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. So,
9 if a patient would
come that has ten failed cycles
10 elsewhere, it is not
at all unlikely that she could
11 become pregnant in our
program or in another
12 program if she
switched programs because laboratory
13 procedures and
clinical procedures are very
14 different from program
to program. So, we felt
15 that at least there
should be a couple of cycles
16 done by our own
program if the patient came from
17 elsewhere.
18 The average
number of previous cycles in
19 these patients is well
over four. These patients
20 have recurrent
implantation failure. So, they come
21 in. They do not become pregnant. We put multiple
22 embryos back. They have a good response to
23 follicular stimulation
so they make a lot of eggs
24 but they do not become
pregnant. They have normal
25 fertilization
rates. They also all had recurrent
120
1 poor embryo
morphology. However, there was one
2 exception to
that. There was one patient that had
3 normal fertilization
but zygote block. The zygotes
4 basically fall apart
in fragments and other zygotes
5 would never even do
that. They would just stay.
6 Fertilized as they
are, they would never divide.
7 So, one of the 28
patients did not have poor embryo
8 morphology. She simply did not have developing
9 embryos.
10 A number of
these patients were male
11 factor patients and it
is important to realize that
12 when you get poor
embryo development, some of that
13 may be caused by the
male factor. The sperm may be
14 the cause of abnormal
development, particularly
15 because the sperm
brings in the centriole that is
16 obviously crucial for
division. The centriole in
17 the human is inherited
through the maternal line.
18 It is possible, and
being suggested by Jonathan Van
19 Blerkom that men that
have abnormal centriole
20 function. Certainly, we have found that in some
21 subsets of men there
are high rates of mosaicism,
22 indicating that there
are problems with division
23 and, therefore, their
infertility is correlated
24 with embryonic
failure.
25 When I say
nine male factors, it really
121
1 means that they had
abnormal semen. There could
2 have been other male
factors as well with normal
3 semen. There can be patients that have normal
4 sperm but they can
still be infertile. Five of
5 these patients had
repeated miscarriages. So, five
6 of them had been
implanted before but always
7 miscarried.
8 So, we did
33 attempts. Two did not have
9 viable embryos for
transfer; 21 transfers and 13
10 clinical
pregnancies. There were more clinical
11 pregnancies from this
patient group, and the reason
12 for that is that in
order to do the cytoplasmic
13 transfer we only used
ten percent or so of the
14 cytoplasm of a donor
egg. So, we actually use
15 donor eggs several
times. We go into the same
16 donor egg of two
times. Twice. We go in there
17 twice, and sometimes
more if only a few donor eggs
18 are availability. Most donors are good stimulators
19 so they will have a
good response to follicular
20 stimulation and will
make a lot of eggs. So, the
21 procedure yields a lot
of eggs that are not used.
22 What we offer to our
patients is that those eggs
23 are injected by sperm
from the male partner and
24 that embryos resulting
from this are frozen for
25 later use. So, it is not only cytoplasmic transfer
122
1 procedure, it is also
an egg donation cycle. There
2 are patients that
don't come back for another
3 attempt of ooplasmic
transplantation or they are
4 discouraged to do
that, and then they come back for
5 frozen embryos from
the donor eggs that were
6 injected with the
sperm from the husbands.
7 So, the data
I am showing here is clean
8 data. These are pregnancies that occurred from
9 transferring embryos
that were derived from
10 ooplasmic
transplantation. But if the patients
11 have failed, some of
them may have another chance
12 using the frozen
embryos.
13 There was a
firs trimester miscarriage.
14 There was an XO
pregnancy. Obviously, these are
15 fairly common, the
single most common chromosomal
16 anomaly in early
pregnancy. This happened at the
17 end of '98. A few months later we had a twin
18 pregnancy and one of
the fetuses on amnio was
19 diagnosed as XO as
well. We published this a few
20 years ago.
21 With that
information, we returned to the
22 internal review board
to let other patients that
23 are undergoing this
experimental protocol know that
24 this may be a
potential issue. If you look at the
25 statistics, many
statisticians have told me that
123
1 there may be an issue
or there may not be an issue.
2 On twin was
born and also a quadruplet was
3 born. This is one of two patients where there was
4 a very clear
improvement in embryo morphology.
5 However, we understand
that we are so biased as
6 embryologists that maybe
we were imagining some of
7 this. So, four embryos were transferred. Of
8 course, in that
respect we should have only
9 transferred two or
so. This was a patient who had
10 five previous attempts
and always had very poor
11 embryos and now,
suddenly, the embryos looked much
12 better. In spite of our advice in the consent form
13 that were given to her
at the time of these
14 products, she did not
elect the selective
15 reduction.
16 Seventeen
babies were born. Pediatric
17 follow-up has been
done only in a proportion of
18 them that we know
of. By that, I mean some of
19 these patients are
from abroad. The issue came up
20 before that really not
all these patients are
21 interested in
follow-up by us, and we have tried to
22 be quite forceful with
them. So, we have been able
23 to do follow-up in 13
of the 17 babies. However,
24 more recently it is
more likely that some of them
25 will refuse further
investigations by us. This is
124
1 not just this
particular group. That is common for
2 all infertility
follow-up, that you lose sight of
3 these patients. Some of them will move and not
4 even leave a return
address.
5 On twin,
this one twin that was born with
6 mixed sex, a boy and a
girl, the boy at 18 months
7 was reported to have
been diagnosed by pervasive
8 developmental
disorder, not of specific origin.
9 The incidence of this
in the recent literature is
10 1/250 to 1/500. This was reported to us in June of
11 last year. That was at the age of 18 months, and
12 we have no good
follow-up. This is just what is
13 going on with this
little boy.
14 One issue
that comes up is, well, does
15 this really work? One way of investigating this is
16 to look at attempt
numbers. You see here, on the
17 left--the colors are
very confusing but on the left
18 you see the first
attempt number here in the
19 general IVF population
that we studied. The second
20 attempt number, the
third, fourth, fifth, based on
21 about 2500
patients. So, you can see in the first
22 attempt number the procedure
rate. In the first
23 attempt number the
success rate is very high but
24 then it significantly
drops, which makes sense
25 because it left us
with a more complex population.
125
1 The third attempt is
also significant because of
2 the high numbers
involved. Then it sort of
3 flattens off.
4 If we look
at the per embryo, it is also
5 marked. This is the incidence of success by
6 embryo. It is well over 30 percent when you come
7 the first time, then
it significantly drops to less
8 than 20 percent the
second time, and again drops
9 significantly the
third time to about 15 percent,
10 14 percent.
11 Now, the
ooplasmic transfer cases are
12 here, in the red bars,
and they have an average of
13 about 4.8 previous
attempts. So, they actually are
14 between these two
bars. That is where they should
15 be. But these patients also contain patients
that
16 have repeated failure
with apparently normal
17 looking embryos. So, you can't really make that
18 comparison strictly
but it is suggestive that at
19 least it worked to
some extent. This is early
20 days, only 28 patients
and 33 cycles.
21 Some
comments about the mitochondria work
22 that we have
done. Spare eggs can be looked at and
23 you can use a stain
for mitochondria and then look
24 if the egg fertilizes
where these mitochondria go
25 to. We found in a number of cases they can go to
126
1 the blastomeres,
sometimes not all blastomeres but
2 it is well
proportioned. There was one indication
3 that they can survive
for at least a few days, but
4 the best was to look
at the polymorphisms in the
5 hypervariable area of
the mitochondrial genome. We
6 did that work
originally with regular sequencing.
7 Se looked at spare
eggs and embryos that were not
8 transferred after
ooplasmic transplantation.
