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
Thursday, May 9, 2002
8:00 a.m.
Hilton Hotel
Gaithersburg, Maryland
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BEEN EDITED OR CORRECTED BUT APPEARS AS RECEIVED FROM THE COMMERCIAL
TRANSCRIBING SERVICE. ACCORDINGLY, THE
FOOD AND DRUG ADMINISTRATION MAKES NO REPRESENTATION AS TO ITS ACCURACY.
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P A R T I C I P A N T S
Daniel R. Salomon, M.D., Acting Chair
Gail Dapolito, Executive Secretary
Rosanna L. Harvey, Committee Management Specialist
Members:
Bruce R. Blazar, M.D., Industry Representative
Katherine A. High, M.D.
Richard C. Mulligan, Ph.D.
Alice H. Wolfson, J.D., Consumer Representative
Alison F. Lawton, Consumer Representative
Mahendra S. Rao, M.D.,
Ph.D.
Temporary Voting Members:
Lori P. Knowles, L.L.B., B.C.L., M.A., LL.M.
Thomas F. Murray, Ph.D.
Robert K. Naviaux, M.D., Ph.D.
Eric A. Shoubridge, Ph.D.
Jonathan Van Blerkom, Ph.D.
Edward A. Sausville, M.D., Ph.D.
Eric A. Schon, Ph.D.
Guests and Guest Speakers:
Robert Casper, M.D., Ph.D.
Susan Lanzendorf, Ph.D., H.C.L.D.
Marina O'Reilly, Ph.D.
Jacques Cohen, Ph.D.
Amy Patterson, M.D.
Stephen M. Rose, Ph.D.
FDA Participants:
Jesse Goodman, M.D.
Philip Noguchi, M.D.
Scott Monroe, M.D.
Mercedes Serabian, M.D.
Jay B. Siegel, M.D.
Deborah Hursh, Ph.D.
Malcolm Moos, M.D.
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C O N T E N T S
PAGE
Session I:
Update Research Program:
Laboratory of Gene Regulation, Amy Rosenberg, M.D. 5
Laboratory of Immunobiology, Ezio Bonvini, Ph.D. 15
Session III:
Welcome and Administrative Remarks,
Daniel Salomon, M.D. 28
Presentation of Certificate of Appreciation to
Dr. Edward Sausville, Jay P. Siegel, M.D. 34
Ooplasm Transfer in Assisted Reproduction:
FDA Introduction
Deborah Hursh, Ph.D. 41
Cytoplasmic Transfer in the Human
Susan Lanzendorf, Ph.D. 48
Question and Answer 61
Ooplasm Transfer
Jacques Cohen, Ph.D. 100
Question and Answer 136
Transmission and Segregation of mitochondria DNA
Eric Shoubridge, Ph.D. 167
Mitochondrial Function and Inheritance Patterns
in Early Human Embryos
Jonathan Van
Blerkom, Ph.D. 199
Question and Answer 224
Ethical Issues in Human Ooplasm Transfer
Experimentation
Lori Plasma Knowles, LL.B. 257
Open Public Hearing:
Jamie Grifo, M.D., American Society
for Reproductive Medicine 278
Pamela Madsen, American Infertility
Association 283
Questions to the Committee 287
4
1 P R
O C E E D I N G S
2 DR.
SALOMON: Welcome this morning to the
3 Biological Response
Modifiers Advisory Committee.
4 I have been
complaining about the lack of titles
5 but at least they had
numbers but ow they don't
6 even have a number
here. Oh yes, we do, meeting
7 number 32. Eventually they will get the idea and
8 give me titles.
9 I am Dan
Salomon. I have the pleasure of
10 chairing the committee
today. What we are going to
11 do this morning is
have about a one-hour open
12 session here that I
guess merges into a closed
13 session at 8:45. Then, there will be a break at
14 9:00 and at 9:00 we
will get into the main topic of
15 the morning. So, a lot of things like introducing
16 the members of the
committee I will save for nine
17 o'clock if you guys
will forgive the lack of pomp
18 and circumstance this
early in the morning. I also
19 reserve the right to
say something totally stupid
20 for the next hour
since I am from California and it
21 is awfully early for
me right now.
22 Without any
further ado, we should get
23 going. It is Amy getting up there, Amy Rosenberg
24 from the Laboratory of
Gene Regulation, to give us
25 an update on research
programs, and that will be
5
1 followed by Ezio
Bonvini, from the Laboratory of
2 Immunobiology.
3
Update Research Program
4
Laboratory of Gene Regulation
5 DR. ROSENBERG: I am actually the Director
6 of the Division of
Therapeutic Proteins, and I am
7 here to speak for Ed
Max and Serge Beaucage, who
8 are members of the
Laboratory of Gene Regulation
9 who, unfortunately,
could not be here today.
10 This is a
follow-up to the site visit and
11 I will run through the
follow-up for Dr. Max first.
12 Dr. Max works with
three research scientists, as
13 you can see here. The non-research
14 responsibilities of a
laboratory include primary
15 review responsibility
for several cytokines and
16 thrombolytics and
anticoagulants. They
17 additionally provide
expert consultation on issues
18 of molecular biology,
particularly quantitative PCR
19 assays and
immunoglobulin genes. In addition, Dr.
20 Max performs a lot of
administrative functions. He
21 is the associate
director for research in OTRR and,
22 as well, he organizes
semina series; he chairs the
23 research coordinating
committee; and he manages the
24 CBER library.
25 The projects
that are ongoing in his
6
1 laboratory, two were
primarily dealt with in the
2 site visit, mechanisms
of immunoglobulin isotype
3 switching and
characterization of the human 3'
4 immunoglobulin heavy
chain enhancer complex.
5 The mission
relevance of the research is
6 listed here. Regarding gene regulation, FDA
7 regulates strategies
to alter gene expression.
8 Basically, we have a
lot of products being produced
9 by knock-in
technology. Insulators are now
10 becoming increasingly
important in transgenic
11 animals. Regarding
isotype switching, there is a
12 little more activity,
in fact. There are specific
13 strategies to have TH2
to TH1 switches. So,
14 increasing IgG,
decreasing IgE to protect against
15 allergic type
reactions. Additionally, our
16 division regulates
several agents that are known to
17 directly affect
isotype switching, cytokines IL4,
18 TGF-beta and CD40
ligand. As we all fervently
19 believe, good basic
science enables appropriate
20 regulation.
21 Dealing with
the first project, mechanisms
22 of immunoglobulin
isotype switching, this is just
23 to remind you that
isotype switching involves a
24 switch recombination
event which juxtaposes VDJ
25 segments with
downstream constant regions of
7
1 different isotype
genes.
2 The first
aspect of this project involves
3 a study of the Ku
protein complex, how does this
4 participate in
immunoglobulin gene recombination?
5 Ku protein has been
found to be key in sealing
6 double-stranded DNA
breaks, and it is found that
7 during isotype
switching this protein increases in
8 B cells and that
knockout mice that are deficient
9 for Ku seal DNA breaks
inappropriately. Since the
10 site visit, this
laboratory has cloned additional
11 breakpoints in tumors
from Ku knockouts that they
12 are trying to
characterize to clarify the role of
13 Ku in sealing these
double-stranded breaks.
14 The second aspect of this project involves
15 characterization or
identification of the role of
16 the ATM proteins in
switch recombination. This is
17 a collaboration with
Dr. Hodes at NCI. They found
18 that the ATM knockout
mice show a defect in isotype
19 switch recombination
intrinsic to B cells, and
20 since the site visit
they have basically adapted
21 their assay to become
really a quantitative assay
22 so that they can more
accurately measure the degree
23 of switch
recombination.
24 Regarding
the second project, which is the
25 characterization of
the human 3' IgH enhancer
8
1 complex, there are
many aspects that they are
2 investigating, one,
the genomic neighborhood. That
3 aspect has been
completed. The human IgH 3'
4 enhancer complex in
humans resulting from a
5 duplication event that
causes large segments to be
6 duplicated so that
downstream of C-alpha 1 and
7 C-alpha 2 constant
regions the laboratory
8 characterized these
nearly identical enhancer
9 complexes, each
composed of a strong enhancer
10 designated HS12, which
are flanked by two weaker
11 enhancers, HS3 and
HS4. Both HS12 enhancers are
12 flanked by inverted
repeats.
