1
DEPARTMENT OF HEALTH AND HUMAN SERVICES
FOOD AND DRUG ADMINISTRATION
CENTER FOR BIOLOGICS EVALUATION AND RESEARCH
BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE
OPEN SESSION
Meeting #32
Friday, May 10, 2002
8:10 a.m.
Hilton Hotel
Gaithersburg, Maryland
2
PARTICIPANTS
Daniel R. Salomon, M.D., Acting Chair
Gail Dapolito, Executive Secretary
MEMBERS
Katherine A. High, M.D.
Richard C. Mulligan, Ph.D.
Mahendra S. Rao, M.D., Ph.D.
Alice J. Wolfson, J.D. (Consumer
Representative)
TEMPORARY VOTING MEMBERS
Martin Dym, M.D.
Jon W. Gordon, M.D., Ph.D.
Thomas F. Murray, Ph.D.
Terence Flotte, M.D.
Eric T. Juengst, Ph.D.
R. Jude Samulski, Ph.D.
GUESTS/GUEST SPEAKERS
Valder Arruda, M.D., Ph.D.
Linda Couto, Ph.D.
Mark Kay, M.D.
Stephen M. Rose, Ph.D.
FDA PARTICIPANTS
Jay P. Siegel, M.D.
Philip D. Noguchi, M.D.
Daniel Takefman, Ph.D.
Anne Pilaro, Ph.D.
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C O N T E N T S
PAGE
Welcome/Administrative Remarks
Dr. Daniel Salomon, Acting Chair 4
Introduction of Committee 5
Conflict of Interest Statement
Gail Dapolito, Executive Secretary 8
FDA Introduction
Potential for Inadvertent Germline Transmission
of Gene Transfer Vectors: FDA Approach for
Patient Follow Up
Daniel Takefman, Ph.D. 13
Guest Presentations
AAV Vector Biology, Jude Samulski, Ph.D. 23
Questions and Answers 46
Germline Transmission by Gene Transfer
Vectors: Assessing the Risk
Jon Gordon, M.D., Ph.D. 61
Questions and Answers 84
A Phase I Trial of AAV-Mediated Liver-Directed
Gene Therapy for Hemophilia B
Mark Kay, M.D., Ph.D. 98
Safety Studies to Support Intrahepatic
Delivery of AAV, Linda Couto, Ph.D. 116
Assessing the Risk of Germline Transmission of
AAV in a Rabbit Model
Valder Arruda, M.D. 130
Questions and Answers 144
Open Public Hearing
Mr. Steven Humes 177
National Hemophilia Foundation
Dr James Johnson, Patient 184
Dr. Kenneth Chahine, Avigen 190
Committee Discussion of Questions 197
4
1 P R
O C E E D I N G S
2
Opening Remarks
3 DR.
SALOMON: Good morning, everybody.
4 Welcome to day two of
the Biological Response
5 Modifiers Advisory
Committee Meeting No. 32. I
6 guess we should call
it 32B. We have got a title.
7 I have been
complaining and I finally got what I
8 wanted a title for
these meetings. This one, this
9 is good - Vector
Pellucida 2002. Not my title,
10 but, you know, you
can't criticize it, I got what I
11 wanted. Thank you.
12 So, welcome
everybody. Today we have
13 changed the scenery
around the table quite a bit.
14 So, to get reoriented,
I think we should go back
15 around again this time
and introduce ourselves, so
16 that both the
audience, as well as each other, has
17 a little sense of who
we are and what we are doing.
18 Just if you
can introduce yourself, we
19 will just go around
the table and give a few
20 sentences on where you
are from and what you do,
21 what kind of expertise
you bring.
22 In front of
you is a button on the thing.
23 It says speaker. If you push it, it turns red.
24 Talk, and then when
you are done, turn it off.
25 Otherwise, there is a
funny feedback. So if I am
5
1 ever looking at you,
gesturing, it means to turn it
2 off. It is one of my big duties.
3 Introduction of Committee
4 DR.
DYM: Martin Dym, Georgetown
5 University. I worked on the testis and
6 specifically on
spermatogonia, which are the male
7 germline stem cells.
8 DR.
FLOTTE: I am Terry Flotte from the
9 University of
Florida. We have been working on AAV
10 biology, AAV vectors
and AAV gene therapy.
11 DR.
JUENGST: I am Eric Juengst. I am in
12 the Department of
Bioethics at Case Western Reserve
13 University and
recently rotated off the RAC is
14 where my last
connection with these issues.
15 DR.
MURRAY: I am Tom Murray. I am from
16 the Hastings Center,
Bioethics, the world's first
17 bioethics research
institute, and my work has been
18 in a variety of
issues, but quite a lot in
19 genetics, parents, and
children.
20 MS.
WOLFSON: I am Alice Wolfson. I am
21 the Consumer
Advocate. In this incarnation, I am a
22 policyholder's lawyer
representing policyholders
23 against their
insurance companies when they don't
24 pay what they are
supposed to pay.
25 In my
previous incarnation, however, I am,
6
1 and was, a women's
health activist and a founder of
2 the National Women's
Health Network.
3 DR.
RAO: My name is Mahendra Rao. I am
4 in the Intramural
Program at the National Institute
5 on Aging. I am also a member of the BRMAC. I work
6 on stem cells, most
parts of the body, I guess.
7 DR.
SALOMON: Jude, we missed you the
8 first time around.
9 DR.
SAMULSKI: I am Jude Samulski from the
10 University of North
Carolina, and work in the area
11 of AAV vectors.
12 DR.
SALOMON: I am Dan Salomon. I have
13 the pleasure of
chairing the committee today. I am
14 from the Scripps
Research Institute in La Jolla,
15 California. I work on cell transplantation,
16 particularly islet
cell transplantation and tissue
17 engineering and
therapeutic gene delivery.
18 MS.
DAPOLITO: Gail Dapolito, Center for
19 Biologics. I am the Executive Secretary of the
20 committee.
21 DR. GORDON: Jon Gordon from Mount Sinai
22 School of
Medicine. I make a lot of transgenic
23 mouse models of
disease and gene therapy for
24 disease. I was on the RAC. I am actually the
25 first person to say
the word "transgenic," if that
7
1 means anything.
2 DR.
SALOMON: It means a lot.
3 DR.
PILARO: I am Anne Pilaro. I am an
4 expert toxicologist in
the Division of Clinical
5 Trials at CBER. I regulate a lot of the gene
6 therapy protocols, in
fact, I think I have 167
7 active right now.
8 DR.
TAKEFMAN: Dan Takefman. I am a gene
9 therapy product
reviewer with the Division of
10 Cellular and Gene
Therapies, CBER.
11 DR.
NOGUCHI: Phil Noguchi. I am director
12 of the Division of
Cell and Gene Therapy at CBER.
13 DR.
SALOMON: Welcome. We will be joined
14 a little bit later by
my colleague to the right,
15 Richard Mulligan from
Harvard Medical School.
16 This is
interesting for two reasons. One
17 is that this is kind
of a revisit to a very
18 important area that
the BRMAC dealt with, not the
19 last time, but I guess
at least two times ago,
20 where we initially
talked about how to address
21 potential regulatory
issues specifically with this
22 Avigen trial, and then
more generally with how to
23 deal with the
potential of infection germline in
24 this case with semen.
25 We got into
the whole discussion about
8
1 semen versus infecting
the motile sperm and what
2 was the evidence, if
any, that you could really
3 infect the germline,
the spermatogonia, or infect
4 the sperm themselves,
and very much tried to deal
5 with some of the
practical issues of what you would
6 demand of any company
of a sponsor in doing this
7 kind of research, and
to do it in such a way that
8 you wouldn't put an
unnecessary hold that could
9 therefore interrupt a
very important trial unless
10 there was awfully good
evidence.
11 It is also
very interesting in that it is
12 an interesting theme
for the two days. In some way
13 I am sorry that some
of you weren't here yesterday
14 where there we were
really talking about another
15 kind of germline
transfer issue, the injection of
16 ooplasm into oocytes
for infertile women, but it is
17 an interesting thing
now to go on to the idea of
18 potentially doing
something like this through
19 therapeutic gene
delivery.
20 We have to
read the conflict of interest.
21 Gail.
22 Conflict
of Interest Statement
23 MS.
DAPOLITO: I would just like to read
24 for the public record,
the conflict of interest
25 statement for today's
meeting.
9
1 Pursuant to the authority granted under
2 the Committee charter,
the Director of FDA Center
3 for Biologics
Evaluation and Research has appointed
4 Drs. Terence Flotte,
Jon Gordon, Eric Juengst,
5 Thomas Murray, Daniel
Salomon, and Jude Samulski as
6 temporary voting
members for the discussions
7 regarding issues
related to germline transmission
8 of gene therapy
vectors.
9 Dr. Salomon
serves as the Acting Chair for
10 today's session.
11 To determine if any conflicts of
interest
12 existed, the Agency
reviewed the submitted agenda
13 and all financial
interests reported by the meeting
14 participants. As a result of this review, the
15 following disclosures
are being made:
16 In
accordance with 18 U.S.C. 208, Drs.
17 Terence Flotte,
Jonathan Gordon, Daniel Salomon,
18 and Jude Samulski were
granted waivers permitting
19 them to participate
fully in the committee
20 discussions. Dr. Richard Mulligan was granted a
21 limited waiver for
this discussion which permits
22 him to participate in
the committee discussion
23 without a vote. Dr. Katherine High recused herself
24 from this committee
meeting.
25 In regards
to FDA's invited guests, the
10
1 Agency has determined
that services of these guests
2 are essential. The following interests are being
3 made public to allow
meeting participants to
4 objectively evaluate
any presentation and/or
5 comments made by the
guests related to the
6 discussions of issues
of germline transmission of
7 gene therapy vectors.
8 Dr. Valder
Arruda is employed by the
9 University of
Pennsylvania. He is involved in the
10 studies of
adeno-associated virus vectors. Dr.
11 Stephen Rose is
employed by the Office of
12 Biotechnology
Activities, NIH.
13 In the event
that the discussions involve
14 other products or
firms not already on the agenda,
15 for which FDA's
participants have a financial
16 interest, the
participants are aware of the need to
17 exclude themselves
from such involvement, and their
18 exclusion will be noted
for the public record.
19 With respect
to all other meeting
20 participants, we ask
in the interest of fairness
21 that you state your
name, affiliation, and address
22 any current or
previous financial involvement with
23 any firm whose product
you wish to comment upon.
24 Copies of
these waivers addressed in this
25 announcement are
available by written request under
11
1 the Freedom of
Information Act.
2 As a final
note, as a courtesy to the
3 committee discussants
and your neighbors in the
4 audience, we ask that
cell phones and pagers be put
5 in silent mode.
6 Thanks.
7 DR.
SALOMON: Thank you, Gail.
8 What we will
do here is begin with an FDA
9 introduction from Dan
Takefman, will kind of walk
10 us through some of the
key issues that the FDA
11 wants to answer. Remember that part of the dynamic
12 here is that we are an
FDA Advisory Committee.
13 There will
be times when we all, certainly
14 myself as a scientist,
get really interested in
15 some scientific
question, but at some point you
16 will have to forgive me
if we steer away from that
17 since, if we are not
really answering the FDA's
18 question, then, we are
not doing what we are
19 supposed to be doing
here.
20 In the
meantime, though, obviously, to the
21 extent that any of
these scientific issues are
22 relevant to answering
the questions, you know, you
23 obviously are here and
your expertise is greatly
24 welcomed.
25 I guess the
other thing, as long as I am
12
1 giving an introduction
on that score, I will just
2 say that we are going
to try and come to consensus
3 on some of these
questions, but in some instances,
4 there is no consensus,
and there is no effort here
5 on my part to force
this group into consensus, so
6 well-articulated,
minority opinions or even just
7 where we go, I am
sorry, but there is no way we can
8 agree on it, that's
the kind of information that we
9 need to pin down.
10 So it is
important for us to make sure
11 that we have
represented everything as evenly as
12 possible for the
community. The last thing I will
13 say to the audience is
that I feel you also are
14 participants in this
meeting. This is an open
15 public meeting. That mike in the center is open. I
16 welcome all of you, if
you have something to say,
17 to come up during the
meeting during discussion and
18 make your points, and
we will definitely be here to
19 listen to them and try
and make sure that we do an
20 adequate discussion of
this.
21 Dan, you are
on.
22
FDA Introduction
23 Potential for
Inadvertent Germline Transmission of
24 Gene Transfer Vectors: FDA Approach for
Patient
25
Follow Up
13
1
Daniel Takefman, Ph.D.
2 DR.
TAKEFMAN: Thank you. I would like to
3 welcome the committee
and speakers, and thank
4 everyone for
participating in today's meeting.
5 [Slide.
6 The topic
for today is the discussion of
7 potential for
inadvertent germline transmission of
8 gene transfer vectors,
and as Dan said, this has
9 been a topic of
previous discussions and public
10 meetings. Today, we will be discussing the finding
11 of vector sequences in
patient semen and to discuss
12 FDA's current approach
for patient follow up.
13 [Slide.
14 Concerns
regarding inadvertent germline
15 transmission, or IGLT,
are twofold.
16 Societal/ethical
concerns are based on previous
17 public discussions and
publications in which
18 deliberate germline
alteration has been deemed
19 unacceptable.
20
Additionally, there are potential adverse
21 biological effects,
such as genetic disorders,
22 birth defects, and
lethality to developing fetus,
23 just to list a few
which are also of concern.
24 [Slide.
25 What is the
likelihood that IGLT would be
14
1 deleterious? Well, retroviruses have been used as
2 tools to investigate
the role of certain genes
3 which are important in
development. I refer to, in
4 this slide, data
involving retroviral insertion to
5 the germline of mice
and as a specific example, a
6 retrovirus was used to
infect a murine blastocyst.
7 In this case, this
infection resulted in a mouse
8 strain with a lethal
embryonic mutation, which was
9 induced by proviral
insertion into the alpha-1
10 collagen gene. This mutation was recessive, so
11 that the phenotypic
effect required homozygosity.
12 [Slide.
13 So data
exist suggesting that in the case
14 of retroviruses,
deliberate insertion into the
15 germline may be
deleterious, but what about data
16 from preclinical
animal studies regarding the
17 ability of gene
transfer vectors to transmit to the
18 germline?
19 Well, the
FDA does require biodistribution
20 studies with gene
transfer vectors in relevant
21 animal models. These biodistribution studies,
22 performed in support
of clinical trials, have shown
23 evidence of vector
dissemination to gonadal tissue.
24 However, in
most studies, vector sequences
25 have not been detected
in semen samples, and the
15
1 point I need to make
in regards to these
2 preclinical studies is
that they are not always
3 predictive of human
experience.
4 A case in
point is today's topic in which
5 vector sequences were
found in semen from clinical
6 trial subjects,
however, initial preclinical
7 studies, such as those
done in dogs, demonstrated
8 no detectable vector
in semen.
9 Again,
certainly in today's case, animal
10 studies are not always
predictive.
11 [Slide.
12 I would like
to give an update on the kind
13 of current active gene
transfer INDs we currently
14 have in file just to
give you an idea of what is
15 being used in the
clinic.
16 You can see
here in regards to retroviral
17 vectors, they are
predominantly being used in ex
18 vivo types of gene
transfer studies, while
19 adenoviral vectors and
plasmids are often being
20 used in direct in vivo
type of administrations.
21 You will
notice here with AAV vectors,
22 compared to other
systems, FDA has seen relatively
23 few gene transfer
INDs. Of the few we have, they
24 are primarily in vivo,
localized injection type of
25 administrations.
16
1 [Slide.
2 I would like
to go over some of the
3 factors that FDA
considers important for assessing
4 risks of inadvertent
germline transmission of gene
5 transfer vectors.
6 Certainly,
integration potential of the
7 vectors is important
to consider. Of the current
8 vectors being used in
the clinic, FDA is
9 considering both
retroviral and AAV vectors as
10 vectors with potential
to integrate. Certainly
11 with retroviruses, as
well as lentiviral vectors,
12 they are known to have
efficient abilities to
13 integrate and host
genomes.
14 In terms of
AAV vectors, this system is
15 not as clearly worked
out as in other systems, such
16 as retroviruses. FDA is currently considering AAV
17 vectors as having a
low, but potential to integrate
18 in vivo, and I
specifically refer here to a couple
19 of papers from Nakai's
lab in which he showed low
20 levels of integration
in mouse livers.
21 [Slide.
22 The risk of
inadvertent germline
23 transmission is also
likely highly dependent upon
24 route of
administration. An ex vivo gene transfer
25 would likely represent
a minimal risk in terms of
17
1 IGLT, while at the
other end of the spectrum, a
2 systemic injection
would represent a relatively
3 higher risk in terms
of transfer to the germline
4 via hematogenous
spread.
5 [Slide.
6 As Dr.
Salomon mentioned, IGLT has been a
7 topic of discussion, and
I would like to go over
8 some of the previous
public discussions in order to
9 put today's meeting in
a little perspective.
10 Beginning
with the March 1999 RAC meeting,
11 here, there was a
focused discussion on preclinical
12 data which
demonstrated gonadal distribution. It
13 was the consensus from
this meeting that despite
14 this preclinical data,
the probability of
15 inadvertent germline
transmission occurring during
16 a gene transfer
clinical trial was low.
17 However,
further discussion became
18 necessary at the
November 2000 BRMAC meeting. At
19 this meeting, we heard
data from a trial which
20 involved I.V.
administration of a gammaretroviral
21 vector which contained
the factor VIII gene for
22 treatment of
hemophilia A.
23 I should
point out this was the first
24 trial under IND which
involved I.V. administration
25 of a gammaretroviral
vector. Data was presented in
18
1 which 1 out 12
subjects treated had vector
2 sequences transiently
present in semen.
3 In the one
patient, vector sequences were
4 detected at only one
time point by DNA-PCR.
5 [Slide.
6 Then, at a
recent meeting of the RAC, a
7 trial was presented,
which will also be presented
8 today, which involved
an AAV vector, which contains
9 the factor IX gene for
the treatment of hemophilia
10 B. This is the first trial under IND which
11 involved
administration of an AAV vector into the
12 hepatic artery.
13 Data was
presented in which vector
14 sequences were found
in semen of the first two
15 patients treated. The first patient had positive
16 PCR signal at multiple
time points for up to 10
17 weeks post
administration, and the implication here
18 is that all patients
treated in this trial may test
19 positive for vector
sequences in semen samples.
20 [Slide.
21 So to
summarize some of the consensus from
22 these public
discussions, there was a consensus
23 from the RAC meeting
on preclinical data that the
24 probability of
inadvertent germline transmission is
25 low and that the use
of a fertile subject
19
1 population was
acceptable.
2 From the
BRMAC meeting, the committee
3 agreed with FDA's
approach to institute a clinical
4 hold when vector
sequences are detected in semen
5 samples from study
subjects.
6 There was a
consensus from both the RAC
7 and the BRMAC that
there is a need to determine if
8 vector is associated with
sperm cells. Using
9 fractionation methods,
such as density separation,
10 potential
contaminating transduced white blood
11 cells can be removed
from sperm cell fractions.
12 You are going to hear
more later on from Avigen on
13 their fractionation assays.
14 [Slide.
15 I would like
to turn now to FDA's approach
16 for patient follow up,
which has been modified in
17 response to these
public discussions and from data
18 regarding this current
trial.
19 Prior to
initiation of the trial, of
20 course, if during
preclinical animal studies,
21 vector is found in
gonadal tissue, this finding and
22 the potential for
germline alterations should be
23 included in informed
consent documents.
24 [Slide.
25 As for FDA's
current approach for patient
20
1 follow up, if semen
from clinical trial subjects
2 tests positive for
vector sequences, the clinical
3 trial will be allowed
to continue, however, FDA
4 will request timely
follow-up testing of
5 fractionated
semen. As has been in the case in the
6 past, barrier
contraception is requested until
7 three consecutive
samples test negative.
8 [Slide.
9 Now, if the
motile sperm fraction tests
10 positive for vector
sequences, FDA will institute a
11 clinical hold and
subject enrollment will be
12 stopped until it is
determined that the signal from
13 the motile sperm
fraction is transient, and
14 specifically, we are
asking for serial fractionated
15 samples to test
negative three times over three
16 consecutive monthly
intervals.
17 [Slide.
18 I would like
to turn now to some of the
19 concerns that FDA
has. Specifically, the finding
20 of vector sequences in
semen may become more
21 common. Certainly with subject from trials
22 involving systemic or
intrahepatic administration
23 of AAV, such as in
this trial, every patient
24 treated might have
vector sequences found in semen
25 samples.
21
1
Additionally, we have new vector classes
2 on the horizon, such
as lentiviral vectors, which
3 we know have a high
potential to integrate, and
4 there is also new
production technologies which
5 allow for higher titer
viruses to be produced and
6 new clinical
applications of gene delivery systems
7 designed to increase
transduction efficiency, all
8 of which may make the
detection of vector sequences
9 in subject semen more
prevalent in future clinical
10 trials.
11 [Slide.
12 Of
particular concern, the fact that
13 patient follow up is
difficult with certain
14 populations. Obviously, there are technical
15 limitations in the
ability to monitor women and
16 certain men who are
unable to repeatedly supply
17 adequate samples. There is technical limitations
18 to monitor these
subject populations for evidence
19 of germline
alterations.
20 The specific
concern will be re-presented
21 in the form of a
question to the committee for
22 discussion in the
afternoon session.
23 [Slide.
24 To
summarize, FDA's primary concern of
25 inadvertent germline
transmission of gene transfer
22
1 vectors is with
systemic administration of
2 integrating vectors.
3 A clinical
hold is instituted only if
4 vector sequences are
detected in motile sperm
5 fractions, and the
inability to monitor certain
6 patient populations is
a concern and warrants
7 further discussion.
8 I will end
here and just remind everyone
9 that there is a number
of background talks and
10 still data on the
clinical trial and preclinical
11 studies to be
presented, so I would request that we
12 limit the majority of
discussion of patient follow
13 up until the afternoon
session, but I will be happy
14 to answer a few
questions at this time for
15 clarification.
16 DR. SALOMON: Thank you, Dan.
17 Are there
any questions from the committee
18 to the FDA regarding
the overall umbrella charge
19 that we have for
today? Okay.
20 The next are
two presentations. It is a
21 pleasure to start with
Jude Samulski from the
22 University of North
Carolina to talk to us about
23 the biology of AAV
vectors.
24
Guest Presentations
25
AAV Vector Biology
23
1
Jude Samulski, Ph.D.
2 DR.
SAMULSKI: It is a pleasure to be
3 here. I want to thank Daniel for asking me to come
4 up. He requested that I give some type of overview
5 of AAV biology and try
to focus a little bit on our
6 understanding of the
potential for integration and
7 mechanisms.
8 I think what
I am going to do is offer you
9 an opinion of a
consensus of what we think is
10 happening in the
field, point you in the direction
11 of probably papers
that are relevant, that start to
12 show trends that are
happening, but more than
13 likely I am going to
end up with the conclusion
14 that Daniel has
already described, is that AAV is
15 somewhere on that
curve as a vector that can
16 integrate, the
efficiency is not well established,
17 but the potential is
there.
18 I will start
off by introducing you to the
19 life cycle of this virus. In the laboratory, an
20 AAV particle can have
a lytic component or a latent
21 component, so we refer
to it as a biphasic life
22 cycle.
23 It has been
established that it is
24 dependent on a helper
virus in order to go through
25 a productive lytic
cycle, and in this setting, the
24
1 virus goes in,
reproduces, and progeny comes back
2 out.
3 What was
established in the laboratory in
4 the early seventies
was that if you took AAV
5 particles and put them
in cells in the absence of
6 the helper, you could
see this persistence, what
7 was referred to as
"latency," and in this setting,
8 it was determined that
the virus was establishing
9 an integration event
in the chromosome, and in this
10 integration event, it
appeared to be targeting,
11 meaning it was going
to a specific locus in the
12 human genome.
13 This was all
done in vitro and tissue
14 culture cells, and to
complete the biological life
15 cycle, if you take
these cells and now superinfect
16 them with adenovirus,
AAV has the ability to come
17 back out of the
chromosome and reenter its lytic
18 component.
19 So in the
laboratory, it was established
20 the mechanism in which
we could argue how AAV,
21 which was found in
nature in clinical isolates of
22 adenovirus, how these
two would co-persist, but we
23 could also explain a
question of what is the
24 consequences of AAV
infecting the cell in the
25 absence of its
helper. Is that genetic suicide?
25
1 That answer was no,
the virus has a mechanism of
2 persistence.
3 I should
argue that there is absolutely
4 zero data of AAV
integration in humans. This is
5 all established in
vitro, and it is inferred that
6 this mechanism can
take place.
7 I should also mention that the early
8 studies of AAV showing
up in clinical isolates, it
9 has only been isolated
in adenovirus, although
10 herpes can supply the
same helper function. There
11 has never been a
clinical isolate of herpes that
12 has had a
contamination of AAV.
13 So what you
should be asking yourself is
14 that we can mimic a
paradigm in tissue culture and
15 substitute other
viruses, but what appears to be
16 out there in nature is
this co-relationship. This
17 was established in
vitro, and it is presumed that
18 this can also happen
in vivo.
19 The genome
is fairly simple. It is about
20 5,000 base pairs, and
what is of importance today
21 is paying a little bit
of attention to what is
22 referred to as the Rep
genes and the inverted
23 terminal repeats of
the virus, which are the
24 origins of
replication, the packaging signal, and
25 what appear to be the
break points that join
26
1 recombination events
with the chromosome.
2 Of the Rep
genes that are made, it has
3 been shown that it is
the large Rep proteins, Rep
4 78 and 68, that appear
to be responsible for the
5 integration
events. I just want to point out that
6 in AAV, these are
identical proteins. They only
7 differ by a splice
variate, and in the absence of
8 adenovirus, this is
the dominant protein that you
9 see in the presence of adenovirus. This comes on
10 first and then it
switches over to Rep 68.
11 They all
have enzymatically identical
12 activities. They bind
to the AAV terminal repeat
13 and what is called a
Rep binding element. They
14 have a site-specific,
strand-specific endonuclease
15 activity where they
can nick this molecule, and
16 they have helicase
activity which allows it to
17 unravel to DNA.
18 So we see a
relationship with the Rep
19 proteins were the key
element on the virus, which
20 is the origin of
replication, showing that it has a
21 binding site, a
nicking site, and enzymatic
22 activities to allow
this virus to replicate.
23 So the first
evidence of AAV integrating
24 site specifically was
generated in Ken Burns' lab
25 in 1996, and in this
study, what they did was
27
1 pulled out some
junctions, sequenced the junctions,
2 and went back and used
those sequences as probes.
3 This is just
a representative example from
4 our lab that shows
that if you look at your
5 chromosome 19 locus in
a control cell, it is about
6 a 2.6 kilobase fragment,
but after you integrate
7 and establish
independent clones, you can find
8 variance that show
evidence that the chromosome
9 sequence now has a
rearrangement suggestive of an
10 insertion, and some of
these are multiple fragments
11 showing that there is
amplification and
12 rearrangement.
13 If you take
a blot like this and strip off
14 the chromosome 19
probe and then come back with the
15 viral probe, you can
see there is co-segregation of
16 these viral sequences
with these chromosome 19
17 rearranged, so this
was the data that said there
18 was a preferred site
of integration, a
19 rearrangement of
chromosome 19 and a
20 co-localization of
these sequences with chromosome
21 19 sequences.
22 Ken Burns
and others looked in detail to
23 bring to try to
understand why was this virus going
24 to this specific
locus, and from that study came
25 the following
information.
28
1 There is an
identical Rep binding site and
2 a nicking site located
on human chromosome 19, so
3 what we had was a
mechanism that is virtually of
4 viral origin sitting
on chromosome 19, that gave a
5 putative reason for
why this site is preferred as
6 an integration locus
over any other sequence in the
7 human genome.
8 What I
should point out is that further
9 studies have shown
that not only is the Rep binding
10 required, the spacing
between this binding site to
11 the nicking site and
the nicking site itself, so if
12 you take these
sequences and count them up, there
13 are over 15 base
pairs.
14 It is argued
that a sequence over 15
15 nucleotides is only
represented one time in the
16 human genome. This is probably why this virus is
17 only targeting this
locus. This element is present
18 in about 200,000
copies in the human genome, which
19 would argue that the
Rep protein is sitting on lots
20 of spots on the human
chromosome, but it is only
21 when it is this
context that it can initiate the
22 event to promote the
integration step.
23 So we have a
model and a mechanism that is
24 being supported both
in vitro and in vivo.
25 A group in
Italy went on to show that the
29
1 site has an open
chromatin confirmation and that it
2 is not a closed site,
so it is not a site that is
3 unaccessible. All of these things are beginning to
4 support the type of
DNA structure that AAV needs to
5 see in order to go
into the chromosome.
6 A number of
labs, including our own, have
7 gone after looking at
these integration events, and
8 most of you are pretty
well aware, that if you look
9 at retroviral
integration event, it is a fair
10 precise cut and paste
mechanism in which it cuts
11 the chromosome,
integrates its genome, and there is
12 like a 3 to 5
nucleotide duplication on either
13 side.
14 When you
looked at these AAV proviral
15 structures, what we
saw was there were a lot of
16 tandem repeats,
amplification events, and all of
17 these things were
supporting a type of integration
18 that was completely
different than the
19 well-characterized
retrovirus integration.
20 This has
been consistent both in cell
21 lines, as well as
episomal integration events, as
22 well as in vitro
systems, so there is a mechanism
23 for integration that
is not consistent with a cut
24 and paste. It is referred to as a non-homologous
25 amplification
mechanism.
30
1 Our lab and
others went on to look at the
2 break points between
the viral terminal repeat,
3 which I showed you has
this origin activity, and
4 this hairpin
structure, and the junctions between
5 that and chromosome
19.
6 What you can
see was there was very little
7 fidelity and
conserving the integrity of the
8 terminal repeat. You would get break points that
9 were scattered
throughout these hairpins, and these
10 are just positioned
here on the sequence to give
11 you an impression that
there is no fixed break
12 point between the
viral sequence and the chromosome
13 19. They cluster around this hairpin element,
but
14 other than that, you
can virtually find break
15 points throughout
these sequences.
16 If you look
at that from a biological
17 point of view, it
again suggests that AAV may have
18 a problem in retaining
its integrity as a virus if
19 it's indiscriminately
breaking these hairpins and
20 going into the
chromosome, but this virus has a
21 phenomenal ability of
carrying out a step code gene
22 correction.
23 There is
technically two copies of every
24 sequence in the
hairpin, and since there is two
25 hairpins, there is the
total of four copies on the
31
1 virus, so between all
of these copies, the virus
2 will gene convert back
and forth and regenerate
3 these sequences with
fair efficiency, so you always
4 get a wild-type virus
coming back out even though
5 what is integrated in
the chromosome may be
6 somewhat fragmented.
7 Because the
virus also integrates in what
8 appears to be
head-to-tail concatemers, it is
9 preserving the
integrity of these hairpins
10 internally, and again
allowing it to use it as a
11 template to amplify
and come back out of the
12 chromosome.
13 So to get to
the mechanism, Matt Weitzman
14 in Roland Owens' lab
did an experiment in the early
15 nineties that said
that they could show that the
16 Rep protein of AAV
could form a complex between the
17 terminal repeat of the
virus and this
18 pre-integration site.
19 Again, this
made logical sense because
20 there was the same Rep
binding element on both of
21 these sequences. This
is just an illustration from
22 Sam Young's data
showing the Rep protein bound to
23 the terminal repeats
of an AAV vector. It has an
24 extremely high
affinity for the sequence and a Rep
25 complex binding to the
same element on chromosome
32
1 19. It was data like this and other that began
to
2 propose a model that
the virus express its Rep
3 protein, it binds to
this element on chromosome 19.
