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DEPARTMENT OF HEALTH AND HUMAN SERVICES

FOOD AND DRUG ADMINISTRATION

CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE

OPEN SESSION

Meeting #32

Friday, May 10, 2002

8:10 a.m.

Hilton Hotel

Gaithersburg, Maryland

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PARTICIPANTS

Daniel R. Salomon, M.D., Acting Chair

Gail Dapolito, Executive Secretary

MEMBERS

Katherine A. High, M.D.

Richard C. Mulligan, Ph.D.

Mahendra S. Rao, M.D., Ph.D.

Alice J. Wolfson, J.D. (Consumer

Representative)

TEMPORARY VOTING MEMBERS

Martin Dym, M.D.

Jon W. Gordon, M.D., Ph.D.

Thomas F. Murray, Ph.D.

Terence Flotte, M.D.

Eric T. Juengst, Ph.D.

R. Jude Samulski, Ph.D.

GUESTS/GUEST SPEAKERS

Valder Arruda, M.D., Ph.D.

Linda Couto, Ph.D.

Mark Kay, M.D.

Stephen M. Rose, Ph.D.

FDA PARTICIPANTS

Jay P. Siegel, M.D.

Philip D. Noguchi, M.D.

Daniel Takefman, Ph.D.

Anne Pilaro, Ph.D.

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C O N T E N T S

PAGE

Welcome/Administrative Remarks

Dr. Daniel Salomon, Acting Chair 4

Introduction of Committee 5

Conflict of Interest Statement

Gail Dapolito, Executive Secretary 8

FDA Introduction

Potential for Inadvertent Germline Transmission

of Gene Transfer Vectors: FDA Approach for

Patient Follow Up

Daniel Takefman, Ph.D. 13

Guest Presentations

AAV Vector Biology, Jude Samulski, Ph.D. 23

Questions and Answers 46

Germline Transmission by Gene Transfer

Vectors: Assessing the Risk

Jon Gordon, M.D., Ph.D. 61

Questions and Answers 84

A Phase I Trial of AAV-Mediated Liver-Directed

Gene Therapy for Hemophilia B

Mark Kay, M.D., Ph.D. 98

Safety Studies to Support Intrahepatic

Delivery of AAV, Linda Couto, Ph.D. 116

Assessing the Risk of Germline Transmission of

AAV in a Rabbit Model

Valder Arruda, M.D. 130

Questions and Answers 144

Open Public Hearing

Mr. Steven Humes 177

National Hemophilia Foundation

Dr James Johnson, Patient 184

Dr. Kenneth Chahine, Avigen 190

Committee Discussion of Questions 197

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

2 Opening Remarks

3 DR. SALOMON: Good morning, everybody.

4 Welcome to day two of the Biological Response

5 Modifiers Advisory Committee Meeting No. 32. I

6 guess we should call it 32B. We have got a title.

7 I have been complaining and I finally got what I

8 wanted a title for these meetings. This one, this

9 is good - Vector Pellucida 2002. Not my title,

10 but, you know, you can\'t criticize it, I got what I

11 wanted. Thank you.

12 So, welcome everybody. Today we have

13 changed the scenery around the table quite a bit.

14 So, to get reoriented, I think we should go back

15 around again this time and introduce ourselves, so

16 that both the audience, as well as each other, has

17 a little sense of who we are and what we are doing.

18 Just if you can introduce yourself, we

19 will just go around the table and give a few

20 sentences on where you are from and what you do,

21 what kind of expertise you bring.

22 In front of you is a button on the thing.

23 It says speaker. If you push it, it turns red.

24 Talk, and then when you are done, turn it off.

25 Otherwise, there is a funny feedback. So if I am

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1 ever looking at you, gesturing, it means to turn it

2 off. It is one of my big duties.

3 Introduction of Committee

4 DR. DYM: Martin Dym, Georgetown

5 University. I worked on the testis and

6 specifically on spermatogonia, which are the male

7 germline stem cells.

8 DR. FLOTTE: I am Terry Flotte from the

9 University of Florida. We have been working on AAV

10 biology, AAV vectors and AAV gene therapy.

11 DR. JUENGST: I am Eric Juengst. I am in

12 the Department of Bioethics at Case Western Reserve

13 University and recently rotated off the RAC is

14 where my last connection with these issues.

15 DR. MURRAY: I am Tom Murray. I am from

16 the Hastings Center, Bioethics, the world\'s first

17 bioethics research institute, and my work has been

18 in a variety of issues, but quite a lot in

19 genetics, parents, and children.

20 MS. WOLFSON: I am Alice Wolfson. I am

21 the Consumer Advocate. In this incarnation, I am a

22 policyholder\'s lawyer representing policyholders

23 against their insurance companies when they don\'t

24 pay what they are supposed to pay.

25 In my previous incarnation, however, I am,

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1 and was, a women\'s health activist and a founder of

2 the National Women\'s Health Network.

3 DR. RAO: My name is Mahendra Rao. I am

4 in the Intramural Program at the National Institute

5 on Aging. I am also a member of the BRMAC. I work

6 on stem cells, most parts of the body, I guess.

7 DR. SALOMON: Jude, we missed you the

8 first time around.

9 DR. SAMULSKI: I am Jude Samulski from the

10 University of North Carolina, and work in the area

11 of AAV vectors.

12 DR. SALOMON: I am Dan Salomon. I have

13 the pleasure of chairing the committee today. I am

14 from the Scripps Research Institute in La Jolla,

15 California. I work on cell transplantation,

16 particularly islet cell transplantation and tissue

17 engineering and therapeutic gene delivery.

18 MS. DAPOLITO: Gail Dapolito, Center for

19 Biologics. I am the Executive Secretary of the

20 committee.

21 DR. GORDON: Jon Gordon from Mount Sinai

22 School of Medicine. I make a lot of transgenic

23 mouse models of disease and gene therapy for

24 disease. I was on the RAC. I am actually the

25 first person to say the word \"transgenic,\" if that

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1 means anything.

2 DR. SALOMON: It means a lot.

3 DR. PILARO: I am Anne Pilaro. I am an

4 expert toxicologist in the Division of Clinical

5 Trials at CBER. I regulate a lot of the gene

6 therapy protocols, in fact, I think I have 167

7 active right now.

8 DR. TAKEFMAN: Dan Takefman. I am a gene

9 therapy product reviewer with the Division of

10 Cellular and Gene Therapies, CBER.

11 DR. NOGUCHI: Phil Noguchi. I am director

12 of the Division of Cell and Gene Therapy at CBER.

13 DR. SALOMON: Welcome. We will be joined

14 a little bit later by my colleague to the right,

15 Richard Mulligan from Harvard Medical School.

16 This is interesting for two reasons. One

17 is that this is kind of a revisit to a very

18 important area that the BRMAC dealt with, not the

19 last time, but I guess at least two times ago,

20 where we initially talked about how to address

21 potential regulatory issues specifically with this

22 Avigen trial, and then more generally with how to

23 deal with the potential of infection germline in

24 this case with semen.

25 We got into the whole discussion about

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1 semen versus infecting the motile sperm and what

2 was the evidence, if any, that you could really

3 infect the germline, the spermatogonia, or infect

4 the sperm themselves, and very much tried to deal

5 with some of the practical issues of what you would

6 demand of any company of a sponsor in doing this

7 kind of research, and to do it in such a way that

8 you wouldn\'t put an unnecessary hold that could

9 therefore interrupt a very important trial unless

10 there was awfully good evidence.

11 It is also very interesting in that it is

12 an interesting theme for the two days. In some way

13 I am sorry that some of you weren\'t here yesterday

14 where there we were really talking about another

15 kind of germline transfer issue, the injection of

16 ooplasm into oocytes for infertile women, but it is

17 an interesting thing now to go on to the idea of

18 potentially doing something like this through

19 therapeutic gene delivery.

20 We have to read the conflict of interest.

21 Gail.

22 Conflict of Interest Statement

23 MS. DAPOLITO: I would just like to read

24 for the public record, the conflict of interest

25 statement for today\'s meeting.

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1 Pursuant to the authority granted under

2 the Committee charter, the Director of FDA Center

3 for Biologics Evaluation and Research has appointed

4 Drs. Terence Flotte, Jon Gordon, Eric Juengst,

5 Thomas Murray, Daniel Salomon, and Jude Samulski as

6 temporary voting members for the discussions

7 regarding issues related to germline transmission

8 of gene therapy vectors.

9 Dr. Salomon serves as the Acting Chair for

10 today\'s session.

11 To determine if any conflicts of interest

12 existed, the Agency reviewed the submitted agenda

13 and all financial interests reported by the meeting

14 participants. As a result of this review, the

15 following disclosures are being made:

16 In accordance with 18 U.S.C. 208, Drs.

17 Terence Flotte, Jonathan Gordon, Daniel Salomon,

18 and Jude Samulski were granted waivers permitting

19 them to participate fully in the committee

20 discussions. Dr. Richard Mulligan was granted a

21 limited waiver for this discussion which permits

22 him to participate in the committee discussion

23 without a vote. Dr. Katherine High recused herself

24 from this committee meeting.

25 In regards to FDA\'s invited guests, the

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1 Agency has determined that services of these guests

2 are essential. The following interests are being

3 made public to allow meeting participants to

4 objectively evaluate any presentation and/or

5 comments made by the guests related to the

6 discussions of issues of germline transmission of

7 gene therapy vectors.

8 Dr. Valder Arruda is employed by the

9 University of Pennsylvania. He is involved in the

10 studies of adeno-associated virus vectors. Dr.

11 Stephen Rose is employed by the Office of

12 Biotechnology Activities, NIH.

13 In the event that the discussions involve

14 other products or firms not already on the agenda,

15 for which FDA\'s participants have a financial

16 interest, the participants are aware of the need to

17 exclude themselves from such involvement, and their

18 exclusion will be noted for the public record.

19 With respect to all other meeting

20 participants, we ask in the interest of fairness

21 that you state your name, affiliation, and address

22 any current or previous financial involvement with

23 any firm whose product you wish to comment upon.

24 Copies of these waivers addressed in this

25 announcement are available by written request under

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1 the Freedom of Information Act.

2 As a final note, as a courtesy to the

3 committee discussants and your neighbors in the

4 audience, we ask that cell phones and pagers be put

5 in silent mode.

6 Thanks.

7 DR. SALOMON: Thank you, Gail.

8 What we will do here is begin with an FDA

9 introduction from Dan Takefman, will kind of walk

10 us through some of the key issues that the FDA

11 wants to answer. Remember that part of the dynamic

12 here is that we are an FDA Advisory Committee.

13 There will be times when we all, certainly

14 myself as a scientist, get really interested in

15 some scientific question, but at some point you

16 will have to forgive me if we steer away from that

17 since, if we are not really answering the FDA\'s

18 question, then, we are not doing what we are

19 supposed to be doing here.

20 In the meantime, though, obviously, to the

21 extent that any of these scientific issues are

22 relevant to answering the questions, you know, you

23 obviously are here and your expertise is greatly

24 welcomed.

25 I guess the other thing, as long as I am

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1 giving an introduction on that score, I will just

2 say that we are going to try and come to consensus

3 on some of these questions, but in some instances,

4 there is no consensus, and there is no effort here

5 on my part to force this group into consensus, so

6 well-articulated, minority opinions or even just

7 where we go, I am sorry, but there is no way we can

8 agree on it, that\'s the kind of information that we

9 need to pin down.

10 So it is important for us to make sure

11 that we have represented everything as evenly as

12 possible for the community. The last thing I will

13 say to the audience is that I feel you also are

14 participants in this meeting. This is an open

15 public meeting. That mike in the center is open. I

16 welcome all of you, if you have something to say,

17 to come up during the meeting during discussion and

18 make your points, and we will definitely be here to

19 listen to them and try and make sure that we do an

20 adequate discussion of this.

21 Dan, you are on.

22 FDA Introduction

23 Potential for Inadvertent Germline Transmission of

24 Gene Transfer Vectors: FDA Approach for Patient

25 Follow Up

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1 Daniel Takefman, Ph.D.

2 DR. TAKEFMAN: Thank you. I would like to

3 welcome the committee and speakers, and thank

4 everyone for participating in today\'s meeting.

5 \[Slide.

6 The topic for today is the discussion of

7 potential for inadvertent germline transmission of

8 gene transfer vectors, and as Dan said, this has

9 been a topic of previous discussions and public

10 meetings. Today, we will be discussing the finding

11 of vector sequences in patient semen and to discuss

12 FDA\'s current approach for patient follow up.

13 \[Slide.

14 Concerns regarding inadvertent germline

15 transmission, or IGLT, are twofold.

16 Societal/ethical concerns are based on previous

17 public discussions and publications in which

18 deliberate germline alteration has been deemed

19 unacceptable.

20 Additionally, there are potential adverse

21 biological effects, such as genetic disorders,

22 birth defects, and lethality to developing fetus,

23 just to list a few which are also of concern.

24 \[Slide.

25 What is the likelihood that IGLT would be

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1 deleterious? Well, retroviruses have been used as

2 tools to investigate the role of certain genes

3 which are important in development. I refer to, in

4 this slide, data involving retroviral insertion to

5 the germline of mice and as a specific example, a

6 retrovirus was used to infect a murine blastocyst.

7 In this case, this infection resulted in a mouse

8 strain with a lethal embryonic mutation, which was

9 induced by proviral insertion into the alpha-1

10 collagen gene. This mutation was recessive, so

11 that the phenotypic effect required homozygosity.

12 \[Slide.

13 So data exist suggesting that in the case

14 of retroviruses, deliberate insertion into the

15 germline may be deleterious, but what about data

16 from preclinical animal studies regarding the

17 ability of gene transfer vectors to transmit to the

18 germline?

19 Well, the FDA does require biodistribution

20 studies with gene transfer vectors in relevant

21 animal models. These biodistribution studies,

22 performed in support of clinical trials, have shown

23 evidence of vector dissemination to gonadal tissue.

24 However, in most studies, vector sequences

25 have not been detected in semen samples, and the

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1 point I need to make in regards to these

2 preclinical studies is that they are not always

3 predictive of human experience.

4 A case in point is today\'s topic in which

5 vector sequences were found in semen from clinical

6 trial subjects, however, initial preclinical

7 studies, such as those done in dogs, demonstrated

8 no detectable vector in semen.

9 Again, certainly in today\'s case, animal

10 studies are not always predictive.

11 \[Slide.

12 I would like to give an update on the kind

13 of current active gene transfer INDs we currently

14 have in file just to give you an idea of what is

15 being used in the clinic.

16 You can see here in regards to retroviral

17 vectors, they are predominantly being used in ex

18 vivo types of gene transfer studies, while

19 adenoviral vectors and plasmids are often being

20 used in direct in vivo type of administrations.

21 You will notice here with AAV vectors,

22 compared to other systems, FDA has seen relatively

23 few gene transfer INDs. Of the few we have, they

24 are primarily in vivo, localized injection type of

25 administrations.

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1 \[Slide.

2 I would like to go over some of the

3 factors that FDA considers important for assessing

4 risks of inadvertent germline transmission of gene

5 transfer vectors.

6 Certainly, integration potential of the

7 vectors is important to consider. Of the current

8 vectors being used in the clinic, FDA is

9 considering both retroviral and AAV vectors as

10 vectors with potential to integrate. Certainly

11 with retroviruses, as well as lentiviral vectors,

12 they are known to have efficient abilities to

13 integrate and host genomes.

14 In terms of AAV vectors, this system is

15 not as clearly worked out as in other systems, such

16 as retroviruses. FDA is currently considering AAV

17 vectors as having a low, but potential to integrate

18 in vivo, and I specifically refer here to a couple

19 of papers from Nakai\'s lab in which he showed low

20 levels of integration in mouse livers.

21 \[Slide.

22 The risk of inadvertent germline

23 transmission is also likely highly dependent upon

24 route of administration. An ex vivo gene transfer

25 would likely represent a minimal risk in terms of

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1 IGLT, while at the other end of the spectrum, a

2 systemic injection would represent a relatively

3 higher risk in terms of transfer to the germline

4 via hematogenous spread.

5 \[Slide.

6 As Dr. Salomon mentioned, IGLT has been a

7 topic of discussion, and I would like to go over

8 some of the previous public discussions in order to

9 put today\'s meeting in a little perspective.

10 Beginning with the March 1999 RAC meeting,

11 here, there was a focused discussion on preclinical

12 data which demonstrated gonadal distribution. It

13 was the consensus from this meeting that despite

14 this preclinical data, the probability of

15 inadvertent germline transmission occurring during

16 a gene transfer clinical trial was low.

17 However, further discussion became

18 necessary at the November 2000 BRMAC meeting. At

19 this meeting, we heard data from a trial which

20 involved I.V. administration of a gammaretroviral

21 vector which contained the factor VIII gene for

22 treatment of hemophilia A.

23 I should point out this was the first

24 trial under IND which involved I.V. administration

25 of a gammaretroviral vector. Data was presented in

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1 which 1 out 12 subjects treated had vector

2 sequences transiently present in semen.

3 In the one patient, vector sequences were

4 detected at only one time point by DNA-PCR.

5 \[Slide.

6 Then, at a recent meeting of the RAC, a

7 trial was presented, which will also be presented

8 today, which involved an AAV vector, which contains

9 the factor IX gene for the treatment of hemophilia

10 B. This is the first trial under IND which

11 involved administration of an AAV vector into the

12 hepatic artery.

13 Data was presented in which vector

14 sequences were found in semen of the first two

15 patients treated. The first patient had positive

16 PCR signal at multiple time points for up to 10

17 weeks post administration, and the implication here

18 is that all patients treated in this trial may test

19 positive for vector sequences in semen samples.

20 \[Slide.

21 So to summarize some of the consensus from

22 these public discussions, there was a consensus

23 from the RAC meeting on preclinical data that the

24 probability of inadvertent germline transmission is

25 low and that the use of a fertile subject

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1 population was acceptable.

2 From the BRMAC meeting, the committee

3 agreed with FDA\'s approach to institute a clinical

4 hold when vector sequences are detected in semen

5 samples from study subjects.

6 There was a consensus from both the RAC

7 and the BRMAC that there is a need to determine if

8 vector is associated with sperm cells. Using

9 fractionation methods, such as density separation,

10 potential contaminating transduced white blood

11 cells can be removed from sperm cell fractions.

12 You are going to hear more later on from Avigen on

13 their fractionation assays.

14 \[Slide.

15 I would like to turn now to FDA\'s approach

16 for patient follow up, which has been modified in

17 response to these public discussions and from data

18 regarding this current trial.

19 Prior to initiation of the trial, of

20 course, if during preclinical animal studies,

21 vector is found in gonadal tissue, this finding and

22 the potential for germline alterations should be

23 included in informed consent documents.

24 \[Slide.

25 As for FDA\'s current approach for patient

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1 follow up, if semen from clinical trial subjects

2 tests positive for vector sequences, the clinical

3 trial will be allowed to continue, however, FDA

4 will request timely follow-up testing of

5 fractionated semen. As has been in the case in the

6 past, barrier contraception is requested until

7 three consecutive samples test negative.

8 \[Slide.

9 Now, if the motile sperm fraction tests

10 positive for vector sequences, FDA will institute a

11 clinical hold and subject enrollment will be

12 stopped until it is determined that the signal from

13 the motile sperm fraction is transient, and

14 specifically, we are asking for serial fractionated

15 samples to test negative three times over three

16 consecutive monthly intervals.

17 \[Slide.

18 I would like to turn now to some of the

19 concerns that FDA has. Specifically, the finding

20 of vector sequences in semen may become more

21 common. Certainly with subject from trials

22 involving systemic or intrahepatic administration

23 of AAV, such as in this trial, every patient

24 treated might have vector sequences found in semen

25 samples.

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1 Additionally, we have new vector classes

2 on the horizon, such as lentiviral vectors, which

3 we know have a high potential to integrate, and

4 there is also new production technologies which

5 allow for higher titer viruses to be produced and

6 new clinical applications of gene delivery systems

7 designed to increase transduction efficiency, all

8 of which may make the detection of vector sequences

9 in subject semen more prevalent in future clinical

10 trials.

11 \[Slide.

12 Of particular concern, the fact that

13 patient follow up is difficult with certain

14 populations. Obviously, there are technical

15 limitations in the ability to monitor women and

16 certain men who are unable to repeatedly supply

17 adequate samples. There is technical limitations

18 to monitor these subject populations for evidence

19 of germline alterations.

20 The specific concern will be re-presented

21 in the form of a question to the committee for

22 discussion in the afternoon session.

23 \[Slide.

24 To summarize, FDA\'s primary concern of

25 inadvertent germline transmission of gene transfer

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1 vectors is with systemic administration of

2 integrating vectors.

3 A clinical hold is instituted only if

4 vector sequences are detected in motile sperm

5 fractions, and the inability to monitor certain

6 patient populations is a concern and warrants

7 further discussion.

8 I will end here and just remind everyone

9 that there is a number of background talks and

10 still data on the clinical trial and preclinical

11 studies to be presented, so I would request that we

12 limit the majority of discussion of patient follow

13 up until the afternoon session, but I will be happy

14 to answer a few questions at this time for

15 clarification.

16 DR. SALOMON: Thank you, Dan.

17 Are there any questions from the committee

18 to the FDA regarding the overall umbrella charge

19 that we have for today? Okay.

20 The next are two presentations. It is a

21 pleasure to start with Jude Samulski from the

22 University of North Carolina to talk to us about

23 the biology of AAV vectors.

24 Guest Presentations

25 AAV Vector Biology

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1 Jude Samulski, Ph.D.

2 DR. SAMULSKI: It is a pleasure to be

3 here. I want to thank Daniel for asking me to come

4 up. He requested that I give some type of overview

5 of AAV biology and try to focus a little bit on our

6 understanding of the potential for integration and

7 mechanisms.

8 I think what I am going to do is offer you

9 an opinion of a consensus of what we think is

10 happening in the field, point you in the direction

11 of probably papers that are relevant, that start to

12 show trends that are happening, but more than

13 likely I am going to end up with the conclusion

14 that Daniel has already described, is that AAV is

15 somewhere on that curve as a vector that can

16 integrate, the efficiency is not well established,

17 but the potential is there.

18 I will start off by introducing you to the

19 life cycle of this virus. In the laboratory, an

20 AAV particle can have a lytic component or a latent

21 component, so we refer to it as a biphasic life

22 cycle.

23 It has been established that it is

24 dependent on a helper virus in order to go through

25 a productive lytic cycle, and in this setting, the

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1 virus goes in, reproduces, and progeny comes back

2 out.

3 What was established in the laboratory in

4 the early seventies was that if you took AAV

5 particles and put them in cells in the absence of

6 the helper, you could see this persistence, what

7 was referred to as \"latency,\" and in this setting,

8 it was determined that the virus was establishing

9 an integration event in the chromosome, and in this

10 integration event, it appeared to be targeting,

11 meaning it was going to a specific locus in the

12 human genome.

13 This was all done in vitro and tissue

14 culture cells, and to complete the biological life

15 cycle, if you take these cells and now superinfect

16 them with adenovirus, AAV has the ability to come

17 back out of the chromosome and reenter its lytic

18 component.

19 So in the laboratory, it was established

20 the mechanism in which we could argue how AAV,

21 which was found in nature in clinical isolates of

22 adenovirus, how these two would co-persist, but we

23 could also explain a question of what is the

24 consequences of AAV infecting the cell in the

25 absence of its helper. Is that genetic suicide?

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1 That answer was no, the virus has a mechanism of

2 persistence.

3 I should argue that there is absolutely

4 zero data of AAV integration in humans. This is

5 all established in vitro, and it is inferred that

6 this mechanism can take place.

7 I should also mention that the early

8 studies of AAV showing up in clinical isolates, it

9 has only been isolated in adenovirus, although

10 herpes can supply the same helper function. There

11 has never been a clinical isolate of herpes that

12 has had a contamination of AAV.

13 So what you should be asking yourself is

14 that we can mimic a paradigm in tissue culture and

15 substitute other viruses, but what appears to be

16 out there in nature is this co-relationship. This

17 was established in vitro, and it is presumed that

18 this can also happen in vivo.

19 The genome is fairly simple. It is about

20 5,000 base pairs, and what is of importance today

21 is paying a little bit of attention to what is

22 referred to as the Rep genes and the inverted

23 terminal repeats of the virus, which are the

24 origins of replication, the packaging signal, and

25 what appear to be the break points that join

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1 recombination events with the chromosome.

2 Of the Rep genes that are made, it has

3 been shown that it is the large Rep proteins, Rep

4 78 and 68, that appear to be responsible for the

5 integration events. I just want to point out that

6 in AAV, these are identical proteins. They only

7 differ by a splice variate, and in the absence of

8 adenovirus, this is the dominant protein that you

9 see in the presence of adenovirus. This comes on

10 first and then it switches over to Rep 68.

11 They all have enzymatically identical

12 activities. They bind to the AAV terminal repeat

13 and what is called a Rep binding element. They

14 have a site-specific, strand-specific endonuclease

15 activity where they can nick this molecule, and

16 they have helicase activity which allows it to

17 unravel to DNA.

18 So we see a relationship with the Rep

19 proteins were the key element on the virus, which

20 is the origin of replication, showing that it has a

21 binding site, a nicking site, and enzymatic

22 activities to allow this virus to replicate.

23 So the first evidence of AAV integrating

24 site specifically was generated in Ken Burns\' lab

25 in 1996, and in this study, what they did was

27

1 pulled out some junctions, sequenced the junctions,

2 and went back and used those sequences as probes.

3 This is just a representative example from

4 our lab that shows that if you look at your

5 chromosome 19 locus in a control cell, it is about

6 a 2.6 kilobase fragment, but after you integrate

7 and establish independent clones, you can find

8 variance that show evidence that the chromosome

9 sequence now has a rearrangement suggestive of an

10 insertion, and some of these are multiple fragments

11 showing that there is amplification and

12 rearrangement.

13 If you take a blot like this and strip off

14 the chromosome 19 probe and then come back with the

15 viral probe, you can see there is co-segregation of

16 these viral sequences with these chromosome 19

17 rearranged, so this was the data that said there

18 was a preferred site of integration, a

19 rearrangement of chromosome 19 and a

20 co-localization of these sequences with chromosome

21 19 sequences.

22 Ken Burns and others looked in detail to

23 bring to try to understand why was this virus going

24 to this specific locus, and from that study came

25 the following information.

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1 There is an identical Rep binding site and

2 a nicking site located on human chromosome 19, so

3 what we had was a mechanism that is virtually of

4 viral origin sitting on chromosome 19, that gave a

5 putative reason for why this site is preferred as

6 an integration locus over any other sequence in the

7 human genome.

8 What I should point out is that further

9 studies have shown that not only is the Rep binding

10 required, the spacing between this binding site to

11 the nicking site and the nicking site itself, so if

12 you take these sequences and count them up, there

13 are over 15 base pairs.

14 It is argued that a sequence over 15

15 nucleotides is only represented one time in the

16 human genome. This is probably why this virus is

17 only targeting this locus. This element is present

18 in about 200,000 copies in the human genome, which

19 would argue that the Rep protein is sitting on lots

20 of spots on the human chromosome, but it is only

21 when it is this context that it can initiate the

22 event to promote the integration step.

23 So we have a model and a mechanism that is

24 being supported both in vitro and in vivo.

25 A group in Italy went on to show that the

29

1 site has an open chromatin confirmation and that it

2 is not a closed site, so it is not a site that is

3 unaccessible. All of these things are beginning to

4 support the type of DNA structure that AAV needs to

5 see in order to go into the chromosome.

6 A number of labs, including our own, have

7 gone after looking at these integration events, and

8 most of you are pretty well aware, that if you look

9 at retroviral integration event, it is a fair

10 precise cut and paste mechanism in which it cuts

11 the chromosome, integrates its genome, and there is

12 like a 3 to 5 nucleotide duplication on either

13 side.

14 When you looked at these AAV proviral

15 structures, what we saw was there were a lot of

16 tandem repeats, amplification events, and all of

17 these things were supporting a type of integration

18 that was completely different than the

19 well-characterized retrovirus integration.

20 This has been consistent both in cell

21 lines, as well as episomal integration events, as

22 well as in vitro systems, so there is a mechanism

23 for integration that is not consistent with a cut

24 and paste. It is referred to as a non-homologous

25 amplification mechanism.

30

1 Our lab and others went on to look at the

2 break points between the viral terminal repeat,

3 which I showed you has this origin activity, and

4 this hairpin structure, and the junctions between

5 that and chromosome 19.

6 What you can see was there was very little

7 fidelity and conserving the integrity of the

8 terminal repeat. You would get break points that

9 were scattered throughout these hairpins, and these

10 are just positioned here on the sequence to give

11 you an impression that there is no fixed break

12 point between the viral sequence and the chromosome

13 19. They cluster around this hairpin element, but

14 other than that, you can virtually find break

15 points throughout these sequences.

16 If you look at that from a biological

17 point of view, it again suggests that AAV may have

18 a problem in retaining its integrity as a virus if

19 it\'s indiscriminately breaking these hairpins and

20 going into the chromosome, but this virus has a

21 phenomenal ability of carrying out a step code gene

22 correction.

23 There is technically two copies of every

24 sequence in the hairpin, and since there is two

25 hairpins, there is the total of four copies on the

31

1 virus, so between all of these copies, the virus

2 will gene convert back and forth and regenerate

3 these sequences with fair efficiency, so you always

4 get a wild-type virus coming back out even though

5 what is integrated in the chromosome may be

6 somewhat fragmented.

7 Because the virus also integrates in what

8 appears to be head-to-tail concatemers, it is

9 preserving the integrity of these hairpins

10 internally, and again allowing it to use it as a

11 template to amplify and come back out of the

12 chromosome.

13 So to get to the mechanism, Matt Weitzman

14 in Roland Owens\' lab did an experiment in the early

15 nineties that said that they could show that the

16 Rep protein of AAV could form a complex between the

17 terminal repeat of the virus and this

18 pre-integration site.

19 Again, this made logical sense because

20 there was the same Rep binding element on both of

21 these sequences. This is just an illustration from

22 Sam Young\'s data showing the Rep protein bound to

23 the terminal repeats of an AAV vector. It has an

24 extremely high affinity for the sequence and a Rep

25 complex binding to the same element on chromosome

32

1 19. It was data like this and other that began to

2 propose a model that the virus express its Rep

3 protein, it binds to this element on chromosome 19.

