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 that was
14 matured in vivo. There were 17 control eggs that
15 received no
cytoplasmic transfer, and after
16 insemination 53
percent of these fertilized.
17 Cytoplasmic transfer,
47 percent of these
18 transferred. We did see a little bit higher rate,
19 since these were cytoplasmic
transfers and the
20 injection of a single
sperm having three prime
21 nuclei suggests that
there was damage to the
22 spindle in these eggs.
23 In
conclusion, we feel that cytoplasmic
24 transfer, if performed
clinically, should move
25 forward cautiously and
with the full consent of the
61
1 patients. Just to give you some of the feelings of
2 the patients, should
this procedure be found to not
3 be harmful to the
offspring and studies continue,
4 we do have many
patients out there who are not
5 bothered by the fact
that their offspring would
6 have the genetic
material of another person because
7 for these patients the
only other recourse is to
8 use donor eggs. So, in that case, their children
9 would have none of
their genetic material. So,
10 having some of their
genetic material appeals to
11 them, and a lot of
patients would pick this
12 procedure over going
to the donor egg. Thank you.
13
Question and Answer
14 DR.
SALOMON: Thank you, Dr. Lanzendorf.
15 This initial
presentation is open for questions and
16 discussion. There are so many different kinds of
17 questions here and
you, of course, get the
18 privilege of being the
fist one. One of the things
19 that is going to come
up is if you go to an IND,
20 then in this whole
area the big question is always
21 going to be
preclinical work and models. So, let
22 me make the first
question here a little bit about
23 these primate studies.
24 The primate
studies were done in 1990, and
25 then the first
clinical report you made was seven
62
1 years later, in 1997.
2 DR.
LANZENDORF: Right.
3 DR.
SALOMON: Maybe at some point you
4 could kind of explain
to us in the seven years, but
5 specifically for the
primate studies, can you make
6 me understand this a
little bit better because it
7 will be important
later in our discussions for is
8 this a good model
because then one might focus on
9 such a model. To the extent it is not a good
10 model, one should be
cautious.
11 DR.
LANZENDORF: Right.
12 DR.
SALOMON: So, the question I would
13 have specifically is
what defines this model as a
14 model for infertility?
15 DR. LANZENDORF: The non-human primate as
16 a model?
17 DR.
SALOMON: Yes. Essentially, you had
18 these oocytes. I am assuming, just guessing, that
19 you cultured them in
vitro for a while and, the
20 longer they were in
vitro, they became less and
21 less viable. So, when you implanted the
22 controls--I am not
saying you did, I guess this
23 wasn't your study, but
when they implanted the
24 oocytes and they
didn't get a successful pregnancy
25 and they managed to
salvage 13 percent with
63
1 cytoplasmic transfer
from a fresh egg--is that
2 right?
3 DR.
LANZENDORF: Right.
4 DR.
SALOMON: So, it was the culture of
5 the oocytes for X
number of days or weeks that
6 caused them to lose
their viability?
7 DR.
LANZENDORF: When you take immature
8 eggs from a primate, a
monkey or a human, and they
9 haven't completed the
maturational process within
10 the ovaries, they have
to complete it in a dish and
11 that usually takes
about 24 hours, sometimes 48
12 hours. These eggs historically are not as
13 developmentally
competent as eggs that had
14 completed maturation
in the body. Does that make
15 sense? Before we go in to remove an egg from a
16 patient we try to time
it so that when we are
17 taking these eggs out
they are already mature. So,
18 just the whole aspect
of collecting immature eggs
19 for in vitro
fertilization, monkey or human, has
20 always posed a problem
when these eggs are not as
21 competent.
22 That early
study that was published in
23 1990 was not looking
at cytoplasmic transfer as a
24 way to cure this
problem. It was trying to look at
25 what is the
problem. What is it about immature
64
1 eggs that they don't
do well? So, they said, well,
2 if we put some
cytoplasm from one that was matured
3 in vitro into this
egg, will it do better? And, it
4 did. So, that 1990 report was never, from what I
5 understand, a report
to say let's go out there and
6 start doing
cytoplasmic transfer. You know, I
7 don't think the Jones
Institute looked at it as
8 though, oh, we can
cure these immature eggs from
9 this problem and let's
start doing this in
10 patients. So, that is why when you talk about the
11 seven years--you know,
I don't think any of us even
12 considered doing it as
a procedure to help
13 infertile couples.
14 DR.
SALOMON: I appreciate that
15 clarification. Sort of the follow-up then is 13
16 percent were
successful pregnancies with this
17 procedure.
18 DR.
LANZENDORF: Right.
19 DR.
SALOMON: Again, were there a whole
20 lot of miscarriages
and other problems in the other
21 87 percent?
22 DR.
LANZENDORF: I don't know, but having
23 done monkey IVS and
worked with monkey IVS and used
24 it as a model, I can
say that a lot of times doing
25 in vitro fertilization
in fertile monkeys is a
65
1 hundred times harder
than doing it in a group of
2 infertile human
patients. You know, monkeys are
3 somewhat difficult to
work with during in vitro
4 fertilization. There are sites around the United
5 States, primate
centers and places like that, who
6 have got it down to a
fine art and I do believe
7 that the non-human
primate is the model that should
8 be looked at. But, again, it is a very difficult
9 procedure but there
are places in the United States
10 that do it quite well
and I believe could do these
11 experiments.
12 DR.
SALOMON: Richard?
13 DR.
MULLIGAN: Just to go back to the data
14 set, between the 1990
report and 1997, can you
15 characterize what is
the complete data set? Or,
16 can some expert tell
us? I assume there have been
17 other things that were
done, repeats from the 1990
18 experiment?
19 DR.
LANZENDORF: No, there was nothing
20 ever done.
21 DR. MULLIGAN: So, the wealth of
22 information about the
potential of this comes from
23 that 1990 experiment?
24 DR.
LANZENDORF: Right. Again, that was
25 not an experiment
exploring cytoplasm transfer. It
66
1 was trying to look at
is it the cytoplasm the
2 problem? Is it the nucleus that is the problem?
3 Is it the monkey's
uterus that is the problem? So,
4 it was just a basic
study trying to look at what is
5 the problem with
immature eggs; it was never a
6 cytoplasmic transfer
procedure. So, it was never
7 pursued as an
experimental design to continue.
8 DR.
MULLIGAN: Just for perspective, how
9 many actual eggs were
in that group that resulted
10 in 13 percent
pregnancy?
11 DR.
LANZENDORF: I have no idea. I was
12 not there and I don't
believe I brought the article
13 with me. I am sorry.
14 DR.
SAUSVILLE: And when one speaks of a
15 sham procedure in this
case, which comes up both in
16 the monkey experiments
and in some of the more
17 recent data, does sham
mean withdrawal from
18 something else--
19 DR.
LANZENDORF: Right.
20 DR.
SAUSVILLE: --in the donor egg and
21 manipulation of the
recipient egg? Or is it
22 saline? Could you give us a little bit of
23 background about what
the exact shams and controls
24 are?
25 DR.
LANZENDORF: Well, in our lab a sham,
67
1 an actual control
would be one that was just put on
2 the stage of the
microscope, that would have seen
3 the effects of the
change in temperatures and
4 moving around and
being put into dishes. A sham
5 injection is one in
which, at least in experiments
6 I was involved with,
we would draw up culture media
7 and use that to inject
into the egg. So, the egg
8 was actually seeing
the movement of substance, the
9 puncture of the needle
and things like that. You
10 know, in some of the
experiments the sperm was
11 injected also, in some
it wasn't. That wasn't part
12 of the design. But we tried to keep it exactly
13 like the actual
procedure without the transfer of
14 the cytoplasm in a
sham.
15 DR.
SAUSVILLE: But a key point is that
16 the culture medium is
what constituents the sham
17 injection. Isn't that correct?
18 DR.
LANZENDORF: Yes.
19 DR.
SAUSVILLE: And that, of course, has
20 145 millimolar of
sodium chloride as opposed to
21 what is inside.
22 DR.
LANZENDORF: Right.
