical practices in following the protocol.  That's a sponsor's responsibility and periodically either for cause, but most commonly at the time of licensing, we inspect to ensure that that, in fact, the trial had been adequately monitored, or more importantly we judge the success of the monitoring by ensuring that the documentation do support the fact that the data are of high quality and that the patient's welfare and rights were appropriately protected.
What we have not done and again, I'm speaking Agency-wide, what is pioneering about this effort is we have not asked sponsors to tell us up front for our review how they go about doing that and have not reviewed those activities, rather we've trusted that they do okay and then post hoc at the end when they come in for licensure, we inspect to ensure that we can trust the data and also again, checking for patient protection.
As many of us, Dr. Zoon and myself sat in discussions with senior officials at NIH and at the Department and in the period of the winter of 1999 and 2000 and looking at some of the things that we had discovered at some of these inspections and some of the concerns that were being read and also the loss or significant loss of public confidence in the ability of medical researchers to protect patient safety and welfare and rights, particularly potentially in the area of gene therapy, we began to look at what could be done to better assess the situation and better determine where the problems were, improve the status of events and also potentially if appropriate, restore public confidence.  
So this approach of asking sponsors to describe their monitoring techniques represents, if you will, a pilot effort, something the Agency has not engaged in before to any significant extent.  We did require it, but on the other hand, recognizing the novelty of this and the difficulties of responding as well as reviewing to these data, we implemented it with a certain amount of flexibility.  We were asking for a lot of data and then we asked for it in a two to three page summary.  We weren't highly specific and I suppose aside from the fact that we had good reason to expect something better than we got, we also had good reason to expect that we didn't know exactly what we were asking for and that sponsors didn't know exactly what to provide, simply because of the nature that this was something new and we were -- so what developed was an interactive process to get at what we felt would be the most important information to know and what sponsors and the most important thing for sponsors to do.
Now part of what we discovered is that there were a subset of sponsors just as we discussed somewhat about academic sponsors involved in the manufacture of products this morning that some academic investigators involved in the conduct of clinical trials, the concept of quality assurance and quality control and independent oversight of their activities was a relatively new concept which isn't to say that they weren't doing good clinical trials or safe trials or protecting patients, but the concept of independent oversight and documentation in some of these same principles which is what we have traditionally looked to for assurances that that happens, was relatively new and so the answers we got back, I'll make a long story short, but the answers we got back to the initial round of questions, as you'll hear more of soon, reflected a broad range of to some extent lack of clarity on our part, but also of just not understanding what the issue was.  You know, quality assurance, I thought that was the FDA's job or the IRB's job or something like that.  And so we've got that -- if we didn't get back substantive and workable and reassuring responses on people within a couple months of the three months' deadline, there were clinical holds.  So there should be no suggestion here that three quarters of the people haven't responded a year later and they're still conducting trials.  That's not what's happened.  But what has happened is first of all a lot of their responses indicated that they were describing systems that they've had implemented since receiving the letter or since the headlines in gene therapy, so a lot of this involved implementation of new systems to ensure quality control and a lot of it involved -- well, they respond, but they maybe missed some points we were interested in and we'd get back to them and say we really want to hear more about how you're doing this and so forth.
So to say three quarters is incomplete is true.  On the other hand, there's been 100 percent review of these responses and those trials that are on-going are in a position that we're comfortable with where they are.
DR. SALOMON:  Okay, without any further discussion -- I appreciate that clarification.  We'll get back to that because I have some questions on that and I think Ed had a comment.  If you'll accept my apologies then for interrupting, Patricia.  
DR. KEEGAN:  My concern is just that I hesitate to give numbers where I don't have firm numbers on some of these issues.  But at any rate, those areas where we found that again some of the plans on more detailed failed work, I'm sorry -- go back a slide.  Go ahead.  All right.
This is actually a summary of the description of the monitoring procedures that were described there.  We found that there was a lot of variability across the board that monitoring visits might vary from weekly to annual, that monitoring visits in some instances were not tied to the calendar, but were tied to patient accrual.  That was a relatively uncommon situation.  More often it was really tied to a calendar.  That the proportion of patients' records that were reviewed and verified for accuracy also ranged, and it was variable.  It ranged from 10 to 100 percent.  Again, in some instances it also varied by the phase of the study or the size of the study.
Next slide.  In terms of the concerns that we had where people still needed to doa little bit more work, there were failures.  Probably the most frequent was failure to describe actually the individual who was responsible for directing the investigational drug product to make clear whose job that was.  Sometimes there was also a failure to put in details about the procedure itself.  Failure to describe the procedure for removal of investigators who failed to adhere to the protocol is written in the GCPs.
Next.  No procedure described to ensure that the modifications were reported to the FDA.  Again, not that it may not be happening, but that they didn't describe the procedure.  No procedure to describe for verification the study information against the source documents or for how they maintain the study records and not providing a procedure to ensure that the safety reports are filed to the IND.  This last one is the only one that raised just a little bit of concern in that that was one of the few where it wasn't simply a lack of information, but where there was some -- in some instances some misconceptions on the part of the investigator, that if they filed it to the IRB, the IRB would send it to us.  Or if they put it to MedWatch, it would end up in their IND.  And in those instances we did make sure that people were contacted and understood that they had it wrong and what they needed to do to correct that immediately.
In terms of the clinical monitoring staff, again, a variety of arrangements that this basically covers the waterfront here.  Frequently, particularly if you're a sponsor-investigator, it's a research nurse or team of nurses who report to the investigator.  Also, at academic sites and this seems to be a relatively recent phenomenon that many sites now have a clinical site team that reports to some individual at that study site, for instance, an administrator, and that they perform a service for investigators at that site to do monitoring and auditing, that there's monitoring staff that's employed directly by a commercial industry sponsor, that there are contract research organizations which perform this either for sponsor investigators or commercial sponsors or sometimes it will be a combination of the above, particularly again for the smaller biotech companies or even for larger biotech companies that they will have their own staff and it sometimes also employed the services of a CRO.
Next slide.  In terms of training and qualification of the monitoring staff, this seems to be fulfilled primarily by training as a health professional.  In some instances commercial sponsors and CROs also have developed their own predominantly on-site separate training programs for the individuals who do monitoring for them.
Next slide.  The concerns in the clinical monitoring program that rose to our review that are -- and again, this is a rare instance.  I think there's actually a very limited number of sponsors, I want to say or one or two, who transferred monitoring obligations to the CRO, but failed to maintain a copy, so they weren't able to give us much in the way of a summary of the CRO's procedures for fulfilling the obligations.  However, they did verify that they had reviewed those procedures at the time of the contract and felt that they fulfilled all their criteria for monitoring.
And the other which I believe you've heard about before is the fact that there are sponsor investigators directly supervising the monitoring staff which raises concerns about the ability of a monitor to implement corrective action for somebody who is her direct supervisor.
Next slide.  In terms of commercial sponsors, again, we found that there's been a problem very limited, but a few commercial sponsors who have acquired other industry-sponsored or academic programs where there wasn't any details about monitoring and they don't really have much information about studies conducted prior to their acquisition of the studies and that raised a whole other set of questions about how much background work they needed to do to investigate particularly older studies.
In terms of the impression, and again, this is for the 200 INDs which we have, at least, preliminarily looked at and had discussions, most of the sponsors have staff identified to perform monitoring and auditing.  There is a variable frequency of monitoring and a variable amount or extent of data verification, how many search records are evaluated, how many patients' records of the proportion to patients in a trial.  And again, variable degree of independence between the clinical monitors and the investigators.
The impact of the variations in the conduct and organizational structure of the monitoring programs on adherence to GMPs is not clear from our review.  We don't know if it matters, exactly, whether the frequency or certain types of programs make a difference.  It is clear where we have specifically asked and received a response that there are a number of sponsors who have augmented and approved their programs in the past two years.
Next slide.  With regards to the preclinical and this will be much briefer.  There are 135 INDs where the response has been reviewed and deemed to be completely adequate.  In 119, the sponsors verified that all safety information had been submitted.  For 14, the sponsors actually supplied some additional information and in some instances it was just publications of previously reported information and in others it was actually new information that we had not seen. 
There are two sponsors which have summarized -- it's actually one sponsors with two INDs who summarized additional information, but hasn't provided the raw details and they have been asked and have verified that they will be supplying that shortly.
There are 39 INDs or master files where there's the responses were incomplete and they've been asked to clarify what exactly they meant by their response.  The most common was well, it's not applicable to my file and we didn't often know what precisely they meant by that, meaning it's not applicable because I did it or it's not applicable because I don't have any animal studies or what, so we have asked for additional information and there are 16 that remain where I don't have the results of the review yet, where they're under review and I'm not sure if they were adequate or what the actual outcome was.
The majority of sponsors appear to be in compliance with the applicable regulations for submission of the animal safety studies and the only question raised by reviewers was the ones where sponsors said all our information is contained in our cross reference file and it was sort of a parenthetical by our staff that they were not certain to what extent all IND sponsors are completely aware of everything that's contained in the master file.  They certainly don't have any right to be aware of everything and so on occasion our response is we hope that you have that in writing, that you'll be aware and have that in confirmation that all animal studies are being appropriately reported.
Next slide.  That's it.
DR. SALOMON:  Excellent.  Then I'd like to go forward without any more discussion to Dr. Salewski, Chief of the Bioresearch Monitoring Branch who is going to talk about the exact overview of the subset centers that we've done on site.  And then we have a series of questions that I think are clearly extraordinarily important to this discussion this afternoon.
MR. SALEWSKI:  When I was asked to present to this advisory committee I asked to see the roster of the members and I didn't recognize anybody's name, so I decided a brief overview of the Bioresearch Monitoring Program might be helpful to everybody involved.  
The purpose of the Bioresearch Monitoring Program is to ensure the integrity and quality of the data that's submitted to the Agency in support of a marketing permit.  That includes INDs, NDAs, IDEs and ensure that the rights and welfare of the human subjects are protected.
In FDA, each of the five centers has an active Bioresearch Monitoring Program.  Currently, it's coordinated by the Office of Enforcement.  That will change relatively soon.  There's a new office in the Office of the Commissioner called the Office of Human Research Trials where all of the programs under Bioresearch Monitoring will be oversight responsibility and coordination responsibility will be transferred to that office, except for the Good Laboratory Practice Program which will remain in the Office of Regulatory Affairs.  And all Bioresearch Monitoring Programs are conducted by field investigators, occasionally accompanied by an expert from the Center when we feel the need for that expertise.
There are four programs associated with the Bioresearch Monitoring Program and as you can see we have oversight of product development from the animal testing stage through the clinical trials associated with marketing applications.
When do we become involved with biologics?
We mostly get involved, most of our work is associated with license applications.  On occasion we do get referrals from CBER staff when they have concerns about how a study is being conducted or how they're not getting appropriate responses from sponsors of clinical investigators and after a while they'll come to us and ask us to help them correct the situation.  Sometimes other centers, if they find they have a problem with the clinical investigator or an IRB or a sponsor, they'll notify us in case we have any protocols being conducted by those people or research being conducted, so if we have concerns we could also go out and take a look.
Also, recently, I mean in the last two or three years, we've had a real upswing in complaints.  We get complaints from sponsors about clinical investigators.  We get complaints from IRBs about sponsors and clinical investigators.  And I have consumers up there and by consumers I mean participants in the clinical trials or their relatives.  They felt they'd been mistreated or not -- didn't get the appropriate test article, so they come to us those kind of complaints and ask us to resolve any issues.  And also we get a lot of complaints from former employees of sponsors and IRBs and clinical investigators who at the time they were working for them thought they were doing the right thing, but once they had to find other employment, they decided it wasn't quite right, so they thought they'd let FDA know.
And then there's the routine surveillance.  We haven't really conducted much of that over the years until recently for the gene therapy initiative was our first real routine surveillance try.  A typical cycle for a BLA in our center, a Bioresearch Monitoring Representative is part of the committee, the licensing committee.  This is just a typical overview of it and the committee member discusses with the medical review officers and the scientific review officers and the statisticians what their concerns are for the trials, what trial sites they think they'd like to go see.  We develop an assignment.  We send the assignment out to the field.  The field will go out and od the investigation.  They'll write up an EIR which is an Establishment Inspection Report.  They'll send that to my group.  We'll evaluate the EIR.  We're write the appropriate correspondence and then after we get all the inspection reports associated with the license application, we'll develop a summary document which will provide to the licensing committee detailing what we found at each of the clinical sites and with the recommendation to either accept the data or reject the data from one or all the sites.
Next.  How do we go about selecting the sites?  Basically, we'll sit down with the reviewers and see what their concerns are.  The goal that we shoot for is that we try to get the sites that have treated at least 50 percent of the patient population.  Sometimes we can't do that because there are some trials that are huge like the TPA trial had 60,000 subjects treated at over 500 sites.  So we couldn't quite do that.  And there are other trials where they've treated maybe 110 subjects at 87 sites.  So we don't have the resources to do that.  So we'll get together with the statisticians and come up with some kind of scheme to do our inspection with.
