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