23 DR.
SAUSVILLE: So, a small amount
24 actually then could
result in a market change--
25 DR.
LANZENDORF: Right. We realize that
68
1 probably our shams
should actually do worse than
2 cytoplasmic transfer
because of these things being
3 dumped into them.
4 DR.
SAUSVILLE: And they did, right?
5 DR.
LANZENDORF: And they did.
6 DR.
SALOMON: Dr. Monroe?
7 DR.
MONROE: I have a question about the
8 relevance of the
monkey experiment that we have
9 been addressing and
the type of patient who might
10 be a recipient of this
procedure. It seems to me
11 that in the monkey
studies the question was the
12 issue of immature
eggs.
13 DR.
LANZENDORF: Right.
14 DR.
MONROE: It wasn't a question of
15 people for whom that
wasn't necessarily the problem
16 but just had poor
embryo development. Is that the
17 correct interpretation? So, they are very
18 different questions
that we would be addressing.
19 DR.
LANZENDORF: Right. Those three
20 patients, the people
that we think could be helped
21 from this procedure,
we really don't know what is
22 wrong with their eggs
but they are typically young
23 patients. They do well on retrieval. They stem
24 well. They get a large number of eggs. That is
25 what usually happens
with this age group. They
69
1 fertilize find but
then, after being in culture for
2 a couple of days, they
usually would not even be
3 recognizable as an
embryo--total fragmentation. We
4 use a grading scale of
one to five, one being the
5 best and five the
worst, and they were typically
6 all five. In the cases where we would see that
7 transfer would have
been pointless but usually
8 patients like a
transfer even if they are told that
9 it is probably pointless. So, there is something
10 inherent about those
patients' eggs that is the
11 problem and whether it
is a cytoplasmic thing we
12 don't know, but it is
something we see over and
13 over again. The patient who achieved a pregnancy,
14 this happened to her
in like six other stem
15 stimulations and there
was nothing else that we
16 could offer her.
17 DR.
RAO: Two sort of more scientific
18 questions, one was
sort of an extension of what Dr.
19 Sausville asked, and
that is, has there been any
20 comparison with
cytoplasm from any other cell as a
21 control that has been
used in these experiments?
22 DR.
LANZENDORF: From another egg?
23 DR.
RAO: Not just from another egg, from
24 any other cell as a
control?
25 DR.
LANZENDORF: No.
70
1 DR.
RAO: I mean, do you really need
2 oocyte cytoplasm?
3 DR.
LANZENDORF: We have always used
4 oocyte cytoplasm.
5 DR.
RAO: And to your knowledge, there is
6 no data?
7 DR.
LANZENDORF: Not that I know of.
8 DR.
RAO: You showed data where you had
9 pronuclei, right?
10 DR.
LANZENDORF: Right.
11 DR.
RAO: So, there was maybe a high
12 probability of
injury. Were those experiments done
13 with the spindle view
imaging system?
14 DR.
LANZENDORF: No. We got our PolScope
15 at the same time we
got our letter.
16 DR.
NAVIAUX: Just a question about the
17 optics that are being
used. At any time, are the
18 oocytes exposed to
ultraviolet light?
19 DR.
LANZENDORF: No.
20 DR.
NAVIAUX: And the imaging of the
21 PolScope, what are the
physics of that?
22 DR.
LANZENDORF: I am not sure, but it is
23 just a changing of the
wavelength of the light that
24 allows you to see the
spindle. It was initially
25 designed, I think, to
look at the membrane around
71
1 it. We found that by using it we could also see
2 the spindle.
3 DR.
NAVIAUX: Are dyes ever used to image
4 nucleic acid?
5 DR.
LANZENDORF: No. The PolScope is used
6 by some labs pretty
extensively for ICSI. So,
7 there are probably
pretty good pregnancy results
8 for that. I hope I am not getting the PolScope
9 people in
trouble. It is routinely used.
10 DR.
SCHON: PolScope is polarizing optics.
11 It has been around for
fifty years and it is just
12 like a microscope.
13 DR.
NAVIAUX: The basis for that question
14 is that certain types
of mitochondrial dysfunction
15 are responsive to
ultraviolet lights and others are
16 less responsive. But that is not relevant.
17 DR.
SALOMON: Dr. Casper?
18 DR.
CASPER: Susan, do you know if any
19 monkeys were actually
born from the cytoplasmic
20 transfer, from that 13
percent pregnancy rate? If
21 so, are there any
records regarding their health,
22 life span or anything
like that?
23 DR. LANZENDORF: I don't think there are
24 any records at
all. I have the article here. It
25 just talks about
pregnancy rate. It doesn't say
72
1 anything about live
births that I can see.
2 DR.
SALOMON: Dr. Rao?
3 DR.
RAO: Another question, are the donor
4 oocytes tested in any
fashion?
5 DR.
LANZENDORF: Our donor oocytes are
6 eggs from our typical
donor pool. We have an
7 active donor egg program. So, somebody coming into
8 the program to donate
their eggs for a pregnancy in
9 another couple have
extensive screening,
10 psychological as well
as medical, and we do
11 genetics testing and
things like that.
12 DR.
RAO: Does that include mitochondria?
13 DR.
LANZENDORF: No, it does not include
14 mitochondrial
diseases, no. But they are tested.
15 DR.
SALOMON: So, another question, you
16 know, in this perfect
position to answer all these
17 questions at the
beginning of the day, not all
18 necessarily that you
have to defend, but you used
19 the term "embryo
quality" a couple of times. If
20 you will excuse my
ignorance, can you educate me a
21 little bit about what
do you do objectively to
22 determine embryo
quality?
23 DR.
LANZENDORF: Embryo quality is just
24 basically all
morphological. No one has devised
25 some kind of
biochemical marker to say this embryo
73
1 is better than that
embryo, but typically you start
2 out with the one cell;
then you have two, then
3 four; and you see that
beautiful clover leaf kind
4 of pattern going on
there. When you start seeing
5 poor quality embryos
you will see that the cleavage
6 divisions aren't
equal. Some of the blastomeres
7 are very large, some
are very small. There are
8 other things called
cytoplasmic blebs and fragments
9 that start forming and
these things can take over
10 the entire--all the
blastomeres just start
11 fragmenting and people
think this is some kind of
12 apoptosis that is
going on.
13 Through the
years we have seen that when
14 you transfer four
perfect four grade cells with no
15 fragmentations, the
implantation rate is
16 considerably high than
if you were to transfer five
17 totally fragmented,
very poor embryos. Very
18 rarely, if ever, would
you see a pregnancy there.
19 So, we are even
confident telling these patients
20 you don't want to
undergo the transfer or pay for
21 the transfer; your
chances of getting pregnant with
22 these three grade five
embryos is zero. So, it is
23 an assessment. It is not always correct. A lot of
24 times we put three
grade one embryos and a patient
25 doesn't get pregnant,
or we put some very poor
74
1 quality embryos and
the patient does get pregnant.
2 So, it is not 100
percent. But when you see a
3 patient come through
six, seven times and every
4 single time they have
very, very poor quality
5 embryos it becomes
something about this patient.
6 You know, what can we
do to improve this? Doctors
7 will try changing
stimulation protocols and it
8 doesn't work. We have a certain class of patients
9 and this is their
problem, and they are told to go
10 to donor egg.
11 DR.
SALOMON: Just to summarize, if you
12 have a good
relationship with your technologists
13 you have a sense of
confidence in this subjective
14 reading--
15 DR.
LANZENDORF: Oh, yes.
16 DR. SALOMON: --of good and bad embryos.
17 DR.
LANZENDORF: Yes.
18 DR.
SALOMON: I mean, just to show you
19 that you are not alone
in that area, I am
20 interested in islet
transplantation and we are
21 similarly clueless
about an objective determination
22 of a quality islet
preparation, and that is a major
23 area now focused for
research in a program that I
24 am involved in.
25 DR.
LANZENDORF: Right.
75
1 DR.
SALOMON: So, it is not unusual.
2 DR.
SCHON: These patients who have gone
3 through six or seven
times and have always had
4 these poor quality
embryos, are they consistently
5 poor quality from day
one to fertilization onward,
6 or is it sort of an
abrupt change, let's say, on
7 day two or three?