9 Then we
looked at amniocentesis. That was
10 actually quite
frustrating because it is not easy
11 to get good cells
there for this type of work. In
12 a couple of these
babies we have been able to look
13 at the time of
delivery and obtained placental
14 tissue by being
present at delivery, and also
15 obtained fetal cord
blood.
16 If you look
at the incidence of
17 heteroplasmy, and I
must say this again, this is
18 heteroplasmy in the
hypervariable area, maybe we
19 should distinguish
that from other forms of
20 heteroplasmy because
these are extremely common in
21 the general
population. Spare embryos, about half
22 of them, after a few
days of culture, showed these
23 polymorphisms so that
you can basically confirm
24 that mitochondria were
present from the donor.
25 On
amniocentesis we did only ten, and
127
1 three of them sere
positive. So, mitochondria were
2 present from the donor
in amniotic cells. In the
3 placenta it was
3/13. We are only looking to
4 obtain blood at the
first year from those babies
5 that were positive at
the time of delivery, and two
6 have been tested thus
far and are still positive
7 for donor
mitochondria.
8 Recent work
by Carol Brenner--because this
9 is done by sequencing
which, I think most agree, is
10 not that sensitive a
method--recent work by Carol
11 Brenner has shown
using molecular beacon for
12 hypervariable
locations, using this work it has
13 been found that up to
as much as 50 percent of the
14 mitochondria in the
blood at the time of birth
15 would be positive for
the donor. So, when we
16 inject 10 percent, it
certainly doesn't mean that
17 there will always be
10 percent, but no doubt in
18 some children there
will likely be a trend to
19 homoplasmy and in
others maybe a consistent
20 heteroplasmy.
21 The word
heritable was used this morning
22 by Dr. Hursh, and I do
object to that because there
23 is no proof at all
that this is heritable, but it
24 is certainly
possible. No proof so far.
25 Here are the
three famous words, germline
128
1 genetic modification,
used by J.C. Barritt in the
2 publication last
year. There were four authors on
3 this paper. The three other authors do not agree
4 with this
wording. So, it only appeared in an
5 abstract; it didn't
appear in the regular text. We
6 don't agree because we
don't think that it is
7 modification. It is a kind of difference, change,
8 or maybe I don't have
the right word. It is
9 different from what
has ever happened but in my
10 opinion it is not
germline genetic modification.
11
Mitochondrial diversity, not all that
12 dissimilar from this,
occurs in the hypervariable
13 area in 10-15 percent
of normal humans. This is
14 recent work from more
sensitive assays by Tully and
15 coworkers. I must reiterate that the hypervariable
16 area is a non-coding
region.
17 One issue
that hasn't come up today yet is
18 that maybe this is a
technique that places at risk
19 the transfer of
mitochondrial disease.
20 Mitochondria are
maternally inherited so in egg
21 donation you have
mitochondria 100 percent from the
22 donor. There are no known cases of mitochondrial
23 disease after egg
donation. Certainly, when you
24 use ten percent of the
mitochondria from the donor,
25 would there then be
suddenly an indication that
129
1 there is an increased
risk factor of mitochondrial
2 disease?
3 I will
quickly go through the risks, the
4 potential risk
factors. Mechanical damage has been
5 raised as a risk
factor. While it is an ICSI
6 derived procedure, the
survival rate with this
7 procedure was better
than 90 percent. However, it
8 is slightly higher, in
our lab at least, than the
9 average damage rate to
eggs after ICSI just
10 injecting a sperm.
11 Cytoplasmic
transfer, the fertilization
12 rate is over 65
percent. So, we think that is a
13 normal fertilization
rate. With ICSI there have
14 been 100,000 babies born. The pre-implantation
15 development with ICSI
seems like IVF and the
16 malformation rate
seems like IVF. Certainly, with
17 the bare minimum
results we have, we think that the
18 malformation rate from
our procedure also resembles
19 that of IVF.
20 So, what are
other risks potentially to
21 offspring? Inadvertent transfer has been raised as
22 a potential
issue. If you have unique organelles
23 you don't want to
transfer those and boost them.
24 Like the centriole
sperm derived, centriole is
25 separately placed in
the cytoplasm. The sperm is
130
1 intactly placed in the
cytoplasm. So, it is
2 unlikely you will lose
that.
3 Avoid the
spindle, and if you cannot avoid
4 it there should be
cytokinetic analysis. So, one
5 thing we do is every
egg, every donor egg from
6 which cytoplasm has
been taken, we give it to the
7 cytogeneticist that is
specialized in single-cell
8 cytogenetics to
confirm that the chromosomes are
9 still there. In two cases we couldn't confirm this
10 in two eggs and the
next day we, indeed, saw things
11 that we call
subnuclei. These are basically small
12 nuclei that were
present in the periphery of the
13 egg, not in the middle
but in the periphery,
14 confirming that the
cytogeneticist was right. So,
15 it is a good thing to
have a cytogeneticist around,
16 otherwise one should
do very detailed study of the
17 zygote, or one could
use a microscope that will
18 visualize the spindle
at the time of piercing, as
19 Dr. Lanzendorf has
done.
20 Enhanced
survival has been raised as a
21 potential risk to the
offspring. The embryo is now
22 better and, therefore,
you will get higher
23 implantation rates and
implantation of embryos that
24 would have normally,
under normal IVF/ICSI
25 conditions not have
been implanted.
131
1 Aneuploidy
is common. Aneuploidy is the
2 issue that has raised
a lot of concern in this
3 particular group of
patients. Aneuploidy is
4 common. It has been found that this is enhanced in
5 ICSI by one percent,
more or less one percent. The
6 most common anomaly
that is found in ICSI and also
7 in the natural
population is XO. This is exactly
8 what we found in two
patients that this in early
9 pregnancy.
10 Then
heteroplasmy, is that a risk to the
11 offspring? Well, we have confirmed three
12 polymorphisms in three
births. We think that these
13 are common in the
population, or similar
14 polymorphisms are common
in the population. In
15 general though,
heteroplasmy is very common in
16 early human embryos
when we studied this
17 experimentally in the
spare material. From the
18 animal
experimentation, there is no evidence of
19 risk between outbred
individuals in the same
20 species. There are clearly anomalies that have
21 been shown in the
literature when you don't use the
22 same species, or when
you use highly inbred
23 individuals of the
same species.
24 What are the risks to the mother? An
25 elevated incidence of
chromosomal anomaly should be
132
1 considered a risk, if
there is such a thing. There
2 is no statistical
evidence for this so far. As I
3 said, aneuploidy is
extremely common, and XO is the
4 most common form.
5 What cell
issues can there be? Should
6 there be donor
screening? If we do that for this
7 procedure, I don't
know the complexity of that. I
8 don't know the cost
factors associated with it.
9 Abnormal
zygotes, fertilized eggs, I used
10 the mitochondria from
there to inject back into
11 another zygote. But that has been done. It has
12 been reported by a
group in Taiwan. I think this
13 was raised here
before, can you maybe look at other
14 cells and get
mitochondria from other cells? That
15 has been done as
well. If I have a little bit of
16 time later, I will get
back to that. Actually,
17 there has ben one
abstract, where mitochondria were
18 taken from granulosis
cells, the cells that
19 surround oocytes. These were then injected into
20 the patient's eggs.
21 We videotape
the whole procedure for later
22 evidence that we
transferred the cytoplasm. Can
23 one use frozen
oocytes? We have not used frozen
24 oocytes. The disadvantage of our procedure is that
25 you have to
simultaneously stimulate and monitor
133
1 the patient and the
recipient and retrieval and
2 maturation of the egg
has to occur on the same day.