4 In vitro, Rob Cotton showed that this is

5 sufficient to start a synchronized single-stranded

6 DNA replication. So now you have this region of

7 chromosome 19 serving as an origin. Since the Rep

8 protein is terminally attached to this chromosomal

9 sequence, and you can reinitiate, we feel that

10 there is a number of initiation events that are

11 taking place on this region of chromosome 19.

12 It should be understood that there is an

13 enzyme called Fen-1 which is a host enzyme, that

14 actually repairs this type of repeated initiation

15 event, however, if you have a hairpin or a protein

16 attached to this, it doesn\'t have the ability to

17 correct these sequences.

18 So what happens is you see recombination

19 events taking place to resolve these molecules. It

20 has been suggested that the AAV genome, which has

21 Rep, allows for Rep-Rep tethering mechanism, as

22 Weitzman showed, and at this point it is all host

23 enzymes that are involved in inserting this

24 sequence into the host genome, and this type of

25 tandem repeat, head-to-tail type of format.

33

1 This is data that was provided to me by

2 Regina Hildabraun. It is not published. It is

3 coming out in a journal Virology. She has

4 developed a real-time PCR assay to look at the

5 efficiency of AAV viruses to go to chromosome 19.

6 It is a PCR assay that look at the terminal repeat

7 and a locus on chromosome 19.

8 What I think is important to see here is

9 that she can score integration events taking place

10 over the first 72 hours or so, but the most

11 important thing is that the wild-type virus, which

12 she is seeing an integration event for about 1,000

13 particles, so it is suggest about 0.1 percent of

14 all the AAV virus is capable of carrying out

15 integration.

16 This is completely different than like the

17 retroviruses where it is 100 percent integration.

18 As Daniel said, there is a propensity for

19 the virus to integrate. The efficiency is what

20 needs to be look at in this setting.

21 This is a paper that was published by

22 Ernst Winocour. I think this is of importance

23 because what I am going to suggest to you is this

24 is another parvovirus called minute virus in mice.

25 It\'s an autonomous parvovirus. Nowhere is its life

34

1 cycle does it establish latency. It has no

2 mechanism. There has never been any data

3 supporting it.

4 But what Ernst was able to do was show

5 that these viruses also have terminal repeats, they

6 also have Rep-like proteins, and that he could take

7 an episome substrate and show that this virus could

8 also integrate into a target sequence if the Rep

9 protein on this minute virus was present and if the

10 subsequent sequences were available.

11 So what I think this is suggesting is that

12 the parvoviruses have proteins that are involved in

13 replication that are able to carry out nicking and

14 helicase activity on substrates. In the case of

15 minute virus of mice, there is no target in the

16 genome.

17 In the case of AAV, there is an origin

18 identical to AAV sitting on chromosome 19. So the

19 question may be, does AAV really set up a latency

20 or is this an interaction between Rep proteins and

21 target sequences, and 1 percent begins to suggest

22 that it is not a very efficient mechanism.

23 I am going to shift gears and now talk to

24 you about vectors because I think this is where

25 most of the interest is. In the laboratory, a

35

1 number of people generate vectors by different

2 procedures.

3 In our lab, we use plasmids to start to

4 make the vector, so now we only retain the terminal

5 repeats. The gene of interest is in the middle.

6 You have a helper plasmid carrying the Rep and

7 capture genes, and another plasmid carrying the

8 essential sequences from adenovirus to activate all

9 of these steps.

10 What happens when all of these are in the

11 cell, you produce a single virus particle, which is

12 an AAV particle carrying the foreign gene of

13 interest. If you take these viruses and put them

14 in tissue culture cells, and put them under

15 selection, what you see is if you go to the

16 chromosome 19 region and look at individual clones

17 that had the vector integrated in the human genome,

18 you don\'t see a significant rearrangement under

19 chromosome 19 sequence.

20 So unlike wild type where it appeared that

21 70 to 90 percent of the integrations were targeting

22 this locus, the vectors have lost this ability to

23 go to chromosome 19. It has been shown by a number

24 of labs that if you add Rep back to this reaction,

25 these vectors will go to chromosome 19 and

36

1 integrate.

2 So it is fairly well established now that

3 AAV vectors have no targeting capacity and that

4 what they do have is the capacity to integrate into

5 the chromosome under these selected conditions.

6 This is an approach that Charley Yang took

7 in the lab about seven years ago, in which he made

8 AAV vectors that were carrying a plasmid origin and

9 ampicillin sequence, as well as a selectable

10 mechanism to look at selection in eukaryotic cells.

11 He made this into a virus, allowed it to

12 integrate into the chromosome, and he used enzymes

13 that were cut outside of the viral DNA, closed this

14 up into a circle, and pulled out these so-called

15 cellular junctions, and when he characterized

16 these, he came up with the following results.

17 The break points of the terminal repeat

18 and the chromosome were almost identical to what we

19 saw with wild type. They clustered around the

20 hairpin structure, but there was no defined break

21 point in any of these vectors.

22 When we looked at the location that they

23 were going into, they appeared to be random on

24 chromosome 17, 7, 1. We had two examples of it

25 integrating on chromosome 2. But what we were

37

1 seeing was that all of the characteristics of

2 integration were identical to wild type. It is

3 just that their targeting ability was lost.

4 Instead of going to 19, it was random.

5 If you look at the vectors, they were

6 again consistent with this head-to-tail mechanism

7 and amplification event or rearrangement event. I

8 should mention that David Russell has just

9 published a little paper in Nature Medicine that

10 has shown another clustering of these things pulled

11 out of HeLa cells, and we have generated the exact

12 same information. There is breakage and

13 duplication and some type of random repeats that

14 are being generated.

15 So I want to point out because I think we

16 get misled a lot when we think about AAV\'s

17 integration and that it is something special. This

18 ability to form concatemers is something that was

19 documented a number of years ago by Schimke\'s lab.

20 In fact, if you look at any transgenic animal that

21 has ever been generated, it is always generated in

22 a head-to-tail concatemer formation.

23 If you look at virtually any cell line

24 that is established by plasmids to give stability,

25 it is typically a head-to-tail concatemer, that is

38

1 going into the chromosome. So what we see is that

2 AAV is probably using host enzymes to generate

3 these concatemers that eventually go into the

4 chromosome.

5 As I mentioned to you, without the Rep

6 protein, there is no targeting capability. This

7 integration appears to be random. The insertion

8 that takes place at the integration site is not a

9 cut and paste mechanism, it\'s a deletion,

10 amplification, rearrangement, illegitimate type of

11 recombination.

12 This is just our data showing all of the

13 break points that we have generated both with

14 vectors with wild type AAV as far as the junctions

15 that are generated between the terminal repeats and

16 the chromosome, and you can see that again there

17 are preferred clustering sites, but there is no

18 distinct break point that takes place between AAV

19 molecule and the chromosomal DNA sequence.

20 We concluded from this study that when AAV

21 vectors go into cells, it is cellular recombination

22 pathways that are responsible for the integration

23 of that, and that there is no viral participation

24 in this enzymatic step, it is all carried by

25 cellular recombination.

39

1 If you look at the data that has been

2 generated, it falls under the category of an

3 illegitimate, non-homologous recombination. This

4 would be true if you put in plasmid DNA,

5 oligonucleotides, any piece of DNA that ends up

6 going into the chromosome. It is following a

7 pathway that supported cellular enzymes carrying

8 out the integration step.

9 I want to just summarize this and then I

10 am going to switch to the last third of the talk,

11 which is going to just talk about information

12 generated with vectors in animals.

13 Right now, AAV vectors do not target

14 chromosome 19. They are identical to wild type

15 with respect to the terminal repeat break points.

16 They are essentially identical at this level. The

17 head-to-tail orientation of vector proviruses, you

18 can find tail-to-tail and head-to-head, but this is

19 pretty much the dominant species you will see.

20 They rearrange to chromosome integration

21 site. There is not a cut and paste mechanism.

22 There is always some type of deletion,

23 amplification, and rearrangement that takes place

24 at the integration locus.

25 So by all these criteria, AAV fits the

40

1 conditions of an insertional mutagen. It has the

2 ability to go into the chromosome, and the critical

3 question is at what frequency does it carry out

4 this insertion event.

5 This is where I think we began to

6 accumulate data in the field that drifted us away

7 from all that information that was derived in

8 vitro, and you should understand that the data was

9 derived in vitro was under selected conditions with

10 a gene, such as G418 or neomycin, so that you are

11 only looking at the integration events.

12 In vivo, the first data that began to

13 suggest that this may not be consistent with what

14 was happening in vitro was actually carried out in

15 Terry Flotte\'s lab where they were looking at

16 adeno-associated viruses in monkeys after

17 administration for airway gene delivery.

18 When they characterized this, they saw

19 that the virus was persisting for a period of time

20 and the virus could be rescued completing all of

21 those steps that we talked about in the life cycle,

22 but it was showing up as an episome. There was

23 very little data suggesting that this type of

24 persistence was taking place as an integration

25 event.

41

1 This is a paper that I would like to

2 direct people to, because I think buried in this

3 paper is some really important information. This

4 was a study carried out in Jim Wilson\'s lab where

5 what he virtually did was an in vivo selection like

6 what we do with in vitro selection with G418, in an

7 animal model that had a disease for the liver, so

8 the AAV vector was transducing a gene and to

9 deliver, that he could put a selective pressure on.

10 This selective pressure meant that if this

11 liver was to survive, the virus had to integrate.

12 After it integrated, you could see nodules begin to

13 grow of liver cells. He characterized those

14 nodules. He showed they had integration events in

15 them. They were similar to what I have just

16 described for in vitro.

17 They were tandem repeats, rearrangements,

18 and an illegitimate recombination mechanism, but if

19 you go into the paper and dig at the multiplicity

20 of virus that he was putting into the liver, 1012

21 particles per liver, he was only getting about 0.1

22 percent of the liver cells showing an integration

23 event.

24 So I think what Daniel was referring to is

25 where does AAV fit on this curve of an obligated

42

1 integration event versus the potential to

2 integrate, and this study, under selective

3 pressure, there was a frequency that was derived,

4 which I think may be telling to the type of numbers

5 that may happen in the absence of selection.

6 I point to these last two papers only

7 because it has been characterized in extensive

8 detail in muscle, and I bring up Phil Johnson\'s

9 study because he now has an abstract that is going

10 to be presented as ASGT, where he is showing that a

11 majority of what I think he calls 98.5 percent of

12 all the vectors that are in skeletal muscle are

13 persisting in episomal form.

14 He does a real-time PCR assay. I am not

15 going to try to describe his data, it is written in

16 an abstract form, but I think it is something that

17 the field in general will want to look at and see

18 if this will be something that can be used for

19 other target tissues.

20 But it is consistent with the theme. What

21 I did not talk about here today was any of the data

22 that Mark and Kathy have generated, because I know

23 they are going to speak later and they can tell you

24 specifically what has been derived in their hands,

25 but I think the theme is we see what these vectors,

43

1 they have the propensity to set up a persistence,

2 the data that has been generated in liver, muscle,

3 lung, and brain is that episomal forms that are

4 predominantly seen, but there is always the

5 potential and evidence for integration.

6 This is the last paper that I am going to

7 point you to, and I am going to just mention this

8 because I think this is going to give us a starting

9 place to begin to understand AAV integration in

10 whole animal.

11 Terry Flotte and his lab have generated

12 some data showing that the DNA-dependent protein

13 kinase, the gene that has mutated in SCID mice,

14 seems to have an impact on the molecular phase of

15 AAV genomes.

16 Again, I am going to paraphrase what

17 Terry\'s data says, and he can speak to it in more

18 detail because he has got new data that is a little

19 bit more extensive. It appears that if you knock

20 out this protein kinase, which is involved in

21 immunoglobulin rearrangement as one example of its

22 role in the human cell, the virus appears to

23 integrate more efficiently into the chromosome.

24 This is an enzyme that plays a role in

25 end-to-end joining, and it seems that if you lose

44

1 the ability of these host enzymes to form the

2 so-called concatemer structure that we all

3 characterize, you can see an increase in

4 integration event takes place.

5 So it appears that if you are defective in

6 one pathway, AAV will just follow another host

7 mechanism for persistence, which is an integration

8 mechanism.

9 Again, if there are any specific

10 questions, I will ask you to direct them to Terry

11 where he can give you the details of what is going

12 on, but what this data tells me is that we probably

13 we will be able to identify these so-called

14 cellular recombination pathways that are

15 influencing AAV vectors when they go into so-called

16 non-dividing tissue.

17 I am going to conclude by trying to

18 reemphasize the following points. Wild type and

19 AAV vector integration is not very efficient, and

20 this fairly well documented in vitro. It is

21 something that seems to be a theme that is

22 recurring in vivo.

23 If you look at the ability of the virus to

24 target chromosome 19, it is absolutely dependent on

25 a viral protein called Rep. The mechanism is now

45

1 well understood because they are identical binding

2 sites to facilitate this targeting.

3 AAV vectors, which do not have Rep

4 protein, do not have the ability to go to

5 chromosome 19 into the site-specific manner. If

6 you look at the proviral structure of wild type AAV

7 and vector DNA, they are essentially identical at

8 all levels.

9 The break points and the terminal repeats,

10 the amplification, the concatemerization, and the

11 rearrangement under chromosome sequence is

12 identical whether it\'s on chromosome 19 or randomly

13 inserted throughout the genome.

14 Finally, with the limited number of

15 studies that are being done, it appears that in

16 non-dividing cells in vivo, the AAV vectors exist

17 predominantly in an episomal form, and again, I

18 will conclude.

19 Daniel basically summarized the AAV field

20 by saying it has the propensity to integrate into

21 the chromosome, where it fits on that rheostat as

22 being very efficient or not efficient, I think it

23 is going to be dependent on more studies in vivo in

24 which we can continue to accumulate data.

25 But as of today, what we keep seeing is

46

1 some propensity for this episomal form, but the

2 risk is still there, and I will stop there and take

3 questions.

4 DR. SALOMON: Thank you very much. Very

5 interesting.

6 Q&A

7 I have a couple of questions that kind of

8 occurred to me in the setting of thinking about

9 this thing riskwise. You have been very straight

10 about it. What is interesting is a lot of times

11 when it is introduced for the first time, people

12 talk about OAB, it\'s a parvovirus, it has been in

13 humans for a really long time, and it has been

14 extremely safe in the sense that it is not

15 associated with any known disease entity, and the

16 implication is many times that therefore, AAV gene

17 therapy as a vector is going to be similarly safe.

18 However, I think what you very clearly

19 point out in all the molecular biology that has

20 been done with the vector is that an AAV vector

21 really isn\'t anything like a wild-type AAV in the

22 sense that now what you have got mainly is

23 episomes, it is not integrating in chromosome 19,

24 so there is a lot of assurance that one might take

25 from the first part of the data that it is probably

47

1 not reasonable to carry forward into thinking about

2 AAV vectors.

3 DR. SAMULSKI: Right. I will give

4 opinions on both sides. I think if you look at the

5 biology of the virus, it falls in the biological

6 features, so that we don\'t see significant immune

7 response generated from AAV infections. You don\'t

8 see that with wild type.

9 You don\'t see the virus taking over the

10 host cell as a lytic virus does, so there is

11 consistency in that aspect of saying AAV is more

12 like its features of being non-pathogenic, but I

13 think you only need to hear what Phil and them

14 mentioned at the RAC probably every time AAV is

15 discussed, you know, this is not normal. You are

16 putting in 1012 viruses into a focal injection,

17 hundreds of particles, lots of genomes. This is

18 something that doesn\'t happen in nature, and so it

19 shouldn\'t be considered as the viral life cycle,

20 because in that setting, we can\'t reproduce the

21 viral life cycle. We are not getting a systemic

22 infection that is disseminating and maybe setting

23 up latency.

24 We are inducing an artificial way of

25 getting persistence. So I think you are right on

48

1 the money there. I think what will go back and

2 forth between these systems is how much does the

3 vector mimic wild type. As far as integration they

4 are identical, it is just one is on 19, the other

5 one is random.

6 So there is some ability to go back and

7 forth as to what is happening.

8 DR. SALOMON: So the second question I had

9 was I don\'t know a lot about chromosome 19, so I

10 apologize for what I am certain are stupid

11 questions to the geneticists here, but is it clever

12 that the virus chose this area in chromosome 19, is

13 that a safe area to integrate in that?

14 I guess the follow-up question here would

15 be maybe one thing to think about, has anyone

16 thought about it, is if you add the Rep gene back

17 and let it integrate into a place that we know is

18 safe instead of having all this episomal DNA that

19 we have no idea what it is doing.

20 DR. SAMULSKI: Your question is something

21 that you would discuss at a cocktail hour, why does

22 AAV go to 19. We could say mechanistically, there

23 is a viral origin sitting on 19. Did the virus

24 pick it up from 19 and retrofit it into its life

25 cycle or is that a remnant, some integration event

49

1 that took place who knows when.

2 It is only conserved in monkeys and

3 humans, so it is a sequence that is not found, so

4 there may be some selective pressure for why that

5 took place. Is it a safe site? In tissue culture,

6 we are in HeLa cells, there are 19 chromosomes, 3

7 copies in 19, we can get latency all the time. In

8 vivo, there hasn\'t been the kind of studies you

9 would want to see, and if AAV integrates in 19, is

10 that going to be an adverse event.

11 I would argue 19 in liver cells may not be

12 essential, but 19 in another tissue like neuronal

13 cells may be essential, but to get back to your

14 question, which I think is more directed to what is

15 on that locus, there is no gene located at that

16 region.

17 Michael Linden has argued that there is a

18 transcript that can go through this region that is

19 related to a muscle transcript, but from our and

20 other studies, there has never been an integration

21 event that has disrupted that gene or the potential

22 for the gene, but again, there are all tissue

23 culture cells, so I think it is an interesting

24 biology.

25 When we first saw this, what is clustered

50

1 on chromosome 19 were a lot of genes we would have

2 liked to have seen it go into, the receptor for

3 polio virus, a receptor for a lot of other viruses,

4 and we thought, oh, maybe, AAV will integrate, give

5 the host cell a mechanism of protection from

6 another infections agent, and there would be a

7 reason for why it targets, but this locus is not by

8 those type of genes, although it would have been a

9 nice story. So it is an unknown.

10 DR. SALOMON: I had one last question, and

11 that is when it integrates and then almost sort of

12 kind of does its version of concatemerization in

13 that area \-- that is not quite exactly what

14 happens, but \-- what does it do to the promotor

15 regions in the ITR, is the payload gene still

16 promoted, or does it destroy the promoter region,

17 so you basically have dead genes there?

18 DR. SAMULSKI: AAV is not like the

19 retrovirusus where it has a promoter, a strong

20 promoter in the LTR. It has promoter-like

21 activity, but all the cassettes have the promoter

22 built in between the terminal repeats, and so the

23 gene remains intact, the break points seem to be in

24 this buffering area in the terminal repeats.

25 So, again, all of these things are skewed.

51

1 They are put under selection so you insert the

2 genes that go in intact, and they rescue them out.

3 We can only see the products that E. coli will

4 tolerate, so you have to realize that head-to-head

5 and tail-to-tail formations are not very stable in

6 E. coli, so we are getting a biased opinion every

7 time we pull these out.

8 The PCR reaction is extremely biased

9 because that is Mother Nature\'s best primer, it\'s

10 an 80 percent GC hairpin structure. If you try to

11 prime through that region, you will generate

12 deletions, so we even think a lot of our data

13 showing break points is an artifact of pulling out

14 junctions.

15 The only data that begins to support that

16 if you have a real controlled Rep expression, you

17 don\'t see as much amplification rearrangement. The

18 group in Italy put the Rep gene on the regulatable

19 promoter, and they actually dosed in the amount of

20 Rep, and what they was the integrations were more

21 well behaved.

22 So I would say that we have not been able

23 to mimic what probably the virus does very well,

24 but we can score all the downstream events. It

25 goes in a chromosome, it looks like this, and so

52

1 forth.

2 So I would be hesitant about taking my

3 opinion about this field and turning it into this

4 is the fact of all it all happened.

5 For the vectors where there is no Rep, and

6 you do see the integration, it is cellular

7 mechanisms that are putting it into the chromosome.

8 DR. SALOMON: Dr. Rao and then Dr.

9 Mulligan.

10 DR. RAO: Is there any evidence of

11 mobilization of the integrated thing, wild-type

12 infection?

13 DR. SAMULSKI: That is a good point.

14 There is the risk of mobilization if you get an

15 added infection and a wild-type AAV infection, so

16 you need a two-hit kinetics to move the vector out

17 of the chromosome.

18 In the laboratory, if you do those

19 experiments, wild-type dominates the product that

20 comes out, because there are more elements that

21 ensure packaging, and they are not in the vectors,

22 but you do mobilize it if you get a two-hit

23 kinetic.

24 DR. RAO: Is there a rough percentage on

25 that? I know wild-type predominates, but \--

53

1 DR. SAMULSKI: Wild-type plate

2 90-something percent of all the virus that comes

3 out, and if you cycle it, it is the only virus that

4 you see. The vector doesn\'t compete very well in

5 that setting, but the risk is there, in an in vivo

6 setting.

7 DR. MULLIGAN: In the in vivo case, the

8 integration question is complicated by all the free

9 copies, and I think it is important that people

10 that are not experts here get a sense of if you had

11 very efficient integration in the sense that you

12 had one copy for large number of cells, but then

13 you had hundreds of unintegrated copies, that would

14 confuse your interpretation, so can you

15 characterize for people how you get at the issue,

16 that is, if you just look at the sum of

17 unintegrated copies, and that is a large number,

18 and then the sum of integrated copies, and that is

19 a small number, then, one conclusion is that you

20 have mainly unintegrated gene transfer, but in

21 principle, on a cell-by-cell basis, you could have

22 very efficient integration, while on top of it you

23 could have a large amount of unintegrated copies.

24 Now, in vitro, I know that is not the case

25 because you can actually directly assess that, but

54

1 how have the various tests actually ruled out that

2 that is not the case?

3 DR. SAMULSKI: I think that is a good and

4 hard question. I think Mark has generated data

5 that begins to look at that where he has put virus

6 in hepatocytes, and he will probably discuss this,

7 and then did a partial hepatectomy to let the liver

8 cells grow, and tried to score how many of those

9 regenerated liver cells still carry a copy

10 suggesting that that fraction had integration, and

11 the ones that lost it were primarily episomal.

12 I will let him describe that, but I don\'t

13 think there is any good way to assess that

14 question.

15 DR. MULLIGAN: I would think that now that

16 there is these, in human cells, outlaw PCR

17 approaches, the question is can you actually

18 directly calculate the total absolute number of

19 integrations independent of how much total DNA is

20 there?

21 DR. SAMULSKI: I don\'t know how I would do

22 that. I think this is what Phil Johnson is doing

23 in his abstract. He is looking at ALU real-time

24 PCR going across genomes and stuff like that.

25 DR. MULLIGAN. Has anyone looked, like

55

1 Ernest Whittaker, like his system if you have an

2 adeno-infection or HIV infection, and you all of a

3 sudden do an AAV infection, is the propensity for

4 integration of AAV into, say, HIV, a higher

5 integration because it\'s unintegrated initially

6 than it would be to go in the chromosome?

7 DR. SAMULSKI: I think that is another

8 good question, that is, if you are in a cell that

9 has substrates, what is the fate of AAV to those

10 substrates, will it go into them, or a more

11 preferred event. I don\'t think anyone has an

12 answer to that, but it\'s a good question. It is

13 something that has got to begin to be looked at.

14 I think I would like to just emphasize

15 that AAV in the early days was put in the bone

16 marrow stem cells with a lot of efficiency, and

17 then it was shown that as you tried to amplify

18 these cells, they weren\'t very good and I think it

19 was speaking directly to the fact that it wasn\'t

20 integrating and therefore, you could transduce them

21 and get positive cells, but once they are asked to

22 divide, you lost that.

23 So I think why AAV has been such a niche

24 virus for the so-called non-dividing cells is

25 because is can set up this persistence. I think

56

1 the integration frequency is probably going to be

2 determined by do non-dividing cells carry out

3 illegitimate recombination, at what rate compared

4 to a dividing cell. That is going to be an

5 important number that is going to influence the

6 outcome in these type of studies.

7 DR. GORDON: I have a couple of very quick

8 questions that are just simple factual answers.

9 Where in the life cycle of AAV does the

10 uncoating of the genome take place? That is one.

11 The second question is you said that when you add

12 Rep back to the vectors, then, you get chromosome

13 19 integration again. How is it added back, as a

14 gene or as a protein?

15 DR. SAMULSKI: The answer to the first

16 question is the parvovirus are argued to go into

17 the nucleus and uncoat to release their DNA into

18 the nucleus. There is probably a capsic component

19 still associated with the virus that is sitting on

20 those terminal repeats that either prevents it

21 from, you know, being naked DNA, but at the same

22 time may recruit other factors to the origin.

23 As far as the second question that you had

24 \-- I forgot it already \--

25 DR. GORDON: Adding Rep back.

57

1 DR. SAMULSKI: That\'s my senior moment

2 there.

3 Rep protein has been added both as

4 plasmids, as physical protein injectate, and as

5 inducible protein in the cell line, and all of

6 those will take vectors and allow it to go to

7 chromosome 19.

8 The last thing I will mention is that both

9 the Italian group and our lab have generated a

10 mouse that carries the chromosome 19 locus, and in

11 our case, it is sitting on the X chromosome. When

12 we put wild-type virus into that, it goes to that

13 chromosome 19 locus even though it\'s on the X

14 chromosome, again suggesting it\'s the cis elements

15 that are driving where it goes, and not that it

16 happened to be on 19 in humans, and stuff like

17 that.

18 DR. DYM: I think you alluded to my

19 question, but i am going to ask it anyways. Can

20 you clarify or comment on the ability of the AAV to

21 get into dividing cells versus non-dividing cells,

22 and, of course, in the testis, the spermatogonia

23 are very actively dividing, the sperm are not.

24 DR. SAMULSKI: I think there is no

25 difference between AAV going into dividing or

58

1 non-dividing cells. If the receptor is present, it

2 will bind, and then I think the mechanism for

3 internalization is clathrin-coated pits, endosome

4 release, and traffic.

5 If you can carry out those steps, it is

6 indistinguishable whether it\'s a dividing cell or

7 non-dividing cell. In the very early days, it was

8 suggested that AAV preferred dividing cells, but

9 that was in vitro looking at selection and

10 therefore you were biasing the system.

11 I think once people went in vivo, they

12 realized that all of that was probably misleading a

13 little bit.

14 DR. MULLIGAN: You didn\'t mention about

15 other AAV serotypes, so in principle, the

16 efficiency of the intervention would depend upon

17 just the virus titer.

18 Do you have any sense that AAV-1, for

19 instance, which in muscle is much, much more

20 efficient, would potentially be better at infecting

21 germ cells?

22 DR. SAMULSKI: I think Richard\'s point is

23 a really interesting one because we and others have

24 seen that the other serotypes have better propisms,

25 are more efficient. The question is what are their

59

1 integration mechanisms.

2 The only one that we have data on is Type

3 4. Type 4, which is camana monkeys, will target

4 monkey cells and integrate, will target human cells

5 and integrate in the chromosome 19, so the

6 wild-type virus will capitulate exactly what the

7 human virus is.

8 The other four, 1, 3, and 5, it is

9 unknown, but they are so homologous, about 80 to 90

10 percent homologous, they all bind to the terminal

11 repeats, they all can package each other\'s DNA.

12 Chances are they will do the same type of

13 integration.

14 There are differences in these terminal

15 repeats if you look at them. Type 5 is different

16 than Type 2, and if that is a substrate, that may

17 be more prone for recombination enzymes, you may

18 see an integration frequency that is different.

19 DR. MULLIGAN: I just meant the capsid,

20 looking at risk for germline infection, if it

21 happens just proportionately, it much better

22 infects that cell and even though integration is

23 very efficient, then you get more efficiency.

24 DR. SAMULSKI: I misunderstood. I think

25 if the virus has a more efficient tropism in those

60

1 kind of cells, chances are the integration

2 frequency is going to be higher. That is kind of a

3 given.

4 DR. SALOMON: Sort of a follow-up question

5 here is \-- and you may have answered this, and I

6 apologize if you did \-- if you have a cell that is

7 actively dividing or is activated, let\'s say, so it

8 has a lot of open chromatin structures, it is more

9 likely to integrate in that setting than in, let\'s

10 say, a stable cell that is not activated?

11 Obviously, where I am going is in, you

12 know, if you had an injury or inflammation, or

13 something, are those areas in which the rules might

14 be different?

15 DR. SAMULSKI: Sure. I think that is

16 exactly what the data are supporting. This virus

17 looks for open chromatin contacts. Events that

18 were scored appeared to be in genes, promoter

19 regions in the gene. I think they are all because

20 of the same reason, these were open chromatin. If

21 it\'s condensed chromatin, there is probably no

22 mechanism, because again it\'s a cellular event and

23 it is going to be acting on cellular regions of the

24 DNA, better accessible.

25 DR. SALOMON: That was great. Thank you.

61

1 DR. SAMULSKI: Thank you.

2 DR. SALOMON: Very useful.

3 The second presentation is on germline

4 transmission by gene transfer vectors and some

5 thoughts on assessing the risk from John Gordon,

6 Mount Sinai School of Medicine.

7 Germline Transmission by Gene Transfer Vectors

8 Assessing the Risk

9 Jon Gordon, M.D., Ph.D.

10 DR. GORDON: I was asked to talk a little

11 bit about not necessarily what we are doing to

12 address this problem in my own lab, but just to

13 talk about what I think are the points of

14 susceptibility for germline integration of vectors

15 into various gametogenic cells and to review the

16 literature on it, so that is what I will do.

17 I am not an embryologist by profession,

18 and I don\'t wear the lot on spermatogenesis either,

19 but we have a spermatogonium expert in the audience

20 in case I make a mistake, so that will be good.

21 The ontogeny of gametes in relation to

22 their susceptibility to gene insertion. Primordial

23 germ cells are the cells that ultimately arise to

24 both eggs and sperm, and these arise in the yolk

25 sac or the epiblast in the mouse at about three

62

1 weeks\' gestation in the human.

2 There aren\'t a very great number of those.

3 They then migrate by ameboid movement through the

4 dorsal mesentery to the genital ridge. During this

5 migration process, they also multiply. These cells

6 are quite easily identified because they stain very

7 strongly for alkaline phosphatase.

8 They arrive to the genital ridges that may

9 be the end of five weeks\' gestation in the human.