But basically, the higher the number of subjects at a site, the more likely we are to go and inspect that site.  Also, the geographical distribution plays a part in our selection.  If a license application has 10 clinical sites, six of which are in California, we may end up only doing one or two of the sites in California.  Also, we look at the inspectional history of the clinical investigator.  We'll not only look at our database, but we'll contact the Center for Drugs and look at their database to see if that person had been inspected before and if he has, what type of inspection, what kind of problems did they find at the site.  If it was a violative inspection it's most likely we'll go back and look at that clinical investigator to see if he's changed the way he's conducted trials.  If the reviewers note inconsistencies in data such as too many adverse reactions at one clinical site or not enough adverse reactions at one clinical site or if the data is being driven by one clinical site, we'll basically go see those places.
What we do because the field, there's many other things other than wait for a Bioresearch Monitoring Inspection assignment.  They also do blood banks.  They also do warehouse inspections.  So instead of going in there cold, we like to give our investigators some information so that when they go into a clinical site they know what they're looking and they know what they're looking for.  We tell them what the product is, how it was developed, who the sponsor is obviously, what patient population this product is being given in and what the expected outcomes of the trial are.  We ask them to look at adverse events, see if the protocol was followed, see if all the subjects met inclusion criteria and not exclusion criteria, see if the blinding was maintained throughout the study.  We checked to make sure that the appropriate dose was given at the appropriate time frame and did they meet their end points.
And after they go through all of this, after they perform their inspection, they'll sit down with the clinical investigator and go through with them before they leave which we call a close out, before they close out the inspection they'll sit down with the clinical investigator and discuss with them the findings, this is what we found that you didn't follow your protocol, you included several people who met the exclusion criteria.  And they'll discuss it with them and they'll make this part of their report that they send to us.  After they leave, they'll write up this EIR.  They'll send it to us.  We'll classify it.  We have basically three classifications, no action indicated, voluntary action indicated, where there are several violations of the regulations, but the violations really didn't affect the data from the study or violate the subjects' rights or welfare.  Then there's official action indicated where it's met a threshold, where the data has been affected by their conduct in the study.
What we do is we will issue correspondence.  We have basically two types.  One is the untitled letter which goes to the NAI investigations and the voluntary action indicated investigations.  We'll write to the clinical investigator or the sponsor and say this is what we found at your site, how do you plan to correct it in the future?  And then we have titled letters.  One is a warning letter where we say this information, the violations here are affected.  What happened at your site?  You have 15 days to tell us how you're going to correct this or tell us why we're wrong in our assessment.
Then we have this Notice of Initiation of Disqualification proceedings and the opportunity to explain, commonly called the NINPO.  By the way, it took the Agency 14 months to come up with that name and you know it's a good name because nobody likes it.
And this is where a clinical investigator will get this notice once he meets the threshold of deliberately violating the regulations repeatedly.  It's "or repeatedly violating the regulations or submitting fraudulent data to FDA or to the sponsors."  We'll initiate that disqualification procedure and of course, they have the opportunity to explain and if we accept their explanation the matter will be dropped.  If we don't, we go ahead.  We proceed with a Part 16 hearing.
What can we do as far as administrative actions?  We can recommend that the data not be accepted to support the application.  We can recommend that they refused to file the BLA or put the IND on clinical hold or terminate the IND.  In compliance, we can't actually do those.  We make recommendations because it's the scientific review staff that makes the determination of whether to place someone on clinical hold or terminate the IND.
However, as far as disqualification goes, we have the authority to go ahead, go forward with disqualification or the application integrity policy issues.  But we do that in conjunction and the support to the medical and scientific staff at our centers.  We don't go off on our own and do this.  It's a joint decision, it's just that we end up with the work of doing it. 
Okay.  And now, the gene therapy inspections.  After the inspections of -- in Philadelphia and in Boston, the Center was concerned about the state of gene therapy investigations in the United States.  So what we decided to do was take a randomized sample.  At the time, there wre 211 active INDs.  We -- after consultation with our statisticians we determined that a number, 30, would be appropriate and so we selected 30 INDs in a randomized fashion and we extracted every principal investigator doing a study in each of those INDs.  We ended up with 24 sponsors and 70 clinical investigators.  So we basically issued 70 assignments to look at how clinical investigations were being done.  
The breakdown is here.  As you expect, most of them are independent with only six commercial sponsors and as you would expect the commercial sponsors had the most clinical investigators associated with their INDs at 46 and I thought you might be interested in this.  We asked the field to do these inspections within 60 days.  We didn't quite meet that time frame, but the field spent over 4,000 hours doing these clinical investigations.  That meant they spent between three business days and 26 business days in the clinical labs, in the doctor's office, looking at their records with an average of 75 hours and that's equivalent to four and a half -- what we call full-time equivalents in the Agency.
And what we found was this.  Washouts are places where they hadn't started treating subjects yet, so out of these 70 clinical sites, 11 of them were washouts.  The classifications are broken down there which we're pleased to see that there are only three really violative inspections and again, just so you know, voluntary actions, we found some regulations of the regulations, but they didn't reach a threshold where we take an action.  An official action indicated where there was only three of those, again, where we actually took administrative actions.
And for the commercial sponsors, this is the breakdown of the left most column is the breakdown within those four to six.  The overall is within all the gene therapy inspections.  So as you can see, there was most of them had some violations of the regulations, but not enough to warrant an action.
The government, of course, we do a better job.
(Laughter.)
Next.  And the independent clinical investigators which actually kind of surprised me, they were doing very well.  Our inspection, I guess I should clarify.  Our inspection just looked at how they performed their clinical trial.  We didn't look at monitoring.  We just looked at this is your protocol, did you follow it?  Did you do all the appropriate paperwork?  Did you notify people of adverse reactions?  You kept count of your drugs and your patients?  That's all we did.  We wanted a snapshot to see what was going on.
And this is a comparison of what we find, in general, as compared to -- with the gene therapy inspections.  As you can see, that's fiscal years.  Fiscal Year 2000 includes the gene therapy inspections and the one below that is without the gene therapy inspections and you can see that on average, even though these were Phase 1 and Phase 2 studies, the investigators were doing a fairly decent job on following the protocol and taking care of the patients' rights and welfare.
Next slide.  And what we found the most common violations that we found and the most popular one was not to follow the protocol.  That includes things like enrolling subjects who didn't meet the entrance criteria.  Not giving the appropriate does or at the appropriate time.  Not doing appropriate lab work, etcetera.  And then there was problems with the consent forms and lack of supporting data for the case report form entries, etcetera.  As you can see, and these are basically in line with what we find in our normal course of business.  They're no different than anybody else.  There's no surprises.  The surprise for me was that they were so good, actually and that was pleasant.  
I think that's it.  Do you have any questions?
DR. SALOMON:  Joe, just two quick things.  What's a washout?
MR. SALEWSKI:  A washout is when they hadn't started treated subjects.
DR. SALOMON:  Okay, and then the last thing, I just want to make sure I understood this right.  Under GT inspections, a comparison, I think your third to the last slide.
MR. SALEWSKI:  Okay.
DR. SALOMON:  So GT was gene therapy and 2000 total was just all of your bio actions?
MR. SALEWSKI:  Yes.
DR. SALOMON:  In the year 2000?
MR. SALEWSKI:  Yes.
DR. SALOMON:  Good.  I understand.  Okay, I think we better delve into this before -- there are a series of three questions that I've been given to generate discussion on what is clearly a very important issue.
Before I bring up the questions which take the group's discussion in specific directions that I'm going to try and hold you to, is there anyone who feels that they just have to make a brief, underline the word brief, comment overall?  I mean I've certainly taken the liberty and I won't deny anyone else on the committee to do that.  But if -- so I know that -- do you want to --
MS. LAWTON:  I actually had one question for the presenter and that was I was interested to know with this comparison for the gene therapy trials that were audited compared to the other trials, do you have a feel for the ratio of kind of Phase 1-2 trials that you looked at compared to Phase 3 trials normally?
MR. SALEWSKI:  Normally, that comparison was what we usually look at are Phase 3 trials.  So these being Phase 1, Phase 2, they turn out very well compared to what we see.
DR. SALOMON:  Okay, any other questions?  All right.  So I'd like to go on record as saying that it is really, the message is reassuring as I hear it based on the data today that after all the publicity on -- and concerns by the public that the rate of serious violations and conduct done during what's clearly a very rigorous review with hours spent at each center is actually half or less the general violation rate, depending on how you did it.  And I think that's pretty remarkable.
MR. SALEWSKI:  I just want to add that the Philadelphia sites and the Boston sites weren't included in the gene therapy results.  It was totally different.  
DR. SALOMON:  Right, well, that certainly wouldn't have been random either.
(Laughter.)
In fact, if they had been I think we'd have to start all over with the idea of how you randomize this which we've let you go on.  Okay.
So the questions, the first question is really a critical one and it's going to take a little bit of reading to set the stage for, so forgive me.  So the regulations acknowledge that the sponsor of an IND may also be the clinical investigator.  In that case, they're referred to as a sponsor investigator.  The FDA wants us to consider, however, that it's difficult to understand how a sponsor investigator is capable of performing certain required tasks and it's evident that the experience recently in gene therapy has had everybody take a much harder look at this.
Specifically, the regulations impose that an IND sponsor or sponsor investigator who discovers that an investigator is not complying with the signed agreement, that the general investigational plan or the requirements of this part are applicable to parts, blah, blah, blah -- that this investigator now should promptly secure compliance or discontinue shipments of the investigational new drug to that investigator and end the investigator's participation in the investigation.  
Well, the obvious point here is is that if you're the investigator, it's kind of a discussion in the mirror.  
(Laughter.)
And that's obviously an issue of major concern.  
Secondly, a sponsor shall select a monitor qualified by training and experience to monitor the progress of the investigation.  Now here we realize that in practice that has meant that that monitor is typically a research nurse or a research technician employed fully by the investigators or sponsor/investigator and we've already begun -- Dr. O'Fallon, for example, pointed out to us the obvious problem with that.  These people work for us.  They want to please us and in fact, something that -- no, it was Joe mentioned I thought was really, really critical and that was that an increasing number of complaints are from research nurses or monitors who had left the employ of the investigators and now are complaining to the FDA.  I think that was definitely something worth repeating.  
So please discuss the relative merits of various approaches to the oversight monitoring.  So given the potential concerns with monitoring programs in which the monitors directly report to the 
sponsor/ investigator, I think that's what I've just articulated, should these be discouraged?
If such a program is utilized, we should discuss what, if any, additional elements or safeguards could be employed to ensure adequate oversight and minimize conflicts of interest issues, etcetera.
There's a second part of this, but let's start with that.
DR. SAUSVILLE:  So I think this is really a proverbial fox and henhouse sort of question and I think that one approach that might bear some thinking is that the institutions, either universities or hospitals or what have you which are the sites at which these studies are conducted, might be in the position of serving that bridging or intermediary role.  My own view is that to have a research nurse work for the investigator who's studying the entity that the research nurse is monitoring and if that's the closed loop is that that needs to be strongly discouraged, if not actually made -- I hesitate to use the word illegal, that's not our role, but I mean at least in some way made not a normative procedure.  I think that the institution which is at one level another type of sponsor of the research should be charged with putting in place a monitoring system for the studies that it undertakes by its investigators, that the cost of that is going to be figured into the indirect costs, either for grants or for other funding arrangements and that the monitoring service, in essence, report to the institution.  The institution is then in the position of serving as an ultimate watchdog who would hopefully balance the fox and henhouse relationship.  
I don't know that that actually has been put into practice, but that strikes me as one model in which we might get around some of these issues.
DR. SALOMON:  That was very nicely stated, so let me just make sure that -- so one possible reaction here that Ed has articulated very nicely is that we just advise the FDA that this is not an acceptable relationship in the future and then -- and that's one thing we should just decide.  That doesn't necessarily mean then what it is we should suggest in its place, but we should parse this out that one comment is this is not an acceptable thing.
Now the second thing, also well articulated, is that we should allow the institution to use indirect funds and other resources within the institution to provide that service for investigators within that institution.  I see those as two different things, both very important for us to discuss.  
Dick?
DR. CHAMPLIN:  Just one thing, the obvious thing here.  The research nurse job actually isn't monitoring and the research nurse's fundamental job is to be conducting the research, generally screening patients, eligibility, etcetera, collecting data, making sure that the samples are collected and that the credence given according to the protocol.
DR. SALOMON:  That's monitoring.