3 That is not
simple. That is actually quite a
4 challenge. So, using frozen oocytes would be an
5 advantage but oocyte
freezing by itself is an
6 experiment we feel,
therefore, we stayed away from
7 this.
8 We do the
chromosome screen of the eggs
9 that are used. Of course, before you transfer the
10 embryo you could also
do another chromosome screen.
11 We have stayed away
from that but we have that
12 technology because
these embryos are often not well
13 formed, and are
already challenged by the procedure
14 and taking another
cell out of the embryo before it
15 is transferred may be
detrimental in this
16 particular group of
embryos, not necessarily in
17 other groups of
embryos.
18 So, what
further non-clinical
19 experimentation should
be done? Well, we should
20 look at costs. I am not sure that the primate
21 model is a good model
for human reproduction but
22 others probably
dispute that. The mouse model we
23 are using. Although there are profound genetic and
24 profound differences
with the human, that is more
25 affordable and results
are very rapidly obtained.
134
1 The issue
with ooplasmic transplantation
2 and the way we have
done it and Dr. Lanzendorf's
3 group has done it is
that that is just one
4 particular
application. There is a host of
5 applications that are
waiting that, in one way or
6 another, involve
ooplasmic transplantation, not
7 necessarily for the
same purpose as I have
8 described here. One of them is treating
9 mitochondrial
disease. You could replace the whole
10 cytoplasm or ooplasm
of a donor egg in a patient
11 that is at risk of
transferring mitochondrial
12 disease to
offspring. That is one potential
13 application.
14 There are
other applications as well,
15 avoiding aneuploidy by
going into very immature
16 eggs and changing the
regulation of how miosis
17 occurs by trying to
maintain regular ploidy rather
18 than aneuploidy. It is obviously under cytoplasmic
19 control. So, if you were to do this early, at
20 least in theory we
believe you could avoid
21 aneuploidy. That would be important particularly
22 since aneuploidy is
the biggest problem area in our
23 field. There are other applications as well.
24 Here are the
two babies that had benign
25 heteroplasmy. This picture was taken two years ago
135
1 so they are almost
four years old and they are both
2 doing fine.
3 Finally,
just a few words about
4 transferring
mitochondria, this was reported in an
5 abstract last year. This was shortly after
6 September 11 so I was
waiting in the room for that
7 particular
presentation but they never came to the
8 country and this
meeting was very poorly attended
9 because this was only
a few weeks after September
10 11. Anyhow, the abstract argues that there is a
11 single course for
ooplasmic problems, and that is
12 the mitochondria. There is absolutely no
13 confirmation for
that. Mitochondria obviously may
14 have a higher rate of
mutation but there is no
15 proof that this is the
only problem. They used
16 somatic mitochondria
which is an interesting idea,
17 but the isolation
process could be an issue, for
18 instance formation of
free radicals.
19 Age-related
mutation should be considered
20 since these are
mitochondria from somatic cells and
21 may have, or very
likely will have age-related
22 mutations. They are also replicating mitochondria.
23 What will happen in
the recipient cells? That is
24 an interesting
question that will come up.
25 Mitochondria in eggs
do not replicate. They do
136
1 that after
implantation. So, they are actually
2 somewhat dormant in
that respect. Somatic
3 mitochondria are very
different. Somatic
4 mitochondria have
multiple mitochondrial genomes
5 per mitochondrion for
instance, whereas oocyte
6 mitochondria only have
one genome. So, they are
7 very different
although they seem similar.
8 That is all
I have to present. Thank you.
9
Question and Answer
10 DR.
SALOMON: Thank you very much, Dr.
11 Cohen. Obviously with the changes in this
12 morning's schedule we
are not quite following the
13 time line here but
this is such an extraordinarily
14 rich presentation in
terms of questions that I
15 think we are just
going to have to spend some time
16 to address these. I think this and Dr.
17 Lanzendorf's are kind
of pivotal. So, I do realize
18 that we are not on
time but we will deal with this
19 in a little bit.
20 I have a lot
of questions but let me just
21 start with one little
part and then turn it over to
22 some of the others, as
I am sure I won't be alone.
23 You know, the one
theme that we picked up in Dr.
24 Lanzendorf's
presentation is what is the basic
25 science background for
doing this? Then we will go
137
1 on to talk about what
is the clinical evidence for
2 doing this, and you
have given us a lot to think
3 about.
4 So, going
back to the basic science
5 evidence of it, you
presented two kinds of basic
6 science arguments for
ooplasm transfer, i.e., kind
7 of a rationale. One was this PED phenotype. The
8 other was some data on
Mad2 mRNA transcript numbers
9 and maternal age. Again, it is okay if it is not
10 convincing but I
didn't find that either of those
11 was clear to me or
convincing.
12 With respect
to the PED gene phenotype, I
13 didn't understand how
you related slow and fast
14 embryos back to a PED
gene phenotype, and then how
15 that had anything to
do with ooplasm transfer.
16 Similarly, you implied
that gene arrays and other
17 technologies have
shown differences in gene
18 expression as a
function of maternal age in terms
19 of implantation
failures, and that certainly makes
20 sense to me in some of
the functional genomics we
21 do in angiogenic stem
cells. But how do you relate
22 a change in transcript
numbers to transferring
23 10-15 percent of
ooplasm? I mean, what evidence is
24 there that 10-15
percent of ooplasm transfer
25 provides an increase
in, in your example, Mad2 mRNA
138
1 transcripts, and does
that increase them to a level
2 that is equal to more
successful implantation
3 phenotype? So, I guess those are the kinds of
4 questions I would like
you to address since those
5 are your arguments.
6 DR.
COHEN: I have a short memory so I
7 will start with the
last one, why ten percent? It
8 seems so little. If it was a blood cell it would
9 be little, but the
human egg is the largest cell
10 that exists. It is an enormous volume and it is
11 known that you can
lose 75 percent of the volume
12 and still get a
human. So, 75 percent of the
13 volume can be
destroyed and since you have to have
14 some unique organelles
like chromosomes and a
15 centriole, it is
likely that you can reduce that
16 volume even
further. So, ten percent is not little
17 at all, and we have
calculated it is about 10,000
18 mitochondria for
instance. So, it is a huge
19 amount. That is considerably higher than the
20 number of mitochondria
in mouse eggs for instance
21 that are smaller.
22 So, coming
back to the PED, I think what
23 is different in other
developmental sequences is
24 that in mammalian
fertilization early development
25 the embryonic genome
is not active yet. It is all
139
1 dependent on what is
present in the egg. So, when
2 you sequentially look
at a transcript like actin
3 and you look at it one
day and the next day and the
4 next day, you will see
it diminished to levels that
5 you could almost call
starving, if that would be
6 the right word for it,
but it really dramatically
7 diminishes and then at
the activation of the genome
8 the embryo starts
taking care of all this and you
9 can see that going up.
10 So, what
this shows is that these levels
11 of expression are so
different between cells of the
12 same stage that it is
maybe not direct evidence but
13 it is likely that there
is a physiological
14 difference between
these individuals. I think Mad2
15 is very likely because
there it is a spindle
16 regulating factor and
it is related to maternal
17 age, and we know that
in maternal age not only is
18 there an increase in
aneuploidy but the typical
19 non-disjunction form
of aneuploidy in mosaicism is
20 also related to
maternal age in the human in early
21 development. So, I think it is very plausible.
22 In PED, in
the mouse at least, a human
23 homolog has never been
found. I was just
24 indicating that there
is a phenotypic similarity.
25 We are looking for
human homologs and they are
140
1 probably in the HLA
system.
