DEPARTMENT OF HEALTH AND HUMAN SERVICES
FOOD AND DRUG ADMINISTRATION
CENTER FOR BIOLOGICS EVALUATION AND RESEARCH
BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE
Friday, May 10, 2002
Daniel R. Salomon, M.D., Acting Chair
Gail Dapolito, Executive Secretary
Katherine A. High, M.D.
Richard C. Mulligan, Ph.D.
Mahendra S. Rao, M.D., Ph.D.
Alice J. Wolfson, J.D. (Consumer
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.
Valder Arruda, M.D., Ph.D.
Linda Couto, Ph.D.
Mark Kay, M.D.
Stephen M. Rose, Ph.D.
Jay P. Siegel, M.D.
Philip D. Noguchi, M.D.
Daniel Takefman, Ph.D.
Anne Pilaro, Ph.D.
C O N T E N T S
Dr. Daniel Salomon, Acting Chair 4
Introduction of Committee 5
Conflict of Interest Statement
Gail Dapolito, Executive Secretary 8
Potential for Inadvertent Germline Transmission
of Gene Transfer Vectors: FDA Approach for
Patient Follow Up
Daniel Takefman, Ph.D. 13
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
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
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,
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
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
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
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.
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
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
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
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.
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
25 I guess the
other thing, as long as I am
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
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.
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.
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
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.
25 What is the
likelihood that IGLT would be
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.
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
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
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.
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
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.
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
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.
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
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.
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.
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
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.
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.
25 As for FDA's
current approach for patient
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.
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.
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
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
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.
24 To summarize, FDA's primary concern of
25 inadvertent germline
transmission of gene transfer
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
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
AAV Vector Biology
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
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
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
1 virus goes in, reproduces, and progeny comes back
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
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?
1 That answer was no, the virus has a mechanism of
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
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
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
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
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
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
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
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
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
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
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
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.
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
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
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
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
1 number of people generate vectors by different
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
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
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
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
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
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
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
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
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
24 So I think what Daniel was referring to is
25 where does AAV fit on
this curve of an obligated
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,
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
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
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
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
1 some propensity for this episomal form, but the
2 risk is still there, and I will stop there and take
4 DR. SALOMON: Thank you very much. Very
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
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
persistence. So I think you are right
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
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
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
25 When we
first saw this, what is clustered
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.
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
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
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.
10 DR. RAO: Is there any evidence of
11 mobilization of the integrated thing, wild-type
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
24 DR. RAO: Is there a rough percentage on
25 that? I know wild-type predominates, but --
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
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
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
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
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.
MULLIGAN. Has anyone looked, like
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
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 --
GORDON: Adding Rep back.
1 DR. SAMULSKI: That's my senior moment
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
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
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
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
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
1 kind of cells, chances are the integration
2 frequency is going to be higher. That is kind of a
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.
SALOMON: That was great. Thank you.
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
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.
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
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
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
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
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
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
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
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
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
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
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
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
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,
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.
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
1 spermatogenesis, and, by the way, also concentrates
2 androgens to very high levels in this region of the
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
1 genetically transduced.
2 DR. SALOMON: Thank you very much, Jon.
3 That was excellent.
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
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
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
13 I think we should at least begin to study
14 this because there are tens of thousands of cycles
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
25 Well, of
course, these new mitochondrial
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
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
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
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
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
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
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
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
23 So it would only be speculation on my
24 part, I don't know.
SALOMON: Dr. Dym and then Dr. Rao.
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
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
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
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.
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
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
because, as he said, that is an
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.
20 DR. SALOMON: We will go ahead and get
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
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.
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.
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
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
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
24 Now, this was a very useful technology,
25 but it does not
quantify how much integration
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
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.
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.
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.
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
1 before and follow the procedure to cover the
2 patients from any type of bleeding.
3 Subjects are observed for at least 24
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
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.
24 DR. KAY: I am really sorry. There was a
25 mix-up about
transferring the slides, so I
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
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
24 Now, the first subject that was treated is
25 a 63-year-old male
with severe factor IX
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.
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
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
25 His liver
function studies and prothrombin
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
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
15 The important issue here, too, is that
16 this patient did not have detectable factor IX
17 inhibitor by Bethesda assay.
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
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
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
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
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
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
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
1 subjects to practice barrier contraception until
2 three consecutive monthly semen samples are
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
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
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
25 Patient 2, like Patient 1, had totally
1 normal LFTs, no elevations related to the vector
3 Renal function, not shown with the first
4 patient, but were also normal in the second patient
6 Again, the CBC including the platelet
7 counts were normal. There was no elevation with
8 vector administration
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
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
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
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,
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
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.
individual has supernormal sperm
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
1 have obtained from this Patient No. 2.
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
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
25 We have used
five different species -
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
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
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
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.
24 We also did a gonadal distribution study
25 in dogs. In this
study, three normal dogs were
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
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.
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.
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.
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
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
24 This is the results of that study. What
25 you can is that in 2
of the 4 injected animals, we
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