DR. CHAMPLIN:  Well, that is actually doing the study and collecting the data.  Now monitoring is a second function that is -- it is the oversight function that that role is being done correctly.  So I think it's a misinterpretation to say that the research nurse shouldn't actually work for the investigator.  There should be a second layer where someone else who is not primarily involved in the protocol is, in fact, monitoring and I don't disagree with the concept that it should be an institutional function because the institution, of course, does take responsibility for the conduct of research activities carried on within its jurisdiction.
DR. SALOMON:  Okay, so that's fair.  What Dick's clarifying is it's not that there's something wrong with the research nurse.  There should be research nurses, but as long as they're identified with actually the conduct and perhaps supervision of materials flowing around, that's all a good function, but the monitor.  There has to be a position now that we refer to as a monitor which actually is an important point here.
DR. CHAMPLIN:  A fundamental --
DR. SALOMON:  That person can't work for the sponsor investigator.
DR. CHAMPLIN:  A fundamental principal of quality assurance is you don't inspect yourself or monitor yourself, that there has to be an independent entity and that serves that function, so the people conducting the trial shouldn't be monitoring themselves, but some other individual within the organization should have that function.
DR. SALOMON:  That's good.  That's a refinement.  
DR. PATTERSON:  You actually started to talk about the issue that I wanted to bring up.  I think it would be helpful if the committee came to a common understanding of what is meant by independence.  Are we talking about independence from a reporting relationship?  Independence of financial ties?  And harkening back, actually, there's a good analogy I think from Mary Malarkey's presentation this morning, the independence of the QC, the testing unit, from the production unit and the QC unit has -- although it may be employed by the sponsor, it has an authority to override in some instances their decision may trump.  And I think trying to figure out in terms of clinical trial oversight what those relationships are or are not.  Even in the situation that Ed described, one could argue that there may be some institutional conflicts of interest and ultimately the people reporting to the institution are employed by them.  So I'm having some difficulty understanding what it means by independence.
DR. SAUSVILLE:  I mean I guess my view about that is that the unit that does monitoring might work for and actually obviously might be employed to a certain extent either -- certainly at least in a contractual sense by the university or by the institution.  But I think that the nature of the relationship should be that they are empowered to make their decisions quite independently from the decision making structure that runs the clinical trial and now how one exactly sets that up I guess would obviously bear some thought, but the general principle would harken exactly to what you said.  This needs to be viewed almost as an Inspector General or some type of function that is quite independent from the actual operation of the trial.
DR. SALOMON:  The problem here though is what follows and that is what -- as Amy points out, what is independent?  So an institution, how independent is an institution of its investigators?  Now an institution will often hold the patent on the product that the institutional investigator is testing, so there already there's a -- it's very common these days and that's a major thing.  They may even hold stock in the company that the investigator started to run to do these trials and we have examples of that right now.
DR. SAUSVILLE:  But that's exactly why, I think that they would be vested in, as it were, getting this right.  Because I think that if the monitoring agent were actually independent in the sense that I mean in the limit case they were actually a company that was hired for this purpose.  And at one level they're going to get paid whether or not there's a patent ultimately resulting in a product or not.  I mean the nature of their relationship is that they are contracted for it.
DR. SALOMON:  Right, but one of the recent cases, I believe the facts are correct, at least as I know them from the newspapers is that one of the CROs that was contracted had a stock position, an ownership position in the company.
DR. SAUSVILLE:  That clearly then fits into what was brought up before.  That's the -- that type of CRO should be intrinsically disqualified from this role.
DR. SALOMON:  So how do you generate a CRO in a university or in an institution, a research institution that can do that?
DR. CHAMPLIN:  For example, the University of Pennsylvania has a vested interest to be sure that they don't have regularities in future gene therapy or other clinical research studies.  The institution's interest is to remain in business as a clinical research center and it is clearly in their best interest to avoid these kind of events, so that they have a natural interest, to be sure that the clinical research is done appropriately, far exceeding any gains that they have from any individual product being successful or not, so I think that there's much more confidence there at least in my mind than perhaps a small biotech company in monitoring their own clinical trial where they have a much greater financial interest in its success or failure.
DR. SIEGEL:  It's worth nothing that although closely related, there is a distinction to be made and I think Amy is right.  The issue of what independence plays is very complex, but there is a distinction to be made between the independence, 
vis-a-vis functional independence and reporting responsibility versus the issue of financial conflict of interest.  They're both very important.  It's worth knowing that in the long history of drug development that clinical trials are monitored by the sponsors which are usually pharmaceutical companies and which have a tremendous financial interest in that trial and I think for the most part, but not always that financial interest points toward their ensuring that they get the best, highest quality data and the highest quality trial and good patient protection, but not always, but -- and that -- but what differed from some of the cases we're talking about, well, the levels of financial conflict of interest differ, but another thing that does differ is this issue that those monitors are not working for or with the investigator and the FDA actually has had to tighten up its regulations in this area, but the sponsor has an obligation and is expected to dismiss the -- to act independently and to dismiss the investigator when he's not acting well or to correct those actions or dismiss them as it says in our regulation.
So conceivably, a university such as you're suggesting Dr. Sausville, there may be some financial interest.  I imagine there's always some level of financial interest, sometimes more if they own stock in the company, but it's not -- but on the other hand it might well be very different if you have study nurse monitors who are reporting to and hired by and working for the dean's office than if you had study nurse monitors who are reporting to working for and hired by the principal investigator that are actually monitoring what the investigator does.  And as to your question, just one quick comment, you did ask has this been done, is this being done.  We're seeing a growing number of institutions, particularly those institutions that either by OHRP or FDA, or the press, have had some bad publicity about their clinical trials, but a growing number of institutions building clinical trial oversight programs, we've got report a number of them are occurring in gene therapy and in your handouts, there are some concerns about are they intensive enough, trained enough and so forth.  We think it's an interesting direction to look in.  We're all in agreement with I think the original sound advice, the first thing this committee said, you can't very well monitor what you're doing yourself.  
I should say one more thing to put this in context.  All of these issues are being broadly discussed throughout the country, academia, throughout the department, throughout the agency.  There's new policy under development.  It's a bigger question than gene therapy, but it has -- a lot of the questions arose from gene therapy and I say that as a matter of context because on the one hand, this committee and it's advice isn't going to directly lead to a decisive decision, but on the other hand, I think we recognize very importantly that decisions that we make in this area and I and others in the room are quite involved in the committees that will be making decisions in this area, are -- can only be made with really a lot with feedback from the patient and scientific communities.  We can come up with all sorts of rules about what universities and researchers can do and I assure you from past experience that we're quite capable of coming up with roles that don't work.  And so -- we really are interested in this discussion.
DR. SALOMON:  So, so far what I think we've already -- just the way the discussion goes, then unless someone ants to stop here, let me just capture one thought that's clear, that we are advising you that the sponsor should not employ the monitor, the investigator sponsor should not work -- well, that's actually interesting.  The monitor shouldn't work for the investigators if there's a sponsor and let's say six institutions under that sponsor, nor should a sponsor/investigator at a single institution, either that an academic or biotech, in either case a monitor should never work for the investigator or the investigator/sponsor.  I think we've all said that.  So that's good.  We've got that settled.
Then the discussion is going toward who then is far enough away or independent enough to equal -- noble enough to take on the responsibilities of monitoring it, right, and trying to be practical here.  		MS. MEYERS:  It's supposed to be the IRB.  And the IRB's responsibility is not just to approve protocols, but to monitor the conduct of the research.  		DR. SAUSVILLE:  That's not correct.  I mean -- right.  IRBs certainly receive reports about adverse events.  They judge protocol consents and are very active in human protection aspect, but IRBS, at least in the places that I have been have not involved themselves with the shall we say the technical management, how the clinical trial is being conducted.  That's just not their role.
MS. MEYERS:  Then they're not obeying the common rule.
DR. SIEGEL:  IRBs are charged with monitoring the progress of a trial.  
MS. MEYERS:  It's HHS.
DR. SIEGEL:  I think there's a broad range of interpretations as to what that means.  What we're talking about here and the problem -- part of the problem here is the use of the word monitoring.  Because we talk about trials being monitored by data safety monitoring boards or monitoring committees and there's been -- who often, you know, in most cases are in no position to know whether the data they're looking at are exactly the same as what's in the patient's chart or whether the -- for a consent form to sign.  They're monitoring, but they're not doing site monitoring.  It's an unfortunate duality of the use of the terms.  IRBs are responsible for monitoring either because of interpretation or because of staffing.  Most IRBs practice that by at least once a year, reviewing the safety reports and adverse events.  Most IRBs do not, but they certainly are authorized to.  I doubt many at all are staffed to and I'm not even sure that -- they are one of the options, but to actually do what we're talking about, going out and actually looking at what's going on.
DR. SALOMON:  I think what we have to realize here is the reality.  The reality is that over the last several years, because of the concerns that have been raised, there's just been an explosion of awareness, followed by a near explosion of requirements.  And there's no IRB that I know of that's in any type of position to do this.  They'll look at the trial initially.  They'll look at the consensus initially.  They do not have people that go out and monitor 25 percent of my consents.  They will get my -- if I have a series of adverse events, they get reported immediately.
MS. MEYERS:  But if you don't report your adverse event, they don't know about it, do they.
DR. SALOMON:  That's right.
MS. MEYERS:  That's why they have to do the monitoring.
DR. SALOMON:  That's why what IRBS now are demanding.
MS. MEYERS:  They don't have the money to do it and if HHS understands this, they would put the extra money in the grant funds to --
DR. SALOMON:  We're getting there Abbey.  What we're saying is that the conventional IRB set up in reality is not set up to do this.  That's all we're saying.  We're not saying that an IRB or an arm of the IRB that we now might name a monitoring group or an institutional data safety monitoring board for trials isn't appropriate.  I think that's where the group is going actually or is trying to get us there.
MS. MEYERS:  But it would be appropriate if the funds were there.
MS. LAWTON:  But surely one alternative that we're talking about is if the IRBs are able to see that there's an independent monitor assigned to a study that would give them that competence in the same way as we're talking about.
DR. SALOMON:  That's right and that monitor would report to the IRB and that's a very appropriate -- the IRB then would be linked integrally with the whole system.
MS. MEYERS:  But when we say independent that again gets back to the thing what happens when the institution owns the company or the stock in the company or a patent on the product?
DR. SIEGEL:  Of course, the IRB also is an arm of the institution so it's no more independent than an institutional monitoring group that isn't part of the IRB.
MS. LAWTON:  Well, Greg Koski is suggesting that IRBs should not be from an institution, but they should be regional.
DR. NOGUCHI:  Dan, I would like to just make one correction.  Actually, for this area, other than the product requirements, these are not new requirements.  There's not an explosion on new requirements.  There is a vast understanding that there are a lot of requirements that a lot of people didn't realize were there.
(Laughter.)
DR. SALOMON:  The correction is accepted.		
DR. NOGUCHI:  As part of the government, we will add requirements when necessary, but what amounts to what we're talking about is not new ones.
MS. MEYERS:  They've been there since 1960.
DR. SAUSVILLE:  But that illustrates the education and outreach function that was alluded to this morning.  I mean the idea that many -- to me, the statistics were certainly encouraging, as you say, that things weren't worse than they were.  The other way of looking at this is 50 percent of the trials had a problem.
DR. CHAMPLIN:  My institution has actually such a body, an opposite protocol research that is linked with the IRB and they have, in fact, taken on the job of monitoring INDs that don't have another sponsor in terms of an outside pharmaceutical company or what have you.  And in our past experience we found, in fact, the most egregious errors did occur in the unmonitored single investigator type of projects where there was no one supervising that activity.  And clearly this type of approach gives a second look to the conduct of all studies and it's certainly been positive and I think that that model is probably the most reasonable one.
There is realistically no way you can get beyond the institution and have some outside entity now monitoring things without really getting into a very complex logistics that's probably not at all realistic.  And I think that as long as there's conflict of interest observation within an institution, those people monitoring and the IRB have no vested interest in the product or the company that's being monitored, I don't really view that there's a problem there.  I really don't see any large institutions looking to push something inappropriately for their own financial gain.
DR. SALOMON:  Okay, so let's take what's Dick saying and explore this a little bit because it still is how much distance do we have to go that stays reasonable, it can be done practically and yet is done properly.  Now Abbey mentioned something that's very interesting, the new head of -- is it OBA?  OHRP, right.  
The new head, he came out to La Jolla and we met with him and then he gave a talk and in his talk he specifically mentioned something Abbey raised and that was he is suggesting that there be professional paid regional IRBs so that in our area where we have Scripps, UCSD and Salk, for example, and a couple other smaller programs, that we would all have one IRB and that could fulfill this sort of -- just as a counterpoint, there is some discussion going on and I don't think that we necessarily need to settle that, but I think that the committee has spoken pretty clearly here that it can't be someone who works directly for the investigator and/or directly linked back to the sponsor and it could be done -- right now most of us feel it could be reasonably be done in the institution.  That's good, you disagree.  That it could be done within the institution if there was a data safety monitoring board study monitoring group that answered to the traditional IRB.