8 DR.
LANZENDORF: It is usually the first
9 cleavage division.
10 DR. SCHON: So, at the first cell division
11 you start seeing these
abnormalities, but these
12 multiple patients that
were selected for
13 cytoplasmic transfer
and had had consistently poor
14 embryo quality up to
that point on multiple
15 attempts, was there
any attempt to see whether or
16 not the embryos could
be put back earlier, let's
17 stay at the one cell
stage or at the two cell stage
18 before this
fragmentation occurred to divorce the
19 notion that there was
an embryo problem versus the
20 ability of that
particular patient's embryo to
21 survive in culture?
22 DR.
LANZENDORF: The patient who got
23 pregnant, I believe
but I can't say for certain she
24 had a ZIFT
procedure. I mean, this patient was
25 hell-bent on getting
pregnant and eery time she
76
1 came she was going to
do something different to try
2 to improve her
chances. So, we are talking about
3 three patients and I
know I could look this up for
4 you in their records,
but I feel pretty confident
5 that even those
procedures would not have helped
6 them, and I believe
that one had tried other
7 procedures.
8 DR.
SALOMON: Dr. Murray and then Dr.
9 Mulligan.
10 DR.
MURRAY: Thank you. Dr. Lanzendorf,
11 in your presentation
the last point you made was a
12 kind of empirical
claim with a moral punch line.
13 You said that most
patients having to choose
14 between a donor egg
and cytoplasmic transfer would
15 not be bothered with
the fact that the child may
16 have genetic material
from the mitochondria of the
17 egg donor. In ethics we are as intensely focused
18 on the text as
scientists are focused on data. So,
19 it would be very
helpful to know, if not now and
20 you could submit
later, exactly what question the
21 patients were
responding to and what information
22 they had been given
about the significance and
23 risks of getting
heteroplasmy for example.
24 DR.
LANZENDORF: Well, before the two
25 pregnancies from
Jacques Cohen's lab, we would talk
77
1 to the patients about what it would mean to
have
2 mitochondria from
somebody else, and that there
3 mitochondrial diseases
and things like that.
4 Again, at that point
we were more concerned about
5 transfer of nuclear
material after being reassured
6 by a mitochondria
person that mitochondria would
7 not be transferred,
but we did always have it in
8 the consent form. Then after those pregnancies
9 became evident, we
immediately amended our consent
10 form to talk about the
two children who had been
11 born. I don't believe that we did any patients
12 after that because
that was soon after we received
13 the letter.
14 DR.
MURRAY: Did your mitochondrial expert
15 not inform you about
the possibility of
16 heteroplasmy?
17 DR.
LANZENDORF: No, he didn't. Well,
18 that is what we went
to ask him about because one
19 of the things we were
interested in was looking at
20 transferring
mitochondria from one egg to the
21 other. We actually had a patient who came to us
22 also with a
mitochondrial disease and wanted us to
23 do nuclear transfer
for her so that her nucleus
24 could be put into an
egg with normal cytoplasm.
25 So, we also explored
with her being able to take
78
1 just a small amount of
cytoplasm from a normal
2 donor egg, and we were
assured from our person we
3 talked to that that
much transfer of cytoplasm
4 would not affect the
egg. It would not be passed
5 on to the progeny, and
things like that.
6 DR.
MURRAY: They were wrong.
7 DR.
LANZENDORF: We initially approached
8 this as wanting it to
be the mitochondria that
9 provided the benefit.
10 DR.
MURRAY: So, you got incorrect--
11 DR.
LANZENDORF: Oh, yes.
12 DR.
MURRAY: I don't know what the
13 protocol is. This is my first meeting with the
14 committee, but I would
appreciate it if you could
15 give us at some point
the actual question asked on
16 which you based this
particular conclusion.
17 DR.
LANZENDORF: Well, it was just sitting
18 down, talking to patients,
consenting patients and,
19 you know, we do a
weekly lecture, an egg class
20 where embryologists
just sit around the table and
21 we present slides,
similar to these, and show them
22 the kind of thing and,
you know, patients
23 immediately jump up
and, "oh, I don't have to go to
24 a donor egg. I can possibly have my genetic
25 material in my
child." Then you say, "well,
but
79
1 there is the chance of
mitochondrial transfer." "I
2 don't care about
that." "Well, it may change
the
3 way the baby
looks." You know, those are the
4 things that an
infertile couple are thinking about.
5 DR.
MURRAY: You have a mitochondrial
6 genome and a nuclear
genome that comes into balance
7 in some way that we
don't understand. So, really
8 part of the issue is
not simply having somebody
9 else's
mitochondria. The issue is whether that
10 mitochondrial DNA, in
its interactions with that
11 woman's nuclear DNA,
is going to draw you into a
12 new aspect of being
that you would otherwise not
13 have had the
possibility of encountering. So, I
14 think there is a
complexity there.
15 DR.
LANZENDORF: Right, and at that time
16 we did not understand
the complexity so we would
17 most definitely change
the way we talk to the
18 patient, get more
information, explain to them more
19 about the role of
mitochondria and things like
20 that. But I still believe that should this
21 procedure receive an
IND, there are going to be
22 patients who will be
lining up for it. We get
23 calls weekly from all
over the world wanting the
24 procedure.
25 DR. SALOMON:
Along the same line as the
80
1 ethics aspect of it,
what does it mean that when
2 you went back to the
couple that had the twins that
3 they just said, forget
it; we don't want to know
4 anything. Again, I am not in your field but that
5 kind of concerns me
that either they weren't really
6 prepared for the
experimental nature of the
7 procedure or they
don't really appreciate how
8 important it would be
to test their children.
9 DR.
LANZENDORF: Right.
10 DR.
SALOMON: Or, is this really such an
11 emotional issue and,
of course, we know it is such
12 an emotional issue
that this is going to be a very
13 difficult problem
going forward in these studies,
14 that the parents
really are not going to want you
15 to come near their
kids.
16 DR.
LANZENDORF: This is information that
17 I obtained from a
medical director, and I can go
18 back to the medical
director, or maybe you can go
19 back to the medical
director and explain why you
20 think it is important,
that these things occur and
21 maybe the couple can
be brought back in and talked
22 to again. But when the letter went out and, of
23 course, when I found
out about this meeting I asked
24 would she consider
having her children evaluated.
25 He said, no, I just
saw them last week and
81
1 mentioned it and they
had no interest in it; they
2 couldn't care less if
their kids have mitochondria
3 from somebody
else. They are perfectly normal and
4 they are happy and,
no, they don't want to be
5 bothered. So, whether
it is the medical director or
6 not, making it a big
enough issue--I don't know.
7 DR.
SALOMON: What I think this tells us
8 is it is just as an
insight that as we go forward
9 in this area, part of
what happens is educating the
10 whole process and how
you do clinical trials in
11 cutting edge
technologies.
12 DR.
LANZENDORF: Right.
13 DR.
SALOMON: In a gene therapy trial, for
14 example, we couldn't
expect any of our patients
15 afterwards to be
surprised that we have come
16 forward to them and
want to see whether or not--I
17 mean, even though
these are not minor issues, as
18 Jay is hand waving to
me, in any clinical trial it
19 is really important of
course, and I think it does
20 reflect part of what
is going to happen to this
21 whole area as we get
more used to thinking of it in
22 these terms.
23 DR.
LANZENDORF: Right.
24 DR.
SALOMON: Dr. Sausville?
25 DR.
SAUSVILLE: Actually, before my
82
1 question I just have a
comment. I would simply
2 state that people have
wildly different takes on
3 what their view of reasonability
is in terms of
4 going after this. It is well documented in my own
5 field that in cancer
susceptibility testing that
6 some people just don't
want to know.
7 DR.
LANZENDORF: Right.
8 DR. SAUSVILLE: And one has to respect
9 that. Actually, the reason I was pushing down the
10 button is that I
wanted to actually return a little
11 bit to the data that
was in your presentation,
12 specifically the more
recent experiments of Dr.