2 DR.
SALOMON: But I am just pointing out
3 to you that to make
your case what you need to do
4 is show us that if you
transmitted 10-15 percent of
5 the ooplasm that
therein would be contained enough
6 messenger RNA from
Mad2 to alter the
7 transcriptorsome of
the recipient in such a way
8 that at least you
wouldn't have to demonstrate in
9 the first set of
experiments that it was
10 functional, but just
demonstrate that even
11 numerically the
transcriptorsome would be altered
12 significantly enough
to bring it into a range.
13 Then, of course, the
next set of experiments would
14 be to show that it is
functional.
15 DR.
COHEN: Would you give me permission
16 to do this in the
human?
17 DR.
SALOMON: We will get back to that,
18 but I think what we
are all trying to do is
19 respectfully sit here
and say, okay, what is the
20 data? What is the data basic? What is the data in
21 animal studies and
what is the data in clinical? I
22 was just starting with
the basic. You have made a
23 very intelligent start
by saying, okay, look, here
24 are changes in
transcriptosome, changes in
25 messenger RNA
levels. My response is, okay, you
141
1 know, I am following
you but I am saying it is not
2 convincing. I mean, you have to give us a little
3 bit more to justify
this at this basic level. If
4 the data is not there,
the data is not there.
5 DR. VAN
BLERKOM: Just to clarify
6 something, you are not
saying that the embryo from
7 fertilization to,
let's say, the four-cell stage is
8 transcriptionally
inactive, are you?
9 DR.
COHEN: No, it is not. There is some
10 leakage, yes.
11 DR. VAN
BLERKOM: Because, in fact, things
12 like actin, etc. are
made off maternal--
13 DR.
COHEN: Sure.
14 DR. VAN
BLERKOM: Even in the mouse where
15 it had been earlier
thought that major genome
16 activation occurred
around the two-cell stage, in
17 fact it has been
brought back earlier to the
18 pronuclear stage. In fact, there is probably
19 embryonic genomic
activation very early, but the
20 major genomic
activation, that is the major switch
21 from the maternal
stores to a whole embryonic
22 program is probably at
about the four- to
23 eight-cell stage, but
it is not transcriptionally
24 inactive.
25 DR.
COHEN: Yes, thank you for explaining.
142
1 DR.
NAVIAUX: How long would you expect to
2 be able to detect
transferred RNA in the embryo?
3 What is the half-life?
4 DR.
COHEN: The half-life is very short I
5 think.
6 DR.
NAVIAUX: Would you expect it to be
7 equivalent to the RNA
already in the oocyte?
8 DR. COHEN: The experiment that hasn't
9 been done is to take
an oocyte and then take one of
10 the two-cell
blastomeres and then take one of the
11 other cells of the
two-cell blastomere and look
12 sequentially like
that. It is done by indirect, by
13 looking at populations
and then comparing the
14 different stages. It is very clear that it
15 diminishes from stage
to stage. It is very
16 sensitive. It diminishes very rapidly.
17 DR. NAVIAUX:
I was trying to get a feel
18 for the window of
opportunity for other potential
19 genetic events to
occur from the transferred
20 nucleic acid,
including potentially the
21 retrotransposition of
this.
22 DR.
COHEN: I have no evidence for that.
23 It is certainly
possible.
24 DR.
SALOMON: Dr. Sausville, Dr. Mulligan
25 and Dr. Van Blerkom.
143
1 DR.
SAUSVILLE: The concern I have about
2 the direction of the
conversation that is happening
3 now and, again, I
congratulate you on a very
4 thoughtful
presentation but I think it does
5 highlight one of the
issues, that mitochondria have
6 been put on the table
as one explanation for a
7 benefit. I guess we are going to hear more about
8 mitochondrial
physiology in which, hopefully, there
9 will be some clear and
direct evidence that
10 mitochondria might do
such a thing.
11 But we have just heard of another class of
12 molecules, your
presentation brought up a
13 particular class of
mRNAs, forgetting the whole
14 issue of mRNA in
general. I mean, this points to a
15 key difficulty that I
think we have in thinking
16 about this in that one
of the components of an IND
17 is actually a
definition of what actually is the
18 substance under
investigation in an IND. I am a
19 little concerned, even
if one believes there is an
20 effect and we heard
earlier this morning that there
21 really isn't any
evidence that there is an effect,
22 is how we would define the potential basis for
23 investigational
activity with this. Are we going
24 to have ooplasm that
has a particular type of mRNA
25 or a particular number
of mitochondria or a
144
1 particular class of
mitochondrial genomes? I would
2 be interested in your
thoughts on how one would
3 define, in essence,
the focus of the IND
4 application in this
regard.
5 DR.
COHEN: I asked that question to the
6 FDA representatives a
few months ago and I didn't
7 get an answer because
I don't think they understand
8 that either.
9 DR.
SALOMON: I think that is why we are
10 here.
11 DR.
COHEN: Yes, so I wouldn't know how to
12 do this. I have no idea.
13 DR.
SAUSVILLE: Well, if you don't--
14 DR.
COHEN: Personally, I have not
15 experienced this IND
process. Looking at the IND
16 process, it is so
different, the psychology of it
17 is so different from
this type of typical medical
18 intervention approach
that it is extremely
19 difficult to come up
with a solution.
20 DR.
SIEGEL: Just from a historical
21 perspective, there are
certainly plenty of
22 precedents in
biological development in particular
23 for products whose
active ingredients are not well
24 identified. Some of the earliest biologics,
25 regulated as
biologics, were horse antisera and,
145
1 you know anti-venoms
and toxins and so forth. Of
2 course, over the last
couple of decades the field
3 has moved to much more
highly purified products
4 which are, therefore,
easier to ensure that you
5 don't have unwanted
materials and where you can
6 quantitate what you
have. We certainly support
7 that area of
development, but there is nothing
8 about an IND process
per se that requires that you
9 have a handle on what
component it is of what you
10 are testing that is
the potential active component.
11 DR.
SAUSVILLE: Ah. But, on the other
12 hand, my understanding
is--and those are good
13 examples
actually--that despite that lack of
14 definition there is,
nonetheless, a very precise
15 assay that will tell
you that your material is
16 functioning as you
think it is functioning.
17 Correct?
18 DR.
SIEGEL: That is right, and we
19 certainly require by
the time of licensure a
20 potency assay. That is required by regulation and
21 that requires
development of information. In fact,
22 it is the case for
many that we now have under IND.
23 However, the
development of the potency assay often
24 occurs concurrent with
the early clinical studies
25 because it requires
identification of markers that
146
1 can be measured that,
hopefully, then can be
2 validated to be
predictive of the desired clinical
3 effect.
4 DR.
SAUSVILLE: So, that then actually
5 does play back to the
question I asked. You
6 pointed to the
limitations appropriately of the
7 animal models that are
around for this type of
8 work. Nonetheless, it would seem that such models
9 might be the place to
begin to develop this type of
10 information that could
be a basis for conveying
11 confidence at the very
least, forgetting the IND
12 process, that you
would be able to advise a
13 particular patient
that the procedures that are in
14 place are likely to be
productive of some normative
15 standard of activity
through the process.
16 DR.
COHEN: Yes, and I think that the body
17 of literature is not
enormous, but particularly the
18 work of Larry Smith is
very convincing and this is
19 done in outbred mice
going through 15 generations
20 with apparently normal
development, normal growth.
21 What else are you
looking for?
22 DR.
SAUSVILLE: I would like to know what
23 conveys that normal
growth. What is the physical
24 basis of that normal
growth?
25 DR.
COHEN: That is more than a
textbook.