Now if someone doesn't agree with that, tell me.
MS. LAWTON:  So if I can comment on that you said that the monitor cannot be directly linked with the sponsor and I disagree with that because as long as it's not an investigator/sponsor IND, clearly the sponsor --
DR. SALOMON:  I didn't mean to imply that, right.  If the sponsor is a company and they have six investigators and hire a sponsor at the company -- a monitor at the company to go around and see -- that's okay.
MS. LAWTON:  Maybe if I can also just comment, based on -- we had discussions this morning about quality control of operations and clearly the reporting structure and the independence of that quality control group on the operations side, this is exactly the same issue for clinical and I would say that you can set up, just like all of the drug companies, biotech companies have had to do, you should be able to set that up in an institution as long as you have the right processes and accountability, etcetera for that to work, but it's how that's done.  But there is a model there for it to work.
DR. SALOMON:  Okay.
DR. SIEGEL:  Well, yes.  Part of that appears to be the most problem working -- whether it's sponsor investigator or not is -- when you're talking about working is having a reporting system where the monitoring is to someone in the company independent of the investigator, but unfortunately one of the areas we've run into problems where the investigator is, in fact, the CEO or the principal stockholder of the company and he's investigating his own product and then it is probably pretty hard for somebody within that company to have the level of independence needed.
DR. SALOMON:  Jay, that's an interesting question.  If I'm -- the word "sponsor" how is that defined?  If I'm the CEO of the company and the investigator, is that a sponsor investigator?  A lot of times I'm not the CEO, right?  The cute thing is I'm on the scientific board and I tell everyone I don't get any money from the company which is, of course, baloney, but that's how we play it.
DR. SIEGEL:  It's probably fair to say that most of the pertinent FDA regulations were written at a time when some of the sorts of arrangements, product development and research were not fully considered and so that's why you would read in the regulation that you're responsible for monitoring your own activities and taking actions against yourself if you don't do them well.  Doesn't sort of make a lot of sense in that context.  But it was really written with a view to other contexts.
Technically, the sponsor who signs as the sponsor when they file with the FDA and takes on, therefore, the requirements under regulations and guidance and responsibilities of the sponsor, but frankly in some sense that's almost a non-answer.  That is the true answer but we can see the same trial with the same monitoring or what appears to be identical trials monitored where the sponsor is the National Cancer Institute, the Director of the National Cancer Institute, the lab chief in the National Cancer Institute or the principal investigator, but they may well have the same oversight mechanisms and the same thing in business.  You could see out of the same group where the sponsor might be the university, an institute within a university, the head of that institute.  So in some sense, although we talk about it as the sponsor investigator, more to the point is what Pat was getting at was really what the structures and where the true responsibility lies and that's where we're trying to grow our understanding of is figuring out how to address this.
DR. SALOMON:  So trying to grapple with what you were saying and what Jay is saying, in the spirit of the discussion, we don't want a monitor who works for any broad sense of that term, works for the investigator.  And therefore, if the investigator is a stakeholder in the company which is the sponsor, that is also a violation.
MS. LAWTON:  Isn't there two separate issues here?  On the one hand we're talking financial involvement and I think that's one way you could look at how independent do they need to be because for most of us in industry now, there's the guidance on financial disclosure of investigators and we have standard procedures on how we would check that and how we'd make a decision on using investigators.  So that would be one thing.  But then the other one is the example that you gave, Jay, where you have all of the different levels, the investigator, the institution, etcetera, all reporting into the same place, not necessarily  the financial issue of the investigator themselves.
DR. SALOMON:  Richard, do you want to make a comment?
DR. MULLIGAN:  Yes, I thought maybe if we kept it to the industry issue, it actually may be more helpful.  I think it's getting more complicated with 
-- the industry has a history and I think it might be helpful to analyze.  They have a monitoring system.  What are the strengths and weaknesses of that system and what is the perceived level of independence of that monitoring system.  I think the answer is it's complicated and if you looked at it from the academic point of view you'd say this sort of relationship would be unacceptable, but if you looked at it from an industry point of view, this is the standard by which monitoring occurs.  And that being the case, you're really talking about almost simply an organizational distinction between the two.  It's not really who works for who or whatever, but it's an organization, a safety board or monitoring board.  It's almost a title.  I think at the end of the day as far as you're going to get from the point of view of truly conceptually what's independent.  I'd like someone to comment on the industry standard, maybe Jay, how you look at that because I think you've really got to resolve the industry standard before you go to academic.
DR. SALOMON:  But Richard, can I make a comment.  To me, the problem with this analogy to the industry standard, maybe I don't have it quite right, but what I'm listening is, see, in industry the monitors are paid for, work for, work within the industry within the business, right?  Drug Company XYZ has a monitoring group.  The critical thing though is that they are not working for the person doing the study in the clinic, the investigator.
To  me, the problem here that we've been dealing with isn't a problem with the sponsor having the monitors work for them, it's the problem of the investigator having the monitors work for them.
DR. MULLIGAN:  I'm not sure that I would agree with that, but I think that the issues of independence and separateness are comparable issues, however you want to look at it.  That is, the monitors are within the company.  They have all the interest in seeing things move ahead.
I still agree with what you say, but I think that at the end of the day in the academic context, all you really are going to end up being able to do is to have a separate organization and name, a name, a body and I think the issue of who they report to, obviously they should report directly to the principal investigator, but they're going to work for the IRB or they're going to work for the Dean's Office or something.  I don't think that that distinction is going to be all that keen.
DR. SALOMON:  That was fine and the weakness that got brought up that I was trying to address in exception was the situation in which we said no, we don't need a separate institutional group because the sponsor hired the monitor.  But in the case in which the investigator in the academic institution has a relationship with the company, i.e., on their scientific advisory board, the inventor, the starting scientist, whatever, then in that case, the fact that the monitor was hired by the sponsor could be perceived by the public as getting around our recommendations that there be an independent --
DR. MULLIGAN:  I agree.  I think there probably then is a consensus that if you don't have the monitor hired by the investigator, if you have it institutionally, however that would be, that's clear what we want to have, right?
DR. SAUSVILLE:  I actually would like to pursue the thought -- I think there is two different sorts of model, at least two, implicit in this, in that when you look at the industrial model where the company that's conducting even the early phase trials is going to be the company that ultimately hopes to file a BLA.  There, it's in the company's interest to have a very rigorous review and reporting on its investigators because ultimately as we just heard there's going to be an inspection process that they're going to have to run as a gauntlet.
In contrast, that what we call academic investigator or the investigator/sponsor, however we want to call this, it's very unusual for that incident.  I don't think it's ever happened that those individuals then would actually go for the BLA.  The initial start off is generally designed to hand off at some level these initial observations to somebody else.  It's a big company, small company, some other company.  And that's where, I think, there really is a difference because at one level the responsibility at that point is going to be out of their hands.  And so what we're talking about is these very early Phase 1 and Phase 2 endeavors of ensuring that the investigator to monitoring relationship on every level doesn't compromise obviously safety, but also produces a coherent body of data that then is actually, if there's value to it, able to be moved to an actual production orientation.  So I do think there are a couple of different levels, as it were, which investigators related to so-called sponsors in this process.  And it is unique to gene therapy, different than what we call drug role.
DR. SALOMON:  Dick?
DR. CHAMPLIN:  I don't know how uniform this now is around the country, but most institutions have evolved conflict of interest policies that would preclude principal investigators on a protocol of having an equity or large-scale interest in the sponsoring company and that that clearly is a healthy thing in terms of that potential conflict of interest in that often in the Phase 1 phase is that had indicated there is no company and at that point, you might perceive the investigator having potential conflicts, but at least once a company is involved, I think that that policy of precluding equity and interest by the investigator is a prudent one.
DR. SAUSVILLE:  You have evolved.  I mean obviously it's been a reactive process and I think part of the reason we're here, actually, is the events that those changes have evolved, as you say.
DR. SALOMON:  Abbey.
MS. MEYERS:  There was a two-day conference on conflict of interest last summer.  It was co-sponsored by FDA and NIH and Secretary Shalala was very, very interested in what it said.  But basically I think that everybody agreed with that conclusion, that if somebody, an investigator has an equity interest in a product or a patent, that investigator should not be involved in the clinical trials because it would have the appearance of a possible bias in the data.
DR. SIEGEL:  There was such a conference and Greg Koski is leading a departmental group that is following up on that.  
The American Society for Gene Therapy issued, I'm not sure you'd call it a policy, I guess it's a policy, but it's not enforceable in any real sense, but saying you shouldn't do this.  The FDA has regulations.  They're more focused on assuring data quality and so they focused really on Phase III clinical trials and they don't outlaw such agreements, but what they do is indicate that all such agreements have to be reported in detail to the FDA and that we can toss out the data on that basis, so at the time of a license application.  So for those efficacy trials, they probably have had a chilling effect on using investigators with financial conflicts.
I'm not sure though, in the type of discovery phases of research that we're talking about, Dr. Champlin, I'm not sure that there's that much consistency across academic centers.  I think there are, while there are some that have been those sorts of relationships, there are others that, in fact, as best I can tell, encourage their investigators to have  cooperative agreements with industry and at least so rumors go.  And I'm not sure there is yet a consensus on this issue in the academic community.
DR. CHAMPLIN:  I think certainly in the Phase 1, as Ed indicated, the goal is to establish preliminary data that would then justify an outside corporation from licensing a developing technology.  So in the context of generating that preliminary data, obviously, the investigator, the inventor has an incentive to make that product as successful.
But I don't see the institution at that point having a major bias that they're going to support in any way anything other than the highest quality research and so having the oversight at the level of the Dean's Office or the IRB, Office of Protocol Research or what have you on an institutional level, I don't see as any major conflict, and I can see as the most practical way to deal with this issue.
DR. SALOMON:  Michael. 
DR. O'FALLON:  I think we've always had a situation where highly successful and therefore influential investigators, whether they had connections with industries, they had a lot more influence than the institution than normal RO1 kinds of guys and so we can't solve that problem.  The problem is a personal problem.
I think we have to make a suggestion that some administrative, some process through the administration of the institution perhaps through the IRB which is already in existence, clearly would have to be enhanced.  I agree, our IRB is absolutely swamped and all of the people are volunteers, quote unquote.
I think we're starting to micromanage the situation here.
DR. SALOMON:  And again, I think that's now -- we don't have to solve all these issues.
DR. NOGUCHI:  You're right.  You don't want to solve them all, but I bring everybody back to the basic finding that is really driving us here.  Although we've discussed about what we did since the University of Pennsylvania incident, what that clearly indicated is that the regulations that the FDA has is, in most part good, but there are situations that need to be dealt with regarding human subject protection, period.
There are models from both the industry side, from the academic side.  There are newer models that are being tried.  All of them have strengths and all of them have weaknesses, but the fact of the matter is if we agree that many of the innovations in gene therapy come from the academic situation, what is their piratical approach that we can really take toward that and I think that while we certainly all feel differently about whether one has a better or less advantage, I would just encourage people to try to look to the fact that FDA, in fact, is not making any specific requirements.  We have suggested that this may be a useful area for CROs, but as you've noted, CROs are not without their own problems.  We've noted that academics have their own set of problems in terms of who reports to who, and yet there are strengths in the situation as well in terms of vigor and energy and other academic freedoms that are useful in the discussion.
Voicing all the advantage and disadvantages is an absolute requirement, what you've been doing, but then the real challenge is going to be everybody's opinion aside, depending on where they come from, that this might be better or worse.  For the current situation how can we move ahead?
DR. SALOMON:  Okay, so let me stop and try again to summarize what I think the committee is telling you today, with the same idea, step in and tell me you disagree.  So I think what we all seem to be agreeing on is that there has to be a monitor for any study.  There has to be no relationship, there can be no direct relationship between that monitor and the investigator or investigators.  And that should extend back as far as the sponsor, so a sponsor could hire a monitor and  monitor trials by investigators with the exception if an investigator is part of the company that that would be considered a violation of the basic understanding.  That the monitoring in an academic institution should be done by a separate group within the institution, acknowledging the limitations that we've discussed in detail that yes, at an institutional level there is a potential conflict of interest with institutional holding of patents, etcetera, but that the nobility of the institution is great enough vis-a-vis the monitoring obligations, particularly with federal oversight, RAC and FDA that it's acceptable and pragmatic, and that that organization should report to the IRB or be the IRB in some new iteration of what an RIB is.  But I think frankly, to get people's heads around in academia, you're better off talking about it as a separate organization because if you try and say the IRB can do it everyone is going to get hysterical. 