10 During this period, the cells are unprotected, that

11 is, they are not within the capsule of a gonad, and

12 they are mitotically active, allowing infection by

13 agents that require mitotic activity. We will

14 return to this point of what agents may require it.

15 Fetal gene therapy must take this risk

16 into account, and the RAC had a sort of mock fetal

17 gene therapy protocol presented one time, and this

18 issue has to be raised.

19 Now, female gametes, which are of a little

20 bit less interest today, but they are important, of

21 course, they become oogonia, and they divide by

22 mitosis until about 5 months or a little longer to

23 generate several million oogonial cells. At this

24 point, many begin to die, while others become

25 primary oocytes.

63

1 Primary oocytes enter meiosis, a complete

2 crossing over, and then they stop. The chromatids

3 remain associated, but crossing over is completely.

4 Then, they are surrounded by follicle cells in what

5 are called primordial follicles.

6 Once they are in the primordial follicle,

7 they become relatively inaccessible because you

8 have to get through the layer of follicle cells,

9 which is a single cell layer basically at this

10 point, in order to reach the egg, which is sitting

11 at the end of crossing over in the so-called

12 dicteate \[ph\] stage.

13 They sit in this stage until the follicle

14 begins to develop towards ovulation, and there is

15 some hypothesis that this long term association of

16 the chromatids has something to do with chromosome

17 nondisjunction in older eggs.

18 Now, at puberty, the follicle develops in

19 response to FSH from the pituitary. Numerous

20 follicle cells surrounding the oocyte are within

21 the follicle wall, and they begin to produce

22 glycoprotein \"egg shell,\" the zona pellucida.

23 So, as the egg is developing, then, the

24 number of follicle cells that sit between the egg

25 and the outside world increase, the wall of the

64

1 follicle becomes a consolidated structure, and the

2 zona pellucida is laid down. This is a glycoprotein

3 human egg shell, mammalian egg shell, very hard to

4 penetrate.

5 As the follicle matures, meiosis resumes,

6 and one resumes, and as the first polar body is

7 released, the chromosomes then move to a metaphase

8 of the second meiotic division, and that is how

9 they are found after ovulation.

10 To enter the egg, genes must past through

11 the follicle wall, they have to get through or

12 between the follicle cells around the egg, and then

13 they have to get through the zona.

14 We would regard the egg as a non-meiotic

15 cell at this point.

16 At ovulation, the egg is in metaphase II

17 and is surrounded by the zona and the granulosa

18 cell layer. Some of the cells are ovulated with

19 the egg.

20 Although immunoglobulin molecules will

21 pass through the zona, there is no evidence that

22 naked DNA or viruses will do so. There have been

23 experiments at least with retroviruses that have no

24 viruses that I am aware of where very high amounts

25 have been put onto zona intact eggs, and then lacZ

65

1 staining look for later in cleavage, for example,

2 without seeing anything.

3 After fertilization, MII is completed with

4 release of the second polar body formation and

5 formation of the female pronucleus.

6 Now, micromanipulation to assist

7 reproduction can assist genetic material in by

8 passing the zona. I just would like to make the

9 point here of two contrasting papers in the

10 literature, one by an Italian group in I believe

11 now the late eighties, in which they asserted that

12 if you performed in vitro fertilization with

13 plasmid DNA sitting in the medium, about 30 percent

14 of the mice born were positive for transgene

15 sequences.

16 The plasmid they happened to use in this

17 case was a commercially available SV40-based vector

18 and to prove that they had integration in these

19 mice, they cloned the material back out of the

20 mouse genome and sequenced the vector material that

21 was in the mouse genome.

22 The published sequences contain nothing

23 junctional, they were all internal sequences to a

24 commercially published sequence. They also did a

25 so-called MBO1/DPN1 digest to show that the

66

1 material was in mammalian cells and was therefore

2 digestible with I believe it\'s MBO1, if I don\'t

3 them in backwards order, and the only problem with

4 this southern blot showing disappearance of this

5 band was that the southern blot did not include the

6 molecular weight size that the band was originally

7 in.

8 It stopped before you could get that high

9 up on the gel, which wasn\'t very high, I might add,

10 about 4.3 kb.

11 So, needless to say, there were a few

12 weaknesses in this publication. Nonetheless, it

13 made the cover of Cell and was accompanied by a

14 very exuberant editorial saying that this had

15 something to do with evolution, plasmids jumping

16 into gametes out there in the ocean where fish have

17 ex vivo fertilization, for example, and multiple

18 labs tried to repeat this work and 2,300 mice were

19 produced in a number of labs, we tried it too,

20 could not reproduce this work even using the

21 identical reagents, and no one makes transgenic

22 mice this way even though it is a heck of a lot

23 easier than microinjection.

24 However, if you do another experiment, and

25 that is, mix plasmid DNA with sperm, as was done

67

1 before but now inject the sperm into the egg, so

2 now you are bypassing the zona with a microneedle,

3 and the sperm and DNA around it go into the egg, a

4 significant percentage of the mice are transgenic,

5 and that is a reproducible result.

6 So, in humans, if we think about

7 micromanipulation, and this is something I have

8 been asserting in an editorial that I have in

9 press, we have to think about the fact that the

10 environment had better be clean, because we can get

11 DNA in by that method.

12 My opinion of what occurs here is that the

13 pronucleus forms quickly after the sperm is

14 injected, DNA gets entrapped into it, and it is

15 pretty much the same as microinjecting DNA into a

16 pronucleus.

17 Now, another interesting point is there is

18 there papers indicating that retroviruses and

19 lentiviruses will infect MII oocytes, which are not

20 meiotic reactive, but which do not have a nuclear

21 membrane. The chromosomes are sitting at a

22 metaphase of the second meiotic division to produce

23 transgenic cattle, monkeys, and mice.

24 I think these papers are very interesting,

25 but there is one slight problem with the assertion

68

1 that it is the non-meiotic MII oocyte that is the

2 target, and that is, of course, that if you soak

3 MII oocytes in the vector, and then fertilize them,

4 there are still going to be vector around after

5 fertilization, and it is not really possible to

6 completely clean them and then fertilize them to

7 show that you had no vector around at

8 fertilization, so it is possible in my view that

9 fertilization occurred and then these vectors went

10 in.

11 But, nonetheless, you can get MII oocytes

12 transduced with retroviruses and in mice, now

13 lentiviruses from David Baltimore\'s lab, and again

14 this raises an issue in clinical in vitro

15 fertilization where the zona is opened not

16 infrequently, either for injecting sperm, for

17 biopsying embryos, and so on.

18 Now, male gametes. Now, in the male, the

19 primordial germ cell step is the same. They get to

20 the genital ridges as before, but them they become

21 dormant where they are contained within sex cords.

22 They sex cords are like the future seminiferous

23 tubules of the testis, they remain this way.

24 The sex cords have a membranous barrier

25 between them and the outside world, but this is

69

1 much less protected structure than it becomes after

2 puberty. The cells are mitotically inactive and

3 relatively unprotected.

4 At puberty, these PGC\'s become

5 spermatogonia and begin dividing. Type A

6 spermatogonia are renewable stem cells that produce

7 more Type A spermatogonia, but they can also

8 produce Type B spermatogonia, and those are

9 committed to meiosis.

10 It has been shown, mainly by Ralph

11 Brimster\'s lab, that spermatogonia can be

12 transduced with retroviruses and lentiviruses, I

13 believe are correct now. This is one in vitro and

14 it is not clear how efficiently one could

15 accomplish this in an intact testis with intact

16 spermatogenesis. Perhaps our colleague in the

17 audience, an expert on spermatogonia, can speak to

18 that, but it clearly is biologically possible to

19 transduce them even though it is not very easy.

20 Generally, they are put back into a testis

21 that doesn\'t have its own spermatogenesis, so that

22 you can sort of have a natural selection for those

23 cells exposed to the vectors in the outside world,

24 and you can get transgenic mice that way.

25 Now, when meiosis beings and the

70

1 spermatogonia are formed also, the testis becomes

2 organized the seminiferous tubules. Pre-meiotic

3 cells are at the tubule periphery where agents can

4 get to them, but they will have to get through the

5 tubule wall, but theoretically, they could be

6 reached from a hematogenous spread to the

7 seminiferous tubule.

8 However, Sertoli cells, situated within

9 the seminiferous tubules, form tight junctions that

10 sequester meiotic cells behind what is called the

11 \"blood testis barrier,\" so actually not a barrier

12 between the blood and meiotic cells, it is between

13 the Sertoli calls and the meiotic cells.

14 Sperm move toward the lumen of the tubule

15 as they complete meiosis and morphological

16 transformation. Now, this barrier is needed, of

17 course, because it doesn\'t occur because these

18 meiosis-specific proteins don\'t appear until after

19 puberty, and therefore they are potential

20 immunogens, so this has to be a immunologically

21 privileged site, and that is the rationale for

22 having the blood testis barrier.

23 Meiotic cells are difficult to access

24 except retrograde through sex ducts. You can

25 inject vectors into the epididymis, for example,

71

1 and find them in the testis. So someone is

2 undergoing, for example, prostate gene therapy, it

3 is not at all impossible that one could get vectors

4 moving retrograde back up and thereby get to the

5 cells that are behind the blood testis barrier.

6 Male gametes. Now, sperm maturation or

7 spermiogenesis, is characterized by a loss of most

8 cytoplasm, replacement of the histones by much

9 tighter binding protamines, and near complete

10 cessation of gene expression. I say \"near\" because

11 there are a few post-meiotically expressed genes.

12 Again, what you have to realize is that

13 the idea of sexual reproduction is to give all

14 gametes an equal chance of getting to the egg, and

15 if you have postmeiotic gene expression could have

16 allelic variance which would give sperm an

17 advantage theoretically, and so the organism does

18 everything possible to prevent that.

19 As meiosis begins, actually, once Type B

20 spermatogonia become committed, these cytoplasmic

21 bridges remain between the cells. These are very

22 large and they allow even mRNA size molecules to

23 pass from one cell to another, so allelic

24 variations between spermatogenic cells, those

25 differences are minimized in terms of their

72

1 potential impact on spermatogenesis, and then late

2 in spermiogenesis, there are a few genes active,

3 but mainly there are the chromatin is very tightly

4 condensed and very difficult to access.

5 I should point out parenthetically there

6 that there have been papers from Anderson\'s lab way

7 back when, showing that retroviruses like open

8 chromatin in preference \-- or DNA hypersensitive

9 chromatin \-- in preference to highly condensed

10 chromatin.

11 The nucleus then becomes surrounded by

12 what I would call the giant lysosome, the acrosome,

13 contains lytic enzymes for presumably digesting

14 your way through the zona in fertilization, and it

15 is difficult to access DNA in the sperm head.

16 Now, again, I would say that there are

17 some papers saying that this has been done

18 successfully. There is a paper from France saying

19 that pig sperm can be transduced with adenovirus.

20 This paper found lacZ expression in cleaving

21 embryos after exposing sperm to adenovirus, and

22 then found piglets that had mRNA-derived by RT-PCR

23 that had mRNA derived from adenovirus in multiple

24 tissues of these piglets.

25 Now, I would just analyze this paper a

73

1 little bit for your benefit, if I might. The lacZ

2 vector used in that paper was a vector that was

3 received from another laboratory and which had a

4 nuclear localization signal. So the lacZ should

5 have been in the nucleus of these embryo cells, and

6 indeed, when we have used such things on embryos,

7 we see the nucleus stain.

8 However, the pig embryo is loaded with

9 lipids, and they are basically black. You can\'t

10 see the nucleus in a pig embryo, and if you want to

11 inject a pronucleus in a pig to make transgenic

12 pigs, you have to centrifuge the embryo to get the

13 lipid out of the way, so you can even see the

14 structures.

15 So, in the photograph showing lacZ

16 staining of these embryos, there were black embryos

17 that were exposed to the vector, and there were

18 slightly less black embryos that were not exposed

19 to the vector, and the nucleus was not visible in

20 either case.

21 The staining for lacZ was done for 15 days

22 in this experiment, and I would assert to you from

23 my own work with lacZ staining that you could stain

24 your teeth if you did it for 15 days.

25 The staining was on the zona. There is no

74

1 reason why there should be staining on the zona,

2 but we have used lacZ staining on embryos with

3 adenovectors on zona-free embryos just exposing the

4 embryo, we never seen staining, not on zona-free,

5 but, for example, injecting it under the zona, we

6 never see zona staining.

7 These people found RT-PCR-positive tissues

8 in all three germ layers of the piglets born, that

9 is, ectoderm, mesoderm, and endodermal derivatives.

10 Now, this vector was replication-defective. The

11 only possible way to be in all three germ layers is

12 if it integrated and got replicated.

13 However, their southern blots were

14 negative. To me, that is a very incongruous

15 result, so I don\'t believe the result, let me just

16 give you my own opinion there.

17 We tried this in mice and could not repeat

18 it, at least in mice. However, I think this paper

19 and the other paper with the sperm-mediated plasmid

20 transfer speaks to one of the sort of difficult

21 problems for the FDA, I believe. These are

22 published data and it is very difficult to say, oh,

23 well, that\'s great, but it is not a good paper, so

24 we will just ignore it. It is very difficult to

25 ignore it when people say they are doing these

75

1 kinds of things successfully, then, one has to step

2 in and address it.

3 Male gametes continued. Now, the mature

4 sperm on route to release can be exposed to vectors

5 via fluid from the seminal vesicle, prostate, and

6 in the urethra, a small amount of urine, as well,

7 although maybe you are uncomfortable to see or hear

8 that, it\'s true.

9 Virus found in the ejaculate could be from

10 any of these four sources or from the sperm

11 themselves if somehow it got there, and I should

12 say that one could imagine all also that the cells

13 that line the sex ducts could be received vector

14 from the bloodstream and then pass it on

15 theoretically to sperm although I think that is

16 very unlikely.

17 As vectors diversify, though, we can\'t

18 completely rule that out. Reports of successful

19 transduction of mature sperm are difficult to

20 repeat, and I have already discussed that.

21 Male gametes continued. When sperm bind

22 to the zona, they undergo the acrosome reaction.

23 The acrosome reaction is fusion of the outer

24 acrosome membrane. You remember the acrosome is

25 the giant lysosome. The best way to think of this,

76

1 as I have told my family, it seems to work on them,

2 if a fist put in a pillow, a soft pillow, and that

3 put into a garbage bag.

4 Now, the soft pillow is the acrosome, and

5 the fist is the nucleus, so the nuclear membrane is

6 coming in contact with the inner acrosomal

7 membrane. Then, you have the feathers, which is

8 the acrosomal contents, then, the outer acrosomal

9 membrane, the other side of the pillow, and then

10 that is right underneath the plasma membrane, the

11 plastic bag.

12 Well, if you slash open the plastic bag

13 and the outer side of the pillow, and sew those

14 seams together, you will release all the feathers

15 to the outside. The acrosome reaction occurs, and

16 the bottom line of that is a lot of the sperm

17 plasma membrane is lost.

18 So even passive association of genetic

19 material with the membrane, a lot of it can be

20 lost. However, often the entire sperm is

21 incorporated into the egg and the plasma membrane

22 and components associated with the tail may still

23 be there, so it is possible to passively get it in,

24 I think.

25 Now, shortly after fertilization, sperm

77

1 head decondenses to form the male pronucleus. DNA

2 replication begins. Genetic material that enters

3 the egg with sperm, as I pointed out, from these

4 microinjection of sperm experiments, you can have a

5 relatively highly frequent integration.

6 Now, the early embryo, I wanted to mention

7 it because of my allusions to IVF, the early embryo

8 cleaves within the protective zone until

9 implantation, when hatching occurs. Now, hatching

10 and implementation virtually occur concomitantly

11 under normal circumstances, so the embryo is

12 difficult to access even though it has to get out

13 of the zona.

14 However, micromanipulation can open the

15 zona and expose the embryo to gene transfer agents

16 for more extended periods. Take, for example, the

17 many thousands of IVF cycles that go on every year

18 where the zona is open to theoretically assist

19 hatching. In my opinion, assisted hatching is of

20 debatable effectiveness, but there have been some

21 papers that embryos from older women implant more

22 frequently if you open the zona, and what happens

23 there is you may open the zone at the four-cell

24 stage, put it in the uterus and it sits there until

25 the blastocyst stage and then implants, and so now

78

1 you have the naked cells of the zona opened embryo

2 sitting there where agents that may be in there

3 from the woman being infected with something, from

4 the lab technician who had gene therapy, from

5 whatever source, have a much greater time period in

6 which they could get to the embryo.

7 The embryo is quite easily transduced by a

8 variety of agents, retroviruses being the first one

9 done by Yenish in the early seventies, recombinant

10 retroviruses in the mid-eighties, controversy

11 whether adenoviruses integrate. Our own lab did

12 one where we did early embryos with adenovirus, and

13 what we found was adenovirus was very toxic, so if

14 you put enough in to be sure of getting

15 transduction, the embryos were all killed. If you

16 put in so little that none of the embryos were

17 killed, you had no transduction, but if you have

18 sort of an intermediate level, then, very rarely

19 you can see PCR-positive tail biopsies in offspring

20 that is clearly a mosaic integration.

21 So it is possible to infect embryos, and

22 as IVF becomes more and more interested in zona

23 opening, let me give you another example,

24 pre-implantation genetic diagnosis. You may have

25 heard the speech of Frances Collins at the ASGT

79

1 meeting in California where he went on about

2 pre-implantation genetic diagnosis and result of

3 finding out things from the genome project, for

4 example.

5 Well, pre-implantation genetic diagnosis

6 requires first injection of the sperm because if

7 you do regular IVF, there is hundreds of sperm that

8 are still around and many bound to the zona. When

9 you then biopsy the embryo for PCR, if one of those

10 other sperm gets into your PCR reaction, you are

11 looking for one molecule here, that is, or two

12 molecules, to genotype the embryo, an extraneous

13 sperm is unacceptable, so you have to do ICSI, that

14 is, intra-cytoplasmic sperm injection.

15 Well, that opens the zona, and as I

16 pointed out before, it is very easy to make

17 transgenic mice if you do ICSI with DNA in the

18 medium.

19 Then, you go back later and open the zona

20 again, but this time a much bigger hole, so that

21 you can take a cell off to do genetic diagnosis,

22 and so I think from the point of view of germline

23 transmission, it is much more risky thing to do

24 than just tell the women to get pregnant. She will

25 have a 75 percent chance then of having a baby that

80

1 hasn\'t have genetic disease in the case of

2 recessive genetic disease. She has a 100 percent

3 change of getting pregnant, of course, while in

4 pre-implantation genetic diagnosis, her chances are

5 only 20 percent. It is going to cost her nothing

6 to get pregnant, while in pre-implantation genetic

7 diagnosis, it costs about \$15,000 to get pregnant.

8 Then, she has no risk of all these other things,

9 which, of course, in pre-implantation genetic

10 diagnosis, she has.

11 I might also add that she has to be

12 superovulated for pre-implantation genetic

13 diagnosis. There have been deaths from

14 hyperstimulation syndrome. There have been

15 problems with surgical retrieval of oocytes. I was

16 a little angry with Frances for always saying that

17 instead of saying how about just doing prenatal

18 diagnosis and doing an abortion in the quarter of

19 cases where it is necessary.

20 I just thought I would give you a few

21 pictures here. There is spermatogenesis in a

22 normal testis. Actually, it is a seminiferous

23 tubule that we injected with adenovirus vector, and

24 the periphery of the less mature sperm cells. As

25 you see, you move towards the periphery, the sperm

81

1 heads become condensed and you can see tails, and

2 so on.

3 Then, they are released into the lumen of

4 the tubule and then may go out. I said there is

5 minimal cytoplasm on sperm, but a normal variant in

6 sperm is a so-called cytoplasmic droplet, which

7 kind of like hangs behind the mid-piece of the

8 sperm, so there can be a significant amount of

9 cytoplasm in ejaculated sperm.

10 Here is a developing egg. I was pointing

11 out to you the barriers of penetration of this

12 structure for its virovector. Here is the DA

13 nucleus. You can\'t see the incipient zona

14 pellucida, but there is a very white band around as

15 it is beginning to form, many follicle cells

16 around, and then the follicle wall. So it is

17 difficult to get there.

18 This is some experiments we did when

19 injecting adenovirus vector into the ovary at

20 unbelievable concentrations against any for lacZ.

21 You can see that this vector didn\'t want to get

22 into the follicle. The eggs didn\'t make it through

23 frozen section, so we have done

24 immunohistochemistry to show that the follicle is

25 not penetrated.

82

1 Here is injection directly into the

2 seminiferous tubule. My contention is that we

3 should do provocative experiments that tell us

4 whether or not it is biologically possible to

5 transduce these cells, because in the future, gene

6 therapy will be promulgated, vectors will

7 diversify, their tropisms will change, their

8 structures will change, the methods of

9 administrations will change, and the number of

10 people treated will grow, so we need to know can

11 these things actually get in, not we need to design

12 experiments not to show ourselves as they probably

13 won\'t happen. We need to do experiments to tell us

14 whether or not it can happen, so that we can write

15 the proper consent forms.

16 When we do adenovirus vectors into

17 seminiferous tubules directly in a procedure we

18 call seminiferous tubule cannulation, we see a lot

19 of staining for lacZ, this is immunohistochemical,

20 in the periphery, and it looks as if Sertoli cells

21 are the transduced cells.

22 This is a Sertoli cell. It is sort of

23 anchored to the periphery of the tubule and extends

24 its way in. The Sertoli cell surrounds the

25 spermatogenic cell and sort of helps it complete

83

1 spermatogenesis, and, by the way, also concentrates

2 androgens to very high levels in this region of the

3 testis.

4 We are doing this test to ask ourselves

5 can we transduce these intermediate cells that are

6 behind the blood testis area by injecting vector

7 directly into an intact seminiferous tubule. We

8 believe that this suggests no, but we think we need

9 to go to nucleic acid hybridization to really know

10 because especially like for AAV, which has a

11 delayed expression, we need to know where the

12 genetic material actually is.

13 This is just a view of the acrosome

14 reaction. This is the acrosome. With those

15 enzymes for getting through the zona pellucida, the

16 main one is a proteolytic enzyme acrosome, and I

17 hate to say this, but there is a paper from Japan

18 where acrosome was knocked out and the mice were

19 completely fertile. It has never been repeated,

20 but everybody believes it. That is rather a shock,

21 I must say.

22 You can see how much of the plasmid memory

23 can be lost in the acrosome reaction.

24 That is the summary them of where

25 gametogenesis is more or less susceptible to being

84

1 genetically transduced.

2 DR. SALOMON: Thank you very much, Jon.

3 That was excellent.

4 Q&A

5 It is interesting that yesterday, we were

6 talking about a procedure that came very close to

7 what you just described, so what they are doing it

8 taking infertile oocytes from the presumed patient

9 or from the infertile mother, and taking normal

10 donor oocytes and injecting the sperm \-- it\'s ICSI

11 \-- but also ooplasm from the normal oocyte donor.

12 One of the issues that we discussed in

13 detail was the potential of chromosomal DNA

14 fragments being injected with the ICSI, and you

15 have now given additional evidence. We were

16 concerned of recombination potential, the gene

17 delivery.

18 DR. GORDON: Well, let me just say that I

19 wrote an editorial to Fertility and Sterility,

20 which is in press, but I haven\'t received galleys

21 yet, and therefore, there is some concerns about it

22 being released to the committee and then, of

23 course, to the public yet.

24 But I list all these procedures of

25 micromanipulation and their potential risks for

85

1 inadvertent germline Transmission. I makes some

2 suggestions about what might be done to sort of do

3 quality control in IVF labs. That would at least

4 address this issue proactively.

5 I mean should we multiplex PCR media in

6 which we do micromanipulation just to make sure

7 there is not DNA in there, or should we discuss

8 whether or not practitioners of this forms of IVF,

9 we should at least know that they haven\'t had 1015

10 retroviruses put into them the day before for gene

11 therapy for something, which could happen down the

12 road.

13 I think we should at least begin to study

14 this because there are tens of thousands of cycles

15 done.

16 Now, in terms of the papers of ooplasm

17 transfer, I have a written editorial published, in

18 which I say that the use of germline gene

19 manipulation \-- unfortunately, these people did

20 this mitochondrial DNA analysis on newborns who had

21 received ooplasmic transfer, and the found the DNA

22 of the donor cytoplasm in the newborn\'s bloodstream

23 \-- they called this the first germline gene

24 transfer.

25 Well, of course, these new mitochondrial

86

1 DNAs were not transmitted through the germline yet,

2 so it was a little bit of a loose use of the term,

3 and remember that if it is mitochondria, you can

4 always get rid of it is you just allow the person

5 to be a male who has received all of that, because

6 sperm mitochondria are not transmitted to the next

7 generation.

8 There was a very interesting paper where

9 sperm mitochondria were injected into an egg and

10 destroyed and then liver mitochondria were injected

11 and weren\'t destroyed, so it seems like the egg

12 knows how to find sperm mitochondria, distinguish

13 them from others and destroy them.

14 So that type of gene transfer if not

15 germline in my opinion, and although these people

16 wanted notoriety for using that phrase, I am not

17 sure they got the one they were looking for, but in

18 any case, that is very easy to thwart. All you have

19 to do is make sure that it\'s only male reproduction

20 after that.

21 DR. SALOMON: This is very interesting but

22 we are going to have to stop, because that, we

23 discussed yesterday. Too bad you weren\'t here.

24 I have one quick question and then we will

25 start from the panel. In terms of interpreting

87

1 experiments where you say we looked at gene

2 transfer with adenoviral vectors, they were all

3 adeno that you showed us this time, no AAV, right?

4 It got into the Sertoli cells, for

5 example, it didn\'t get into the spermatogonia, and

6 from what I looked at, those were spermatogonia,

7 not the more mature spermatids, right, because you

8 were showing right at the edge there \--

9 DR. GORDON: Some maturing, yes, it looked

10 like there might have been spermatogonia. That

11 slide does not rule out. That slide shows that we

12 can certainly get a ton of vector there, which I

13 believe is important. I think provocative tests

14 need to be done, not bloodstream injections where

15 we will never find the cells that got exposed.

16 DR. SALOMON: The specific question I had

17 is at some point, you point out very well that the

18 DNA in the developing sperm condenses and

19 transcription diminishes dramatically to almost

20 stopping, and I certainly have no expertise in

21 exactly when in the cycle that is happening, but it

22 would seem to me that particularly, experiments

23 done with mature sperm in which you tried to do

24 something that required transcription as the

25 measure of whether you got gene delivered would be

88

1 a failure because there is no transcription going

2 on, so even if you got gene in, to just take sperm,

3 incubate it with AAV vector or adenovector or any

4 vector, and then show this is not lacZ positive

5 wouldn\'t mean anything.

6 Did I miss something along the line?

7 DR. GORDON: Well, I am not so sure how

8 much transcription is needed to get that to occur.

9 I mean you are more a vectorologist than myself,

10 but it would seem to me that if you get a vector

11 into the head of the sperm, that the sperm could

12 then fertilize the egg, and then it would

13 decondense into a pronucleus and development would

14 begin, and any vectors that were in there could

15 then act as if they had just infected a dividing

16 cell line.

17 So, if you could get the sperm to carry it

18 in, you wouldn\'t have to transduce the sperm,

19 integrate it into the sperm head, but you could

20 certainly get viruses into the embryo by that

21 method theoretically.

22 DR. SALOMON: Right. So if you want to

23 test it, you would have to test it several steps

24 down the line, that you have delivered whatever you

25 carried in, got transcription again, make the

89

1 beta-galactoside gene, then, you do the colored

2 substrate. I am just trying to understand. From

3 what you are saying, if you took just mature sperm

4 and incubated them with a vector, and that might

5 even occur in the \-- there is probably a lot of

6 transcription going on in the spermatogonia,

7 though, right?

8 DR. GORDON: Yes.

9 DR. SALOMON: That must be a metabolically

10 active cell.

11 DR. GORDON: Yes.

12 DR. SALOMON: So this would probably not

13 be a criticism of studies done on the first things

14 you showed.

15 DR. GORDON: Well, here is what I did. I

16 exposed sperm to adenovirus vectors, made sure that

17 they got exposed to is, 10, 100 virions per cell,

18 and then I did in vitro fertilization with those

19 same sperm.

20 Then, the embryos that those sperm

21 conceived were evaluated for expression. The other

22 thing we did was we allowed fetuses to be produced

23 or newborns and we evaluated them by PCR.

24 Now, my opinion is there were a lot of

25 experiments that preceded those in which animals

90

1 were injected in their brain with adenovirus and

2 then bred. Well, you know, there is 300 million

3 sperm in a mouse ejaculate, and you are looking at

4 10 of them when you look at 10 pups. So that is

5 statistically not satisfying.

6 But if you have an in vitro system where

7 every cell is exposed and then you have a way of

8 assessing whether it got in, I think that you are

9 doing much more to really answer the question.

10 DR. FLOTTE: I had sort of a natural

11 history question. I was wondering if you had any

12 thoughts about human endogenous retrovirus

13 sequences in our genome and what is the most likely

14 access that those originally had to the human germ

15 line.

16 Then, a follow-up question, do you think

17 there is any significance to the fact that we don\'t

18 find human endogenous AAV sequences in the genome?

19 DR. GORDON: The first question. Well,

20 there is a tiny little sort of moment of

21 accessibility I think at hatching of the embryo in

22 vivo. The embryo has to hatch out and then

23 implant, and it is naked. That could be a point

24 where a person who had a lot of viremia or a lot of

25 virus in interstitial uterine fluid that you could

91

1 get one in.

2 I must say that in mice, retrovirus-like

3 sequences are also found endogenously in the

4 genome. That, to me, would be a logical place to

5 think of it occurring. It is very hard to imagine

6 it occurring. You could also think of a viremic

7 male having it get into a spermatogonia.

8 I mean now that it has been shown that you

9 can get it into spermatogonia, at least in vitro,

10 it might be much less probable in vitro, but if you

11 have 30 million centuries to work on it, you know,

12 you may see it. So this is exactly the point, of

13 course, about provocative testing, too.

14 So that is my view. Now, what is the

15 significance of not finding a virus, I mean I

16 really can\'t say anything about that. It could be

17 a combination of factors - I haven\'t looked enough,

18 the virus has too low an integration frequency,

19 there is not a biological setting in which there is

20 good access of a virus at a susceptible point, you

21 know, ontogeny, such as uterine fluid at a time of

22 implantation.

23 So it would only be speculation on my

24 part, I don\'t know.

