1

 

                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

                                                                 2

 

                              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.

                                                                 3

 

                            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

                                                                 4

 

  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

                                                                 5

 

  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,

                                                                 6

 

  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

                                                                 7

 

  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

                                                                 8

 

  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.

                                                                 9

 

  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

                                                                10

 

  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

                                                                11

 

  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

                                                                12

 

  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

                                                                13

 

  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

                                                                14

 

  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

                                                                15

 

  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.

                                                                16

 

  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

                                                                17

 

  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

                                                                18

 

  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

                                                                19

 

  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

                                                                20

 

  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.

                                                                21

 

  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

                                                                22

 

  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

                                                                23

 

  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

                                                                24

 

  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? 

                                                                25

 

  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

                                                                26

 

  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.

                                                                28

 

  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