13 Brown.
14 DR.
LANZENDORF: That was a small amount
15 of work that a
clinical fellow did before he
16 departed. It has not been published. We thought
17 the numbers were too
low to even publish. So, it
18 was just an effort of
going through my files,
19 trying to find
information that I thought--
20 DR.
SAUSVILLE: And I appreciate your
21 candor in showing us
the preliminary nature of the
22 data, but I did want
to try and go back to I guess
23 the three slides that
talk about the difference
24 between controls and
shams. So, I guess,
25 recognizing the
numbers are small in terms of
83
1 statistics, the slides
that have the fertilization
2 results, lead me
through the clear evidence that
3 there is even a
suggestion of an effect of the
4 cytoplasmic transfer
as opposed to the sham
5 procedure. I am showing my ignorance in the field.
6 DR. LANZENDORF: Evidence that it helped?
7 DR.
SAUSVILLE: Right.
8 DR.
LANZENDORF: There was no evidence.
9 DR.
SAUSVILLE: Right, so one has to be
10 concerned,
therefore--and maybe we will hear from
11 other speakers--that
the underpinnings either
12 historically or
currently are somewhat
13 questionable.
14 DR.
LANZENDORF: Right, I agree.
15 DR.
SAUSVILLE: I wanted to make sure I
16 wasn't missing anything.
17 DR.
SALOMON: I guess I get to be blunt.
18 Why would you do
this? I don't get it.
19 DR.
LANZENDORF: Why would we do the
20 procedure?
21 DR.
SALOMON: Yes, I mean I don't see any
22 data, and it is very
early in the day and this is
23 not my field, but so
far from what you presented, I
24 wouldn't imagine doing
this.
25 DR.
LANZENDORF: That small study that I
84
1 presented at the end,
again, was trying to
2 reproduce that first
study with immature eggs.
3 When we are doing this
procedure for patients, for
4 the patients that we
did it wasn't an immature egg
5 issue. Again, when I said it didn't help, it was
6 not helping immature
eggs. To me, there is no data
7 out there yet that
shows that it does or does not
8 help mature eggs.
9 DR.
SALOMON: What is the data that it
10 helps? I mean, you showed us data from the older
11 mothers. Right?
12 DR.
LANZENDORF: Right.
13 DR.
SALOMON: And that, you said, didn't
14 show any
difference. Right? Then the second thing
15 you showed us was the
data from three women who had
16 had a history of
non-successful implantation and
17 pregnancy. Right?
I hope I am using the right
18 terms. One of those gave birth to the twins.
19 DR.
LANZENDORF: Right.
20 DR.
SALOMON: Was that just a statistical
21 blip? Or, that one set of three, is that the data?
22 DR.
LANZENDORF: That is why we need more
23 data. I mean, was it just her time? If it had
24 been a regular IVF she
could have got pregnant.
25 So, it may have just been her time. I am not
85
1 saying that any of
this supports that the procedure
2 actually does
something.
3 DR.
SCHON: One of the peculiarities of
4 the IVF field is that
it is largely patient driven,
5 and if somebody put on
the internet, for example,
6 that extracts of
dentine were found to improve
7 pregnancy rates, I
would venture to say that people
8 from all over the
world would be calling and asking
9 for that procedure to
be done. That is the history
10 of this field. Many things are done without any
11 evidence-based
medicine traditionally used in other
12 studies or without any
validation and that is why
13 we are here
today. That is part of the nature of
14 this field from day
one.
15 DR. VAN
BLERKOM: Your comment about some
16 patients may go
through nine cycles before being
17 successful. You described a particular pattern of
18 severe dysmorphology
in embryonic development in
19 patients that you
thought this might help. Is it
20 possible that patients
who show significant
21 consistent
dysmorphology in embryonic development
22 nonetheless become
pregnant after six, seven,
23 eight, nine cycles?
24 DR.
LANZENDORF: No, I would have to pull
25 out the stats.
86
1 DR. VAN
BLERKOM: We just don't know the
2 answer?
3 DR.
LANZENDORF: No. We can maybe find
4 out. There are programs out there with thousands
5 and thousands of
patients and, you know, it might
6 be interesting to
look. Of those patients who
7 finally got pregnant
after their ninth attempt, did
8 they have a history of
poor morphology.
9 DR.
SCHON: I can answer that from my
10 experience. We had a patient from Israel who had
11 18 attempts at IVF in
Israel and all failed. I
12 think this was about
six years ago. Her 19th
13 attempt in our program
and she had twins.
14 DR.
LANZENDORF: It could have been the
15 program.
16 DR.
SCHON: It could have been the program
17 or it could have been
something else. That is the
18 point. When you have consistent failures, the
19 question is are the
failures consistent with your
20 program or are they
from other programs. So, are
21 the objective criteria
that you use and someone
22 else uses the same?
23 DR.
LANZENDORF: Right.
24 DR.
SCHON: That is really the problem
25 because if you are
evaluating performance of
87
1 embryos in vitro from
different programs, there is
2 no standard objective
criteria. It is empirical.
3 So, what looks bad to
you may not look so bad to
4 somebody else; and
what looks terrible to you may
5 not look terrible to
somebody else. And, that is
6 part of the problem in
this field. It is
7 empirically driven.
8 DR.
LANZENDORF: Right, but it could have
9 been the method of
transfer that finally got her
10 pregnant, if the way
they were transferring changed
11 over time or something
like that.
12 DR.
RAO: Maybe this will sound naive, but
13 in your opinion then
what kinds of cases would you
14 actually look at for
cytoplasm transfer?
15 DR. LANZENDORF: Cases where there is
16 documented poor
morphology over repeated IVF
17 attempts, where the
patient was younger than 40
18 years of age is what I
think should be looked at.
19 One of the reasons we
included the 40 and over in
20 the study is because
many of the patients who are
21 trying to achieve a
pregnancy are of that age
22 group, and you could
not convince them that you
23 didn't think it would
work for them. We have done
24 this in eight patients. Still we have patients who
25 want to do it even
though we have shown that, but I
88
1 think we need to stop
focusing on that age group.
2 DR.
RAO: Let me extend that, poor
3 morphology in a young
age group, where you mature
4 the eggs in culture?
5 DR.
LANZENDORF: No, in vivo.
6 DR.
RAO: In vivo, and you will then
7 select those eggs and
look at those which have poor
8 morphology.
9 DR.
LANZENDORF: You do the cytoplasm
10 procedure on all of
the eggs at the time of
11 fertilization.
12 DR.
RAO: You just do it on all and then
13 just pick the best.
14 DR. LANZENDORF: Yes, and on the day of
15 transfer, what we
typically do with any patient is
16 we decide how many
will be transferred, and then
17 transfer the ones with
the best morphology.
18 DR.
MULLIGAN: I actually have a different
19 question but just in
response to his point, I am
20 still missing the line
of reasoning for the context
21 in which you say that
this might be the most
22 useful. I mean, you said that basically there is
23 really no data out there,
yet when you are asked,
24 well, what specific
context would you think this
25 would be most useful
in, is that completely
89
1 independent of the
fact that there is no data?
2 DR.
LANZENDORF: That is my hypothesis.
3 DR.
MULLIGAN: And the hypothesis is that
4 ooplasm could be
useful but you would agree that
5 there is no data?
6 DR.
LANZENDORF: I agree.
7 DR. MULLIGAN: Just scientifically, I find
8 it a little odd that
that 1990 study just kind of
9 disappeared. Does anyone know what happened to the
10 people who did
this? That is, did they do this and
11 then have a train
wreck or something?
12 DR.
LANZENDORF: Dr. Flood is practicing
13 IVF in Virginia Beach,
down the street from us. I
14 could try to talk to
her. Three of the other
15 people are not in this
country. Gary Hodgins is
16 retired for medical
reasons.
17 DR.
MULLIGAN: You know, scientifically,
18 usually when something
like this does happen there
19 is a paper and you
could look at something and say
20 that is very
interesting. If you see no report in
21 the next four or five
years, certainly in my field,
22 it means
something. So, I am just curious. It
23 would probably be very
useful to try to track these
24 people and see. Can you do literature searches?