And I think that's pretty much specific.  And I should just say from personal experience when we submitted our grant in March for our retroviral gene therapy program, I set up a DSMB within Scripps that none of whom obviously, they're independent, and we brought in several people from UCSD, so it's not even just institutional.  I set up a super DSMB at the City of Hope, so that they were totally non-institutional and they report to the DSMB that reports to the IRB that reports to the three IRBs that reports to the GCRC which has an Executive Advisory Board and an IRB.  So I mean -- I think that's what's happening in academia.  I think we're getting the message.
MS. LAWTON:  If I can just say a couple of things to that.  First of all, I still want to come back to a DSMB as separate from what we're talking about currently on monitoring, so I don't think we should make that comparison.  It's very different activities that we're talking about here.
I think there is one additional level that you could add on if you wanted to to add some level of kind of comfort around the independence of the monitoring group reporting separately to the institution and under GCPs which is basically what we're talking about here, you also have the need to audit and you could have a totally independent auditing group that that institution also has to ensure because to monitor the independence, if you like, of their monitoring group.  I mean it sounds complicated, but this is basic GCPs that we're talking about.  It's just how you set it up for those institutions.
DR. MULLIGAN:  I was just going to say that I think you did summarize things very well.  I think the CRO that Phil mentioned is something you didn't add, that that could be an alternative approach to it, right?
DR. SALOMON:  I agree.  A CRO could be done.  I guess I'm sort of nervous about saying anything about CROs.  I'd hate it to get all the way turned around, that now every academician has to hire a CRO because I can just see that being terrible.
DR. SAUSVILLE:  You can just add that to the part of the different --
DR. SALOMON:  I agree completely.
DR. SAUSVILLE:  It relates to the size of the place.  I'm sure, M.D. Anderson is large enough, so to speak, that it could empower some panel to do this.  I can imagine smaller places that might actually need to look outside themselves.  The general principal is the end result.  How you get there, there are different solutions to.
DR. SALOMON:  Fair enough, but that CRO could report to your IRB and that could be the institutional link in that case.
DR. SIEGEL:  See there, the reporting issue, I'm glad you've commented on that because we have at various times hypothesized that perhaps sponsor/investigators who hired CROs are getting more independent feedback than those who hire their study nurse to do the monitoring, but in fact, if the CRO is reporting back only to the investigator, and in fact, we've seen a problem related to that sort of structure, some rather serious problem, so -- 
DR. SALOMON:  But on that face, you've violated it.
DR. SIEGEL:  They could hire a CRO who then could report to somebody who has independent authority such as an IRB.
DR. SALOMON:  No.  The point here is that again, the CRO, just to keep it simple, Jay, the CRO should not be hired by the investigator.  Just like the -- in the concept that we've given you, the monitor should not be hired by or work for the investigator.
DR. SIEGEL:  I'm sorry, we're discussing solutions for the sponsor/investigator trial and there's nobody to hire the CRO but for the sponsor.
DR. SALOMON:  No, the IRB can hire the CRO.
DR. SAUSVILLE:  I mean the -- I'm going to return to the point that the institution is the platform on which all this is occurring and we've certainly seen the institution does get tarred by the brush of whatever difficulties emerge.  So it would seem to me that they should be, the institution should be and I used the word before, empowered, to really step in here and -- I mean it's true that the CRO could be hired by the sponsor/investigator, if you want to use that term, but the reporting goes back to the institution which ultimately gives the investigator the license to proceed.
DR. SIEGEL:  Right, so you're not suggesting then, if you're talking about within the institution that -- you're not suggesting a preference as to whether an institution has its own internal employees who are an independent monitoring office or IRB employees or whether they hire a CRO?
DR. SAUSVILLE:  How they do it, one can imagine different solutions.
DR. SALOMON:  That was the point Richard was making to me and I thought it was well taken.  But the point again should be if I'm the investigator, I don't hire the CRO directly.  The CRO should be hired by the IRB or the institutional group, what you want to call it, you know, the monitoring -- institutional monitoring board, the IMB.  Great.
(Laughter.)
DR. CHAMPLIN:  One plea to try to make this as simple as one can do it.  This is an unfunded mandate at the moment, another hurdle that the Phase 1 investigator has to cope with to get an idea off the ground and this is becoming an increasingly onerous task and so to not pile on anything other than trying to empower the institutional IRB or monitoring board I think is probably where we should draw the line today.
DR. SALOMON:  Abbey and then Amy.
MS. MEYERS:  I just want to make the comment because somebody mentioned FDA's regulations for conflict of interest.  I want to say it's the most ridiculous thing I have ever read.  I read it about a month ago and it's about a paragraph long and it says that the investigator has to report any kind of financial stake he has in the product or something, so and then the sponsor puts that information into a file and keeps it in his file until the drug or the product is going through the approval process and then FDA has the right to say we'd like to see that file.  So the investigator says I own $100,000 of stock in your company and they put it into a file, you see.  The patient never finds out.  Nobody knows about it unless after the product is going through the approval process, then you ask about it.  It's a ridiculous rule and it should be up front and it should be in the informed consent document.
DR. SALOMON:  Actually, all our informed consent documents have that very specifically addressed, Abbey in that you -- item 16 of the Scripps informed consent is the investigator does or does not and if the answer is yes to this question, explain the financial interest.
MS. MEYERS:  That's wonderful that your institution says that.  I have never seen an informed consent document with a paragraph about that.
DR. SIEGEL:  Let me comment on that and I don't want to stand here as a defender or an attacker of the rule in its entirety.  I'm sure that each and every one of us could design a different rule that we'd like better.
It's important to understand in viewing that rule that its intent was not, which isn't to say it shouldn't have been, but its intent was not and it's clearly -- its outcome is not to optimize or ensure protection of patients from financial conflicts of interest.  The design of the rule reflected desire to ensure the integrity and quality of the data that support determinations of safety and efficacy for marketing.  That's why and -- which isn't to say that the first isn't as important a goal, but however, there's a resource issue, of course, in what the FDA does in terms of conflict of interest and of course, as I know you understand very well, the oversight of patient protection is a complex interaction that involves, of course, IRBs, FDA, NIH, so I will agree with you 100 percent that that rule doesn't do what needs to be done in terms of consent and patient protection, whether that should be a different FDA rule or whether in fact we need something that has a scope well beyond the FDA is, I think, is an important issue that of course, we're not going to discuss here.  But I do want to say viewed from the perspective of how can you protect patient rights, yes, you can say that's a ridiculous rule, but the rule is there for a purpose and it does appear to have had some significant roles in achieving that purpose in the sense that even though we don't check until after the Phase 3 trial is done, we have some rather consistent response from industry that before -- when they learn of these conflicts of interest, before they start the Phase III trial, the vast majority of them will select another investigator or ask for divestiture because they realize that they're placing themselves at great risk if they use that investigator.
MS. MEYERS:  Don't you think FDA should know about this in advance, if not after the fact, but in advance?
DR. SALOMON:  What I want to do just because of time issues stay on track here.  The second of the two parts here, I think we've really pretty much discussed.  There is a little bit of a twist and sometimes I'm accused of missing the twist and going on, one of the twists you could put here is should we advise the FDA specifically on what they should do in terms of monitoring the institutional monitoring board, the IMB?
(Laughter.)
Now I don't know whether that twist was there, maybe I've just gotten paranoid over the years, but Dick?
DR. CHAMPLIN:  I think for an institution like say the University of Pennsylvania, their role, their job is to teach students, to generate knowledge and academic activity.  Clearly -- and to advance any sort of pharmaceutical or gene therapy product is a very minor consideration for them relative to their reputation for honesty, integrity and their overall value to the community.
And so that I say there's real incentive for an institution to do anything other than do the best possible job of monitoring the quality of their clinical research because that's what their reputation depends upon.  And so I see them as the white knights, perhaps, in dealing with this issue in the future.
DR. CHAMPLIN:  I think that we've come to an agreement on the committee with what -- the premise of what you're saying is that the institution is noble enough to do this right and that's the premise of the institutional monitoring board.  The question, I guess I was just trying to make sure we didn't leave and go on to the next one without making sure you guys didn't want -- is that in a way, that could be a whole lot of stuff could go on and then you could find out you had an incompetent, not an ignoble institutional monitoring board.  And so I guess the question about be probably the FDA does want to have some sort of program in practice that does review the institutional monitoring boards, not every year, but on some sort of a basis.
DR. CHAMPLIN:  Actually, the FDA inspected us this week.  Spent a week at M.D. Anderson, reviewing our IRB.  And we passed, I'm happy to say, but there is a process already in place for just that function.
DR. SALOMON:  And if that's considered adequate, then we can move on.
MS. LAWTON:  Yes, the only comment that I would have on that is that it's my understanding, yes, we've just been through these inspections because of gene therapy, but there is not the resources at FDA to routinely do those types of auditing.  So what we're saying is that we're actually -- we are relying on the institutions to do that, to play that role appropriately.  And that's fine if that's what we leave it at, but I don't think we should assume, especially for Phase 1-2 trials, you also heard it's more common to do audits of Phase 3 trials and so it's very unlikely that these institutions will be reviewed and audited for that role that we're now saying they should play.
DR. SIEGEL:  Well, that's right, but what I heard Dr. Champlin say and I think it's right, it's not exactly that the institutions are noble, but that it's, in fact, in their self-interest to do this right.  And I think my perspective of the experience of the last couple of years with academic institutions is that while that's clearly true, some have not realized that and some -- which is to say they don't have well functional IRBs or well functional clinical monitoring or oversight and may not realize how much that's in their disinterest until they go through experiences such as five or six major academic medical centers have gone through in the past year or two and I won't name names, but we all know who they are anyhow, at which time and I've talked to a number of university deans and presidents and they all seem to think that, in fact, it is in their interest to do these oversight programs much better, that the harm to prestige and the financial harms as well can be huge.  		So I think that a lot of what is needed is also education and discussion and networking and university-sharing experiences and learning from each other and learning from industry and from professional groups and whatever and --
DR. SALOMON:  My point, Jay, in follow up to what Dick was saying is if tomorrow we now institute a guidance that institutional monitoring boards need to be set up at all the institutions around the country, which is kind of what we're advising, something like that or these different alternatives, all I'm trying to say is that if you then think you've got the problem solved, I just question that and there should be some sort of a process then that monitors these institutional monitoring boards.  That's all I'm saying.
DR. SIEGEL:  No more than having commercial sponsors do the monitoring, solves the problem, there has to be some sort of oversight function.
DR. SALOMON:  Right, particularly while it's new.
MS. LAWTON:  Sorry, can I just ask a question because one way you say you're checking now is new INDs and annual reports, etcetera, that it's a requirement to document for you how the monitoring will be done and the organizational structure involved in that, so that's one way that you could actually look very easily to see what is in place from these institutions when an IND is filed.  And you could go back and do that retrospectively as well, if you needed to.
DR. SIEGEL:  Right, indeed.
DR. CHAMPLIN:  I was just saying that, thinking that this isn't unlike other things that the FDA does in terms of setting standards and expectations.  You don't inspect every blood bank every year, but you set standards that blood banks need to comply to and you would inspect some to ensure that, in fact, those things are being carried out.  This would be the same principle.  You set standards on what institutional review should be and then institutions are held to that standard when they're occasionally inspected.
DR. SALOMON:  Amy and then Abbey.
MS. MEYERS:  I have to say that people -- that's the way so many people got HIV and hepatitis.  All right, we can't allow this to happen anymore,  with gene therapy especially because it's going to go right down the tubes if there are more deaths and more abuses of the system.  And we have to do something more carefully because the institutions are not the white knights.  The University of Pennsylvania was not a white knight and OHRP has gone in and closed down university after university for all of their clinical trials because the abuses were so bad.  So the government has got to step in and it has got to be much stronger than it's ever been in the past.
DR. SALOMON:  Amy?
DR. PATTERSON:  My comment sounds awfully mundane after that.  I was going to perhaps offer a segue to Question 2.  I think Dan gave an excellent summary about 15 minutes ago, but I think that Alison Lawton's comment about keeping in mind that there's a clear distinction between a clinical trial monitor and a monitoring board and I think the dialogue is continue to muddy those different roles and responsibilities and I want to put in a plea to the committee when you're answering Question 2 to make sure you're very clear about what you're referring to when you're using the term monitoring because I think it will have a big impact on the utility of your advice to FDA, to distinguish a clinical trial monitor from a DSMB.
DR. SALOMON:  Good.  Abbey, does anybody want to comment specifically on -- you did, I know, I know.
I think then we can move on to Question 2 which Amy has done a good job of sort of setting the stage for.  So the regulations and guidance indicate monitoring should be adequate to ensure data integrity and protection of patients' rights and welfare, but they don't describe either the frequency of monitoring or the extent, the proportion of the patients enrolled, sampling, for example.  In some institutional monitoring programs, a randomly selected sampling of active studies are monitored during the year.  It's conceivable that over several years, some studies might never be monitored during the conduct of the trial and only I guess retrospectively.