4 In vitro,
Rob Cotton showed that this is
5 sufficient to start a
synchronized single-stranded
6 DNA replication. So now you have this region of
7 chromosome 19 serving
as an origin. Since the Rep
8 protein is terminally
attached to this chromosomal
9 sequence, and you can
reinitiate, we feel that
10 there is a number of
initiation events that are
11 taking place on this
region of chromosome 19.
12 It should be
understood that there is an
13 enzyme called Fen-1
which is a host enzyme, that
14 actually repairs this type
of repeated initiation
15 event, however, if you
have a hairpin or a protein
16 attached to this, it
doesn't have the ability to
17 correct these
sequences.
18 So what
happens is you see recombination
19 events taking place to
resolve these molecules. It
20 has been suggested
that the AAV genome, which has
21 Rep, allows for
Rep-Rep tethering mechanism, as
22 Weitzman showed, and
at this point it is all host
23 enzymes that are
involved in inserting this
24 sequence into the host
genome, and this type of
25 tandem repeat,
head-to-tail type of format.
33
1 This is data
that was provided to me by
2 Regina Hildabraun. It is not published. It is
3 coming out in a
journal Virology. She has
4 developed a real-time
PCR assay to look at the
5 efficiency of AAV
viruses to go to chromosome 19.
6 It is a PCR assay that
look at the terminal repeat
7 and a locus on
chromosome 19.
8 What I think
is important to see here is
9 that she can score
integration events taking place
10 over the first 72
hours or so, but the most
11 important thing is
that the wild-type virus, which
12 she is seeing an
integration event for about 1,000
13 particles, so it is
suggest about 0.1 percent of
14 all the AAV virus is
capable of carrying out
15 integration.
16 This is
completely different than like the
17 retroviruses where it
is 100 percent integration.
18 As Daniel
said, there is a propensity for
19 the virus to
integrate. The efficiency is what
20 needs to be look at in
this setting.
21 This is a
paper that was published by
22 Ernst Winocour. I think this is of importance
23 because what I am
going to suggest to you is this
24 is another parvovirus
called minute virus in mice.
25 It's an autonomous
parvovirus. Nowhere is its life
34
1 cycle does it
establish latency. It has no
2 mechanism. There has never been any data
3 supporting it.
4 But what
Ernst was able to do was show
5 that these viruses
also have terminal repeats, they
6 also have Rep-like
proteins, and that he could take
7 an episome substrate
and show that this virus could
8 also integrate into a
target sequence if the Rep
9 protein on this minute
virus was present and if the
10 subsequent sequences
were available.
11 So what I
think this is suggesting is that
12 the parvoviruses have
proteins that are involved in
13 replication that are
able to carry out nicking and
14 helicase activity on
substrates. In the case of
15 minute virus of mice,
there is no target in the
16 genome.
17 In the case
of AAV, there is an origin
18 identical to AAV
sitting on chromosome 19. So the
19 question may be, does
AAV really set up a latency
20 or is this an
interaction between Rep proteins and
21 target sequences, and
1 percent begins to suggest
22 that it is not a very
efficient mechanism.
23 I am going
to shift gears and now talk to
24 you about vectors
because I think this is where
25 most of the interest
is. In the laboratory, a
35
1 number of people
generate vectors by different
2 procedures.
3 In our lab, we
use plasmids to start to
4 make the vector, so
now we only retain the terminal
5 repeats. The gene of interest is in the middle.
6 You have a helper
plasmid carrying the Rep and
7 capture genes, and
another plasmid carrying the
8 essential sequences
from adenovirus to activate all
9 of these steps.
10 What happens
when all of these are in the
11 cell, you produce a
single virus particle, which is
12 an AAV particle
carrying the foreign gene of
13 interest. If you take these viruses and put them
14 in tissue culture
cells, and put them under
15 selection, what you
see is if you go to the
16 chromosome 19 region
and look at individual clones
17 that had the vector
integrated in the human genome,
18 you don't see a
significant rearrangement under
19 chromosome 19
sequence.
20 So unlike
wild type where it appeared that
21 70 to 90 percent of
the integrations were targeting
22 this locus, the
vectors have lost this ability to
23 go to chromosome 19.
It has been shown by a number
24 of labs that if you
add Rep back to this reaction,
25 these vectors will go
to chromosome 19 and
36
1 integrate.
2 So it is
fairly well established now that
3 AAV vectors have no
targeting capacity and that
4 what they do have is
the capacity to integrate into
5 the chromosome under
these selected conditions.
6 This is an approach that Charley Yang took
7 in the lab about seven
years ago, in which he made
8 AAV vectors that were
carrying a plasmid origin and
9 ampicillin sequence,
as well as a selectable
10 mechanism to look at
selection in eukaryotic cells.
11 He made this
into a virus, allowed it to
12 integrate into the
chromosome, and he used enzymes
13 that were cut outside
of the viral DNA, closed this
14 up into a circle, and
pulled out these so-called
15 cellular junctions, and when he characterized
16 these, he came up with
the following results.
17 The break
points of the terminal repeat
18 and the chromosome
were almost identical to what we
19 saw with wild
type. They clustered around the
20 hairpin structure, but
there was no defined break
21 point in any of these
vectors.
22 When we
looked at the location that they
23 were going into, they
appeared to be random on
24 chromosome 17, 7,
1. We had two examples of it
25 integrating on
chromosome 2. But what we were
37
1 seeing was that all of
the characteristics of
2 integration were
identical to wild type. It is
3 just that their targeting ability was lost.
4 Instead of going to
19, it was random.
5 If you look
at the vectors, they were
6 again consistent with
this head-to-tail mechanism
7 and amplification
event or rearrangement event. I
8 should mention that
David Russell has just
9 published a little
paper in Nature Medicine that
10 has shown another
clustering of these things pulled
11 out of HeLa cells, and
we have generated the exact
12 same information. There is breakage and
13 duplication and some
type of random repeats that
14 are being generated.
15 So I want to
point out because I think we
16 get misled a lot when
we think about AAV's
17 integration and that
it is something special. This
18 ability to form
concatemers is something that was
19 documented a number of
years ago by Schimke's lab.
20 In fact, if you look
at any transgenic animal that
21 has ever been
generated, it is always generated in
22 a head-to-tail
concatemer formation.
23 If you look
at virtually any cell line
24 that is established by
plasmids to give stability,
25 it is typically a
head-to-tail concatemer, that is
38
1 going into the
chromosome. So what we see is that
2 AAV is probably using
host enzymes to generate
3 these concatemers that
eventually go into the
4 chromosome.
5 As I
mentioned to you, without the Rep
6 protein, there is no
targeting capability. This
7 integration appears to
be random. The insertion
8 that takes place at
the integration site is not a
9 cut and paste
mechanism, it's a deletion,
10 amplification,
rearrangement, illegitimate type of
11 recombination.
12 This is just
our data showing all of the
13 break points that we
have generated both with
14 vectors with wild type
AAV as far as the junctions
15 that are generated
between the terminal repeats and
16 the chromosome, and
you can see that again there
17 are preferred
clustering sites, but there is no
18 distinct break point
that takes place between AAV
19 molecule and the
chromosomal DNA sequence.
20 We concluded
from this study that when AAV
21 vectors go into cells,
it is cellular recombination
22 pathways that are
responsible for the integration
23 of that, and that
there is no viral participation
24 in this enzymatic
step, it is all carried by
25 cellular
recombination.
39
1 If you look
at the data that has been
2 generated, it falls
under the category of an
3 illegitimate,
non-homologous recombination. This
4 would be true if you
put in plasmid DNA,
5 oligonucleotides, any
piece of DNA that ends up
6 going into the
chromosome. It is following a
7 pathway that supported
cellular enzymes carrying
8 out the integration step.
9 I want to
just summarize this and then I
10 am going to switch to
the last third of the talk,
11 which is going to just
talk about information
12 generated with vectors
in animals.
13 Right now,
AAV vectors do not target
14 chromosome 19. They are identical to wild type
15 with respect to the
terminal repeat break points.
16 They are essentially
identical at this level. The
17 head-to-tail
orientation of vector proviruses, you
18 can find tail-to-tail
and head-to-head, but this is
19 pretty much the
dominant species you will see.
20 They
rearrange to chromosome integration
21 site. There is not a
cut and paste mechanism.
22 There is always some
type of deletion,
23 amplification, and
rearrangement that takes place
24 at the integration
locus.
25 So by all
these criteria, AAV fits the
40
1 conditions of an
insertional mutagen. It has the
2 ability to go into the
chromosome, and the critical
3 question is at what
frequency does it carry out
4 this insertion event.
5 This is
where I think we began to
6 accumulate data in the
field that drifted us away
7 from all that
information that was derived in
8 vitro, and you should
understand that the data was
9 derived in vitro was
under selected conditions with
10 a gene, such as G418
or neomycin, so that you are
11 only looking at the
integration events.
12 In vivo, the
first data that began to
13 suggest that this may
not be consistent with what
14 was happening in vitro
was actually carried out in
15 Terry Flotte's lab
where they were looking at
16 adeno-associated
viruses in monkeys after
17 administration for
airway gene delivery.
18 When they
characterized this, they saw
19 that the virus was
persisting for a period of time
20 and the virus could be
rescued completing all of
21 those steps that we
talked about in the life cycle,
22 but it was showing up
as an episome. There was
23 very little data
suggesting that this type of
24 persistence was taking
place as an integration
25 event.
41
1 This is a
paper that I would like to
2 direct people to,
because I think buried in this
3 paper is some really
important information. This
4 was a study carried
out in Jim Wilson's lab where
5 what he virtually did
was an in vivo selection like
6 what we do with in
vitro selection with G418, in an
7 animal model that had
a disease for the liver, so
8 the AAV vector was
transducing a gene and to
9 deliver, that he could put a selective
pressure on.
10 This
selective pressure meant that if this
11 liver was to survive,
the virus had to integrate.
12 After it integrated,
you could see nodules begin to
13 grow of liver cells. He characterized those
14 nodules. He showed they had integration events in
15 them. They were similar to what I have just
16 described for in
vitro.
17 They were
tandem repeats, rearrangements,
18 and an illegitimate
recombination mechanism, but if
19 you go into the paper
and dig at the multiplicity
20 of virus that he was
putting into the liver, 1012
21 particles per liver,
he was only getting about 0.1
22 percent of the liver
cells showing an integration
23 event.
24 So I think
what Daniel was referring to is
25 where does AAV fit on
this curve of an obligated
42
1 integration event
versus the potential to
2 integrate, and this
study, under selective
3 pressure, there was a
frequency that was derived,
4 which I think may be
telling to the type of numbers
5 that may happen in the
absence of selection.
6 I point to
these last two papers only
7 because it has been
characterized in extensive
8 detail in muscle, and
I bring up Phil Johnson's
9 study because he now
has an abstract that is going
10 to be presented as
ASGT, where he is showing that a
11 majority of what I
think he calls 98.5 percent of
12 all the vectors that
are in skeletal muscle are
13 persisting in episomal
form.
14 He does a
real-time PCR assay. I am not
15 going to try to
describe his data, it is written in
16 an abstract form, but
I think it is something that
17 the field in general
will want to look at and see
18 if this will be
something that can be used for
19 other target tissues.
20 But it is
consistent with the theme. What
21 I did not talk about
here today was any of the data
22 that Mark and Kathy
have generated, because I know
23 they are going to
speak later and they can tell you
24 specifically what has
been derived in their hands,
25 but I think the theme
is we see what these vectors,
43
1 they have the
propensity to set up a persistence,
2 the data that has been
generated in liver, muscle,
3 lung, and brain is
that episomal forms that are
4 predominantly seen,
but there is always the
5 potential and evidence
for integration.
6 This is the
last paper that I am going to
7 point you to, and I am
going to just mention this
8 because I think this
is going to give us a starting
9 place to begin to
understand AAV integration in
10 whole animal.
11 Terry Flotte
and his lab have generated
12 some data showing that
the DNA-dependent protein
13 kinase, the gene that
has mutated in SCID mice,
14 seems to have an
impact on the molecular phase of
15 AAV genomes.
16 Again, I am
going to paraphrase what
17 Terry's data says, and
he can speak to it in more
18 detail because he has
got new data that is a little
19 bit more
extensive. It appears that if you knock
20 out this protein
kinase, which is involved in
21 immunoglobulin
rearrangement as one example of its
22 role in the human
cell, the virus appears to
23 integrate more efficiently
into the chromosome.
24 This is an
enzyme that plays a role in
25 end-to-end joining,
and it seems that if you lose
44
1 the ability of these
host enzymes to form the
2 so-called concatemer
structure that we all
3 characterize, you can
see an increase in
4 integration event
takes place.
5 So it
appears that if you are defective in
6 one pathway, AAV will
just follow another host
7 mechanism for
persistence, which is an integration
8 mechanism.
9 Again, if
there are any specific
10 questions, I will ask
you to direct them to Terry
11 where he can give you
the details of what is going
12 on, but what this data
tells me is that we probably
13 we will be able to
identify these so-called
14 cellular recombination
pathways that are
15 influencing AAV
vectors when they go into so-called
16 non-dividing tissue.
17 I am going
to conclude by trying to
18 reemphasize the
following points. Wild type and
19 AAV vector integration
is not very efficient, and
20 this fairly well
documented in vitro. It is
21 something that seems
to be a theme that is
22 recurring in vivo.
23 If you look
at the ability of the virus to
24 target chromosome 19,
it is absolutely dependent on
25 a viral protein called
Rep. The mechanism is now
45
1 well understood
because they are identical binding
2 sites to facilitate
this targeting.
3 AAV vectors,
which do not have Rep
4 protein, do not have
the ability to go to
5 chromosome 19 into the
site-specific manner. If
6 you look at the
proviral structure of wild type AAV
7 and vector DNA, they
are essentially identical at
8 all levels.
9 The break
points and the terminal repeats,
10 the amplification, the
concatemerization, and the
11 rearrangement under
chromosome sequence is
12 identical whether it's
on chromosome 19 or randomly
13 inserted throughout
the genome.
14 Finally,
with the limited number of
15 studies that are being
done, it appears that in
16 non-dividing cells in
vivo, the AAV vectors exist
17 predominantly in an
episomal form, and again, I
18 will conclude.
19 Daniel
basically summarized the AAV field
20 by saying it has the
propensity to integrate into
21 the chromosome, where
it fits on that rheostat as
22 being very efficient
or not efficient, I think it
23 is going to be
dependent on more studies in vivo in
24 which we can continue
to accumulate data.
25 But as of
today, what we keep seeing is
46
1 some propensity for
this episomal form, but the
2 risk is still there,
and I will stop there and take
3 questions.
4 DR.
SALOMON: Thank you very much. Very
5 interesting.
6
Q&A
7 I have a
couple of questions that kind of
8 occurred to me in the
setting of thinking about
9 this thing riskwise.
You have been very straight
10 about it. What is interesting is a lot of times
11 when it is introduced
for the first time, people
12 talk about OAB, it's a
parvovirus, it has been in
13 humans for a really
long time, and it has been
14 extremely safe in the
sense that it is not
15 associated with any
known disease entity, and the
16 implication is many
times that therefore, AAV gene
17 therapy as a vector is
going to be similarly safe.
18 However, I
think what you very clearly
19 point out in all the
molecular biology that has
20 been done with the
vector is that an AAV vector
21 really isn't anything
like a wild-type AAV in the
22 sense that now what
you have got mainly is
23 episomes, it is not
integrating in chromosome 19,
24 so there is a lot of
assurance that one might take
25 from the first part of
the data that it is probably
47
1 not reasonable to
carry forward into thinking about
2 AAV vectors.
3 DR.
SAMULSKI: Right. I will give
4 opinions on both
sides. I think if you look at the
5 biology of the virus,
it falls in the biological
6 features, so that we
don't see significant immune
7 response generated
from AAV infections. You don't
8 see that with wild
type.
9 You don't
see the virus taking over the
10 host cell as a lytic
virus does, so there is
11 consistency in that
aspect of saying AAV is more
12 like its features of
being non-pathogenic, but I
13 think you only need to
hear what Phil and them
14 mentioned at the RAC
probably every time AAV is
15 discussed, you know,
this is not normal. You are
16 putting in 1012
viruses into a focal injection,
17 hundreds of particles,
lots of genomes. This is
18 something that doesn't
happen in nature, and so it
19 shouldn't be
considered as the viral life cycle,
20 because in that
setting, we can't reproduce the
21 viral life cycle. We are not getting a systemic
22 infection that is
disseminating and maybe setting
23 up latency.
24 We are
inducing an artificial way of
25 getting
persistence. So I think you are right
on
48
1 the money there. I
think what will go back and
2 forth between these
systems is how much does the
3 vector mimic wild
type. As far as integration they
4 are identical, it is
just one is on 19, the other
5 one is random.
6 So there is
some ability to go back and
7 forth as to what is
happening.
8 DR.
SALOMON: So the second question I had
9 was I don't know a lot
about chromosome 19, so I
10 apologize for what I
am certain are stupid
11 questions to the
geneticists here, but is it clever
12 that the virus chose
this area in chromosome 19, is
13 that a safe area to
integrate in that?
14 I guess the
follow-up question here would
15 be maybe one thing to
think about, has anyone
16 thought about it, is
if you add the Rep gene back
17 and let it integrate
into a place that we know is
18 safe instead of having
all this episomal DNA that
19 we have no idea what
it is doing.
20 DR. SAMULSKI: Your question is something
21 that you would discuss
at a cocktail hour, why does
22 AAV go to 19. We could say mechanistically, there
23 is a viral origin
sitting on 19. Did the virus
24 pick it up from 19 and
retrofit it into its life
25 cycle or is that a
remnant, some integration event
49
1 that took place who
knows when.
2 It is only
conserved in monkeys and
3 humans, so it is a
sequence that is not found, so
4 there may be some
selective pressure for why that
5 took place. Is it a safe site? In tissue culture,
6 we are in HeLa cells,
there are 19 chromosomes, 3
7 copies in 19, we can
get latency all the time. In
8 vivo, there hasn't
been the kind of studies you
9 would want to see, and
if AAV integrates in 19, is
10 that going to be an
adverse event.
11 I would
argue 19 in liver cells may not be
12 essential, but 19 in
another tissue like neuronal
13 cells may be
essential, but to get back to your
14 question, which I
think is more directed to what is
15 on that locus, there
is no gene located at that
16 region.
17 Michael
Linden has argued that there is a
18 transcript that can go
through this region that is
19 related to a muscle
transcript, but from our and
20 other studies, there
has never been an integration
21 event that has
disrupted that gene or the potential
22 for the gene, but
again, there are all tissue
23 culture cells, so I
think it is an interesting
24 biology.
25 When we
first saw this, what is clustered
50
1 on chromosome 19 were
a lot of genes we would have
2 liked to have seen it
go into, the receptor for
3 polio virus, a
receptor for a lot of other viruses,
4 and we thought, oh,
maybe, AAV will integrate, give
5 the host cell a
mechanism of protection from
6 another infections
agent, and there would be a
7 reason for why it
targets, but this locus is not by
8 those type of genes,
although it would have been a
9 nice story. So it is an unknown.
10 DR. SALOMON: I had one last question, and
11 that is when it
integrates and then almost sort of
12 kind of does its
version of concatemerization in
13 that area -- that is
not quite exactly what
14 happens, but -- what
does it do to the promotor
15 regions in the ITR, is
the payload gene still
16 promoted, or does it
destroy the promoter region,
17 so you basically have
dead genes there?
18 DR.
SAMULSKI: AAV is not like the
19 retrovirusus where it
has a promoter, a strong
20 promoter in the
LTR. It has promoter-like
21 activity, but all the
cassettes have the promoter
22 built in between the
terminal repeats, and so the
23 gene remains intact,
the break points seem to be in
24 this buffering area in
the terminal repeats.
25 So, again,
all of these things are skewed.
51
1 They are put under
selection so you insert the
2 genes that go in
intact, and they rescue them out.
3 We can only see the
products that E. coli will
4 tolerate, so you have
to realize that head-to-head
5 and tail-to-tail
formations are not very stable in
6 E. coli, so we are
getting a biased opinion every
7 time we pull these
out.
8 The PCR
reaction is extremely biased
9 because that is Mother
Nature's best primer, it's
10 an 80 percent GC
hairpin structure. If you try to
11 prime through that
region, you will generate
12 deletions, so we even
think a lot of our data
13 showing break points
is an artifact of pulling out
14 junctions.
15 The only
data that begins to support that
16 if you have a real
controlled Rep expression, you
17 don't see as much
amplification rearrangement. The
18 group in Italy put the
Rep gene on the regulatable
19 promoter, and they
actually dosed in the amount of
20 Rep, and what they was
the integrations were more
21 well behaved.
22 So I would
say that we have not been able
23 to mimic what probably
the virus does very well,
24 but we can score all
the downstream events. It
25 goes in a chromosome,
it looks like this, and so
52
1 forth.
2 So I would
be hesitant about taking my
3 opinion about this
field and turning it into this
4 is the fact of all it
all happened.
5 For the
vectors where there is no Rep, and
6 you do see the integration,
it is cellular
7 mechanisms that are
putting it into the chromosome.
8 DR.
SALOMON: Dr. Rao and then Dr.
9 Mulligan.
10 DR.
RAO: Is there any evidence of
11 mobilization of the
integrated thing, wild-type
12 infection?
13 DR.
SAMULSKI: That is a good point.
14 There is the risk of
mobilization if you get an
15 added infection and a
wild-type AAV infection, so
16 you need a two-hit
kinetics to move the vector out
17 of the chromosome.
18 In the
laboratory, if you do those
19 experiments, wild-type
dominates the product that
20 comes out, because
there are more elements that
21 ensure packaging, and
they are not in the vectors,
22 but you do mobilize it
if you get a two-hit
23 kinetic.
24 DR.
RAO: Is there a rough percentage on
25 that? I know wild-type predominates, but --
53
1 DR. SAMULSKI: Wild-type plate
2 90-something percent
of all the virus that comes
3 out, and if you cycle
it, it is the only virus that
4 you see. The vector doesn't compete very well in
5 that setting, but the
risk is there, in an in vivo
6 setting.
7 DR.
MULLIGAN: In the in vivo case, the
8 integration question
is complicated by all the free
9 copies, and I think it
is important that people
10 that are not experts
here get a sense of if you had
11 very efficient
integration in the sense that you
12 had one copy for large
number of cells, but then
13 you had hundreds of
unintegrated copies, that would
14 confuse your
interpretation, so can you
15 characterize for
people how you get at the issue,
16 that is, if you just
look at the sum of
17 unintegrated copies,
and that is a large number,
18 and then the sum of
integrated copies, and that is
19 a small number, then,
one conclusion is that you
20 have mainly
unintegrated gene transfer, but in
21 principle, on a
cell-by-cell basis, you could have
22 very efficient
integration, while on top of it you
23 could have a large
amount of unintegrated copies.
24 Now, in
vitro, I know that is not the case
25 because you can
actually directly assess that, but
54
1 how have the various
tests actually ruled out that
2 that is not the case?
3 DR.
SAMULSKI: I think that is a good and
4 hard question. I think Mark has generated data
5 that begins to look at
that where he has put virus
6 in hepatocytes, and he
will probably discuss this,
7 and then did a partial
hepatectomy to let the liver
8 cells grow, and tried
to score how many of those
9 regenerated liver
cells still carry a copy
10 suggesting that that
fraction had integration, and
11 the ones that lost it
were primarily episomal.
12 I will let
him describe that, but I don't
13 think there is any
good way to assess that
14 question.
15 DR.
MULLIGAN: I would think that now that
16 there is these, in
human cells, outlaw PCR
17 approaches, the
question is can you actually
18 directly calculate the
total absolute number of
19 integrations
independent of how much total DNA is
20 there?
21 DR.
SAMULSKI: I don't know how I would do
22 that. I think this is what Phil Johnson is doing
23 in his abstract. He is looking at ALU real-time
24 PCR going across
genomes and stuff like that.
25 DR.
MULLIGAN. Has anyone looked, like
55
1 Ernest Whittaker, like
his system if you have an
2 adeno-infection or HIV
infection, and you all of a
3 sudden do an AAV
infection, is the propensity for
4 integration of AAV
into, say, HIV, a higher
5 integration because
it's unintegrated initially
6 than it would be to go
in the chromosome?
7 DR.
SAMULSKI: I think that is another
8 good question, that
is, if you are in a cell that
9 has substrates, what
is the fate of AAV to those
10 substrates, will it go
into them, or a more
11 preferred event. I don't think anyone has an
12 answer to that, but
it's a good question. It is
13 something that has got
to begin to be looked at.
14 I think I
would like to just emphasize
15 that AAV in the early
days was put in the bone
16 marrow stem cells with
a lot of efficiency, and
17 then it was shown that
as you tried to amplify
18 these cells, they
weren't very good and I think it
19 was speaking directly
to the fact that it wasn't
20 integrating and
therefore, you could transduce them
21 and get positive
cells, but once they are asked to
22 divide, you lost that.
23 So I think
why AAV has been such a niche
24 virus for the
so-called non-dividing cells is
25 because is can set up
this persistence. I think
56
1 the integration
frequency is probably going to be
2 determined by do
non-dividing cells carry out
3 illegitimate
recombination, at what rate compared
4 to a dividing
cell. That is going to be an
5 important number that
is going to influence the
6 outcome in these type
of studies.
7 DR.
GORDON: I have a couple of very quick
8 questions that are
just simple factual answers.
9 Where in the
life cycle of AAV does the
10 uncoating of the
genome take place? That is one.
11 The second question is
you said that when you add
12 Rep back to the
vectors, then, you get chromosome
13 19 integration
again. How is it added back, as a
14 gene or as a protein?
15 DR.
SAMULSKI: The answer to the first
16 question is the
parvovirus are argued to go into
17 the nucleus and uncoat
to release their DNA into
18 the nucleus. There is probably a capsic component
19 still associated with
the virus that is sitting on
20 those terminal repeats
that either prevents it
21 from, you know, being
naked DNA, but at the same
22 time may recruit other
factors to the origin.
23 As far as
the second question that you had
24 -- I forgot it already
--
25 DR.
GORDON: Adding Rep back.
57
1 DR.
SAMULSKI: That's my senior moment
2 there.
3 Rep protein
has been added both as
4 plasmids, as physical
protein injectate, and as
5 inducible protein in
the cell line, and all of
6 those will take
vectors and allow it to go to
7 chromosome 19.
8 The last
thing I will mention is that both
9 the Italian group and
our lab have generated a
10 mouse that carries the
chromosome 19 locus, and in
11 our case, it is
sitting on the X chromosome. When
12 we put wild-type virus
into that, it goes to that
13 chromosome 19 locus
even though it's on the X
14 chromosome, again
suggesting it's the cis elements
15 that are driving where
it goes, and not that it
16 happened to be on 19
in humans, and stuff like
17 that.
18 DR.
DYM: I think you alluded to my
19 question, but i am
going to ask it anyways. Can
20 you clarify or comment
on the ability of the AAV to
21 get into dividing
cells versus non-dividing cells,
22 and, of course, in the
testis, the spermatogonia
23 are very actively
dividing, the sperm are not.
24 DR.
SAMULSKI: I think there is no
25 difference between AAV
going into dividing or
58
1 non-dividing
cells. If the receptor is present, it
2 will bind, and then I
think the mechanism for
3 internalization is
clathrin-coated pits, endosome
4 release, and traffic.
5 If you can
carry out those steps, it is
6 indistinguishable
whether it's a dividing cell or
7 non-dividing
cell. In the very early days, it was
8 suggested that AAV
preferred dividing cells, but
9 that was in vitro
looking at selection and
10 therefore you were
biasing the system.
11 I think once
people went in vivo, they
12 realized that all of
that was probably misleading a
13 little bit.
14 DR.
MULLIGAN: You didn't mention about
15 other AAV serotypes,
so in principle, the
16 efficiency of the
intervention would depend upon
17 just the virus titer.
18 Do you have
any sense that AAV-1, for
19 instance, which in
muscle is much, much more
20 efficient, would
potentially be better at infecting
21 germ cells?
22 DR.
SAMULSKI: I think Richard's point is
23 a really interesting
one because we and others have
24 seen that the other
serotypes have better propisms,
25 are more
efficient. The question is what are
their
59
1 integration
mechanisms.
2 The only one
that we have data on is Type
3 4. Type 4, which is camana monkeys, will target
4 monkey cells and
integrate, will target human cells
5 and integrate in the
chromosome 19, so the
6 wild-type virus will
capitulate exactly what the
7 human virus is.
8 The other
four, 1, 3, and 5, it is
9 unknown, but they are
so homologous, about 80 to 90
10 percent homologous,
they all bind to the terminal
11 repeats, they all can
package each other's DNA.
12 Chances are they will
do the same type of
13 integration.
14 There are
differences in these terminal
15 repeats if you look at
them. Type 5 is different
16 than Type 2, and if
that is a substrate, that may
17 be more prone for
recombination enzymes, you may
18 see an integration
frequency that is different.
19 DR.
MULLIGAN: I just meant the capsid,
20 looking at risk for
germline infection, if it
21 happens just
proportionately, it much better
22 infects that cell and
even though integration is
23 very efficient, then
you get more efficiency.
24 DR.
SAMULSKI: I misunderstood. I think
25 if the virus has a
more efficient tropism in those
60
1 kind of cells, chances
are the integration
2 frequency is going to
be higher. That is kind of a
3 given.
4 DR.
SALOMON: Sort of a follow-up question
5 here is -- and you may
have answered this, and I
6 apologize if you did
-- if you have a cell that is
7 actively dividing or
is activated, let's say, so it
8 has a lot of open chromatin structures, it
is more
9 likely to integrate in
that setting than in, let's
10 say, a stable cell
that is not activated?
11 Obviously,
where I am going is in, you
12 know, if you had an
injury or inflammation, or
13 something, are those
areas in which the rules might
14 be different?
15 DR.
SAMULSKI: Sure. I think that is
16 exactly what the data
are supporting. This virus
17 looks for open
chromatin contacts. Events that
18 were scored appeared
to be in genes, promoter
19 regions in the
gene. I think they are all because
20 of the same reason,
these were open chromatin. If
21 it's condensed
chromatin, there is probably no
22 mechanism, because
again it's a cellular event and
23 it is going to be
acting on cellular regions of the
24 DNA, better
accessible.
25 DR.
SALOMON: That was great. Thank you.
61
1 DR.
SAMULSKI: Thank you.
2 DR.
SALOMON: Very useful.
3 The second
presentation is on germline
4 transmission by gene
transfer vectors and some
5 thoughts on assessing
the risk from John Gordon,
6 Mount Sinai School of
Medicine.
7 Germline
Transmission by Gene Transfer Vectors
8
Assessing the Risk
9 Jon
Gordon, M.D., Ph.D.
10 DR.
GORDON: I was asked to talk a little
11 bit about not
necessarily what we are doing to
12 address this problem
in my own lab, but just to
13 talk about what I
think are the points of
14 susceptibility for
germline integration of vectors
15 into various
gametogenic cells and to review the
16 literature on it, so
that is what I will do.
17 I am not an
embryologist by profession,
18 and I don't wear the
lot on spermatogenesis either,
19 but we have a
spermatogonium expert in the audience
20 in case I make a
mistake, so that will be good.
21 The ontogeny
of gametes in relation to
22 their susceptibility
to gene insertion. Primordial
23 germ cells are the
cells that ultimately arise to
24 both eggs and sperm,
and these arise in the yolk
25 sac or the epiblast in
the mouse at about three
62
1 weeks' gestation in
the human.
2 There aren't
a very great number of those.
3 They then migrate by ameboid movement through the
4 dorsal mesentery to
the genital ridge. During this
5 migration process,
they also multiply. These cells
6 are quite easily
identified because they stain very
7 strongly for alkaline
phosphatase.