147
1 I mean, that is the
whole field of early
2 embryology. You are looking at an extremely
3 difficult process that
is hindered by all sorts of
4 factors in terms of
how we can study it. I am as
5 curious as you
are. So, I appreciate the concern,
6 but that is looking at
the oocyte like a product;
7 let's understand the
product, and I think what is
8 being attempted here
is to take something this
9 complicated and then
put it in the form of IND. I
10 have no idea how to do
that.
11 DR.
SALOMON: I think we will return to
12 that this
afternoon. I think the issue that has
13 ben well articulated
now is what--I mean, we can
14 always take every one
of these questions and get
15 down to these really
big, fundamental scientific
16 questions and we all
know around the table that you
17 are not going to know
every single thing about how
18 you create a normal
embryo before you do these
19 studies. No one is holding you to that sort of a
20 standard. But it will be really interesting to
21 talk about what it is
we want to know, and what
22 kind of scientific
questions will be answered even
23 while perhaps certain
clinical studies are going on
24 just to make sure that
there is development along
25 the right lines in the
field. Dr. Mulligan?
148
1 DR.
MULLIGAN: Can you give us a sense of
2 how you test for
fragmentation of either
3 mitochondrial DNA or
nuclear DNA and then transfer?
4 In principle, if you
such out the cytoplasm there
5 is some chance for
fragmentation of both of those
6 DNAs, and it would be,
I think, very important to
7 see if that does occur
because once you have kind
8 of disrupted the normal
mitochondrial architecture
9 it is like doing gene
transfer, that is, it is like
10 injecting fragments of
DNA and there is every
11 expectation that there
would be uptake by the
12 chromosomal DNA like
normally occurs. So, have you
13 looked at ways in a
single cell?
14 DR.
COHEN: I would be more concerned
15 about it in the
isolation process of mitochondria,
16 but here is a package
of cytoplasm that is moved
17 from one cell to
another cell within seconds in a
18 synchronous
fashion. So, I don't think that
19 concern is really
valid. It would certainly be
20 valid I think in the
work that was done by the
21 Taiwanese where
mitochondria were isolated and then
22 processed in ways we
don't know yet, but they were
23 processed, isolated
from granulosis cells and then
24 injected into the
recipient cells. I think there
25 that is a concern
because you do true isolation
149
1 process of an
organelle. In our case we are
2 transferring cytoplasm
intact.
3 DR.
MULLIGAN: Yes, but I thought you said
4 there is a risk of
actually getting contamination.
5 DR.
COHEN: Sure.
6 DR.
MULLIGAN: So, in principle that has
7 the potential for
fragmentation, and isn't that key
8 to see whether or not
there are detectable bits and
9 pieces of genomic DNA?
10 DR.
COHEN: No, we have just done
11 classical
cytogenetics. We looked for whole
12 chromosomes; we have
not looked for bits.
13 DR.
MULLIGAN: You mentioned that you have
14 a good cytogeneticist
who can detect things, that
15 is, the most gross
assay for a microbiologist to be
16 able to detect things
much easier.
17 DR.
COHEN: Yes.
18 DR. VAN
BLERKOM: Maybe you could clear up
19 some points on what
you said. As I recall, in the
20 initial births the
amount of DNA that was
21 detectable was a trace
amount.
22 DR.
COHEN: There was nothing in the
23 original, right.
24 DR. VAN
BLERKOM: Now you are saying that
25 Carol has seen up to
50 percent. Was that from the
150
1 original samples using
another assay, or is the
2 mitochondrial DNA
expanding?
3 DR.
COHEN: No, they are all the same
4 samples and, I am
sorry, I just gave you the wrong
5 answer because in the
first births we were not able
6 to confirm
heteroplasmy; we found a homoplasmy
7 condition. In the births since then, with regular
8 sequencing, we found
levels, we found levels up to
9 20 percent.
10 DR. VAN
BLERKOM: At birth?
11 DR.
COHEN: At birth. That includes the
12 placenta. Placenta seems to be always higher.
13 With the new method
those same samples were
14 reassayed and there we
found levels up to 50
15 percent.
16 DR. VAN
BLERKOM: So, it is very likely a
17 sensitivity
issue. So, you don't have evidence
18 that there is an
expansion of the mitochondria from
19 birth.
20 DR.
COHEN: No, I don't have evidence of
21 it yet but I have
always been interested in that.
22 DR. VAN
BLERKOM: The other question then
23 is if you look at the
process of cytoplasm
24 transfer, which I
don't think is an issue related
25 to mitochondrial damage
just from the logistics of
151
1 the transfer process,
in at attempt to standardize,
2 and I know you have
done this so maybe you should
3 talk about the data
where you have actually taken
4 the same amount of
cytoplasm from different
5 portions of eggs and
then counted the number of
6 mitochondria, and
there are differences.
7 DR.
COHEN: Yes.
8 DR. VAN
BLERKOM: So, maybe you can talk a
9 little bit about the
extent of differences that you
10 get that is location
dependent, and how that my
11 reflect on what you
are putting back, what you know
12 and don't know about
the magnitude of the donated
13 mitochondria.
14 DR.
COHEN: The procedure is standard,
15 however, ooplasm
differs from egg to egg. There
16 are physical
properties that are different. So,
17 you want to pick up
cytoplasm just using suction.
18 It is certainly not comparable
from one cell to the
19 other. So, in some cases the procedure differs
20 from other cases. Also, the cytoplasm is not
21 sampled
statically. I should have brought a
22 videotape. It is actually sampled throughout the
23 whole area opposite
the polar body rather than one
24 area. It is a good, valid point. It is known that
25 the egg is very
dissimilar from area to area so we
152
1 try to sample a
relatively large area of the egg.
2 We have also varied
the amount of cytoplasm that we
3 transfer. All I can say about that is that if you
4 look at the higher
amounts, the higher volumes of
5 cytoplasm that has
been transferred, the more
6 likely it is that the
procedure is unsuccessful,
7 for reasons I don't
understand but that is what the
8 finding was.
9 DR.
SCHON: Just a clarification, Dr.
10 Mulligan, I am
gathering that the question about
11 fragmentation--I won't
talk about the nuclear
12 transposition events,
but at least for the
13 mitochondrial DNA
transposition events, my guess is
14 that, first, there
would be very few fragmentation
15 events to begin
with. It is a tiny molecule. It
16 is stuck in nucleoids
inside the mitochondria. If
17 you visualize what is
going on, it probably
18 wouldn't happen that
frequently. Let's say it
19 does, and it does go
into the nucleus, first, the
20 worry would not be
whether that transfected DNA
21 would actually do
something because it has a
22 different genetic
code. Whether it would transpose
23 into some other gene,
it may but again the likely
24 hood would be low
because there are at least a
25 thousand and maybe
more nuclear embedded
153
1 pseudogenes of
mitochondrial DNA to begin with so
2 it would probably go
in by homologous recombination
3 into places that are
genetically quiescent--I don't
4 know how else to put
it. So, it could happen but I
5 wouldn't give a huge
probability for it.
6 DR.
MULLIGAN: Yes, I would think the risk
7 would be cytoplasmic
DNA actually integrating in
8 the incorrect
location. I would very much doubt
9 that you would get
what you say, homologous
10 integration into
pseudogenes or mitochondrial
11 sequences. So, it would be the risk of insertions
12 comparable to a
retrovirus insertion. It is like
13 thinking of injecting
a plasmid DNA. I guess what
14 I didn't know is what
the chances that the intact
15 mitochondria would
actually, by whatever vortex
16 when you are trying to
suck out the cytoplasm, with
17 there is damage such
that you would actually get,
18 you know, naked
DNA. But the other half of it, of
19 course, was the
nuclear DNA which I think would be
20 much more likely to
have the same potential for
21 integrating in some incorrect
location. And, I
22 think it is very, very
tough from all we know with
23 gene transfer to
assess the efficiency of the
24 process. It is very amazing how different
25 approaches to gene
transfer can dramatically give
154
1 you different
efficiency. So, even several
2 molecules, you know,
if they are given by a fancy
3 method like this, this
could be the most efficient
4 method we have
relative to other systems.