13 So, they
went on to study the functional
14 motifs in HS12 and
other 3' enhancers. The have
15 identified functional
motifs in the enhancers by
16 sequence conservation
between the human enhancers
17 and the murine
homologs. They have performed in
18 vivo footprinting
using LM-PCR, and they have
19 performed transient
transfections with luciferase
20 reporter constructs
that are driven by enhancers
21 mutated in putative
functional motifs.
22 Regarding
this aspect, since the site
23 visit the laboratory
has used DNA swan protection
24 as an alternative
technique for in vivo
25 footprinting. They have extended the footprinting
9
1 analysis outside the
evolutionary conserved cores
2 of the HS12 and HS4
areas, and they have
3 constructed and tested
additional reporter plasmid
4 containing DNA outside
the core enhancers.
5 With regard
to the response of this
6 enhancer complex to
IL4 and CD40 ligand, it is
7 found that these are
factors, which are TH2
8 stimuli, actually
inhibited the action of the HS12
9 enhancer in the germinal center B cell
lines.
10 Other enhancers, an
endogenous one here, were
11 unaffected. Since the site visit they have
12 investigated candidate
IL4 or CD40 responsive
13 elements in the HS12
enhancer by constructing
14 reporter plasmid
driven by multimerized candidate
15 enhancer motifs.
16 Regarding
the last project, looking at
17 locus control region
function in chromatin, they
18 found that there is a
CPG island within a cluster
19 of DNA swan
hypersensitivity sites that showed the
20 activity of gene
insulators. So, the level of
21 transcription in the
normal situation is here. If
22 you have gene
insulators it cuts down dramatically,
23 and these CPG islands
as well cut down dramatically
24 on transcription. So, since the site visit they
25 have constructed
additional plasmid to define the
10
1 active insulator
element. They are also searching
2 for a possible
homologous insulator downstream of
3 the murine enhancers.
4 Additional
studies in progress involve
5 chromatin
immunoprecipitation studies to identify
6 transcription factors
found to be enhancers in
7 vivo, and they are
using single cell assays for the
8 3' enhancer function
using stable transfectants of
9 GFP constructs. That is the follow-up on the Max
10 lab.
11 DR.
SALOMON: Thank you, Amy. I feel bad
12 for Alice since she is
an attorney and she came in
13 a little late, she is
going to have trouble with
14 the test questions on
enhancer.
15 [Laughter]
16 We will try
and help you through it. The
17 next is from the
representing the laboratory of
18 immunobiology.
19 DR.
ROSENBERG: No, I have to give
20 follow-up on Dr.
Beaucage. I am sorry. So, the
21 laboratory of Dr.
Beaucage, he works with five
22 postdoctoral
fellows. His regulatory
23 responsibilities
include primary review of
24 hematologic products,
enzyme replacement therapies,
25 anti-cancer enzymes
and thrombolytics. He provides
11
1 expert consultation on all of the nucleotide
2 diagnostic kits with
the Center's Office of Blood.
3 He has large
responsibility for helping to draft
4 the guidance for
industry on submission of CMC
5 information for
synthetic oligonucleotides. He has
6 also performed some
inspections regarding
7 hematologic products
and thrombolytics.
8 Overview of
his program--as you know, he
9 is an oligonucleotide
chemist, and he is
10 responsible in large
part for development of the
11 phosphoramidite method
so he has three major
12 efforts. The first is effects in development of
13 deoxyribonucleotide
cyclic anacylphosphoramidetes
14 and stereo-controlled
synthesis of oligonucleotide
15 phosphorofioates for
potential therapeutic
16 applications.
17 Essentially,
since the site visit the
18 group has optimized
the coupling efficiency of
19 deoxynucleoside cyclic
anacylphosphoramidites to
20 enable synthesis of
nuclease-resistant P
21 stereo-defined
oligonucleotides containing all four
22 nucleotides. They found that pryrrolidin and DBU
23 are the preferred
bases for efficient coupling of
24 deoxyribonucleotide
acylphosphoramidites
25 uncontrolled for GLAS,
which is important for
12
1 potential applications
for microarray. They
2 published a paper in
the Journal of the American
3 Chemical Society,
describing the development of a
4 simple NMR method to
determine the absolute
5 configuration of
deoxyribonucleotide
6 phosphoramidites at
phosphorus, and the findings,
7 again, have appeared
in the Journal. They are also
8 working to improve the
resistance of CPG
9 oligonucleotides to
nuclease activities by using
10 P-stereo defined
oligos.
11 The second
effort involves efforts towards
12 the discovery of
phosphodiester protecting groups
13 for potential
applications to large-scale
14 production of
alphalation free therapeutic
15 oligonucleotides and
to the synthesis of
16 oligonucleotides on
microarrays. They found that
17 the 3-NN-dimethyl
carboxymedopropryl group--this
18 group right here, is a
novel phosphate
19 thiophosphate
protecting group for solid phase
20 synthesis that has
recently been developed. The
21 monomers which are
required are easily prepared
22 from inexpensive raw
materials. The protecting
23 group can be removed
from the oligonucleotides
24 under the basic
conditions that are used
25 standardly, and, thus,
it is actually a very
13
1 convenient protecting
group. But, most
2 importantly, the
thermolytic properties of the
3 protecting group are
particularly attractive to the
4 synthesis of DNA
oligonucleotides on microarrays
5 because it minimizes
exposure of the arrays to the
6 harsh nucleophilic
conditions used for
7 oligonucleotide
protection. So, these conditions
8 are actually quite
mild and favorable.
9 The third
effort is involved in the
10 development of
thermophilic 5'hydoxyl protecting
11 groups for nucleoside
or nucleotides for synthesis
12 of, again, DNA oligos
on microarrays. The
13 thermolytic phosphate
protecting groups described
14 in the site visit
report have been applied to the
15 protecting group in
the 5'hydroxyl of nucleosides
16 as carbonates, but
this was found to be quite
17 impractical. Recently the laboratory has
18 discovered that the
5'O and methyl, 1 phenylmethyl
19 oxycarbinol protecting
group can be thermolytically
20 cleaved from
nucleosides in aqueous ethanol within
21 10 minutes at 90
degrees. Here is the loss of this
22 protecting group.
23
Interestingly enough, this forms a
24 fluorescent byproduct
and it permits the accurate
25 determination of the
D-protection deficiency. The
14
1 protecting group
appears to be stable in organic
2 solvents at ambient
temperature, which also again
3 makes it increasingly
attractive to the synthesis
4 of oligonucleotides on
microarrays. That is the
5 follow-up for the
Beaucage lab.
6 DR.
SALOMON: I think someone should get
7 the message back to
them that you have represented
8 them really remarkably
well. That was a beautiful
9 presentation of not your own laboratory
efforts. I
10 think anybody who
didn't know that would have had a
11 clue that this wasn't
your own work.
12 DR.
ROSENBERG: That is because they
13 didn't ask questions.
14 [Laughter]
15 Thank you
very much, Dan, I do appreciate
16 it.
17 DR.
SALOMON: It is also a representation
18 of the kind of quality
work going on at the FDA.
19 My only regret is
there aren't enough people in the
20 audience that should
hear that kind of thing
21 because that is
something that we should have saved
22 for the end of day
when there are a lot of people
23 here. The next presentation is from Ezio Bonvini,
24 the Laboratory of Immunobiology,
Division of
25 Monoclonal Antibodies.
15
1
Laboratory of Immunobiology
2 DR.
BONVINI: Thank you very much. I
3 would like to thank
Dr. Salomon and the members of
4 the advisory
committee.
5 My duty
today is to summarize the work
6 that we have done, and
the focus of my laboratory
7 is on the regulation
of phospholipase C-gamma
8 activation in immune
cells. The laboratory is
9 operationally divided
into two inter-related units,
10 one focusing on the
coupling of C-gamma-1 to the
11 antigen receptor TMB
cells. The second, which is
12 headed by Dr.
Rellahan, looks at the control of
13 phospholipase C
activation, and in particular the
14 control mediated by a
complex molecule called
15 C-Cbl.
16
Recapitulating the functional division, we
17 have two interacting
units, one that I coordinate
18 which is currently
made up of a research assistant,
19 Karen DeBell, and a
postdoctoral fellow, Carmen
20 Serrano. I would also like to acknowledge past
21 postdoctoral members
of the laboratory that, in one
22 way or another, have
contributed to this project,
23 and they have actually
all left and found
24 employment elsewhere.