25 DR. SALOMON: Dr. Dym and then Dr. Rao.

92

1 DR. DYM: I had a couple of questions, but

2 first I will thank you also for a lucid

3 presentation. I will just comment briefly that

4 there are a number of people who are using in vivo

5 approaches, as I think you know, to get viruses

6 into the spermatogonia through the seminiferous

7 tubular lumens. Brimster is one and there was a

8 paper by Blanchard & Vokalhyde in Biology of

9 Reproduction in 1997.

10 Again, they showed that it only went into

11 the Sertoli cells, but Brimster and a number of

12 others, actually, five or six labs, in monkeys and

13 in rodents and in cattle, are using this

14 seminiferous tubule injection or ret-A testis

15 injection. It is in vivo, but it is not practical.

16 I mean you can\'t put it in that way normally.

17 But this leads me to my second question

18 having to do with barriers. You mentioned

19 barriers. I do believe there are barriers from

20 your work and from other people\'s work, and that is

21 why probably virus in a muscle or systemic virus

22 may not get into the spermatogonia, but this is in

23 normal animals or maybe in normal people, but the

24 barriers actually break down when there is a

25 diseased person or a diseased animal.

93

1 I am just wondering if you know anything

2 about that and if, when the barriers break down.

3 Actually, another thought came to mind. For

4 example, in AIDS patients, the barriers are broken

5 down and the virus, which is circulating in the

6 blood, let\'s say, from a man who has gotten

7 infected via needle, the virus is in the blood, and

8 then eventually it breaks down and gets into the

9 closed lumen or semen compartments, whether it is

10 testis or epididymis, but it does get across the

11 barrier, so viruses do get across in diseased

12 conditions.

13 Some of these patients you are talking

14 about might have a breakdown of the barrier.

15 DR. GORDON: I am glad you actually

16 mentioned that because I think it is worth some

17 comment. First of all, I think viruses might be

18 able to break the barrier and then go through. I

19 mean viruses can hurt cells, and if you flood cells

20 with them, you might get a weakening of a barrier

21 by the very action of the virus.

22 Then, there are disease states. Disease

23 states are exposed internal portion of the

24 seminiferous tubules to the outside, I think

25 intuitively are not likely to be so flagrant as to

94

1 raise the risk significantly just because I think

2 that would have a big impact on spermatogenesis,

3 too, but I did want to say that there are ways \--

4 well, the FDA speaker was point out that localized

5 injection is less risky than perhaps systemic

6 injection, but I think one exception should be

7 taken to that, and that is injections into things

8 like the prostate, which by no means is an inactive

9 area of research, so I do agree that while these

10 barriers exist, one cannot predict from that

11 intuition that in all of the settings of gene

12 therapy, where a vector\'s ability to cross barriers

13 may vary, or a vector\'s ability to violate the

14 barrier and get in on their own may vary, where

15 disease states may vary.

16 So biologically, these barriers exist, but

17 I think it is quite true that you can by no means

18 be guaranteed that they are going to protect you

19 completely, and provocative testing is needed.

20 DR. RAO: You give a very nice summary, at

21 least for me, in terms of understanding that there

22 is great protection of the male and female gametes.

23 So, let\'s say you do, in fact, a patient

24 with adeno-associated virus at some titer, 1011,

25 and now see adeno-associated virus in ejaculate.

95

1 What would you speculate as which cell was infected

2 and does it have to actually be an integration

3 event that you are seeing this one year later?

4 DR. GORDON: No, I don\'t think it has to

5 be an integration. A year later is really a long

6 time. But weeks later, as what happened in this

7 case that probably prompted this discussion, could

8 be in anything, could be seen in the fluid

9 component, could be in other cells, there is always

10 a few white cells perhaps, could be in the debris

11 that would slough off from endothelium, not at all

12 necessarily in sperm, and even if it came out with

13 sperm, that doesn\'t mean it is in them. It could

14 be just on them, and washing them could take care

15 of it, or IVF could take care of it.

16 I think it is reasonable if a sperm

17 fraction in infractionated semen is positive to

18 step back and say, well, now, a red flag has been

19 risen. If you find it in whole semen it really

20 could be from any variety of sources.

21 DR. DYM: Just one more comment maybe in

22 relation to what you said. You know, those of us

23 who work in the testis, and there are many of us

24 working on spermatogonia who are actually trying to

25 infect and transduce the spermatogonia and the germ

96

1 cells, we never think of doing it in the sperm, we

2 always think of doing it in the spermatogonia as

3 the only permanent way.

4 I think that maybe addresses some point

5 that you made. That would be permanent, you know,

6 generation after generation after generation. It\'s

7 an eternal cell, it\'s an immortal cell, the

8 spermatogonia. The sperm dies.

9 DR. RAO: The reason I asked the question

10 was one needs to evaluate, when you are looking at

11 any kind of risk, as to where the virus particle is

12 present, and that is an important thing that we

13 need to clarify if you are going to say that you

14 detected in the sperm or in the ejaculate where is

15 it really going to be present.

16 From what we heard, it is unlikely to be

17 present in the sperm per se, at least in the sperm

18 DNA, and given what we have heard about integration

19 events, maybe it is unlikely to be present in the

20 spermatogonia, but we need to know it. It is best

21 to ask the expert directly.

22 DR. GORDON: Well, I just would say that

23 if you found it in semen a year later, I would be a

24 little more worried that it got into is

25 spermatogonium because, as he said, that is an

97

1 immortal cell. Spermatogenesis proceeds in waves,

2 and if you get it into any cell that is not the

3 Type A spermatogonium, you may have its appearance,

4 but then it will disappear.

5 That is why people are trying to do

6 spermatogonia, but I must add that there are a

7 number of papers in the literature, none of which I

8 believe, but there is man of them saying that you

9 can get DNA into mature sperm by a variety of

10 methods - opening the epididymis and giving it an

11 electrical shock with your biorad electroparator,

12 people will say that works. I mean you should see

13 those data, they are so pathetic, but nonetheless,

14 they are published, so what can you say, the data

15 are published.

16 DR. SALOMON: I would like to call this

17 session to the break. We will see everybody back

18 in 10 minutes.

19 \[Recess.\]

20 DR. SALOMON: We will go ahead and get

21 started.

22 This portion of the session, we are going

23 to have a series of presentations from Avigen and

24 then from the University of Pennsylvania.

25 The next two speakers will provide us some

98

1 specific information on the AAV vector from Avigen.

2 The first speaker is Mark Kay. Welcome.

3 A Phase I Trial of AAV-Mediated Liver-Directed

4 Gene Therapy for Hemophilia B

5 Mark Kay, M.D., Ph.D.

6 DR. KAY: Thank you.

7 What I would like to do is summarize our

8 Phase I trial of AAV-mediated liver-directed gene

9 therapy for hemophilia B, which is a collaborative

10 effort between many investigators at Stanford, the

11 Children\'s Hospital, Philadelphia, and Avigen.

12 \[Slide.

13 Today\'s focus are issues pertaining to the

14 inadvertent germline transmission of AAV vector and

15 what I would like to do is summarize data related

16 the clinical trial to date.

17 \[Slide.

18 There has been some discussion about

19 integration of AAV in the liver, and although Jude

20 suggested that I was going to show data about

21 integration, I actually have those slides, but not

22 in this particular talk, so let me just summarize

23 where things are and give some explanation.

24 We know that, in general, if you inject

25 reasonable high doses of AAV into mice that you can

99

1 get something in the neighborhood of 50 percent of

2 hepatocytes that are stably modified with AAV. In

3 some situations, it might be slightly higher or

4 lower.

5 Now, it turns out that if you give these

6 regular doses of AAV into mice, the vector genomes

7 actually get into almost 100 percent of the

8 hepatocyte nuclei, but over time, most of those

9 single stranded genomes are lost and here is only a

10 small proportion of cells that remain with stably

11 transduced vector genomes

12 Now, the proportion of integrated genomes

13 is actually small. Generally, it is actually less

14 than 5 percent. I think the definitive evidence

15 that AAV integrated in liver was a study done in

16 collaboration with Linda Couto and Hikiyuki \[ph\]

17 Nikai, where they actually were able to clone out

18 integration junctions, so basically within the

19 vector, they put bacterial origins of replication

20 and then were able to take genomic DNA, put them

21 back in the bacteria, and clone out the covalent

22 linkage of the vector where it integrated into the

23 genome.

24 Now, this was a very useful technology,

25 but it does not quantify how much integration

100

1 actually occurred. So we have recently published

2 on studies where we have injected AAV into animals

3 and we wait for a period of time until there is

4 stable transduction, and then what we actually do

5 is a hepatectomy.

6 Now liver cells will equally regenerate,

7 such that each cell divides once or twice, and as a

8 result, DNA genomes that are not associated with

9 centromeres or telimeres are lost, and we have

10 positive and negative controls for this, and what

11 we find is that in most situations, the amount of

12 integrated genomes, of the stable genomes is very

13 small, it is usually less than 5 or 10 percent of

14 the double-stranded vector DNA.

15 Now, gene expression from the integrated

16 forms, which again is small, and the episomal

17 forms, parallels the proportion of vector DNA in

18 each state, so if you do a partial hepatectomy and

19 you look at the amount of vector genomes before and

20 after, you get around 90 to 95 percent reduction

21 both in gene expression and in number of genomes,

22 again indicating that most of the expression comes

23 from the episomal forms.

24 There is no detectable increase in the

25 proportion of integrated genomes over time, and

101

1 very recently we have tried to push these animals,

2 giving them extremely high doses in the range of

3 1014 to 1015 per kilo, and we do not increase the

4 proportion of integrated genomes.

5 The proportion of transduced cells with

6 integrated genomes is small and most integrates

7 that when we have actually molelecularly analyzed

8 them are 1 or 2 copy genomes.

9 \[Slide.

10 The clinical trial objective is to test

11 the hypothesis that AAV mediated liver-directed

12 gene transfer is safe; characterize the human

13 immune response to the transgene product and to the

14 vector; determine whether germline transmission of

15 vector occurs following hepatic administration; and

16 determine dose capable of producing clinically

17 relevant factor IX levels in the blood.

18 \[Slide.

19 It\'s a Phase I open-label, dose escalation

20 safety trial of AAV Human Factor IX administration

21 by infusion into the hepatic artery.

22 \[Slide.

23 The vector is infused into the liver via a

24 balloon occlusion catheter placed in the hepatic

25 artery, and Factor IX protein is administered

102

1 before and follow the procedure to cover the

2 patients from any type of bleeding.

3 Subjects are observed for at least 24

4 hours

5 \[Slide.

6 This is the dose escalation plan of the

7 trial as it is written. The dose in vector genomes

8 is 2 x 1011 per kilogram. The observed levels in

9 mice is somewhere between undetectable and 1

10 percent.

11 Importantly, is that when you get into the

12 second cohort, we were at a dose of 1 x 1012 per

13 kilo, and in dogs that were given a similar, not

14 identical vector, levels in the range of 4 to 12

15 percent are achieved.

16 These levels of Factor IX would result in

17 a substantial improvement in the clinical course

18 with the individuals going from a severe phenotype

19 to that of a much milder phenotype. So this would

20 be somewhere in an efficacious range, so the point

21 is that at doses within this trial, we are at

22 efficacious doses in a dog model of hemophilia.

23 \[Pause.\]

24 DR. KAY: I am really sorry. There was a

25 mix-up about transferring the slides, so I

103

1 apologize.

2 This was just an introductory slide about

3 hemophilia, basically that it is a very well

4 understood disease and with sustained levels of 1

5 percent, you can get a therapeutic response, and we

6 do have very good animal models which are the dogs.

7 Now, this is basically what I said, that

8 we have actually been able, we and others and more

9 recently Kathy High\'s group, has gotten reasonably

10 high and therapeutic levels of canine factor IX in

11 dogs reaching 4 to 12 percent. I won\'t go through

12 this again

13 \[Slide.

14 This is just a photograph of a patient who

15 is being treated here. As I said, it is through

16 the hepatic artery and they go into the invasive

17 radiology suite. A catheter is inserted into the

18 femoral artery and it is cannulated into the

19 hepatic artery, which can be followed by

20 fluoroscopy here, and then the vector is placed on

21 an infusion pump, as shown here, and then

22 administered at a specific rate into the patient

23 \[Slide.

24 Now, the first subject that was treated is

25 a 63-year-old male with severe factor IX

104

1 deficiency. Status/post bilateral knee

2 replacements 5 years prior to the procedure. He is

3 HIV-negative. He was HCV-positive, but his HCV

4 viral load by PCR was negative on multiple

5 occasions several years apart. Per our protocol,

6 these patients are considered to have spontaneously

7 cleared HCV, and do not require liver evaluation

8 before being enrolled into the trial.

9 He is the father of 3 and he has a

10 grandson with hemophilia.

11 \[Slide.

12 The first procedure was done in August of

13 last year. He received 2 x 1011 vector genomes per

14 kilogram. No complications. He was discharged

15 home to his referring hemophilia treatment center

16 after five days

17 \[Slide.

18 This is a summary of his clinical data

19 baseline before the procedure and afterwards out to

20 week 24. The important point here is that his CBCs

21 have all been within normal limits including

22 platelet counts, which have been an issue with some

23 of the adenovirus trials

24 \[Slide.

25 His liver function studies and prothrombin

105

1 times have also remained normal, as shown here.

2 His ALT and AST are normal, and they remained

3 normal throughout the 24-week period for which he

4 has bee monitored.

5 So the hepatic administration of this

6 vector in this patient did not appear to have any

7 liver injury

8 \[Slide.

9 The coagulation data for this first

10 patient is shown here. His factor IX levels have

11 basically remained at a subtherapeutic or

12 nontherapeutic level. This basically is

13 background. Remember that these patients do treat

14 themselves.

15 The important issue here, too, is that

16 this patient did not have detectable factor IX

17 inhibitor by Bethesda assay.

18 \[Slide.

19 One of the aspects of the protocol is to

20 monitor the different body fluids for vector

21 shedding and, of course, the reason why we are here

22 today. This just is a very simplified diagram of

23 the PCR assay that is done by Deb Leonards\' group

24 at the University of Pennsylvania

25 \[Slide.

106

1 This shows the actual sequence of the

2 vector and the PCR primers are depicted here as a

3 control for the PCR reaction itself. Some of the

4 samples are spiked with very small plasmid numbers

5 of a second vector that has the same sequences for

6 the primers, but there has been a deletion of 97

7 base pairs, so one can distinguish between the

8 spiked copy, if you will, and the vector copy

9 \[Slide.

10 This just shows an example of one of the

11 gels of this analysis here. This is the baseline

12 sample here. This is the spiked sample below, and

13 this is day seven of a body fluid where you can see

14 both the spiked and the actual vector band shown

15 here. So this gives you an idea of the PCR

16 studies. Some of these will be discussed again in

17 more detail with some of the preclinical studies.

18 If we look at the vector sequences by PCR,

19 in the different body fluids here, in the first

20 patient, again, we see transient vector DNA up

21 until week 2 in the serum, transiently for a couple

22 of days in saliva, there was none in urine and

23 stool, and white blood cell pellet was done at week

24 12, but that was negative

25 \[Slide.

107

1 This is what was somewhat of a surprise to

2 us based on dog studies we had done. In fact, when

3 we did look at his vector DNA in semen, we did find

4 that there was DNA present in his semen, but it was

5 transient and it slowly fell off over a period, and

6 after week 12, has remained persistently negative.

7 Now, these samples are performed in

8 triplicate in 1-microgram DNA samples. When we did

9 get positivity in these first couple of samples, we

10 went to a fractionation procedure to try to

11 fractionate out the motile sperm fraction from the

12 seminal fluid sample and the pellet.

13 Now, in this motile sperm fraction, we

14 were only able to get 220 nanograms of DNA, so it

15 wasn\'t the 1 microgram, but this amount of DNA was

16 PCR-negative in this individual.

17 I also want to point out the sensitivity

18 of the assay is less than 1 copy per 30,000 haploid

19 genomes or, in other words, 1 copy per 30,000

20 sperm.

21 Now, as a result of this result, we did

22 make some changes in the consent form related to

23 the issue of informing the patients about this

24 result, and basically, what it says the study

25 subjects shall be adult males who are 18 years of

108

1 age or older.

2 The first patient treated under this

3 protocol was very shown by very sensitive

4 techniques to have vector in his semen for as long

5 as 10 weeks after treatment. Although the vector

6 was not found in the sperm fraction, the

7 significance of this finding is unclear, and all

8 patients are strongly urged to use barrier birth

9 control devices, condoms, until the patient is

10 informed that semen has been clear of vector for at

11 least three months.

12 The investigators will notify you when it

13 is safe to stop barrier methods of birth control.

14 The consequences of gene transfer, the germline

15 cells are unknown, but could potentially result in

16 serious birth defects or fetal death or other

17 unanticipated health consequences, such as cancer,

18 in the offspring due to the disruption of normal

19 genes by the transferred DNA. If you are

20 considering having children in the future, it is

21 recommended that you bank sperm before beginning

22 the procedure to ensure a source of sperm that is

23 free of contamination with the vector.

24 The reason for storing semen is that it is

25 possible that if the sperm cells do take up the

109

1 vector during the procedure, it may or may not

2 result in life-long changes to the sperm. The

3 investigators will provide you with information on

4 sperm banking and this one is for Stanford at

5 Stanford University or at your home institution.

6 This opportunity will be provided to you at no

7 additional expense.

8 So the point here is that we urge the

9 individuals to undergo a barrier contraception, we

10 talk about the risk in this first patient, and the

11 fact that we will sperm bank in case they are

12 considering or uncertain about future childbearing.

13 Now, because of this issue of finding, at

14 least in the first patient, transient AAV vector

15 sequences in the semen, we amended the plan to

16 address this issue of inadvertent germline

17 transmission, and the protocol was changed, so that

18 semen collection was done as a baseline, and then

19 at weeks 1, 8, 12, 16, or possible more.

20 Now, the idea was, and the plan is, that

21 beginning at 8 weeks, the sample is then

22 fractionated and total semen and motile fractions

23 are analyzed for vector genomes by PCR. If the

24 8-week motile sperm fraction is negative, we would

25 be allowed to proceed to the next dose cohort. All

110

1 subjects to practice barrier contraception until

2 three consecutive monthly semen samples are

3 negative.

4 So, although we will test and fractionate

5 through week 16, the question is we continue if

6 there haven\'t been three successive negative semen

7 samples

8 \[Slide.

9 Subject 2 was a 48-year-old male with

10 severe hemophilia B. He had a bilateral knee

11 replacement in 1999 and elbow replacement in 2001.

12 He is HIV-positive and HCV-positive. He

13 underwent a liver biopsy and was shown to have

14 minimal fibrosis and based on criteria in the

15 protocol, was allowed to be included in the study.

16 He had a non-Hodgkin\'s large cell lymphoma

17 in 1986, was treated, had a relapse in 1996, and

18 was treated and he is on medications for his HIV

19 \[Slide.

20 The procedure was performed in January,

21 the end of January of this year, received the same

22 dose as the first patient. No complications. Went

23 back after 7 days

24 \[Slide.

25 Patient 2, like Patient 1, had totally

111

1 normal LFTs, no elevations related to the vector

2 \[Slide.

3 Renal function, not shown with the first

4 patient, but were also normal in the second patient

5 \[Slide.

6 Again, the CBC including the platelet

7 counts were normal. There was no elevation with

8 vector administration

9 \[Slide.

10 Now, with the second patient, again, we

11 see no evidence of inhibitors, and we have also

12 noticed that there is a question of whether there

13 is any detectable factor IX in this patient. The

14 week 8 and week 12 samples were obtained at least

15 14 days prior to factor IX administration, and

16 there are some low levels of factor IX here

17 detectable, but again it is unclear whether this is

18 really and truly from gene transfer. I just wanted

19 to point out that this is the data to date. So it

20 is still questionable

21 \[Slide.

22 Now, when we looked at his body fluids,

23 the saliva was positive for a slightly longer

24 period of time, up to one week. His serum was also

25 positive up to four weeks, which again was two

112

1 weeks longer than the first patient.

2 Unlike the first patient, we did see

3 transient positivity in the urine, but only out

4 until day 2, and he also has had some positive

5 stool samples, as well

6 \[Slide.

7 Now, this is where we are with the semen

8 analysis for the vector DNA. He has remained

9 positive up through week 14, but let me talk about

10 the total semen first.

11 The total semen, the signal of the PCR has

12 started to diminish, similarly to what we have seen

13 in Patient 1. If you remember Patient 1, he was

14 persistently negative after week 12, and the week

15 14 sample, which we just obtained this week,

16 although it was positive, the signal appears to be

17 weak, so it appears to be going down in

18 concentration, although this is not an absolutely

19 quantitative assay.

20 Now according to the protocol, we were

21 supposed to fractionate his week 8 sample into the

22 fractions that I discussed earlier, to look at the

23 motile sperm fraction, but it turns out that this

24 individual has ejaculate volumes that are well

25 below half a ml. When the sample went to the lab,

113

1 it has got to be fractionated within about 30

2 minutes or so, and when they got the sample, the

3 lab said, you know, based on our SOP that we have,

4 and the one that is provided in the protocol, this

5 volume is not adequate to fractionate, so it wasn\'t

6 fractionated.

7 Well, we went back, and after discussions

8 with FDA and our colleagues, we realized that there

9 are standard operating procedures in these clinical

10 laboratories to fractionate low-volume ejaculates,

11 and this then was attempted on the week 14 sample.

12 But unfortunately, the DNA recovery from

13 this week 14 sample was such that it would only be

14 possible to run triplicate samples of 300 nanograms

15 per ml, and based on our changes in the protocol,

16 which we have just sent to the FDA, this would be a

17 fractionated sample that we would not analyze. So

18 the fractionated sample with 300 nanograms in it

19 was not analyzed by PCR.

20 It has turned out that although it is

21 simple in theory, it has been difficult, a little

22 more difficult than we had anticipated doing these

23 fractionation procedures and getting the kinds of

24 DNA recoveries that one would want.

25 This individual has supernormal sperm

114

1 counts so although his volume is low, it appears

2 that spermatogenesis in this individual appears to

3 be normal because his counts are well above normal.

4 It also turns out that there are lots of

5 rules and regulations in the labs that do the

6 fractionation. In fact, we are learning that many

7 of these labs are not allowed to fractionate

8 HIV-positive samples, which has also led to some of

9 the difficulty in getting these specimens

10 fractionated at will.

11 So based on this, we have added new

12 exclusion criteria. We realize that this

13 individual has an issue with ejaculate volume, but

14 with normal sperm counts, that is very, very rare

15 and unusual, but because of this in this patient,

16 we have added an additional exclusion criteria to a

17 revised protocol.

18 First of all, we state in there that an

19 exclusion issue are related to patients who are

20 unwilling to provide required semen samples, and

21 patients that are unable to provide semen samples

22 of adequate semen volume, which we define at 1 1/4

23 ml sperm count, and we define the cutoff at 20 x

24 106 sperm per ml, and with motility of greater than

25 50 percent. Again, this was based on the data we

115

1 have obtained from this Patient No. 2.

2 \[Slide.

3 So, in conclusion, I can say that Subjects

4 1 and 2 have tolerated the procedure well, vector

5 DNA is present transiently and total semen from

6 Subject 1, not present in the motile sperm fraction

7 at week 3, albeit the sample that was analyzed was

8 220 nanograms, not the desired 1 microgram.

9 We have much limited data in Subject 2

10 although the signal is going down, we still haven\'t

11 detected a sample that has been negative, and

12 currently, based on what has been approved, that

13 the enrollment of the subjects at the mid-dose

14 proceeds only if Subject 2 shows absence of signal

15 in the motile sperm fraction.

16 So, in summary, what I would like to say

17 is that clinical studies demonstrate safety and

18 long-term efficacy of AAV factor IX in the liver in

19 the large animal model of hemophilia. We think

20 that this is really the impetus to move forward.

21 The initial clinical studies indicate that

22 this gene transfer strategy can be safety

23 translated into human subjects, and we strongly

24 believe that the completion of the Phase I study is

25 required for valid risk-benefit analysis of the

116

1 strategy.

2 We would like to present a proposal to you

3 of what we would see as a reasonable route of

4 moving forward, but before we do that, there will

5 be two additional speakers who are going to present

6 the preclinical data studies that have been done to

7 try to address this issue, what has been done, the

8 data to date, future studies in a number of

9 different animal settings.

10 Thank you.

11 DR. SALOMON: Thank you very much.

12 We won\'t have any questions until after

13 the second speaker.

14 This second talk is from Linda Couto of

15 Avigen entitled Safety Studies to Support

16 Intrahepatic Delivery of AAV.

17 Safety Studies to Support Intrahepatic Delivery

18 of AAV

19 Linda Couto, Ph.D.

20 DR. COUTO: I am going to describe a

21 series of preclinical studies that were performed

22 to evaluate the safety of delivering AAV to the

23 hepatic artery

24 \[Slide.

25 We have used five different species -

117

1 mice, rats, dogs, rabbits, and monkeys to assess

2 the toxicology and biodistribution, but today, I am

3 going to limit my talk just to the biodistribution

4 studies that are relevant to inadvertent germline

5 transmission

6 \[Slide.

7 I am going to summarize the studies in

8 rats, dogs, and monkeys, and then Valder Arruda is

9 going to present some more recent data in rabbits,

10 which appear to be probably the best model for

11 studying inadvertent germline transmission.

12 However, before discussing the

13 biodistribution data, I just want to point out that

14 in all of these five species, we haven\'t seen any

15 toxicology at doses up to 1 x 1013 vector genomes

16 per kilogram, which is 50-fold higher than our

17 starting clinical dose.

18 This is the biodistribution study that was

19 performed in rats. In this study there were five

20 groups of animals. One group was treated with the

21 excipient. One group was treated with an AAV null

22 vector, which does not contain a transgene. Then,

23 there were three groups of animals that were

24 injected with increasing doses of an AAV factor IX

25 vector from 1 x 1011 per kilogram to 1 x 1013 per

118

1 kilogram.

2 So what you can see is that at 50 days

3 post-injection, we saw a good gene transfer to the

4 liver, so at the low dose we were seeing about 1

5 copy per 60 cells in the liver, and at the high

6 dose we were seeing about 1 copy per 1 to 2 cells.

7 At this time point, we also did see vector

8 dissemination to the gonads at least in some of the

9 animals. At the low dose we didn\'t see any

10 dissemination, but at the high dose we saw about 1

11 copy per 1,700 cells, so this was about 1,000-fold

12 lower than the gene transfer we were seeing in the

13 liver.

14 At this time point, we were also seeing

15 vector in the blood, however, by day 92

16 post-injection, we no longer detected any sequences

17 in the blood, and the level of gene transfer to the

18 liver and the gonads had decreased.

19 So, at the 92-day time point, we were

20 seeing about 1 vector copy per 4 cells in the

21 liver, and only about 1 copy per 4,000 cells in the

22 gonads, but only in the highest dosed animals.

23 \[Slide.

24 We also did a gonadal distribution study

25 in dogs. In this study, three normal dogs were

119

1 injected with AAV null vector at doses ranging from

2 3.7 to 7 x 1012 vectors genomes per kilogram, and

3 in this study, the vector was delivered using the

4 method that we are using in the clinic. So, a

5 catheter was inserted into the femoral artery and

6 then using fluoroscopic guidance was advanced to

7 the hepatic artery where the vector was infused.

8 Then, semen samples were collected at various times

9 post-injection.

10 In addition to the semen samples, we also

11 looked at toxicology parameters and also looked at

12 gonadal tissue at the time of sacrifice.

13 In this experiment, we used the AAV null

14 vector, which contains a promotor list transgene.

15 The reason for using this was just to prevent any

16 CTL response, eliminating the transduced cells.

17 \[Slide.

18 So, these are the results of PCR analysis

19 of the dog semen. The lower panel here represents

20 an ethidium bromide stain gel of the PCR products,

21 and over here on the right you can see that the

22 level of sensitivity is about 100 copies per

23 microgram. At this level of sensitivity, there is

24 no evidence of vector sequences in any of the dogs

25 at any of the time points out to day 90.

120

1 We also did a southern blot of this gel,

2 and increased the sensitivity down to 10 copies per

3 microgram, which is 1 copy per 30,000 haploid

4 genomes, and again we are not seeing any detection

5 of sequences in the semen of these dogs.

6 We also performed PCR on gonadal tissue

7 and again we didn\'t see any evidence of

8 dissemination to the gonads in these animals.

9 \[Slide.

10 More recently we have looked at toxicology

11 and biodistribution in the non-human primates, and

12 in this study we have treated 6 cynomolgus monkeys,

13 2 animals were treated with the excipient, 2

14 animals got a factor IX vector at a dose of 7 x

15 1012 into the hepatic artery, and another 2 animals

16 received the same dose of vector via the portal

17 vein.

18 This study was designed as a toxicology

19 study, but we tried to get some limited

20 biodistribution study by harvesting the liver and

21 the gonads and doing PCR analysis when the animals

22 were sacrificed at day 135

23 \[Slide.

24 This is the results of that study. What

25 you can is that in 2 of the 4 injected animals, we

121

1 saw gene transfer to the liver. It is not really

2 clear why only 2 of the 4 animals worked, but what

3 we can say is that in those 2 animals, gene

4 transfer was relatively efficient, so 1 of the

5 animals that got the vector via hepatic artery, we

6 saw vector genomes at about 1 vector sequence per 3

7 cells, and in the other animal we saw 1 to 2 vector

8 sequences per cell.

9 What we also saw was, you know, despite

10 this high level of gene transfer to the liver, we

11 did not detect any sequences in the gonads, and the

12 level of sensitivity in this particular PCR assay

13 was 1 copy per 40,000 diploid cells.

14 \[Slide.

15 We also took advantage of this non-human

16 primate testes to ask the question whether any of

17 the cells in the testes had the receptor for AAV,

18 which Jude Samulski\'s group had previously reported

19 to be heparan sulfate proteoglycan.

20 So what we are looking at here is a

21 stained section of the non-human private testes,

22 and the heparan sulfate proteoglycan is stained and

23 nuclei are stained blue with DAPI. What you can

24 clearly see is that the receptor, heparan sulfate

25 proteoglycan is present in the basement membranes

122

1 surrounding the seminiferous tubules, but none of

2 the spermatogenic cells are staining positive to

3 HSPG, suggesting that these cells would be

4 non-permissive for AAV infection.