25 Did they eve publish
anything on this?
90
1 DR.
LANZENDORF: No, I know they didn't.
2 I was doing my post
doc somewhere else so I had
3 very little
information.
4 DR. VAN
BLERKOM: These were probably
5 clinical fellows doing
a paper for clinical
6 fellowship.
7 DR.
LANZENDORF: Right.
8 DR. VAN
BLERKOM: But it was preceded in
9 the '80s and '70s by
work in mice and other
10 species, by the way,
and it was really designed in
11 the mouse to look at
cell cycle regulation, cell
12 cycle checks which led
to the discovery of factors
13 involved in the
maturation of their egg and their
14 timing. So, these guys just looked at it in the
15 monkey, again looking
for whether or not
16 cytoplasmic factors
from one stage would induce
17 maturation or assist
maturation in other eggs.
18 That is all. There is a precedent for this type of
19 work in mouse and lots
of other invertebrates.
20 DR.
MULLIGAN: At that point, was there
21 impact upon the work?
22 DR. VAN
BLERKOM: No.
23 DR.
MULLIGAN: No one really read the
24 paper or thought it
was interesting?
25 DR. VAN
BLERKOM: No, there was no point
91
1 to it. I mean, it was just a confirmation that as
2 in the mouse, as in
starfish, as in sea urchins
3 there are factors in
the cytoplasm that are
4 spatially and
temporally distinct and are involved
5 in miotic maturation
of the egg, period.
6 DR.
SALOMON: I was told by Gail that
7 there is someone in
the audience that wanted to
8 make a comment. If so, I didn't want to exclude
9 them. If you could please identify yourself?
10 DR.
WILLADSEN: I am Steen Willadsen. I
11 work as a consultant
at St. Barnabas, the Institute
12 of Reproductive
Medicine and Science. It was
13 actually something
else I wanted to comment on.
14 It was the
statement from, I think,
15 Jonathan Van Blerkom
that the IVF work is patient
16 driven. I don't basically disagree with that. So
17 is cancer
treatment. But he then went on to say
18 that all sorts of
things were being offered that
19 had no scientific
background, or at least suggested
20 that. I would disagree with that. I would
21 disagree that all
sorts of things are being
22 offered. I don't think there are that many things
23 that are being
offered.
24 Since I have
the microphone, I think I
25 should say also that
the people on the committee
92
1 are very much
concerned about how clinical trials
2 should be
conducted. Therefore, you focus on
3 whether all the things
are in place for that when
4 you hear about
research. Therefore, it sounds
5 strange and looks like
a big jump, here we go from
6 experiments with
monkeys and then nine years later,
7 or whenever it is,
suddenly it happens in humans
8 and looks to you as if
the duck hasn't been moving,
9 so to speak, but in
fact there has been a lot of
10 paddling going
on. The first mammalian cloning
11 experiments were
successful were in 1984 or 1985
12 and, yet, Dolly was in
1996 and in between it
13 looked like it had
kind of gone dead. Not at all.
14 There was plenty of
work going on, but that doesn't
15 mean that it would be
worth publishing. It might
16 be for you because you
are interested in the whole
17 process of how this is
controlled; what steps
18 should be taken from
the administrative level. But
19 that is not how
research is done in basic
20 embryology. Thank you.
21 DR.
SALOMON: Thank you. Well, you have
22 to understand we look
forward and we ask our
23 questions to discover
what has been going on that
24 has not been
published, as well as what has been
25 published. The question, if you remember, that was
93
1 asked was what
happened between 1990 and 1997 and
2 if there were things
going on that weren't
3 published that were
pertinent, that is the time to
4 hear about them. We certainly understand the fact
5 that much goes on that
doesn't come to the public.
6 But now when you want
to step up and start doing
7 clinical trials, it is
time to think about those
8 things.
9 I want to
thank Dr. Lanzendorf. You have
10 shouldered a bigger
responsibility--
11 DR.
LANZENDORF: Thank you.
12 DR. SALOMON: Oh, I am sorry, there is
13 someone else from the
audience.
14 DR.
MADSEN: I Pamela Madsen. I am the
15 executive director of
the American Infertility
16 Association and I do
represent the patients, and I
17 am a former patient
and a former infertile person.
18 It is an
echo but I decided the echo
19 should come from the
patient organization in
20 response to the
gentleman from St. Barnabas. Yes,
21 it is patient
driven. I was going to use the exact
22 same model of the
cancer patient who doesn't have
23 hope. These patients, you have to be clear, are
24 looking for certain
technologies. There isn't
25 anything else being
offered to them and you really
94
1 need to be clear about
that. These patient groups
2 are looking for these
technologies. IVF is not
3 working for them and
their only other hope, if they
4 want to experience a
pregnancy, is donor egg. That
5 is all they have and
you need to be clear about
6 that.
7 You also
really need to be clear that when
8 you are looking at
small data sets, and I am not a
9 clinician, not a
doctor or a scientist so forgive
10 me, these are very
small data sets because you have
11 stopped the research
and, as patients, we want to
12 see the research. We want there to be bigger data
13 sets, and there are
lots of patients who are very
14 eager to have a chance
at this research. We need
15 to continue and I
thought you should hear that
16 again from a patient
as well as the clinicians.
17 Thank you.
18 DR.
SALOMON: I appreciate that.
19 Certainly, one of the
things I want to reiterate
20 here is that anyone
who is here today, part of your
21 responsibility is to
make sure that we are being
22 appropriately
sensitive to all the public
23 stakeholders in this
area as we venture into this
24 conversation, both to
have a sense of how it is
25 practiced in the
clinical field--you know, I said
95
1 in your experience do
you feel comfortable and your
2 answer was, yes, you
do. That is the kind of thing
3 that we need to hear
and be reassured on, and the
4 same thing from
patient advocacy groups and
5 research advocacy
groups. If you feel like we have
6 veered off a line that
is sensitive to the state of
7 this field, then it is
very appropriate to get up
8 and remind us.
9 Again, thank
you very much, Dr.
10 Lanzendorf. That was excellent; a good start. We
11 will take now a
ten-minute break and start again.
12 [Brief
recess]
13 DR.
SALOMON: We can get started. Before
14 we go on with the
regular scheduled presentations,
15 it is a special
pleasure to introduce Kathy Zoon,
16 who is--I know I will
blow this--the director of
17 CBER. My only concern was not to promote her high
18 enough!
19 DR.
ZOON: Dan, thank you and the
20 committee very much
for giving me an opportunity to
21 come here today. I apologize that I couldn't be
22 here this morning to
speak to you but we were
23 working on some budget
issues at FDA. I know you
24 can understand that.
25 I would
like, in a few minutes, to give
96
1 the committee and the
interested parties in the
2 audience an update on
CBER's proposal for a new
3 office at the Center
for Biologics. This new
4 office has the
proposed title of the Office of
5 Cell, Tissue and Gene
Therapy Products, something
6 very close to the
heart of this committee. One
7 might ask why is CBER
doing this. CBER is doing
8 this because there are
many issues regarding
9 tissues and the
evolution of cell and cell
10 therapies and gene
therapies that we see as an
11 increasing and
expanding growth area for our
12 Center. Rather than reacting when it gets ahead of
13 us, CBER has always
taken the position of being
14 proactive, trying to
establish an organizational
15 structure and
framework so that we can be ready to
16 deal with
tissue-engineered products, regular
17 cellular products,
banked human tissues, repro
18 tissues and, of
course, the topic of today,
19 assisted reproductive
tissues.
20 We have
gotten the go-ahead from Deputy
21 Commissioner Crawford
and Secretary Thompson to
22 proceed on this
office, and we are very much
23 engaging in the
communities of all affected people,
24 especially our
committee who has had to deal with
25 so many issues to get
your feedback and advice
97
1 because we want to do
this right. We want to make
2 sure that we have as
much input when we go in to
3 finalizing the structure
and functions of this
4 office to do the very
best job we can. We
5 recognize that this
will be an evolution for all of
6 us because we are
still evolving with our tissue
7 regulations as rules,
as well as the sciences
8 surrounding cellular
therapies and tissue
9 engineering, and we
very much understand that but
10 we believe it is time
to be prepared and move
11 forward and get ready
for this area.