25 Dr. Rellahan
has one permanent staff
16
1 member, Dr. Laurie
Graham, a lab associate, and she
2 also enjoys the
benefit of a number of students who
3 have actually
contributed during the summer to her
4 project.
5 Now, we do
what we do for a number of
6 reasons. The laboratory has the regulatory
7 responsibility for
monoclonal antibodies and
8 protein directed
against T-cells for the purpose of
9 immune suppression or
immunomodulation. More and
10 more so, these
antibodies interact with surface
11 receptors that
interfere either in signalling
12 blockade or signalling
manipulation with the
13 purpose of
immunomodulation. Furthermore, signal
14 transvection targeting
can be used as surrogate for
15 potency of
biologics. A number of biologics and a
16 number of monoclonal
antibodies, also trigger a
17 number of adverse
events to undesired signaling.
18 Another fundamental
reason is the familiarity with
19 the knowledge base and
technology.
20 The focus on
PLC-gamma, PLC-gamma
21 regulates calcium
mobilization in a variety of
22 cells, including
immune cells, and I don't think I
23 need to go any further
for this audience but
24 calcium is a critical
component in control for
25 transcriptional
activation through a number of
17
1 elements, one of which
is an important element,
2 calcineurin
phosphatase as a target for a number of
3 drugs; the other path
being calcium dependent
4 proteinases. The duration of the effects of the
5 flux of calcium
controls a number of cellular
6 responses with a
prolonged calcium flux being a
7 requirement for
immunocompetence. As I said
8 earlier, a number of
calcium-dependent pathways are
9 a target of
immunosuppressive structures which
10 include cyclosporin A,
among others.
11 Again, I
don't think I can go through the
12 data in detail, but
what I would like to give you
13 is a flavor for how
complex PLC-gamma is. This is
14 the molecule which is
a cytoplasmic molecule which
15 contains a number of
separate domains. The
16 molecules need to be
recruited to the surface where
17 the substrate where
PTdinsP, a lipid, resides, and
18 needs to undergo
presumably a confirmation or
19 modification to bridge
together the X and Y domains
20 of the catalytic
subdomain.
21 Our focus
has been largely on the
22 cytochromology 2
domain, which are individual
23 domains which are
known to interact with calcium
24 and phosphorolytic
protein and the cytochromology 3
25 domains which are
known to interact with the
18
1 protein rich
region. When we started these
2 investigations, the
mechanism of activation of
3 PLC-gamma was largely
unknown or misinterpreted, I
4 should say, so we
focused on this largely because
5 by their own nature we
thought they were
6 responsible for
targeting phospholipase C-gamma
7 with a number of
regulatory proteins. So, we
8 pursued this by
mutational analysis of the enzyme,
9 and recently we
obviously focused on a number of
10 other domains but I
will not go into any of this.
11 This enzyme
is regulated by
12 phosphorylation, and
there are at least four known
13 targets in
phosphorylation, here in yellow, and
14 that is also another
focus of our investigation but
15 we use studies of
phosphorylation somewhat as a
16 surrogate marker for
activation.
17 So, I will
briefly summarize the results
18 of our studies, which
have all been published, and
19 I will split them
vertically into the different
20 domains. The cytochromology of amino-2 terminal
21 domain is the most
critical domain in the
22 activation of
PLC-gamma-1 in T and B cells. This
23 domain is required in
sufficient phosphorylation.
24 It is required for
membrane translocation and this
25 requirement, we think,
is required for activation
19
1 because its activation
correlates with the degree
2 of
phosphorylation. What this domain does
is bind
3 a number of adapters
which were recently
4 discovered. One is Lat which we identified in
5 collaboration with
Larry Samuelson. The other is
6 BLnk which we
identified in collaboration with Tom
7 Korozaky, who actually
cloned it. The
8 cytochromology to the
C domain appeared to be
9 dispensable for
phosphorylation of membrane
10 translocation,
although it is required for
11 activation in vivo,
and the function of this domain
12 is largely unknown,
but since the site visit report
13 we have gained quite a
number of insights and this
14 is a very critical
domain to investigate as it
15 pertains to the
ability of PLC-gamma to couple to a
16 number of different
pathways, including
17 co-stimulatory
pathways, and to a function of
18 PLC-gamma that is
independent of this catalytic
19 activity.
20 The
cytochromology 3 domain appears to be
21 dispensable
phosphorylation, however, enhances
22 membrane
translocation, and I will provide a
23 summary at the end of
how it does that, and by
24 virtue of its
announcement of membrane
25 translocation,
enhanced activation of the enzyme in
20
1 vivo. Its function, we have identified binding to
2 the protocol gene
C-Cbl and Art Wizer's group, one
3 of the leaders in the
field, has shown that the
4 domain binds with
Lp-76, another adaptive molecule.
5 Of course, I
don't have the time to go
6 through all the
details but I just want to
7 summarize again some
of the milestones that we have
8 achieved since we
started this project. With
9 respect to PLC
coupling to the receptor, we
10 reported initially
that PLC-gamma-1 SS-2 domain was
11 critical for coupling
it to the T-cell receptor.
12 Then, we explored the
role of cytochromology domain
13 of PLC-gamma coupling
to the B cell receptor.
14 Recently we have
focused on the ability of membrane
15 raft, which are a
microdomain, to function at the
16 microdomain that
segregates PLC-gamma and other
17 molecules for their
regulators, and we have shown
18 that
recompartmentalization of PLC-gamma to this
19 microdomain is, in
itself, sufficient to lead to
20 PLC-gamma activation,
activation of the cells and
21 IL-2 separation.
22 With respect
to the negative regulation of
23 PLC-gamma, which is
the focus of Dr. Rellahan's
24 research, we have
shown that C-Cbl inhibits
25 TCR-induced 81
activation, a reporter gene whose
21
1 activation depends on
raft and isoglycerol, and
2 isoglycerol is under
the control of PLC-gamma.
3 PLC-gamma-1 binds
C-Cbl in its HS-3 domain and
4 C-Cbl exerts
inhibitory function, however, it
5 transforms a
counterpart of C-Cbl-70Z-3 Cbl which
6 lacks the ability of
C-Cbl molecule to ubiquinate
7 the target
protein. This molecule, 76-C-Cbl,
8 activates PLC-gamma
and does so through a
9 differential pathway,
a pathway which is not shared
10 completely by the T
cell receptors, suggesting the
11 possibility of
regulation of PLC-gamma through an
12 alternate mechanism of
activation.
13 Rather than
going through data, I would
14 like to give you a
model that will try to summarize
15 our findings with
those of other laboratories and
16 put everything
together.
17 This is a
schematic TCR receptor. The TCR
18 receptor interacts
with the antigen it encounters
19 of antigen presenting
cells. Now, in the membrane
20 of many cells,
including T cells, it is
21 homogeneous. Depicted here in red are rats which
22 contain a number of
different molecules, including
23 the Lck which is
brought together through the
24 T-cell receptor by the
action of the antigen into
25 the raft. The rafts contain an adaptor molecule,
22
1 called raft, which we
have shown to interact with
2 phospholipase C. This occurs subsequent to
3 phosphorylation of Lck
of the CD3 molecules which
4 are associated with
the alpha and beta chain of the
5 T cell receptor. Following phosphorylation, a
6 cytoplasmic kinase
called Zap 70 is recruited, and
7 it is the Zap 70 that
phosphorylates these other
8 transmembrane adapters
into the rat.
9 This is the
signal that tells PLC-gamma,
10 which is a cytoplasmic
enzyme which is
11 constitutively bound
to the Lck-76 through the
12 SSS-3 domain. That is the signal to recruit
13 PLC-gamma through the
amino termini cytochromology
14 to this adaptor. This interaction is further
15 stabilized by the
presence of Gads, a second
16 adaptor molecule,
which interacts with Lck-76 and,
17 in turn, interacts
with the cytochromology-2
18 domain. That explains the contribution of the
19 cytochromology-3 domain
to stabilize the
20 interaction of
PLC-gamma to the membrane.
21 PLC-gamma in
the raft compartment can be
22 phosphorylated by a
number of kinases which are
23 either present in the
raft compartment, such as
24 RLK, or recruited to
the raft compartment via the
25 action of another
specialized phosphorylated lipid
23
1 PIP-3, such as
ITK. These are a member of the TAK
2 family of kinase which
are a member of the
3 subfamily of kinase,
although their mechanism of
4 regulation is
different. The contribution of Lck
5 and RLK in our hands
shows that it leads to
6 phosphorylation of
PLC-gamma-1 which presumably
7 induces a confirmation
of modification of PLC-gamma
8 and the ability of
PLC-gamma to activate and
9 mobilize calcium.