8 They arrive to the genital ridges
that may
9 be the end of five
weeks' gestation in the human.
10 During this period,
the cells are unprotected, that
11 is, they are not
within the capsule of a gonad, and
12 they are mitotically active,
allowing infection by
13 agents that require
mitotic activity. We will
14 return to this point
of what agents may require it.
15 Fetal gene
therapy must take this risk
16 into account, and the
RAC had a sort of mock fetal
17 gene therapy protocol
presented one time, and this
18 issue has to be
raised.
19 Now, female
gametes, which are of a little
20 bit less interest
today, but they are important, of
21 course, they become
oogonia, and they divide by
22 mitosis until about 5
months or a little longer to
23 generate several
million oogonial cells. At this
24 point, many begin to
die, while others become
25 primary oocytes.
63
1 Primary
oocytes enter meiosis, a complete
2 crossing over, and
then they stop. The chromatids
3 remain associated, but
crossing over is completely.
4 Then, they are
surrounded by follicle cells in what
5 are called primordial
follicles.
6 Once they
are in the primordial follicle,
7 they become relatively
inaccessible because you
8 have to get through
the layer of follicle cells,
9 which is a single cell
layer basically at this
10 point, in order to
reach the egg, which is sitting
11 at the end of crossing
over in the so-called
12 dicteate [ph] stage.
13 They sit in
this stage until the follicle
14 begins to develop
towards ovulation, and there is
15 some hypothesis that
this long term association of
16 the chromatids has
something to do with chromosome
17 nondisjunction in
older eggs.
18 Now, at
puberty, the follicle develops in
19 response to FSH from
the pituitary. Numerous
20 follicle cells
surrounding the oocyte are within
21 the follicle wall, and
they begin to produce
22 glycoprotein "egg
shell," the zona pellucida.
23 So, as the
egg is developing, then, the
24 number of follicle
cells that sit between the egg
25 and the outside world
increase, the wall of the
64
1 follicle becomes a
consolidated structure, and the
2 zona pellucida is laid
down. This is a glycoprotein
3 human egg shell,
mammalian egg shell, very hard to
4 penetrate.
5 As the
follicle matures, meiosis resumes,
6 and one resumes, and
as the first polar body is
7 released, the
chromosomes then move to a metaphase
8 of the second meiotic
division, and that is how
9 they are found after
ovulation.
10 To enter the
egg, genes must past through
11 the follicle wall,
they have to get through or
12 between the follicle
cells around the egg, and then
13 they have to get
through the zona.
14 We would
regard the egg as a non-meiotic
15 cell at this point.
16 At
ovulation, the egg is in metaphase II
17 and is surrounded by
the zona and the granulosa
18 cell layer. Some of the cells are ovulated with
19 the egg.
20 Although
immunoglobulin molecules will
21 pass through the zona,
there is no evidence that
22 naked DNA or viruses
will do so. There have been
23 experiments at least
with retroviruses that have no
24 viruses that I am
aware of where very high amounts
25 have been put onto
zona intact eggs, and then lacZ
65
1 staining look for
later in cleavage, for example,
2 without seeing
anything.
3 After
fertilization, MII is completed with
4 release of the second
polar body formation and
5 formation of the
female pronucleus.
6 Now,
micromanipulation to assist
7 reproduction can
assist genetic material in by
8 passing the zona. I just would like to make the
9 point here of two
contrasting papers in the
10 literature, one by an
Italian group in I believe
11 now the late eighties,
in which they asserted that
12 if you performed in
vitro fertilization with
13 plasmid DNA sitting in
the medium, about 30 percent
14 of the mice born were
positive for transgene
15 sequences.
16 The plasmid
they happened to use in this
17 case was a
commercially available SV40-based vector
18 and to prove that they
had integration in these
19 mice, they cloned the
material back out of the
20 mouse genome and
sequenced the vector material that
21 was in the mouse
genome.
22 The
published sequences contain nothing
23 junctional, they were
all internal sequences to a
24 commercially published
sequence. They also did a
25 so-called MBO1/DPN1
digest to show that the
66
1 material was in
mammalian cells and was therefore
2 digestible with I
believe it's MBO1, if I don't
3 them in backwards
order, and the only problem with
4 this southern blot
showing disappearance of this
5 band was that the
southern blot did not include the
6 molecular weight size
that the band was originally
7 in.
8 It stopped
before you could get that high
9 up on the gel, which
wasn't very high, I might add,
10 about 4.3 kb.
11 So, needless
to say, there were a few
12 weaknesses in this
publication. Nonetheless, it
13 made the cover of Cell
and was accompanied by a
14 very exuberant
editorial saying that this had
15 something to do with
evolution, plasmids jumping
16 into gametes out there
in the ocean where fish have
17 ex vivo fertilization,
for example, and multiple
18 labs tried to repeat
this work and 2,300 mice were
19 produced in a number
of labs, we tried it too,
20 could not reproduce
this work even using the
21 identical reagents,
and no one makes transgenic
22 mice this way even
though it is a heck of a lot
23 easier than
microinjection.
24 However, if
you do another experiment, and
25 that is, mix plasmid
DNA with sperm, as was done
67
1 before but now inject
the sperm into the egg, so
2 now you are bypassing
the zona with a microneedle,
3 and the sperm and DNA
around it go into the egg, a
4 significant percentage
of the mice are transgenic,
5 and that is a
reproducible result.
6 So, in
humans, if we think about
7 micromanipulation, and
this is something I have
8 been asserting in an
editorial that I have in
9 press, we have to
think about the fact that the
10 environment had better
be clean, because we can get
11 DNA in by that method.
12 My opinion
of what occurs here is that the
13 pronucleus forms
quickly after the sperm is
14 injected, DNA gets
entrapped into it, and it is
15 pretty much the same
as microinjecting DNA into a
16 pronucleus.
17 Now, another
interesting point is there is
18 there papers
indicating that retroviruses and
19 lentiviruses will
infect MII oocytes, which are not
20 meiotic reactive, but
which do not have a nuclear
21 membrane. The chromosomes are sitting at a
22 metaphase of the
second meiotic division to produce
23 transgenic cattle,
monkeys, and mice.
24 I think
these papers are very interesting,
25 but there is one
slight problem with the assertion
68
1 that it is the
non-meiotic MII oocyte that is the
2 target, and that is,
of course, that if you soak
3 MII oocytes in the
vector, and then fertilize them,
4 there are still going
to be vector around after
5 fertilization, and it
is not really possible to
6 completely clean them
and then fertilize them to
7 show that you had no
vector around at
8 fertilization, so it
is possible in my view that
9 fertilization occurred
and then these vectors went
10 in.
11 But, nonetheless, you can get MII
oocytes
12 transduced with
retroviruses and in mice, now
13 lentiviruses from
David Baltimore's lab, and again
14 this raises an issue
in clinical in vitro
15 fertilization where
the zona is opened not
16 infrequently, either
for injecting sperm, for
17 biopsying embryos, and
so on.
18 Now, male
gametes. Now, in the male, the
19 primordial germ cell
step is the same. They get to
20 the genital ridges as
before, but them they become
21 dormant where they are
contained within sex cords.
22 They sex cords are
like the future seminiferous
23 tubules of the testis,
they remain this way.
24 The sex
cords have a membranous barrier
25 between them and the
outside world, but this is
69
1 much less protected
structure than it becomes after
2 puberty. The cells are mitotically inactive and
3 relatively
unprotected.
4 At puberty,
these PGC's become
5 spermatogonia and
begin dividing. Type A
6 spermatogonia are
renewable stem cells that produce
7 more Type A
spermatogonia, but they can also
8 produce Type B
spermatogonia, and those are
9 committed to meiosis.
10 It has been
shown, mainly by Ralph
11 Brimster's lab, that
spermatogonia can be
12 transduced with
retroviruses and lentiviruses, I
13 believe are correct
now. This is one in vitro and
14 it is not clear how
efficiently one could
15 accomplish this in an
intact testis with intact
16 spermatogenesis. Perhaps our colleague in the
17 audience, an expert on
spermatogonia, can speak to
18 that, but it clearly
is biologically possible to
19 transduce them even
though it is not very easy.
20 Generally,
they are put back into a testis
21 that doesn't have its
own spermatogenesis, so that
22 you can sort of have a
natural selection for those
23 cells exposed to the
vectors in the outside world,
24 and you can get
transgenic mice that way.
25 Now, when
meiosis beings and the
70
1 spermatogonia are
formed also, the testis becomes
2 organized the
seminiferous tubules. Pre-meiotic
3 cells are at the
tubule periphery where agents can
4 get to them, but they
will have to get through the
5 tubule wall, but
theoretically, they could be
6 reached from a hematogenous
spread to the
7 seminiferous tubule.
8 However,
Sertoli cells, situated within
9 the seminiferous
tubules, form tight junctions that
10 sequester meiotic
cells behind what is called the
11 "blood testis
barrier," so actually not a barrier
12 between the blood and
meiotic cells, it is between
13 the Sertoli calls and
the meiotic cells.
14 Sperm move
toward the lumen of the tubule
15 as they complete
meiosis and morphological
16 transformation. Now, this barrier is needed, of
17 course, because it
doesn't occur because these
18 meiosis-specific
proteins don't appear until after
19 puberty, and therefore
they are potential
20 immunogens, so this
has to be a immunologically
21 privileged site, and
that is the rationale for
22 having the blood
testis barrier.
23 Meiotic
cells are difficult to access
24 except retrograde
through sex ducts. You can
25 inject vectors into
the epididymis, for example,
71
1 and find them in the
testis. So someone is
2 undergoing, for
example, prostate gene therapy, it
3 is not at all
impossible that one could get vectors
4 moving retrograde back
up and thereby get to the
5 cells that are behind
the blood testis barrier.
6 Male
gametes. Now, sperm maturation or
7 spermiogenesis, is
characterized by a loss of most
8 cytoplasm, replacement
of the histones by much
9 tighter binding
protamines, and near complete
10 cessation of gene
expression. I say "near"
because
11 there are a few
post-meiotically expressed genes.
12 Again, what
you have to realize is that
13 the idea of sexual
reproduction is to give all
14 gametes an equal
chance of getting to the egg, and
15 if you have
postmeiotic gene expression could have
16 allelic variance which
would give sperm an
17 advantage
theoretically, and so the organism does
18 everything possible to
prevent that.
19 As meiosis
begins, actually, once Type B
20 spermatogonia become
committed, these cytoplasmic
21 bridges remain between
the cells. These are very
22 large and they allow
even mRNA size molecules to
23 pass from one cell to
another, so allelic
24 variations between
spermatogenic cells, those
25 differences are
minimized in terms of their
72
1 potential impact on
spermatogenesis, and then late
2 in spermiogenesis,
there are a few genes active,
3 but mainly there are
the chromatin is very tightly
4 condensed and very
difficult to access.
5 I should
point out parenthetically there
6 that there have been
papers from Anderson's lab way
7 back when, showing
that retroviruses like open
8 chromatin in
preference -- or DNA hypersensitive
9 chromatin -- in
preference to highly condensed
10 chromatin.
11 The nucleus then becomes surrounded by
12 what I would call the
giant lysosome, the acrosome,
13 contains lytic enzymes
for presumably digesting
14 your way through the
zona in fertilization, and it
15 is difficult to access
DNA in the sperm head.
16 Now, again,
I would say that there are
17 some papers saying
that this has been done
18 successfully. There is a paper from France saying
19 that pig sperm can be
transduced with adenovirus.
20 This paper found lacZ
expression in cleaving
21 embryos after exposing
sperm to adenovirus, and
22 then found piglets
that had mRNA-derived by RT-PCR
23 that had mRNA derived
from adenovirus in multiple
24 tissues of these
piglets.
25 Now, I would
just analyze this paper a
73
1 little bit for your
benefit, if I might. The lacZ
2 vector used in that
paper was a vector that was
3 received from another
laboratory and which had a
4 nuclear localization
signal. So the lacZ should
5 have been in the
nucleus of these embryo cells, and
6 indeed, when we have
used such things on embryos,
7 we see the nucleus
stain.
8 However, the
pig embryo is loaded with
9 lipids, and they are
basically black. You can't
10 see the nucleus in a
pig embryo, and if you want to
11 inject a pronucleus in
a pig to make transgenic
12 pigs, you have to
centrifuge the embryo to get the
13 lipid out of the way,
so you can even see the
14 structures.
15 So, in the
photograph showing lacZ
16 staining of these
embryos, there were black embryos
17 that were exposed to
the vector, and there were
18 slightly less black embryos
that were not exposed
19 to the vector, and the
nucleus was not visible in
20 either case.
21 The staining
for lacZ was done for 15 days
22 in this experiment,
and I would assert to you from
23 my own work with lacZ
staining that you could stain
24 your teeth if you did
it for 15 days.
25 The staining
was on the zona. There is no
74
1 reason why there
should be staining on the zona,
2 but we have used lacZ
staining on embryos with
3 adenovectors on
zona-free embryos just exposing the
4 embryo, we never seen
staining, not on zona-free,
5 but, for example,
injecting it under the zona, we
6 never see zona staining.
7 These people
found RT-PCR-positive tissues
8 in all three germ
layers of the piglets born, that
9 is, ectoderm,
mesoderm, and endodermal derivatives.
10 Now, this vector was
replication-defective. The
11 only possible way to
be in all three germ layers is
12 if it integrated and
got replicated.
13 However,
their southern blots were
14 negative. To me, that is a very incongruous
15 result, so I don't
believe the result, let me just
16 give you my own
opinion there.
17 We tried
this in mice and could not repeat
18 it, at least in
mice. However, I think this paper
19 and the other paper
with the sperm-mediated plasmid
20 transfer speaks to one
of the sort of difficult
21 problems for the FDA,
I believe. These are
22 published data and it
is very difficult to say, oh,
23 well, that's great,
but it is not a good paper, so
24 we will just ignore
it. It is very difficult to
25 ignore it when people
say they are doing these
75
1 kinds of things
successfully, then, one has to step
2 in and address it.
3 Male gametes
continued. Now, the mature
4 sperm on route to
release can be exposed to vectors
5 via fluid from the
seminal vesicle, prostate, and
6 in the urethra, a
small amount of urine, as well,
7 although maybe you are
uncomfortable to see or hear
8 that, it's true.
9 Virus found
in the ejaculate could be from
10 any of these four
sources or from the sperm
11 themselves if somehow
it got there, and I should
12 say that one could
imagine all also that the cells
13 that line the sex
ducts could be received vector
14 from the bloodstream
and then pass it on
15 theoretically to sperm
although I think that is
16 very unlikely.
17 As vectors
diversify, though, we can't
18 completely rule that
out. Reports of successful
19 transduction of mature
sperm are difficult to
20 repeat, and I have
already discussed that.
21 Male gametes
continued. When sperm bind
22 to the zona, they
undergo the acrosome reaction.
23 The acrosome reaction
is fusion of the outer
24 acrosome
membrane. You remember the acrosome is
25 the giant
lysosome. The best way to think of
this,
76
1 as I have told my
family, it seems to work on them,
2 if a fist put in a
pillow, a soft pillow, and that
3 put into a garbage
bag.
4 Now, the
soft pillow is the acrosome, and
5 the fist is the
nucleus, so the nuclear membrane is
6 coming in contact with
the inner acrosomal
7 membrane. Then, you have the feathers, which is
8 the acrosomal
contents, then, the outer acrosomal
9 membrane, the other
side of the pillow, and then
10 that is right
underneath the plasma membrane, the
11 plastic bag.
12 Well, if you
slash open the plastic bag
13 and the outer side of
the pillow, and sew those
14 seams together, you
will release all the feathers
15 to the outside. The acrosome reaction occurs, and
16 the bottom line of
that is a lot of the sperm
17 plasma membrane is
lost.
18 So even
passive association of genetic
19 material with the
membrane, a lot of it can be
20 lost. However, often the entire sperm is
21 incorporated into the
egg and the plasma membrane
22 and components
associated with the tail may still
23 be there, so it is
possible to passively get it in,
24 I think.
25 Now, shortly
after fertilization, sperm
77
1 head decondenses to
form the male pronucleus. DNA
2 replication
begins. Genetic material that enters
3 the egg with sperm, as
I pointed out, from these
4 microinjection of
sperm experiments, you can have a
5 relatively highly frequent
integration.
6 Now, the
early embryo, I wanted to mention
7 it because of my
allusions to IVF, the early embryo
8 cleaves within the
protective zone until
9 implantation, when
hatching occurs. Now, hatching
10 and implementation
virtually occur concomitantly
11 under normal
circumstances, so the embryo is
12 difficult to access
even though it has to get out
13 of the zona.
14 However,
micromanipulation can open the
15 zona and expose the embryo
to gene transfer agents
16 for more extended
periods. Take, for example, the
17 many thousands of IVF
cycles that go on every year
18 where the zona is open
to theoretically assist
19 hatching. In my opinion, assisted hatching is of
20 debatable
effectiveness, but there have been some
21 papers that embryos
from older women implant more
22 frequently if you open
the zona, and what happens
23 there is you may open
the zone at the four-cell
24 stage, put it in the uterus
and it sits there until
25 the blastocyst stage
and then implants, and so now
78
1 you have the naked
cells of the zona opened embryo
2 sitting there where
agents that may be in there
3 from the woman being
infected with something, from
4 the lab technician who
had gene therapy, from
5 whatever source, have
a much greater time period in
6 which they could get
to the embryo.
7 The embryo is
quite easily transduced by a
8 variety of agents,
retroviruses being the first one
9 done by Yenish in the
early seventies, recombinant
10 retroviruses in the
mid-eighties, controversy
11 whether adenoviruses
integrate. Our own lab did
12 one where we did early
embryos with adenovirus, and
13 what we found was
adenovirus was very toxic, so if
14 you put enough in to
be sure of getting
15 transduction, the
embryos were all killed. If you
16 put in so little that
none of the embryos were
17 killed, you had no
transduction, but if you have
18 sort of an
intermediate level, then, very rarely
19 you can see
PCR-positive tail biopsies in offspring
20 that is clearly a
mosaic integration.
21 So it is
possible to infect embryos, and
22 as IVF becomes more
and more interested in zona
23 opening, let me give
you another example,
24 pre-implantation
genetic diagnosis. You may have
25 heard the speech of
Frances Collins at the ASGT
79
1 meeting in California
where he went on about
2 pre-implantation
genetic diagnosis and result of
3 finding out things
from the genome project, for
4 example.
5 Well, pre-implantation genetic
diagnosis
6 requires first
injection of the sperm because if
7 you do regular IVF,
there is hundreds of sperm that
8 are still around and
many bound to the zona. When
9 you then biopsy the
embryo for PCR, if one of those
10 other sperm gets into
your PCR reaction, you are
11 looking for one
molecule here, that is, or two
12 molecules, to genotype
the embryo, an extraneous
13 sperm is unacceptable,
so you have to do ICSI, that
14 is, intra-cytoplasmic
sperm injection.
15 Well, that
opens the zona, and as I
16 pointed out before, it
is very easy to make
17 transgenic mice if you
do ICSI with DNA in the
18 medium.
19 Then, you go
back later and open the zona
20 again, but this time a
much bigger hole, so that
21 you can take a cell
off to do genetic diagnosis,
22 and so I think from
the point of view of germline
23 transmission, it is
much more risky thing to do
24 than just tell the
women to get pregnant. She will
25 have a 75 percent
chance then of having a baby that
80
1 hasn't have genetic
disease in the case of
2 recessive genetic
disease. She has a 100 percent
3 change of getting
pregnant, of course, while in
4 pre-implantation
genetic diagnosis, her chances are
5 only 20 percent. It is going to cost her nothing
6 to get pregnant, while
in pre-implantation genetic
7 diagnosis, it costs
about $15,000 to get pregnant.
8 Then, she has no risk
of all these other things,
9 which, of course, in
pre-implantation genetic
10 diagnosis, she has.
11 I might also
add that she has to be
12 superovulated for
pre-implantation genetic
13 diagnosis. There have been deaths from
14 hyperstimulation
syndrome. There have been
15 problems with surgical
retrieval of oocytes. I was
16 a little angry with
Frances for always saying that
17 instead of saying how
about just doing prenatal
18 diagnosis and doing an
abortion in the quarter of
19 cases where it is
necessary.
20 I just
thought I would give you a few
21 pictures here. There is spermatogenesis in a
22 normal testis. Actually, it is a seminiferous
23 tubule that we
injected with adenovirus vector, and
24 the periphery of the
less mature sperm cells. As
25 you see, you move
towards the periphery, the sperm
81
1 heads become condensed
and you can see tails, and
2 so on.
3 Then, they
are released into the lumen of
4 the tubule and then
may go out. I said there is
5 minimal cytoplasm on
sperm, but a normal variant in
6 sperm is a so-called
cytoplasmic droplet, which
7 kind of like hangs
behind the mid-piece of the
8 sperm, so there can be
a significant amount of
9 cytoplasm in
ejaculated sperm.
10 Here is a
developing egg. I was pointing
11 out to you the
barriers of penetration of this
12 structure for its
virovector. Here is the DA
13 nucleus. You can't see the incipient zona
14 pellucida, but there
is a very white band around as
15 it is beginning to
form, many follicle cells
16 around, and then the
follicle wall. So it is
17 difficult to get
there.
18 This is some
experiments we did when
19 injecting adenovirus
vector into the ovary at
20 unbelievable concentrations
against any for lacZ.
21 You can see that this
vector didn't want to get
22 into the
follicle. The eggs didn't make it
through
23 frozen section, so we
have done
24 immunohistochemistry
to show that the follicle is
25 not penetrated.
82
1 Here is
injection directly into the
2 seminiferous
tubule. My contention is that we
3 should do provocative
experiments that tell us
4 whether or not it is
biologically possible to
5 transduce these cells,
because in the future, gene
6 therapy will be
promulgated, vectors will
7 diversify, their
tropisms will change, their
8 structures will
change, the methods of
9 administrations will
change, and the number of
10 people treated will
grow, so we need to know can
11 these things actually
get in, not we need to design
12 experiments not to
show ourselves as they probably
13 won't happen. We need to do experiments to tell us
14 whether or not it can
happen, so that we can write
15 the proper consent
forms.
16 When we do
adenovirus vectors into
17 seminiferous tubules
directly in a procedure we
18 call seminiferous
tubule cannulation, we see a lot
19 of staining for lacZ,
this is immunohistochemical,
20 in the periphery, and
it looks as if Sertoli cells
21 are the transduced
cells.
22 This is a
Sertoli cell. It is sort of
23 anchored to the
periphery of the tubule and extends
24 its way in. The Sertoli cell surrounds the
25 spermatogenic cell and
sort of helps it complete
83
1 spermatogenesis, and,
by the way, also concentrates
2 androgens to very high
levels in this region of the
3 testis.
4 We are doing
this test to ask ourselves
5 can we transduce these
intermediate cells that are
6 behind the blood
testis area by injecting vector
7 directly into an
intact seminiferous tubule. We
8 believe that this
suggests no, but we think we need
9 to go to nucleic acid
hybridization to really know
10 because especially
like for AAV, which has a
11 delayed expression, we
need to know where the
12 genetic material
actually is.
13 This is just
a view of the acrosome
14 reaction. This is the acrosome. With those
15 enzymes for getting
through the zona pellucida, the
16 main one is a
proteolytic enzyme acrosome, and I
17 hate to say this, but
there is a paper from Japan
18 where acrosome was
knocked out and the mice were
19 completely
fertile. It has never been repeated,
20 but everybody believes
it. That is rather a shock,
21 I must say.
22 You can see
how much of the plasmid memory
23 can be lost in the
acrosome reaction.
24 That is the
summary them of where
25 gametogenesis is more
or less susceptible to being
84
1 genetically
transduced.
2 DR.
SALOMON: Thank you very much, Jon.
3 That was excellent.
4
Q&A
5 It is
interesting that yesterday, we were
6 talking about a
procedure that came very close to
7 what you just
described, so what they are doing it
8 taking infertile
oocytes from the presumed patient
9 or from the infertile
mother, and taking normal
10 donor oocytes and
injecting the sperm -- it's ICSI
11 -- but also ooplasm
from the normal oocyte donor.
12 One of the
issues that we discussed in
13 detail was the
potential of chromosomal DNA
14 fragments being
injected with the ICSI, and you
15 have now given
additional evidence. We were
16 concerned of
recombination potential, the gene
17 delivery.
18 DR.
GORDON: Well, let me just say that I
19 wrote an editorial to
Fertility and Sterility,
20 which is in press, but
I haven't received galleys
21 yet, and therefore,
there is some concerns about it
22 being released to the
committee and then, of
23 course, to the public
yet.
24 But I list
all these procedures of
25 micromanipulation and
their potential risks for
85
1 inadvertent germline
Transmission. I makes some
2 suggestions about what
might be done to sort of do
3 quality control in IVF
labs. That would at least
4 address this issue proactively.
5 I mean
should we multiplex PCR media in
6 which we do
micromanipulation just to make sure
7 there is not DNA in
there, or should we discuss
8 whether or not
practitioners of this forms of IVF,
9 we should at least
know that they haven't had 1015
10 retroviruses put into
them the day before for gene
11 therapy for something,
which could happen down the
12 road.
13 I think we
should at least begin to study
14 this because there are
tens of thousands of cycles
15 done.
16 Now, in
terms of the papers of ooplasm
17 transfer, I have a
written editorial published, in
18 which I say that the
use of germline gene
19 manipulation -- unfortunately,
these people did
20 this mitochondrial DNA
analysis on newborns who had
21 received ooplasmic
transfer, and the found the DNA
22 of the donor cytoplasm
in the newborn's bloodstream
23 -- they called this
the first germline gene
24 transfer.
25 Well, of
course, these new mitochondrial
86
1 DNAs were not
transmitted through the germline yet,
2 so it was a little bit
of a loose use of the term,
3 and remember that if
it is mitochondria, you can
4 always get rid of it
is you just allow the person
5 to be a male who has
received all of that, because
6 sperm mitochondria are
not transmitted to the next
7 generation.
8 There was a
very interesting paper where
9 sperm mitochondria
were injected into an egg and
10 destroyed and then
liver mitochondria were injected
11 and weren't destroyed,
so it seems like the egg
12 knows how to find
sperm mitochondria, distinguish
13 them from others and
destroy them.
14 So that type
of gene transfer if not
15 germline in my
opinion, and although these people
16 wanted notoriety for
using that phrase, I am not
17 sure they got the one
they were looking for, but in
18 any case, that is very
easy to thwart. All you have
19 to do is make sure
that it's only male reproduction
20 after that.
21 DR.
SALOMON: This is very interesting but
22 we are going to have
to stop, because that, we
23 discussed yesterday.
Too bad you weren't here.
24 I have one
quick question and then we will
25 start from the
panel. In terms of interpreting
87
1 experiments where you
say we looked at gene
2 transfer with
adenoviral vectors, they were all
3 adeno that you showed
us this time, no AAV, right?
4 It got into
the Sertoli cells, for
5 example, it didn't get
into the spermatogonia, and
6 from what I looked at,
those were spermatogonia,
7 not the more mature
spermatids, right, because you
8 were showing right at
the edge there --
9 DR.
GORDON: Some maturing, yes, it looked
10 like there might have
been spermatogonia. That
11 slide does not rule
out. That slide shows that we
12 can certainly get a
ton of vector there, which I
13 believe is
important. I think provocative tests
14 need to be done, not
bloodstream injections where
15 we will never find the
cells that got exposed.
16 DR.
SALOMON: The specific question I had
17 is at some point, you
point out very well that the
18 DNA in the developing
sperm condenses and
19 transcription
diminishes dramatically to almost
20 stopping, and I
certainly have no expertise in
21 exactly when in the
cycle that is happening, but it
22 would seem to me that
particularly, experiments
23 done with mature sperm
in which you tried to do
24 something that
required transcription as the
25 measure of whether you
got gene delivered would be
88
1 a failure because
there is no transcription going
2 on, so even if you got
gene in, to just take sperm,
3 incubate it with AAV
vector or adenovector or any
4 vector, and then show
this is not lacZ positive
5 wouldn't mean
anything.
6 Did I miss
something along the line?
7 DR. GORDON: Well, I am not so sure how
8 much transcription is
needed to get that to occur.
9 I mean you are more a
vectorologist than myself,
10 but it would seem to
me that if you get a vector
11 into the head of the
sperm, that the sperm could
12 then fertilize the
egg, and then it would
13 decondense into a
pronucleus and development would
14 begin, and any vectors
that were in there could
15 then act as if they
had just infected a dividing
16 cell line.
17 So, if you
could get the sperm to carry it
18 in, you wouldn't have
to transduce the sperm,
19 integrate it into the
sperm head, but you could
20 certainly get viruses
into the embryo by that
21 method theoretically.
22 DR. SALOMON: Right. So if you want to
23 test it, you would
have to test it several steps
24 down the line, that
you have delivered whatever you
25 carried in, got
transcription again, make the
89
1 beta-galactoside gene,
then, you do the colored
2 substrate. I am just trying to understand. From
3 what you are saying,
if you took just mature sperm
4 and incubated them
with a vector, and that might
5 even occur in the --
there is probably a lot of
6 transcription going on
in the spermatogonia,
7 though, right?
8 DR.
GORDON: Yes.
9 DR.
SALOMON: That must be a metabolically
10 active cell.
11 DR. GORDON: Yes.
12 DR.
SALOMON: So this would probably not
13 be a criticism of
studies done on the first things
14 you showed.
15 DR.
GORDON: Well, here is what I did. I
16 exposed sperm to
adenovirus vectors, made sure that
17 they got exposed to
is, 10, 100 virions per cell,
18 and then I did in
vitro fertilization with those
19 same sperm.
20 Then, the
embryos that those sperm
21 conceived were
evaluated for expression. The other
22 thing we did was we
allowed fetuses to be produced
23 or newborns and we
evaluated them by PCR.
24 Now, my
opinion is there were a lot of
25 experiments that
preceded those in which animals
90
1 were injected in their
brain with adenovirus and
2 then bred. Well, you know, there is 300 million
3 sperm in a mouse
ejaculate, and you are looking at
4 10 of them when you
look at 10 pups. So that is
5 statistically not
satisfying.
6 But if you
have an in vitro system where
7 every cell is exposed
and then you have a way of
8 assessing whether it
got in, I think that you are
9 doing much more to
really answer the question.
10 DR.
FLOTTE: I had sort of a natural
11 history question. I was wondering if you had any
12 thoughts about human
endogenous retrovirus
13 sequences in our
genome and what is the most likely
14 access that those
originally had to the human germ
15 line.
16 Then, a
follow-up question, do you think
17 there is any
significance to the fact that we don't
18 find human endogenous
AAV sequences in the genome?
19 DR.
GORDON: The first question. Well,
20 there is a tiny little
sort of moment of
21 accessibility I think
at hatching of the embryo in
22 vivo. The embryo has to hatch out and then
23 implant, and it is
naked. That could be a point
24 where a person who had
a lot of viremia or a lot of
25 virus in interstitial
uterine fluid that you could
91
1 get one in.
2 I must say
that in mice, retrovirus-like
3 sequences are also
found endogenously in the
4 genome. That, to me, would be a logical place to
5 think of it
occurring. It is very hard to imagine
6 it occurring. You could also think of a viremic
7 male having it get
into a spermatogonia.
8 I mean now
that it has been shown that you
9 can get it into
spermatogonia, at least in vitro,
10 it might be much less
probable in vitro, but if you
11 have 30 million
centuries to work on it, you know,
12 you may see it. So this is exactly the point, of
13 course, about
provocative testing, too.
14 So that is
my view. Now, what is the
15 significance of not
finding a virus, I mean I
16 really can't say
anything about that. It could be
17 a combination of
factors - I haven't looked enough,
18 the virus has too low
an integration frequency,
19 there is not a
biological setting in which there is
20 good access of a virus
at a susceptible point, you
21 know, ontogeny, such as
uterine fluid at a time of
22 implantation.