12 So, my plea
at this point is, please,
13 provide the advice;
certainly, those in the
14 audience as well that
have an interest in this
15 area. We are very much interested in hearing from
16 you. There are two e-mail addresses for those who
17 might wish to do it
through e-mail. It is
18
zoon@CBER.FDA.gov. Then, Sherry
Lard who is the
19 associate for quality
assurance and ombudsman at
20 FDA is also taking
comments in case people prefer
21 to remain anonymous
because that is important. Her
22 e-mail address is
lard@CBER.FDA.gov. If you prefer
23 not to e-mail and you
prefer to call, the numbers
24 are on the HHS
directory off the web site, if you
25 want to find any of
us.
98
1 We are very
happy and very pleased that
2 this committee would
deliberate and think about
3 this, and I will be
looking forward. The time line
4 for this new office,
we hope to have as many
5 comments as possible
by the end of May. We would
6 like to finalize the
structure and functional
7 statements probably in
June, and then work on the
8 issues that are
administrative to moving the office
9 forward, and are
looking forward to an
10 implementation date of
October 1, which is the
11 beginning of the
fiscal year. So, just to give you
12 a sense of the
dynamics and the organization. It
13 is a goal. We are hoping that we can achieve this
14 goal and that is where
we are focused on.
15 So, I am
very happy to have the
16 opportunity today to
be here and present this
17 proposal to you, as
well as receive your feedback.
18 Thank you.
19 DR.
SALOMON: Thank you very much, Dr.
20 Zoon. Tomorrow when we have some time because I
21 see today as being
very busy, we will try and find
22 some time as a group
to discuss this just as an
23 initial thing, because
I am interested in some
24 thoughts that everyone
has. That is not to mean
25 that anything else
can't go on informally or
99
1 formally otherwise.
2 Just one
question, it is a pretty big
3 deal, how often do you
guys make new offices like
4 this?
5 DR.
ZOON: We sometimes create new
6 offices. In fact, over the past probably three
7 years we have elevated
the Division of
8 Biostatistics and
Epidemiology, which is
9 responsible for our
statistical reviews at the
10 Center as well as for
overseeing adverse events, we
11 have elevated that
office, led by Dr. Susan
12 Ellenberg, to an
office level. Most recently, we
13 broke out our
information technology group, which
14 was an office under an
office, as a separate
15 office. This one is more complicated because it
16 takes the experiences
in both the Office of
17 Therapeutics that is
relevant and the Office of
18 Blood that had a lot
of the tissue programs and
19 tissue activities, and
moving people together as
20 appropriate. So, this is a much bigger
21 reorganization, more
complex. The last big one we
22 did was in 1993.
23 DR.
SALOMON: That is more what I was
24 thinking. I mean, my initial response is that this
25 is a remarkable
recognition of where this field has
100
1 gone in the last five
to ten years. We are talking
2 now about such a myriad
of studies going from
3 neural stem cells to
xenotransplantation to islet
4 transplantation to
gene therapy of various sorts,
5 all of which have been
major touchstones for public
6 comment and regulatory
concerns. So, I think this
7 is a really big deal
and we appreciate the
8 opportunity to hear
about it and also to give you
9 some input
constructively while it is being
10 developed. Thank you, Dr. Zoon.
11 It is my
pleasure to introduce Dr. Jacques
12 Cohen, from the
Institute for Reproductive Medicine
13 and Science of St.
Barnabas, and to get back to
14 today's topic of
ooplasm transfer. Dr. Cohen?
15
Ooplasm Transfer
16 DR.
COHEN: Good morning. Thank you, Mr.
17 Chairman. Thank you for your kind invitation.
18 For my
presentation I will follow or try
19 to follow the
guidelines for questions that the
20 BRMAC has asked in
this document that I found in my
21 folder. But I will deviate from it now and then.
22 First of
all, I would like to acknowledge
23 three individuals, two
of them are here, that have
24 been crucial for this
work, Steen Willadsen who,
25 about twelve years ago
or so, suggested that there
101
1 could be potential
clinical applications for
2 cytoplasmic
replacement or ooplasmic
3 transplantation; Carol
Brenner who has done a lot
4 of the molecular
biology, microgenetics of this
5 work, together with
Jason Barret; and Henry Malter
6 who has been involved
in the last three or four
7 years.
8 I would like
to backtrack a little bit
9 after Susan
Lanzendorf's presentation and, first of
10 all, look at all the
different oocyte deficits that
11 exist. The most important one is aneuploidy.
12 Aneuploidy is
extremely common in early human
13 embryos and oocytes,
is highly correlated with
14 maternal age, as I
will show you. It is the most
15 common problem in our
field.
16 Chromosome
breakage is not that
17 well-known, not that
well studied but is also very
18 common. I am not just thinking about the risk of
19 transmitting of
translocations but also about
20 spontaneous chromosome
breakage that occurs in
21 oocytes and embryos.
22 Gene
dysfunction is being studied,
23 particularly now that
tools are being made
24 available.
25 But we have to keep in mind a couple of
102
1 things here. When we study these phenomena there
2 are a couple of things
that are important to know.
3 First of all, there is
no government funding. So,
4 it is all paid out of
the clinical work. Secondly,
5 we can only study
these phenomena in single cells
6 because we have really
only single cells available
7 to us. Thirdly, genomic activation is delayed.
8 But that, I mean the
finding that the early human
9 embryo is really an
egg that is on automatic. It
10 is not activated
yet. Expression by the new genome
11 hasn't occurred
yet. In the human it is considered
12 to occur between four
to eight cell stages, three
13 days after
fertilization. This is important
14 because when we talk
about ooplasmic
15 transplantation we
truly try to affect the period
16 that occurs before
genomic activation.
17 Here is the
correlation between aneuploidy
18 and implantation. On the horizontal axis you see
19 maternal age. This finding is pretty old now.
20 This was based on
doing fluorescence in situ
21 hybridization in
embryos, in embryos that were
22 biopsied and the
single cells taken out. This was
23 done by Munne and
coworkers many years ago now. At
24 that time, they were
only able to do two or three
25 chromosome probes,
molecular probes to assess
103
1 chromosome. So, the rate of aneuploidy is pretty
2 clear and it seemed to
us, and many others, that
3 this correlation is so
apparent that you couldn't
4 do anything with
ooplasm or cytoplasm because in
5 the mature egg
aneuploidy was already present,
6 particularly
correlated with maternal age, and that
7 problem was so obvious
that not much else could be
8 done.
9 But a lot of
data has been gathered since
10 this. Particularly what has been done is to do
11 embryo biopsy, take a
cell out at the four to eight
12 cell stage. If you look at the implantation rate
13 here, in the green
bars and, again, on the
14 horizontal axis you
see the maternal age here, you
15 can see that
implantation--which is defined as one
16 embryo being
transferred giving fetal heart beat,
17 the implantation rate
diminishes significantly with
18 maternal age.
19 What you see
in the orange bars is what
20 happens or will happen
if one does aneuploidy
21 testing. It shows that in the older age groups you
22 will get an increase
in implantation because
23 embryos that are
affected by aneuploidy are now
24 selected out. They have been diagnosed. You can
25 take those triploid or
trisomic or monosomic
104
1 embryos out and put
them aside so that you only
2 transfer diploid
embryos.
3 The thing though is that this is not
a
4 straight line. What we had really hoped is that we
5 would have a very high
rate of success regardless
6 of age per
embryo. That is not the case. If you
7 use egg donors and you
put embryos back in women of
8 advanced maternal age,
you will find that this is a
9 straight line. So, if you use eggs and embryos
10 that come from eggs
from donors that are younger
11 than 31, younger than
30 you will find that the
12 recipient now behaves
like a young woman.
13 So, what is
different here is that it is
14 not just the
aneuploidy that is causing this
15 difference, but also
there is this huge discrepancy
16 still that must be
related to other causes, other
17 anomalies that are
present in the egg and,
18 therefore, in the
embryo that should be studied.