10 Our data
showed that if we artificially
11 target PLC-gamma
through the lipid raft we
12 basically bypass this
entire initial phase,
13 although Lck and RLK
are still required, presumably
14 because of their
contribution to the
15 phosphorylation. Artificially targeted PLC-gamma
16 to the raft
compartment is phosphorylated and is
17 active bypassing the
receptor entirely. So, this
18 is a dominant,
positive variant of the PLC-gamma.
19 What
happened with the negative
20 regulation, initial
phase is the same and PLC-gamma
21 is interacting with
the Lck-76. C-Cbl binds to the
22 SU-3 domain of
PLC-gamma very much in the manner
23 seen with Lck-76. So, there is probably
24 competition by a
mechanism which we still don't
25 understand. C-Cbl is also phosphorylated in
24
1 response to activation
of the T cell receptor and
2 that leads to
inhibition of PLC-gamma presumably
3 via a mechanism of
ubiquitilation. We are still
4 investigating this,
however, data that confirm that
5 this may be the case is that the variant to
73-Z
6 C-Cbl, and we now have
data with another variant
7 that is Ub-ligase
deficient, which results in the
8 dephosphorylation of
PLC-gamma by a mechanism that
9 we still do not know
but that does not require
10 Lck-76, and that leads
to the activation of
11 PLC-gamma by a
mechanism that is independent of the
12 T-cell receptor. So, we believe that C-Cbl and
13 Lck-76 and the
equilibrium between the two
14 coordinate the
assembly of the complex that in one
15 case is activatory and
in the other case is
16 inhibitory.
17 As far as
our future plan, we will
18 continue to
investigate the role of PLC-gamma-1 and
19 gamma-2 as a second
isozyme present preferentially
20 in B-cells and in
other hematopoietic cells where
21 gamma-1 is
ubiquitously present in all cells. We
22 will focus further
between these two enzymes and
23 other pathways in the
co-stimulatory activation of
24 T cells.
25 I mentioned
earlier the function that the
25
1 function of the SS2
domain is still unknown and we
2 have obtained quite a
bit of new exciting results
3 on the function of this domain and its
coupling to
4 a number of different
molecules, but the bottom
5 line that I want to
give you is that domain
6 regulates the
intrinsic activity of PLC-gamma by
7 intermediate
intermolecular interaction which
8 regulates its opening
up and the availability of
9 the other
subdomains. So, it is a fundamental
10 mechanism of
regulation.
11 We will
continue, of course, to
12 investigate the role
and mechanism of
13 phosphorylation of
PLC-gamma. What the enzymes are
14 that phosphorylate the
PLC-gamma are largely
15 unknown. We have a candidates are, as I mentioned
16 earlier, but what the
different candidates do in
17 terms of individual
residues, and there are at
18 least four and mostly
likely five residues, and
19 what is the role of
the individual residue is still
20 quite unclear.
21 Because we
have made a dominant positive,
22 we have now also
developed a dominant negative
23 PLC-gamma, and we will
certainly ask the question
24 of the role of
PLC-gamma development by using
25 transgenic
technology. Finally, and I am not going
26
1 to dwell on this, but
we are using technology to
2 recompartmentelize
PLC-gamma intracellularly by a
3 condition of
mechanism.
4 With respect
to the role of C-Cbl again,
5 C-Cbl is probably a
threshold for activation, and
6 the impact of C-Cbl on
the co-stimulatory signal is
7 the ability of the
cell to behave as naive or
8 memory will be
investigated. We are going to
9 generate some
C-Cbl-deficient lines and we are
10 going to try to do
that by a number of different
11 strategies. As I said, we have some new data on
12 the C-Cbl-mediated
with the delineation of
13 PLC-gamma-1. I can tell you that it is
14 ubiquitilated. The role of C-Cbl in this remains
15 to be determined but
we have evidence that by using
16 Ub-ligase to inhibit
the C-Cbl negative cells is,
17 in fact, the case.
18 Finally, we
will try, as I said earlier,
19 to generate some C-Cbl
deficient cell line using
20 interferon RNA and
that will help us in the study
21 of kinetics in mice
for PLC-gamma activation.
22 I just want
to leave you with the number
23 of individuals who
have contributed in one way or
24 another with
particular reagents and a number of
25 collaborators that we have worked with whom I
would
27
1 like to acknowledge
for their help in this. And, I
2 will be glad to take
any questions.
3 DR.
SALOMON: That was a very nice
4 presentation and good
work, and also my same
5 comments, that I wish
more people could see the
6 kind of quality work
that is going on in the FDA,
7 oftentimes, with a lot
less support not because of
8 your fault or the FDA
support but just because of
9 the budget constraints
than we are used to in
10 academia. It is excellent.
11 The part
that is confusing me here,
12 besides the fact that
I really am still asleep, is
13 that we now have to
switch officially to a closed
14 session to vote on
accepting the report. Gail will
15 make sure that the
right people have to leave.
16 Anyway, we will see
you again very shortly.
17 [Whereupon,
the open session was recessed
18 to continue in closed
session and reconvene in open
19 session at 9:15 a.m.]
28
1 P R
O C E E D I N G S
2 Welcome
and Administrative Remarks
3 DR.
SALOMON: If we can get everybody to
4 sit down we will start
the main show, I guess we
5 should say. For the larger group here now, this is
6 meeting number 32 of
the Biological Response
7 Modifiers Advisory
Committee. My name is Dan
8 Salomon. I have the pleasure to chair the meeting
9 this morning. What we usually do at the start, as
10 in many big committee
meetings where a lot of us
11 don't know each other
initially--we will certainly
12 get to know each other
as the day goes on, is just
13 to go around the table
and introduce yourself, and
14 make a couple of quick
sentences about what your
15 interests are and your
scientific expertise. We
16 can start at that end
of the table. Dr. Casper?
17 DR.
CASPER: Hi. I am Bob Casper, am a
18 professor of
obstetrics and gynecology and
19 physiology at the
University of Toronto, and I am
20 head of the Division
of the Reproductive Sciences.
21 I have clinically been
involved st in vitro
22 fertilization for
several years, and our laboratory
23 at the present time
has an interest in
24 mitochondrial research
involving aging of human
25 oocytes. We have also been doing some work with
29
1 mitochondrial transfer
experiments in mice.
2 DR.
SALOMON: There is a button here that
3 you push and then you
have to remember to turn it
4 off, otherwise there
will be feedback.
5 DR.
KNOWLES: Thank you. I am Lori
6 Knowles. I am from the Hastings Center. I have a
7 background in
international law and policy, and I
8 am principal
investigator right now of an
9 international project
on reprogenetic regulation
10 and affects, and also
do work in international stem
11 cell policy.
12 DR.
NAVIAUX: I am Bob Naviaux, from the
13 Mitochondrial
Metabolic Disease Center at the
14 University of
California, San Diego. My basic work
15 is in mitochondrial
DNA replication, and we also
16 have interest in
inborn errors of metabolism and
17 adult and childhood
mitochondrial disorders.
18 DR.
SHOUBRIDGE: I am Eric Shoubridge. I
19 am a professor at
McGill University in the
20 Departments of Human
Genetics and Neurology and
21 Neurosurgery. I have a research lab at the
22 Montreal Neurological
Institute and our laboratory
23 is interested in the
basis of mitochondrial
24 disease, the molecular
basis, and we are interested
25 in basic, fundamental
aspects of mitochondrial
30
1 genetics.
2 DR.
SCHON: My name is Eric Schon. I am a
3 professor of genetics
and development in the
4 Department of
Neurology at Columbia University, and
5 I do everything that
Eric Shoubridge does.
6 [Laughter]
7 DR. VAN
BLERKOM: Jon Van Blerkom. I am
8 from the University of
Colorado, Molecular Biology
9 Department, and I am
also in clinical practice in
10 in vitro
fertilization, for about twenty years.
11 DR.
MURRAY: I am Tom Murray. I am from
12 the Hastings Center
these days, after fifteen years
13 of medical schools,
most recently Case Western
14 Reserve
University. My research has been
broadly
15 in the field of ethics
and medicine and the life
16 sciences, and I have
done a lot of work on
17 reproductive
technologies, genetics and parents and
18 children.