5 DR.
SCHON: Then, could I just comment to
6 Dr. Sausville? I actually think that trying to
7 figure out the exact
active ingredient, if you
8 will, of the ooplasm
may well wind up being a
9 bottomless pit. It is the ooplasm itself that may
10 actually be doing
it. There is not evidence that
11 it is
mitochondria. If you were to merely
just put
12 in mitochondria or
some subfractionation element,
13 you might get nothing
also. I think there is so
14 much synergism going
on that merely doing pair-wise
15 analyses, each alone
might give no outcome whereas
16 ooplasm, where we have
no evidence that there is
17 outcome yet, might
give an outcome, and it should
18 be borne in mind.
19 DR.
RAO: Just a couple of clarifications
20 for what you talked
about. You made a point about
21 saying you disagreed
with germline transmission.
22 Was that because it
hasn't been tested in germinal
23 cells or is it because
you want to wait for F2? I
24 mean, what is the
reason?
25 DR.
COHEN: Also the modification. It is
155
1 not a modification and
it has not been proven to be
2 heritable.
3 DR.
RAO: So, because it is not heritable.
4 DR.
COHEN: Not proven to be heritable.
5 DR.
RAO: The second question was on the
6 point that you made
about somatic mitochondria, was
7 this ooplasmic
mitochondria, and you said one big
8 difference was in the
rate of cell division. But
9 do you think there is
any other major difference?
10 The other point you
made was about it is a multiple
11 genome. Did you mean that it is because it had
12 inherited mutations
and that is why it was more
13 than one genome?
14 DR.
COHEN: Yes, it is all those things.
15 There are multiple
genomes and mitochondria from
16 somatic cells, anywhere
from two to ten I think.
17 In eggs the ratio is
very close to one. So, that
18 is different. The other difference is that
19 mitochondria and
oocytes and embryos do not
20 replicate, whereas
somatic mitochondria do. So,
21 that would be a
different control situation. It is
22 an interesting
suggestion.
23 DR.
RAO: The last question is that there
24 seems to be a
suggestion that there won't be a
25 whole lot of
mitochondrial transfer that would have
156
1 occurred, at least it
was a surprising result that
2 you had in
mitochondrial transfer. What is the
3 basis? I mean, I am not absolutely sure why people
4 thought that you would
not get mitochondrial
5 transfer and maybe you
can tell me.
6 DR.
COHEN: Well, if I had this discussion
7 several years ago it
may have been a different
8 story, but we use it
as a marker. We are just
9 interested to see what
happened to these
10 mitochondria, and this
is the outcome of it. But
11 it was the advantage
of hindsight. You are totally
12 right, I mean, it is
not surprising.
13 DR.
CASPER: I want to go back to Dr.
14 Mulligan's point again. We do have some experience
15 in creating
mitochondrial preparations from
16 granulosis cells, from
mouse embryonic stem cells,
17 from human umbilical
cord blood to hematopoietic
18 stem cells and also
from human leukemia cell line,
19 and it is actually
quite easy to do it. There are
20 some technical issues
that took us a while to
21 actually figure out,
but morphologically at least
22 when you look at the
preparations they seem to be
23 pretty pure, intact
mitochondria. So, the actual
24 morphology at least of
the mitochondria looks
25 normal. We have injected these mitochondrial
157
1 preparations into
mouse oocytes and zygotes.
2 There is a
stain of mice called FVB mice
3 that have a
mitochondrial defect and oocytes
4 fragment in vitro, and
with both granulosis cell,
5 so somatic cell
mitochondrial injections and with
6 stem cell
mitochondrial injections we have been
7 able to prevent at
least 50 percent of the
8 fragmentation rate in
those oocytes. We have also
9 injected mitochondria
into mouse zygotes and we
10 have found that,
contrary to there being any
11 detrimental effect, it
does seem to advance or
12 speed up the rate of
blastocyst formation in those
13 mice.
14 Those are
preliminary results so far but
15 we certainly didn't
see any detrimental effect
16 unless we actually let
the mitochondrial
17 preparations sit for a
while on the bench, and then
18 what we think is
happening is that you are starting
19 to get leakage and
cytochrome C which could
20 actually be
detrimental at that point. So,
21 certainly from a
cytoplasmic transfer point of
22 view, I don't think
you are going to damage the
23 mitochondria at all
because we are actually
24 mechanically
disrupting the cell membrane of these
25 cells and centrifuging
the contents to separate out
158
1 the mitochondria, and
we don't seem to do any
2 damage to the
mitochondria in that situation.
3 Let me
comment on the prior comment. I
4 think you would have
been surprised had there been
5 homoplasmy; you would
have expected heteroplasmy.
6 In fact, we were the
group that analyzed Dolly for
7 heteroplasmy and we
did not find it. It was
8 homoplasmic. Those were sheep, and if you look at
9 cows, they are
heteroplasmic all over the place.
10 So, it is the
expectation to be heteroplasmic and
11 it is something to
worry about.
12 DR.
SAUSVILLE: You referred to the
13 experience with ICSI,
which I interpret to be
14 intracytoplasmic sperm
implantation. Is that
15 correct?
16 DR.
COHEN: Injection.
17 DR.
SAUSVILLE: Injection. Just from a
18 sort of standard
practice of this field, what would
19 be the expected rate
of major abnormalities
20 resulting from ICSI as
a process?
21 DR.
COHEN: In the literature there is a
22 range from 2 percent
to nine percent. But a larger
23 study, a study from
the Belgium group who
24 originated the
procedure, with 3000 babies born, I
25 think there was 3.4
percent, something around
159
1 there, and showed a
significant increase in the
2 rate of XOs.
3 DR. SAUSVILLE: But still that rate didn't
4 go beyond a three
percent sort of range?
5 DR.
COHEN: No.
6 DR.
SAUSVILLE: Thank you.
7 DR.
COHEN: There is one publication that
8 shows a rate of nine
percent, but it was the same
9 in the ICF population
that was studied. That was a
10 recent paper in The
Journal of Medicine, in March.
11 It was based on a
small sample size but that is the
12 only really high rate
I know of.
13 DR. SALOMON: Dr. Moos?
14 DR.
MOOS: First a comment on several of
15 the remarks that have
dealt with the
16 characterization of
the active principle. Cell
17 biologists and
biochemists have been fractionating
18 very complex systems
for well over a hundred years
19 to see what part does
what, and we are nowhere near
20 the bottom of the
pit. Nevertheless, even though
21 we are shy of finding
out where is the final proton
22 and what it does, we
have amassed a tremendous
23 amount of very useful
information.
24 So, I submit
that a sensible way to look
25 at it is to do the
sorts of experiments that are
160
1 feasible and
reasonable not just to enhance our
2 understanding or to
prove that this is good and
3 that is bad, but to
allow us to be able to develop
4 some sense of what is
necessary to keep consistent
5 for a product to
perform in a way that we can
6 understand and
predict.
7 A specific
question that extends a point
8 that was raised by Dr.
Salomon and yourself, Dr.
9 Cohen, since you
brought up specific mRNA
10 transcripts, has
anyone evaluated whether injection
11 simply of RNAs
encoding some of the candidate genes
12 you mentioned or pools
of candidate genes has a
13 beneficial effect on
embryo quality?