In those programs where selection of studies for monitoring occurs annually such that a study could accrue patients up to one year before the first monitoring study. 
I guess what they're asking us is if we've agreed in the first part that we have to have an institutional monitoring board, how -- what kind of a guideline, what do we expect from that institutional monitoring board which of course is the same thing as if our institutional review board hires a CRO, it's still the CRO is becoming our institutional monitoring board.  So if everyone is okay with the concept of an IMB, just so we have the right -- we're all talking about the same thing.
MS. LAWTON:  I guess I'm not because now I'm getting confused as to whether you're looking at the IMB as more of a DSMB type or is the IMB overseeing --
DR. SALOMON:  IMBC is what we've been talking about all along.  It is a monitor.  It's not a data safety monitoring board.
MS. LAWTON:  Okay.
DR. SALOMON:  It's monitoring the trials.  It's -- I mean maybe we should define, if you want, exactly what monitoring means.  Why don't you start, Alison?
MS. LAWTON:  I can go there.  I just think maybe we shouldn't use the phrase an IMB because I think that's what's confusing it.  I think what we all are in agreement, that we're talking about monitoring and that's separate from an DSMB.  Monitoring is going in and checking source verification of the data that's put in the case report forms.  We routinely do that 100 percent, source verification, you know, making sure adverse events reported, etcetera, that type of monitoring.
DR. SALOMON:  What do you call the group in your company that does that?
MS. LAWTON:  That is part of the clinical operations group, that's from the company that would go in.  We would have clinical monitors for every single study assigned every site that's involved in that study.
DR. CHAMPLIN:  This was usually done for a licensing trial, but not necessarily every trial that's being done with a new product.
MS. LAWTON:  I disagree with that very strongly.  We monitor every single study regardless of what phase of development.
DR. SAUSVILLE:  I just -- maybe this is in the spirit of what was being stated, I mean we've used this term IMB or monitoring board.  I actually think that's being more complicated than it has to be.  Studies, as was stated, are monitored routinely in a Phase 1 and Phase 2 context, at least by what, for example, studies of NCI sponsors.
And one could imagine that an institution, if the reporting structure, and this gets back to what we said before of the people who are doing the monitoring is separate from the investigator, it doesn't need to be dressed up as a board or anything.  I think there are well established ways of source verifying adverse events, reporting, etcetera.  
If you feel that we want to layer on this notion that there would be an auditing function or a monitoring function, that's going in, I think, a potentially difficult direction.  I think that as long as the general principles are stated, how -- either companies or institutions solve this, I think, to use the analogy that Dick made before, FDA should set the standards and obviously when these things are called into question in the normal following up of things then if things aren't being done, then it will make itself apparent.  And that's where it would stop.
DR. SALOMON:  Yes, I have no problem with any of that stuff.  I guess I was -- remember, I initially came up with the IMB just to have a word and we congratulated me initially for having quickly -- it just shows you why you can never come quickly with a word because it doesn't work that way.
I like the idea now of the OCM, the Office of Clinical Monitoring.
(Laughter.)
Just kidding.  Anyway, the bottom line here is that it's not -- I just wanted to stop us from talking about that being an invisible add-on tomorrow to the IRB, that's all I was trying to get across, but it could be just two or three individuals given some space somewhere who are in charge of monitoring all these programs.
So if we do that, how often should these people be monitoring?  Are we talking about weekly, every single patient enrolled, some sort of a guideline for if it's a 100-patient trial or a 
10-patient trial that it would be different?  
MS. MEYERS:  In my mind, I'm thinking this is going to be the people who go in there and check that the adverse events have been reported to the IRB and to NIH, the RAC, and to FDA, just to make sure that the paperwork is right and that nothing is being kept secret.  So I don't think they'd be needed more than twice a year to go in and check and make sure that all those adverse events have been --
DR. SAUSVILLE:  To me, it's an 
accrual-based issue.  I mean if you have a very active trial, they're going to have to be working all the time.  If you have relatively infrequent accrual they don't have to be doing things all the time.  So I mean that's going to be -- generally, there's a percentage type basis, 10 percent, 20 percent of the charts get looked at, that's on the high end.  Two percent is on the low end.  And people probably sort themselves out somewhere in between.
DR. SALOMON:  There's certainly -- there's one more question and there's more discussion that we could have.  We're at a point here and particularly because of some issues that need to be done today, cannot be done tomorrow, and particularly with Dr. Sausville who needs to leave some time around 6, so I'm going to end this discussion here.  I think that I've summarized it more than once.  I don't think you need to hear me do this again.  I'm sure you're all relieved.  If we haven't solved everything, I'm willing to at the discretion of my colleagues here bring this up again tomorrow and I'd like to end here for the moment and go on to the end here which is we need to present the CBER intramural research programs and then have -- we need to do that quickly enough to have some time to close the session and have some discussion with Dr. Sausville who chaired that.
DR. SIEGEL:  You needn't feel badly about not solving everything, let me just say that.  That wasn't the goal, as I indicated.  This is an intensive, but on-going and not overnight process of relooking.  The whole structures of oversight of clinical research and patient protection and I think the perspectives of this committee are a very important part of that and we appreciate the discussion and I'm sure we'll be talking with you more about it in the future.
DR. SALOMON:  This part is still public.  It represents the on-going FDA process of site visiting and review of internal research programs and we have --
DR. SIEGEL:  Yes.  I am on the agenda, that is, in lieu of Katy Stein, the Director of the Division of Monoclonal Antibodies who is unable to be here today.
And in the interest of time and also because it's really not terribly essential to the process, I'll keep my remarks very brief.  As we're entering into the overview of Dr. Marjorie Shapiro, the role of the division director and my role is just to provide a little bit of framework.  The Division of Monoclonal Antibodies is one of the three 
product-oriented divisions in my office, along with Phil's Division of Cell and Gene Therapy and Division of Therapeutic Proteins and then we have a Clinical Trials Division that Karen directs and an Applications Review and Policy Division.  And it has as its name would imply oversight of monoclonal antibodies, both for diagnostic and therapeutic use, as well as some closely related products built in monoclonal antibody backgrounds.  The science in this field and the technology in this field have been expanding and burgeoning rapidly as many of you know with tremendous advances and the technologies for engineerings these antibodies, designing them, selecting them and producing them and applying them to various diseases and as such they've represented as many as half of the new products that we've reviewed and this division plays a very important role, both in review of those products and in setting the policies and procedures for that class of products.
Dr. Shapiro works within the Laboratory of Molecular and Developmental Immunology in that division and is one of our investigator-reviewers and I'll leave it at that.
DR. SALOMON:  It's my understanding now that we'll get a brief presentation.
DR. SHAPIRO:  Good afternoon.  I'm going to try to shorten my remarks, so if things don't go as smoothly as they might have, it's in the interest of time.
My interest has been in my lab has been in studying the contribution of individual germ line light changings to the diversity of the antibody repertoire and we've shown that genes that are fully functional in terms of their ability to recombine don't always get used in a pre-immune repertoire.  And from this observation, we then went on to start another project because we're beginning to see antibodies derived from new and exciting technologies in the field such as the humanized mass and fate displaced library* (T6S1-beg.)  And they have a vast potential to produce both safer and perhaps more efficacious antibodies.  But there may be potential implications that we don't understand about these products, such as they don't undergo the normal selection process that an antibody that's produced in a human or a mouse might go through.
I'm going to briefly skip through this.  This is my slide of B cell development which I hope you all are aware of.
Next slide, please.  Basically, B cell development hinges on the rearrangement of heavy chain and light chain genes, expression of various forms of the B cell receptor on the cell surface, lead to a variety of processes including allelic exclusion in the pre-B cell, receptor editing, apoptosis and so on as you go on through development.
Next slide, please.  This is a picture taken from a paper from Hans Zackov's group which mapped the entire three megabase murine light chain region.  There are 141 individual genes which are represented by the mice.  Mice here of the same color are within the same light chain family.  We've been particularly interested in the three gene family which is shown there in the oval, the Vk10C family.  Two members of this family are seen in a variety of immune responses to both T development and independent antigens in several different kinds of inbred mice, but the Vk10C gene has never been seen in a mature antibody and we've been investigating why.
So the next two slides show the results of our studies.  Next slide, please.  
The first paper we published on this we showed that the Vk10C is structurally functional and is capable of recombination, that messenger RNA is present in the spleen at 100 to 1,000 fold lower levels than that of the utilized genes Vk10A and B, and an in vitro model using a reporter gene assay, we show that the Vk10C promoter is less efficient in 
pre-B cells than the Vk10A promoter.
Now we've done some site-directed mutagenesis of the three nucleotides that are different between the A and C promoters and we show that if you change one nucleotide that would be near the transcription initiation site, in the Vk10C gene and change that to the Vk10A nucleotide, we can restore the efficiency.
We then went on and tried some EMSA, electromobility shift assays and those results were inconclusive, so we're sort of at a dead end for now with this aspects.
Next slide, please.  A more recent paper we published we showed that the Vk10C gene is completely accessible to the recombination machinery.  It's equally accessible or even more accessible than the Vk10B gene based on a readout of germ line transcripts, that the gene recombines at the same frequency as other family members and the most interesting observation was that as a B cell matures from a pre-B cell through the mature B cell stage in the periphery, you selectively lose productive Vk10C rearrangements.  
So the next slide shows some possible reasons for Vk10C expression.  The first is that the promoter is inefficient in pre-B cells and because of this you may not get enough light chain protein expressed to pair with heavy chain to put a mature immunoglobulin on the cell surface.
Another possibility is the light chain protein doesn't pair well with heavy chains and again, you wouldn't get immunoglobin expressed on the surface.  In both cases, this cell would remain functionally a pre-B cell because it wouldn't have any mature immunoglobulin on the surface, so light chain would continue to recombine until it made a good light chain of some other gene from some other family and that would be a reason for losing a Vk10C rearrangement.
The third possibility is that Vk10C can pair with heavy chain, but when it gets put on the surface it undergoes a negative selection event.  In such a case, again, the immature B cell which is still in the bone marrow, a negative selection event would either lead to apoptosis or again receptor editing where a light chain recombination would continue and again you would lose the light chain gene.
So next slide.  At the time of the site visit last October, I had these slides about future directions and I want to spend a little bit of time discussing what we've done with these proposed experiments at that time.
The first experiment, again, is to get back to this inefficiency of the Vk10C promoter.  So we thought rather than trying to stick with the in vitro assay and the gel shifts, we would try to do a real time PCR in freshly isolated pre-B cells.  And an outline of this experiment is shown on the next slide.  All the nucleotides we had used which were specific for the Vk10A, B and C genes in all our other experiments were not appropriate or the constraints of a real time PCR assay, so we had to go back and develop new primers and probes to do this.  We've developed a five prime primer and two different three prime primers which are shown up there on the white that would give us amplicons of 163 and 177 base pairs.  And we have three probes each specific for the Vk10A, B and C genes, all contained from within the CDR1 region.  And the Vk10C probe differs from A by two nucleotides.  It differs from B by 4 and A and B differ from each other by two nucleotides.  The other thing we had to do was generate appropriate plasmid to use as controls to work out the conditions.  So we now have done all this and we're starting to do the experiments to work out the right PCR cycle conditions and temperatures and everything.  So once we work that out we'll go and we'll sort for pre-B cells and do the experiment and hopefully we'll get an informative answer.
Next slide, please.  The second future direction was to look, to examine this question of can Vk10C pair with heavy chains.  And the way we propose to do this is to put a Vk10CJk1 rearrangement in phage display vector and then clone in PCR of polyclonal heavy chain rearrangements from LPS stimulated spleen cells.  We haven't started this yet.  It depends on our ability to make a phage display library and I'll get to that with the second project.
Next slide, please.  The third question that we wanted to explore is maybe Vk10C is negatively selected.  The experiment design on the bottom, right now it looks like we may not end up doing it based on results we've gotten from the first experiment.  The second experiment and I'll discuss the outline of it in a minute, but in the next slide, I'm going to show you results of, we've examined the usage of the Vk10C in autoimmune mice.  The reason for doing this is we thought because autoimmune mice are deficient in getting rid of heavy light chain pairs that would be negatively selected in a normal background, perhaps if this was the case we would see increased expression of this gene in mice of autoimmune backgrounds.  But as you can see the top 6 mouse strains have the autoimmune background and the last row there is the Vk10 frequency of Vk10C rearrangements in the spleen and you really don't see a significant difference from C57BL/6 and BALB/c mice which have normal backgrounds.  So from this experiment it's looking like Vk10C is not negatively selected.
Next slide, please.  The second experiment to address is to look at another kind of recombination called RS recombination.  The top line depicts a germ line kappa locus.  The greenish box would be the constant region and downstream of that, the black and white box is something called an RS element.  It's 10s of KBs downstream.  And also in the middle that little black triangle is an isolated heptomer which is part of the recombination signal sequence of antibodies.