19 DR.
RAO: My name is Mahendra Rao, and I
20 am a section chief in
stem cell biology at the
21 National Institute of
Aging, and I am a member of
22 this committee. My interests are in embryonic stem
23 cells and adult stem
cells.
24 DR.
MULLIGAN: I am Richard Mulligan. I
25 am from the Harvard
Medical School, Children's
31
1 Hospital. I am a stem cell person and a gene
2 transfer person, and a
member of BRMAC.
3 DR.
SALOMON: I am Dan Salomon. I am from
4 the Scripps Research
Institute and my lab is doing
5 cell transplantation,
tissue engineering,
6 angiogenesis and
therapeutic gene delivery.
7 MS.
DAPOLITO: Gail Dapolito, Center for
8 Biologics, executive
secretary.
9 DR.
SAUSVILLE: Ed Sausville. I am the
10 associate director of
NCI's Division of Cancer
11 Treatment and
Diagnosis, with responsibility for
12 the development of our
therapeutics program, and
13 our interest is in the
preclinical studies leading
14 to the approval for
INDs for drugs and biologics.
15 MS.
WOLFSON: Alice Wolfson. I am the
16 consumer
representative on the committee. I am
an
17 attorney specializing
in policy holder
18 representation, with
particular emphasis on
19 disability policy
holders and their struggles with
20 their insurance
companies. I have a strong
21 interest in
health. I am a founder of the National
22 Women's Health
Network, and I am particularly
23 interested in the
social effects of postponing
24 fertility as well as
the social effects of not
25 postponing fertility
and I think it may have, along
32
1 with the scientific
elements in it, the beginnings
2 of a possibility of a
resurgence of another wing of
3 the women's movement.
4 DR.
ROSE: I am Stephen Rose. I am from
5 the National Institute
of Health, Office of
6 Biotechnology
Activities, deputy director for the
7 recombinant DNA
program.
8 DR. MONROE: I am Scott Monroe. I am from
9 the Division of
Reproductive and Neurologic Drug
10 Products at CDER. I am an
11
obstetrician/gynecologist and a reproductive
12 endocrinologist.
13 DR.
SERABIAN: I am Mercedes Serabian. I
14 am an expert
toxicologist with the Office of
15 Therapeutics in the
Division of Clinical Trials,
16 and I will be part of
the review team at CBER that
17 will be reviewing
these INDs when they come in.
18 DR. MOOS: I am Malcolm Moos, from the
19 Division of Cellular
Gene Therapy at the FDA. My
20 research interests are
cell and tissue
21 specification and
patterning, and I am also
22 concerned with review
of cellular products,
23 primarily that have to
do with that general
24 biological area.
25 DR.
HURSH: I am Deborah Hursh. I am also
33
1 a cellular product
reviewer in the Division of Cell
2 and Gene Therapy, and
I have a research lab
3 studying developmental
biology and signal
4 transduction.
5 DR.
NOGUCHI: I am Phil Noguchi. I am the
6 director of the
Division of Cell and Gene Therapy,
7 where we see these and
other novel technologies and
8 continually struggle
with doing the right thing.
9 [Laughter]
10 DR.
SIEGEL: I am Jay Siegel. I direct
11 the Office of
Therapeutics Research and Review at
12 the Center for
Biologics, FDA.
13 DR.
SALOMON: I welcome all of you. I
14 think one of the
privileges of being on the
15 committee and
certainly chairing it is the chance
16 to interact with
experts at each of these sessions
17 that take me into
areas that are often new to me,
18 and today is
definitely one of those areas. It is
19 a fantastically
important discussion that we are
20 going to have that has
a lot of implications on
21 what is going to
happen over the next several
22 years. So, I specifically feel a lot of
23 responsibility to this
particular session and how
24 we go forward.
25 There will
be some more comments later on
34
1 that, just simple
administrative things. My job,
2 obviously, is to stay
on time and also to get the
3 questions the FDA
answered and keep everybody on
4 track. So, if you will forgive me sometimes
5 playing my administrative
role which sometimes
6 includes being
rude. I apologize in advance.
7 The button
thing, we have all been through
8 it. It gets to be a real problem with feedback
and
9 also with the
transcriber. So, if I ever sort of
10 look at you and kind
of point to the button, it is
11 just to let you
know. I think that is the major
12 thing. I want to try and keep track of sort of
13 what we are going to
do next so you will sort of
14 know where we are going.
15 What we will
do now is a presentation of
16 the certificate of
appreciation to Dr. Ed
17 Sausville, with some
more comments to follow that.
18 Then Gail Dapolito has
some official things to read
19 into the record and then
we will start the full
20 session with Dr.
Hursh.
21 Presentation
of Certificate of Appreciation
22 DR.
SIEGEL: It is indeed an honor, tinged
23 with regret at his
departure but an honor to speak
24 of the many services
that Dr. Sausville has
25 provided to us through
his participation in BRMAC
35
1 in recent years, and
to thank you for them. Those
2 of you on the
committee, of course, are aware of
3 his many thoughtful
contributions to the
4 deliberations to this
committee. Some of you may
5 be somewhat less aware
of his many contributions as
6 a representative of
BRMAC to the Oncological Drugs
7 Advisory Committee and
other FDA committees to
8 which we have taken
products for consideration of
9 approval, as well as
contributions to our lab
10 evaluation and site
visiting program.
11 We ask a
lot, as you know, of BRMAC
12 members. It ranges from discussion of the issues
13 regarding
manufacturing a product, viral purity,
14 protein stability,
immunogenicity, and so forth,
15 and how we should
focus on safety. The issues of
16 clinical testing of a
product; what is the
17 appropriate trial
design to get the answers we need
18 and what to make of
the answers when those trials
19 are done; and, of
course, as you heard this morning
20 the issues of
evaluating our research programs and
21 how to make sure that
they are tied in intimately
22 to our mission and our
goals and are of the highest
23 quality.
24 We choose
experts in each and all of these
25 areas to help us in
our functions, but it is rare
36
1 that we have an
expert--rare both inside the agency
2 and outside but very
much appreciated when we have
3 someone such as Dr.
Sausville who really is the
4 regulatory expert
triple threat, who integrates an
5 understanding of the
clinical evaluation of the
6 basic science, of the
research needed to support
7 that, and can
participate in an integrated
8 assessment in any of
those areas, understanding the
9 implications for the
others. That is what you have
10 done for us for these
several years and it is very
11 much appreciated. Thank you very much.
12 [Applause]
13 DR.
GOODMAN: I know Dr. Zoon and I really
14 second that and
appreciate the tremendous breadth
15 of expertise Dr.
Sausville has brought. I was
16 going to stress the
same thing. From what I have
17 understood and seen,
this translational ability
18 between the laboratory
and the clinical setting,
19 and an understanding
of product development, those
20 things are just
extremely important and we really
21 appreciate it. We look forward to continuing to
22 call on you and get
your input and help. Thanks
23 very, very much. So, we have a nice certificate
24 and plaque.
25 [Applause]
37
1 DR.
SALOMON: I can't not make my own
2 personal comments,
having been together with Ed on
3 this committee for
four years. I don't know how
4 many of you have seen
the movie "The Scorpion
5 King." I guess is depends on how old your kids
6 are, but the actor in
it is called "The Rock"
7 because I suppose he
is a professional wrestler as
8 well. But I really think that he is competing with
9 the real
"rock" who is Ed Sausville.
On any
10 committee like this
you have to have a rock. I
11 mean, you have to have
the one guy who you can
12 always turn to, even
though everything has gone to
13 shreds, and he just
hits it right on the head. You
14 have to shut up and
listen to him whenever he says
15 anything. Really, whenever there has been any kind
16 of issue here, he is
one of the people that I come
17 to at the break and
say, "you know, Ed, what the
18 heck do we do
now?" And, he always has good
19 advice. This is not good at all, to have Ed
20 leaving and all I can
do is say I will always be
21 dragging you back
here, and he is really, really
22 going to be a loss to
the committee. Thank you.
23 Gail?
24 MS.
DAPOLITO: I would like to read the
25 meeting
statement. This announcement is part of
38
1 the public record for
the May 9, 2002 Biological
2 Response Modifiers
Advisory Committee meeting.