5 What I have just shown you has

6 demonstrated that at least in some animal species,

7 we do see dissemination of AAV vector to gonads,

8 and although we didn\'t see dissemination of vector

9 to the semen in dogs, Valder Arruda will show some

10 data demonstrating that we do get vector

11 dissemination to semen of rabbits, and Mark Kay

12 also just presented our data from the clinical

13 trial demonstrating that we are seeing vector

14 dissemination in human patients.

15 So there certainly is the risk for both

16 horizontal and vertical germline transmission.

17 What I would like to present now are some studies

18 that we have been working on and also some

19 published work that addresses the risk of AAV

20 dissemination in both horizontal and vertical

21 transmission.

22 The first study is a paper from Philip

23 Moray\'s \[ph\] lab looking at vector shedding in a

24 number of biological fluids, and then I will

25 present the development of a cell-based infectivity

123

1 assay, so that we can now begin to look at

2 biological activity of AAV in semen samples.

3 Then, I will also address the issue of

4 vertical transmission by describing an experiment

5 that we have initiated in collaboration with Dr.

6 Jon Gordon to see whether AAV can infect murine

7 sperm cells

8 \[Slide.

9 So, the study that was published in the

10 Journal of Molecular Therapy last December from

11 Philip Moray\'s group is shown on this slide. They

12 injected 8 monkeys with an AAV-Epo vector at doses

13 ranging from 5 x 108 to 1 x 1010 infectious units

14 per kilogram, and their vector had a particle

15 infectivity ratio of about 100.

16 This vector was inject intramuscularly and

17 then at various time points post-injection, a

18 number of body fluids, such as serum, feces, urine,

19 saliva, lacrimal and nasal, but not semen, were

20 evaluated both by PCR for vector sequences and

21 using a replication center assay to look for

22 biologically active AAV.

23 In addition, they looked in the peripheral

24 blood mononuclear cells for vector sequences

25 \[Slide.

124

1 This is a figure from their paper, which

2 shows the results of the replication center assay.

3 In this assay, cells are coinfected with AAV and

4 the helper virus for AAD, adenovirus. Following

5 incubation for several days, the cells are

6 harvested and filtered onto a nylon membrane and

7 then harbodized to a radioactive probe.

8 So what we are looking here is the ability

9 of AAV in the presence of its helper virus, to both

10 infect and replicate in this cell.

11 The panel on the lefthand slide shows the

12 controls. This is AAV that has been spiked just

13 into media, and you can detect 1,000 down to 1

14 infectious unit. However, when the AAV is spiked

15 into either serum, feces, urine, the level of

16 sensitivity in the assay decreased about 10- to

17 100-fold.

18 On the righthand portion of the slide is

19 the results of testing the serum from two of the

20 monkeys, and you can see that 30 minutes

21 post-injection there is evidence of biologically

22 active AAV in the serum, and you can also detect

23 some activity one day and two days post-injection,

24 but by five days post-injection, there is no longer

25 any biological activity in the serum

125

1 \[Slide.

2 This slide just summarizes the results

3 from all 8 monkeys. The red bars indicate a

4 30-minute time point. The yellow bars represent a

5 one-day time point, and the blue bars represent the

6 two-day time point.

7 Basically, you can see that in all of the

8 animals, by three to four days post-injection,

9 there is no longer any biologically active AAV in

10 the serum. They also tested other body fluids, but

11 they only found activity in the serum.

12 \[Slide.

13 Finally, they also looked for AAV

14 sequences in peripheral blood mononuclear cells,

15 and surprisingly, they were able to detect this

16 signal out to 10 to 15 months post-injection. So

17 vector sequences can be persistently detected in

18 the peripheral blood mononuclear cells

19 \[Slide.

20 So, just to summarize their data, AAV

21 vector sequences are detected in all body fluids by

22 PCR for approximately 6 days. I didn\'t show you

23 this, but they did also demonstrate that the PCR

24 signal is due to packaged AAV sequences rather than

25 free DNA.

126

1 They also demonstrated that biologically

2 active AAV was detected in serum for 48 to 72

3 hours, suggesting that the risk of horizontal

4 transmission is limited to a short period of time

5 post-injection.

6 Finally, they also concluded that vector

7 sequences can be detected in the PBMCs for as long

8 as 10 to 15 months following an intramuscular

9 administration

10 \[Slide.

11 After discussions with the FDA and also

12 following the December RAC meeting, it became clear

13 that it was important to develop an assay, so that

14 we could detect or try to detect biologically

15 active AAV in semen samples, so this just

16 schematically illustrates the assay that we have

17 been developing.

18 Basically, it is similar to the

19 replication center assay that I just described,

20 however, the readout of replication in this case

21 relies on a quantitative PCR assay rather than a

22 hybridization.

23 So basically HeLa cells which express the

24 AAR Rep and Cap genes are incubated in the presence

25 of 100 microliters of semen with increasing doses

127

1 of an AAV factor IX vector and with adenovirus.

2 Then, 72 post-injection the cells are

3 harvested, DNA extracted, and subjected to

4 quantitative PCR

5 \[Slide.

6 This just depicts a typical result from

7 this assay where the intensity of the red color is

8 meant to represent the amount of PCR amplification

9 detected in the well. So in the case of just

10 spiking vector into media, you can see that we can

11 detect as few as 10 to 50 vector genomes per well,

12 however, when the AAV is spiked into semen, the

13 level of sensitivity of the assay decreases about

14 10-fold, so that now the lowest dose that results

15 in an amplification signal is 500 vector genomes

16 per well per 100 microliters or 5,000 vector

17 genomes per ml of semen.

18 So what we intend to do with patient and

19 animal semen is two different assays. First of

20 all, we will simply extract DNA from the sample and

21 do quantitative PCR to determine the number of

22 vector genome per ml, and in addition, we will take

23 a portion of the sample, run the infectivity assay

24 to determine the infectious units per ml of semen,

25 and then we will be able to monitor and compare the

128

1 kinetics of clearance of both the physical and the

2 infectious particles.

3 \[Slide.

4 To address the issue of vertical

5 transmission, as I mentioned, we have initiated a

6 collaboration with Dr. Gordon. In this case, what

7 we propose to do is expose murine sperm cells to

8 very high doses of AAV.

9 We feel this is a very rigorous test of

10 whether AAV can actually transduce sperm although a

11 very non-natural situation, but this slide just

12 illustrates the steps that are being taken.

13 First of all, murine sperm are isolated

14 and then exposed to an AAV factor IX vector at an

15 MOI of about 1,000. The sperm are used in an in

16 vitro fertilization, and then the fertilized

17 oocytes are implanted into pseudopregnant females.

18 The fetuses will be harvested 10 to 12

19 days post-gestation, DNA will be extracted and

20 subjected to southern blot analysis, and what we

21 will be looking for is single copy AAV factor IX

22 sequences in genomic DNA, and if we are able to

23 detect one copy per diploid genome, that will be

24 used as evidence of vertical germline transmission

25 \[Slide.

129

1 Just to summarize what I have just been

2 discussing, first of all, the extent of vector

3 dissemination to animal tissues correlates with

4 dose and decreases with time.

5 Following intrahepatic delivery of AAV,

6 vector is either absent from gonadal tissue, which

7 was the case in the dogs and the non-human

8 primates, or present at levels 1,000-fold lower

9 than liver, as in the case with the rats, and in

10 this case it clears with time.

11 The studies in the non-human primate

12 suggest that AAV in serum is not infectious after

13 72 hours, but vector signal can be detected in

14 PMBCs for up to 10 months after an intramuscular

15 administration.

16 The AAV receptor, HSPG, is not expressed

17 on non-human primate spermatogonial cells,

18 suggesting that these cells may not be infected by

19 AAV.

20 Finally, we believe that the data is

21 consistent with hematogenous dissemination of

22 vector to gonads with clearance over time.

23 \[Slide.

24 So the issues that we are continuing to

25 address are, first of all, is there infectious

130

1 virus in semen, and as I mentioned, we have

2 developed an infectivity assay which we intend to

3 use both on humans and animal semen samples.

4 Another question is are the vector

5 sequences in semen associated with the motile

6 sperm, other cells, or the seminal fluid, and as

7 Mark Kay mentioned, we have begun fractionating the

8 human semen samples, and we have also begun doing

9 this with rabbit samples, as you will hear in the

10 next presentation.

11 Another question that we are trying to

12 answer is can AAV infect mature sperm cells, and we

13 have initiated a study using IVF to demonstrate or

14 not demonstrate this in animal models.

15 I will stop there.

16 DR. SALOMON: Thank you very much, Linda.

17 I was thinking of doing the next talk and

18 then discussing all three of the talks as a group.

19 Would that be okay with everybody? I got the

20 feeling Avigen was kind of packaging this as a

21 group.

22 Assessing the Risk of Germline Transmission of

23 AAV in a Rabbit Model

24 Valder Arruda, M.D.

25 DR. ARRUDA: I would like to talk now

131

1 about preclinical studies, address the issue of

2 biodistribution following injection of an AAV

3 vector into rabbits as a model to analyze the

4 inadvertent germline transmission

5 \[Slide.

6 Animals in these studies were injected

7 with the same vector to be used in clinical trial.

8 The doses ranged from 1 x 1011 to 1 x 1013 vg/kg.

9 Semen is collected at serial time points

10 after injections, we intend to fractionate the

11 semen, analyze the total semen and fractions by PCR

12 developed by the human specimens, as Dr. Kay said

13 before.

14 \[Slide.

15 Although when we talking collection of

16 semen for rabbits, I like just to mention that the

17 method we are using is the natural method, using an

18 artificial vagina that has an advantage, it

19 provides an uncontaminated sample for each animal.

20 However, this method has a disadvantage.

21 The animal requires to be trained to do this

22 procedure, and this has some implication, as you

23 will hear later on during my talk.

24 \[Slide.

25 When we talk about semen, we talk actually

132

1 a marker, what happens in both genital and urinary

2 tract. Actually, 70 percent of what we call

3 ejaculate comes from the seminal vesicle, 20

4 percent from the prostate gland, and only 5 percent

5 from tests and ducts, and a small portion from

6 accessory glands.

7 Also, although spermatozoa is the main

8 cellular component of semen, there are other cells

9 that is special for our case is really important to

10 know, and these cells are present normal in fertile

11 donors like leucocytes, epithelial cells, immature

12 germline cells, and enucleated cytoplasm, and this

13 can be around the cells.

14 Also, for rabbits, are commonly found

15 debris in gel. Gel especially comes from ejaculate

16 of young animals. Together, this explains the

17 reasons why we would like to fractionate the total

18 semen before we save it in aliquot to analyze the

19 total semen, we go for fractionation to obtain the

20 motile sperm and seminal fluid and the normal type

21 \[Slide.

22 What we have up to now is actually 3

23 cohort of animals that has been injected in total

24 27 rabbits.

25 The first cohort consists of 12 animals.

133

1 They were 5 months old at the time of injection.

2 Although sexually mature, these animals were not

3 experienced in semen collection, so we are

4 restricted to analyze only later time points.

5 It was necessary to go back and look for

6 experienced animals. At this point, we could get 3

7 animals. They were 18 months old and semen was

8 collected weekly.

9 More recently, we have a group of 12

10 rabbits, median age are around 20 months, and these

11 we obtained from retired breeders.

12 What I am going to start to show to you is

13 the result of the 10 in the second cohort followed

14 by the third cohort, and only the later time point

15 for the very first group we inject.

16 \[Slide.

17 For all these animals, serum sample was

18 collected 24 hours injections for the 8 and up to 7

19 days. For all of them, we have augmented vector

20 sequences by the PCR.

21 Typically, each sample that has been

22 analyzed for each animal are represented here. We

23 run assay in triplicate with just semen and one

24 spiked experiment to exclude PCR inhibition.

25 As you can see here, this is the first

134

1 experienced rabbits that we inject. At the low

2 dose, no signal was detected, in the triplicate

3 experiment, one single band out of triplicate in

4 the mid-dose cohort, and the higher dosed animal,

5 three out of three. This higher dose, although it

6 is not a qualitative assay, is close to 10 cups of

7 vector plasmid.

8 \[Slide.

9 So this table shows the serial time points

10 from the three experiments, rabbits ranged for 7

11 days following injection up to 115 days. Each

12 assay, as you can see here, was run in triplicate

13 in the yellow line, the semen that was detected as

14 a positive signal. For the lower dose animal, we

15 never detected any signal during this period. For

16 the mid-dose animal, signal has been detected up to

17 day 22, for the higher dose, up to day 44

18 \[Slide.

19 We attempt to fractionate the rabbit semen

20 and the optimal fractionation actually depend on

21 the size and shape of the sperm, as well as the pH

22 of the semen. At the very first time point, we use

23 parameters worked out for human semen, and actually

24 reagents for human semen, and when you look under

25 the microscope, we saw a lot of agglutinations,

135

1 cell debris. You can see that even after fall in

2 fractionation, you concentrate fraction of motile

3 sperm, but it still has a lot of debris.

4 \[Slide.

5 When one compares germ cells for human and

6 rabbits, they are different, so the volume of

7 ejaculate is smaller in rabbits, and we anticipate

8 that this would be a problem for fractionation as

9 for humans, although the density of the sperm in

10 rabbits is higher, the characteristics of this

11 sperm is different. They are pretty much the same

12 total length, but the distribution is different.

13 \[Slide.

14 So, we talked with people at this company,

15 Nidacon, and they actually in-house some reagents

16 to use for rabbits. We didn\'t use if that was

17 really helpful or not, so we just took a chance and

18 we used the reagents that have been developed for

19 rabbits. Not only the grade had changed, but also

20 the centrifugation conditions changed.

21 After that, we improved the fractionation,

22 but occasionally, we still detect 1 or 2 percent of

23 cells other than motile sperm.

24 \[Slide.

25 So these results are from the first three

136

1 experienced animals. The top animal is what I

2 showed before. So these are the points that we are

3 able to fractionate the semen in these animals.

4 As you can see, the motile sperm analysis

5 shows a positive signal in the mid-dose animal.

6 The high-dose animal, at this point, the volume was

7 not enough to allow fractionation. It was just 200

8 microliters. So, we saved it only for the total

9 semen analysis.

10 After day 7, the second time point was day

11 22, and all the animals turns out to be negative,

12 and up to here, we use the human protocol, and

13 after this, the rabbit protocol, but after that, as

14 you can see, no signal has been identified by the

15 same PCR reaction.

16 For the normal type sperm, seminal fluids,

17 again, we have seen signal positive for the

18 mid-dose group and from the higher dose group, and

19 again, for low-dose animal, we have never been able

20 to detect

21 \[Slide.

22 Now, I will show the third cohort. these

23 are 12 rabbits, experience rabbits, and we have

24 only two time points. It is important here that we

25 have time point 7 - 15 days, 15 days we didn\'t have

137

1 for the very first group, we just skipped to day

2 22nd.

3 This is the total semen. You can see that

4 three animals on the low dose cohort was positive

5 at 7 days, but became negative at 15 days. For the

6 mid-dose and the high-dose, these animals are still

7 positive although decrease in numbers at the normal

8 type sperm fraction, also we can see that the

9 higher the dose, the higher the number of animals

10 positive up to this early time point.

11 The motile sperm analysis, we have not

12 observed any positive signal for the lower dose

13 animal, a positive signal for the mid-dose and

14 high-dose, and again I would say that at this

15 point, the positive didn\'t change much from 7 to 15

16 days. We still collect today, actually, the day

17 21.

18 The last group, these are the first cohort

19 that we inject that we inexperienced at that time,

20 so it took us like a couple weeks to train these

21 animals and now they are able to provide the

22 specimen.

23 So we have here, we collect semen for

24 groups that were injected a week apart. That is

25 why we have this range of days, from the low, mid,

138

1 and high dose, they are persistent negatively until

2 day 132.

3 It is interesting as Dr. Couto showed

4 before, in non-human primates, one can detect

5 peripheral blood mononuclear blood cells positive

6 at late time points following AAV injection. Here,

7 we also have been able to detect that these

8 animals, they present positive signal in their

9 peripheral white blood cells, and the top panel

10 shows, at the same time, which corresponds to three

11 months following injection, the total semen are

12 negative.

13 I am not going to go into detail into the

14 rabbit experiment, this is just to represent a

15 schematic, a very simplistic overview to say that

16 these are numbers of days that get usually a

17 spermatogen cycle in rabbits takes up to 42 days.

18 Initially, the stem cell, it is outside

19 the protected area, so outside the blood-testis

20 barrier. After day 16, cross the blood barrier,

21 came to spermatocytes, and takes up to 10 days from

22 the mature cells, spermatozoa, to get to the semen.

23 So, you assume that the stem cell has been

24 exposed to a vector at day zero. The first time

25 point that one animal should show up a positive

139

1 signal in the semen started at day 52, maybe with a

2 peak at day 58, and after that, you have the

3 steady-state signal.

4 If you put back the three cohorts of

5 animals we inject so far, we can tell the

6 following. We detect PCR-positive signal in total

7 semen or some fractions, 7, 15, 20, and 44 days.

8 The PCR becomes negative, the old sample tests

9 after day 50. This is for the first three cohorts

10 of animals, and this is for the very first 12

11 animals that we start collecting at day 86 up to

12 day 1 to 132.

13 If we consider that the rabbits

14 spermatogenesis single sites 44, 42, 46 days, at

15 this time point that we analyze, we will be able to

16 analyze at least two to three sites of the total

17 rabbit spermatogenesis.

18 Although it doesn\'t look like we are

19 transducing any immature or stem cell at this point

20 following only two or three sites of

21 spermatogenesis, there has been talk before here

22 was that possibility that actually the vector cross

23 the basal compartment, cross the blood-testis

24 barrier, and gets into more mature cells at this

25 point.

140

1 I even should skip this, but I will try to

2 do what has been said before that may not work, and

3 didn\'t work, that is was put multi-sperm of these

4 rabbits in culture. This is just to show \-- I hope

5 you can appreciate these are spermatozoas, and

6 these we still found some cells into the motile

7 sperm fraction, and unfortunately, by this computer

8 thing, we cannot make the picture come out.

9 What we did, we exposed these cells to

10 AAV2 under a CMV control expressing a GFP. The MOI

11 used a range from 1 to 5,000, and the committee has

12 a cut that we provide, shows that only that cell

13 that I identify here, it looks like a bean or

14 something like that, actually turns out to be

15 positive for GFP. Any other, the motile cells were

16 positive.

17 So, initially, for the muscle trial, we

18 performed pretty much a similar series of

19 experiments, used the same model, the rabbit, and I

20 would just like to summarize this. This actually

21 has been published in 2001, and what we are able to

22 identify following intramuscular injection of AAV2

23 into rabbits was the following.

24 We performed a series of IF staining and

25 FISH analysis shows that we can detect signal from

141

1 the vector, and this is localized in the vessel

2 wall and the testicular basement membrane, which

3 are rich structures for heparan sulfate

4 proteoglycan, which there is no receptor for AAV

5 serotype 2.

6 The detectable signal especially in the

7 gonads disappears with time. It is important to

8 remember that in this cohort of rabbits, no semen

9 signal was ever identified, and also into the

10 gonads, we neither detected any intracellular

11 signal when you analyze animals following 7, 36,

12 and 90 days.

13 This is just to represent what we believe

14 that the signal, this is IF staining, what you call

15 localization for the AAV capsid and for heparan

16 sulfate proteoglycan on the vessel wall in the

17 testis basement membrane.

18 So, in the last experiment that I would

19 like to show is attempt to transduce not mature

20 spermatozoa, but immature spermatozoa. In this we

21 have murine cells in culture in which murine

22 spermatogonia and Sertoli cells were co-cultured.

23 We transduced again with AAV2 under

24 control of the CMV promotor expressing lacZ at the

25 MOI 5000, and we stain for x-gal. Here, it showed

142

1 the signal. Just before, I should say, that we

2 identified the spermatogonia by immunostaining with

3 a monoclonal antibodies to germ cell nuclei

4 antigen.

5 This is the result. At the bottom is the

6 mouse spermatogonia and Sertoli cells that give

7 this kind of reddish signal. In contrast, if you

8 take the same transduced model, the fibroblasts or

9 human \[inaudible\] it turns blue.

10 I would like to conclude that intravenous

11 administration of the dose of AAV up to 1 x 1013 in

12 rabbits results in transient detectable signal in

13 semen in a dose-dependent manner.

14 PRC positivity of the semen persists up to

15 day 44 in that cohort, that we have follow-up for

16 almost 100 days revealed no positive signal, which

17 is a duration of 2 or 3 times of the rabbit

18 spermatogenesis.

19 Vector signal can be detected in

20 peripheral mononuclear blood cells for at least

21 three months in rabbits in contrast to non-human

22 primates, we know that this can go up to 10 months,

23 but the vector is not biologically active after day

24 7.

25 In ongoing experiments, you can predict

143

1 that we will continue follow up with kinetic

2 clearance, determination of anatomic localization

3 of signal as a function of the vector dose

4 following intravascular injection.

5 To determine whether AAV infectivity is

6 detected in rabbit semen, we are followed by the

7 experiments that Dr. Couto has before. As well, we

8 would like to determine whether receptor for AAV2

9 is present in mice, rabbit, and human spermatozoa.

10 That could give us some idea.

11 I will stop here.

12 DR. SALOMON: I want to make sure we all

13 understand where we are going. What we are going

14 to do now is have a discussion of the three talks

15 that came, followed by people who have been invited

16 to speak in the public hearing.

17 We had talked about moving that out of

18 order, but I am told that is not proper, but to

19 reassure everybody that we will discuss the

20 questions to the committee after that, so that

21 everything will be on the table before we get to

22 the questions.

23 I think there are a number of interesting

24 issues raised by the these three presentations, and

25 I would like to put those open for some discussion.

144

1 Dr. Dym.

2 Q&A

3 DR. DYM: I had a question for Dr. Couto,

4 please, just a clarification, and maybe I didn\'t

5 understand some things, but when you showed very

6 elegantly that the receptor for AAV is around the

7 seminiferous tubule and in the interstitial spaces,

8 not inside the tubule, but then you didn\'t show AAV

9 in the gonads.

10 Isn\'t there a discrepancy there?

11 Shouldn\'t it show up in gonad if the receptor is

12 there, or did I miss something?

13 DR. COUTO: That particular section was an

14 animal that was not even injected with an AAV

15 vector, so we were just strictly looking at to tell

16 whether the receptor for AAV is even present in a

17 non-human primate testis.

18 DR. DYM: But in your other monkey tissue,

19 didn\'t you say it is not detectable in testis?

20 DR. COUTO: The AAV sequences are not

21 detectable in the gonadal tissue, correct, by PCR.

22 DR. DYM: So, wouldn\'t they be there,

23 because the receptor is there? I am missing

24 something.

25 DR. HIGH: Can I clarify that question?

145

1 So, the answer to your very perceptive question, if

2 you look in the Molecular Therapy paper, there is

3 actually a FISH analysis of a rabbit testis from an

4 animal sacrificed at day 7, after injection with

5 AAV, and in that, you can see tracking in the same

6 location that Dr. Couto showed in the non-human

7 primate testis.

8 You can see AAV vector detected by FISH

9 analysis in the same location along the testicular

10 basement membrane and actually around the vessel

11 wall, as well. You can detect that at day 7, but

12 by longer time points, which were presented in

13 several of the studies that Dr. Couto did, both in

14 rabbits and in other species, as well, if you look

15 at later time points like 50 days after or 100 days

16 or 135 days, you don\'t see AAV vector any longer in

17 the gonadal tissue.

18 So, your point is correct, and if you look

19 early on, you can see that, and that has been

20 published in that Molecular Therapy study or day 7,

21 but at later time points you don\'t see it.

22 DR. SALOMON: I have a couple questions.

23 Going back to the very beginning, I posed a

24 question about if sperm were not transcriptionally

25 active, then, how do you interpret an experiment

146

1 where you put in a CMV-GFP vector?

2 DR. ARRUDA: I don\'t think that is the

3 ideal experiment. We just want to see if one could

4 transduce motile sperm in culture. There are some

5 people that say they can, in fact, transduce some

6 mature spermatozoa using more aggressive ways. We

7 do not expect anything else. It wasn\'t a surprise

8 that the results were negative, but I think the

9 best way to answer your question is to perform an

10 experiment that Dr. Couto is doing with transducer

11 cells in culture, and then you do in vitro

12 fertilization and see the outcome.

13 DR. SALOMON: I certainly don\'t think that

14 the evidence that you didn\'t get GFP expression

15 really addressed anything.

16 DR. ARRUDA: I agree.

17 DR. SALOMON: If it had been positive, I

18 agree it would have been important, but I don\'t

19 know what is the point in showing it as negative.

20 DR. ARRUDA: I agree with that, and also

21 it has been published that one can detect lacZ in

22 this spermatozoa.

23 DR. SALOMON: What I would like to hear

24 some discussion of is whether the experiment that

25 Dr. Couto presented, and I guess that is going to

147

1 be done with you, Jon, is the best study.

2 I have some concerns about that. It is a

3 very good study. It seems to me that it is really

4 almost going over the top, which you said yourself,

5 Jon, was what you should do. So I would like to

6 hear your comment on that as a point of discussion.

7 It seems to me you have a reagent,

8 however, that would also be extremely useful, and

9 that would be to take your AAV-CMV promoter GFP and

10 put it into the rabbit, and then actually trace GFP

11 expression in different compartments particularly

12 in this case, of course, in the spermatogonia, I

13 mean so you could do it at different \-- I don\'t

14 need to tell you all the different variations of

15 that, but that seems to me to be the most

16 physiologic experiment.

17 DR. GORDON: I want to make a brief

18 comment on that. It is no surprise that sperm will

19 not express genes put into them, but that doesn\'t

20 mean that genes aren\'t in there, and couldn\'t be

21 expressed late, just to reemphasize the strategy of

22 doing this IVF.

23 The other comment i would make is if there

24 were AAV-CMV lacZ, I haven\'t looked for GFP in

25 embryos, although I am sure it can be done, but if

148

1 there were lacZ vectors, where I know I can look,

2 you could look at thousands of cells in a very

3 short time after exposing sperm to such a vector,

4 and just simply stainings on an intact embryo. We

5 did that for adenovirus, it worked really well, and

6 that would be a very nice protection against

7 contamination when one harvests fetuses and the

8 strategy that we are taking, which is of major

9 concern to us, and which has been discussed amongst

10 us over the last few weeks.

11 DR. COUTO: There is another experiment

12 that we have thought of doing, and it is based on

13 an experiment that Bob Braun\'s lab has done with

14 adenovirus, where they had an adenovirus that had

15 an expression cassette that has a protamine

16 promoter hooked up to a lacZ vector.

17 In that case, they are doing a natural

18 route of administration, and then looking to see

19 whether all of the progenitor spermatogonia,

20 spermatocytes, et cetera, in an entire seminiferous

21 tubule turn blue over the course of time. That is

22 another experiment that would not only look at the

23 mature sperm, but also the immature sperm.

24 DR. SALOMON: The idea there is to use the

25 protamine promoter as kind of a tissue-specific

149

1 promoter, so that is even cooler, as would say in

2 California.

3 Jon, the question that I had for you was

4 the experiment that they are talking about where

5 you essentially culture the sperm with 1,000 MOI

6 and then you go and do IBF. Isn\'t that just going

7 to have a bunch of DNA coating the sperm? Don\'t we

8 already know the results of this experiment before

9 they do it?

10 DR. GORDON: Meaning you think it would be

11 positive. Well, I can just say that we did that

12 with adenovirus. The rationale of this \-- and it

13 was not positive \-- the rationale for this is that

14 if AAV arrives to semen, then, it can expose motile

15 sperm, and in the rabbit, motile sperm seemed to be

16 associated with AAV.

17 So the question then is can these carry

18 the genomes into the embryo via the natural

19 fertilization process. As I pointed out in my

20 talk, that is not easy to do, and we certainly did

21 not find that to be the case with adenovirus, even

22 at 100 viruses per cell, so we would not predict it

23 to be positive, mostly because of the investments

24 of the sperm are mostly lost en route through the

25 zona, and so on.

150

1 So we don\'t expect it to be positive. If

2 it\'s positive, we have to look at why that is true,

3 whether it is really transduction or whether it is

4 so much AAV in our IBF prep that we couldn\'t get

5 rid of it all, something like that.

6 DR. SALOMON: So, Jon, I have to ask the

7 stupid question. So why did your DNA experiments

8 work when you coated the sperm, so why does that

9 work?

10 DR. GORDON: That works only if you load

11 the sperm into a microneedle, push the microneedle

12 through the zona, then through the plasma membrane

13 of the egg, and then insert the sperm with the DNA

14 around it directly into the cytoplasm. That works.

15 I say to you that I don\'t believe the Cell paper

16 which said that just mixing it with DNA and doing

17 IBF works, since no one seems to be able to repeat

18 it including me.

19 DR. SALOMON: Thank you for that

20 clarification.

21 DR. KAY: I just want to make a comment

22 that even under the very worse scenario, where you

23 do get carrying in of the single-stranded genome

24 into the embryo, at very low copy number, even one

25 copy, the chances that that single-stranded genome

151

1 is going to become double-stranded is very low.

2 DR. GORDON: Another final comment about

3 the thing I said about exposing them and then

4 looking for expression in embryos, that is a

5 problem with the kinetics of AAV genome activation.

6 It takes a long time, as I understand it, to

7 actually turn the genes on, and so the experiment

8 is a little bit less easy to do with that, as are

9 experiments with protamine promoters, and so on,

10 with AAV, just because it takes a long time to turn

11 the genome on.

12 DR. SALOMON: Dr. Rao.

13 DR. RAO: This is for Dr. Arruda. There

14 are two things which weren\'t absolutely clear to me

15 in your presentation. When you showed the

16 spermatogonia in culture and you looked at AAV

17 infection with the high MOI of infection, there

18 were some infection. You showed 293\'s at the same

19 time?

20 DR. ARRUDA: Yes. We have two cell lines

21 as control. Those are 293 cells with human cell

22 line and the murine cell line, the fibroblasts,

23 that was positive.

24 DR. RAO: But the spermatogonia were not?

25 DR. ARRUDA: The murine spermatogonia was

152

1 not. That experiment was murine spermatogonia.

2 DR. RAO: And when you see positivity in

3 the rabbit motile sperm, fractionated sperm, where

4 do you think the virus is there? I mean you

5 fractionate the rabbit sperm.

6 DR. ARRUDA: Yes.

7 DR. RAO: And you take the motile

8 fraction, which you now have purified.