Now what can happen is two kinds of recombination here, either a germine V gene, the green gene on the left can recombine through its recombination signal sequence directly with the RS element downstream of Ck in which case you would delete the constant region and any VJ join which would have occurred.  And this is a way to inactivate a kappa allele which may have had a nonproductive rearrangement or may be negatively selected for some reason and might prepare the cell to go on and arrangement the lambda locus which usually occurs after kappa rearrangement, but not all the time.
A second kind of recombination would recombine the isolated heptomer in the entron to the RS element downstream of the constant region.  Again, this would inactivate this locus, but it would leave a VJ join intact.  Both of these kind of recombinations are seen in 74 percent of lambda positive cells and 12 percent of kappa positive cells.  Twenty-five percent of these rearrangements of the Type B which leaves the VJ join intact.  And in earlier studies, people have shown that about 47 percent of these VJ joins are in frame which would indicate -- they took that to indicate that these good rearrangements perhaps were eliminated because of the negative selection process.
So we've designed primers and are working out the conditions now in the lab that would amplify specifically Vk10 rearrangements to this RS element and again, we have the primers that we've used in the past that are specific for the three genes.  And we would like to ask the question, do we see a higher frequency, a significantly higher frequency of Vk10C in frame or productive rearrangements in this kind of recombination than the others.  If it's higher, then this could be taken as evidence of negative selection.  If it's not higher, then it would be consistent with our studies in autoimmune mice in that Vk10C is not negatively selected.
Next slide, please.  This slide, long term future directions for continuing this study.  There are 20 other genes that are functional in terms of that they don't have obvious mutations that would preclude them from recombining or undergoing splicing or being expressed in any other way, but there have also been no antibodies seen that have used these genes.  So we're interested in seeing if the phenomenon of Vk10C is specific to that or if we can find a common reason for why these 20 genes that have been maintained in the repertoire over the years are still available.
And we'd also very much like to get to the level of studying the accessibility of this locus at the level of chromatin.  Hopefully that will come in the near future.
So we'll skip the next couple of slides in the interest of time.  This is my -- we're going directly to the next project.  No, go back one slide, please.
I mentioned before that we have these two new technologies that have vast potential to make antibodies, especially phage display, to make antibodies against antigens that are not good immunogens in vivo.  So you can target a lot more things and we see a lot of potential there.  But phage display libraries do not undergo any kind of normal selection process.  It's totally in vitro.  So you could get heavy light chain pairs that would never come out in a human.  So we asked the question is the phage display repertoire normal.  And so what we did was we immunized the mouse with tetanus toxoid.  We made hybridomas from half the spleen and we made messenger RNA from the other half of the spleen to generate a phage display library.  Now as I said, last October, we would like to be as good as regulated industry at making a phage display library, but we're not there yet.  We initially had some trouble with the initial vectors that we chose.  We have since gotten a new vector.  We were having problems with both having high background levels and low efficiencies.  When I get back into the lab next week, hopefully we will find out that we've solved those problems and we can generate the library because that is a main goal of ours.
Actually, one of these slides I skipped.  Maybe we could just go back one slide, please.  What I wanted to say is antibodies are inherently immunogenic.  We do have a lot of experience now with licensed products.  Our murine products, the whole antibodies, you can see that 55 to greater than 80 percent of patients make an immune response to it.  When you remove the constant region, that drops down to a pretty good level, similar to what you see for chimeric and humanized antibodies.  While we don't have a lot of experience with phage display antibodies, there are some hints that they may be more immunogenic than one would have predicted.
Can we go forward two slides, please?  So while we haven't made the phage display library yet, we have analyzed our hybridomas and most of our hybridomas bind to the fragment C portion of tetanus toxin and so this summary slide is a little bit more complicated than when I presented it in the fall because we've done some more studies and we're still trying to sort them out.  But what we did was we generated 11 fragment C specifics antibodies and two other antibodies, the 18.2.12.6 and the 18.1.7 were generated at CBER in the 1980s and we included those in our analysis.  So we grouped them by the VHVL pairs that they express and then we did ELISAs, cross-blocking ELISAs to show that they recognize four unique epitodes on fragment C.  We then set up an ELISA to show if these monoclonals could block fragment C from binding gangliocyte which is how tetanus binds to neurons and gets inside cells.  And the 18.2.12.6 had been previously shown to enhance binding.  In our hands, it did the same.  All the other antibodies blocked binding except for the one on the bottom, 72B9.  
Then last summer we started a collaboration with Elaine Neale and Karen Bateman of the National Institute for Child Health.  They have a spinal cord neuron assay and so we put our antibodies in our system to see if they could block the activity of tetanus toxin on spinal cord neurons.  And everything worked the same as in our GT1B binding ELISA except for the second antibody, 35F7 and the last one, 72B9, where in the spinal cord neuron assay the results were the opposite with what we saw in our GT1B binding ELISA.  So we wanted to explore why this happened and we looked at the buffer components and it turns out that the pH has an influence on our GT1B binding ELISA.  It didn't change the results of the other antibodies, but for those two that didn't get consistent results, when we started out with our antibodies and a lower pH buffer, then the results of our ELISA were more consistent with the spinal cord neuron assay.  And we still don't understand this completely, but that's the data that we have so far.
So next slide, for our future directions, obviously, the phage display library is on the top of our list.  
Next slide.  We wanted to do in vivo protection assays with our antibodies and affinity measurement.  Before we start these those we realize that even though we grew our antibodies in reduced serum medium, bovine serum, bovine IGG has an antitetanus component to it.  So we went back and we rederived all our hybridomas in serum-free medium and we're purifying them now.  And so we'll get to doing these studies.
But we've spent a lot of time in the last year trying to map the epitopes which we thought would be straight forward and that's also been a problem for us.  
Conventional wisdom has it that if your antibodies recognize an antigen on Western Blot, then they recognize linear epitopes.  So we contracted with a company -- next slide -- which would map our antibodies on a series of overlapping peptides and these are the profiles.  The top two rows and then the panel on the bottom right show the profiles after the isotope controls have been subtracted out.  The panel on the bottom left is a gamma 1 control.  I didn't have room for the gamma 2 control here.  And you can see that we really don't have any good binding.  The peaks you see in the middle two panels, all the way on the right, were also the major peaks in all the antibodies before the back-on was subtracted and if you look -- actually, next slide, please.
Okay, if you look here that peptide falls in this little cavity here in between these two loops, so you wouldn't think that that peptide, that area would be available for binding to antibodies.  And indeed, when we had the peptide made, we couldn't show direct binding of the antibodies to that peptide.  So this -- these data weren't informative to us other than to tell us perhaps that the conventional wisdom didn't hold true here and perhaps we have confirmational epitopes.
So that we have some other colleagues in the Office of Vaccines that also study tetanus and they have made a series of amino acid substitution mutants and a deleton mutant in this part of fragment C and in the next slide, this is data that we just generated in the last week.  I see all my symbols didn't translate.  
I didn't name the mutants, didn't specify the mutants because they haven't been published yet, but what we did was we compared their binding relative to wild type fragment C.  And in all cases, we didn't have any antibody where it bound fragment C and then the mutant, it didn't bind at all. It either increased the binding or decreased the binding or what here is shown as the squares.  It just stayed the same.  So like I said, we just got this data in the last week and we needed to sit down and look at it.  Maybe it will be informative, but at a first glance, we may not be able to figure out what the epitopes of these antibodies are.  There is one more thing we could try, but we've been trying for a year, so I don't know.
Next slide, please.  In our future directions, we have about half a dozen or so antibodies that don't bind fragment C that we want to do similar assays with.  We've also rederived these in ceoprime medium and are purifying them.  So we'll get those experiments done.
The last slide is our long-term future directions which at this point we haven't begun to even think about yet.  And I'd like to acknowledge on the next slide, I have two people in my lab, Sean Fitzsimmons and Kathy Clark who have done all the work, our collaborators at the Institute for Child Health who did the spinal cord neuron assays, Heather Louch and Willie Vanno of OVRR who provided us with mutants.
Thank you.
(Applause.)
DR. SALOMON:  The tradition has not been to clap for presentations, but I think that that tradition isn't appropriate when someone is presenting their data.
DR. SHAPIRO:  Thank you.
DR. SALOMON:  Data takes precedence, nicely done.
Phil?
DR. NOGUCHI:  Like Jay, I'll be very brief.    
I direct the Division of Cellular and Gene Therapies where you have, over the last, quite a number of occasions been reviewing problems that we have in terms of product development related to cell therapies, gene therapies and the like.
I'd like to present here now, Dr. Tom Eggerman who is a member of the Laboratory of Molecular Tumor Biology.
DR. EGGERMAN:  Thank you, Phil.  Good afternoon.  For my site visit review I will be presenting a talk about the identification and characterization of binding proteins for high density lipoproteins, usually referred to for short HDL.  Before proceeding to the data I want to give some background information regarding the importance of HDL, describe what HDL is, what its role is in lipid metabolism and then review some aspects about known HDL binding proteins.
Heart disease is the number one cause of death in this country and atherosclerosis is a major contributor to heart disease.  Several risk factors have been identified for atherosclerosis, among them are low levels of high density lipoproteins and this data is from the Framingham Heart Study and in this figure, the relative risk of developing coronary artery disease in men is shown to decrease as the levels of HDL increase.  
In contrast, levels of low density lipoprotein cholesterol, so-called bad cholesterol, LDL cholesterol, there's an increased relative risk of coronary artery disease.
With the initial national cholesterol education efforts, the emphasis was on ways of reducing total and LDL cholesterol.  More recently, the emphasis has expanded, recognizing the importance of HDL cholesterol, in fact, most major drug companies are now developing drugs, specifically targeting for elevation of HDL cholesterol.
Clinical studies such as the VA HDL Intervention Trial are suggesting that raising HDL cholesterol levels is also beneficial and for this reason because the therapeutic interventions are so limited, it's critical and HDL metabolism be more fully understood and so that the best therapeutic interventions can be developed.
The characteristics of high density lipoproteins are shown in this figure.  These lipoproteins exist as articles with a diameter of 5 to 12 nanometers with a lipid core and hydrophilic surface of principally protein and phospholipid.  Their size is 1.7 to 3.6 times 10^5 daltons and the lipid consists of 5 to 10 percent of triglycerides, 15 to 25 percent cholesterol and 20 to 30 percent of phospholipids.  The remaining 35 to 60 percent is protein.
Next.  There are several proteins called apolipoproteins which can be found associated with HDL and provide structural integrity to these particles.  The two major ones are Apolipoprotein A-I or ApoA-I for short with a mass of 28,000 daltons and Apolipoprotein A-II which exists as a dimer and has a mass of 17,000 daltons.  Since these proteins exist on the surface of lipid particles, it is thought that they are also ligands involved in mediating many of the HDL effects.
Next slide, please.  HDL is known to have at least two functions, the well known involvement in lipid metabolism, especially cholesterol transport and the lesser known function of acting as a carrier for lipid soluble materials.  An example of this is lipopolysaccharaides or LPS.  I will be presenting data demonstrating that these two functions can potentially overlap.
A synopsis of the metabolism of HDL is shown in this figure.  The primary function of HDL is to mediate cholesterol movement from the periphery to the liver, the so-called reverse cholesterol transport.  The initial source of HDL called nascent HDL is from the liver and intestine where newly synthesized ApoA-I is released with only a small amount of lipid. 
In the presence of peripheral cells, nascent HDL is able to accumulate free cholesterol from a plasma membrane.  A candidate binding protein is supposed to be responsible for this activity is called ABCA-I or ATP binding cassette I protein which mediates cholesterol translocation from intracellular stores to the plasma membrane.  A defect in this protein causes Tangiers Disease which is associated with very low levels of HDL cholesterol.  It is thought that there could be other HDL binding sites on the surface that could have other functions such as signalling.  As I had mentioned previously, another function for HDL is carrying the lipid soluble molecules such as LPS and this is demonstrated here in this slide.  SRB-I or scavenger receptor B-I is found on the liver as well as steroidogenic tissues including the adrenal gland, ovaries and testes and takes up cholesterol estrorich particles including HDL.  As a scavenger receptor, it can recognize multiple ligands and also it will take up multiple types of lipids.  Other HDL receptors have been proposed and may exist on the liver or potentially other cells that are involved in the uptake of HDL particles.
The identified binding proteins that I showed on the previous slide, the ABCA-I and the SRB-I have KDs that correspond to a relatively affinity site.  From the literature, including our own lab, statute analysis of HDL binding indicates that there is a higher affinity site or sites so as yet to be identified, HDL binding proteins must exist to correspond to these higher affinity binding sites.
There also have been several observations suggesting additional HDL binding proteins.  These include what's called the retroendocytosis of HDL particles where HDL particles are taken up by the cell or liver and then resecreted.  The uptake of HDL remnant particles after metabolism, signal transduction as I mentioned earlier and also LPS uptake.