3 Pursuant to
the authority granted under
4 the Committee Charter,
the director of FDA Center
5 for Biologies
Evaluation and Research has appointed
6 Ms. Lori Knowles and
Drs. Thomas Murray, Robert
7 Naviaux, Eric Schon,
Eric Shoubridge, Daniel
8 Salomon and Jonathan
Van Blerkom as temporary
9 voting members for the
discussions on issues
10 related to ooplasm
transfer in assistive
11 reproduction. In addition, Dr. Salomon serves as
12 the acting chair for
this meeting.
13 To determine
if any conflicts of interest
14 existed, the agency
reviewed the submitted agenda
15 and all financial
interests reported by the meeting
16 participants. In regards to FDA's invited guests,
17 the agency has
determined that the services of
18 these guests are
essential. The following
19 interests are being
made public to allow meeting
20 participants to
objectively evaluate any
21 presentation and/or
comments made by the guests
22 related to the
discussions and issues related to
23 ooplasm transfer in
assisted reproduction.
24 Dr. Robert
Casper is employed by the
25 University of Toronto
in the Division of
39
1 Reproductive Science
at Mt. Sinai Hospital in
2 Toronto. Dr. Jacques Cohen is employed by the St.
3 Barnabas Medical
Center. Dr. Susan Lanzendorf is
4 employed by the
Eastern Virginia Medical School at
5 the Jones Institute of
Reproductive Medicine. Drs.
6 Amy Patterson, Marina
O'Reilly and Stephen Rose are
7 employed by the Office
of Biotechnology Activities,
8 NIH.
9 In the event
that the discussions involve
10 other products or
firms not already on the agenda
11 for which FDA
participants have a financial
12 interest, the
participants are aware of the need to
13 exclude themselves
from such involvement and their
14 exclusion will be
noted for the public record.
15 With respect
to all other meeting
16 participants, we ask
in the interest of fairness
17 that you state your
name, affiliation, and address
18 any current or
previous financial involvement with
19 any firm whose product
you wish to comment upon.
20 Thank you.
21 DR.
SALOMON: Thank you, Gail. Before we
22 officially get
started, let me just make a couple
23 of quick
comments. That is, the task we have
here
24 is to begin now,
through about four o'clock this
25 afternoon at which
point we will have gone through
40
1 a series of
presentations on this issue of ooplasm
2 transfer that clearly
touch on some absolutely
3 major areas, we
encourage you to ask questions and
4 to set the stage for
critical discussions which I
5 will try to keep on
time, but also it is so
6 important that these
critical discussions develop
7 that we will have to
be a little flexible about how
8 that goes, leading up
to a discussion at 4:00 of
9 specific questions
that have been put together by
10 the FDA that will
frame issues the FDA wants input
11 from us on regarding
developing an IND process for
12 this field.
13 The only
other comment I want to make to
14 all of you is get your
thoughts out on the table.
15 There is no need to
force an agreement on anybody.
16 You are more than
welcome to articulate and defend
17 a minority
opinion. I don't believe my job here is
18 to come up with some
absolute consensus. My job is
19 to identify where
consensus can be reached,
20 however, as well as to
have you help us figure out
21 where there isn't
consensus and perhaps other
22 additional efforts in
those areas are coming.
23 We have to
make sure that when we are
24 done--I feel very
strongly--that we can say to the
25 public that this was
an open, balanced discussion
41
1 of the issues. That is a major responsibility.
2 If, in the middle of
discussions, somebody goes,
3 you know, we are
really missing this piece and we
4 just don't have it
here today, then that should go
5 into the record as
well because I think that is
6 part of being fair to
the whole field.
7 With respect
to the audience, I feel you
8 are as much part of
this discussion as we are. It
9 is a little harder to
control you so you will have
10 to forgive that, but
you are certainly not just
11 welcome but encouraged
to step up at key points of
12 the discussion and
bring your expertise and your
13 viewpoints to it. The rules are simply to keep it
14 brief and identify
yourself, and realize that with
15 the competition to try
to keep everything on time I
16 will also have to
manage that. But very much,
17 please feel part of
the discussion that will take
18 place today.
19 That is
basically it and I am really
20 looking forward to any
discussion that follows and
21 the diversity of
expertise we have here. With that
22 introduction, Dr.
Hursh?
23 Ooplasm
Transfer in Assisted Reproduction
24
FDA Introduction
25 DR.
HURSH: I would also like to welcome
42
1 the participants and
the audience to this meeting
2 of the Biological
Response Modifiers Advisory
3 Committee.
4 This is day
one of a two-day meeting of
5 the Biological
Response Modifiers Advisory
6 Committee. On this first day we will discuss
7 ooplasm transfer in
the treatment of female
8 infertility. On the second day the topic will be
9 potential germline
transmission during gene
10 therapy. We have chosen to link these two topics
11 as both of them deal
with the transfer of genetic
12 material go gametes,
sperm and eggs.
13 This has
occurred in the case of ooplasm
14 transfer and is a potential
inadvertent risk of
15 gene therapy. In both cases heritable genetic
16 modifications will be
produced. While the FDA and
17 the Recombinant DNA
Advisory Committee have
18 discussed some of
these issues previously, FDA felt
19 it was timely to have
further open public
20 discussion on the
subject of gene transfer in
21 gametes in light of
the evidence of new mechanisms,
22 such as the
manipulation of oocytes by which germ
23 cells can be
genetically modified.
24 Since
today's discussion is focused on
25 ooplasm transfer, I
will limit the rest of my
43
1 remarks to that
topic. We will hear about this in
2 much greater detail
from our first two speakers
3 but, in brief, in
ooplasm transfer 5 percent to 15
4 percent of an
unfertilized egg cytoplasm, which is
5 called ooplasm, is
transferred from a donor into a
6 recipient, and is then
fertilized in vitro.
7 Recipients are women
who have been unable to
8 conceive through
conventional in vitro
9 fertilization. The cytoplasm of an oocyte is
10 considered specialized
and it contains proteins,
11 messenger RNAs, small
molecules and organelles. It
12 is not clear which of
these components is the
13 putative active
component of ooplasm, but it is
14 with one of these
organelles, the mitochondria,
15 that we will be
primarily concerned with.
16 Most of you
are probably aware that
17 mitochondria are the
powerhouse of a cell, the site
18 where aerobic
respiration, the production of energy
19 using oxygen
occurs. But they have other
20 functions. They are involved in fatty acid
21 metabolism,
intracellular ion balance and
22 programmed cell death.
23 As you can
see on the schematic diagram
24 here, they are a very
specialized subcellular
25 structure, membrane
bound, and each cell has many,
44
1 many mitochondria to
support the energy
2 requirements of that
cell. I would like to draw
3 your attention to the
little squiggle in the middle
4 because that is one of
the issues about
5 mitochondria that
concerns us here. Perhaps the
6 most important feature
for our purposes is that,
7 due to their supposed
evolution from primitive
8 bacteria, mitochondria
contain their own genome.
9 The
mitochondrial genome is very small.
10 It is only about
17,000 base pairs as opposed to
11 several billion for
the human genome. However, it
12 has 37 distinct
genes. Unrelated individuals have
13 distinct genotypes of
mitochondria, so distinct
14 that they can be used
by forensic biologists to
15 establish relatedness
among human beings. The
16 mitochondrial DNA,
while small, is very important
17 because mutations
associated with mitochondrial DNA
18 result in human
disease. While I realize you
19 cannot read what is in
the balloons, the point of
20 the schematic diagram
here is this is the circular
21 mitochondrial genome
and each one of these balloons
22 represents positions
of mapped mitochondrial
23 mutations that result
in human disease.
24 Mitochondria
obey unusual rules of
25 inheritance. In mammals, after fertilization, the
45
1 mitochondria
contributed by the sperm are
2 apparently
destroyed. Therefore, the only
3 population of
mitochondria in a developing embryo
4 and in the resultant
progeny come from the pool
5 existing in the oocyte
prior to fertilization.
6 In general,
oocytes therefore get all of
7 their mitochondria
from the mother and that
8 mitochondria is a
homogeneous pool of a single
9 genetic type. This is a condition that is called
10 homoplasmy. This is the more common situation in
11 human oocytes. Having two distinct genetic forms,
12 two distinct pools of
mitochondria is less common
13 and this is referred
to as heteroplasmy. While
14 heteroplasmy is
unusual with wild type
15 mitochondria, it is
actually seen in people who
16 have mitochondrial
disease where you can have a
17 population of mutant
and a population of wild type
18 mitochondria
co-existing in the same cell.