23 So it would
only be speculation on my
24 part, I don't know.
25 DR.
SALOMON: Dr. Dym and then Dr. Rao.
92
1 DR.
DYM: I had a couple of questions, but
2 first I will thank you
also for a lucid
3 presentation. I will just comment briefly that
4 there are a number of
people who are using in vivo
5 approaches, as I think
you know, to get viruses
6 into the spermatogonia
through the seminiferous
7 tubular lumens. Brimster is one and there was a
8 paper by Blanchard
& Vokalhyde in Biology of
9 Reproduction in 1997.
10 Again, they
showed that it only went into
11 the Sertoli cells, but
Brimster and a number of
12 others, actually, five
or six labs, in monkeys and
13 in rodents and in
cattle, are using this
14 seminiferous tubule
injection or ret-A testis
15 injection. It is in vivo, but it is not practical.
16 I mean you can't put
it in that way normally.
17 But this
leads me to my second question
18 having to do with
barriers. You mentioned
19 barriers. I do believe there are barriers from
20 your work and from
other people's work, and that is
21 why probably virus in
a muscle or systemic virus
22 may not get into the
spermatogonia, but this is in
23 normal animals or
maybe in normal people, but the
24 barriers actually
break down when there is a
25 diseased person or a
diseased animal.
93
1 I am just
wondering if you know anything
2 about that and if,
when the barriers break down.
3 Actually, another
thought came to mind. For
4 example, in AIDS
patients, the barriers are broken
5 down and the virus,
which is circulating in the
6 blood, let's say, from
a man who has gotten
7 infected via needle,
the virus is in the blood, and
8 then eventually it
breaks down and gets into the
9 closed lumen or semen
compartments, whether it is
10 testis or epididymis,
but it does get across the
11 barrier, so viruses do
get across in diseased
12 conditions.
13 Some of
these patients you are talking
14 about might have a
breakdown of the barrier.
15 DR.
GORDON: I am glad you actually
16 mentioned that because
I think it is worth some
17 comment. First of all, I think viruses might be
18 able to break the
barrier and then go through. I
19 mean viruses can hurt
cells, and if you flood cells
20 with them, you might
get a weakening of a barrier
21 by the very action of
the virus.
22 Then, there
are disease states. Disease
23 states are exposed
internal portion of the
24 seminiferous tubules
to the outside, I think
25 intuitively are not
likely to be so flagrant as to
94
1 raise the risk
significantly just because I think
2 that would have a big
impact on spermatogenesis,
3 too, but I did want to
say that there are ways --
4 well, the FDA speaker
was point out that localized
5 injection is less risky
than perhaps systemic
6 injection, but I think
one exception should be
7 taken to that, and
that is injections into things
8 like the prostate,
which by no means is an inactive
9 area of research, so I
do agree that while these
10 barriers exist, one cannot predict from that
11 intuition that in all
of the settings of gene
12 therapy, where a
vector's ability to cross barriers
13 may vary, or a
vector's ability to violate the
14 barrier and get in on
their own may vary, where
15 disease states may
vary.
16 So
biologically, these barriers exist, but
17 I think it is quite
true that you can by no means
18 be guaranteed that
they are going to protect you
19 completely, and
provocative testing is needed.
20 DR.
RAO: You give a very nice summary, at
21 least for me, in terms
of understanding that there
22 is great protection of
the male and female gametes.
23 So, let's
say you do, in fact, a patient
24 with adeno-associated
virus at some titer, 1011,
25 and now see
adeno-associated virus in ejaculate.
95
1 What would you
speculate as which cell was infected
2 and does it have to
actually be an integration
3 event that you are
seeing this one year later?
4 DR.
GORDON: No, I don't think it has to
5 be an
integration. A year later is really a
long
6 time. But weeks later, as what happened in this
7 case that probably
prompted this discussion, could
8 be in anything, could
be seen in the fluid
9 component, could be in
other cells, there is always
10 a few white cells
perhaps, could be in the debris
11 that would slough off
from endothelium, not at all
12 necessarily in sperm,
and even if it came out with
13 sperm, that doesn't
mean it is in them. It could
14 be just on them, and
washing them could take care
15 of it, or IVF could
take care of it.
16 I think it is reasonable if a sperm
17 fraction in
infractionated semen is positive to
18 step back and say,
well, now, a red flag has been
19 risen. If you find it in whole semen it really
20 could be from any
variety of sources.
21 DR.
DYM: Just one more comment maybe in
22 relation to what you
said. You know, those of us
23 who work in the
testis, and there are many of us
24 working on
spermatogonia who are actually trying to
25 infect and transduce
the spermatogonia and the germ
96
1 cells, we never think
of doing it in the sperm, we
2 always think of doing
it in the spermatogonia as
3 the only permanent
way.
4 I think that maybe addresses some point
5 that you made. That would be permanent, you know,
6 generation after
generation after generation. It's
7 an eternal cell, it's
an immortal cell, the
8 spermatogonia. The sperm dies.
9 DR.
RAO: The reason I asked the question
10 was one needs to
evaluate, when you are looking at
11 any kind of risk, as
to where the virus particle is
12 present, and that is
an important thing that we
13 need to clarify if you
are going to say that you
14 detected in the sperm
or in the ejaculate where is
15 it really going to be
present.
16 From what we
heard, it is unlikely to be
17 present in the sperm
per se, at least in the sperm
18 DNA, and given what we
have heard about integration
19 events, maybe it is
unlikely to be present in the
20 spermatogonia, but we
need to know it. It is best
21 to ask the expert
directly.
22 DR.
GORDON: Well, I just would say that
23 if you found it in
semen a year later, I would be a
24 little more worried
that it got into is
25 spermatogonium
because, as he said, that is an
97
1 immortal cell. Spermatogenesis proceeds in waves,
2 and if you get it into
any cell that is not the
3 Type A spermatogonium,
you may have its appearance,
4 but then it will
disappear.
5 That is why
people are trying to do
6 spermatogonia, but I
must add that there are a
7 number of papers in
the literature, none of which I
8 believe, but there is
man of them saying that you
9 can get DNA into
mature sperm by a variety of
10 methods - opening the
epididymis and giving it an
11 electrical shock with your biorad
electroparator,
12 people will say that
works. I mean you should see
13 those data, they are
so pathetic, but nonetheless,
14 they are published, so
what can you say, the data
15 are published.
16 DR. SALOMON: I would like to call this
17 session to the
break. We will see everybody back
18 in 10 minutes.
19 [Recess.]
20 DR.
SALOMON: We will go ahead and get
21 started.
22 This portion
of the session, we are going
23 to have a series of
presentations from Avigen and
24 then from the
University of Pennsylvania.
25 The next two
speakers will provide us some
98
1 specific information
on the AAV vector from Avigen.
2 The first
speaker is Mark Kay. Welcome.
3 A Phase I Trial
of AAV-Mediated Liver-Directed
4 Gene
Therapy for Hemophilia B
5 Mark Kay, M.D., Ph.D.
6 DR.
KAY: Thank you.
7 What I would
like to do is summarize our
8 Phase I trial of
AAV-mediated liver-directed gene
9 therapy for hemophilia
B, which is a collaborative
10 effort between many
investigators at Stanford, the
11 Children's Hospital,
Philadelphia, and Avigen.
12 [Slide.
13 Today's
focus are issues pertaining to the
14 inadvertent germline
transmission of AAV vector and
15 what I would like to
do is summarize data related
16 the clinical trial to
date.
17 [Slide.
18 There has
been some discussion about
19 integration of AAV in
the liver, and although Jude
20 suggested that I was
going to show data about
21 integration, I
actually have those slides, but not
22 in this particular
talk, so let me just summarize
23 where things are and
give some explanation.
24 We know
that, in general, if you inject
25 reasonable high doses
of AAV into mice that you can
99
1 get something in the
neighborhood of 50 percent of
2 hepatocytes that are
stably modified with AAV. In
3 some situations, it
might be slightly higher or
4 lower.
5 Now, it
turns out that if you give these
6 regular doses of AAV
into mice, the vector genomes
7 actually get into
almost 100 percent of the
8 hepatocyte nuclei, but
over time, most of those
9 single stranded
genomes are lost and here is only a
10 small proportion of
cells that remain with stably
11 transduced vector
genomes
12 Now, the
proportion of integrated genomes
13 is actually
small. Generally, it is actually less
14 than 5 percent. I think the definitive evidence
15 that AAV integrated in
liver was a study done in
16 collaboration with
Linda Couto and Hikiyuki [ph]
17 Nikai, where they
actually were able to clone out
18 integration junctions,
so basically within the
19 vector, they put
bacterial origins of replication
20 and then were able to
take genomic DNA, put them
21 back in the bacteria,
and clone out the covalent
22 linkage of the vector
where it integrated into the
23 genome.
24 Now, this
was a very useful technology,
25 but it does not
quantify how much integration
100
1 actually
occurred. So we have recently published
2 on studies where we
have injected AAV into animals
3 and we wait for a
period of time until there is
4 stable transduction,
and then what we actually do
5 is a hepatectomy.
6 Now liver
cells will equally regenerate,
7 such that each cell
divides once or twice, and as a
8 result, DNA genomes
that are not associated with
9 centromeres or
telimeres are lost, and we have
10 positive and negative
controls for this, and what
11 we find is that in
most situations, the amount of
12 integrated genomes, of
the stable genomes is very
13 small, it is usually
less than 5 or 10 percent of
14 the double-stranded
vector DNA.
15 Now, gene
expression from the integrated
16 forms, which again is
small, and the episomal
17 forms, parallels the
proportion of vector DNA in
18 each state, so if you
do a partial hepatectomy and
19 you look at the amount
of vector genomes before and
20 after, you get around
90 to 95 percent reduction
21 both in gene
expression and in number of genomes,
22 again indicating that
most of the expression comes
23 from the episomal
forms.
24 There is no
detectable increase in the
25 proportion of
integrated genomes over time, and
101
1 very recently we have
tried to push these animals,
2 giving them extremely
high doses in the range of
3 1014 to 1015 per kilo,
and we do not increase the
4 proportion of
integrated genomes.
5 The
proportion of transduced cells with
6 integrated genomes is
small and most integrates
7 that when we have
actually molelecularly analyzed
8 them are 1 or 2 copy
genomes.
9 [Slide.
10 The clinical
trial objective is to test
11 the hypothesis that
AAV mediated liver-directed
12 gene transfer is safe;
characterize the human
13 immune response to the
transgene product and to the
14 vector; determine whether
germline transmission of
15 vector occurs
following hepatic administration; and
16 determine dose capable
of producing clinically
17 relevant factor IX
levels in the blood.
18 [Slide.
19 It's a Phase
I open-label, dose escalation
20 safety trial of AAV
Human Factor IX administration
21 by infusion into the
hepatic artery.
22 [Slide.
23 The vector
is infused into the liver via a
24 balloon occlusion
catheter placed in the hepatic
25 artery, and Factor IX
protein is administered
102
1 before and follow the
procedure to cover the
2 patients from any type
of bleeding.
3 Subjects are
observed for at least 24
4 hours
5 [Slide.
6 This is the
dose escalation plan of the
7 trial as it is
written. The dose in vector genomes
8 is 2 x 1011 per
kilogram. The observed levels in
9 mice is somewhere
between undetectable and 1
10 percent.
11 Importantly,
is that when you get into the
12 second cohort, we were
at a dose of 1 x 1012 per
13 kilo, and in dogs that
were given a similar, not
14 identical vector,
levels in the range of 4 to 12
15 percent are achieved.
16 These levels
of Factor IX would result in
17 a substantial
improvement in the clinical course
18 with the individuals
going from a severe phenotype
19 to that of a much
milder phenotype. So this would
20 be somewhere in an
efficacious range, so the point
21 is that at doses
within this trial, we are at
22 efficacious doses in a
dog model of hemophilia.
23 [Pause.]
24 DR.
KAY: I am really sorry. There was a
25 mix-up about
transferring the slides, so I
103
1 apologize.
2 This was
just an introductory slide about
3 hemophilia, basically
that it is a very well
4 understood disease and
with sustained levels of 1
5 percent, you can get a
therapeutic response, and we
6 do have very good
animal models which are the dogs.
7 Now, this is
basically what I said, that
8 we have actually been
able, we and others and more
9 recently Kathy High's
group, has gotten reasonably
10 high and therapeutic
levels of canine factor IX in
11 dogs reaching 4 to 12
percent. I won't go through
12 this again
13 [Slide.
14 This is just
a photograph of a patient who
15 is being treated
here. As I said, it is through
16 the hepatic artery and
they go into the invasive
17 radiology suite. A
catheter is inserted into the
18 femoral artery and it
is cannulated into the
19 hepatic artery, which
can be followed by
20 fluoroscopy here, and
then the vector is placed on
21 an infusion pump, as
shown here, and then
22 administered at a
specific rate into the patient
23 [Slide.
24 Now, the
first subject that was treated is
25 a 63-year-old male
with severe factor IX
104
1 deficiency. Status/post bilateral knee
2 replacements 5 years
prior to the procedure. He is
3 HIV-negative. He was HCV-positive, but his HCV
4 viral load by PCR was
negative on multiple
5 occasions several
years apart. Per our protocol,
6 these patients are
considered to have spontaneously
7 cleared HCV, and do
not require liver evaluation
8 before being enrolled
into the trial.
9 He is the
father of 3 and he has a
10 grandson with
hemophilia.
11 [Slide.
12 The first
procedure was done in August of
13 last year. He received 2 x 1011 vector genomes per
14 kilogram. No complications. He was discharged
15 home to his referring
hemophilia treatment center
16 after five days
17 [Slide.
18 This is a
summary of his clinical data
19 baseline before the
procedure and afterwards out to
20 week 24. The important point here is that his CBCs
21 have all been within
normal limits including
22 platelet counts, which
have been an issue with some
23 of the adenovirus
trials
24 [Slide.
25 His liver
function studies and prothrombin
105
1 times have also
remained normal, as shown here.
2 His ALT and AST are
normal, and they remained
3 normal throughout the
24-week period for which he
4 has bee monitored.
5 So the
hepatic administration of this
6 vector in this patient
did not appear to have any
7 liver injury
8 [Slide.
9 The
coagulation data for this first
10 patient is shown here.
His factor IX levels have
11 basically remained at
a subtherapeutic or
12 nontherapeutic
level. This basically is
13 background. Remember that these patients do treat
14 themselves.
15 The
important issue here, too, is that
16 this patient did not
have detectable factor IX
17 inhibitor by Bethesda
assay.
18 [Slide.
19 One of the aspects
of the protocol is to
20 monitor the different
body fluids for vector
21 shedding and, of
course, the reason why we are here
22 today. This just is a very simplified diagram of
23 the PCR assay that is
done by Deb Leonards' group
24 at the University of
Pennsylvania
25 [Slide.
106
1 This shows
the actual sequence of the
2 vector and the PCR
primers are depicted here as a
3 control for the PCR
reaction itself. Some of the
4 samples are spiked
with very small plasmid numbers
5 of a second vector
that has the same sequences for
6 the primers, but there
has been a deletion of 97
7 base pairs, so one can
distinguish between the
8 spiked copy, if you
will, and the vector copy
9 [Slide.
10 This just
shows an example of one of the
11 gels of this analysis
here. This is the baseline
12 sample here. This is the spiked sample below, and
13 this is day seven of a
body fluid where you can see
14 both the spiked and
the actual vector band shown
15 here. So this gives you an idea of the PCR
16 studies. Some of these will be discussed again in
17 more detail with some
of the preclinical studies.
18 If we look
at the vector sequences by PCR,
19 in the different body
fluids here, in the first
20 patient, again, we see
transient vector DNA up
21 until week 2 in the
serum, transiently for a couple
22 of days in saliva,
there was none in urine and
23 stool, and white blood
cell pellet was done at week
24 12, but that was
negative
25 [Slide.
107
1 This is what was somewhat of a
surprise to
2 us based on dog
studies we had done. In fact, when
3 we did look at his
vector DNA in semen, we did find
4 that there was DNA
present in his semen, but it was
5 transient and it slowly
fell off over a period, and
6 after week 12, has
remained persistently negative.
7 Now, these
samples are performed in
8 triplicate in
1-microgram DNA samples. When we did
9 get positivity in
these first couple of samples, we
10 went to a
fractionation procedure to try to
11 fractionate out the
motile sperm fraction from the
12 seminal fluid sample
and the pellet.
13 Now, in this
motile sperm fraction, we
14 were only able to get
220 nanograms of DNA, so it
15 wasn't the 1
microgram, but this amount of DNA was
16 PCR-negative in this
individual.
17 I also want
to point out the sensitivity
18 of the assay is less
than 1 copy per 30,000 haploid
19 genomes or, in other
words, 1 copy per 30,000
20 sperm.
21 Now, as a
result of this result, we did
22 make some changes in
the consent form related to
23 the issue of informing
the patients about this
24 result, and basically,
what it says the study
25 subjects shall be
adult males who are 18 years of
108
1 age or older.
2 The first
patient treated under this
3 protocol was very
shown by very sensitive
4 techniques to have
vector in his semen for as long
5 as 10 weeks after
treatment. Although the vector
6 was not found in the
sperm fraction, the
7 significance of this
finding is unclear, and all
8 patients are strongly
urged to use barrier birth
9 control devices,
condoms, until the patient is
10 informed that semen
has been clear of vector for at
11 least three months.
12 The
investigators will notify you when it
13 is safe to stop
barrier methods of birth control.
14 The consequences of
gene transfer, the germline
15 cells are unknown, but
could potentially result in
16 serious birth defects
or fetal death or other
17 unanticipated health
consequences, such as cancer,
18 in the offspring due
to the disruption of normal
19 genes by the
transferred DNA. If you are
20 considering having
children in the future, it is
21 recommended that you
bank sperm before beginning
22 the procedure to
ensure a source of sperm that is
23 free of contamination
with the vector.
24 The reason
for storing semen is that it is
25 possible that if the
sperm cells do take up the
109
1 vector during the
procedure, it may or may not
2 result in life-long
changes to the sperm. The
3 investigators will
provide you with information on
4 sperm banking and this
one is for Stanford at
5 Stanford University or
at your home institution.
6 This opportunity will be provided to you at
no
7 additional expense.
8 So the point
here is that we urge the
9 individuals to undergo
a barrier contraception, we
10 talk about the risk in
this first patient, and the
11 fact that we will sperm bank in case they are
12 considering or
uncertain about future childbearing.
13 Now, because
of this issue of finding, at
14 least in the first
patient, transient AAV vector
15 sequences in the
semen, we amended the plan to
16 address this issue of
inadvertent germline
17 transmission, and the
protocol was changed, so that
18 semen collection was
done as a baseline, and then
19 at weeks 1, 8, 12, 16,
or possible more.
20 Now, the
idea was, and the plan is, that
21 beginning at 8 weeks,
the sample is then
22 fractionated and total
semen and motile fractions
23 are analyzed for
vector genomes by PCR. If the
24 8-week motile sperm
fraction is negative, we would
25 be allowed to proceed
to the next dose cohort. All
110
1 subjects to practice
barrier contraception until
2 three consecutive
monthly semen samples are
3 negative.
4 So, although we will test and
fractionate
5 through week 16, the
question is we continue if
6 there haven't been
three successive negative semen
7 samples
8 [Slide.
9 Subject 2
was a 48-year-old male with
10 severe hemophilia
B. He had a bilateral knee
11 replacement in 1999
and elbow replacement in 2001.
12 He is
HIV-positive and HCV-positive. He
13 underwent a liver
biopsy and was shown to have
14 minimal fibrosis and
based on criteria in the
15 protocol, was allowed
to be included in the study.
16 He had a
non-Hodgkin's large cell lymphoma
17 in 1986, was treated,
had a relapse in 1996, and
18 was treated and he is
on medications for his HIV
19 [Slide.
20 The
procedure was performed in January,
21 the end of January of
this year, received the same
22 dose as the first
patient. No complications. Went
23 back after 7 days
24 [Slide.
25 Patient 2, like Patient 1, had totally
111
1 normal LFTs, no
elevations related to the vector
2 [Slide.
3 Renal
function, not shown with the first
4 patient, but were also normal in the second
patient
5 [Slide.
6 Again, the
CBC including the platelet
7 counts were
normal. There was no elevation with
8 vector administration
9 [Slide.
10 Now, with the second patient, again,
we
11 see no evidence of
inhibitors, and we have also
12 noticed that there is
a question of whether there
13 is any detectable
factor IX in this patient. The
14 week 8 and week 12
samples were obtained at least
15 14 days prior to
factor IX administration, and
16 there are some low
levels of factor IX here
17 detectable, but again
it is unclear whether this is
18 really and truly from
gene transfer. I just wanted
19 to point out that this
is the data to date. So it
20 is still questionable
21 [Slide.
22 Now, when we
looked at his body fluids,
23 the saliva was
positive for a slightly longer
24 period of time, up to
one week. His serum was also
25 positive up to four
weeks, which again was two
112
1 weeks longer than the
first patient.
2 Unlike the
first patient, we did see
3 transient positivity
in the urine, but only out
4 until day 2, and he
also has had some positive
5 stool samples, as well
6 [Slide.
7 Now, this is
where we are with the semen
8 analysis for the
vector DNA. He has remained
9 positive up through week 14, but let me talk
about
10 the total semen first.
11 The total
semen, the signal of the PCR has
12 started to diminish,
similarly to what we have seen
13 in Patient 1. If you remember Patient 1, he was
14 persistently negative
after week 12, and the week
15 14 sample, which we
just obtained this week,
16 although it was
positive, the signal appears to be
17 weak, so it appears to
be going down in
18 concentration,
although this is not an absolutely
19 quantitative assay.
20 Now
according to the protocol, we were
21 supposed to
fractionate his week 8 sample into the
22 fractions that I
discussed earlier, to look at the
23 motile sperm fraction,
but it turns out that this
24 individual has
ejaculate volumes that are well
25 below half a ml. When the sample went to the lab,
113
1 it has got to be
fractionated within about 30
2 minutes or so, and when they got the sample,
the
3 lab said, you know,
based on our SOP that we have,
4 and the one that is
provided in the protocol, this
5 volume is not adequate
to fractionate, so it wasn't
6 fractionated.
7 Well, we went back, and after
discussions
8 with FDA and our
colleagues, we realized that there
9 are standard operating
procedures in these clinical
10 laboratories to
fractionate low-volume ejaculates,
11 and this then was
attempted on the week 14 sample.
12 But
unfortunately, the DNA recovery from
13 this week 14 sample
was such that it would only be
14 possible to run
triplicate samples of 300 nanograms
15 per ml, and based on
our changes in the protocol,
16 which we have just
sent to the FDA, this would be a
17 fractionated sample
that we would not analyze. So
18 the fractionated
sample with 300 nanograms in it
19 was not analyzed by
PCR.
20 It has
turned out that although it is
21 simple in theory, it
has been difficult, a little
22 more difficult than we
had anticipated doing these
23 fractionation
procedures and getting the kinds of
24 DNA recoveries that
one would want.
25 This
individual has supernormal sperm
114
1 counts so although his
volume is low, it appears
2 that spermatogenesis
in this individual appears to
3 be normal because his
counts are well above normal.
4 It also
turns out that there are lots of
5 rules and regulations
in the labs that do the
6 fractionation. In fact, we are learning that many
7 of these labs are not
allowed to fractionate
8 HIV-positive samples,
which has also led to some of
9 the difficulty in
getting these specimens
10 fractionated at will.
11 So based on
this, we have added new
12 exclusion
criteria. We realize that this
13 individual has an
issue with ejaculate volume, but
14 with normal sperm
counts, that is very, very rare
15 and unusual, but
because of this in this patient,
16 we have added an
additional exclusion criteria to a
17 revised protocol.
18 First of
all, we state in there that an
19 exclusion issue are
related to patients who are
20 unwilling to provide
required semen samples, and
21 patients that are
unable to provide semen samples
22 of adequate semen
volume, which we define at 1 1/4
23 ml sperm count, and we
define the cutoff at 20 x
24 106 sperm per ml, and
with motility of greater than
25 50 percent. Again, this was based on the data we
115
1 have obtained from
this Patient No. 2.
2 [Slide.
3 So, in
conclusion, I can say that Subjects
4 1 and 2 have tolerated
the procedure well, vector
5 DNA is present
transiently and total semen from
6 Subject 1, not present
in the motile sperm fraction
7 at week 3, albeit the
sample that was analyzed was
8 220 nanograms, not the
desired 1 microgram.
9 We have much
limited data in Subject 2
10 although the signal is
going down, we still haven't
11 detected a sample that
has been negative, and
12 currently, based on
what has been approved, that
13 the enrollment of the
subjects at the mid-dose
14 proceeds only if
Subject 2 shows absence of signal
15 in the motile sperm
fraction.
16 So, in
summary, what I would like to say
17 is that clinical
studies demonstrate safety and
18 long-term efficacy of
AAV factor IX in the liver in
19 the large animal model
of hemophilia. We think
20 that this is really
the impetus to move forward.
21 The initial
clinical studies indicate that
22 this gene transfer
strategy can be safety
23 translated into human
subjects, and we strongly
24 believe that the
completion of the Phase I study is
25 required for valid
risk-benefit analysis of the
116
1 strategy.
2 We would
like to present a proposal to you
3 of what we would see
as a reasonable route of
4 moving forward, but
before we do that, there will
5 be two additional
speakers who are going to present
6 the preclinical data
studies that have been done to
7 try to address this
issue, what has been done, the
8 data to date, future
studies in a number of
9 different animal
settings.
10 Thank you.
11 DR.
SALOMON: Thank you very much.
12 We won't
have any questions until after
13 the second speaker.
14 This second
talk is from Linda Couto of
15 Avigen entitled Safety
Studies to Support
16 Intrahepatic Delivery
of AAV.
17 Safety Studies
to Support Intrahepatic Delivery
18
of AAV
19
Linda Couto, Ph.D.
20 DR.
COUTO: I am going to describe a
21 series of preclinical
studies that were performed
22 to evaluate the safety
of delivering AAV to the
23 hepatic artery
24 [Slide.
25 We have used
five different species -
117
1 mice, rats, dogs,
rabbits, and monkeys to assess
2 the toxicology and
biodistribution, but today, I am
3 going to limit my talk
just to the biodistribution
4 studies that are
relevant to inadvertent germline
5 transmission
6 [Slide.
7 I am going
to summarize the studies in
8 rats, dogs, and
monkeys, and then Valder Arruda is
9 going to present some
more recent data in rabbits,
10 which appear to be
probably the best model for
11 studying inadvertent
germline transmission.
12 However,
before discussing the
13 biodistribution data,
I just want to point out that
14 in all of these five
species, we haven't seen any
15 toxicology at doses up
to 1 x 1013 vector genomes
16 per kilogram, which is
50-fold higher than our
17 starting clinical
dose.
18 This is the
biodistribution study that was
19 performed in
rats. In this study there were five
20 groups of
animals. One group was treated with the
21 excipient. One group was treated with an AAV null
22 vector, which does not
contain a transgene. Then,
23 there were three
groups of animals that were
24 injected with
increasing doses of an AAV factor IX
25 vector from 1 x 1011
per kilogram to 1 x 1013 per
118
1 kilogram.
2 So what you
can see is that at 50 days
3 post-injection, we saw
a good gene transfer to the
4 liver, so at the low
dose we were seeing about 1
5 copy per 60 cells in
the liver, and at the high
6 dose we were seeing
about 1 copy per 1 to 2 cells.
7 At this time
point, we also did see vector
8 dissemination to the
gonads at least in some of the
9 animals. At the low
dose we didn't see any
10 dissemination, but at
the high dose we saw about 1
11 copy per 1,700 cells,
so this was about 1,000-fold
12 lower than the gene
transfer we were seeing in the
13 liver.
14 At this time
point, we were also seeing
15 vector in the blood,
however, by day 92
16 post-injection, we no
longer detected any sequences
17 in the blood, and the
level of gene transfer to the
18 liver and the gonads
had decreased.
19 So, at the
92-day time point, we were
20 seeing about 1 vector
copy per 4 cells in the
21 liver, and only about
1 copy per 4,000 cells in the
22 gonads, but only in
the highest dosed animals.
23 [Slide.
24 We also did
a gonadal distribution study
25 in dogs. In this
study, three normal dogs were
119
1 injected with AAV null
vector at doses ranging from
2 3.7 to 7 x 1012
vectors genomes per kilogram, and
3 in this study, the
vector was delivered using the
4 method that we are
using in the clinic. So, a
5 catheter was inserted
into the femoral artery and
6 then using
fluoroscopic guidance was advanced to
7 the hepatic artery
where the vector was infused.
8 Then, semen samples
were collected at various times
9 post-injection.
10 In addition
to the semen samples, we also
11 looked at toxicology
parameters and also looked at
12 gonadal tissue at the
time of sacrifice.
13 In this
experiment, we used the AAV null
14 vector, which contains
a promotor list transgene.
15 The reason for using
this was just to prevent any
16 CTL response,
eliminating the transduced cells.
17 [Slide.
18 So, these
are the results of PCR analysis
19 of the dog semen. The lower panel here represents
20 an ethidium bromide
stain gel of the PCR products,
21 and over here on the
right you can see that the
22 level of sensitivity
is about 100 copies per
23 microgram. At this level of sensitivity, there is
24 no evidence of vector
sequences in any of the dogs
25 at any of the time
points out to day 90.
120
1 We also did
a southern blot of this gel,
2 and increased the
sensitivity down to 10 copies per
3 microgram, which is 1 copy
per 30,000 haploid
4 genomes, and again we
are not seeing any detection
5 of sequences in the
semen of these dogs.
6 We also
performed PCR on gonadal tissue
7 and again we didn't
see any evidence of
8 dissemination to the
gonads in these animals.
9 [Slide.
10 More
recently we have looked at toxicology
11 and biodistribution in
the non-human primates, and
12 in this study we have
treated 6 cynomolgus monkeys,
13 2 animals were treated
with the excipient, 2
14 animals got a factor
IX vector at a dose of 7 x
15 1012 into the hepatic
artery, and another 2 animals
16 received the same dose
of vector via the portal
17 vein.
18 This study
was designed as a toxicology
19 study, but we tried to
get some limited
20 biodistribution study
by harvesting the liver and
21 the gonads and doing
PCR analysis when the animals
22 were sacrificed at day
135
23 [Slide.
24 This is the
results of that study. What
25 you can is that in 2
of the 4 injected animals, we
121
1 saw gene transfer to
the liver. It is not really
2 clear why only 2 of
the 4 animals worked, but what
3 we can say is that in
those 2 animals, gene
4 transfer was
relatively efficient, so 1 of the
5 animals that got the
vector via hepatic artery, we
6 saw vector genomes at
about 1 vector sequence per 3
7 cells, and in the
other animal we saw 1 to 2 vector
8 sequences per cell.
9 What we also
saw was, you know, despite
10 this high level of
gene transfer to the liver, we
11 did not detect any
sequences in the gonads, and the
12 level of sensitivity
in this particular PCR assay
13 was 1 copy per 40,000
diploid cells.
14 [Slide.
15 We also took
advantage of this non-human
16 primate testes to ask
the question whether any of
17 the cells in the testes
had the receptor for AAV,
18 which Jude Samulski's
group had previously reported
19 to be heparan sulfate
proteoglycan.
20 So what we
are looking at here is a
21 stained section of the
non-human private testes,
22 and the heparan
sulfate proteoglycan is stained and
23 nuclei are stained
blue with DAPI. What you can
24 clearly see is that
the receptor, heparan sulfate
25 proteoglycan is
present in the basement membranes
122
1 surrounding the
seminiferous tubules, but none of
2 the spermatogenic
cells are staining positive to
3 HSPG, suggesting that
these cells would be
4 non-permissive for AAV
infection.