9 DR. ARRUDA: Yes.

10 DR. RAO: You think there are no

11 contaminating cells, right? And you see by PCR

12 that there is some positivity, right?

13 DR. ARRUDA: That\'s correct.

14 DR. RAO: Where do you think that is

15 coming from?

16 DR. ARRUDA: If you remember, even when we

17 use what we call the optimal protocol, it is a list

18 developed for rabbits, reagents for rabbits, we

19 still see some debris, which you don\'t know which

20 kind of cells are those, and also no motile sperm

21 cells, other than, any other, so it is a more

22 concentrated fraction of motile sperm up to 87 or

23 95 percent, but we still see some of those.

24 Just from technical reasons like if the

25 animal has very little, urinated during the

153

1 procedure, you are contaminated, you don\'t see that

2 unless the thing turns out to be yellowish, things

3 like this.

4 DR. SALOMON: Dr. High.

5 DR. HIGH: I just want to underscore the

6 point that you raised in that question, because it

7 is really critical to the kind of analysis that we

8 are trying to do, and I am not sure we are really

9 on the right track.

10 That is, that what semen fractionation

11 does is enrich for motile sperm, but it doesn\'t

12 really exclude all other cell types, so when you

13 take semen fractionation and couple it with a

14 procedure like PCR, which will certainly detect

15 small amounts of contaminating material that may

16 not be from motile sperm, then, have we really come

17 up with the best test. So your point is well made.

18 DR. DYM: This is for Dr. Kay. It is sort

19 of a comment, maybe a question related to maybe why

20 it seems difficult to get the vectors into the

21 germline or into the gonads.

22 When you put it into the liver, it, of

23 course, goes straight into the liver, maybe a

24 little bit goes via the artery, gastroduodenal

25 artery to the upper part of the GI tract, and then

154

1 it goes into the capillary spaces, and so on, then

2 back to the heart, back to the aorta.

3 Now, those testicular arteries, I know

4 them very well in the human also. They are tiny,

5 little things, and it may not just get down there

6 again.

7 DR. KAY: I actually would say that it

8 probably does, and let me explain how the liver

9 flow works. First of all, the catheter is

10 ballooned at a point that is past the

11 gastroduodenal, so there shouldn\'t be backflow into

12 that artery directly.

13 However, even by clamping off the hepatic

14 artery, most of the blood flow through the liver

15 still occurs because 60 percent of blood flow

16 through the liver is through the portal

17 vasculature.

18 So, what we suspect happens is that you

19 get actually infusion into the liver, and that you

20 actually get washing into the venous side through

21 the portal circulation into the vena cava, and then

22 you get disseminated flow.

23 If you look at animals in biodistribution

24 studies that have got an hepatic artery or portal

25 vein, or what have you, you do find the vector in

155

1 other tissues, but at very low concentrations. To

2 the best that we can tell, at a reasonable rate,

3 and, you know, we can define that statistically or

4 not, the only other tissue that we have really seen

5 anything that would be even suggestive of

6 transduced cells is a rare positive cell in the

7 spleen.

8 DR. DYM: Do you know then the clearance

9 of this?

10 DR. KAY: No, we don\'t know that. That is

11 something that we are actually working on in animal

12 models. It is almost impossible to do in mice, and

13 we have developed some surgical techniques in the

14 rat where we have actually clamped off the vena

15 cava in the portal vein and then just infusing the

16 liver, let it sit at different dwell times and then

17 release the clamp, and then are looking at how much

18 gets into the rest of the circulation.

19 I think the question is how much of it

20 gets into the liver on the first pass, and things

21 like that, and we don\'t have a definitive answer

22 for that, but we are doing studies as best we can

23 to try to address it.

24 DR. SALOMON: Dr. Gordon.

25 DR. GORDON: I just wanted to reemphasize

156

1 again this discussion with Dr. High that just

2 preceded that, if the motile sperm fractionation

3 results in the detection of this material, that

4 doesn\'t mean that it is on the sperm, but it

5 certainly means that the sperm could come in

6 contact with it. That is really the issue in these

7 IBF strategies.

8 If the sperm can come in contact with it,

9 does that mean that they can carry it in and cause

10 vertical germline transmission, and that is why I

11 feel it is necessary to make sure that you do that

12 experiment.

13 I can tell you that with adenovirus they

14 don\'t. I think it is hard to do that, and I don\'t

15 think it will happen with AAV either, but a

16 discussion of exactly where it is sitting in the

17 motile sperm fraction isn\'t really that relevant.

18 You know that that means it could come in contact

19 with motile sperm.

20 DR. SALOMON: Do we know that there is any

21 heparan sulfate proteoglycans on the sperm itself?

22 I know that they showed the picture that the

23 spermatogonia and the seminiferous tubules seemed

24 to be negative.

25 DR. GORDON: I don\'t know the answer to

157

1 that.

2 DR. SALOMON: I guess that follows up to

3 me a question. That is, you showed the HSG

4 expression in the seminiferous tubule as a point

5 like reassuring us that the target wasn\'t there. I

6 am not an expert on AAV, so I defer to my

7 colleagues on this.

8 I mean is that it? I mean there is not

9 other cell attachment molecule? I mean if so, that

10 would seem to be rather unique since every time I

11 think that is it, there is always something else.

12 Jude? Obviously, I am looking at you.

13 DR. SAMULSKI: It has been shown that FGF

14 will also bind to virus, and we know that alpha-V,

15 beta 5 is like a co-receptor, so it is not an all

16 or none scenario, but it is a good indicator if

17 those cells are likely to take virus up. You

18 typically heparan. It is not just heparan itself

19 either, it\'s high sulfonated heparan, so there is

20 different kinds of heparan.

21 DR. MULLIGAN: The infection point in the

22 rabbit, how does the rabbit compare for AAV2

23 relative to other kinds of cells? That is

24 obviously, the model is only as good as how

25 sensitive it is. Is there a way you have tried to

158

1 look at a range of different rabbit kinds of cells

2 versus other kinds of cells, human cells to see if

3 rabbit cells are equally, in a general way,

4 susceptible to infection? There is definitely in

5 the AAV serotype business, great differences, not

6 only species differences, but also obviously,

7 tissue differences?

8 DR. ARRUDA: We have some idea. As I

9 said, we inject the same vector that has been used

10 in the clinical trials, so it is expressing human

11 factor IX, and the cohort of animals, that is of

12 the highest dose, we are able to detect human

13 factor IX in the rabbit plasma.

14 So what this tells us is that I can say

15 how it transduce efficiently in liver cells.

16 DR. MULLIGAN: Comparable to the amount

17 you have?

18 DR. ARRUDA: No, it\'s lower than because

19 the major difference is that we did I.V. infusion,

20 not deliver into the hepatic artery, and if you do

21 these in the same animal, you see 5 or even less

22 expression follow I.V. That is why we have to go

23 into the hepatic artery.

24 DR. MULLIGAN: If you just compare I.V.

25 and the amounts versus the rabbit, is it

159

1 comparable?

2 DR. ARRUDA: I would say not, because the

3 mouse, we inject \-- you can correct me \-- some of

4 this, like C57, they respond very well to I.V.

5 infusion compared with even other strains.

6 DR. KAY: I think there is a complicating

7 factor and that using human factor IX in a rabbit,

8 and do the rabbits develop inhibitors? I mean

9 because you get a slow rise of expression over

10 time, you may never hit the peak level, and in

11 mice, you know, it is very dependent on different

12 strains.

13 DR. MULLIGAN: I know it\'s complicated,

14 but I mean that ultimately the question is whether

15 or not there is a way to have a sense of whether or

16 not the rabbit is as susceptible. I mean the

17 argument in a way goes to your favor in that the

18 animal models for AAV may not be that good because

19 of the differences, like with the VSVGs, a

20 pseudotype, you can infect all kinds of different,

21 1,000 tissues.

22 AAV seems somewhat different as a vector

23 system because there are such big differences from

24 species to species and tissue and tissue. So it is

25 not clear whether the rabbit would be a better or

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1 worse system, but it would be nice to have a sense

2 of typical tissues that people attempt to do

3 transduction with, is it comparable.

4 DR. SALOMON: So you want to see data in a

5 rabbit, for example, showing that intrahepatic

6 artery injection has a somewhat similar

7 transduction efficiency.

8 DR. MULLIGAN: LacZ and muscle, in a

9 rabbit muscle, looking at the number of positive

10 muscles \-- Jude, you must have done this sort of

11 thing. No?

12 DR. SAMULSKI: Officially, we have not

13 done this experiment.

14 DR. MULLIGAN: How about unofficially?

15 DR. SAMULSKI: Unofficially, we haven\'t

16 done it either.

17 \[Laughter.\]

18 DR. KAY: Can I raise the issue of

19 hematogenous spread again? I mean this vector,

20 unlike the retroviruses and other vectors that are

21 being used, that have a potential to integrate, are

22 not pseudotyped, and they basically represent the

23 capsid of the wild-type virus.

24 During wild-type infection, there is going

25 to be some hematogenous spread although I am not

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1 sure that anyone knows what the concentration is,

2 and yet we haven\'t had any evidence of the AAV

3 sequences into the human germline, unlike what has

4 happened with most mammals with retroviruses.

5 So, I think, in nature, that there is some

6 hematogenous spread of the wild-type virus, yet it

7 hasn\'t been detected in our germline.

8 Any comment on that?

9 DR. MULLIGAN: I want to switch back to

10 this infection question. In the case of doing in

11 vitro infections, there are different kinds of

12 cells, my impression was that people often with AAV

13 use very, very high multiplicities of infection

14 like 10,000 to 1, or 50,000 to 1.

15 Do you feel comfortable that you have

16 really, in the in vitro rabbit infections, really

17 dosed, put on a virus to potentially detect

18 something?

19 The question was, when you do in vitro

20 infections, different kinds of cells, what is the

21 maximum multiplicity of infection that you use to

22 see if something could be infected? Is it 5,000 or

23 is it 50,000?

24 DR. SAMULSKI: In our hands, we have seen

25 things like certain fibroblasts or real refractory,

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1 and you need about 100,000 particles to it

2 transduced.

3 DR. MULLIGAN: That was my impression. So

4 the question is, have you really, with all the

5 vagaries of the system, have you really given it

6 the greater shot, unless I got that wrong, you did

7 5,000 was your multiplicity of infection? Was

8 there any reason you didn\'t test 10 times that?

9 No.

10 DR. SALOMON: Trying to get some sort of

11 themes going here, it seems to me at least three

12 different things could be discussed. The first

13 would be the idea that the adenovirus associates

14 with the sperm or with \-- well, actually with the

15 sperm itself, and therefore, would be carried into

16 the female and might then enter the egg at the time

17 of the sperm\'s fusion, and inadvertently deliver

18 the genetic material from the virus. That\'s one

19 possibility.

20 I think that that possibility, Dr.

21 Gordon\'s and your experiment would address, so that

22 is a good thing, we don\'t have any data yet, but it

23 sounds like you have got that on track.

24 The second possibility would be that the

25 sperm are carrying the virus, and I am not

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1 convinced that any of the tests we have seen so far

2 adequately address that. I am not trying to be

3 overcritical either, because I can see how

4 difficult some of these studies are to do, and

5 commend you for doing things like figuring out how

6 to fractionate rabbit sperm, and it shows how

7 careful you are trying to be.

8 But it still seems to me that when you are

9 dealing with literally millions of sperm in a

10 typical ejaculate, and you are doing PCR studies

11 that were sensitive down to 1 in 30,000, that this

12 is not going to work, I mean that that is not very

13 convincing, and specific studies of looking at in

14 vivo expression, or whether you use the GMP, the

15 CMD promoter or the protamine promoter, something

16 along those lines haven\'t been done yet.

17 I don\'t think that we really know the

18 answer to that part. I guess a third thing that

19 occurs to me is that regardless of the germline

20 transfer question, if semen of male patients had

21 got the vector for weeks and weeks, your patient at

22 the lowest dose is 14 weeks positive in semen, is

23 that going to get transferred to vaginal cells and

24 other cells in mothers?

25 We all know through bitter experience with

164

1 HIV that that is a portal to the blood, as well.

2 You know, how likely is it even if we just focus on

3 the semen positivity, to which there is no

4 conflict, right, I mean we all agree there is semen

5 positivity, are we going to see a lot of the

6 partners infected and how does that impact on

7 issues in terms of doing these studies?

8 DR. COUTO: One thing that we are trying

9 to do to address that is with the development of

10 this infectivity assay, at least try to demonstrate

11 that there possibly is, even if it\'s there, it\'s

12 not infectious.

13 There may be vector sequences there in the

14 semen, but after a couple of days, maybe it\'s no

15 longer infectious, so that is one thing that would

16 address that.

17 DR. SALOMON: So your assay would \--

18 infectious, though \-- you are saying won\'t

19 replicate if you add helper virus and wild-type

20 adeno for the Rep and Cap genes, but it doesn\'t

21 really address whether it just delivers the payload

22 gene, right? How likely will it be? Tell me if I

23 am being dumb.

24 But I mean how likely would it be that a

25 positive semen that actually has adenoviral capsid,

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1 that you inject it earlier in the hepatic artery,

2 will, when injected during sexual intercourse into

3 the female, just transfer it to vagina mucosa?

4 DR. COUTO: If it is able to infect the

5 cells and you provide adenoviral help, it should be

6 able to replicate, and that is really what we are

7 asking in that assay, so I think we can rule that

8 out.

9 DR. KAY: The Mollier \[ph\] data suggested

10 that at least in the high end injected in the

11 blood, although the vector DNA was detected for a

12 long period of time, the biological activity of

13 those particles diminished to undetectable levels

14 after a very short time.

15 So what I think Linda is trying to say,

16 just to reiterate, is that there is a reasonable

17 chance that the DNA or the particle could stick

18 around, but it may not be actually infectious or

19 able to transduce a cell.

20 If it went into like, say, a vaginal

21 epithelial cell one time, you would never see that.

22 I mean it would be a single transduction event, it

23 wouldn\'t replicate. I guess the question is if it

24 was carried in on the sperm into an oocyte, what is

25 the chance of naked DNA or DNA that is partially

166

1 exposed could get through the zona pellucida during

2 a fertilization event.

3 DR. GORDON: All I can say to that is I

4 don\'t think it has ever been seen in the literature

5 despite claims to the contrary.

6 DR. SALOMON: I wasn\'t trying to make

7 things too complex. I guess I was just saying that

8 we are really not discussing just the Avigen factor

9 IX study even though that is on the table here, my

10 feeling here is we are discussing just in general

11 issues here. I greatly respect Avigen being here

12 and presenting it, because it is always great to

13 have a specific study to focus on, but we also

14 don\'t want to lose sight of the fact that there are

15 bigger issues here.

16 So I am saying that a lot of different

17 clinical trials could come along following

18 potential success in the Avigen trial, and I

19 certainly do wish you the best with this one.

20 Those could deliver gene payloads that could be a

21 lot more serious than delivering some extra factor

22 IX to a woman inadvertently, so that is really all

23 I am trying to say is if you start delivering \--

24 oh, who knows, I don\'t want to make stuff up \--

25 but, you know, just a gene payload that might be

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1 toxic, whether that would be delivered to the

2 vagina of the woman and produce some problem there.

3 Again, I am not trying to make that a big

4 killer issue, but it seems like from everything I

5 have heard that it is still theoretically possible.

6 DR. MULLIGAN: One thing that Jon\'s \--

7 what do you call them, provocative experiment \--

8 would test is, in principle, whether or not any

9 AAV, since that is the worst case, we are soaking

10 things with the AAV, and then you are doing the

11 most efficient means of sex, maybe not the most

12 efficient, but you are doing it so you are opening

13 up as much AAV as possible, so you could look for

14 things in addition to the integrated sequences.

15 I think I saw that you were going to test

16 only for integrated sequences. After the in vitro

17 fertilization experiment, it might be useful to,

18 since you have the material, to look for whether or

19 not there is AAV. Presumably, if it wasn\'t

20 integrated, it would be dramatically diluted, but

21 it would be interesting to see if you could detect

22 it, because that would address in a sense the worst

23 case of whether or not, during sexual intercourse,

24 you can transfer AAV, and it can persist maybe as

25 an unintegrated form, but these is some infection.

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1 DR. GORDON: We are doing that.

2 DR. SAMULSKI: I have a question, and it

3 is more a curiosity. It seems that the so-called

4 debris and other contaminants are a major

5 contributor to the positive results, and I am

6 wondering if people here have felt that the efforts

7 to purify these different fractions have been

8 exhaustively done, because it seems that when you

9 move to other reagents, whether they are oligos or

10 plasmids, this is going to come up over and over

11 again.

12 If there is more energy put into the first

13 step of the assay, of collecting and fractionating,

14 will we move away from these long risk things and

15 get into a better assay that is going to tell us

16 there is something worth paying attention to.

17 Again, I turn it back this way, because when you

18 hear someone say this was optimized for rabbits,

19 and this was optimized for humans, does that mean

20 it has been done for 20 years and optimized, or is

21 just gives them the result they need to get

22 something away from something.

23 DR. GORDON: I just want to very briefly

24 comment on that. Even a fraction of motile sperm

25 is a very heterogeneous population of cells. I

169

1 mean some of them have two heads. Some of them

2 have a huge cytoplasmic droplet, which can be close

3 to the volume of a sperm.

4 So when you actually try to do an

5 absolutely totally pure separation of motile sperm

6 from everything else with a similar density,

7 similar parameters of measurement, similar

8 configuration, it is very, very difficult, and I

9 think that if you try to solve this problem that

10 way, by getting a golden fractionation procedure,

11 you are going to be chasing your tail for a long

12 time, not that I want to introduce other tails into

13 the discussion.

14 DR. JUENGST: So, thinking generically,

15 kind of at the policy level, I think I learned two

16 things this morning that increased my sense that

17 there are risks here. The first one was the idea

18 that it is not just the integration of a factor IX

19 gene in a harmless place on chromosome 19, but the

20 random integration of genes from the episome

21 presence of the vector.

22 The second was the increased risk even if

23 through natural fertilization, it looks low with

24 artificial means of fertilization, ICSI and

25 infertility techniques, so it looks like the

170

1 patients who are at greatest risk, hypothetically

2 speaking, worst case scenario, would be gene

3 therapy patients who then had fertility problems

4 and needed to go to a fertility clinic.

5 DR. SALOMON: To Jude\'s question, the

6 other way around here would be there is still no

7 evidence that these vectors are getting into the

8 spermatogonia, so if you could do enough really

9 well designed, basic, preclinical work, you might

10 be able to make a good case that you just monitor

11 the semen, and not be obsessing about all this

12 purification, et cetera, you know, if you could

13 convince yourself that it wasn\'t specifically being

14 carried in the germline package of the sperm.

15 DR. GORDON: Let me just say that I think

16 that is a very important point because if you

17 cannot transduce the spermatogonia, then, when the

18 semen are clear, you can feel that they will be

19 clear, and that not another wave of spermatogenesis

20 will provide more positive sperm to the ejaculate.

21 DR. SALOMON: Certainly, the data

22 presented today still do not give us any cause \--

23 you know, there is no smoking gun yet that these

24 are being delivered to germline cells.

25 DR. MULLIGAN: Jon\'s work wouldn\'t address

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1 the worst case for trying to get an earlier

2 precursor infected, right? I mean that is, you

3 could think of the same Jon kind of approach where

4 you would put in as much AAV into exactly right

5 time and location, to do the same sort of worst

6 case, and that probably would be the ultimate worst

7 case.

8 DR. SALOMON: Yes. In fact, that was the

9 point I was making, too, earlier. There was the

10 issue of whether it got in or didn\'t, but his

11 experiment addressed the latter, right, where it

12 was just attached to the outside.

13 But the experiments haven\'t been done yet

14 or designed yet or proposed yet to do the ones that

15 we both suggested, and that is, prove yes or no,

16 whether it gets into the spermatogonia, and if you

17 could get out of that, then, you could make the FDA

18 and the sponsor\'s life a lot easier.

19 DR. GORDON: Well, I just want to say

20 again that we have an abstract today at ASGT, in

21 which we are developing this technique of perfusing

22 intact seminiferous tubules with very high

23 concentrations of vector. I showed some of the

24 stuff from adeno expressing lacZ, and again that

25 would be a very highly provocative test.

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1 It doesn\'t seem to disturb the

2 spermatogenesis much, if at all, and that, with

3 nucleic acid hybridizations, you wouldn\'t have to

4 rely on promoters and vectors with delayed

5 expressions, which is AAV, would I think be a good

6 standard to arrive to.

7 DR. NOGUCHI: Just to follow up a little

8 bit on how provocative you be, Jon, wouldn\'t the

9 most and even more provocative state be to expose

10 sperm to AAV, and then immediately do ICSI, and

11 then look at the outcomes of that?

12 DR. GORDON: I think that would work

13 because then all the natural barriers to getting it

14 in would be circumvented, but I do emphasize those

15 are natural barriers and that is an artifactual

16 situation, however, as I was saying before in my

17 talk, my official talk, I mean there is a lot of

18 clinical activity where these barriers are

19 bypassed, and I think that we should begin to be

20 interested in that subject, and I don\'t think that

21 is the subject for this meeting, but I think it is

22 a subject that the FDA needs to begin to get

23 interested in.

24 DR. MULLIGAN: I like Phil\'s approach

25 because then it is really more directed an

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1 integration question. It is like doing a

2 transgenic system where you dump in more and more

3 AAV in exactly the right \-- for something that

4 happened, and you can see whether it does or

5 doesn\'t happen.

6 DR. NOGUCHI: It actually pertains in a

7 way, based on the discussion here, to this

8 experiment, as well, if, in fact, you have the

9 presence of vector even if it\'s not integrated, but

10 it is around, it could coat the sperm or it could

11 be attached to the sperm. That is the equivalent

12 of what we are talking about. You have a vector, a

13 sperm, a union with an egg, and things.

14 So I think they are two different things.

15 One is, is there integration into the actual person

16 being treated, and then the other part, can there

17 be a transmission by other than biological means,

18 but just by pure mechanical. That is an issue that

19 pertains, and it is related also to the question of

20 how much sensitivity is enough if we are going to

21 be talking about barrier contraception as a means

22 to mitigate this period of washout, you know, how

23 much washout is enough.

24 DR. SALOMON: I guess as long as we are at

25 the most provocative experiment discussion, I mean

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1 you could simply inject the AAV into the egg, and

2 then with the lacZ, and ask then in like a mouse

3 embryo or in a chicken embryo or in a rabbit

4 embryo, where it was distributed, and ask the

5 question whether there is some unusual integration

6 or whether it just quickly segregated.

7 DR. GORDON: Just a brief comment on that.

8 We have another abstract at ASGT, about a

9 adenovirus injection directly into embryo, and what

10 we were doing was asking the question, does this

11 intricate cycle of virus on coating and

12 translocation of the genome to the nucleus, are

13 these obligate steps for expression, which has

14 always been assumed, but never been proven.

15 Now, the one-celled embryo appears not to

16 have a receptor for adenovirus from our

17 experiments, and so what we did was injected the

18 virus directly into the cytoplasm and said, well,

19 we will bypass the endosome and see what we can

20 get.

21 We never lacZ expression under those

22 circumstances. We then say, well, let\'s help the

23 virus even more, we will put it right into the

24 pronucleus, and we have done that. In that case,

25 we see low rates of embryos that express which

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1 appear consistent with perhaps viral genomes that

2 have been partially shredded by freezing and

3 thawing.

4 To confirm that, we took the viral genome

5 and just injected the pure DNA and got a very

6 similar result. So in adeno, it doesn\'t appear

7 that the virus can actually do its thing if it\'s

8 not allowed to go through the regular cycle of

9 infection, but AAV, which is why I asked about

10 uncoating, when it uncoats, if you put that

11 directly in the pronucleus, it is a single-stranded

12 genome, but there is very active repair mechanisms

13 in the pronucleus, and all of that, I should think

14 it would work, but I don\'t think it has ever been

15 tried. I would be happy to try it if someone wants

16 me to try it.

17 DR. SAMULSKI: So, we actually did those

18 experiments, and if you remove the zona pellucida

19 and inject the virus, you can get blastocysts to

20 turn blue, so it will transduce those cells. It

21 doesn\'t work with adenovirus, just like you said,

22 for the same reason. When it comes out of

23 endosomes, there is a proteolytic cleavage that is

24 responsible for the virus on coat, and if you don\'t

25 go through that pathway, it won\'t go through.

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1 DR. KAY: Is it integrated?

2 DR. SAMULSKI: We only did in vitro and

3 carried them out and were able to show that it

4 would transduce those cells, and when we started to

5 collaborate with our colleagues to implant them

6 back in for embryos, the postdoc left, and all of

7 this stuff stopped, so we didn\'t do any more on it.

8 DR. GORDON: What was the helper for that?

9 DR. SAMULSKI: There was no helper. We

10 were simply trying to find a better way of making

11 transgenic animals using AAV as a way of delivering

12 genes, and showed that once could physically put it

13 in, it would transduce those cells, so I think

14 Phil\'s question is partially answered, you will get

15 it in, and it will work by some mechanical mean.

16 And far as the stability, integration, all

17 of that stuff, there are no answers at all. It was

18 just lacZ.

19 DR. MULLIGAN: What was the number of

20 virus particles?

21 DR. SAMULSKI: It was extremely high. We

22 were putting in about 1010. For a number of

23 reasons, it partially was \-- well, we could talk

24 about it later.

25 DR. SALOMON: Any other discussions here?

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1 What the committee should feel at this

2 point is that just in terms of science presented,

3 that we are comfortable with the main issues, and

4 then we will do the public comment and go on to

5 answering the specific questions from the FDA.

6 Open Public Hearing

7 DR. SALOMON: What I would like to do is

8 introduce the public comment. Five minutes have

9 been allotted to each of three speakers.

10 The first speaker that I would like to

11 invite up is Mr. Steven Humes of the National

12 Hemophilia Foundation.

13 MR. HUMES: Good afternoon. My name is

14 Steven Humes and I am the Director of Research at

15 the National Hemophilia Foundation, hereinafter

16 referred to as NHF.

17 NHF is a not-for-profit organization

18 dedicated to improving the quality of life for all

19 individuals with hemophilia and other bleeding

20 disorders. Today, we thank the members of the

21 Biological Response Modifiers Advisory Committee

22 for allowing us the opportunity to provide

23 testimony on recent reports of the presence of

24 adeno-associated virus, or AAV, in the seminal

25 fluid of individuals participating in a hemophilia

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1 factor IX liver-directed gene transfer trial.

2 There are at least 10 proteins in our

3 blood, which must work in a precise sequence to

4 make the blood clot. A deficiency in any one of

5 these proteins can lead to abnormal bleeding.

6 Hemophilia A is caused by a deficiency of or defect

7 in a clotting protein known as factor VIII. A

8 deficiency of or defect in clotting factor IX

9 causes hemophilia B. Both forms of hemophilia and

10 other bleeding disorders are X-chromosome linked

11 recessive genetic disorders. In the United States,

12 there are approximately 17,000 individuals living

13 with a diagnosis of hemophilia.

14 Hemophilia manifests itself by easy

15 bruisability and recurrent bleeding into joints and

16 muscles as well as bleeding intra-abdominally and

17 into the central nervous system. The severity of

18 an individual\'s hemophilia is determined by the

19 amount of circulating clotting factor. The

20 majority of individuals affected with hemophilia

21 have severe disease.

22 Individuals with severe hemophilia

23 typically have eight to 10 bleeding episodes each

24 month. This chronically recurrent hemorrhaging

25 causes disability, persistent pain, and sometimes

179

1 death.

2 In the past three decades, significant

3 advances have occurred in the treatment of

4 hemophilia with the development of plasma-derived

5 and then recombinant clotting factor products.

6 While the development of these new products has

7 proved enormously beneficial, persons with

8 hemophilia continue to face many difficulties that

9 affect their quality of life.

10 Prior to the development of viral

11 inactivation technologies, many individuals with

12 hemophilia were infected with HIV and hepatitis

13 through their unwitting use of contaminated

14 clotting factor products. The HIV epidemic has

15 cost this community dearly, causing the deaths of

16 over 6,000 hemophilia patients, their spouses,

17 partners, and children.

18 Today, an additional 2,200 continue to

19 live with HIV and its complications. It is

20 estimated that more than 70 percent of all persons

21 with hemophilia have been exposed to hepatitis C.

22 While the development of recombinant factor

23 significantly improved safety, it is sometimes in

24 shortage and also an extraordinarily expensive

25 medicine, especially for individuals with severe

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1 hemophilia who must treat frequent bleeding

2 episodes or who self-infuse prophylactically as

3 often as three times per week.

4 Because of the many challenges facing this

5 community and the limitations of current treatment

6 modalities, we look to gene therapy as the most

7 promising approach to cure hemophilia. To this

8 end, NHF has funded numerous gene therapy projects

9 and five scientific workshops on gene therapy, and

10 two-day gene therapy symposium is planned for our

11 annual meeting in October 2002.

12 We believe that research into a monogenic

13 disorder such as hemophilia may also lead to

14 progress in the treatment of more complex

15 disorders, such as multi-gene inherited disorders,

16 as well as cancer.

17 In 1996, an NIH report on gene therapy

18 recognized hemophilia as one of the most likely

19 disorders for which gene therapy will succeed. We

20 believe that this statement is as true today as it

21 was six years ago.

22 In the fall of 2001, vector

23 biodistribution studies from a factor IX deficiency

24 gene therapy trial noted the presence of AAV vector

25 in the semen of a trial participant. That study

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1 also noted that while vector was present in the

2 seminal fluid, there was no evidence of

3 transduction of sperm.

4 On November 17, 2001, NFH\'s Medical and

5 Scientific Advisory Council, or MASAC, reviewed

6 this issue and drafted its Recommendation No. 127,

7 which was approved by the NHF Board of Directors

8 the following day, and is attached to this document

9 that you have before you.

10 The recommendation requests, in reviewing

11 such unexpected findings, that the Recombinant DNA

12 Advisory Committee, or RAC, and the Food and Drug

13 Administration consider the risks to the trial

14 participant and, following appropriate analysis,

15 allow trials to proceed if such risks can be

16 mitigated.

17 NHF believes that a case-by-case

18 evaluation of unexpected findings will permit

19 improvements in safety and efficacy while enabling

20 continued pursuit of improved treatments for

21 hemophilia.

22 NHF believes that the AAV factor IX

23 liver-directed gene transfer trial currently being

24 conducted should continue. As Steven Faust, a

25 person with severe hemophilia and co-chairman of

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1 NHF\'s Advocacy Committee, stated this January

2 before the RAC, we see no inherent risks in these

3 findings that might cause additional risk to the

4 trial subjects.