19 In studies
of heteroplasmy it has been
20 observed that
mitochondrial genotypes can be
21 partitioned unequally
among tissues, and I believe
22 we will hear a great
deal more about this from one
23 of our speakers this
morning, Dr. Eric Shoubridge.
24 So, what
happens after ooplasm transfer?
25 If there are
mitochondria transferred during
46
1 ooplasm transfer, what
is the result? In March of
2 2001, a laboratory of
Dr. Jacques Cohen reported
3 that two children born
after the ooplasm transfer
4 protocol were
heteroplasmic, which means the
5 genotypes of both the
ooplasm donor and the mother
6 could be detected in
their tissues. These children
7 were approximately one
year old at the time of this
8 analysis, so this was
a persistent heteroplasmy
9 that had been
maintained.
10 At the time
of Dr. Cohen's publication the
11 FDA was already
considering action in the area of
12 ooplasm transfer. The report of heteroplasmy
13 raised our concerns,
as did information in two
14 pregnancies occurring
after ooplasm transfer
15 resulted in fetuses
with Turner's syndrome, a
16 condition where there
is only one X chromosome.
17 In addition,
despite the fact that Dr.
18 Cohen refers to this
as an experimental protocol
19 that should not be
widely used, we felt that it was
20 beginning to spread
rapidly into clinical practice
21 in the United States
by 2001. There were at least
22 23 children born in
the United States after using
23 ooplasm transfer. Three United States clinics had
24 published on this
procedure and we, at FDA, were
25 able to find five
additional clinics that were
47
1 advertising this
procedure on the internet.
2 FDA had
concerns about whether we
3 understood all the
ramifications of this procedure
4 and whether we
understood its safety in particular,
5 and reacted by sending
letters to practitioners who
6 were identified by
publications on ooplasm transfer
7 or by advertisements
offering the procedure. We
8 advised practitioners
that we would now require the
9 submission of an
investigational new drug
10 application, or IND,
to the agency and its
11 subsequent review to
continue to treat new
12 patients. After the letter was issued we had
13 telephone
conversations with several practitioners
14 who wanted to know
more about the IND submissions
15 procedure.
16 After these
conversations FDA felt this
17 topic would be well
served by open public
18 transparent discussion
of the ooplasm transfer
19 procedure and the data
behind it, hence this
20 meeting. The major issue we, at FDA, are trying to
21 achieve consensus on
at this advisory committee
22 meeting is are
preclinical and clinical data
23 supporting the safety
and efficacy of ooplasm
24 transfer sufficient to
justify the risks of
25 clinical trials? If additional data are needed,
48
1 what types of data
would be the most informative,
2 what model systems,
what size studies?
3 FDA's tasks in regulating new therapies is
4 to weigh risks and
benefits and to determine what
5 safeguards need to be
in place to ensure the safety
6 of human
subjects. That is what we will do with
7 ooplasm transfer. While the FDA welcomes
8 discussion with all
interested parties, our topic
9 today is very
limited. We will, therefore, limit
10 today's discussion to
the science behind ooplasm
11 transfer and not
extend that discussion to FDA's
12 jurisdiction in
general, FDA's proposed rules for
13 the regulation of
human cells and tissues and other
14 assisted reproductive
technologies. Thank you very
15 much.
16 DR.
SALOMON: Thank you, Deborah. Unless
17 there are any pressing
questions, I think the
18 purpose of that was
clearly just to set the stage
19 for what is to
follow. What I would like to do is
20 invite Dr. Susan
Lanzendorf to present cytoplasmic
21 transfer in the human
oocyte. She is from the
22 Jones Institute of
Reproductive Medicine.
23
Cytoplasmic Transfer in the Human
24 DR.
LANZENDORF: I have come here today to
25 share some of the
experiences that we have
49
1 encountered at the
Jones Institute with the
2 procedure of cytoplasm
transfer in the human.
3 Cytoplasmic
transfer was first considered
4 at the Jones Institute
back in 1990 when an
5 investigator, a
clinical fellow, Flood et al.,
6 reported that the
developmental potential of
7 oocytes to mature in
vitro can be increased by
8 injecting with the
cytoplasm of oocytes matured in
9 vivo. This was performed in the monkey model.
10 This study
found that 13 percent of the
11 injected oocytes
resulted in pregnancies while none
12 of the sham-injected
or non-surgical controls
13 resulted in a
pregnancy. The investigators felt
14 that this suggested
that factors may be present
15 within the cytoplasm
that control genetic,
16 maturational and/or
developmental properties.
17 Then, in
1997, Cohen and coworkers
18 reported the first
human pregnancy from the
19 transfer of cytoplasm
from donor eggs. They
20 reported that the goal
of the procedure was to
21 provide healthy
cytoplasmic factors to the eggs of
22 the patients who
repeatedly produce embryos of poor
23 quality.
24 We were very
interested in this report.
25 We see a lot of
patients who come through in vitro
50
1 fertilization who
repeatedly fail to achieve a
2 pregnancy and many
times we are at a loss on how to
3 continue treatment in these patients who just
don't
4 seem to get
pregnant. So, we approached our
5 institutional review
board to see if we could
6 investigate this
procedure.
7 We decided
to look at two groups of
8 patients, in one of
which the wife is 40 years of
9 age or older, or in
couples who have had at least
10 two previous IVF
attempts which resulted in only
11 poor quality
embryos. In in vitro fertilization we
12 have found that when
you transfer embryos that have
13 an ideal morphology
they result in a higher
14 pregnancy rate than
those who have less than an
15 ideal morphology. So, this was an attempt to try
16 to improve this and,
hopefully, increase the
17 pregnancy rate.
18 Again, we
put this to the institutional
19 review board and we
requested permission to do this
20 with 15 consenting
patients. We worked very hard
21 on our consent form,
being that this was a
22 procedure where very, very
little was known. So,
23 of course, we tried to
emphasize to the patients
24 the risks that they
might encounter, including that
25 the effect of the
procedure on the couple's eggs or
51
1 their ability to
establish a pregnancy totally
2 unknown. What is also unknown is if the procedure
3 would increase the
risk of obstetric complications,
4 or if the thawed donor
eggs would even survive. I
5 should point out here
that we used frozen and
6 thawed donor eggs for
our procedure. So, we
7 emphasized to the
patient that if the thawed eggs
8 didn't survive the
procedure would not be performed
9 and they may not get a
transfer. In addition, the
10 patient's eggs may not
survive the procedure or
11 they may fail to
fertilize and develop normally and
12 they would not obtain
a transfer.
13 We also
emphasized the risk to the
14 offspring. It is not known if the procedure would
15 increase risk of
obstetric complications or fetal
16 abnormalities. The eggs could be damaged in some
17 way that could affect
the offspring. And, there
18 was the possibility
that genetic material could be
19 transferred from the
egg donor to the patient's
20 eggs and it is unknown
if this could adversely
21 affect the offspring.
22 In our
consent form we did break this out
23 into talking and
making clear to the patient that
24 there are two types of
genetic material, DNA from
25 the nucleus of the egg
and the DNA from the
52
1 mitochondria. So, we were careful to make them
2 understand that the
two different possibilities of
3 genetic material could
be transferred.
4 The consent
form also stressed that
5 because the procedure
is so new there is no way to
6 determine what the
exact risks are, or at what rate
7 the risks occur. In our other consent forms we try
8 to say, you know, we
have seen a 50 percent
9 survival rate, or we
have seen a 60 percent
10 pregnancy rate but we
couldn't even do this with
11 this procedure because
it is so new so we
12 emphasized this to them.
13 It was also
recommended that all of the
14 patients who achieve a
pregnancy have an
15 amniocentesis
regardless of their age. Then, of
16 course, the boiler
plate other risks that cannot be
17 identified at that
time.
18 This is just
to show you quickly how we
19 perform the
procedure. Again, we used
20 frozen-thawed donor
eggs so the donor eggs that
21 contributed the
cytoplasm were collected and
22 cryopreserved at a
previous state. Then, when the
23 patient came through
on the day of their aspiration
24 and cytoplasm
transfer, the donor eggs were thawed.