5 What I have
just shown you has
6 demonstrated that at
least in some animal species,
7 we do see
dissemination of AAV vector to gonads,
8 and although we didn't
see dissemination of vector
9 to the semen in dogs,
Valder Arruda will show some
10 data demonstrating
that we do get vector
11 dissemination to semen
of rabbits, and Mark Kay
12 also just presented
our data from the clinical
13 trial demonstrating
that we are seeing vector
14 dissemination in human
patients.
15 So there certainly is the risk for
both
16 horizontal and
vertical germline transmission.
17 What I would like to
present now are some studies
18 that we have been
working on and also some
19 published work that
addresses the risk of AAV
20 dissemination in both
horizontal and vertical
21 transmission.
22 The first
study is a paper from Philip
23 Moray's [ph] lab
looking at vector shedding in a
24 number of biological
fluids, and then I will
25 present the
development of a cell-based infectivity
123
1 assay, so that we can
now begin to look at
2 biological activity of
AAV in semen samples.
3 Then, I will
also address the issue of
4 vertical transmission
by describing an experiment
5 that we have initiated
in collaboration with Dr.
6 Jon Gordon to see
whether AAV can infect murine
7 sperm cells
8 [Slide.
9 So, the
study that was published in the
10 Journal of Molecular
Therapy last December from
11 Philip Moray's group
is shown on this slide. They
12 injected 8 monkeys
with an AAV-Epo vector at doses
13 ranging from 5 x 108
to 1 x 1010 infectious units
14 per kilogram, and
their vector had a particle
15 infectivity ratio of
about 100.
16 This vector
was inject intramuscularly and
17 then at various time
points post-injection, a
18 number of body fluids,
such as serum, feces, urine,
19 saliva, lacrimal and
nasal, but not semen, were
20 evaluated both by PCR
for vector sequences and
21 using a replication
center assay to look for
22 biologically active
AAV.
23 In addition,
they looked in the peripheral
24 blood mononuclear
cells for vector sequences
25 [Slide.
124
1 This is a
figure from their paper, which
2 shows the results of
the replication center assay.
3 In this assay, cells
are coinfected with AAV and
4 the helper virus for
AAD, adenovirus. Following
5 incubation for several
days, the cells are
6 harvested and filtered
onto a nylon membrane and
7 then harbodized to a
radioactive probe.
8 So what we
are looking here is the ability
9 of AAV in the presence
of its helper virus, to both
10 infect and replicate
in this cell.
11 The panel on
the lefthand slide shows the
12 controls. This is AAV that has been spiked just
13 into media, and you
can detect 1,000 down to 1
14 infectious unit. However, when the AAV is spiked
15 into either serum,
feces, urine, the level of
16 sensitivity in the
assay decreased about 10- to
17 100-fold.
18 On the
righthand portion of the slide is
19 the results of testing
the serum from two of the
20 monkeys, and you can
see that 30 minutes
21 post-injection there
is evidence of biologically
22 active AAV in the
serum, and you can also detect
23 some activity one day
and two days post-injection,
24 but by five days
post-injection, there is no longer
25 any biological
activity in the serum
125
1 [Slide.
2 This slide
just summarizes the results
3 from all 8
monkeys. The red bars indicate a
4 30-minute time
point. The yellow bars represent a
5 one-day time point,
and the blue bars represent the
6 two-day time point.
7 Basically,
you can see that in all of the
8 animals, by three to
four days post-injection,
9 there is no longer any
biologically active AAV in
10 the serum. They also tested other body fluids, but
11 they only found
activity in the serum.
12 [Slide.
13 Finally,
they also looked for AAV
14 sequences in
peripheral blood mononuclear cells,
15 and surprisingly, they
were able to detect this
16 signal out to 10 to 15
months post-injection. So
17 vector sequences can
be persistently detected in
18 the peripheral blood
mononuclear cells
19 [Slide.
20 So, just to
summarize their data, AAV
21 vector sequences are detected
in all body fluids by
22 PCR for approximately
6 days. I didn't show you
23 this, but they did
also demonstrate that the PCR
24 signal is due to
packaged AAV sequences rather than
25 free DNA.
126
1 They also
demonstrated that biologically
2 active AAV was
detected in serum for 48 to 72
3 hours, suggesting that
the risk of horizontal
4 transmission is
limited to a short period of time
5 post-injection.
6 Finally,
they also concluded that vector
7 sequences can be
detected in the PBMCs for as long
8 as 10 to 15 months
following an intramuscular
9 administration
10 [Slide.
11 After
discussions with the FDA and also
12 following the December
RAC meeting, it became clear
13 that it was important
to develop an assay, so that
14 we could detect or try
to detect biologically
15 active AAV in semen
samples, so this just
16 schematically
illustrates the assay that we have
17 been developing.
18 Basically,
it is similar to the
19 replication center
assay that I just described,
20 however, the readout
of replication in this case
21 relies on a
quantitative PCR assay rather than a
22 hybridization.
23 So basically
HeLa cells which express the
24 AAR Rep and Cap genes
are incubated in the presence
25 of 100 microliters of
semen with increasing doses
127
1 of an AAV factor IX
vector and with adenovirus.
2 Then, 72
post-injection the cells are
3 harvested, DNA
extracted, and subjected to
4 quantitative PCR
5 [Slide.
6 This just
depicts a typical result from
7 this assay where the
intensity of the red color is
8 meant to represent the
amount of PCR amplification
9 detected in the
well. So in the case of just
10 spiking vector into media,
you can see that we can
11 detect as few as 10 to
50 vector genomes per well,
12 however, when the AAV
is spiked into semen, the
13 level of sensitivity
of the assay decreases about
14 10-fold, so that now
the lowest dose that results
15 in an amplification
signal is 500 vector genomes
16 per well per 100
microliters or 5,000 vector
17 genomes per ml of
semen.
18 So what we
intend to do with patient and
19 animal semen is two
different assays. First of
20 all, we will simply
extract DNA from the sample and
21 do quantitative PCR to
determine the number of
22 vector genome per ml,
and in addition, we will take
23 a portion of the
sample, run the infectivity assay
24 to determine the infectious
units per ml of semen,
25 and then we will be
able to monitor and compare the
128
1 kinetics of clearance
of both the physical and the
2 infectious particles.
3 [Slide.
4 To address
the issue of vertical
5 transmission, as I
mentioned, we have initiated a
6 collaboration with Dr.
Gordon. In this case, what
7 we propose to do is
expose murine sperm cells to
8 very high doses of
AAV.
9 We feel this
is a very rigorous test of
10 whether AAV can
actually transduce sperm although a
11 very non-natural
situation, but this slide just
12 illustrates the steps
that are being taken.
13 First of
all, murine sperm are isolated
14 and then exposed to an
AAV factor IX vector at an
15 MOI of about 1,000.
The sperm are used in an in
16 vitro fertilization,
and then the fertilized
17 oocytes are implanted
into pseudopregnant females.
18 The fetuses
will be harvested 10 to 12
19 days post-gestation,
DNA will be extracted and
20 subjected to southern
blot analysis, and what we
21 will be looking for is
single copy AAV factor IX
22 sequences in genomic
DNA, and if we are able to
23 detect one copy per
diploid genome, that will be
24 used as evidence of
vertical germline transmission
25 [Slide.
129
1 Just to summarize
what I have just been
2 discussing, first of
all, the extent of vector
3 dissemination to
animal tissues correlates with
4 dose and decreases
with time.
5 Following
intrahepatic delivery of AAV,
6 vector is either absent
from gonadal tissue, which
7 was the case in the
dogs and the non-human
8 primates, or present
at levels 1,000-fold lower
9 than liver, as in the
case with the rats, and in
10 this case it clears
with time.
11 The studies
in the non-human primate
12 suggest that AAV in
serum is not infectious after
13 72 hours, but vector
signal can be detected in
14 PMBCs for up to 10
months after an intramuscular
15 administration.
16 The AAV
receptor, HSPG, is not expressed
17 on non-human primate
spermatogonial cells,
18 suggesting that these
cells may not be infected by
19 AAV.
20 Finally, we
believe that the data is
21 consistent with
hematogenous dissemination of
22 vector to gonads with clearance over time.
23 [Slide.
24 So the
issues that we are continuing to
25 address are, first of
all, is there infectious
130
1 virus in semen, and as
I mentioned, we have
2 developed an
infectivity assay which we intend to
3 use both on humans and
animal semen samples.
4 Another
question is are the vector
5 sequences in semen
associated with the motile
6 sperm, other cells, or
the seminal fluid, and as
7 Mark Kay mentioned, we
have begun fractionating the
8 human semen samples,
and we have also begun doing
9 this with rabbit
samples, as you will hear in the
10 next presentation.
11 Another
question that we are trying to
12 answer is can AAV
infect mature sperm cells, and we
13 have initiated a study
using IVF to demonstrate or
14 not demonstrate this
in animal models.
15 I will stop
there.
16 DR.
SALOMON: Thank you very much, Linda.
17 I was
thinking of doing the next talk and
18 then discussing all
three of the talks as a group.
19 Would that be okay
with everybody? I got the
20 feeling Avigen was kind
of packaging this as a
21 group.
22 Assessing the
Risk of Germline Transmission of
23 AAV
in a Rabbit Model
24
Valder Arruda, M.D.
25 DR.
ARRUDA: I would like to talk now
131
1 about preclinical
studies, address the issue of
2 biodistribution
following injection of an AAV
3 vector into rabbits as
a model to analyze the
4 inadvertent germline
transmission
5 [Slide.
6 Animals in
these studies were injected
7 with the same vector
to be used in clinical trial.
8 The doses ranged from
1 x 1011 to 1 x 1013 vg/kg.
9 Semen is
collected at serial time points
10 after injections, we
intend to fractionate the
11 semen, analyze the
total semen and fractions by PCR
12 developed by the human
specimens, as Dr. Kay said
13 before.
14 [Slide.
15 Although
when we talking collection of
16 semen for rabbits, I
like just to mention that the
17 method we are using is
the natural method, using an
18 artificial vagina that
has an advantage, it
19 provides an
uncontaminated sample for each animal.
20 However, this method has a
disadvantage.
21 The animal requires to
be trained to do this
22 procedure, and this
has some implication, as you
23 will hear later on
during my talk.
24 [Slide.
25 When we talk
about semen, we talk actually
132
1 a marker, what happens
in both genital and urinary
2 tract. Actually, 70 percent of what we call
3 ejaculate comes from
the seminal vesicle, 20
4 percent from the
prostate gland, and only 5 percent
5 from tests and ducts,
and a small portion from
6 accessory glands.
7 Also,
although spermatozoa is the main
8 cellular component of
semen, there are other cells
9 that is special for our case is really
important to
10 know, and these cells
are present normal in fertile
11 donors like
leucocytes, epithelial cells, immature
12 germline cells, and
enucleated cytoplasm, and this
13 can be around the
cells.
14 Also, for
rabbits, are commonly found
15 debris in gel. Gel especially comes from ejaculate
16 of young animals.
Together, this explains the
17 reasons why we would
like to fractionate the total
18 semen before we save
it in aliquot to analyze the
19 total semen, we go for
fractionation to obtain the
20 motile sperm and
seminal fluid and the normal type
21 [Slide.
22 What we have
up to now is actually 3
23 cohort of animals that
has been injected in total
24 27 rabbits.
25 The first
cohort consists of 12 animals.
133
1 They were 5 months old
at the time of injection.
2 Although sexually
mature, these animals were not
3 experienced in semen
collection, so we are
4 restricted to analyze
only later time points.
5 It was
necessary to go back and look for
6 experienced
animals. At this point, we could get 3
7 animals. They were 18
months old and semen was
8 collected weekly.
9 More
recently, we have a group of 12
10 rabbits, median age
are around 20 months, and these
11 we obtained from
retired breeders.
12 What I am
going to start to show to you is
13 the result of the 10
in the second cohort followed
14 by the third cohort,
and only the later time point
15 for the very first
group we inject.
16 [Slide.
17 For all
these animals, serum sample was
18 collected 24 hours
injections for the 8 and up to 7
19 days. For all of them, we have augmented vector
20 sequences by the PCR.
21 Typically,
each sample that has been
22 analyzed for each
animal are represented here. We
23 run assay in
triplicate with just semen and one
24 spiked experiment to
exclude PCR inhibition.
25 As you can
see here, this is the first
134
1 experienced rabbits
that we inject. At the low
2 dose, no signal was
detected, in the triplicate
3 experiment, one single
band out of triplicate in
4 the mid-dose cohort,
and the higher dosed animal,
5 three out of
three. This higher dose, although it
6 is not a qualitative
assay, is close to 10 cups of
7 vector plasmid.
8 [Slide.
9 So this
table shows the serial time points
10 from the three
experiments, rabbits ranged for 7
11 days following
injection up to 115 days. Each
12 assay, as you can see
here, was run in triplicate
13 in the yellow line,
the semen that was detected as
14 a positive
signal. For the lower dose animal, we
15 never detected any
signal during this period. For
16 the mid-dose animal,
signal has been detected up to
17 day 22, for the higher
dose, up to day 44
18 [Slide.
19 We attempt
to fractionate the rabbit semen
20 and the optimal
fractionation actually depend on
21 the size and shape of
the sperm, as well as the pH
22 of the semen. At the very first time point, we use
23 parameters worked out
for human semen, and actually
24 reagents for human
semen, and when you look under
25 the microscope, we saw
a lot of agglutinations,
135
1 cell debris. You can see that even after fall in
2 fractionation, you
concentrate fraction of motile
3 sperm, but it still
has a lot of debris.
4 [Slide.
5 When one
compares germ cells for human and
6 rabbits, they are
different, so the volume of
7 ejaculate is smaller
in rabbits, and we anticipate
8 that this would be a
problem for fractionation as
9 for humans, although
the density of the sperm in
10 rabbits is higher, the
characteristics of this
11 sperm is
different. They are pretty much the
same
12 total length, but the
distribution is different.
13 [Slide.
14 So, we
talked with people at this company,
15 Nidacon, and they
actually in-house some reagents
16 to use for
rabbits. We didn't use if that was
17 really helpful or not,
so we just took a chance and
18 we used the reagents
that have been developed for
19 rabbits. Not only the grade had changed, but also
20 the centrifugation
conditions changed.
21 After that,
we improved the fractionation,
22 but occasionally, we
still detect 1 or 2 percent of
23 cells other than
motile sperm.
24 [Slide.
25 So these
results are from the first three
136
1 experienced
animals. The top animal is what I
2 showed before. So these are the points that we are
3 able to fractionate
the semen in these animals.
4 As you can
see, the motile sperm analysis
5 shows a positive
signal in the mid-dose animal.
6 The high-dose animal,
at this point, the volume was
7 not enough to allow
fractionation. It was just 200
8 microliters. So, we saved it only for the total
9 semen analysis.
10 After day 7,
the second time point was day
11 22, and all the
animals turns out to be negative,
12 and up to here, we use
the human protocol, and
13 after this, the rabbit
protocol, but after that, as
14 you can see, no signal
has been identified by the
15 same PCR reaction.
16 For the
normal type sperm, seminal fluids,
17 again, we have seen
signal positive for the
18 mid-dose group and
from the higher dose group, and
19 again, for low-dose
animal, we have never been able
20 to detect
21 [Slide.
22 Now, I will
show the third cohort. these
23 are 12 rabbits,
experience rabbits, and we have
24 only two time
points. It is important here that we
25 have time point 7 - 15
days, 15 days we didn't have
137
1 for the very first
group, we just skipped to day
2 22nd.
3 This is the
total semen. You can see that
4 three animals on the
low dose cohort was positive
5 at 7 days, but became
negative at 15 days. For the
6 mid-dose and the
high-dose, these animals are still
7 positive although
decrease in numbers at the normal
8 type sperm fraction,
also we can see that the
9 higher the dose, the
higher the number of animals
10 positive up to this
early time point.
11 The motile
sperm analysis, we have not
12 observed any positive
signal for the lower dose
13 animal, a positive
signal for the mid-dose and
14 high-dose, and again I
would say that at this
15 point, the positive
didn't change much from 7 to 15
16 days. We still collect today, actually, the day
17 21.
18 The last
group, these are the first cohort
19 that we inject that we
inexperienced at that time,
20 so it took us like a
couple weeks to train these
21 animals and now they
are able to provide the
22 specimen.
23 So we have
here, we collect semen for
24 groups that were
injected a week apart. That is
25 why we have this range
of days, from the low, mid,
138
1 and high dose, they
are persistent negatively until
2 day 132.
3 It is
interesting as Dr. Couto showed
4 before, in non-human
primates, one can detect
5 peripheral blood
mononuclear blood cells positive
6 at late time points
following AAV injection. Here,
7 we also have been able
to detect that these
8 animals, they present
positive signal in their
9 peripheral white blood
cells, and the top panel
10 shows, at the same
time, which corresponds to three
11 months following
injection, the total semen are
12 negative.
13 I am not
going to go into detail into the
14 rabbit experiment,
this is just to represent a
15 schematic, a very
simplistic overview to say that
16 these are numbers of
days that get usually a
17 spermatogen cycle in
rabbits takes up to 42 days.
18 Initially,
the stem cell, it is outside
19 the protected area, so
outside the blood-testis
20 barrier. After day 16, cross the blood barrier,
21 came to spermatocytes,
and takes up to 10 days from
22 the mature cells,
spermatozoa, to get to the semen.
23 So, you
assume that the stem cell has been
24 exposed to a vector at
day zero. The first time
25 point that one animal
should show up a positive
139
1 signal in the semen
started at day 52, maybe with a
2 peak at day 58, and
after that, you have the
3 steady-state signal.
4 If you put
back the three cohorts of
5 animals we inject so
far, we can tell the
6 following. We detect PCR-positive signal in total
7 semen or some
fractions, 7, 15, 20, and 44 days.
8 The PCR becomes
negative, the old sample tests
9 after day 50. This is for the first three cohorts
10 of animals, and this
is for the very first 12
11 animals that we start
collecting at day 86 up to
12 day 1 to 132.
13 If we
consider that the rabbits
14 spermatogenesis single
sites 44, 42, 46 days, at
15 this time point that
we analyze, we will be able to
16 analyze at least two
to three sites of the total
17 rabbit
spermatogenesis.
18 Although it
doesn't look like we are
19 transducing any
immature or stem cell at this point
20 following only two or
three sites of
21 spermatogenesis, there
has been talk before here
22 was that possibility
that actually the vector cross
23 the basal compartment,
cross the blood-testis
24 barrier, and gets into
more mature cells at this
25 point.
140
1 I even should
skip this, but I will try to
2 do what has been said
before that may not work, and
3 didn't work, that is
was put multi-sperm of these
4 rabbits in
culture. This is just to show -- I hope
5 you can appreciate
these are spermatozoas, and
6 these we still found
some cells into the motile
7 sperm fraction, and
unfortunately, by this computer
8 thing, we cannot make
the picture come out.
9 What we did,
we exposed these cells to
10 AAV2 under a CMV control
expressing a GFP. The MOI
11 used a range from 1 to
5,000, and the committee has
12 a cut that we provide,
shows that only that cell
13 that I identify here,
it looks like a bean or
14 something like that,
actually turns out to be
15 positive for GFP. Any other, the motile cells were
16 positive.
17 So,
initially, for the muscle trial, we
18 performed pretty much
a similar series of
19 experiments, used the
same model, the rabbit, and I
20 would just like to
summarize this. This actually
21 has been published in
2001, and what we are able to
22 identify following
intramuscular injection of AAV2
23 into rabbits was the
following.
24 We performed
a series of IF staining and
25 FISH analysis shows
that we can detect signal from
141
1 the vector, and this
is localized in the vessel
2 wall and the
testicular basement membrane, which
3 are rich structures
for heparan sulfate
4 proteoglycan, which
there is no receptor for AAV
5 serotype 2.
6 The
detectable signal especially in the
7 gonads disappears with
time. It is important to
8 remember that in this
cohort of rabbits, no semen
9 signal was ever
identified, and also into the
10 gonads, we neither
detected any intracellular
11 signal when you
analyze animals following 7, 36,
12 and 90 days.
13 This is just
to represent what we believe
14 that the signal, this
is IF staining, what you call
15 localization for the
AAV capsid and for heparan
16 sulfate proteoglycan
on the vessel wall in the
17 testis basement
membrane.
18 So, in the
last experiment that I would
19 like to show is
attempt to transduce not mature
20 spermatozoa, but
immature spermatozoa. In this we
21 have murine cells in
culture in which murine
22 spermatogonia and
Sertoli cells were co-cultured.
23 We
transduced again with AAV2 under
24 control of the CMV
promotor expressing lacZ at the
25 MOI 5000, and we stain
for x-gal. Here, it showed
142
1 the signal. Just before, I should say, that we
2 identified the
spermatogonia by immunostaining with
3 a monoclonal
antibodies to germ cell nuclei
4 antigen.
5 This is the
result. At the bottom is the
6 mouse spermatogonia
and Sertoli cells that give
7 this kind of reddish
signal. In contrast, if you
8 take the same
transduced model, the fibroblasts or
9 human [inaudible] it
turns blue.
10 I would like
to conclude that intravenous
11 administration of the
dose of AAV up to 1 x 1013 in
12 rabbits results in
transient detectable signal in
13 semen in a
dose-dependent manner.
14 PRC
positivity of the semen persists up to
15 day 44 in that cohort,
that we have follow-up for
16 almost 100 days
revealed no positive signal, which
17 is a duration of 2 or
3 times of the rabbit
18 spermatogenesis.
19 Vector
signal can be detected in
20 peripheral mononuclear
blood cells for at least
21 three months in
rabbits in contrast to non-human
22 primates, we know that
this can go up to 10 months,
23 but the vector is not
biologically active after day
24 7.
25 In ongoing
experiments, you can predict
143
1 that we will continue
follow up with kinetic
2 clearance,
determination of anatomic localization
3 of signal as a
function of the vector dose
4 following
intravascular injection.
5 To determine
whether AAV infectivity is
6 detected in rabbit
semen, we are followed by the
7 experiments that Dr.
Couto has before. As well, we
8 would like to
determine whether receptor for AAV2
9 is present in mice,
rabbit, and human spermatozoa.
10 That could give us
some idea.
11 I will stop
here.
12 DR.
SALOMON: I want to make sure we all
13 understand where we
are going. What we are going
14 to do now is have a
discussion of the three talks
15 that came, followed by
people who have been invited
16 to speak in the public
hearing.
17 We had
talked about moving that out of
18 order, but I am told
that is not proper, but to
19 reassure everybody
that we will discuss the
20 questions to the
committee after that, so that
21 everything will be on
the table before we get to
22 the questions.
23 I think
there are a number of interesting
24 issues raised by the
these three presentations, and
25 I would like to put
those open for some discussion.
144
1 Dr. Dym.
2
Q&A
3 DR.
DYM: I had a question for Dr. Couto,
4 please, just a
clarification, and maybe I didn't
5 understand some
things, but when you showed very
6 elegantly that the
receptor for AAV is around the
7 seminiferous tubule
and in the interstitial spaces,
8 not inside the tubule,
but then you didn't show AAV
9 in the gonads.
10 Isn't there
a discrepancy there?
11 Shouldn't it show up
in gonad if the receptor is
12 there, or did I miss
something?
13 DR.
COUTO: That particular section was an
14 animal that was not
even injected with an AAV
15 vector, so we were
just strictly looking at to tell
16 whether the receptor
for AAV is even present in a
17 non-human primate
testis.
18 DR.
DYM: But in your other monkey tissue,
19 didn't you say it is
not detectable in testis?
20 DR.
COUTO: The AAV sequences are not
21 detectable in the
gonadal tissue, correct, by PCR.
22 DR.
DYM: So, wouldn't they be there,
23 because the receptor
is there? I am missing
24 something.
25 DR.
HIGH: Can I clarify that question?
145
1 So, the answer to your
very perceptive question, if
2 you look in the
Molecular Therapy paper, there is
3 actually a FISH
analysis of a rabbit testis from an
4 animal sacrificed at
day 7, after injection with
5 AAV, and in that, you
can see tracking in the same
6 location that Dr.
Couto showed in the non-human
7 primate testis.
8 You can see AAV vector detected by FISH
9 analysis in the same
location along the testicular
10 basement membrane and
actually around the vessel
11 wall, as well. You can detect that at day 7, but
12 by longer time points,
which were presented in
13 several of the studies
that Dr. Couto did, both in
14 rabbits and in other
species, as well, if you look
15 at later time points
like 50 days after or 100 days
16 or 135 days, you don't
see AAV vector any longer in
17 the gonadal tissue.
18 So, your
point is correct, and if you look
19 early on, you can see
that, and that has been
20 published in that
Molecular Therapy study or day 7,
21 but at later time
points you don't see it.
22 DR.
SALOMON: I have a couple questions.
23 Going back to the very
beginning, I posed a
24 question about if
sperm were not transcriptionally
25 active, then, how do
you interpret an experiment
146
1 where you put in a
CMV-GFP vector?
2 DR.
ARRUDA: I don't think that is the
3 ideal experiment. We just want to see if one could
4 transduce motile sperm
in culture. There are some
5 people that say they
can, in fact, transduce some
6 mature spermatozoa
using more aggressive ways. We
7 do not expect anything
else. It wasn't a surprise
8 that the results were
negative, but I think the
9 best way to answer
your question is to perform an
10 experiment that Dr.
Couto is doing with transducer
11 cells in culture, and
then you do in vitro
12 fertilization and see
the outcome.
13 DR.
SALOMON: I certainly don't think that
14 the evidence that you
didn't get GFP expression
15 really addressed
anything.
16 DR.
ARRUDA: I agree.
17 DR.
SALOMON: If it had been positive, I
18 agree it would have
been important, but I don't
19 know what is the point
in showing it as negative.
20 DR.
ARRUDA: I agree with that, and also
21 it has been published
that one can detect lacZ in
22 this spermatozoa.
23 DR.
SALOMON: What I would like to hear
24 some discussion of is
whether the experiment that
25 Dr. Couto presented,
and I guess that is going to
147
1 be done with you, Jon,
is the best study.
2 I have some
concerns about that. It is a
3 very good study. It seems to me that it is really
4 almost going over the
top, which you said yourself,
5 Jon, was what you
should do. So I would like to
6 hear your comment on
that as a point of discussion.
7 It seems to
me you have a reagent,
8 however, that would
also be extremely useful, and
9 that would be to take
your AAV-CMV promoter GFP and
10 put it into the
rabbit, and then actually trace GFP
11 expression in
different compartments particularly
12 in this case, of
course, in the spermatogonia, I
13 mean so you could do
it at different -- I don't
14 need to tell you all
the different variations of
15 that, but that seems
to me to be the most
16 physiologic
experiment.
17 DR. GORDON: I want to make a brief
18 comment on that. It is no surprise that sperm will
19 not express genes put
into them, but that doesn't
20 mean that genes aren't
in there, and couldn't be
21 expressed late, just
to reemphasize the strategy of
22 doing this IVF.
23 The other
comment i would make is if there
24 were AAV-CMV lacZ, I
haven't looked for GFP in
25 embryos, although I am
sure it can be done, but if
148
1 there were lacZ
vectors, where I know I can look,
2 you could look at
thousands of cells in a very
3 short time after
exposing sperm to such a vector,
4 and just simply
stainings on an intact embryo. We
5 did that for adenovirus,
it worked really well, and
6 that would be a very
nice protection against
7 contamination when one
harvests fetuses and the
8 strategy that we are
taking, which is of major
9 concern to us, and
which has been discussed amongst
10 us over the last few
weeks.
11 DR.
COUTO: There is another experiment
12 that we have thought
of doing, and it is based on
13 an experiment that Bob
Braun's lab has done with
14 adenovirus, where they
had an adenovirus that had
15 an expression cassette
that has a protamine
16 promoter hooked up to
a lacZ vector.
17 In that
case, they are doing a natural
18 route of
administration, and then looking to see
19 whether all of the
progenitor spermatogonia,
20 spermatocytes, et
cetera, in an entire seminiferous
21 tubule turn blue over
the course of time. That is
22 another experiment
that would not only look at the
23 mature sperm, but also
the immature sperm.
24 DR.
SALOMON: The idea there is to use the
25 protamine promoter as
kind of a tissue-specific
149
1 promoter, so that is
even cooler, as would say in
2 California.
3 Jon, the question that I had for you was
4 the experiment that
they are talking about where
5 you essentially
culture the sperm with 1,000 MOI
6 and then you go and do
IBF. Isn't that just going
7 to have a bunch of DNA
coating the sperm? Don't we
8 already know the
results of this experiment before
9 they do it?
10 DR.
GORDON: Meaning you think it would be
11 positive. Well, I can just say that we did that
12 with adenovirus. The rationale of this -- and it
13 was not positive --
the rationale for this is that
14 if AAV arrives to
semen, then, it can expose motile
15 sperm, and in the
rabbit, motile sperm seemed to be
16 associated with AAV.
17 So the
question then is can these carry
18 the genomes into the
embryo via the natural
19 fertilization
process. As I pointed out in my
20 talk, that is not easy
to do, and we certainly did
21 not find that to be
the case with adenovirus, even
22 at 100 viruses per
cell, so we would not predict it
23 to be positive, mostly
because of the investments
24 of the sperm are
mostly lost en route through the
25 zona, and so on.
150
1 So we don't expect it to be
positive. If
2 it's positive, we have
to look at why that is true,
3 whether it is really
transduction or whether it is
4 so much AAV in our IBF
prep that we couldn't get
5 rid of it all,
something like that.
6 DR.
SALOMON: So, Jon, I have to ask the
7 stupid question. So why did your DNA experiments
8 work when you coated
the sperm, so why does that
9 work?
10 DR.
GORDON: That works only if you load
11 the sperm into a microneedle, push the
microneedle
12 through the zona, then
through the plasma membrane
13 of the egg, and then
insert the sperm with the DNA
14 around it directly
into the cytoplasm. That works.
15 I say to you that I don't
believe the Cell paper
16 which said that just
mixing it with DNA and doing
17 IBF works, since no
one seems to be able to repeat
18 it including me.
19 DR.
SALOMON: Thank you for that
20 clarification.
21 DR.
KAY: I just want to make a comment
22 that even under the
very worse scenario, where you
23 do get carrying in of
the single-stranded genome
24 into the embryo, at
very low copy number, even one
25 copy, the chances that
that single-stranded genome
151
1 is going to become
double-stranded is very low.
2 DR.
GORDON: Another final comment about
3 the thing I said about
exposing them and then
4 looking for expression
in embryos, that is a
5 problem with the
kinetics of AAV genome activation.
6 It takes a long time,
as I understand it, to
7 actually turn the
genes on, and so the experiment
8 is a little bit less
easy to do with that, as are
9 experiments with
protamine promoters, and so on,
10 with AAV, just because
it takes a long time to turn
11 the genome on.
12 DR.
SALOMON: Dr. Rao.
13 DR.
RAO: This is for Dr. Arruda. There
14 are two things which
weren't absolutely clear to me
15 in your
presentation. When you showed the
16 spermatogonia in
culture and you looked at AAV
17 infection with the
high MOI of infection, there
18 were some
infection. You showed 293's at the same
19 time?
20 DR.
ARRUDA: Yes. We have two cell lines
21 as control. Those are 293 cells with human cell
22 line and the murine
cell line, the fibroblasts,
23 that was positive.
24 DR.
RAO: But the spermatogonia were not?
25 DR.
ARRUDA: The murine spermatogonia was
152
1 not. That experiment was murine spermatogonia.
2 DR.
RAO: And when you see positivity in
3 the rabbit motile
sperm, fractionated sperm, where
4 do you think the virus
is there? I mean you
5 fractionate the rabbit
sperm.
6 DR.
ARRUDA: Yes.