19 So, the
question, and the question is
20 raised very well by
FDA, is there evidence of an
21 ooplasmic
deficit? Dr. Lanzendorf mentioned
22 already
fragments. These are blebs that are
23 produced by the
embryos. Both Jonathan Van Blerkom
24 and our group have
described a number of different
25 types of fragmentation
that have probably different
105
1 origins and causes.
2 The lower
panel basically shows what you
3 see in the upper panel
but now the fragments are
4 highlighted. These fragments in this case, here,
5 occur at a relatively
low incidence but you can
6 score this. Trained embryologists are able to
7 score this quite well,
and proficiency tests have
8 to be in place to make
sure that this is done
9 reliably.
10 There are different fragmentation types.
11 Some of them are
benign and some of them are
12 detrimental. All depend on the type of
13 fragmentation and the
amount of fragments that are
14 present. There are some as well that may not be
15 cytoplasmic in origin,
for example, there is
16 multinucleation that
can occur in cells of early
17 embryos. All these are scored by embryologists.
18 If we look
at this fragmentation
19 phenomenon, here,
again, on the horizontal axis you
20 see how many fragments
there are in an embryo and
21 that is scored from
zero to 100. One hundred means
22 that there is not a
single cell left; all the cells
23 are now
fragmented. Zero means there is not a
24 single fragment that
is seen. Then, there are
25 scores in between.
106
1 Clinically,
we know that you can get
2 fragmentation up to 40
percent, like here, and you
3 can still get maybe an occasional embryo
that is
4 viable but all the
viability is here, on the left.
5 When we looked at gene
expression in spare embryos
6 that are normal; they
have been put aside and
7 patients have
consented to this research, when we
8 look in these embryos,
we are finding now that
9 certain genes are
highly correlated with these
10 morphologic phenomena
and are related to the number
11 of transcripts of
certain genes that are present in
12 the cytoplasm of the oocyte and are present
in the
13 cytoplasm of the early
embryo.
14 You can see
here, in this particular gene,
15 there is a very clear
correlation and a very badly,
16 morphologically poor
embryo is here, on the right,
17 have more transcripts
of this gene in the cells.
18 There were a
couple of genes that were
19 looked at. Here is another one that is correlated
20 in a different way
which fits probably in the
21 hypothesis that
fragmentation doesn't have a single
22 course. It shows though that there is a clear
23 basis, at least
looking at fragmentation, that this
24 goes back to the egg
and that the problems are
25 present in the oocyte.
107
1 Another gene
that has been studied for
2 many years now by Dr.
Warner, in Boston, is the
3 gene that she called
the pre-implantation
4 development gene. This gene phenotypically shows
5 high correlation with
speed of development of early
6 embryos. When we looked in the human we could
7 basically--and this is
very well known, you can see
8 all these different
speeds of development,
9 development stages when
you look at static times.
10 In our data
base we separated patients
11 that had different
developmental stages where
12 embryos may be
eight-cell at one point and where
13 sibling embryos would
be seven cells or four cells.
14 We took all those
patients separately and we found
15 1360 patients that had
very uniform rates of
16 development. You can see here if we look at fetal
17 heart beat projected
from single embryos that there
18 is a highly
significant difference in implantation
19 rate.
20 Similar to
the model in the mouse, in the
21 mouse you have fast
embryos and you have slow
22 embryos. The fast embryos implant at a very high
23 frequency and the slow
embryos can implant, it is
24 not an absolute
phenomenon, but they implant at a
25 much lower
frequency. This is under the control of
108
1 ooplasm, like in the
mouse.
2 In the mouse
the gene product is the Qa-2
3 protein and if it
binds to the membrane the embryos
4 will become fast
embryos and you get good
5 development, and if
the protein is absent you get
6 slow embryos, but you
can get implantation but at a
7 lower frequency.
8 Other
cytoplasmic factors have been looked
9 at. Transports have been looked at and now, with
10 the availability of
microarrays and other
11 technologies, we hope
that even though we are only
12 using single cells for these analyses that we
can
13 correlate some of the
expressions of these genes
14 with viability of the
embryo.
15 Here is an
example. This is Mad2, which
16 is a spindle
regulation factor. We have looked at
17 Mad2 and Bob1 and we
have found--I apologize for
18 the graph, it is
pretty unclear, but the maternal
19 age is again on the
horizontal axis and younger
20 women who had many
transcripts present, a
21 significantly lower
number in all the women.
22 Again, this was
measured in the cytoplasm.
23 For this
meeting, for the purpose of
24 studying ooplasmic
transplantation, is the issue of
25 mitochondria
genes. We have been interested in
109
1 this for quite a long
time. Mitochondrial genome
2 is, and I am sure Dr.
Shoubridge will talk about
3 this later in great
detail, is a relatively simple
4 conserved genome, 37
genes. On the top of it, at
5 least in this picture,
there is an area that has
6 high rates of
polymorphisms, the hypervariable
7 area. Adjacent to it is the replication control
8 region.
9 We have
looked at oocytes, in the yellow
10 bars, and embryos, in
the orange bars, and compared
11 mitochondrial DNA
rearrangements. I have to
12 mention that these are
not potentially normal
13 materials because
these cells are derived from eggs
14 that do not fertilize
or from eggs that do not
15 mature or abnormally
fertilize, and embryos that
16 develop so abnormally
that they cannot be frozen or
17 transferred. So, this is all from spare material.
18 For obvious reasons,
it is very hard to obtain
19 appropriate control
groups for some of these
20 studies.
21 We found 23
novel rearrangements, and the
22 frequency rate was
astoundingly high. So,
23 mitochondrial DNA
rearrangements occur very
24 frequently in oocytes;
significantly less
25 frequently in
embryos. It has been postulated that
110
1 it is very likely that
there is a block in place
2 that selects abnormal
mitochondria in a way that
3 the corresponding cell
doesn't continue to develop.
4 You can see that
fertilization block here. The
5 spare embryos have
less rearrangements than the
6 oocytes, suggesting
that there is a bottleneck, a
7 sieve in place.
8 We have also looked at single base
pair
9 mutation at 414
logs. This was a publication from
10 Sherver in, I think,
1999, who showed, and I am
11 sure the mitochondria
experts here may not
12 necessarily agree with
that work, but showed that
13 in the natural
population this mutation had a high
14 correlation with
aging.
15 So, we were
interested to look at this.
16 It was quite simple to
study, to look at this
17 particular mutation in
spare human egg and embryo
18 material, again, with
the purpose of identifying
19 cytoplasmic factors
that were involved in the
20 formation of a healthy
embryo. We found that this
21 single base pair
mutation was fairly frequently
22 present in human
oocytes that were derived from
23 women that were older,
37 to 42 years of age, and
24 significantly less
present in women that were
25 younger.
111
1 So, when we
look at the clinical
2 rationale, there is a
knowledge base but it is not
3 necessarily specific
for ooplasmic defects. Of
4 course, we know very
little about ooplasmic
5 defects. So, a rationale for studying potential
6 treatments for each
defect does not exist.
7 The question
is, and this came up actually
8 earlier this morning,
is there a rationale at all
9 to do ooplasmic
transplantation? Well, that is
10 saying that all
ooplasms are the same. Well, they
11 are not. They are all different. So, I think that
12 is the rationale. Not all levels of transcripts,
13 not all proteins and
not all mitochondria are the
14 same in the ooplasm of
different eggs.
15 What animal
experimentation has been done,
16 particularly with the
interest of cytoplasmic
17 transplantation? There is a whole body of
18 research, and a lot of
this work was done not
19 keeping in mind that
there was an interest in doing
20 ooplasmic transplantation clinically, and I
think
21 Jonathan Van Blerkom
said that. This work was done
22 because there were
other issues that needed to be
23 studied, genetic
interest in early development.