14 DR.
COHEN: Obviously none of those
15 studies could be done
in the human at this point.
16 I am not sure that
work like that was done.
17 Certainly interference
with mRNA was done, just the
18 opposite, interfering
with a specific RNA but I am
19 not aware of
injecting.
20 DR.
SALOMON: Dr. Murray and then Dr. Van
21 Blerkom and we will
finish there.
22 DR.
MURRAY: Dr. Sausville's questions
23 about abnormalities
associated with ICSI, I believe
24 one of the studies,
recently published, indicated
25 the risk of low birth
weight was also roughly
161
1 double, and that is
after testing for multiple
2 pregnancies.
3 The question
I have for Dr. Cohen, I am
4 asking for help in
making sense of some of the
5 numbers you presented
about the incidence of
6 heteroplasmy. You gave us a number--we don't have
7 copies of your slides
so this is from
8 memory--something like
evidence of heteroplasmy in
9 10-15 percent in a
hypervariable region in the
10 population. Am I recalling that correctly? The
11 question is if you
were to think about risk,
12 obviously one of the
ways one would think about
13 risk is to say, you
know, does this occur more or
14 less often in the
population that has been exposed
15 to this particular
intervention, ooplasm transfer,
16 than the general
population? I assume the
17 hypervariable region
is a non-coding region. Is
18 that correct?
19 DR.
COHEN: Yes.
20 DR.
MURRAY: Therefore, you know, it may
21 not be clinically
significant. But here we have a
22 heteroplasmy that is
perhaps in a coding region, I
23 assume if you are
doing ooplasm transfer, so
24 wouldn't we want also
to have data that gave us
25 some indication about
heteroplasmy in coding
162
1 regions?
2 DR.
COHEN: That has not been done, and
3 that would be interesting. The work that has been
4 done has all been on
the hypervariable area.
5 DR.
MURRAY: So, the 10-15 percent number
6 doesn't tell us very
much. It doesn't tell me very
7 much.
8 DR. COHEN: No, but one thing that comes
9 out is that it is an
evolving field. Going by the
10 literature, five, six,
seven years ago the
11 incidence was
considered to be--well, there was no
12 number but it was very
rare to see this. So, now
13 with new sensitive
assays it is apparently much a
14 higher frequency.
15 DR.
MURRAY: But again, mutations in
16 hypervariable
non-coding regions are presumably not
17 clinically
significant, whereas what we would be
18 interested in is
evidence of mutations--
19 DR.
COHEN: You shouldn't call it a
20 mutation. It is hypervariable; it is not a
21 mutation.
22 DR.
MURRAY: Fair enough.
23 DR.
SCHON: Can I just say something
24 because I think I can
clear this up? You transfer
25 the whole molecule
when you transfer mitochondrial
163
1 DNA, and you and I
differ at 50 different bases in
2 our mitochondrial
DNA. Some of them happen to be
3 in hypervariable
region and some of them are in
4 coding regions. So, you can't speak about
5 mutations in
mitochondrial DNA as being different.
6 You get the whole
molecule. If I transferred your
7 mitochondrial DNA to
me, I would get 50 different
8 base substitutions on
average. Some of them would
9 be in the non-coding,
some in the coding region.
10 So, the
notion that 15 percent of babies
11 that are born with
heteroplasmy in a hypervariable
12 region is just
wrong. It is wrong. There is no
13 evidence for it at
all. The evidence is that
14 somatic mutations, if
you look at individuals and
15 sample muscle or
heart, for instance, you can find
16 heteroplasmy in about
15 percent of those
17 individuals perhaps at
an extremely low level and
18 it is in a single
cell. It has nothing to do with
19 the germline.
20 DR.
SALOMON: So, it is not a safety
21 issue.
22 DR. VAN
BLERKOM: Just two questions. The
23 donors were not
mitochondrially typed. Right?
24 These were random
donors or did you type the
25 mitochondrial DNA?
164
1 DR.
COHEN: No, we didn't do that, no.
2 DR. VAN
BLERKOM: So, this was after the
3 fact?
4 DR.
COHEN: Yes.
5 DR. VAN
BLERKOM: Then the second
6 question, maybe you
can provide some basis or
7 explanation as to why
transferring a relatively
8 small amount of
cytoplasm would give you what you
9 now see as 50 percent
heteroplasmy, and do you
10 think there is an
upper limit on that? In other
11 words, what is the upper limit?
12 DR.
COHEN: The upper limit is 100
13 percent.
14 DR. VAN
BLERKOM: So, as your techniques
15 for sensitivity
increase, is it possible that, in
16 fact, it will be above
50 percent?
17 DR.
COHEN: It is certainly possible, and
18 it is certainly
possible that there would be a
19 drift over time.
20 DR. VAN
BLERKOM: So, how could you
21 replace this fairly
sizeable replacement?
22 DR. COHEN:
It is an enigma of the
23 bottleneck, the
mitochondrial bottleneck. That is
24 where I think some of
the clues lie. Replication
25 doesn't take place
until implantation of
165
1 mitochondria so the
number of mitochondria that are
2 suggested to be passed
on is relatively small. I
3 think Dr. Schon did
some work on that, and I think
4 it is a very small
percent, less than 0.1 percent
5 of the mitochondria in
the oocyte that will
6 actually make it to
clonal expansion.
7 So, if you
look at it that way, I think
8 mathematically
anything is possible. But it is
9 certainly possible
that there is a positive effect
10 here. Everybody always likes to emphasize negative
11 effects. Maybe there is a positive effect here and
12 these are simply
coming from a population that is
13 more fit. That is a possibility. One thing I
14 think Dr. Murray raised
which is interesting is
15 that we only found
3/13 and it looks very similar
16 to maybe ratios that
you would expect. So, it
17 could just be a chance
phenomenon as well.
18 DR.
SALOMON: Thank you all very much.
19 Even though we are off
schedule, I don't think I
20 would do it any
differently, and that is just part
21 of going into these
very new areas where there are
22 just a lot of really
important issues that I think
23 need to get set on the
table early in order for us
24 to do our job. So, I think this is fine. We will
25 just have to deal with
it a little later, and we
166
1 will. There is no free lunch in life, and
2 certainly not on this
committee.
3 But speaking
of lunch, I am going to make
4 an executive decision
that we go to lunch now and
5 then kind of put all
the mitochondria stuff
6 together after
lunch. It is 12:50 essentially. If
7 we can try and do this
in half an hour and be back
8 here--if you can just
sort of eat and come back, we
9 will start as soon as
possible, as close to 1:20 as
10 possible. Thank you.
11 [Whereupon,
at 12:50 p.m., the proceedings
12 were recessed, to
resume at 1:40 p.m.]
167
1 A F T E R N
O O N P R O C E E D I N G S
2 DR.
SALOMON: If we can sit down again.
3 Not that I am
surprised, this is classic, we should
4 have been back at 1:15
and here we are at 1:45.
5 Anyway, I am sure
there will be a couple of other
6 people bopping in as
we go along but we do need to
7 get started.
8 The next
speaker this afternoon is Dr.
9 Eric Shoubridge, from
the Montreal Neurological
10 Institute, to talk
about transmission and
11 segregation of
mitochondrial DNA. That will be
12 followed by Dr. Van
Blerkom, talking about
13 mitochondrial
function. So, we are going to kind
14 of focus on
mitochondria now.
15
Transmission and Segregation of mtDNA
16 DR.
SHOUBRIDGE: I think my brief here is
17 to tell you a little
bit about what we understand
18 about how
mitochondrial DNA sequence variants get
19 transmitted from
generation to generation, and how
20 they segregate in
somatic cells and in the germline
21 after that.