7 DR.
RAO: And you take the motile
8 fraction, which you
now have purified.
9 DR.
ARRUDA: Yes.
10 DR.
RAO: You think there are no
11 contaminating cells,
right? And you see by PCR
12 that there is some
positivity, right?
13 DR.
ARRUDA: That's correct.
14 DR.
RAO: Where do you think that is
15 coming from?
16 DR.
ARRUDA: If you remember, even when we
17 use what we call the
optimal protocol, it is a list
18 developed for rabbits,
reagents for rabbits, we
19 still see some debris,
which you don't know which
20 kind of cells are
those, and also no motile sperm
21 cells, other than, any
other, so it is a more
22 concentrated fraction
of motile sperm up to 87 or
23 95 percent, but we
still see some of those.
24 Just from
technical reasons like if the
25 animal has very
little, urinated during the
153
1 procedure, you are
contaminated, you don't see that
2 unless the thing turns out to be yellowish,
things
3 like this.
4 DR.
SALOMON: Dr. High.
5 DR.
HIGH: I just want to underscore the
6 point that you raised
in that question, because it
7 is really critical to
the kind of analysis that we
8 are trying to do, and
I am not sure we are really
9 on the right track.
10 That is,
that what semen fractionation
11 does is enrich for
motile sperm, but it doesn't
12 really exclude all other
cell types, so when you
13 take semen
fractionation and couple it with a
14 procedure like PCR,
which will certainly detect
15 small amounts of
contaminating material that may
16 not be from motile
sperm, then, have we really come
17 up with the best
test. So your point is well made.
18 DR.
DYM: This is for Dr. Kay. It is sort
19 of a comment, maybe a
question related to maybe why
20 it seems difficult to
get the vectors into the
21 germline or into the gonads.
22 When you put
it into the liver, it, of
23 course, goes straight
into the liver, maybe a
24 little bit goes via
the artery, gastroduodenal
25 artery to the upper
part of the GI tract, and then
154
1 it goes into the
capillary spaces, and so on, then
2 back to the heart,
back to the aorta.
3 Now, those
testicular arteries, I know
4 them very well in the
human also. They are tiny,
5 little things, and it
may not just get down there
6 again.
7 DR.
KAY: I actually would say that it
8 probably does, and let
me explain how the liver
9 flow works. First of all, the catheter is
10 ballooned at a point
that is past the
11 gastroduodenal, so
there shouldn't be backflow into
12 that artery directly.
13 However,
even by clamping off the hepatic
14 artery, most of the
blood flow through the liver
15 still occurs because
60 percent of blood flow
16 through the liver is
through the portal
17 vasculature.
18 So, what we
suspect happens is that you
19 get actually infusion
into the liver, and that you
20 actually get washing
into the venous side through
21 the portal circulation
into the vena cava, and then
22 you get disseminated
flow.
23 If you look
at animals in biodistribution
24 studies that have got
an hepatic artery or portal
25 vein, or what have
you, you do find the vector in
155
1 other tissues, but at
very low concentrations. To
2 the best that we can
tell, at a reasonable rate,
3 and, you know, we can
define that statistically or
4 not, the only other
tissue that we have really seen
5 anything that would be
even suggestive of
6 transduced cells is a
rare positive cell in the
7 spleen.
8 DR.
DYM: Do you know then the clearance
9 of this?
10 DR.
KAY: No, we don't know that. That is
11 something that we are
actually working on in animal
12 models. It is almost
impossible to do in mice, and
13 we have developed some
surgical techniques in the
14 rat where we have actually
clamped off the vena
15 cava in the portal
vein and then just infusing the
16 liver, let it sit at
different dwell times and then
17 release the clamp, and
then are looking at how much
18 gets into the rest of
the circulation.
19 I think the
question is how much of it
20 gets into the liver on
the first pass, and things
21 like that, and we
don't have a definitive answer
22 for that, but we are
doing studies as best we can
23 to try to address it.
24 DR. SALOMON: Dr. Gordon.
25 DR.
GORDON: I just wanted to reemphasize
156
1 again this discussion
with Dr. High that just
2 preceded that, if the
motile sperm fractionation
3 results in the
detection of this material, that
4 doesn't mean that it
is on the sperm, but it
5 certainly means that
the sperm could come in
6 contact with it. That is really the issue in these
7 IBF strategies.
8 If the sperm
can come in contact with it,
9 does that mean that
they can carry it in and cause
10 vertical germline
transmission, and that is why I
11 feel it is necessary
to make sure that you do that
12 experiment.
13 I can tell
you that with adenovirus they
14 don't. I think it is
hard to do that, and I don't
15 think it will happen
with AAV either, but a
16 discussion of exactly
where it is sitting in the
17 motile sperm fraction
isn't really that relevant.
18 You know that that
means it could come in contact
19 with motile sperm.
20 DR.
SALOMON: Do we know that there is any
21 heparan sulfate
proteoglycans on the sperm itself?
22 I know that they
showed the picture that the
23 spermatogonia and the
seminiferous tubules seemed
24 to be negative.
25 DR.
GORDON: I don't know the answer to
157
1 that.
2 DR.
SALOMON: I guess that follows up to
3 me a question. That is, you showed the HSG
4 expression in the
seminiferous tubule as a point
5 like reassuring us
that the target wasn't there. I
6 am not an expert on
AAV, so I defer to my
7 colleagues on this.
8 I mean is
that it? I mean there is not
9 other cell attachment
molecule? I mean if so, that
10 would seem to be
rather unique since every time I
11 think that is it,
there is always something else.
12 Jude? Obviously, I am looking at you.
13 DR.
SAMULSKI: It has been shown that FGF
14 will also bind to
virus, and we know that alpha-V,
15 beta 5 is like a
co-receptor, so it is not an all
16 or none scenario, but
it is a good indicator if
17 those cells are likely
to take virus up. You
18 typically
heparan. It is not just heparan itself
19 either, it's high
sulfonated heparan, so there is
20 different kinds of
heparan.
21 DR. MULLIGAN: The infection point in the
22 rabbit, how does the
rabbit compare for AAV2
23 relative to other
kinds of cells? That is
24 obviously, the model
is only as good as how
25 sensitive it is. Is there a way you have tried to
158
1 look at a range of
different rabbit kinds of cells
2 versus other kinds of
cells, human cells to see if
3 rabbit cells are
equally, in a general way,
4 susceptible to
infection? There is definitely in
5 the AAV serotype
business, great differences, not
6 only species
differences, but also obviously,
7 tissue differences?
8 DR.
ARRUDA: We have some idea. As I
9 said, we inject the
same vector that has been used
10 in the clinical
trials, so it is expressing human
11 factor IX, and the
cohort of animals, that is of
12 the highest dose, we
are able to detect human
13 factor IX in the
rabbit plasma.
14 So what this
tells us is that I can say
15 how it transduce
efficiently in liver cells.
16 DR.
MULLIGAN: Comparable to the amount
17 you have?
18 DR.
ARRUDA: No, it's lower than because
19 the major difference
is that we did I.V. infusion,
20 not deliver into the
hepatic artery, and if you do
21 these in the same
animal, you see 5 or even less
22 expression follow
I.V. That is why we have to go
23 into the hepatic
artery.
24 DR.
MULLIGAN: If you just compare I.V.
25 and the amounts versus
the rabbit, is it
159
1 comparable?
2 DR.
ARRUDA: I would say not, because the
3 mouse, we inject --
you can correct me -- some of
4 this, like C57, they
respond very well to I.V.
5 infusion compared with
even other strains.
6 DR.
KAY: I think there is a complicating
7 factor and that using
human factor IX in a rabbit,
8 and do the rabbits
develop inhibitors? I mean
9 because you get a slow
rise of expression over
10 time, you may never
hit the peak level, and in
11 mice, you know, it is very dependent on different
12 strains.
13 DR.
MULLIGAN: I know it's complicated,
14 but I mean that
ultimately the question is whether
15 or not there is a way
to have a sense of whether or
16 not the rabbit is as
susceptible. I mean the
17 argument in a way goes
to your favor in that the
18 animal models for AAV
may not be that good because
19 of the differences,
like with the VSVGs, a
20 pseudotype, you can
infect all kinds of different,
21 1,000 tissues.
22 AAV seems
somewhat different as a vector
23 system because there
are such big differences from
24 species to species and
tissue and tissue. So it is
25 not clear whether the
rabbit would be a better or
160
1 worse system, but it
would be nice to have a sense
2 of typical tissues
that people attempt to do
3 transduction with, is
it comparable.
4 DR.
SALOMON: So you want to see data in a
5 rabbit, for example,
showing that intrahepatic
6 artery injection has a
somewhat similar
7 transduction
efficiency.
8 DR.
MULLIGAN: LacZ and muscle, in a
9 rabbit muscle, looking
at the number of positive
10 muscles -- Jude, you
must have done this sort of
11 thing. No?
12 DR.
SAMULSKI: Officially, we have not
13 done this experiment.
14 DR.
MULLIGAN: How about unofficially?
15 DR.
SAMULSKI: Unofficially, we haven't
16 done it either.
17 [Laughter.]
18 DR. KAY: Can I raise the issue of
19 hematogenous spread
again? I mean this vector,
20 unlike the
retroviruses and other vectors that are
21 being used, that have
a potential to integrate, are
22 not pseudotyped, and
they basically represent the
23 capsid of the
wild-type virus.
24 During
wild-type infection, there is going
25 to be some
hematogenous spread although I am not
161
1 sure that anyone knows
what the concentration is,
2 and yet we haven't had
any evidence of the AAV
3 sequences into the
human germline, unlike what has
4 happened with most
mammals with retroviruses.
5 So, I think,
in nature, that there is some
6 hematogenous spread of
the wild-type virus, yet it
7 hasn't been detected
in our germline.
8 Any comment
on that?
9 DR.
MULLIGAN: I want to switch back to
10 this infection
question. In the case of doing in
11 vitro infections,
there are different kinds of
12 cells, my impression
was that people often with AAV
13 use very, very high
multiplicities of infection
14 like 10,000 to 1, or
50,000 to 1.
15 Do you feel
comfortable that you have
16 really, in the in
vitro rabbit infections, really
17 dosed, put on a virus
to potentially detect
18 something?
19 The question
was, when you do in vitro
20 infections, different
kinds of cells, what is the
21 maximum multiplicity
of infection that you use to
22 see if something could
be infected? Is it 5,000 or
23 is it 50,000?
24 DR.
SAMULSKI: In our hands, we have seen
25 things like certain
fibroblasts or real refractory,
162
1 and you need about
100,000 particles to it
2 transduced.
3 DR.
MULLIGAN: That was my impression. So
4 the question is, have
you really, with all the
5 vagaries of the system,
have you really given it
6 the greater shot,
unless I got that wrong, you did
7 5,000 was your
multiplicity of infection? Was
8 there any reason you
didn't test 10 times that?
9 No.
10 DR.
SALOMON: Trying to get some sort of
11 themes going here, it
seems to me at least three
12 different things could
be discussed. The first
13 would be the idea that
the adenovirus associates
14 with the sperm or with
-- well, actually with the
15 sperm itself, and
therefore, would be carried into
16 the female and might
then enter the egg at the time
17 of the sperm's fusion,
and inadvertently deliver
18 the genetic material
from the virus. That's one
19 possibility.
20 I think that
that possibility, Dr.
21 Gordon's and your
experiment would address, so that
22 is a good thing, we
don't have any data yet, but it
23 sounds like you have
got that on track.
24 The second
possibility would be that the
25 sperm are carrying the
virus, and I am not
163
1 convinced that any of
the tests we have seen so far
2 adequately address
that. I am not trying to be
3 overcritical either,
because I can see how
4 difficult some of
these studies are to do, and
5 commend you for doing
things like figuring out how
6 to fractionate rabbit
sperm, and it shows how
7 careful you are trying
to be.
8 But it still
seems to me that when you are
9 dealing with literally
millions of sperm in a
10 typical ejaculate, and
you are doing PCR studies
11 that were sensitive
down to 1 in 30,000, that this
12 is not going to work,
I mean that that is not very
13 convincing, and
specific studies of looking at in
14 vivo expression, or
whether you use the GMP, the
15 CMD promoter or the
protamine promoter, something
16 along those lines
haven't been done yet.
17 I don't
think that we really know the
18 answer to that
part. I guess a third thing that
19 occurs to me is that
regardless of the germline
20 transfer question, if
semen of male patients had
21 got the vector for
weeks and weeks, your patient at
22 the lowest dose is 14
weeks positive in semen, is
23 that going to get
transferred to vaginal cells and
24 other cells in
mothers?
25 We all know
through bitter experience with
164
1 HIV that that is a
portal to the blood, as well.
2 You know, how likely
is it even if we just focus on
3 the semen positivity,
to which there is no
4 conflict, right, I
mean we all agree there is semen
5 positivity, are we
going to see a lot of the
6 partners infected and
how does that impact on
7 issues in terms of
doing these studies?
8 DR.
COUTO: One thing that we are trying
9 to do to address that
is with the development of
10 this infectivity
assay, at least try to demonstrate
11 that there possibly
is, even if it's there, it's
12 not infectious.
13 There may be
vector sequences there in the
14 semen, but after a
couple of days, maybe it's no
15 longer infectious, so
that is one thing that would
16 address that.
17 DR.
SALOMON: So your assay would --
18 infectious, though --
you are saying won't
19 replicate if you add
helper virus and wild-type
20 adeno for the Rep and
Cap genes, but it doesn't
21 really address whether
it just delivers the payload
22 gene, right? How likely will it be? Tell me if I
23 am being dumb.
24 But I mean
how likely would it be that a
25 positive semen that
actually has adenoviral capsid,
165
1 that you inject it
earlier in the hepatic artery,
2 will, when injected
during sexual intercourse into
3 the female, just
transfer it to vagina mucosa?
4 DR.
COUTO: If it is able to infect the
5 cells and you provide
adenoviral help, it should be
6 able to replicate, and
that is really what we are
7 asking in that assay,
so I think we can rule that
8 out.
9 DR.
KAY: The Mollier [ph] data suggested
10 that at least in the
high end injected in the
11 blood, although the
vector DNA was detected for a
12 long period of time,
the biological activity of
13 those particles
diminished to undetectable levels
14 after a very short
time.
15 So what I
think Linda is trying to say,
16 just to reiterate, is
that there is a reasonable
17 chance that the DNA or
the particle could stick
18 around, but it may not
be actually infectious or
19 able to transduce a
cell.
20 If it went
into like, say, a vaginal
21 epithelial cell one
time, you would never see that.
22 I mean it would be a
single transduction event, it
23 wouldn't
replicate. I guess the question is if
it
24 was carried in on the
sperm into an oocyte, what is
25 the chance of naked
DNA or DNA that is partially
166
1 exposed could get
through the zona pellucida during
2 a fertilization event.
3 DR.
GORDON: All I can say to that is I
4 don't think it has
ever been seen in the literature
5 despite claims to the
contrary.
6 DR.
SALOMON: I wasn't trying to make
7 things too
complex. I guess I was just saying that
8 we are really not
discussing just the Avigen factor
9 IX study even though
that is on the table here, my
10 feeling here is we are
discussing just in general
11 issues here. I greatly
respect Avigen being here
12 and presenting it,
because it is always great to
13 have a specific study
to focus on, but we also
14 don't want to lose
sight of the fact that there are
15 bigger issues here.
16 So I am
saying that a lot of different
17 clinical trials could
come along following
18 potential success in
the Avigen trial, and I
19 certainly do wish you
the best with this one.
20 Those could deliver
gene payloads that could be a
21 lot more serious than
delivering some extra factor
22 IX to a woman
inadvertently, so that is really all
23 I am trying to say is
if you start delivering --
24 oh, who knows, I don't
want to make stuff up --
25 but, you know, just a
gene payload that might be
167
1 toxic, whether that
would be delivered to the
2 vagina of the woman
and produce some problem there.
3 Again, I am
not trying to make that a big
4 killer issue, but it
seems like from everything I
5 have heard that it is
still theoretically possible.
6 DR.
MULLIGAN: One thing that Jon's --
7 what do you call them,
provocative experiment --
8 would test is, in
principle, whether or not any
9 AAV, since that is the
worst case, we are soaking
10 things with the AAV,
and then you are doing the
11 most efficient means
of sex, maybe not the most
12 efficient, but you are
doing it so you are opening
13 up as much AAV as
possible, so you could look for
14 things in addition to
the integrated sequences.
15 I think I
saw that you were going to test
16 only for integrated
sequences. After the in vitro
17 fertilization
experiment, it might be useful to,
18 since you have the
material, to look for whether or
19 not there is AAV. Presumably, if it wasn't
20 integrated, it would
be dramatically diluted, but
21 it would be
interesting to see if you could detect
22 it, because that would
address in a sense the worst
23 case of whether or
not, during sexual intercourse,
24 you can transfer AAV,
and it can persist maybe as
25 an unintegrated form,
but these is some infection.
168
1 DR.
GORDON: We are doing that.
2 DR.
SAMULSKI: I have a question, and it
3 is more a
curiosity. It seems that the so-called
4 debris and other
contaminants are a major
5 contributor to the
positive results, and I am
6 wondering if people
here have felt that the efforts
7 to purify these
different fractions have been
8 exhaustively done,
because it seems that when you
9 move to other
reagents, whether they are oligos or
10 plasmids, this is
going to come up over and over
11 again.
12 If there is
more energy put into the first
13 step of the assay, of
collecting and fractionating,
14 will we move away from
these long risk things and
15 get into a better
assay that is going to tell us
16 there is something
worth paying attention to.
17 Again, I turn it back
this way, because when you
18 hear someone say this
was optimized for rabbits,
19 and this was optimized
for humans, does that mean
20 it has been done for
20 years and optimized, or is
21 just gives them the
result they need to get
22 something away from
something.
23 DR.
GORDON: I just want to very briefly
24 comment on that. Even a fraction of motile sperm
25 is a very
heterogeneous population of cells. I
169
1 mean some of them have
two heads. Some of them
2 have a huge
cytoplasmic droplet, which can be close
3 to the volume of a
sperm.
4 So when you
actually try to do an
5 absolutely totally
pure separation of motile sperm
6 from everything else
with a similar density,
7 similar parameters of
measurement, similar
8 configuration, it is
very, very difficult, and I
9 think that if you try
to solve this problem that
10 way, by getting a
golden fractionation procedure,
11 you are going to be
chasing your tail for a long
12 time, not that I want
to introduce other tails into
13 the discussion.
14 DR.
JUENGST: So, thinking generically,
15 kind of at the policy
level, I think I learned two
16 things this morning
that increased my sense that
17 there are risks
here. The first one was the idea
18 that it is not just
the integration of a factor IX
19 gene in a harmless
place on chromosome 19, but the
20 random integration of
genes from the episome
21 presence of the
vector.
22 The second
was the increased risk even if
23 through natural
fertilization, it looks low with
24 artificial means of
fertilization, ICSI and
25 infertility
techniques, so it looks like the
170
1 patients who are at
greatest risk, hypothetically
2 speaking, worst case
scenario, would be gene
3 therapy patients who
then had fertility problems
4 and needed to go to a
fertility clinic.
5 DR.
SALOMON: To Jude's question, the
6 other way around here
would be there is still no
7 evidence that these
vectors are getting into the
8 spermatogonia, so if
you could do enough really
9 well designed, basic,
preclinical work, you might
10 be able to make a good
case that you just monitor
11 the semen, and not be
obsessing about all this
12 purification, et
cetera, you know, if you could
13 convince yourself that
it wasn't specifically being
14 carried in the
germline package of the sperm.
15 DR. GORDON: Let me just say that I think
16 that is a very
important point because if you
17 cannot transduce the
spermatogonia, then, when the
18 semen are clear, you
can feel that they will be
19 clear, and that not
another wave of spermatogenesis
20 will provide more
positive sperm to the ejaculate.
21 DR.
SALOMON: Certainly, the data
22 presented today still
do not give us any cause --
23 you know, there is no
smoking gun yet that these
24 are being delivered to
germline cells.
25 DR.
MULLIGAN: Jon's work wouldn't address
171
1 the worst case for
trying to get an earlier
2 precursor infected,
right? I mean that is, you
3 could think of the
same Jon kind of approach where
4 you would put in as
much AAV into exactly right
5 time and location, to
do the same sort of worst
6 case, and that
probably would be the ultimate worst
7 case.
8 DR. SALOMON: Yes. In fact, that was
the
9 point I was making,
too, earlier. There was the
10 issue of whether it
got in or didn't, but his
11 experiment addressed
the latter, right, where it
12 was just attached to
the outside.
13 But the
experiments haven't been done yet
14 or designed yet or
proposed yet to do the ones that
15 we both suggested, and
that is, prove yes or no,
16 whether it gets into
the spermatogonia, and if you
17 could get out of that,
then, you could make the FDA
18 and the sponsor's life
a lot easier.
19 DR.
GORDON: Well, I just want to say
20 again that we have an
abstract today at ASGT, in
21 which we are
developing this technique of perfusing
22 intact seminiferous
tubules with very high
23 concentrations of
vector. I showed some of the
24 stuff from adeno
expressing lacZ, and again that
25 would be a very highly
provocative test.
172
1 It doesn't
seem to disturb the
2 spermatogenesis much,
if at all, and that, with
3 nucleic acid
hybridizations, you wouldn't have to
4 rely on promoters and
vectors with delayed
5 expressions, which is
AAV, would I think be a good
6 standard to arrive to.
7 DR.
NOGUCHI: Just to follow up a little
8 bit on how provocative
you be, Jon, wouldn't the
9 most and even more
provocative state be to expose
10 sperm to AAV, and then
immediately do ICSI, and
11 then look at the
outcomes of that?
12 DR.
GORDON: I think that would work
13 because then all the
natural barriers to getting it
14 in would be
circumvented, but I do emphasize those
15 are natural barriers
and that is an artifactual
16 situation, however, as
I was saying before in my
17 talk, my official
talk, I mean there is a lot of
18 clinical activity
where these barriers are
19 bypassed, and I think
that we should begin to be
20 interested in that
subject, and I don't think that
21 is the subject for
this meeting, but I think it is
22 a subject that the FDA
needs to begin to get
23 interested in.
24 DR.
MULLIGAN: I like Phil's approach
25 because then it is
really more directed an
173
1 integration
question. It is like doing a
2 transgenic system
where you dump in more and more
3 AAV in exactly the
right -- for something that
4 happened, and you can
see whether it does or
5 doesn't happen.
6 DR.
NOGUCHI: It actually pertains in a
7 way, based on the
discussion here, to this
8 experiment, as well,
if, in fact, you have the
9 presence of vector
even if it's not integrated, but
10 it is around, it could
coat the sperm or it could
11 be attached to the
sperm. That is the equivalent
12 of what we are talking
about. You have a vector, a
13 sperm, a union with an
egg, and things.
14 So I think
they are two different things.
15 One is, is there
integration into the actual person
16 being treated, and
then the other part, can there
17 be a transmission by
other than biological means,
18 but just by pure
mechanical. That is an issue that
19 pertains, and it is
related also to the question of
20 how much sensitivity
is enough if we are going to
21 be talking about
barrier contraception as a means
22 to mitigate this
period of washout, you know, how
23 much washout is
enough.
24 DR.
SALOMON: I guess as long as we are at
25 the most provocative
experiment discussion, I mean
174
1 you could simply
inject the AAV into the egg, and
2 then with the lacZ,
and ask then in like a mouse
3 embryo or in a chicken
embryo or in a rabbit
4 embryo, where it was
distributed, and ask the
5 question whether there
is some unusual integration
6 or whether it just
quickly segregated.
7 DR.
GORDON: Just a brief comment on that.
8 We have another
abstract at ASGT, about a
9 adenovirus injection
directly into embryo, and what
10 we were doing was
asking the question, does this
11 intricate cycle of
virus on coating and
12 translocation of the
genome to the nucleus, are
13 these obligate steps
for expression, which has
14 always been assumed,
but never been proven.
15 Now, the
one-celled embryo appears not to
16 have a receptor for
adenovirus from our
17 experiments, and so
what we did was injected the
18 virus directly into
the cytoplasm and said, well,
19 we will bypass the
endosome and see what we can
20 get.
21 We never
lacZ expression under those
22 circumstances. We then say, well, let's help the
23 virus even more, we
will put it right into the
24 pronucleus, and we
have done that. In that case,
25 we see low rates of
embryos that express which
175
1 appear consistent with
perhaps viral genomes that
2 have been partially
shredded by freezing and
3 thawing.
4 To confirm
that, we took the viral genome
5 and just injected the
pure DNA and got a very
6 similar result. So in adeno, it doesn't appear
7 that the virus can
actually do its thing if it's
8 not allowed to go
through the regular cycle of
9 infection, but AAV,
which is why I asked about
10 uncoating, when it
uncoats, if you put that
11 directly in the
pronucleus, it is a single-stranded
12 genome, but there is
very active repair mechanisms
13 in the pronucleus, and
all of that, I should think
14 it would work, but I
don't think it has ever been
15 tried. I would be happy to try it if someone wants
16 me to try it.
17 DR.
SAMULSKI: So, we actually did those
18 experiments, and if
you remove the zona pellucida
19 and inject the virus,
you can get blastocysts to
20 turn blue, so it will
transduce those cells. It
21 doesn't work with
adenovirus, just like you said,
22 for the same
reason. When it comes out of
23 endosomes, there is a
proteolytic cleavage that is
24 responsible for the
virus on coat, and if you don't
25 go through that
pathway, it won't go through.
176
1 DR.
KAY: Is it integrated?
2 DR. SAMULSKI: We only did in vitro and
3 carried them out and
were able to show that it
4 would transduce those
cells, and when we started to
5 collaborate with our
colleagues to implant them
6 back in for embryos,
the postdoc left, and all of
7 this stuff stopped, so
we didn't do any more on it.
8 DR.
GORDON: What was the helper for that?
9 DR.
SAMULSKI: There was no helper. We
10 were simply trying to
find a better way of making
11 transgenic animals
using AAV as a way of delivering
12 genes, and showed that
once could physically put it
13 in, it would transduce
those cells, so I think
14 Phil's question is
partially answered, you will get
15 it in, and it will
work by some mechanical mean.
16 And far as
the stability, integration, all
17 of that stuff, there
are no answers at all. It was
18 just lacZ.
19 DR.
MULLIGAN: What was the number of
20 virus particles?
21 DR.
SAMULSKI: It was extremely high. We
22 were putting in about
1010. For a number of
23 reasons, it partially
was -- well, we could talk
24 about it later.
25 DR.
SALOMON: Any other discussions here?
177
1 What the
committee should feel at this
2 point is that just in
terms of science presented,
3 that we are
comfortable with the main issues, and
4 then we will do the
public comment and go on to
5 answering the specific
questions from the FDA.
6
Open Public Hearing
7 DR.
SALOMON: What I would like to do is
8 introduce the public
comment. Five minutes have
9 been allotted to each
of three speakers.
10 The first
speaker that I would like to
11 invite up is Mr.
Steven Humes of the National
12 Hemophilia Foundation.
13 MR.
HUMES: Good afternoon. My name is
14 Steven Humes and I am
the Director of Research at
15 the National
Hemophilia Foundation, hereinafter
16 referred to as NHF.
17 NHF is a
not-for-profit organization
18 dedicated to improving
the quality of life for all
19 individuals with
hemophilia and other bleeding
20 disorders. Today, we thank the members of the
21 Biological Response
Modifiers Advisory Committee
22 for allowing us the
opportunity to provide
23 testimony on recent
reports of the presence of
24 adeno-associated
virus, or AAV, in the seminal
25 fluid of individuals
participating in a hemophilia
178
1 factor IX
liver-directed gene transfer trial.
2 There are at
least 10 proteins in our
3 blood, which must work
in a precise sequence to
4 make the blood
clot. A deficiency in any one of
5 these proteins can
lead to abnormal bleeding.
6 Hemophilia A is caused
by a deficiency of or defect
7 in a clotting protein
known as factor VIII. A
8 deficiency of or
defect in clotting factor IX
9 causes hemophilia
B. Both forms of hemophilia and
10 other bleeding
disorders are X-chromosome linked
11 recessive genetic
disorders. In the United States,
12 there are
approximately 17,000 individuals living
13 with a diagnosis of
hemophilia.
14 Hemophilia
manifests itself by easy
15 bruisability and
recurrent bleeding into joints and
16 muscles as well as
bleeding intra-abdominally and
17 into the central
nervous system. The severity of
18 an individual's
hemophilia is determined by the
19 amount of circulating
clotting factor. The
20 majority of
individuals affected with hemophilia
21 have severe disease.
22 Individuals with severe hemophilia
23 typically have eight
to 10 bleeding episodes each
24 month. This chronically recurrent hemorrhaging
25 causes disability,
persistent pain, and sometimes
179
1 death.
2 In the past
three decades, significant
3 advances have occurred
in the treatment of
4 hemophilia with the
development of plasma-derived
5 and then recombinant
clotting factor products.
6 While the development
of these new products has
7 proved enormously
beneficial, persons with
8 hemophilia continue to
face many difficulties that
9 affect their quality
of life.
10 Prior to the
development of viral
11 inactivation
technologies, many individuals with
12 hemophilia were
infected with HIV and hepatitis
13 through their
unwitting use of contaminated
14 clotting factor
products. The HIV epidemic has
15 cost this community
dearly, causing the deaths of
16 over 6,000 hemophilia
patients, their spouses,
17 partners, and
children.
18 Today, an
additional 2,200 continue to
19 live with HIV and its
complications. It is
20 estimated that more
than 70 percent of all persons
21 with hemophilia have
been exposed to hepatitis C.
22 While the development
of recombinant factor
23 significantly improved
safety, it is sometimes in
24 shortage and also an
extraordinarily expensive
25 medicine, especially for
individuals with severe
180
1 hemophilia who must
treat frequent bleeding
2 episodes or who
self-infuse prophylactically as
3 often as three times
per week.
4 Because of the many challenges facing this
5 community and the
limitations of current treatment
6 modalities, we look to
gene therapy as the most
7 promising approach to
cure hemophilia. To this
8 end, NHF has funded
numerous gene therapy projects
9 and five scientific
workshops on gene therapy, and
10 two-day gene therapy
symposium is planned for our
11 annual meeting in
October 2002.
12 We believe
that research into a monogenic
13 disorder such as hemophilia
may also lead to
14 progress in the
treatment of more complex
15 disorders, such as
multi-gene inherited disorders,
16 as well as cancer.
17 In 1996, an
NIH report on gene therapy
18 recognized hemophilia
as one of the most likely
19 disorders for which
gene therapy will succeed. We
20 believe that this
statement is as true today as it
21 was six years ago.
22 In the fall
of 2001, vector
23 biodistribution
studies from a factor IX deficiency
24 gene therapy trial
noted the presence of AAV vector
25 in the semen of a
trial participant. That study
181
1 also noted that while
vector was present in the
2 seminal fluid, there
was no evidence of
3 transduction of sperm.
4 On November
17, 2001, NFH's Medical and
5 Scientific Advisory
Council, or MASAC, reviewed
6 this issue and drafted
its Recommendation No. 127,
7 which was approved by
the NHF Board of Directors
8 the following day, and
is attached to this document
9 that you have before
you.
10 The
recommendation requests, in reviewing
11 such unexpected
findings, that the Recombinant DNA
12 Advisory Committee, or
RAC, and the Food and Drug
13 Administration
consider the risks to the trial
14 participant and,
following appropriate analysis,
15 allow trials to
proceed if such risks can be
16 mitigated.
17 NHF believes
that a case-by-case
18 evaluation of
unexpected findings will permit
19 improvements in safety
and efficacy while enabling
20 continued pursuit of
improved treatments for
21 hemophilia.