24 One of the
papers not mentioned before is
25 some interesting work
done by Muggleton-Harris in
112
1 England, in the '80s,
and they looked at mice that
2 had what is called a
two-cell block. These are
3 mice that when you
culture oocytes, zygotes in
4 vitro, the embryos
will arrest. You can change the
5 environment but they
will not develop further. By
6 taking two-cell
embryos from other strains of mice
7 that do not have this
two-cell block, it was
8 possible by
transferring cytoplasm to move the
9 embryos that were
blocked through the block. I
10 think that has been a
pretty good model for this
11 work. However, this was done, of course, after
12 fertilization and
certainly is something that could
13 be considered.
14 Many
cytoplasmic replacement studies have
15 been done from the
early '80s onwards, particularly
16 Azim Surani's group
who looked at many different
17 kinds of combinations
of cytoplasm and cells with
18 and without
enucleation, different sizes, different
19 techniques. Cytoplasm transfer has been studied in
20 the mouse and in the
monkey, and I will mention the
21 work of Larry Smith,
in Quebec, in Canada, who has
22 created hundreds of
mice from experiments that are
23 very similar to the
cytoplasmic transplantation
24 model in the
human. That work was done in 1992 and
25 is continuing,
hundreds of mice over many different
113
1 generations.
2 Then there
is in vitro work done
3 originally by Doug
Waldenson, in Atlanta, and his
4 work involves mixing
mitochondria of different
5 origins in the same
cell and then studying cell
6 function.
7 In Larry
Smith's lab in Quebec,
8 heteroplasmic mice
have been produced, as I said.
9 These are healthy,
normal mice from karyoplasm and
10 cytoplasm transfer. Karyoplasm is part of the cell
11 that contains a
nucleus and contains a membrane.
12 Cytoplasm is also part
of a cell that is surrounded
13 by a membrane. They combined these in many
14 different ways between
inbred mouse strains with
15 differing
mitochondrial backgrounds because they
16 are interested, like
many others, in mitochondrial
17 inheritance. Many of these animals have been
18 produced over 15
generations apparently without
19 developmental type problems.
20 We did an
experiment in 1995-95. It was
21 published in 1996 by
Levron and coworkers where we
22 looked at cytoplasmic
transfer in mouse zygotes and
23 mouse eggs, using F1
hybrids. We did many
24 different kinds of
combinations and found that in
25 most combinations it
did not really affect
114
1 development except
when very large amounts of
2 cytoplasm were fused
back into the recipient cells.
3 We found in one
scenario a significantly improved
4 situation where zygote
and egg cytoplasm was
5 combined.
6 The hybrid
experiments have been done,
7 which I mentioned
before, for creation of cell
8 hybrids with disparate
nuclei and mitochondrial
9 makeup. It has been done across species and across
10 genes even. Normal mitochondrial function has been
11 obtained in many
scenarios. The only scenarios
12 that in hybrids, as
well as in mouse cytoplasm,
13 karyoplasm studies
that are not potentially normal
14 have always been
obtained across species or
15 subspecies. Of course, those experiments are not
16 really models for
mixing mitochondria of two
17 completely outbred
individuals.
18 We have done
work in the last few years
19 that is similar to
that of Larry Smith's laboratory
20 but with the aim of
looking at the mice in more
21 detail and to see how
fertile they are, for
22 instance. So, here we take a zygote from one F1
23 hybrid and then mix
the karyoplasm containing the
24 zygote nuclei with the
cytoplasm of another zygote.
25 It is a
pretty small group here, 12 mice,
115
1 F1 hybrids. In those there were no apparent
2 problems. The first generation is now 30 months
3 old. We have done one more generation of 13
4 individuals that we
just keep around to look at and
5 until now there have
been no apparent problems.
6 One of the
problems with cytoplasmic
7 transfer work, the
ooplasmic transportation work in
8 the human is the use
of ICSI, intercytoplasmic
9 sperm injection. It is basically taking a very
10 sharp needle and go
into the membrane of the
11 oocyte. That has not been easy in animals, believe
12 it or not, but it
works well in the human, very
13 well. The human egg is very forgiving but it
14 doesn't work well at
all in other species. In the
15 mouse it has taken a
couple of tricks to make it
16 work, and that has
only happened in the last few
17 years. So, we think that we have a better model
18 tentatively to compare
what is done in the human,
19 and to do this in the
mouse. I am not saying that
20 the mouse is the best
model for these studies but
21 it has all sorts of
advantages. It is genetically
22 incredibly well
studied. It has a very fast
23 reproductive cycle,
etc. Here you see some embryos
24 that have a good
survival rate, 90 percent or
25 better, from these
experiments.
116
1 So, what is
the clinical experience? The
2 first time we
approached the internal review board
3 at St. Barnabas was
sometime in 1995. The first
4 experimental clinical
procedures were done in 1996.
5 When first results
were obtained and also when we
6 found the first
indication of benign heteroplasmy
7 and this was in
placenta and in fetal cord blood of
8 two of the babies, we
reported this to the IRB and,
9 of course, had to
inform our patients. I think the
10 question came up
before, do you tell your patients
11 about
heteroplasmy? Well, you can only tell
them
12 about it when you find
it. So, it was only found
13 in 1999, and this is
from this time onwards when it
14 was incorporated in
the consent procedure.
15 Then last
year, after a rash of bad
16 publicity, we went
back to the internal review
17 board but this was
also at the time that the FDA
18 sent us a letter. So, this second review is
19 basically not going
forward because we were asked
20 to hold off until
further resolution.
21 How do we do
this clinical? Well, we made
22 the choice to go for
the mature oocyte and not the
23 immature oocyte. We made the choice for the mature
24 oocyte because there
is incredible experience with
25 IVF as well as
intercytoplasmic sperm injection
117
1 manipulating these
eggs. These are small cells
2 that are genetically
similar to the egg and these
3 can be removed
microsurgically. There is
4 experience with
injecting sperm from male factor
5 infertility
patients. Forty percent of our
6 patients have male
factor infertility, possibly
7 more. So, there are more than 100,000 babies born
8 worldwide from this
ICSI procedure.
9 So, we felt
that what was a better
10 approach possibly than
using the more classical
11 micromanipulation
procedures that involve, for
12 instance, the
formation of cytoblasts and
13 karyoblasts and then
fusion, which we thought was
14 maybe just a little
too much. So, we took
15 cytoplasmic transfer
using ICSI as a model. There
16 are advantages to that
and disadvantages. You
17 could do this also at
the time the zygote is formed
18 and the two-cell is
formed. This has been a
19 clinical pilot
experiment we chose. For the first
20 lot of patients we
chose the mature egg.
21 The
procedure was already shown by Dr.
22 Lanzendorf but
basically you pick up a sperm and
23 then go into the donor
egg. I would like to point
24 out here that the
polar body, right next to it--the
25 human egg is very
asymmetric. It is polarized, and
118
1 the spindle that
obviously under light microscopy
2 and also in this
cartoon is not visible, is located
3 very close to the
polar body. So, the idea is that
4 we should not transfer
chromosomes from the polar
5 body. Therefore, we keep the polar body as far as
6 possible away from the
area where we select our
7 cytoplasm from. Then, when cytoplasm has been
8 absorbed in the
needle, it is immediately deposited
9 into a recipient egg.
10 Pictures
don't tell you very much because
11 they are static, but
here is the sperm cell and
12 then going into the
donor egg, here is the donor
13 egg. The polar body cytoplasm of the sperm is now
14 here, and then is deposited
into a mature recipient
15 egg. When we do this we make videos so that we
can
16 see that cytoplasm has
been transferred, but also
17 in the usual
circumstances the cytoplasm between
18 oocytes is very
different, has a different
19 consistency, different
refraction and, therefore,
20 you can usually
immediately see the amount that is
21 transferred and
injected, and that is highlighted
22 here.
23 We have done
28 patients so far. Five had
24 repeated cycles. three of those became pregnant
25 and had a baby the
first time and challenged their
119
1 luck and came back
again. They were all egg
2 donation candidates.
3 Now, I need to say something about
this.
4 First of all, there
are a lot more patients that
5 want to be candidates
but our feeling and also we
6 agreed that we should
do these patients in-house
7 because there are
tremendous differences in
8 outcomes, clinical outcomes between programs.