5 NHF does support, however, increased

6 patient education and efforts directed at improving

7 the informed consent process, mitigating the risk

8 of potential germline transmission through the use

9 of sperm banking and requiring the use of barrier

10 contraceptive methods, and long-term surveillance

11 of trial subjects via PCR vector dissemination

12 studies.

13 Indeed, we believe that through this

14 surveillance, we might learn valuable information

15 about the natural history of AAV shedding that

16 could prove useful in future gene therapy trials.

17 NHF\'s MASAC has laid out detailed guidelines for

18 the conduct of gene therapy trials in its

19 Recommendation No. 120, dated August 16, 2001, a

20 copy of which is also furnished to you.

21 NHF respectfully suggests that the

22 Advisory Committee consider these guidelines when

23 considering future gene therapy trials.

24 NHF is heartened by the preliminary

25 results of gene transfer in humans. We are further

183

1 encouraged by the prompt review of the RAC and

2 FDA\'s Biological Response Modifiers Advisory

3 Committee to the vector biodistribution studies, as

4 we believe that this phenomenon may occur in other

5 trials.

6 We share your commitment to ensuring

7 patient safety, and appreciate your vigilance on

8 behalf of all persons enrolled in gene therapy

9 clinical trials. On behalf of the bleeding

10 disorders community, we urge your continued support

11 for these trials. If enrollment is further

12 delayed, how will we determine if this gene

13 transfer method offers the promise of a cure?

14 Once again, we thank you for this

15 opportunity to address you on this important

16 matter.

17 DR. SALOMON: Thank you very much, very

18 nicely articulated.

19 I think it is always reasonable to point

20 out when you hear something like that, that it is

21 very important for the committee to consider

22 whatever decisions we make affect a group of

23 stakeholders, in this case the hemophilia

24 community, as well as the public, and that is

25 always important to hear that in these sort of

184

1 public comments and think about it.

2 The next speaker is Dr. James Johnson, who

3 is identified as a hemophilia patient. That must

4 have been one of the first two patients to receive

5 the Avigen vector.

6 Welcome, Dr. Johnson.

7 DR. JOHNSON: Dr. Salomon, Committee, good

8 afternoon. I am Dr. James Johnson. I am from

9 Edmund, Oklahoma. I appreciate the opportunity to

10 speak to you wearing several hats. I am an

11 emergency physician practicing in Cushing,

12 Oklahoma, a husband and a father. I have lived for

13 45 years with hemophilia B, factor IX deficiency.

14 In addition, I was blessed to participate in the

15 Phase I safety trial of the gene therapy program at

16 Children\'s, not this one, but the prior one with

17 the IM injections two years ago.

18 My participation began in May of 2000. As

19 a side note, I dubbed myself Lad Back No. 6, since

20 I was the sixth person in that study.

21 As a 45-year-old living with hemophilia

22 and as a participant in a gene therapy study, I

23 want to express my deepest hope that you will allow

24 the current study to continue. This research is of

25 vital importance to the hemophilia community and to

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1 me personally.

2 When I was born in 1957, it took two years

3 for my parents to receive a diagnosis for the

4 bleeding I had. Because my head was swollen at

5 birth, my parents were told I had hydrocephalus and

6 would be mentally disabled.

7 Once the diagnosis of Christmas disease

8 was made, my parents were told that I wouldn\'t live

9 past 20 years of age. I kind of overdid that.

10 My early treatments consisted of ice

11 packs, splints, slings, and rest. Later, I

12 received infusions of whole blood and fresh-frozen

13 plasma. Finally, when I was 12, I received the

14 first dose of factor concentrate. That was 1970.

15 I promptly came down with hepatitis B, and although

16 I did not know it at the time, hepatitis C.

17 Through the 1970s, I would go to the

18 doctor and often have to be hospitalized when I

19 needed treatment. Finally, in 1979, I started

20 self-infusion, which is now the standard of care.

21 I was one of the blessed minority that was not

22 infected with HIV, but as I said earlier, I do have

23 hepatitis C.

24 Also, I have suffered the ravages of

25 arthritic complications of hemophilia as you might

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1 have noticed when I walked up. When we first went

2 to the hemophilia meetings, my wife said, Jim, they

3 all walk just like you do.

4 As you can imagine, hemophilia has

5 affected every area of my life. Like all

6 hemophiliacs, I have had to deal with educational

7 issues, work and employment issues, and struggles

8 with insurance. From a young age, I knew that I

9 would not be able to hold down jobs that required

10 great physical strength or endurance. Fortunately,

11 I am able to hold down my ER job and even do some

12 extra weekend work.

13 About nine years ago, I found that I had a

14 hemophilic pseudotumor in my abdomen. This was the

15 result of repeated bleeds in the psoas muscle.

16 There have occasionally been rebleeds which are

17 very painful and often require hospitalization.

18 This happened just this past week and for a while

19 it looked like I wouldn\'t be able to make it today.

20 I tell you this not for your sympathy or

21 to act macho, but to let you know that even with

22 today\'s best treatments, problems still arise.

23 It has been said of the hemophilia

24 community that we desire to be cured, we don\'t need

25 to be cured. Everyone is entitled to their own

187

1 opinion, but I believe we need a cure. Sure, there

2 are treatments available, but who likes to have to

3 give himself I.V. injections every time he feels

4 pain come on, or injections for days to try to get

5 over a bleed like the one I had this week?

6 We have treatments for other diseases, but

7 still work very hard for a cure. Does diabetes not

8 need a cure? Does hypertension not need a cure?

9 Does asthma not need a cure?

10 As for the current gene therapy study, the

11 one I was in, I enrolled about the time one of the

12 other programs lost a patient. Once the program

13 started back up, I, along with my 11-year-old

14 daughter and I, all had the procedure explained to

15 us in great detail. We were told of all the known

16 risks, as well as the theoretical risks that they

17 could imagine.

18 We were given ample opportunity for

19 questions. In short, I can say from the

20 perspective of both the study participant and as

21 one who has been involved in doing clinical

22 research myself, their informed consent procedure

23 was impeccable.

24 Every step along the way, I have been

25 informed of any new developments. We are still

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1 given every opportunity to ask questions or check

2 in to see how things are progressing. I have

3 always been and always know I will be able to speak

4 to anyone involved with the program with any

5 concerns or ideas.

6 I have met with all of the people involved

7 with the program at Chop, from Dr. High, the

8 director, to Dr. Mannow, to Amy Chu, the clinical

9 coordinator, and to even lab personnel. They are

10 committed and responsible people.

11 I understand that there is concern about

12 the possibility of the AAV vector being present in

13 the semen of some of the participants. This was

14 one of the risks that was reviewed with us before I

15 participated in the earlier trial.

16 It was always stressed that participation

17 in the study was voluntary, there was obligation to

18 participate or continue even after starting the

19 study. We were informed that I should never expect

20 to father a child after the study because of that

21 risk of AAV infection.

22 This was fine with us as my wife had

23 already had a tubal. Those that might consider

24 future children were given the opportunity for

25 sperm storage. All of my body fluids were tested

189

1 for weeks afterwards. There should be plenty of

2 other subjects like myself who do not plan any

3 further family. They would be able to be in the

4 studies at no risk to anyone else until more is

5 known about germline transmission.

6 This is an extremely important area of

7 study. Germline transmission is likely to be an

8 equal problem for all gene therapy if it turns out

9 to be a continuing problem here. The hemophilia

10 community, after having gone through hepatitis B,

11 HIV-AIDS, and now hepatitis C, is a very tough,

12 resilient, and responsible community.

13 We have been on the forefront of the use

14 of barrier contraception to prevent HIV infection,

15 so the idea of contraception and when appropriate,

16 sperm banking, is not foreign to us. Those that do

17 not like that option will not opt for the clinical

18 trials, as did my own brother.

19 In closing, I want to thank you for the

20 opportunity to speak to you. When I did my senior

21 paper in college, over 20 years ago, I wrote of the

22 potential that gene therapy would one day hold for

23 curing hemophilia. It is here. It is a reality.

24 As a member of the hemophilia community, I

25 ask you to work with the gene therapy program and

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1 the community to make gene therapy research program

2 safe and successful in the least time possible.

3 When my daughter asks me about her

4 children and hemophilia, I want to be able to tell

5 her that we have the answer. Please don\'t make me

6 tell her that we got close, but some minor

7 glitches, whether AAV or AV, stop the program.

8 Therefore, her sons will be at risk for the same

9 difficulties I have gone through.

10 Thank you.

11 DR. SALOMON: Thank you very much, Dr.

12 Johnson.

13 The last speaker in the public comment

14 period is Dr. Kenneth Chahine, Avigen Vice

15 President for Business Development and Intellectual

16 Property.

17 DR. CHAHINE: Good afternoon. My initial

18 goal was to try to bring a literative perspective

19 to the committee, but after the last few speakers,

20 I think that is not something I am going to try to

21 do.

22 My goal here today is to first present

23 what we can reasonably glean from the data

24 presented by my colleagues and also to present the

25 assumptions that form the basis of our proposal to

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1 this committee. In our proposal, also, we keep in

2 mind the questions that the FDA posed to the

3 committee.

4 The second goal is to communicate our

5 proposal and the rationale for that proposal. So,

6 what do we know and what can we reasonably assume?

7 The first point is that the procedure is

8 well tolerated, as Dr. Kay indicated. There have

9 been no risk to the patient apart from this

10 inadvertent germline transmission risk that we are

11 talking about today.

12 The second point, which has been clearly

13 talked about amongst the committee members, is the

14 predictive value of the animal models with respect

15 to inadvertent germline transmission. Clearly,

16 some of the animal models don\'t mimic the human

17 biology, while others may, although even the rabbit

18 dose and clearance times seem to be different from

19 what we are seeing in the first two patients.

20 The one consistent trend, however, is that

21 in all of the animal models, the vector is either

22 not there or it has cleared over time.

23 The third point is that the motile sperm

24 fraction may be positive as the dose increase, and

25 will almost certainly take longer to clear, so we

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1 want to make that assumption. I think it is

2 important for the committee to just look at that,

3 and I think our discussion today has brought that

4 out, that the fractionation procedure may not be

5 adequate to address it.

6 The next point is somewhat of a practical

7 point, is that the current rate and current

8 clinical hold triggers, the Phase I trial is going

9 to take very long to complete, and while in no way

10 does this point alone justify recommendation to

11 continue, it does have practical consequences for

12 Avigen, the scientific community, and the

13 hemophilia population.

14 The next point addresses one of the

15 questions that the FDA posed to the committee, and

16 that is, should the enrollment be limited to

17 patients or subjects which are unable to reproduce.

18 Certainly, that will cause delays given

19 the size of the hemophilia population, but will

20 also, in the male population, talking about males

21 that have undergone a vasectomy, limiting

22 enrollment to this patient population will deprive

23 us, the FDA, the scientific and medical community,

24 of the data that we so desperately need to answer

25 the very question we are here today convened to

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1 address.

2 Finally, higher doses my yield therapeutic

3 levels of factor IX based on the preclinical data.

4 It is important to keep in mind that the primary

5 purpose of this trial is certainly safety, but as

6 Mark Kay pointed out, at the higher doses we do

7 expect, based on the preclinical animal data, to

8 get a dose that is potentially therapeutic.

9 We hope that this discussion in the future

10 will actually focus more on a risk-benefit as

11 opposed to simply the risk, which is what we are

12 discussing today of inadvertent germline

13 transmission.

14 So, keeping these points in mind, why is

15 informed consent a reasonable and prudent safeguard

16 against inadvertent germline transmission?

17 The subjects are already counseled and

18 educated on the potential of not only transient but

19 permanent germline transmission. The patients are

20 already advised to use barrier contraceptives, and

21 as we have discussed here and at the Recombinant

22 Advisory Committee meeting, the risk of inadvertent

23 germline transmission is low.

24 The subjects are constantly monitored for

25 positive semen and positive motile sperm, and

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1 germline transmission, very importantly, can be

2 completely avoided by banking sperm.

3 So, in our opinion, when we take the low

4 risk of inadvertent germline transmission, the

5 small number of subjects in the trial, the active

6 use of barrier contraceptives and the sperm

7 banking, these factors together reduce the risk of

8 inadvertent germline transmission to acceptable

9 levels especially when we weigh the risk against

10 the enormously valuable data we will be able to

11 collect, at no risk to the subject, and the

12 potential benefit to the hemophilia community.

13 So what we propose is the following.

14 That Avigen should continue its assay

15 development and preclinical studies in various

16 animal models. While there is some question about

17 the validity of some of the animal models that we

18 are studying, I hope it is clear that Avigen is not

19 trying to skirt or avoid this issue, but we are

20 aggressively going after an answer.

21 The informed consent should be reviewed

22 and updated as needed to reflect the current data,

23 as we have between the first two patients.

24 The Phase I trial should be allowed to

25 continue regardless of whether the motile sperm

195

1 fraction is positive, and that just goes back to

2 the difficulties that we have had in the motile

3 sperm, and once we get to, let\'s say, the next

4 dose, we are assuming that we are going to actually

5 get some contaminations that are going to give us a

6 positive signal.

7 The subjects should be monitored until

8 three monthly semen samples are negative. That is

9 currently in the protocol already. We should, even

10 though the value of the fractionation procedure is

11 a question, we want to continue to find out and

12 maybe improve, as has been suggested here today.

13 We also want to ask a question that is

14 very important and the committee has raised today,

15 which is whether the vector sequences in the semen

16 actually represent biologically active vector.

17 That is just not very clear.

18 If we can do this and continue the trial,

19 we can use the clinical data to identify predictive

20 inadvertent germline transmission preclinical

21 model, which will help if this trial hopefully goes

22 through later phases, having a predictive model

23 would be very good and clearly for other AAV trials

24 coming in the future.

25 We want to continue to encourage subjects

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1 to bank sperm prior to the treatment, and the

2 subjects will continue to be informed of their

3 semen results and counseled about whether they

4 should continue to use contraceptives.

5 The final point is important because it is

6 one of the questions that was raised by the FDA to

7 this committee, which is what happens if you have

8 persistent germline transmission.

9 We think, and I believe that the committee

10 believes, that the possibility of that is low, we

11 feel like we need to have a contingency, and we

12 believe that if the subjects and the partners are

13 encouraged to undergo counseling by the study

14 physician on a regular basis if the vector is

15 persistent in the motile sperm, that that will

16 mitigate against the risk of transmission.

17 We selected greater than one year. That

18 was somewhat arbitrary. We are certainly open to

19 discussing with the FDA what would be a reasonable

20 time frame, but that is just the one we selected

21 that we thought was reasonable.

22 In closing, we are convinced that there is

23 a solution to the time of the completion of this

24 Phase I trial, and are committed to working with

25 the FDA to find and implement such a solution.

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1 Thank you.

2 DR. SALOMON: Thank you very much.

3 The schedule shows lunch. I think you may

4 have intuited that I wasn\'t planning on lunch, and

5 just go into answering the questions. I just

6 wanted to make sure that that was okay with

7 everybody.

8 Committee Discussion of Questions

9 We have a series of questions. I am very

10 comfortable that we have set most of the

11 intellectual background here to do this. The first

12 question:

13 1. If vector sequences are detected in

14 the motile sperm fraction of clinical trial

15 subjects, the current approach of the FDA is to

16 suspend accrual to the study \-- in other words, put

17 it on a clinical hold, and that is what happened in

18 this case, just to make sure that everybody is

19 clear about that, these guys can\'t go forward right

20 now \-- regarding the persistence of the vector then

21 becomes the criteria upon which the clinical hold

22 is raised or not raised.

23 If they are getting out of 14 weeks and

24 they are still positive in their second patient at

25 the lowest dose, that is how they are calculating

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1 that it would take five years to do the study, and

2 it might even be longer.

3 So I think most of us who have done

4 clinical trials are sympathetic with that being

5 very difficult.

6 Enrollment has been allowed to proceed

7 when there are data to show that it is negative.

8 In other words, three consecutive samples.

9 A. Does the committee agree that a

10 clinical hold is warranted when motile sperm tests

11 positive for vector sequence or should enrollment

12 be allowed to continue with appropriate

13 modification made to consent documents?

14 Discussion?

15 Let me just point out something here.

16 There is a little bit of a load in here because we

17 are talking about, at least for me, this is motile

18 sperm tests. I am underwhelmed with this motile

19 sperm test thing. In other words, I think it is

20 great science, but I am not sure, and welcome

21 discussion on that point, that you really have to

22 do motile sperm tests here, and I think it would be

23 just easier to do semen.

24 Go ahead. You wanted to make a

25 qualification?

199

1 DR. KAY: \[Off mike.\]

2 DR. SALOMON: The point is that you are

3 still on hold. I think that is the main

4 clarification.

5 Yes, Tom.

6 DR. MURRAY: First, just to clarify a

7 factual question. The reason you are underwhelmed

8 by the motile sperm test, I am sorry it\'s unfair to

9 ask Dr. Salomon a question. I begin with a

10 clarification from you.

11 The reason you are underwhelmed with the

12 motile sperm test, maybe I misunderstand the nature

13 of the test, but if I understood it correctly \--

14 the first thing we are after is if we are concerned

15 about germline transmission, we don\'t want to see

16 altered genes to a child, and there are a variety

17 of ways to sort of protect against that.

18 The motile sperm test, as I understood it,

19 was imperfect for a variety of reasons, but most of

20 them had to do with the fact that you might still

21 get AAV DNA even if it wasn\'t going to be, so it\'s

22 a sort of both and test.

23 If it came up negative, would you be

24 pretty reassured that you weren\'t going to get?

25 Okay. But if it came up positive, you still

200

1 weren\'t sure whether it meant that it was

2 infectious or not. Okay. So, I have got that

3 clear. I wanted to be sure I got that clear.

4 Let me start then from the back end. If

5 the concern is to prevent the transmission of

6 altered DNA to offspring, there are a number of

7 ways to try to achieve that. One is to test motile

8 sperm. If it comes up negative three times in a

9 row, we feel pretty comfortable that it is not

10 going to happen.

11 I noticed, by the way, that one of the

12 spokespersons for the hemophilia community actually

13 said about requiring barrier contraception. I am

14 assuming the FDA is not in a position to require it

15 or enforce a requirement for barrier contraception,

16 but I think we need to take that as an expression

17 of the genuine concern on the part of that

18 community.

19 So, a second order would be to again

20 strongly encourage barrier contraception, provide

21 very clear informed consent. Give the kinds of

22 warnings that Dr. Gordon was, I think, alerting us

23 to, say, look, a way around this is not to do ICSI

24 with your current sperm after intervention, and be

25 very clear about the variety of things that they

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1 ought not to do right now, to bank sperm

2 beforehand.

3 I mean there are a lot of things you can

4 do to encourage, to sort of defeat the likelihood

5 that there is going to be any transmission. So we

6 have got at least two protections there. A third

7 is \-- well, think about what if the worst possible

8 scenario happened, we go ahead with the trial, we

9 permit the trial to continue despite being pretty

10 comfortable with the test, despite the promise to

11 do barrier, despite the information given in

12 informed consent, a child is, in fact, conceived

13 and born who is carrying an altered gene here.

14 We need to think about \-- we are not

15 talking about a systemic kind of \-- you know, a

16 change in many, many of the births of many

17 children, we are not talking about an intentional

18 modification of a genome, we are talking about, you

19 know, this is an incidental and unintended

20 consequence.

21 I don\'t have the answer to that, but that

22 is the thing we would be guarding against, how

23 horrible of a mora affront or of a precedent would

24 that be? I don\'t have an answer to that one, but I

25 just want to lay it on the table.

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1 One more challenge before us. There is

2 the Avigen issue, which we need to answer. I guess

3 I should hold off for No. 2, but Question No. 2 is

4 going to say, okay, it ain\'t just men, it\'s also

5 women, and how are we going to think about being

6 aware with women.

7 DR. SALOMON: That is Question 2, and we

8 will get to that.

9 To me, you have raised a couple of really

10 interesting points, so let me go back to this

11 motile sperm thing. The problem I have with any

12 sort of testing strategy in a clinical environment

13 is the more complicated you make it, the more

14 difficult it is, more expensive, more technically

15 challenged, and more often is going to go wrong.

16 I have heard no data that convinces me

17 that if the semen is positive, that the motile

18 sperm could be negative and that this is such a big

19 advantage. In the absence of that data, I continue

20 to be underwhelmed with the need to be doing this

21 motile sperm test, because what happens then is you

22 say the motile sperm test, because of the fact that

23 there is a whole bunch of manipulation, or you get

24 a patient that doesn\'t have a large enough volume,

25 although I am not quite sure why they couldn\'t just

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1 dilute it up to 1 1/2 ml with saline, but anyway \--

2 the bottom line here is if you get into that sort

3 of circumstance, it just tells you why these kind

4 of assay systems are problematic.

5 We would actually have to tell them \-- we

6 would have to have this, I would think rather silly

7 discussion about how to set a quality control for a

8 motile sperm test, because if they let it sit out

9 on the bench for a couple hours and then they do

10 this motile sperm test, and call it negative, and

11 the semen is positive, well, that is bogus.

12 I am just having trouble with this test,

13 and I don\'t think it is a minor issue in terms of

14 how they are going to do this trial.

15 DR. MULLIGAN: Just to directly answer the

16 question, I would say that a clinical hold would

17 not be warranted although I would couple the

18 question in 1A to the first part of 3, and then to

19 answer your question, I think the motile sperm

20 thing is perfectly okay, that if we all think that

21 the likely source of the AAV is probably blood or

22 something, then, the best, you can separate those,

23 I think it helps, and I think there are SOPs,

24 people can come up with something.

25 But 3, I know how you hate to jump to the

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1 next question, but I think it has to do with at

2 what point, when repetitive tests showed positivity

3 would everyone think that there was something so

4 unanticipated that you would actually want to stop

5 the trial?

6 My proposal would be no, let the trial go

7 on, but come up with some, maybe it\'s a year, throw

8 out a year, if after one year there is still

9 positivity, even if that was in the blood

10 mononuclear cells, I think that most people would

11 have grave concerns that something was happening

12 that was not anticipated.

13 DR. SALOMON: I followed you up until that

14 last little throw-in about peripheral blood and

15 mononuclear cells, I mean because I wouldn\'t care

16 that much of the patient integrated into a

17 hematopoietic stem cell and was positive.

18 I think the real issue here is only given,

19 I don\'t know why a year, because it seems like that

20 is a hell of a vector reservoir to think that you

21 could keep shedding detectable vector for 12

22 months?

23 DR. MULLIGAN: That\'s the point. The

24 point is that everything we have heard here

25 suggests that you won\'t have persistence of the

205

1 vector for that amount of time, simply by having it

2 sitting there in some tissue. Therefore, something

3 is happening that is unanticipated if it is

4 persisting at that point, and that could be an

5 arbitrary point, but I think a year is certainly

6 enough time to think that something is happening

7 that shouldn\'t happen, at least we don\'t know what

8 is happening.

9 My point was just that a critic that would

10 say, well, yeah, that could still be not in the

11 motile sperm, I would say still, we would be

12 worried about the patient because something very

13 unanticipated has now happened, that is, if we are

14 able to get hematopoietic stem cells transduced by

15 AAV vectors, and you have AAV integrated, I think

16 everyone would want to know that and would have

17 great concern.

18 DR. NOGUCHI: Just to add more confusion

19 to that particular point, the hemophilia trial for

20 factor VIII, and the Chiron proposal, they did

21 report to the same committee that in a situation in

22 which there was a very fractional and very

23 short-lived positivity in the semen, that, in fact,

24 they were able to detect positive peripheral blood

25 samples for I believe well over a year.

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1 That was the unexpected finding in that

2 particular case. Probably an encouraging finding

3 in terms of at least transduction of a somatic

4 cell, I would guess.

5 DR. SALOMON: I think that everybody in

6 this field is clear that the amount of time that

7 even the episomes persist, and whatever small

8 integration occurs or whatever, in different

9 populations, I think Jude was very good about

10 pointing out that there is some data, but it is not

11 completely tested. Is that a fair characterization

12 of what you said?

13 The fact that it would be around, it could

14 be a positive for this as a gene therapy strategy,

15 I am not concerned about it, I think the issue has

16 to focus on the sperm or the semen as a test. My

17 only issues there are technical, but if it is in

18 the sperm at a year, then, it seems to me it is

19 impossible \-- well, is it impossible that at that

20 point \-- I just can\'t imagine you are shedding

21 viral reservoir any longer. The implication at

22 that point would be that it has been integrated

23 into the germline.

24 DR. MULLIGAN: I think the point of

25 setting some long period at which you would stop

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1 and take another look is you want to address it in

2 a way that was not in the clinical protocol.

3 DR. SALOMON: I am agreeing. I am just

4 bringing up some discussion points on the timing.

5 DR. DYM: This is an unrelated comment,

6 but it is on the same issue. With the subject No.

7 2, who has AIDS, I think is what we were told, and

8 spermatogenesis is markedly reduced in patients

9 with AIDS, and this is shown, of course, in the

10 total semen volume, 200 microliter, 150, 150, it is

11 very unlikely that this particular patient will be

12 fertile. I don\'t know if that is an issue or should

13 be raised.

14 DR. KAY: Just for a point of

15 clarification on the patient No. 2, the patient is

16 HIV-positive, his CD4 count at the time, that last

17 one that we checked, was around 340. He has had

18 children in the past, and based on his total sperm

19 count, he actually has a normal number of sperm in

20 the ejaculate, but the volume is very low, so from

21 what we can get from that, the spermatogenesis

22 itself is normal, but there is something wrong with

23 the ability to make the fluids. The pH of the

24 fluid has been normal, suggesting that he doesn\'t

25 have specific obstruction of a prostate versus

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1 seminal vesicle.

2 DR. DYM: I was interested in this

3 question before that may be relevant. Did you

4 check the size of the testis, does he have a normal

5 testis?

6 DR. MAY: Well, since I am not the

7 clinical treater on this case, obviously, Dr.

8 Glader, who I think had to leave to catch a plane,

9 is the individual who examined him.

10 DR. RAO: I actually wanted to retread

11 what Dr. Salomon and Dr. Mulligan said. I don\'t

12 think that it would be necessary that there should

13 be a clinical hold and that that should be

14 dependent on motile sperm test. There are two

15 problems in my mind with just doing the motile

16 sperm test, is that \-- we have already heard about

17 the problems of vector carryover in semen itself,

18 so even if your motile sperm test was negative, you

19 would still worry about that as an issue.

20 The likelihood from the way the test was

21 presented, is that if your semen is negative, then,

22 your motile sperm fraction is going to be negative.

23 So it doesn\'t seem that we should be focusing on

24 motile sperm test as a specific test, but rather on

25 total semen.

209

1 Do we necessarily need to \-- which was

2 part of Question 3, which you put together \-- it

3 didn\'t seem to me that you should include only

4 patients who are incapable of the production, but

5 there should be a time line and that patient

6 consent, the forms should be modified with an

7 emphasis that this is a problem.

8 DR. JUENGST: I guess my hesitation in

9 originally answering your question was because I

10 was hung up on the qualifier, \"or should enrollment

11 be allowed to continue with appropriate

12 modification made to the consent documents.\"

13 Well, no, that is not enough to modify the

14 consent documents. It is really a change in the

15 protocol because informed consent is not a

16 reasonable and prudent safeguard. Patients behaving

17 appropriately cautiously is a prudent and

18 reasonable safeguard.

19 So they need to be more than simply read a

20 few extra lines in the consent form. It needs to

21 be a real concerted program for making this

22 education and sperm banking available, and that

23 sort of thing.

24 It sounds like what they have been doing.

25 DR. SALOMON: It sounds like that has been

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1 a part of their protocol even a trial or two

2 earlier, which is good. I think we are getting

3 there.

4 Does the committee agree that a clinical

5 hold is warranted when motile sperm tests are

6 positive \-- let\'s just call it now \"semen\" \-- you

7 know, if we could vote on this, but I generally

8 don\'t want to go there, I mean that is a decision I

9 think that the FDA can come back to us. We have

10 had a discussion on this motile sperm versus semen,

11 I am comfortable with the semen.

12 If there is a clinical hold, is it

13 warranted? The committee to me has said no, that I

14 don\'t think you need to put a clinical hold on this

15 trial every time the semen is positive. Does

16 anyone want to take a minority opinion here?

17 DR. NOGUCHI: On that one, we might want

18 to at least have a show of hands to make sure we

19 understand the real kind of sense of the committee.

20 DR. SALOMON: Then, we will poll. Do you

21 want us to give you a specific on the motile sperm

22 versus semen?

23 DR. NOGUCHI: I think that is very

24 important. It certainly seems like there is enough

25 discussion that there is some doubt about the extra

211

1 value of fractionated versus just whole semen. I

2 think it would be worthwhile for that, too.

3 DR. SAMULSKI: If we were going to poll, I

4 think it is probably important that I at least make

5 one or two statements about the vector aspect of

6 it.

7 I am not going to proclaim I know the

8 answers, but I have been working at this for over

9 20 years, and there are definitely trends that show

10 up that are extremely consistent, that you can

11 begin to feel confident about, and the virus

12 integration is a trend that has been consistent

13 from when this was first studied and now going from

14 tissue culture to animal models, it doesn\'t

15 integrate very efficiently.

16 I think people need to understand that and

17 buy into the fact that if we are going to put a lot

18 of virus into people, the potential of integration

19 is virtually nil to it can happen, but it is not a

20 high risk potential, and then if you move away from

21 that question and look at the question of if we

22 have a PCR-positive signal, which is something like

23 10 copies in a sample, and we are talking about 0.1

24 percent of the virus ability to integrate, we are

25 getting down to numbers and the amount of sperm

212

1 that one is going to be transmitting, this is like

2 Star Wars in some ways, trying to calculate what is

3 the frequency of the planets lining up again, and

4 stuff like that.

5 It is so vanishing small, the risk that we

6 are talking about, that from a vector perspective,

7 I think there is no reason at all to put this trial

8 on hold. Where I do have my only concern, and it

9 sounds like it is being addressed, is that Phil

10 brought up, which is if you are mechanically going

11 to have virus tracking along, and you are going to

12 do experiments to see if you can come up with a way

13 of artificially getting this into cells, that is an

14 unknown that needs to be resolved, and I think the

15 onus will fall on the group that is interested to

16 get that data in front of people as soon as

17 possible.