Next slide.  I will now begin presenting some of our data on novel HDL binding proteins that we have identified by modifying a Western blot like assay called the Ligand blot.  We've been able to identify three proteins of 100, 95 and 55 kilodaltons that all bind HDL.  We have identified and characterized these proteins, particularly to determine if they correspond to already known HDL binding proteins.  Because of limited time I'll not be able to review all this work as I did in the October site visit.  I will briefly describe the results for the 100 kilodalton protein and for the 95 kilodalton protein and then present some of the data that we have on the 55 kilodalton protein.
The 100 kilodalton protein after purification and microprotein sequencing turned out to be glucose regulatory protein 94 which had been recently identified as an HDL binding protein in 1999 by another group.  The 95 kilodalton protein was characterized and the results will be coming out shortly in the Journal of Biochemistry and we're still in the process of purifying and attempting to identify this protein.
Regarding the 55 kilodalton protein, we purified the 55 kilodalton protein from plasma membranes.  On this slide the results are shown after the final purification step of two dimensional electrophoresis.  On the left panel with Kalassy stain, we observed a triplet, appropriate size, and in the right panel using this Ligand blot and the ligand that we used with aprolipoprotein A-II, one of the proteins on HDL, we also observed the triplet.  After HPLC purification, the cutout band was then microprotein sequenced.  Six piques were found to be 100 percent homologous with heat shock protein 60 of Hsp60.  In addition, mass spectroscopy fragmentation analysis was consistent with Hsp60.
Next slide, please.  Hsp60 or heat shock protein 60 belongs to a family of heat shock proteins which are inducible or constitutively expressed by prokaryotic and eukaryotic cells.  It's also called the mitochondria matrix molecular chaperon, is involved in the appropriate folding of proteins, about 10 to 20 percent of this protein is found in the cell surface.
Multiple diseases are associated with heat shock protein 60 antibodies such as lupus erythematosus and other autoimmune disease and even Type I diabetes.  These diseases are usually associated with accelerated atherosclerosis.  Antibodies to heat shock protein 60 are positively correlated with the development of atherosclerosis in patients and in animal studies, exposure to microbial heat shock protein 60 is associated with a development of atherosclerosis.
Next slide, please.  In this slide we are demonstrating the specificity of the aprolipoprotein, that's one of the proteins on HDL binding to heat shock protein 60 and what we have is the binding in this assay to the 55 kilodalton protein as evaluated by Ligand blot and would show that in the presence of increasing amounts of heat shock protein antibody, called LK-I, that we're able to see decreasing amounts of ApoA-II binding.
Next slide, please.  It's known that heat shock treatment of cells result in increased heat shock protein 60 expression on the cell surface.  In this slide, we have heat shock treated fibroblasts for 30 minutes at 42 degrees and then evaluated, labeled HDL binding to the cells and comparing specific HDL binding which is shown as the third result in each one of these pictures.
Heat shock treated cells had almost three times as much HDL binding as the control cells suggesting that increased heat shock protein 60 expression is associated with the increased HDL binding.
Next slide, please.  A significant question is whether or not the binding of HDL or 
APoA-II is truly physiologic since heat shock protein 60 is known to bind multiple proteins as part of its mitochondrial matrix chaperon function.  When apolipoprotein A-I, again, this is another protein that's found on high density lipoproteins is added to multiple cell types, including fibroblasts, it's been demonstrated to increase cholesterol release from these cells and this is called cholesterol efflux.
We have evaluated ApoA-I induced cholesterol efflux from human fibroblasts in the presence and absence of these three antibodies to heat shock protein 60 that are each targeted to different epitopes of heat shock protein 60.  All three of them indicate that they're able to decrease cholesterol efflux, suggesting that heat shock protein 60 indeed does have a physiological HDL metabolism.
Next slide, please.  Further work to evaluate the significance of heat shock protein 60 in atherosclerosis includes a collaboration with Steve Epstein who was formerly at the NIH and now is at the Washington Hospital Center and he published a paper earlier this year entitled "Antibodies to Human Heat Shock Protein 60 Associated with the Presence and Severity of Coronary Artery Disease, Evidence for an autoimmune Component of Atherogenesis."  We intended to use this antisera from these same patients to determine if heat shock protein 60 antisera blocks Apolipoprotein binding to heat shock protein 60 and if it also will block cholesterol efflux and then correlate these effects with the presence and severity of coronary artery disease.
As I indicated in the beginning of my talk, HDL has an additional function as a carrier of lipid soluble materials.  This is figure, LPS which originates from the cell wall of gram negative bacteria demonstrates alternative pathways which are catalyzed by an enzyme called LPS binding protein or LBP.  This enzyme has significant homology to various enzymes in lipid metabolism highlighting the similarities between LPS metabolism and lipid metabolism.  The alternative pathways shown on this slide include being bound by CD-14 resulting cellular signal transduction, citokine release through well characterized pathways and the possibility of developing endotoxic shock.
Alternative LPS can bind to HDL which appears to be protective and results in neutralization and perhaps also in being directed to a degradation pathway.
Next slide, please.  The structure of Lipopolysaccharide is shown on this slide.  The major structural aspects are the fatty acid portion at the bottom, the polysaccharide portion which is in the upper part and the phosphate groups. 
These components are also found in different combinations in the normal constituents of lipid particles such as phospholipids, triglycerides and cholesterolestese.  LPS can also be referred to as endotoxin and is associated with a development of septic shock as I said earlier.  Multiple FDA investigative new drug applications have been evaluated, attempting to treat this frequently fatal condition.
The absence of endotoxin in a product is an important FDA criteria for allowing its use in patients.  And recently, the presence of bacteria such as chlamydia have been identified in atherosclerotic plaques and the release of LPS from these plaques is now being suggested as a potential pro-atherogenic factor.
In looking at the literature, a 
20-year-old study published in JCI, evaluated the uptake of labeled Lipopolysaccharide that had been associated with HDL.  They observed in control animals that the highest tissue density of uptake was in the adrenal followed by the ovary and the liver.  These three tissues correspond to the same location where the HDL binding protein SR-BI is localized.  When animals were pre-treated with dexamethasone, a treatment known to significantly downregulate SR-BI, the updated in the adrenal was significantly decreased and almost eliminated.
When animals were pre-treated with ACTH, or adrenocorticotrophic hormone, a treatment known to up regulate SR-BI, the update in the adrenal was increased.  This study suggested to us that SR-BI could be involved in LPS tissue updated.
Next slide, please.  In this first experiment we evaluated the potential binding of Lipopolysaccharide to SR-BI by evaluated labeled LPS in SR-BI overexpressing and control cells.  And the overexpressing cells that are seen in the closed circles demonstrated a much higher level of binding than the control cells suggesting that SR-BI actually could act as a receptor.
Next.  To further evaluate this increased binding observation, we evaluated the uptake of LPS into control and the same SR-BI overexpressing cells.  The overexpressing cells in the open squares demonstrated a significantly increased amount of uptake, suggesting that LPS not only binds to SR-BI, but this increased binding is associated with an increased uptake into the cell.
Next slide, please.  In this slide, LPS update is evaluated using a fluorescently labeled LPS.  This compares SR-BI overexpressing and control cells and one can see that there's a significantly increased amount of fluorescently labeled LPS uptake in the 
SR-BI overexpressing cells.  The location for uptake is consistent with the Golgi apparatus where LPS is normally transported intracellularly.
If LPS binds to SR-BI, one would expect that LPS could compete with HDL and proteins on HDL such as ApolipoproteinA-I.  In this figure, the competition of ^125I labeled HDL is determined in competition with LPS and unlabeled HDL.  LPS is a very effective competitor.  It's in the red open circles and has an IC50 of about 5 micrograms per milliliter.  Unlabeled HDL which is in the open squares is a much less good competitor.
Next slide.  Using labeled Apoliprotein 
A-I, we also looked at competition between LPS and ApoA-I and what we saw in this case was that both LPS and ApoA-I had very similar competition curves and the IC-50 for LPS was around 2 micrograms per milliliter.
We have just begun to evaluate the interaction with the other major HDL binding protein ABCA-l and LPS.  In this slide, the two inhibitors of ABCA-l were used to evaluate LPS uptake.  These two inhibitors are sulfobromophthalein and DIDS.  Both inhibitors significantly decreased uptake, suggesting that ABCA-1 may also be involved in LPS metabolism.
Next slide, please.  Potential model for our results is seen in this slide.  SR-BI, as I mentioned earlier is known to be the primary binding site for cholesterol ester uptake.  How cholesterol ester is then shuttled to intracellular stores, however, is not really known.  Our data suggests that SR-BI may have an additional role as an LPS receptor.  SR-BI's known role as a scavenger receptor that takes up many different lipids from multiple ligands is consistent with this possibility.
In the lower part, I have ABCA-1, a protein that's primarily involved in free cholesterol mobilization from intracellular compartments.  Dr. Bryan Brewer's lab has recently demonstrated that ABCA-1 functions as an intracellular shuttle delivering free cholesterol to the cell surface of intracellular stores.  
Our initial work observing effect on 
ABCA-1 on LPS uptake suggests that ABCA-1 may have a role in LPS metabolism and one potential function could be that ABCA-1 acts an intracellular shuttle to carry LPS intracellularly from the cell surface.
As I mentioned earlier, LPS has a significant signal transduction activity that results in cytokine release and the development of endotoxic shock.  We next evaluated how LPS affects the expression of HDL binding proteins.  In this slide, raw cells which are a mouse macrophage cell line were treated with LPS.  Response of both ABCA-1 on the right and SR-BI on the left demonstrate a dramatic reduction of greater than 80 percent in MR and A levels within 24 hours.  Of particular note is ABCA-1 where the potency of the LPS effect is such that complete ambition occurs at only two nanograms per milliliter.
Next slide, please.  In evaluating response of SR-BI protein to LPS treatment, we used a Western blot analysis for these raw cells.  A similar dose response curve was seen as in the previous slide and this case we're looking at the protein instead of the MRNA and again an 80 percent reduction is seen at a concentration of about 2 nanograms per milliliter.
Next slide, please.  To further evaluate SR-B1 and the effect of LPS, the activity of SR-B1, that is, cholesterol ester uptake was evaluated in response to LPS.  Nearly a complete inhibition is seen of cholesterol ester uptake and these plus the two previous slides demonstrate dramatic reductions of 
SR-B1 and MRNA protein inactivity in response to LPS.
Next slide, please.  Conclusion, we have identified three HDL binding proteins.  The 100 kilodalton protein was determined by micro protein sequencing to be the already known HDL binding protein, glucose regulatory protein 94.  The 95 kilodalton protein has been characterized.  We're still in the process of doing further purification and with the hope of identifying whether this is an established or new protein.
With the 55 kilodalton protein, we've determined this to be heat shock protein 60 by microprotein sequencing.  The potential physiologic involvement of this protein has been demonstrated by heat shock protein 60 antibodies binding, preventing binding of ApoA-II to the protein and inhibiting 
ApoA-I stimulated cholesterol efflux.
In addition, heat shock treatment which increases heat shock protein 60 expression on cell surface increases HDL binding.  The sum of these observations suggests that heat shock protein 60 is a novel HDL binding protein which is important in HDL metabolism that may provide a mechanism to explain the known association between immunity developed against heat shock protein 60 and the development of atherosclerosis.
We also presented data demonstrating an interaction between ABCA-I, one of the two primary well-known HDL binding proteins and LPS.  We demonstrated that inhibitors of ABCA-I decreased LPS uptake and that LPS down regulates the expression of ABCA-I.  Further studies are needed to define this interaction between ABCA-I and LPS to determine what role ABCA-I has.
Next slide, please.  And we've also presented data indicating that there's a significant interaction between the HDL binding protein called 
SR-BI and LPS.  Overexpression of SR-BI results in increased binding and uptake of LPS.  LPS competes with both HDL and ApoA-I with binding and LPS downregulates SR-BI MRNA protein and activity.
Next slide, please.  I want to acknowledge all those who have helped us in these studies and CBER, from my lab, Alexander Bocharov and Shaobin Zhong, also from the Division of Therapeutic Proteins, Ray Donnelly and Harold Dickensheets.  From the NIH, Amy Patterson and her group, including Irina Baranova, Zhigang Chen and Tatiana Vishykanova and from Dr. Brewer's group himself and Alan Remaley.  
Thank you very much.
(Applause.)
DR. SALOMON:  Thank you very much, Tom.  Are there any questions from the committee to either of the presenters?
Well, then what I'd like to do is take a break while we go from a public to a closed session where the presentation of the results of the site review by Dr. Sausville will take place.
Thank you all very much.  Thank you to both the presenters who did a beautiful job and for the rest of you, we'll see you back here in a couple of minutes.
(Whereupon, at 5:20 p.m., the open meeting was adjourned.)