22 NHF believes
that the AAV factor IX
23 liver-directed gene transfer trial currently
being
24 conducted should
continue. As Steven Faust, a
25 person with severe
hemophilia and co-chairman of
182
1 NHF's Advocacy Committee,
stated this January
2 before the RAC, we see
no inherent risks in these
3 findings that might
cause additional risk to the
4 trial subjects.
5 NHF does
support, however, increased
6 patient education and
efforts directed at improving
7 the informed consent
process, mitigating the risk
8 of potential germline
transmission through the use
9 of sperm banking and
requiring the use of barrier
10 contraceptive methods,
and long-term surveillance
11 of trial subjects via PCR vector
dissemination
12 studies.
13 Indeed, we
believe that through this
14 surveillance, we might
learn valuable information
15 about the natural
history of AAV shedding that
16 could prove useful in
future gene therapy trials.
17 NHF's MASAC has laid
out detailed guidelines for
18 the conduct of gene
therapy trials in its
19 Recommendation No.
120, dated August 16, 2001, a
20 copy of which is also
furnished to you.
21 NHF
respectfully suggests that the
22 Advisory Committee
consider these guidelines when
23 considering future
gene therapy trials.
24 NHF is
heartened by the preliminary
25 results of gene
transfer in humans. We are further
183
1 encouraged by the
prompt review of the RAC and
2 FDA's Biological
Response Modifiers Advisory
3 Committee to the
vector biodistribution studies, as
4 we believe that this phenomenon
may occur in other
5 trials.
6 We share
your commitment to ensuring
7 patient safety, and
appreciate your vigilance on
8 behalf of all persons
enrolled in gene therapy
9 clinical trials. On behalf of the bleeding
10 disorders community,
we urge your continued support
11 for these trials. If enrollment is further
12 delayed, how will we
determine if this gene
13 transfer method offers
the promise of a cure?
14 Once again,
we thank you for this
15 opportunity to address
you on this important
16 matter.
17 DR.
SALOMON: Thank you very much, very
18 nicely articulated.
19 I think it
is always reasonable to point
20 out when you hear something like that, that it is
21 very important for the
committee to consider
22 whatever decisions we
make affect a group of
23 stakeholders, in this
case the hemophilia
24 community, as well as
the public, and that is
25 always important to
hear that in these sort of
184
1 public comments and
think about it.
2 The next
speaker is Dr. James Johnson, who
3 is identified as a
hemophilia patient. That must
4 have been one of the
first two patients to receive
5 the Avigen vector.
6 Welcome, Dr.
Johnson.
7 DR.
JOHNSON: Dr. Salomon, Committee, good
8 afternoon. I am Dr. James Johnson. I am from
9 Edmund, Oklahoma. I appreciate the opportunity to
10 speak to you wearing
several hats. I am an
11 emergency physician
practicing in Cushing,
12 Oklahoma, a husband
and a father. I have lived for
13 45 years with
hemophilia B, factor IX deficiency.
14 In addition, I was
blessed to participate in the
15 Phase I safety trial
of the gene therapy program at
16 Children's, not this
one, but the prior one with
17 the IM injections two
years ago.
18 My
participation began in May of 2000. As
19 a side note, I dubbed
myself Lad Back No. 6, since
20 I was the sixth person
in that study.
21 As a
45-year-old living with hemophilia
22 and as a participant
in a gene therapy study, I
23 want to express my
deepest hope that you will allow
24 the current study to
continue. This research is of
25 vital importance to
the hemophilia community and to
185
1 me personally.
2 When I was born in 1957, it took two years
3 for my parents to
receive a diagnosis for the
4 bleeding I had. Because my head was swollen at
5 birth, my parents were
told I had hydrocephalus and
6 would be mentally
disabled.
7 Once the diagnosis of Christmas
disease
8 was made, my parents
were told that I wouldn't live
9 past 20 years of
age. I kind of overdid that.
10 My early
treatments consisted of ice
11 packs, splints,
slings, and rest. Later, I
12 received infusions of
whole blood and fresh-frozen
13 plasma. Finally, when I was 12, I received the
14 first dose of factor
concentrate. That was 1970.
15 I promptly came down
with hepatitis B, and although
16 I did not know it at
the time, hepatitis C.
17 Through the
1970s, I would go to the
18 doctor and often have
to be hospitalized when I
19 needed treatment. Finally, in 1979, I started
20 self-infusion, which
is now the standard of care.
21 I was one of the
blessed minority that was not
22 infected with HIV, but
as I said earlier, I do have
23 hepatitis C.
24 Also, I have
suffered the ravages of
25 arthritic
complications of hemophilia as you might
186
1 have noticed when I
walked up. When we first went
2 to the hemophilia
meetings, my wife said, Jim, they
3 all walk just like you
do.
4 As you can
imagine, hemophilia has
5 affected every area of
my life. Like all
6 hemophiliacs, I have
had to deal with educational
7 issues, work and
employment issues, and struggles
8 with insurance. From a young age, I knew that I
9 would not be able to
hold down jobs that required
10 great physical
strength or endurance. Fortunately,
11 I am able to hold down
my ER job and even do some
12 extra weekend work.
13 About nine
years ago, I found that I had a
14 hemophilic pseudotumor
in my abdomen. This was the
15 result of repeated
bleeds in the psoas muscle.
16 There have
occasionally been rebleeds which are
17 very painful and often
require hospitalization.
18 This happened just
this past week and for a while
19 it looked like I
wouldn't be able to make it today.
20 I tell you
this not for your sympathy or
21 to act macho, but to
let you know that even with
22 today's best
treatments, problems still arise.
23 It has been
said of the hemophilia
24 community that we
desire to be cured, we don't need
25 to be cured. Everyone is entitled to their own
187
1 opinion, but I believe
we need a cure. Sure, there
2 are treatments
available, but who likes to have to
3 give himself I.V.
injections every time he feels
4 pain come on, or
injections for days to try to get
5 over a bleed like the
one I had this week?
6 We have treatments
for other diseases, but
7 still work very hard
for a cure. Does diabetes not
8 need a cure? Does hypertension not need a cure?
9 Does asthma not need a
cure?
10 As for the
current gene therapy study, the
11 one I was in, I
enrolled about the time one of the
12 other programs lost a
patient. Once the program
13 started back up, I,
along with my 11-year-old
14 daughter and I, all
had the procedure explained to
15 us in great
detail. We were told of all the known
16 risks, as well as the
theoretical risks that they
17 could imagine.
18 We were
given ample opportunity for
19 questions. In short, I can say from the
20 perspective of both
the study participant and as
21 one who has been
involved in doing clinical
22 research myself, their
informed consent procedure
23 was impeccable.
24 Every step
along the way, I have been
25 informed of any new
developments. We are still
188
1 given every
opportunity to ask questions or check
2 in to see how things
are progressing. I have
3 always been and always
know I will be able to speak
4 to anyone involved
with the program with any
5 concerns or ideas.
6 I have met
with all of the people involved
7 with the program at
Chop, from Dr. High, the
8 director, to Dr.
Mannow, to Amy Chu, the clinical
9 coordinator, and to
even lab personnel. They are
10 committed and
responsible people.
11 I understand
that there is concern about
12 the possibility of the
AAV vector being present in
13 the semen of some of
the participants. This was
14 one of the risks that
was reviewed with us before I
15 participated in the
earlier trial.
16 It was
always stressed that participation
17 in the study was
voluntary, there was obligation to
18 participate or
continue even after starting the
19 study. We were informed that I should never expect
20 to father a child
after the study because of that
21 risk of AAV infection.
22 This was
fine with us as my wife had
23 already had a
tubal. Those that might consider
24 future children were
given the opportunity for
25 sperm storage. All of my body fluids were tested
189
1 for weeks
afterwards. There should be plenty of
2 other subjects like
myself who do not plan any
3 further family. They would be able to be in the
4 studies at no risk to
anyone else until more is
5 known about germline
transmission.
6 This is an
extremely important area of
7 study. Germline transmission is likely to be an
8 equal problem for all
gene therapy if it turns out
9 to be a continuing
problem here. The hemophilia
10 community, after
having gone through hepatitis B,
11 HIV-AIDS, and now
hepatitis C, is a very tough,
12 resilient, and
responsible community.
13 We have been
on the forefront of the use
14 of barrier
contraception to prevent HIV infection,
15 so the idea of
contraception and when appropriate,
16 sperm banking, is not
foreign to us. Those that do
17 not like that option
will not opt for the clinical
18 trials, as did my own
brother.
19 In closing,
I want to thank you for the
20 opportunity to speak
to you. When I did my senior
21 paper in college, over
20 years ago, I wrote of the
22 potential that gene
therapy would one day hold for
23 curing
hemophilia. It is here. It is a reality.
24 As a member
of the hemophilia community, I
25 ask you to work with
the gene therapy program and
190
1 the community to make
gene therapy research program
2 safe and successful in
the least time possible.
3 When my
daughter asks me about her
4 children and
hemophilia, I want to be able to tell
5 her that we have the
answer. Please don't make me
6 tell her that we got
close, but some minor
7 glitches, whether AAV
or AV, stop the program.
8 Therefore, her sons
will be at risk for the same
9 difficulties I have
gone through.
10 Thank you.
11 DR.
SALOMON: Thank you very much, Dr.
12 Johnson.
13 The last
speaker in the public comment
14 period is Dr. Kenneth
Chahine, Avigen Vice
15 President for Business
Development and Intellectual
16 Property.
17 DR.
CHAHINE: Good afternoon. My initial
18 goal was to try to
bring a literative perspective
19 to the committee, but
after the last few speakers,
20 I think that is not
something I am going to try to
21 do.
22 My goal here
today is to first present
23 what we can reasonably
glean from the data
24 presented by my
colleagues and also to present the
25 assumptions that form
the basis of our proposal to
191
1 this committee. In our proposal, also, we keep in
2 mind the questions
that the FDA posed to the
3 committee.
4 The second
goal is to communicate our
5 proposal and the
rationale for that proposal. So,
6 what do we know and
what can we reasonably assume?
7 The first
point is that the procedure is
8 well tolerated, as Dr.
Kay indicated. There have
9 been no risk to the
patient apart from this
10 inadvertent germline
transmission risk that we are
11 talking about today.
12 The second
point, which has been clearly
13 talked about amongst
the committee members, is the
14 predictive value of the animal models with
respect
15 to inadvertent
germline transmission. Clearly,
16 some of the animal
models don't mimic the human
17 biology, while others
may, although even the rabbit
18 dose and clearance
times seem to be different from
19 what we are seeing in
the first two patients.
20 The one
consistent trend, however, is that
21 in all of the animal
models, the vector is either
22 not there or it has
cleared over time.
23 The third
point is that the motile sperm
24 fraction may be
positive as the dose increase, and
25 will almost certainly
take longer to clear, so we
192
1 want to make that
assumption. I think it is
2 important for the
committee to just look at that,
3 and I think our
discussion today has brought that
4 out, that the
fractionation procedure may not be
5 adequate to address
it.
6 The next
point is somewhat of a practical
7 point, is that the
current rate and current
8 clinical hold
triggers, the Phase I trial is going
9 to take very long to
complete, and while in no way
10 does this point alone
justify recommendation to
11 continue, it does have
practical consequences for
12 Avigen, the scientific
community, and the
13 hemophilia population.
14 The next
point addresses one of the
15 questions that the FDA
posed to the committee, and
16 that is, should the
enrollment be limited to
17 patients or subjects
which are unable to reproduce.
18 Certainly,
that will cause delays given
19 the size of the
hemophilia population, but will
20 also, in the male
population, talking about males
21 that have undergone a
vasectomy, limiting
22 enrollment to this
patient population will deprive
23 us, the FDA, the
scientific and medical community,
24 of the data that we so
desperately need to answer
25 the very question we are
here today convened to
193
1 address.
2 Finally,
higher doses my yield therapeutic
3 levels of factor IX
based on the preclinical data.
4 It is important to keep
in mind that the primary
5 purpose of this trial
is certainly safety, but as
6 Mark Kay pointed out,
at the higher doses we do
7 expect, based on the
preclinical animal data, to
8 get a dose that is
potentially therapeutic.
9 We hope that this discussion in the
future
10 will actually focus
more on a risk-benefit as
11 opposed to simply the
risk, which is what we are
12 discussing today of
inadvertent germline
13 transmission.
14 So, keeping
these points in mind, why is
15 informed consent a
reasonable and prudent safeguard
16 against inadvertent
germline transmission?
17 The subjects
are already counseled and
18 educated on the
potential of not only transient but
19 permanent germline
transmission. The patients are
20 already advised to use
barrier contraceptives, and
21 as we have discussed
here and at the Recombinant
22 Advisory Committee
meeting, the risk of inadvertent
23 germline transmission
is low.
24 The subjects
are constantly monitored for
25 positive semen and
positive motile sperm, and
194
1 germline transmission,
very importantly, can be
2 completely avoided by
banking sperm.
3 So, in our
opinion, when we take the low
4 risk of inadvertent
germline transmission, the
5 small number of
subjects in the trial, the active
6 use of barrier
contraceptives and the sperm
7 banking, these factors
together reduce the risk of
8 inadvertent germline
transmission to acceptable
9 levels especially when
we weigh the risk against
10 the enormously
valuable data we will be able to
11 collect, at no risk to
the subject, and the
12 potential benefit to
the hemophilia community.
13 So what we
propose is the following.
14 That Avigen
should continue its assay
15 development and
preclinical studies in various
16 animal models. While there is some question about
17 the validity of some
of the animal models that we
18 are studying, I hope
it is clear that Avigen is not
19 trying to skirt or
avoid this issue, but we are
20 aggressively going
after an answer.
21 The informed
consent should be reviewed
22 and updated as needed
to reflect the current data,
23 as we have between the
first two patients.
24 The Phase I
trial should be allowed to
25 continue regardless of
whether the motile sperm
195
1 fraction is positive,
and that just goes back to
2 the difficulties that
we have had in the motile
3 sperm, and once we get
to, let's say, the next
4 dose, we are assuming
that we are going to actually
5 get some
contaminations that are going to give us a
6 positive signal.
7 The subjects
should be monitored until
8 three monthly semen
samples are negative. That is
9 currently in the
protocol already. We should, even
10 though the value of
the fractionation procedure is
11 a question, we want to
continue to find out and
12 maybe improve, as has
been suggested here today.
13 We also want
to ask a question that is
14 very important and the
committee has raised today,
15 which is whether the
vector sequences in the semen
16 actually represent
biologically active vector.
17 That is just not very
clear.
18 If we can do
this and continue the trial,
19 we can use the
clinical data to identify predictive
20 inadvertent germline
transmission preclinical
21 model, which will help
if this trial hopefully goes
22 through later phases,
having a predictive model
23 would be very good and
clearly for other AAV trials
24 coming in the future.
25 We want to
continue to encourage subjects
196
1 to bank sperm prior to
the treatment, and the
2 subjects will continue
to be informed of their
3 semen results and
counseled about whether they
4 should continue to use
contraceptives.
5 The final
point is important because it is
6 one of the questions
that was raised by the FDA to
7 this committee, which
is what happens if you have
8 persistent germline
transmission.
9 We think,
and I believe that the committee
10 believes, that the
possibility of that is low, we
11 feel like we need to
have a contingency, and we
12 believe that if the
subjects and the partners are
13 encouraged to undergo
counseling by the study
14 physician on a regular
basis if the vector is
15 persistent in the
motile sperm, that that will
16 mitigate against the
risk of transmission.
17 We selected
greater than one year. That
18 was somewhat
arbitrary. We are certainly open to
19 discussing with the
FDA what would be a reasonable
20 time frame, but that
is just the one we selected
21 that we thought was
reasonable.
22 In closing,
we are convinced that there is
23 a solution to the time
of the completion of this
24 Phase I trial, and are
committed to working with
25 the FDA to find and
implement such a solution.
197
1 Thank you.
2 DR.
SALOMON: Thank you very much.
3 The schedule
shows lunch. I think you may
4 have intuited that I
wasn't planning on lunch, and
5 just go into answering
the questions. I just
6 wanted to make sure
that that was okay with
7 everybody.
8 Committee
Discussion of Questions
9 We have a
series of questions. I am very
10 comfortable that we
have set most of the
11 intellectual
background here to do this. The first
12 question:
13 1. If vector sequences are detected in
14 the motile sperm
fraction of clinical trial
15 subjects, the current
approach of the FDA is to
16 suspend accrual to the
study -- in other words, put
17 it on a clinical hold,
and that is what happened in
18 this case, just to
make sure that everybody is
19 clear about that, these
guys can't go forward right
20 now -- regarding the
persistence of the vector then
21 becomes the criteria
upon which the clinical hold
22 is raised or not
raised.
23 If they are
getting out of 14 weeks and
24 they are still
positive in their second patient at
25 the lowest dose, that
is how they are calculating
198
1 that it would take
five years to do the study, and
2 it might even be
longer.
3 So I think
most of us who have done
4 clinical trials are
sympathetic with that being
5 very difficult.
6 Enrollment
has been allowed to proceed
7 when there are data to
show that it is negative.
8 In other words, three
consecutive samples.
9 A. Does the committee agree that a
10 clinical hold is
warranted when motile sperm tests
11 positive for vector
sequence or should enrollment
12 be allowed to continue
with appropriate
13 modification made to
consent documents?
14 Discussion?
15 Let me just
point out something here.
16 There is a little bit
of a load in here because we
17 are talking about, at
least for me, this is motile
18 sperm tests. I am underwhelmed with this motile
19 sperm test thing. In other words, I think it is
20 great science, but I
am not sure, and welcome
21 discussion on that
point, that you really have to
22 do motile sperm tests
here, and I think it would be
23 just easier to do
semen.
24 Go
ahead. You wanted to make a
25 qualification?
199
1 DR.
KAY: [Off mike.]
2 DR.
SALOMON: The point is that you are
3 still on hold. I think that is the main
4 clarification.
5 Yes, Tom.
6 DR.
MURRAY: First, just to clarify a
7 factual question. The reason you are underwhelmed
8 by the motile sperm
test, I am sorry it's unfair to
9 ask Dr. Salomon a
question. I begin with a
10 clarification from
you.
11 The reason
you are underwhelmed with the
12 motile sperm test,
maybe I misunderstand the nature
13 of the test, but if I
understood it correctly --
14 the first thing we are
after is if we are concerned
15 about germline
transmission, we don't want to see
16 altered genes to a
child, and there are a variety
17 of ways to sort of
protect against that.
18 The motile
sperm test, as I understood it,
19 was imperfect for a
variety of reasons, but most of
20 them had to do with
the fact that you might still
21 get AAV DNA even if it
wasn't going to be, so it's
22 a sort of both and
test.
23 If it came
up negative, would you be
24 pretty reassured that
you weren't going to get?
25 Okay. But if it came up positive, you still
200
1 weren't sure whether
it meant that it was
2 infectious or
not. Okay. So, I have got that
3 clear. I wanted to be sure I got that clear.
4 Let me start
then from the back end. If
5 the concern is to
prevent the transmission of
6 altered DNA to
offspring, there are a number of
7 ways to try to achieve
that. One is to test motile
8 sperm. If it comes up negative three times in a
9 row, we feel pretty
comfortable that it is not
10 going to happen.
11 I noticed, by the way, that one of the
12 spokespersons for the
hemophilia community actually
13 said about requiring
barrier contraception. I am
14 assuming the FDA is
not in a position to require it
15 or enforce a
requirement for barrier contraception,
16 but I think we need to
take that as an expression
17 of the genuine concern
on the part of that
18 community.
19 So, a second
order would be to again
20 strongly encourage
barrier contraception, provide
21 very clear informed
consent. Give the kinds of
22 warnings that Dr.
Gordon was, I think, alerting us
23 to, say, look, a way
around this is not to do ICSI
24 with your current
sperm after intervention, and be
25 very clear about the
variety of things that they
201
1 ought not to do right
now, to bank sperm
2 beforehand.
3 I mean there
are a lot of things you can
4 do to encourage, to
sort of defeat the likelihood
5 that there is going to
be any transmission. So we
6 have got at least two
protections there. A third
7 is -- well, think
about what if the worst possible
8 scenario happened, we
go ahead with the trial, we
9 permit the trial to
continue despite being pretty
10 comfortable with the
test, despite the promise to
11 do barrier, despite
the information given in
12 informed consent, a
child is, in fact, conceived
13 and born who is
carrying an altered gene here.
14 We need to
think about -- we are not
15 talking about a
systemic kind of -- you know, a
16 change in many, many
of the births of many
17 children, we are not
talking about an intentional
18 modification of a
genome, we are talking about, you
19 know, this is an
incidental and unintended
20 consequence.
21 I don't have
the answer to that, but that
22 is the thing we would
be guarding against, how
23 horrible of a mora
affront or of a precedent would
24 that be? I don't have an answer to that one, but I
25 just want to lay it on
the table.
202
1 One more
challenge before us. There is
2 the Avigen issue,
which we need to answer. I guess
3 I should hold off for
No. 2, but Question No. 2 is
4 going to say, okay, it
ain't just men, it's also
5 women, and how are we
going to think about being
6 aware with women.
7 DR. SALOMON: That is Question 2, and we
8 will get to that.
9 To me, you
have raised a couple of really
10 interesting points, so
let me go back to this
11 motile sperm
thing. The problem I have with any
12 sort of testing
strategy in a clinical environment
13 is the more
complicated you make it, the more
14 difficult it is, more
expensive, more technically
15 challenged, and more
often is going to go wrong.
16 I have heard
no data that convinces me
17 that if the semen is
positive, that the motile
18 sperm could be
negative and that this is such a big
19 advantage. In the absence of that data, I continue
20 to be underwhelmed
with the need to be doing this
21 motile sperm test,
because what happens then is you
22 say the motile sperm
test, because of the fact that
23 there is a whole bunch
of manipulation, or you get
24 a patient that doesn't
have a large enough volume,
25 although I am not
quite sure why they couldn't just
203
1 dilute it up to 1 1/2
ml with saline, but anyway --
2 the bottom line here
is if you get into that sort
3 of circumstance, it
just tells you why these kind
4 of assay systems are
problematic.
5 We would
actually have to tell them -- we
6 would have to have
this, I would think rather silly
7 discussion about how
to set a quality control for a
8 motile sperm test,
because if they let it sit out
9 on the bench for a
couple hours and then they do
10 this motile sperm
test, and call it negative, and
11 the semen is positive,
well, that is bogus.
12 I am just
having trouble with this test,
13 and I don't think it
is a minor issue in terms of
14 how they are going to
do this trial.
15 DR.
MULLIGAN: Just to directly answer the
16 question, I would say
that a clinical hold would
17 not be warranted
although I would couple the
18 question in 1A to the
first part of 3, and then to
19 answer your question,
I think the motile sperm
20 thing is perfectly
okay, that if we all think that
21 the likely source of
the AAV is probably blood or
22 something, then, the
best, you can separate those,
23 I think it helps, and
I think there are SOPs,
24 people can come up
with something.
25 But 3, I
know how you hate to jump to the
204
1 next question, but I
think it has to do with at
2 what point, when
repetitive tests showed positivity
3 would everyone think
that there was something so
4 unanticipated that you
would actually want to stop
5 the trial?
6 My proposal would be no, let the
trial go
7 on, but come up with
some, maybe it's a year, throw
8 out a year, if after
one year there is still
9 positivity, even if
that was in the blood
10 mononuclear cells, I
think that most people would
11 have grave concerns
that something was happening
12 that was not
anticipated.
13 DR.
SALOMON: I followed you up until that
14 last little throw-in
about peripheral blood and
15 mononuclear cells, I
mean because I wouldn't care
16 that much of the
patient integrated into a
17 hematopoietic stem
cell and was positive.
18 I think the
real issue here is only given,
19 I don't know why a
year, because it seems like that
20 is a hell of a vector
reservoir to think that you
21 could keep shedding
detectable vector for 12
22 months?
23 DR.
MULLIGAN: That's the point. The
24 point is that
everything we have heard here
25 suggests that you
won't have persistence of the
205
1 vector for that amount
of time, simply by having it
2 sitting there in some
tissue. Therefore, something
3 is happening that is
unanticipated if it is
4 persisting at that
point, and that could be an
5 arbitrary point, but I
think a year is certainly
6 enough time to think
that something is happening
7 that shouldn't happen,
at least we don't know what
8 is happening.
9 My point was
just that a critic that would
10 say, well, yeah, that
could still be not in the
11 motile sperm, I would
say still, we would be
12 worried about the
patient because something very
13 unanticipated has now
happened, that is, if we are
14 able to get
hematopoietic stem cells transduced by
15 AAV vectors, and you
have AAV integrated, I think
16 everyone would want to
know that and would have
17 great concern.
18 DR.
NOGUCHI: Just to add more confusion
19 to that particular
point, the hemophilia trial for
20 factor VIII, and the
Chiron proposal, they did
21 report to the same
committee that in a situation in
22 which there was a very
fractional and very
23 short-lived positivity
in the semen, that, in fact,
24 they were able to
detect positive peripheral blood
25 samples for I believe
well over a year.
206
1 That was the
unexpected finding in that
2 particular case. Probably an encouraging finding
3 in terms of at least
transduction of a somatic
4 cell, I would guess.
5 DR.
SALOMON: I think that everybody in
6 this field is clear
that the amount of time that
7 even the episomes
persist, and whatever small
8 integration occurs or
whatever, in different
9 populations, I think
Jude was very good about
10 pointing out that
there is some data, but it is not
11 completely
tested. Is that a fair characterization
12 of what you said?
13 The fact
that it would be around, it could
14 be a positive for this
as a gene therapy strategy,
15 I am not concerned
about it, I think the issue has
16 to focus on the sperm
or the semen as a test. My
17 only issues there are
technical, but if it is in
18 the sperm at a year,
then, it seems to me it is
19 impossible -- well, is
it impossible that at that
20 point -- I just can't
imagine you are shedding
21 viral reservoir any
longer. The implication at
22 that point would be
that it has been integrated
23 into the germline.
24 DR.
MULLIGAN: I think the point of
25 setting some long
period at which you would stop
207
1 and take another look
is you want to address it in
2 a way that was not in
the clinical protocol.
3 DR.
SALOMON: I am agreeing. I am just
4 bringing up some
discussion points on the timing.
5 DR.
DYM: This is an unrelated comment,
6 but it is on the same
issue. With the subject No.
7 2, who has AIDS, I
think is what we were told, and
8 spermatogenesis is
markedly reduced in patients
9 with AIDS, and this is
shown, of course, in the
10 total semen volume,
200 microliter, 150, 150, it is
11 very unlikely that
this particular patient will be
12 fertile. I don't know
if that is an issue or should
13 be raised.
14 DR.
KAY: Just for a point of
15 clarification on the
patient No. 2, the patient is
16 HIV-positive, his CD4
count at the time, that last
17 one that we checked,
was around 340. He has had
18 children in the past,
and based on his total sperm
19 count, he actually has
a normal number of sperm in
20 the ejaculate, but the
volume is very low, so from
21 what we can get from
that, the spermatogenesis
22 itself is normal, but
there is something wrong with
23 the ability to make
the fluids. The pH of the
24 fluid has been normal,
suggesting that he doesn't
25 have specific
obstruction of a prostate versus
208
1 seminal vesicle.
2 DR.
DYM: I was interested in this
3 question before that
may be relevant. Did you
4 check the size of the
testis, does he have a normal
5 testis?
6 DR.
MAY: Well, since I am not the
7 clinical treater on
this case, obviously, Dr.
8 Glader, who I think
had to leave to catch a plane,
9 is the individual who
examined him.
10 DR.
RAO: I actually wanted to retread
11 what Dr. Salomon and
Dr. Mulligan said. I don't
12 think that it would be
necessary that there should
13 be a clinical hold and
that that should be
14 dependent on motile
sperm test. There are two
15 problems in my mind
with just doing the motile
16 sperm test, is that --
we have already heard about
17 the problems of vector
carryover in semen itself,
18 so even if your motile
sperm test was negative, you
19 would still worry
about that as an issue.
20 The
likelihood from the way the test was
21 presented, is that if
your semen is negative, then,
22 your motile sperm
fraction is going to be negative.
23 So it doesn't seem
that we should be focusing on
24 motile sperm test as a
specific test, but rather on
25 total semen.
209
1 Do we
necessarily need to -- which was
2 part of Question 3,
which you put together -- it
3 didn't seem to me that
you should include only
4 patients who are
incapable of the production, but
5 there should be a time
line and that patient
6 consent, the forms
should be modified with an
7 emphasis that this is
a problem.
8 DR.
JUENGST: I guess my hesitation in
9 originally answering
your question was because I
10 was hung up on the qualifier,
"or should enrollment
11 be allowed to continue
with appropriate
12 modification made to
the consent documents."
13 Well, no,
that is not enough to modify the
14 consent
documents. It is really a change in the
15 protocol because
informed consent is not a
16 reasonable and prudent
safeguard. Patients behaving
17 appropriately
cautiously is a prudent and
18 reasonable safeguard.
19 So they need
to be more than simply read a
20 few extra lines in the
consent form. It needs to
21 be a real concerted
program for making this
22 education and sperm
banking available, and that
23 sort of thing.
24 It sounds
like what they have been doing.
25 DR. SALOMON:
It sounds like that has been
210
1 a part of their
protocol even a trial or two
2 earlier, which is
good. I think we are getting
3 there.
4 Does the
committee agree that a clinical
5 hold is warranted when
motile sperm tests are
6 positive -- let's just
call it now "semen" -- you
7 know, if we could vote
on this, but I generally
8 don't want to go
there, I mean that is a decision I
9 think that the FDA can
come back to us. We have
10 had a discussion on
this motile sperm versus semen,
11 I am comfortable with
the semen.
12 If there is
a clinical hold, is it
13 warranted? The committee to me has said no, that I
14 don't think you need
to put a clinical hold on this
15 trial every time the
semen is positive. Does
16 anyone want to take a
minority opinion here?
17 DR.
NOGUCHI: On that one, we might want
18 to at least have a
show of hands to make sure we
19 understand the real
kind of sense of the committee.
20 DR.
SALOMON: Then, we will poll. Do you
21 want us to give you a
specific on the motile sperm
22 versus semen?
23 DR.
NOGUCHI: I think that is very
24 important. It certainly seems like there is enough
25 discussion that there
is some doubt about the extra
211
1 value of fractionated
versus just whole semen. I
2 think it would be
worthwhile for that, too.
3 DR.
SAMULSKI: If we were going to poll, I
4 think it is probably
important that I at least make
5 one or two statements
about the vector aspect of
6 it.
7 I am not going to proclaim I know the
8 answers, but I have
been working at this for over
9 20 years, and there
are definitely trends that show
10 up that are extremely
consistent, that you can
11 begin to feel
confident about, and the virus
12 integration is a trend
that has been consistent
13 from when this was
first studied and now going from
14 tissue culture to
animal models, it doesn't
15 integrate very
efficiently.
16 I think
people need to understand that and
17 buy into the fact that
if we are going to put a lot
18 of virus into people,
the potential of integration
19 is virtually nil to it
can happen, but it is not a
20 high risk potential,
and then if you move away from
21 that question and look
at the question of if we
22 have a PCR-positive
signal, which is something like
23 10 copies in a sample,
and we are talking about 0.1
24 percent of the virus
ability to integrate, we are
25 getting down to
numbers and the amount of sperm
212
1 that one is going to
be transmitting, this is like
2 Star Wars in some
ways, trying to calculate what is
3 the frequency of the
planets lining up again, and
4 stuff like that.
5 It is so
vanishing small, the risk that we
6 are talking about,
that from a vector perspective,
7 I think there is no
reason at all to put this trial
8 on hold. Where I do have my only concern, and it
9 sounds like it is
being addressed, is that Phil
10 brought up, which is
if you are mechanically going
11 to have virus tracking
along, and you are going to
12 do experiments to see
if you can come up with a way
13 of artificially
getting this into cells, that is an
14 unknown that needs to
be resolved, and I think the
15 onus will fall on the
group that is interested to
16 get that data in front
of people as soon as
17 possible.