25 So, before
we get here what we will have
53
1 done is--this is the
pipet here that we also use to
2 do the donation. This is the egg-holding pipet
3 which just holds the
egg in place. This is the
4 egg. So, prior to getting here we would have got
a
5 drop of sperm and
picked up a sperm from the
6 patient's husband and
loaded it in the pipet. We
7 then take this pipet
with the sperm and insert it
8 into the donor
egg. Then, once in the donor egg,
9 we draw up cytoplasm
that will be transferred.
10 We then move
to the recipient's egg, the
11 patient in this
scenario, and then put that pipet
12 into the egg, inject
that cytoplasm into the egg,
13 along with the
husband's sperm. Actually, what
14 occurs is the
cytoplasm transfer and the
15 utilization of the egg
at the same time.
16 Our results,
we had eight patients in
17 eight cycles who were
40 years of age or over, with
18 an average age of
44. The procedure did not appear
19 to have an effect on
embryo quality. I say "did
20 not appear"
because there are too few numbers of
21 actual embryos to
compare with other embryos to
22 make a significant
conclusion. No pregnancies were
23 established in any of
these eight patients.
24 In the same
40 years or older group, 39
25 eggs were retrieved,
with a mean of 3.2 eggs per
54
1 patient. This is low but is normal in patients in
2 this age group. We had a 54 percent fertilization
3 rate, and this would
be with the cytoplasm transfer
4 occurring at the same
time. To do these
5 procedures, we had to
use cytoplasm from nine donor
6 eggs, and these donors
ranged in age from 25 to 29.
7 Of the donor eggs, 62
percent survived the thaw
8 procedure and were
used.
9 We had three
patients who came through who
10 had a history of poor
quality embryos. Actually,
11 this is the group of
patients that we thought we
12 could really help with
this procedure. We did not
13 go into it thinking
that the older patients would
14 be the ones that would
benefit mostly, and I think
15 the other
investigators who performed this
16 procedure would
probably agree that it is not
17 helping the older aged
couples.
18 So, these
were three patients who had
19 significant history of
poor quality embryos in the
20 past. The age of these patients was 35, 35 and 38.
21 The procedure did
appear to have an effect on
22 embryo quality. To us, the embryos looked much
23 better than those that
we had seen from these same
24 patients
previously. Of those three patients,
one
25 achieved a pregnancy. It was a twin pregnancy that
55
1 was established. That particular patient had
2 undergone six previous
IVF attempts with fresh
3 transfer and three
attempts with cryotransfer and
4 never achieved a
pregnancy.
5 In these
three patients 42 eggs were
6 retrieved, a mean of
14.3 which, as you can see, is
7 much higher than in
the older patients; 62 percent
8 fertilization rate
with the cytoplasm transfer.
9 This is the
information on the donors that provided
10 the eggs, and they had
a 66 percent survival, those
11 three donors.
12 These are
the twins. I have been told
13 that the medical
director has spoken with the
14 couple about having
their twins evaluated
15 genetically for all
the questions that we are here
16 about today. The couple is not interested. They
17 feel their children,
who are now three or four
18 years old, are very
healthy and very normal and
19 they don't want
anything else done with that.
20 We were also
looking at other things when
21 we were doing these
studies and before we received
22 our letter to stop
doing them. One of the things
23 that we were
interested in was the inadvertent
24 transfer of the
nuclear material, the chromosomes
25 from the donor egg
into the recipient egg. I
56
1 should point out here
that would had actually met
2 with a mitochondrial
geneticist at our institution
3 to find out--you know,
we posed this problem of
4 transferred
mitochondria, and ask him did he think
5 we would have a
problem there; did he think that
6 these mitochondria
that we transferred we be passed
7 on. He assured us no, it was too few
mitochondria
8 and it couldn't
happen. So, we really didn't go
9 into it thinking that
that would be the problem.
10 We were more concerned
with accidentally
11 transferring the
nuclear material.
12 So, we
looked at some of the eggs that we
13 had taken cytoplasm
out of using staining. We can
14 actually see the
spindle of the egg, and with this
15 stain we can see the
chromosomes on the spindle.
16 So, we looked at these
eggs that provided the
17 cytoplasm, and this
was published just recently,
18 last year, and the
oocytes that we evaluated
19 resulted from either
clinical cases I just
20 described to you or
research procedures which we
21 are doing.
22 In this case
12 oocytes were thawed but
23 were not used for the
transfer. They weren't
24 needed to provide
cytoplasm so we used those as
25 controls. We had 23 eggs that we thawed which
57
1 survived the donation
procedure. These are the
2 ones that served as
tests.
3 When we did
the staining procedure on
4 these eggs, the
control eggs all demonstrated
5 normal meiotic spindle
but when we looked at the
6 test eggs we found
that 2/23 eggs that provided
7 cytoplasm demonstrated
total dispersion of the
8 chromosomes from the
metaphase plate, and complete
9 disorganization of the
spindles.
10 Of course,
the numbers are very small but
11 there was no
significant difference between the two
12 groups. So, we wondered if this was something to
13 do with the drawing out
of the cytoplasm that
14 potentially disrupts
the spindle. We wondered,
15 since it is a
procedure that is very similar to
16 ICSI, if this would be
the same rate of meiotic
17 spindle damage that
you would see in ICSI oocytes.
18 Because we were worried about this we
19 looked at ways to see
if there were some way we
20 could prevent
this. So, we looked at a new
21 microscope that was on
the market, the PolScope.
22 Having this attached
to your microscope actually
23 lets you visualize,
while you are doing a
24 procedure, the actual
spindle so that you can see
25 the spindle and you
can stay clear of it.
58
1 Here is the
egg, just a small part of the
2 egg, the polar body
and the spindle here. So,
3 while you are doing
the procedure, you are sticking
4 something into the egg
and you can see the spindle
5 and stay clear of
it. This is equipment that is
6 currently used in many
laboratories, including ours
7 now, in which clinical
ICSI cases are performed, or
8 research involving
enucleation where they want to
9 see where the spindle
is so they can take out the
10 nuclear material.
11 We also did
a little work with looking at
12 this from a research
aspect. We had a clinical
13 fellow, Sam Brown, who
wanted to see if the
14 original work of Flood
in 1990, where we used
15 immature eggs, would
have the same ft, cytoplasmic
16 transfer. The idea with it is the developmental
17 failure of human
embryos derived from oocytes
18 matured in vitro may
be due to the deficiency of
19 cytoplasmic
factors. In in vitro fertilization we
20 have found that when patients get a lot of
immature
21 eggs, eggs that need
more time maturing before they
22 can be inseminated,
these eggs do not do as well.
23 So, the idea was to
see if human prophase I oocytes
24 became developmentally
competent after
25 microinjecting them
with the ooplasm of eggs
59
1 matured in vivo within
the body.
2 Sam
hypothesized that such an injection
3 would improve
fertilization and blastocyst
4 development of these
immature eggs. This was just
5 a research
project. None of these eggs were
6 transferred back to
patients. It was with the hope
7 of salvaging immature
eggs. For example a patient
8 who gets all immature
eggs after a retrieval could
9 have this procedure
done and improve her chances of
10 achieving a pregnancy.
11 In the first
part of the experiment looked
12 at the effect of
cytoplasmic transfer from in vivo
13 matured eggs into PI
eggs. So, we had three
14 groups, control eggs
which were put on a stage of
15 the microscope but not
actually injected. We found
16 that 74 percent of
these matured to metaphase II
17 after continued
culture. Sham eggs were eggs that
18 were injected with an
equal amount of media only,
19 not cytoplasm, and we
found that only 50 percent
20 matured to metaphase
II. Cytoplasm transfer eggs
21 that actually had the
procedure, 58 percent matured
22 to metaphase II. So, these findings suggested that
23 injecting a substance
into an egg may have a
24 negative impact on
maturation.
25 We also
inseminated these eggs to see if
60
1 they could be
fertilized, and in the control the 14
2 eggs that matured to
metaphase II we had a 50
3 percent fertilization
rate. Shame injected, we
4 only had 38 percent
fertilization rate. With
5 plasmic transfer four
of the eight fertilized,
6 which was 50
percent. The development after
7 culture was not
remarkable between the three
8 groups. The numbers were very low and similar to
9 what we always see
with immature eggs.
10 We also
looked at the effect of
11 cytoplasmic transfer
on eggs that matured in vitro.
12 They were first
allowed to mature in vitro and then
13 they were given the cytoplasm of an egg