18 But other than that, I am sitting here

19 saying we are really discussing something that is

20 virtually impossible, and I think that value of

21 what can come out of the studies is a lot more

22 important than us trying to talk this tightrope.

23 DR. SALOMON: I very much agree with that,

24 too, and I think I am comfortable that the flow of

25 this committee is going in that direction, as well.

213

1 You brought up one thing, Jude, that I

2 just wanted to come back to, and that is you

3 suggested, I don\'t know if you meant it, additional

4 sophistication on this testing strategy, and that

5 would be do quantitative PCR, and if it is lower

6 than a certain number, even that then could enhance

7 one\'s comfort level. I hadn\'t thought of that,

8 because up until now, we have been talking about

9 positive versus negative.

10 Would you be concerned if it came out as a

11 million copies? I mean these guys are detecting it

12 positive at the lowest levels at 14 weeks.

13 DR. SAMULSKI: So that is where I think it

14 is prudent, and it is not just AAV when you keep

15 dosing biologics, whether they are plasmids of

16 oligos, at some point you are going to have a

17 threshold level where it is going to be

18 unpredictable.

19 I think that is the kind of information I

20 would like to see keep coming out of this trial, he

21 didn\'t just persist longer, but here is how he

22 persisted, it was over 1,000 or 10,000 copies per X

23 amount of time, and stuff like that. That is

24 valuable information.

25 DR. SALOMON: I agree with that, and I

214

1 would say that I was not impressed by a statement

2 that it wasn\'t absolutely quantitative, but the

3 signal was going down. I think today, there are

4 very straightforward ways to do quantitative PCR,

5 so we don\'t have to really, in these settings, to

6 any longer talk about nonquantitative PCR studies.

7 Let\'s do a poll of two questions. One

8 would be the first, and that would be motile sperm

9 versus semen testing, just to get that out of the

10 way.

11 Starting with you Jude, motile sperm or

12 whole semen?

13 DR. SAMULSKI: I think whole semen is

14 adequate.

15 DR. DYM: Yes, same. Not necessary to

16 differentiate between the two.

17 DR. JUENGST: For what it is worth, my

18 layman\'s vote will go with these guys. We don\'t

19 differentiate.

20 DR. MURRAY: I confess I do not understand

21 the merits of the two tests sufficiently to make an

22 intelligent vote, but it is not because I am torn

23 between two alternatives I understand well.

24 MS. WOLFSON: I would have to repeat

25 exactly what Dr. Murray said.

215

1 DR. RAO: I think whole semen.

2 DR. SALOMON: I vote whole semen

3 obviously.

4 I am not certain. Richard gave me what he

5 would say for the second one, but I am not

6 comfortable with that, so we will have to say he is

7 not here.

8 Let\'s do the second question, and that

9 was, does the committee agree that a clinical hold

10 is not warranted any longer with a positive semen

11 test? Jude.

12 DR. SAMULSKI: Not warranted.

13 DR. DYM: A hold is not warranted, I

14 agree.

15 DR. JUENGST: A hold is not warranted.

16 DR. MURRAY: I agree that with the proper

17 additional protections put in place, with Eric\'s

18 earlier caveat about it is not just adding lines to

19 informed consent, we would not require a hold.

20 MS. WOLFSON: Again, I agree with Dr.

21 Murray exactly.

22 DR. RAO: I don\'t think a hold is

23 necessary.

24 DR. SALOMON: And I don\'t agree a hold is

25 necessary either. Richard Mulligan told me that he

216

1 also wanted to say that a hold is not \--

2 MS. DAPOLITO: Dr. Mulligan should be here

3 to vote, but Dr. Salomon can put his comments into

4 the record.

5 DR. SALOMON: Excellent.

6 The next question here would be discuss

7 the implications of detecting vector sequences due

8 to the presence of contaminating transduced PBMC or

9 vector (either free or on the surface of a sperm)

10 in the motile sperm fraction.

11 Now, my sense here is we have really

12 pretty much discussed that. If the FDA comfortable

13 with that? I don\'t see any further discussion as

14 being necessary on that one.

15 Anyone else on the committee?

16 \[No response.\]

17 DR. SALOMON: Okay.

18 Question 2. There are technical

19 limitations in the ability to monitor women and

20 certain men for evidence of germline alterations.

21 One approach to monitoring subjects for germline

22 alteration would be to restrict early clinical

23 development of certain gene transfer products to

24 subjects who have been shown to be capable of

25 repetitively supplying adequate semen samples for

217

1 analysis in order to get good data collection for

2 detecting persistence of vector.

3 DR. MURRAY: That does leave out a certain

4 number of potential \-- about 50 percent of the

5 human population actually, which is not relevant

6 for the Avigen trial, but will be relevant for

7 others.

8 DR. SALOMON: Correct.

9 I am going to try and parse it down. So

10 the first issue would be \-- I think you guys

11 actually had a slide on this, am I mistaken? You

12 had a slide saying that you were redesigning \-- you

13 had to have more than 1 1/2 ml of semen and a

14 certain sperm count, wasn\'t that right?

15 They have already incorporated in their

16 protocol, does anyone disagree with that?

17 DR. SAMULSKI: I think it\'s admirable

18 that they are doing it, but i also agree with Dr.

19 Murray. I don\'t think this is something you want

20 to put in as policy because if other things come

21 down that aren\'t related to hemophilia and the

22 population can\'t be in an inclusion criteria, but

23 based on something like this, I don\'t think that

24 would be a direction we would want to go in.

25 DR. SALOMON: Certainly, men who were

218

1 infertile and would have no danger of germline

2 transmission would be excluded from these studies,

3 so I think I could think of one very good reason

4 not to make it policy in the area.

5 Dr. Rao.

6 DR. RAO: I was just going to add that one

7 reason for worrying about sample size was because

8 we were doing motility experiments and

9 fractionation. Now that the committee seems to

10 have a consensus that we don\'t need to do

11 fractionation, I think the tests can be done with a

12 smaller volume, so I don\'t think that should be an

13 exclusion criteria.

14 DR. SALOMON: All you need now would be

15 enough sperm to get, let\'s say, 1 microgram of DNA

16 \-- I am sorry \-- 3 to 4 micrograms of DNA, so you

17 could do triplicate or quadruplicate at 1

18 microgram, and from my own experience doing Tacman

19 PCR, which we do a lot of in the lab, I would be

20 comfortable with that, as well.

21 So, that would be a sperm count of what?

22 Were you guys counting 30,000 sperm for a microgram

23 of DNA?

24 DR. SAMULSKI: 2 x 106.

25 DR. KAY: Yes, we were using 2 to 3 x 105

219

1 sperm per microgram roughly, and we need 4 or 5

2 micrograms, and then the issue is recovery.

3 DR. SALOMON: Would everyone agree there

4 should be a limit of, let\'s say, no less than 5

5 million total sperm in an ejaculate as criteria for

6 entering the trials at this point, 10 million? All

7 right.

8 DR. KAY: Sold.

9 DR. SALOMON: Any discussion on that

10 point? I think the intention of the committee is

11 clear, and the details we would leave to you.

12 Depending on the amount of data required,

13 much of the early clinical experience with the

14 vector might be limited to this restricted

15 population. A development program requiring

16 extensive characterization of distribution to

17 germline cells and germline alterations might delay

18 the acquisition of adequate safety and efficacy

19 data in other populations, for example, women.

20 I guess we can\'t avoid the very important

21 discussion of where women fit into these trials.

22 So this gets to something that Tom, you introduced

23 to us in your comments, thoughtful comments a few

24 minutes ago, and that is, what is our level of

25 sensitivity here, how big of a deal would it be,

220

1 and that has a lot to do with the next trial that

2 comes along that wants to do the study in women,

3 right?

4 DR. MURRAY: Any disorder that is not

5 x-linked.

6 DR. SALOMON: Or a male that has no sperm

7 or falls below it, we need to think about them, as

8 well.

9 The interesting thing here is I mean we

10 are now up against the international consensus that

11 has been supported by many in the lay public, that

12 the line that no one is ready to cross is

13 intentional? Germline transfer certainly. And

14 unintentional germline transfer is probably far

15 enough across that line that it ought to be avoided

16 at all costs, as well.

17 I am very cognizant of the fact that

18 depending on how this discussion goes, we have to

19 be cautious that we are having an advisory

20 committee advising the FDA that under certain

21 circumstances, it is okay to do germline gene

22 transfer, and I want to put that into context for

23 the committee. You can disagree with it, but that

24 is the question.

25 DR. MURRAY: There is one piece of advice

221

1 I think we could give the FDA. It may not be very

2 useful advice, but that is to \--perhaps you have

3 already done it, \-- but basically to take stock of

4 the best possible methods for evaluating germline

5 alterations in females, looking at animals, looking

6 at \-- we have heard a variety of ways of thinking

7 about looking at such alterations in males, what is

8 the best state-of-the-art in thinking about this

9 with females? Granted that many of those assays

10 are going to be completely unavailable with humans,

11 to think about what would be the most \-- morally

12 permissible and without crossing the boundaries of

13 mistreating human subjects, how to involve women,

14 how to monitor potential germline genetic

15 alterations in women.

16 I don\'t think that is easy, maybe you have

17 done it, but if you haven\'t, I would say it is

18 probably an urgent thing. The one thing I could

19 not recommend \-- we had a tough choice here \-- but

20 one think I clearly could not recommend is that

21 women be excluded from such trials. I suspect

22 national policy would also prohibit us from making

23 such a recommendation.

24 DR. NOGUCHI: Just in terms of that, FDA,

25 unless there is a compelling reason to exclude one

222

1 sex or another or any particular subset of man or

2 woman, we would not use anything other than a very

3 specific reason that is both reasonable and can be

4 defended.

5 DR. JUENGST: Another thing to think about

6 is where along the research time line we would be

7 willing to take those risks of inadvertent

8 transduction. We do take genetic risks with

9 patients when we give them chemotherapy and

10 radiation, and we justify that by saying, well, we

11 are saving their lives, so in a Phase III trial of

12 gene therapy, we could make a very similar

13 argument.

14 Now, how about a Phase I safety study,

15 maybe there is a distinction to be drawn there

16 unless we find ourselves in the situation where you

17 can\'t get to Phase III unless you do the Base I.

18 DR. SALOMON: That\'s a good point. Just

19 to highlight that you are pointing out that the

20 quote, \"We are doing it because we save human

21 lives\" is a clear indication of conviction of

22 efficacy, which would allow you to accept a risk

23 that is very different than in a Phase I or Phase

24 II trial.

25 Dr. Rao.

223

1 DR. RAO: I was just going to say that the

2 general consensus that you want to be able to

3 monitor, it is not an issue of whether it can

4 happen or not happen or the probability, but that

5 you want to have an ongoing monitoring to make sure

6 that there is no evidence of germline transfer, and

7 should we be excluding patients where we can\'t

8 monitor that, and the answer is that I think we are

9 doing that already when you set up a criteria,

10 whether you set it for males when they can\'t have a

11 certain sperm count, or whether you can\'t because

12 you don\'t have any available tests to do that

13 monitoring.

14 The question is do we not need to monitor

15 at all. In my opinion, right now, with the

16 available data, it is not clear because there is

17 still enough not known about the virus in the sense

18 what is happening in blood cells, why do we see

19 persistent expression, is there some specific time

20 at which you see better integration, and so on.

21 So there is a finite, maybe already low

22 probability that there might be germline transfer,

23 but whatever that low probability, at the current

24 situation, with what data is available from animal

25 models, we can\'t say that we should include

224

1 patients where we don\'t have any monitoring.

2 DR. SALOMON: If you think about it, there

3 is a couple different ways this could go. None of

4 us, unless some \-- I can\'t imagine are going to

5 give advice that you want to permanently exclude

6 women from certain kinds of trials, right, why even

7 go there, that\'s impossible, so you can see a

8 couple different ways to try and put this together,

9 and I think the framework is already out on the

10 table, one way is to say in the absence of really

11 good definitive preclinical data that would allow

12 you to say with any sort of confidence it cannot go

13 into the germline, and I think we all agree you

14 cannot say that quite yet, no evidence that it

15 does, but no evidence to say that is can\'t, and a

16 lot of evidence suggests that it ain\'t going to be

17 easy and not likely, particularly with this

18 particular class of vectors.

19 You could say okay, really low

20 possibility, so that takes a lot of the pressure

21 off, but it is not enough. So then you go on and

22 say all right, fine, let\'s go into Phase I trials

23 and let\'s restrict the Phase I trials to subject

24 that we can monitor.

25 I think the company themselves, to their

225

1 credit, have taken that view, and the FDA is

2 comfortable with it, and I think we have just

3 refined it a little bit.

4 Then, the only question left is how much

5 data do we need under our belt before you allow in

6 later phases of the trial, to go into women, pretty

7 much saying, hey, it\'s not happening, and I don\'t

8 want to get into demanding someone give us

9 statistical time, like after 100.3 patients we can

10 do it, but I think I am suggesting to you that

11 maybe the best way to think about this is at a

12 certain point, once they get to a Phase III trial,

13 and there is enough confidence that none of these

14 patients with no evidence of germline transfer in

15 these males that can be monitored, that then you

16 could relax the criteria and cautiously open it up

17 to women.

18 So that would be what I would suggest.

19 MS. CHRISTIANSON: Janet Rose

20 Christianson. QARA Services, formerly with Target

21 Genetics Corporation.

22 A brief comment with regard to selecting

23 people for monitoring in Phase I. I think there

24 has got to be another consideration, and I think

25 that has to do with the route of administration.

226

1 If there is no dissemination, for example, the

2 present study that Target is doing is an oral

3 aerosolized delivery of an AAV vector in cystic

4 fibrosis patients. I think that the way it is

5 delivered, and any of the preclinical data,

6 indicating if there is dissemination to the

7 peripheral blood distal nodes, or whatever, should

8 also have a bearing as to whether or not monitoring

9 of females or nonfemales or whomever, should be

10 part of that whole process. I think that has got

11 to be a point just to consider. Maybe my glucose

12 was low and it\'s intuitively obvious, but I did

13 want to make sure that that point was raised.

14 Thank you.

15 DR. SALOMON: I think that is an excellent

16 point.

17 Dr. Rao.

18 DR. RAO: I actually wanted to add one

19 more piece to the whole monitoring issue, and that

20 was just simply to argue that if the criteria or

21 the worry for which you are excluding patients is

22 because of germline transfer, that perhaps one

23 additional criteria for inclusion is people who

24 would not be, are incapable of germline transfer.

25 DR. SALOMON: The only problem with that

227

1 is, it is kind of a dead end in terms of moving the

2 field forward, because you would never be able to

3 tell whether there was germline transfer, so

4 everyone else would be standing there waiting.

5 DR. RAO: Maybe you shouldn\'t exclude them

6 for whatever reason you want to include them in a

7 study. That is all I was trying to say.

8 DR. MURRAY: In a way, we should be very

9 grateful to the folks from Avigen for their

10 inadvertent finding, because it really forces us to

11 confront \-- I don\'t mean just this committee, by no

12 means do I mean just this committee \-- I mean

13 everybody who thinks about these larger issues of

14 the ethics of research, inadvertent germline

15 transfer, and gender equity in research, and all

16 these things, it warns us about what is probably

17 lurking not too far down the road, and in addition

18 to my off-the-cuff injunction to FDA to sort of

19 think as much as they can, I don\'t think this

20 committee is the group to decide what the right

21 balance is, but in fact, I mean RAC has had a

22 recent history of doing policy conferences.

23 This would be a great topic for a RAC

24 policy conference, in my view, about how to balance

25 the concern about monitoring inadvertent germline

228

1 modification against an issue of gender equity, is

2 it as we think it is, that it would be much more

3 difficult to monitor in women, are we wrong about

4 that? Are there ways of monitoring this in women?

5 I am not aware of any, but, you know, there are

6 some fact questions there science questions, and

7 then how should one sort of strike the right policy

8 balance.

9 One emerging suggestion, I think I have

10 heard, is that you do the Phase I \-- where this is

11 a possibility \-- you do the Phase I on males who

12 have sufficient seminal fluid and sperm that you

13 can test, they make 5 million sperm in an

14 ejaculate.

15 Now already, that creates some issues of

16 gender inequity, I understand the rationale for

17 that, but I think it would be a mistake to rush

18 forward into that without a chance to really

19 reflect on how to balance.

20 There are two good things. We are trying

21 to ensure gender equity and participation in

22 research, and we are trying to ensure that we can

23 get a handle on inadvertent germline gene transfer.

24 There are two good things. Somebody has got to

25 figure out what the right balance or plan is, and

229

1 it is not a thing we are going to do by 3 o\'clock

2 today, and we are not the right party to do that.

3 DR. NOGUCHI: Steve, shall we work on that

4 as a possibility, what Dr. Murray is talking about?

5 DR. ROSE: Certainly, it is something that

6 the RAC has been discussing and will continue to

7 discuss, and it is one of the policy conferences we

8 have been thinking about.

9 DR. NOGUCHI: You are welcome to come,

10 too, Tom, I am sure, and probably most of the rest

11 of the people at the table here.

12 DR. SALOMON: Jon.

13 DR. GORDON: Yes, I am commenting. I

14 have recused myself from this discussion because as

15 a committee member, and I am commenting as a member

16 of the public.

17 I think there are a couple of points. One

18 is that whenever you exclude a certain group of

19 people from a study, regardless of the phase of the

20 study, you at least have to be alert to the

21 introduction of biases in the study, so I think

22 people need to be aware of that.

23 Is it going to be more safe for the people

24 you study or less safe? I don\'t think it is

25 necessarily relevant in the present case, but

230

1 anytime people are excluded in some sort of

2 overarching parameter, then, that is a risk.

3 In terms of the addition of females to

4 these trials in hemophilia, not a likely issue to

5 come up, but as people have point out, autosomal

6 disorders it is, I think the committee might

7 consider recommending that good preclinical tests

8 for female germline transmission be encouraged to

9 be developed.

10 I mean it is not impossible to do that,

11 and there is no reason why, if we have been doing

12 all these things with rabbits and monkeys and all

13 that with the male side, why we couldn\'t also do

14 things on the female side.

15 We have a paper where we looked at adeno

16 at the female side, so there is no reason why that

17 couldn\'t be encouraged by the committee.

18 DR. SALOMON: I would actually not want to

19 go there. I don\'t think as a committee, we want to

20 start even getting into whether women as part of

21 participation in a trial ought to undergo

22 laparoscopy and removal of eggs or ultrasound

23 guided biopsies, and things like that, if that is

24 what you were suggesting. I think those are topics

25 for preclinical investigations and not for creating

231

1 yet more complex and even potentially risky

2 barriers for participation in a trial.

3 DR. GORDON: I guess I wasn\'t clear. I

4 believe, I emphasize preclinical studies in animals

5 that would then give one more confidence that a

6 human could be admitted to a study.

7 DR. NOGUCHI: Just to say that the issue

8 of women is pertinent to this discussion, albeit

9 it, it is an extraordinarily small population,

10 there are handful of women with hemophilia, and for

11 them, especially they are totally out of any of the

12 normal support mechanisms. They may not even know

13 what hemophilia is because it is not something that

14 they normally know about, but eventually when one

15 of these things works, they are a part of the

16 question. We will have the same question as to

17 whether or not it is an unreasonable risk for that

18 population albeit it might be as many as on this

19 hand here.

20 DR. SALOMON: I think that at the end,

21 there is no way \-- again, I welcome everyone to

22 comment \-- from my view, I don\'t see how one can

23 refine this any further in the sense that it has

24 been put very clearly that, on one hand, the

25 concept of germline transfer as a potential in a

232

1 clinical gene therapy trial, there has been a lot

2 of discussion on that, and it is pretty much

3 considered to be a line that the public doesn\'t

4 want us to cross, and I think we have to respect

5 that.

6 At the same time, however, we realize that

7 as we gain experience and information, we can begin

8 to feel more and more confident that that is not

9 occurring even though the risk may never be zero,

10 and, of course, we will get into discussion and so

11 I might as well bring it up, that if you show it

12 doesn\'t happen in the males, does that mean that it

13 won\'t happen in the females, and, of course, female

14 biology is very different than male biology, we all

15 realize that.

16 I think there we need to put more energy,

17 I think as John and others have already said, and

18 to some of the preclinical models anticipating what

19 is around the corner for this field, and that I

20 think a reasonable leadership position from the

21 committee.

22 I guess the last thing we have to talk

23 about, and if there is anything else, please jump

24 in, but the last thing I feel we have to talk about

25 is okay, so we come back in here a year from now,

233

1 and we get presented data from company XYZ now, it

2 is not Avigen any longer, but they did a trial like

3 this and 10 of the first 50 patients are

4 persistently positive in their semen at one year,

5 and so they do an in-situ hybridization on motile

6 sperm on these particular 10 patients, and 8 of

7 them are positive in 10 percent of the sperm. Now

8 what?

9 DR. DYM: I will answer the question by

10 asking a question of the virologists. Does that

11 clearly mean that it is coming from the earlier

12 germ cells, or can the virus persist?

13 DR. SALOMON: We might as well get that

14 question on the table. Jude, do you want to

15 comment on that?

16 DR. SAMULSKI: My feeling would be that it

17 would have to be in a germ cell to persist that

18 long and consistently come up positive, and for it

19 to just persist, it would get diluted with time.

20 All those cells kept dividing. So this would be

21 the same as the trial, they would come down over

22 time, so I think you are now talking about a

23 completely different situation.

24 DR. SALOMON: And they do a testicular

25 biopsy and it is positive in the spermatogonia.

234

1 Now what?

2 DR. JUENGST: It\'s at least time to stop

3 and take stock and look at where the gene is being

4 integrated, you know, study what is happening, if

5 it is consistent, those sort of things.

6 DR. SALOMON: That\'s fine. Remember what

7 Dr. Samulski pointed out very clearly is that there

8 is no evidence that these vectors will integrate in

9 some specific spot. They will integrate in some

10 specific spot. They will integrate in multiple

11 concatemers in many areas.

12 DR. RAO: There is two aspects to this.

13 You don\'t know what you are doing now because the

14 assumptions are wrong in some sense. You assume

15 that there will be a very low probability of

16 integration, there wouldn\'t be germline

17 transmission, and that if it did occur, there will

18 be a clear-cut barrier and it wouldn\'t be 10

19 percent. So that I think is pretty clear.

20 The question then is what do you do with

21 the participants, right? I mean what happens with

22 the 10 patients that were persistently positive and

23 who presumably have germline transmission, and that

24 I think is a very hard question. I don\'t know that

25 the FDA has any authority and whether we can do

235

1 anything after the fact.

2 DR. SALOMON: That, we know the answer to

3 that. They can\'t do anything. But the question

4 would be now, 50 of the 50 patients haven\'t had a

5 bleeding episode in the last six months.

6 DR. MURRAY: So, it worlds.

7 DR. SALOMON: It works.

8 DR. MURRAY: I think this is not a

9 far-fetched hypothetical life here. There are

10 scientists here who understand different vectors

11 that may, in fact, operate very differently even

12 than AAV, if I understand correctly, and some of

13 then might be much more likely to incorporate to

14 work themselves into spermatogonia, and so this

15 scenario with the different vector system might not

16 be so far fetched at all.

17 So you have done right by the committee to

18 ask this extremely difficult question. I don\'t

19 feel at all qualified to answer it right now. I

20 would have a number of other questions. I would

21 want to know, look, we are talking about a

22 potential random, you know, incorporations at some

23 random place in the genome of foreign DNA.

24 I would like to know how many copies

25 integrated, are we talking about 1, are we talking

236

1 about 1,000 in each genome? If it is thousands, it

2 would seem to me that increases the chance that

3 some of these mutations are, in fact, may be

4 pathological. A thousand hits is more than one

5 hit.

6 Do we have any analogies? Are there other

7 bits of DNA that get incorporated into the genome

8 in a similar random fashion, and how do they \-- and

9 spermatogonia, and what we do know about their

10 fate, and what do they know about the impact they

11 might have on the health of any child born. If it

12 is absolutely horrendous, then, that is one thing,

13 if it is, well, it happens all the time, and rarely

14 really leads to any harm, that is another thing.

15 So, there are still a lot of factual

16 questions we would ask. That will help, I think,

17 help us sort out, but you are right, we should be

18 thinking about them now.

19 DR. GORDON: As a member of the public, I

20 would like to sort of suggest that the committee,

21 in facing such a circumstance, should consider this

22 the way other risks of drug treatment are

23 considered. Now, if you give cisplatinum or

24 bleomycin to somebody, you can probably damage

25 their DNA, or adriamycin to them, and there are

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1 precautions to be taken.

2 In the case of germ and a gene transfer,

3 which I think is a little bit exceptional because

4 you provide acquisition of function, not simply

5 alteration in the existing genome, there are

6 precautions to be taken before the procedure is

7 performed, and there are precautions that can be

8 taken if, in the event, such a thing is discovered.

9 If 10 percent of sperm had a new gene in

10 them, that would mean that there is a 10 percent

11 chance that a conceptus would have it, let\'s say,

12 presuming those sperm function equally well, well,

13 there are people carrying recessive traits around

14 where there is a 25 percent chance that there is

15 actually going to be genetic disease, and there are

16 approaches to that problem - pre-implantation,

17 genetic diagnosis, conception followed by abortion.

18 So there are ways of addressing it if it

19 occurs, but I think the committee is well advised

20 to consider that a hold should be placed while

21 those considerations are formalized.

22 DR. SALOMON: I think that is what Dr. Rao

23 said. I mean I realize this is \-- I think the

24 major point that I was getting at was just to

25 introduce the question, and I think there has to be

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1 a limit, and I think that limit for me is that. I

2 mean if the hypothesis is wrong for any vector now,

3 I am not talking about the Avigen trial, then,

4 probably we should put it on hold and there should

5 be discussions at the highest level, whether it be

6 at the RAC, or be here, or in every place.

7 That includes the appropriate science

8 experts, as well as policy people and ethicists,

9 because I think there, we really have crossed a

10 line that has been set for us in gene delivery.

11 I think what is critical to the FDA,

12 though, unless someone wants to disagree with me,

13 is the advice that you better make sure that any

14 trial that you allow to go forward is adequately

15 designed to make sure that you don\'t miss this from

16 happening. I mean if you are going to recognize

17 it, the trial had better be designed and monitored

18 properly enough to make sure we recognize it,

19 because then, you have got to deal with these

20 issues as a reality instead of as a theoretical

21 risk.

22 Any other comments from the committee,

23 from the public? Does the FDA feel like we have

24 answered their questions? Are there any additions

25 or refinements, et cetera, that we should deal

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1 with?

2 DR. NOGUCHI: Once again, on behalf of

3 CBER, I do want to extend our heartfelt gratitude

4 for helping us over our current and future

5 difficult issues that we seem to face on an

6 increasingly more frequent level.

7 I think the discussion yesterday and today

8 is going to enable us to move forward in a much

9 more cohesive and responsive and responsible way,

10 and for that I only can say we are again very

11 thankful.

12 Based on your last comments and the

13 questions you raise, however, Dan, I don\'t think

14 you are going to necessarily be able to get away

15 from this committee that easily, so I am sure we

16 will see you again.

17 DR. SALOMON: There are laws that will

18 govern eventually.

19 Dr. Couto, did you have a comment?

20 DR. COUTO: Well, it is actually a

21 question that I wanted to just ask the committee,

22 because it was raised earlier, and that has to do

23 with the most optimal PCR assay for detecting

24 vector sequences in semen, because it was raised

25 that maybe a better assay would be a quantitative

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1 PCR assay.

2 One of the reasons why we are not doing

3 that now is because the FDA asked us to develop a

4 spiked plasmid that has a deletion in the coating

5 region, and so that we could differentiate between

6 our vector sequence and our spiked sequence.

7 Now, we wouldn\'t be able to do that with a

8 quantitative PCR assay, but as you have seen, most

9 of our other assays, biodistribution studies are

10 all done with quantitative assays, so I guess I

11 would just like a little bit of clarification as to

12 what people think would be the best assay in the

13 clinical sample.

14 DR. SALOMON: There is two answers to you,

15 but I mean there is certainly multiplex PCR where

16 you could design your probe. If they wanted the

17 spiked sample deal going, I mean you could easily

18 do that these days, and I can help you figure out

19 how to do that if you don\'t know.

20 Dr. Rao.

21 DR. RAO: I was just going to add exactly

22 the same thing in some sense is that spiking is a

23 method of quantitation, so it is a quantitative

24 method of estimating what you have against a known

25 standard of DNA, so I don\'t think there is

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1 confusion. I mean you can even do spiked multiplex

2 PCR on a quantitative fashion using Tacman type of

3 assays if you want to.

4 DR. SALOMON: If you did a Tacman assay, I

5 mean just to educate me a little, if you did a

6 well-validated Tacman assay or I mean there is now

7 other technologies, I am not doing a commercial

8 blurb for Tacman, just quantitative PCR based,

9 there is fluorescence, there is Cybergreen, there

10 is a bunch of different ways of doing it.

11 If you did that, what is the spiking thing

12 for?

13 DR. TAKEFMAN: Well, the spiking, we just

14 say run samples without spike, but one sample at

15 least with the spike just to test for inhibitory

16 effects. So you could run QPCR on some of the

17 samples.

18 DR. SALOMON: Just that same control, that

19 was the main reason. I mean sometimes you need

20 spiking because there is endogenous transcripts

21 that are confusing your sample, but that is not the

22 issue here when you are looking at vector.

23 DR. NOGUCHI: No, semen does have a

24 history of sometimes inhibiting viruses. HIV

25 detection in semen actually for many years could

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1 not be done because of inhibition.

2 DR. SALOMON: That point is well taken.

3 We have been suffering with serum and plasma for

4 stuff in my lab, looking at retrovirus, that is

5 well taken.

6 Did we miss anything? I mean is there

7 anything else people want to get on the table here

8 at the last minute? No.

9 Again, I want to thank everyone at the

10 committee table, and Avigen particularly. I hope

11 we haven\'t beaten you up too bad, but I think you

12 are going home with pretty much you were hoping

13 for, and I hope for the community that your studies

14 go safely first and then demonstrate efficacy next,

15 as I think it is clear that your stakeholders need

16 a viable therapy. If it\'s not Avigen, then, let\'s

17 pray it is going to be for somebody else doing gene

18 delivery doing it.

19 Anyway, good luck. Good luck to everyone

20 else out there. Travel safe and be healthy.

21 \[Whereupon, at 2:03 p.m., the proceedings

22 were adjourned.\]

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