18 But other
than that, I am sitting here
19 saying we are really
discussing something that is
20 virtually impossible,
and I think that value of
21 what can come out of
the studies is a lot more
22 important than us
trying to talk this tightrope.
23 DR. SALOMON: I very much agree with that,
24 too, and I think I am
comfortable that the flow of
25 this committee is
going in that direction, as well.
213
1 You brought
up one thing, Jude, that I
2 just wanted to come
back to, and that is you
3 suggested, I don't
know if you meant it, additional
4 sophistication on this
testing strategy, and that
5 would be do
quantitative PCR, and if it is lower
6 than a certain number,
even that then could enhance
7 one's comfort
level. I hadn't thought of that,
8 because up until now,
we have been talking about
9 positive versus
negative.
10 Would you be
concerned if it came out as a
11 million copies? I mean these guys are detecting it
12 positive at the lowest
levels at 14 weeks.
13 DR.
SAMULSKI: So that is where I think it
14 is prudent, and it is
not just AAV when you keep
15 dosing biologics,
whether they are plasmids of
16 oligos, at some point
you are going to have a
17 threshold level where
it is going to be
18 unpredictable.
19 I think that
is the kind of information I
20 would like to see keep
coming out of this trial, he
21 didn't just persist
longer, but here is how he
22 persisted, it was over
1,000 or 10,000 copies per X
23 amount of time, and
stuff like that. That is
24 valuable information.
25 DR.
SALOMON: I agree with that, and I
214
1 would say that I was
not impressed by a statement
2 that it wasn't
absolutely quantitative, but the
3 signal was going
down. I think today, there are
4 very straightforward
ways to do quantitative PCR,
5 so we don't have to
really, in these settings, to
6 any longer talk about
nonquantitative PCR studies.
7 Let's do a
poll of two questions. One
8 would be the first,
and that would be motile sperm
9 versus semen testing,
just to get that out of the
10 way.
11 Starting
with you Jude, motile sperm or
12 whole semen?
13 DR.
SAMULSKI: I think whole semen is
14 adequate.
15 DR.
DYM: Yes, same. Not necessary to
16 differentiate between
the two.
17 DR.
JUENGST: For what it is worth, my
18 layman's vote will go
with these guys. We don't
19 differentiate.
20 DR.
MURRAY: I confess I do not understand
21 the merits of the two
tests sufficiently to make an
22 intelligent vote, but
it is not because I am torn
23 between two
alternatives I understand well.
24 MS.
WOLFSON: I would have to repeat
25 exactly what Dr.
Murray said.
215
1 DR.
RAO: I think whole semen.
2 DR.
SALOMON: I vote whole semen
3 obviously.
4 I am not
certain. Richard gave me what he
5 would say for the
second one, but I am not
6 comfortable with that,
so we will have to say he is
7 not here.
8 Let's do the
second question, and that
9 was, does the
committee agree that a clinical hold
10 is not warranted any
longer with a positive semen
11 test? Jude.
12 DR.
SAMULSKI: Not warranted.
13 DR.
DYM: A hold is not warranted, I
14 agree.
15 DR.
JUENGST: A hold is not warranted.
16 DR.
MURRAY: I agree that with the proper
17 additional protections
put in place, with Eric's
18 earlier caveat about
it is not just adding lines to
19 informed consent, we
would not require a hold.
20 MS.
WOLFSON: Again, I agree with Dr.
21 Murray exactly.
22 DR.
RAO: I don't think a hold is
23 necessary.
24 DR.
SALOMON: And I don't agree a hold is
25 necessary either. Richard Mulligan told me that he
216
1 also wanted to say
that a hold is not --
2 MS.
DAPOLITO: Dr. Mulligan should be here
3 to vote, but Dr.
Salomon can put his comments into
4 the record.
5 DR.
SALOMON: Excellent.
6 The next
question here would be discuss
7 the implications of
detecting vector sequences due
8 to the presence of
contaminating transduced PBMC or
9 vector (either free or
on the surface of a sperm)
10 in the motile sperm
fraction.
11 Now, my
sense here is we have really
12 pretty much discussed
that. If the FDA comfortable
13 with that? I don't see any further discussion as
14 being necessary on
that one.
15 Anyone else
on the committee?
16 [No
response.]
17 DR.
SALOMON: Okay.
18 Question
2. There are technical
19 limitations in the
ability to monitor women and
20 certain men for
evidence of germline alterations.
21 One approach to
monitoring subjects for germline
22 alteration would be to
restrict early clinical
23 development of certain
gene transfer products to
24 subjects who have been
shown to be capable of
25 repetitively supplying
adequate semen samples for
217
1 analysis in order to
get good data collection for
2 detecting persistence
of vector.
3 DR.
MURRAY: That does leave out a certain
4 number of potential --
about 50 percent of the
5 human population
actually, which is not relevant
6 for the Avigen trial,
but will be relevant for
7 others.
8 DR.
SALOMON: Correct.
9 I am going
to try and parse it down. So
10 the first issue would
be -- I think you guys
11 actually had a slide
on this, am I mistaken? You
12 had a slide saying
that you were redesigning -- you
13 had to have more than
1 1/2 ml of semen and a
14 certain sperm count,
wasn't that right?
15 They have
already incorporated in their
16 protocol, does anyone
disagree with that?
17 DR.
SAMULSKI: I think it's admirable
18 that they are doing
it, but i also agree with Dr.
19 Murray. I don't think this is something you want
20 to put in as policy
because if other things come
21 down that aren't
related to hemophilia and the
22 population can't be in
an inclusion criteria, but
23 based on something
like this, I don't think that
24 would be a direction
we would want to go in.
25 DR.
SALOMON: Certainly, men who were
218
1 infertile and would
have no danger of germline
2 transmission would be excluded from these studies,
3 so I think I could
think of one very good reason
4 not to make it policy
in the area.
5 Dr. Rao.
6 DR.
RAO: I was just going to add that one
7 reason for worrying
about sample size was because
8 we were doing motility
experiments and
9 fractionation. Now that the committee seems to
10 have a consensus that
we don't need to do
11 fractionation, I think
the tests can be done with a
12 smaller volume, so I
don't think that should be an
13 exclusion criteria.
14 DR.
SALOMON: All you need now would be
15 enough sperm to get,
let's say, 1 microgram of DNA
16 -- I am sorry -- 3 to
4 micrograms of DNA, so you
17 could do triplicate or
quadruplicate at 1
18 microgram, and from my
own experience doing Tacman
19 PCR, which we do a lot
of in the lab, I would be
20 comfortable with that,
as well.
21 So, that
would be a sperm count of what?
22 Were you guys counting
30,000 sperm for a microgram
23 of DNA?
24 DR.
SAMULSKI: 2 x 106.
25 DR.
KAY: Yes, we were using 2 to 3 x 105
219
1 sperm per microgram
roughly, and we need 4 or 5
2 micrograms, and then
the issue is recovery.
3 DR.
SALOMON: Would everyone agree there
4 should be a limit of,
let's say, no less than 5
5 million total sperm in
an ejaculate as criteria for
6 entering the trials at
this point, 10 million? All
7 right.
8 DR.
KAY: Sold.
9 DR.
SALOMON: Any discussion on that
10 point? I think the intention of the committee is
11 clear, and the details
we would leave to you.
12 Depending on
the amount of data required,
13 much of the early
clinical experience with the
14 vector might be
limited to this restricted
15 population. A development program requiring
16 extensive
characterization of distribution to
17 germline cells and
germline alterations might delay
18 the acquisition of
adequate safety and efficacy
19 data in other
populations, for example, women.
20 I guess we
can't avoid the very important
21 discussion of where
women fit into these trials.
22 So this gets to
something that Tom, you introduced
23 to us in your
comments, thoughtful comments a few
24 minutes ago, and that
is, what is our level of
25 sensitivity here, how
big of a deal would it be,
220
1 and that has a lot to
do with the next trial that
2 comes along that wants
to do the study in women,
3 right?
4 DR.
MURRAY: Any disorder that is not
5 x-linked.
6 DR.
SALOMON: Or a male that has no sperm
7 or falls below it, we
need to think about them, as
8 well.
9 The
interesting thing here is I mean we
10 are now up against the
international consensus that
11 has been supported by
many in the lay public, that
12 the line that no one
is ready to cross is
13 intentional? Germline transfer certainly. And
14 unintentional germline
transfer is probably far
15 enough across that
line that it ought to be avoided
16 at all costs, as well.
17 I am very
cognizant of the fact that
18 depending on how this
discussion goes, we have to
19 be cautious that we
are having an advisory
20 committee advising the
FDA that under certain
21 circumstances, it is
okay to do germline gene
22 transfer, and I want
to put that into context for
23 the committee. You can disagree with it, but that
24 is the question.
25 DR.
MURRAY: There is one piece of advice
221
1 I think we could give
the FDA. It may not be very
2 useful advice, but
that is to --perhaps you have
3 already done it, --
but basically to take stock of
4 the best possible methods for evaluating
germline
5 alterations in
females, looking at animals, looking
6 at -- we have heard a
variety of ways of thinking
7 about looking at such
alterations in males, what is
8 the best
state-of-the-art in thinking about this
9 with females? Granted that many of those assays
10 are going to be
completely unavailable with humans,
11 to think about what
would be the most -- morally
12 permissible and
without crossing the boundaries of
13 mistreating human
subjects, how to involve women,
14 how to monitor
potential germline genetic
15 alterations in women.
16 I don't
think that is easy, maybe you have
17 done it, but if you
haven't, I would say it is
18 probably an urgent
thing. The one thing I could
19 not recommend -- we
had a tough choice here -- but
20 one think I clearly
could not recommend is that
21 women be excluded from
such trials. I suspect
22 national policy would
also prohibit us from making
23 such a recommendation.
24 DR.
NOGUCHI: Just in terms of that, FDA,
25 unless there is a
compelling reason to exclude one
222
1 sex or another or any
particular subset of man or
2 woman, we would not
use anything other than a very
3 specific reason that
is both reasonable and can be
4 defended.
5 DR.
JUENGST: Another thing to think about
6 is where along the research
time line we would be
7 willing to take those
risks of inadvertent
8 transduction. We do take genetic risks with
9 patients when we give
them chemotherapy and
10 radiation, and we
justify that by saying, well, we
11 are saving their
lives, so in a Phase III trial of
12 gene therapy, we could
make a very similar
13 argument.
14 Now, how
about a Phase I safety study,
15 maybe there is a
distinction to be drawn there
16 unless we find
ourselves in the situation where you
17 can't get to Phase III
unless you do the Base I.
18 DR.
SALOMON: That's a good point. Just
19 to highlight that you
are pointing out that the
20 quote, "We are
doing it because we save human
21 lives" is a clear
indication of conviction of
22 efficacy, which would
allow you to accept a risk
23 that is very different
than in a Phase I or Phase
24 II trial.
25 Dr. Rao.
223
1 DR.
RAO: I was just going to say that the
2 general consensus that
you want to be able to
3 monitor, it is not an
issue of whether it can
4 happen or not happen
or the probability, but that
5 you want to have an
ongoing monitoring to make sure
6 that there is no
evidence of germline transfer, and
7 should we be excluding
patients where we can't
8 monitor that, and the
answer is that I think we are
9 doing that already
when you set up a criteria,
10 whether you set it for
males when they can't have a
11 certain sperm count,
or whether you can't because
12 you don't have any
available tests to do that
13 monitoring.
14 The question
is do we not need to monitor
15 at all. In my opinion, right now, with the
16 available data, it is
not clear because there is
17 still enough not known
about the virus in the sense
18 what is happening in
blood cells, why do we see
19 persistent expression,
is there some specific time
20 at which you see
better integration, and so on.
21 So there is
a finite, maybe already low
22 probability that there
might be germline transfer,
23 but whatever that low
probability, at the current
24 situation, with what
data is available from animal
25 models, we can't say
that we should include
224
1 patients where we
don't have any monitoring.
2 DR.
SALOMON: If you think about it, there
3 is a couple different
ways this could go. None of
4 us, unless some -- I
can't imagine are going to
5 give advice that you
want to permanently exclude
6 women from certain
kinds of trials, right, why even
7 go there, that's
impossible, so you can see a
8 couple different ways
to try and put this together,
9 and I think the
framework is already out on the
10 table, one way is to
say in the absence of really
11 good definitive
preclinical data that would allow
12 you to say with any
sort of confidence it cannot go
13 into the germline, and
I think we all agree you
14 cannot say that quite
yet, no evidence that it
15 does, but no evidence
to say that is can't, and a
16 lot of evidence
suggests that it ain't going to be
17 easy and not likely,
particularly with this
18 particular class of
vectors.
19 You could
say okay, really low
20 possibility, so that
takes a lot of the pressure
21 off, but it is not
enough. So then you go on and
22 say all right, fine,
let's go into Phase I trials
23 and let's restrict the
Phase I trials to subject
24 that we can monitor.
25 I think the
company themselves, to their
225
1 credit, have taken
that view, and the FDA is
2 comfortable with it,
and I think we have just
3 refined it a little
bit.
4 Then, the
only question left is how much
5 data do we need under
our belt before you allow in
6 later phases of the
trial, to go into women, pretty
7 much saying, hey, it's
not happening, and I don't
8 want to get into
demanding someone give us
9 statistical time, like
after 100.3 patients we can
10 do it, but I think I
am suggesting to you that
11 maybe the best way to
think about this is at a
12 certain point, once
they get to a Phase III trial,
13 and there is enough
confidence that none of these
14 patients with no
evidence of germline transfer in
15 these males that can
be monitored, that then you
16 could relax the
criteria and cautiously open it up
17 to women.
18 So that
would be what I would suggest.
19 MS. CHRISTIANSON: Janet Rose
20 Christianson. QARA Services, formerly with Target
21 Genetics Corporation.
22 A brief
comment with regard to selecting
23 people for monitoring
in Phase I. I think there
24 has got to be another
consideration, and I think
25 that has to do with
the route of administration.
226
1 If there is no
dissemination, for example, the
2 present study that
Target is doing is an oral
3 aerosolized delivery
of an AAV vector in cystic
4 fibrosis
patients. I think that the way it is
5 delivered, and any of
the preclinical data,
6 indicating if there is
dissemination to the
7 peripheral blood
distal nodes, or whatever, should
8 also have a bearing as
to whether or not monitoring
9 of females or
nonfemales or whomever, should be
10 part of that whole
process. I think that has got
11 to be a point just to
consider. Maybe my glucose
12 was low and it's
intuitively obvious, but I did
13 want to make sure that
that point was raised.
14 Thank you.
15 DR.
SALOMON: I think that is an excellent
16 point.
17 Dr. Rao.
18 DR.
RAO: I actually wanted to add one
19 more piece to the
whole monitoring issue, and that
20 was just simply to
argue that if the criteria or
21 the worry for which
you are excluding patients is
22 because of germline
transfer, that perhaps one
23 additional criteria
for inclusion is people who
24 would not be, are
incapable of germline transfer.
25 DR.
SALOMON: The only problem with that
227
1 is, it is kind of a
dead end in terms of moving the
2 field forward, because
you would never be able to
3 tell whether there was
germline transfer, so
4 everyone else would be
standing there waiting.
5 DR.
RAO: Maybe you shouldn't exclude them
6 for whatever reason
you want to include them in a
7 study. That is all I
was trying to say.
8 DR.
MURRAY: In a way, we should be very
9 grateful to the folks
from Avigen for their
10 inadvertent finding,
because it really forces us to
11 confront -- I don't
mean just this committee, by no
12 means do I mean just
this committee -- I mean
13 everybody who thinks
about these larger issues of
14 the ethics of
research, inadvertent germline
15 transfer, and gender
equity in research, and all
16 these things, it warns
us about what is probably
17 lurking not too far
down the road, and in addition
18 to my off-the-cuff
injunction to FDA to sort of
19 think as much as they
can, I don't think this
20 committee is the group
to decide what the right
21 balance is, but in
fact, I mean RAC has had a
22 recent history of
doing policy conferences.
23 This would
be a great topic for a RAC
24 policy conference, in
my view, about how to balance
25 the concern about
monitoring inadvertent germline
228
1 modification against
an issue of gender equity, is
2 it as we think it is,
that it would be much more
3 difficult to monitor
in women, are we wrong about
4 that? Are there ways of monitoring this in women?
5 I am not aware of any,
but, you know, there are
6 some fact questions
there science questions, and
7 then how should one
sort of strike the right policy
8 balance.
9 One emerging
suggestion, I think I have
10 heard, is that you do
the Phase I -- where this is
11 a possibility -- you
do the Phase I on males who
12 have sufficient
seminal fluid and sperm that you
13 can test, they make 5
million sperm in an
14 ejaculate.
15 Now already,
that creates some issues of
16 gender inequity, I
understand the rationale for
17 that, but I think it
would be a mistake to rush
18 forward into that
without a chance to really
19 reflect on how to
balance.
20 There are
two good things. We are trying
21 to ensure gender
equity and participation in
22 research, and we are
trying to ensure that we can
23 get a handle on
inadvertent germline gene transfer.
24 There are two good
things. Somebody has got to
25 figure out what the
right balance or plan is, and
229
1 it is not a thing we
are going to do by 3 o'clock
2 today, and we are not
the right party to do that.
3 DR.
NOGUCHI: Steve, shall we work on that
4 as a possibility, what
Dr. Murray is talking about?
5 DR.
ROSE: Certainly, it is something that
6 the RAC has been
discussing and will continue to
7 discuss, and it is one
of the policy conferences we
8 have been thinking
about.
9 DR.
NOGUCHI: You are welcome to come,
10 too, Tom, I am sure,
and probably most of the rest
11 of the people at the
table here.
12 DR.
SALOMON: Jon.
13 DR.
GORDON: Yes, I am commenting. I
14 have recused myself
from this discussion because as
15 a committee member,
and I am commenting as a member
16 of the public.
17 I think
there are a couple of points. One
18 is that whenever you
exclude a certain group of
19 people from a study,
regardless of the phase of the
20 study, you at least
have to be alert to the
21 introduction of biases
in the study, so I think
22 people need to be
aware of that.
23 Is it going
to be more safe for the people
24 you study or less
safe? I don't think it is
25 necessarily relevant
in the present case, but
230
1 anytime people are
excluded in some sort of
2 overarching parameter,
then, that is a risk.
3 In terms of
the addition of females to
4 these trials in
hemophilia, not a likely issue to
5 come up, but as people
have point out, autosomal
6 disorders it is, I
think the committee might
7 consider recommending
that good preclinical tests
8 for female germline
transmission be encouraged to
9 be developed.
10 I mean it is
not impossible to do that,
11 and there is no reason
why, if we have been doing
12 all these things with
rabbits and monkeys and all
13 that with the male
side, why we couldn't also do
14 things on the female
side.
15 We have a
paper where we looked at adeno
16 at the female side, so
there is no reason why that
17 couldn't be encouraged
by the committee.
18 DR.
SALOMON: I would actually not want to
19 go there. I don't think as a committee, we want to
20 start even getting
into whether women as part of
21 participation in a
trial ought to undergo
22 laparoscopy and
removal of eggs or ultrasound
23 guided biopsies, and
things like that, if that is
24 what you were
suggesting. I think those are topics
25 for preclinical
investigations and not for creating
231
1 yet more complex and
even potentially risky
2 barriers for participation in a trial.
3 DR.
GORDON: I guess I wasn't clear. I
4 believe, I emphasize
preclinical studies in animals
5 that would then give
one more confidence that a
6 human could be
admitted to a study.
7 DR.
NOGUCHI: Just to say that the issue
8 of women is pertinent
to this discussion, albeit
9 it, it is an
extraordinarily small population,
10 there are handful of
women with hemophilia, and for
11 them, especially they
are totally out of any of the
12 normal support
mechanisms. They may not even know
13 what hemophilia is
because it is not something that
14 they normally know
about, but eventually when one
15 of these things works,
they are a part of the
16 question. We will have the same question as to
17 whether or not it is
an unreasonable risk for that
18 population albeit it
might be as many as on this
19 hand here.
20 DR.
SALOMON: I think that at the end,
21 there is no way --
again, I welcome everyone to
22 comment -- from my
view, I don't see how one can
23 refine this any
further in the sense that it has
24 been put very clearly
that, on one hand, the
25 concept of germline
transfer as a potential in a
232
1 clinical gene therapy
trial, there has been a lot
2 of discussion on that,
and it is pretty much
3 considered to be a
line that the public doesn't
4 want us to cross, and
I think we have to respect
5 that.
6 At the same
time, however, we realize that
7 as we gain experience
and information, we can begin
8 to feel more and more
confident that that is not
9 occurring even though
the risk may never be zero,
10 and, of course, we
will get into discussion and so
11 I might as well bring
it up, that if you show it
12 doesn't happen in the
males, does that mean that it
13 won't happen in the
females, and, of course, female
14 biology is very
different than male biology, we all
15 realize that.
16 I think
there we need to put more energy,
17 I think as John and
others have already said, and
18 to some of the
preclinical models anticipating what
19 is around the corner
for this field, and that I
20 think a reasonable
leadership position from the
21 committee.
22 I guess the
last thing we have to talk
23 about, and if there is
anything else, please jump
24 in, but the last thing
I feel we have to talk about
25 is okay, so we come
back in here a year from now,
233
1 and we get presented
data from company XYZ now, it
2 is not Avigen any
longer, but they did a trial like
3 this and 10 of the
first 50 patients are
4 persistently positive
in their semen at one year,
5 and so they do an
in-situ hybridization on motile
6 sperm on these
particular 10 patients, and 8 of
7 them are positive in 10 percent of the
sperm. Now
8 what?
9 DR.
DYM: I will answer the question by
10 asking a question of
the virologists. Does that
11 clearly mean that it
is coming from the earlier
12 germ cells, or can the
virus persist?
13 DR.
SALOMON: We might as well get that
14 question on the
table. Jude, do you want to
15 comment on that?
16 DR.
SAMULSKI: My feeling would be that it
17 would have to be in a
germ cell to persist that
18 long and consistently
come up positive, and for it
19 to just persist, it
would get diluted with time.
20 All those cells kept
dividing. So this would be
21 the same as the trial,
they would come down over
22 time, so I think you
are now talking about a
23 completely different
situation.
24 DR.
SALOMON: And they do a testicular
25 biopsy and it is
positive in the spermatogonia.
234
1 Now what?
2 DR.
JUENGST: It's at least time to stop
3 and take stock and
look at where the gene is being
4 integrated, you know,
study what is happening, if
5 it is consistent,
those sort of things.
6 DR. SALOMON: That's fine. Remember
what
7 Dr. Samulski pointed
out very clearly is that there
8 is no evidence that
these vectors will integrate in
9 some specific
spot. They will integrate in some
10 specific spot. They will integrate in multiple
11 concatemers in many
areas.
12 DR.
RAO: There is two aspects to this.
13 You don't know what
you are doing now because the
14 assumptions are wrong
in some sense. You assume
15 that there will be a
very low probability of
16 integration, there
wouldn't be germline
17 transmission, and that
if it did occur, there will
18 be a clear-cut barrier
and it wouldn't be 10
19 percent. So that I think is pretty clear.
20 The question
then is what do you do with
21 the participants,
right? I mean what happens with
22 the 10 patients that
were persistently positive and
23 who presumably have
germline transmission, and that
24 I think is a very hard
question. I don't know that
25 the FDA has any
authority and whether we can do
235
1 anything after the
fact.
2 DR.
SALOMON: That, we know the answer to
3 that. They can't do anything. But the question
4 would be now, 50 of
the 50 patients haven't had a
5 bleeding episode in
the last six months.
6 DR.
MURRAY: So, it worlds.
7 DR.
SALOMON: It works.
8 DR.
MURRAY: I think this is not a
9 far-fetched
hypothetical life here. There are
10 scientists here who
understand different vectors
11 that may, in fact,
operate very differently even
12 than AAV, if I
understand correctly, and some of
13 then might be much more
likely to incorporate to
14 work themselves into
spermatogonia, and so this
15 scenario with the
different vector system might not
16 be so far fetched at
all.
17 So you have
done right by the committee to
18 ask this extremely
difficult question. I don't
19 feel at all qualified
to answer it right now. I
20 would have a number of
other questions. I would
21 want to know, look, we
are talking about a
22 potential random, you
know, incorporations at some
23 random place in the
genome of foreign DNA.
24 I would like
to know how many copies
25 integrated, are we
talking about 1, are we talking
236
1 about 1,000 in each
genome? If it is thousands, it
2 would seem to me that
increases the chance that
3 some of these
mutations are, in fact, may be
4 pathological. A thousand hits is more than one
5 hit.
6 Do we have
any analogies? Are there other
7 bits of DNA that get
incorporated into the genome
8 in a similar random
fashion, and how do they -- and
9 spermatogonia, and
what we do know about their
10 fate, and what do they
know about the impact they
11 might have on the
health of any child born. If it
12 is absolutely
horrendous, then, that is one thing,
13 if it is, well, it
happens all the time, and rarely
14 really leads to any
harm, that is another thing.
15 So, there
are still a lot of factual
16 questions we would
ask. That will help, I think,
17 help us sort out, but
you are right, we should be
18 thinking about them
now.
19 DR.
GORDON: As a member of the public, I
20 would like to sort of
suggest that the committee,
21 in facing such a
circumstance, should consider this
22 the way other risks of
drug treatment are
23 considered. Now, if you give cisplatinum or
24 bleomycin to somebody,
you can probably damage
25 their DNA, or adriamycin
to them, and there are
237
1 precautions to be
taken.
2 In the case
of germ and a gene transfer,
3 which I think is a
little bit exceptional because
4 you provide
acquisition of function, not simply
5 alteration in the
existing genome, there are
6 precautions to be
taken before the procedure is
7 performed, and there
are precautions that can be
8 taken if, in the
event, such a thing is discovered.
9 If 10
percent of sperm had a new gene in
10 them, that would mean
that there is a 10 percent
11 chance that a
conceptus would have it, let's say,
12 presuming those sperm
function equally well, well,
13 there are people
carrying recessive traits around
14 where there is a 25
percent chance that there is
15 actually going to be
genetic disease, and there are
16 approaches to that
problem - pre-implantation,
17 genetic diagnosis,
conception followed by abortion.
18 So there are
ways of addressing it if it
19 occurs, but I think
the committee is well advised
20 to consider that a
hold should be placed while
21 those considerations
are formalized.
22 DR. SALOMON:
I think that is what Dr. Rao
23 said. I mean I realize this is -- I think the
24 major point that I was
getting at was just to
25 introduce the
question, and I think there has to be
238
1 a limit, and I think
that limit for me is that. I
2 mean if the hypothesis
is wrong for any vector now,
3 I am not talking about
the Avigen trial, then,
4 probably we should put
it on hold and there should
5 be discussions at the
highest level, whether it be
6 at the RAC, or be
here, or in every place.
7 That
includes the appropriate science
8 experts, as well as
policy people and ethicists,
9 because I think there,
we really have crossed a
10 line that has been set
for us in gene delivery.
11 I think what
is critical to the FDA,
12 though, unless someone
wants to disagree with me,
13 is the advice that you
better make sure that any
14 trial that you allow
to go forward is adequately
15 designed to make sure
that you don't miss this from
16 happening. I mean if you are going to recognize
17 it, the trial had
better be designed and monitored
18 properly enough to
make sure we recognize it,
19 because then, you have
got to deal with these
20 issues as a reality
instead of as a theoretical
21 risk.
22 Any other
comments from the committee,
23 from the public? Does the FDA feel like we have
24 answered their
questions? Are there any additions
25 or refinements, et
cetera, that we should deal
239
1 with?
2 DR.
NOGUCHI: Once again, on behalf of
3 CBER, I do want to
extend our heartfelt gratitude
4 for helping us over
our current and future
5 difficult issues that
we seem to face on an
6 increasingly more
frequent level.
7 I think the
discussion yesterday and today
8 is going to enable us
to move forward in a much
9 more cohesive and
responsive and responsible way,
10 and for that I only
can say we are again very
11 thankful.
12 Based on
your last comments and the
13 questions you raise,
however, Dan, I don't think
14 you are going to
necessarily be able to get away
15 from this committee
that easily, so I am sure we
16 will see you again.
17 DR.
SALOMON: There are laws that will
18 govern eventually.
19 Dr. Couto,
did you have a comment?
20 DR.
COUTO: Well, it is actually a
21 question that I wanted
to just ask the committee,
22 because it was raised
earlier, and that has to do
23 with the most optimal
PCR assay for detecting
24 vector sequences in
semen, because it was raised
25 that maybe a better
assay would be a quantitative
240
1 PCR assay.
2 One of the
reasons why we are not doing
3 that now is because
the FDA asked us to develop a
4 spiked plasmid that
has a deletion in the coating
5 region, and so that we
could differentiate between
6 our vector sequence
and our spiked sequence.
7 Now, we
wouldn't be able to do that with a
8 quantitative PCR
assay, but as you have seen, most
9 of our other assays,
biodistribution studies are
10 all done with
quantitative assays, so I guess I
11 would just like a
little bit of clarification as to
12 what people think
would be the best assay in the
13 clinical sample.
14 DR.
SALOMON: There is two answers to you,
15 but I mean there is
certainly multiplex PCR where
16 you could design your
probe. If they wanted the
17 spiked sample deal
going, I mean you could easily
18 do that these days,
and I can help you figure out
19 how to do that if you
don't know.
20 Dr. Rao.
21 DR.
RAO: I was just going to add exactly
22 the same thing in some
sense is that spiking is a
23 method of
quantitation, so it is a quantitative
24 method of estimating
what you have against a known
25 standard of DNA, so I
don't think there is
241
1 confusion. I mean you can even do spiked multiplex
2 PCR on a quantitative
fashion using Tacman type of
3 assays if you want to.
4 DR.
SALOMON: If you did a Tacman assay, I
5 mean just to educate
me a little, if you did a
6 well-validated Tacman
assay or I mean there is now
7 other technologies, I
am not doing a commercial
8 blurb for Tacman, just
quantitative PCR based,
9 there is fluorescence,
there is Cybergreen, there
10 is a bunch of
different ways of doing it.
11 If you did
that, what is the spiking thing
12 for?
13 DR.
TAKEFMAN: Well, the spiking, we just
14 say run samples
without spike, but one sample at
15 least with the spike
just to test for inhibitory
16 effects. So you could run QPCR on some of the
17 samples.
18 DR.
SALOMON: Just that same control, that
19 was the main
reason. I mean sometimes you need
20 spiking because there
is endogenous transcripts
21 that are confusing
your sample, but that is not the
22 issue here when you
are looking at vector.
23 DR.
NOGUCHI: No, semen does have a
24 history of sometimes
inhibiting viruses. HIV
25 detection in semen
actually for many years could
242
1 not be done because of
inhibition.
2 DR.
SALOMON: That point is well taken.
3 We have been suffering
with serum and plasma for
4 stuff in my lab, looking
at retrovirus, that is
5 well taken.
6 Did we miss
anything? I mean is there
7 anything else people
want to get on the table here
8 at the last
minute? No.
9 Again, I
want to thank everyone at the
10 committee table, and
Avigen particularly. I hope
11 we haven't beaten you
up too bad, but I think you
12 are going home with
pretty much you were hoping
13 for, and I hope for
the community that your studies
14 go safely first and
then demonstrate efficacy next,
15 as I think it is clear
that your stakeholders need
16 a viable therapy. If it's not Avigen, then, let's
17 pray it is going to be
for somebody else doing gene
18 delivery doing it.
19 Anyway, good
luck. Good luck to everyone
20 else out there. Travel safe and be healthy.
21 [Whereupon,
at 2:03 p.m., the proceedings
22 were adjourned.]
23
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