22 Most of what
I am going to talk about is
23 in the mouse model, a
mouse model that we generated
24 in my own lab, but I
will try and relate it as much
25 as I can to the human
experience.
168
1 So, just so
that we are all on the same
2 page, a few people
have mentioned the basic
3 principles of
mitochondriogenics but I just want to
4 go over them very
briefly. It is a 1000 copy
5 genome in most
cells. It is strictly maternally
6 inherited. As has been mentioned, the male
7 contribution gets into
the zygote but it is
8 destroyed by
mechanisms that are still not well
9 understood. The gametes are special cells, if you
10 will, but the oocyte
contains about 100,000 copies,
11 at least 100,000
copies of mitochondrial DNA, and
12 they are thought to be
organized at about one copy
13 per organelle, and the
sperm contains about 100.
14 Germline and
somatic mutations can produce
15 mitochondrial DNA
heteroplasmy. So, at birth, it
16 is thought, that most
individuals that are not
17 carrying a disease
mutation that they have
18 inherited from their
mom are homoplasmic. That is,
19 every single
mitochondrial DNA in the body has the
20 same sequence. Nobody has really looked at this in
21 great detail in
thousands of individuals, but it is
22 thought that most
babies have in their bodies the
23 same sequence in every
cell, in every mitochondria.
24 It is a highly
polymorphic genome so each one of us
25 at this table differs
by about 50 base pairs on
169
1 average between our
mitochondrial DNA sequences.
2 How does it
segregate? It segregates for
3 two reasons. One is that the replication of the
4 genome is not very
tightly linked to the cell
5 cycle. In fact, it is not tightly linked to the
6 cell cycle. What that means is that templates can
7 either replicate or
not during a cell cycle. So,
8 what is controlled in
a cell specific way is the
9 total number of copies
of mitochondrial DNA. So,
10 neurons have different
numbers than muscle cells,
11 than fibroblasts and
cells in the kidney, but they
12 are turning over by
mechanisms that we don't
13 understand even in
post-mitotic cells. The copy
14 number is maintained
but who replicates and who
15 doesn't is not very
well controlled or even
16 understood.
17 So, in cells
that are dividing there is an
18 additional feature,
that mitochondrial DNA is
19 randomly partitioned
at cytokinesis. So, we have
20 two mechanisms that
segregate sequence variants,
21 both in cells that are
mitotic and cells that are
22 post-mitotic. That leads to this process that we
23 are all interested in,
called replicative
24 segregation and the
fact that the mitochondrial
25 genotype you get at
birth, if you happen to be
170
1 heteroplasmic, can be
different in space and can
2 change in time.
3 You already
saw this picture that was
4 produced in a review
by Bill DeMaro, and we know
5 now that mutations in
mitochondrial DNA are
6 important in a large
variety of diseases. There
7 aren't very many
things we can say this, except
8 that they can occur at
any age and affect any
9 tissue. That is sort of the worst case
10 interpretation of this
picture here but, in fact,
11 these diseases
generally affect the central nervous
12 system, the heart and
the skeleton muscle, tissues
13 that rely heavily on
ATP produced oxidatively.
14 The two
questions I want to answer today
15 are how is
mitochondrial DNA transmitted between
16 generations? The second one is what controls the
17 segregation of
mitochondrial DNA sequence variants
18 in different tissues
of the body?
19 It has been
known for some time that
20 mitochondrial DNA
sequence variants segregate
21 rapidly between
generations. This was first
22 established by Bill
Houseworth and his colleagues
23 in pedigrees of
Holstein cows. What I want to show
24 you, which is typical
of the human situation, is a
25 large pedigree that
was published by Neils Larson,
171
1 from Sweden probably
about ten years ago. It is a
2 five generation
pedigree that is segregating a
3 particular mutation in
tRNA. It is a point
4 mutation that is
associated with this phenotype
5 called MERF and it has
these clinical features.
6 There is a
single person that is affected
7 by this diseases in
this five generation pedigree
8 who has all of these
features. What I want to
9 point out here is if
we just look at this line of
10 the maternal lineage
here, the numbers that are
11 associated--and I am
sorry, you can't see them from
12 the back--the numbers
that are beside these are
13 measurements of
heteroplasmy in the blood of these
14 individuals. It turns out for this particular
15 mutation, but it is
not generally true, that what
16 is in the blood
correlates reasonably well with
17 what is in affected
tissues. It is always a little
18 bit lower.
19 What I want
to point out is this mom,
20 here, who had a
daughter with 73 percent of this
21 mutation but a son
with nothing. So, in a single
22 generation there is
nearly complete segregation of
23 this mitochondrial
sequence variant which happens
24 to be pathogenic and
produced a disease.
25 This mom,
here, gave 73 percent to her mom
172
1 and then she had four
boys, one of whom had quite a
2 lot, 88 percent,
enough to produce the disorder,
3 and some who were
asymptomatic even though they
4 were carrying large
proportions of the mutation
5 that produces the
disease phenotype. That is
6 because of this
so-called threshold phenomenon
7 here. These guys were not affected because they
8 didn't have enough
mutant mitochondrial DNAs to
9 produce a biochemical
defect in the cells. So,
10 another principle of
mitochondrial genetics is that
11 you have to exceed a
threshold of mutants in a cell
12 in order to produce a
biochemical and, therefore a
13 clinical, phenotype.
14 It turns out
for the vast majority of
15 mutations that we know
about that that threshold is
16 very high. So, if you have 70 percent or 80
17 percent of these
mutants you can sometimes look
18 completely normal, depending
on how they are
19 distributed.
20 In order to
study this, a postdoc in my
21 lab, Jack Jenuth,
decided to make a mouse model.
22 There are no known
natural heteroplasmic variants
23 in the inbred mouse
population that we know about
24 so we had to construct
one. The way we constructed
25 it was much along the
same lines that we have been
173
1 talking about earlier
today in humans. We found
2 two different common
inbred strains of mice, one
3 which is called BALB
and one which is called NZB,
4 that happen to differ
at about 100 base pairs, 100
5 nucleotides between
the two genomes. We simply
6 made, and we did this
in both directions, a
7 cytoblast from one of
them. We injected that under
8 the zona pelucida of
the zygote here, and then we
9 electrofused.
10 We don't
know exactly how much cytoplasm
11 we have put in here,
but probably something on the
12 order of 10-15
percent, which are the numbers which
13 have been bandied
around today. We fully expected
14 to get transmission of
this mitochondrial DNA that
15 we put in here. In fact, we did.
16 So, we did
this in a large number of
17 animals. I just want to point out that these are
18 the amino acid
substitutions that are predicted by
19 the sequence
differences between these two strains.
20 Here is NZB and here
is another so-called old and
21 inbred strain which is
the same as BALB. They are,
22 for the most part, at
non-conserved sites in
23 evolution, and for the
most part conservative
24 substitutions at those
sites. So, in short,
25 polymorphisms. The only one that is not is this
174
1 cystine for what is
either an arginine or a leucine
2 in this particular
one, here. The rest look pretty
3 much like
polymorphisms.
4 So, we thought we were putting in
neutral
5 sequence
variants. I must say, this is exactly
6 kind of parallel to
the situation in ooplasmic
7 transferred
humans. You are putting in a
8 mitochondrial DNA that
might differ at 50 or 100
9 positions in the whole
genome. You are putting in
10 the whole genome and
this is very different than
11 mutations that arise
in the germline or somatic
12 cells where you will
get a single mutation on the
13 same haplotype background. So, it is quite a
14 different situation.
15 This is the
first litter we got from one
16 of our founders. We isolated several female
17 founders. I can't remember exactly the range
18 because it is a few
years ago that we got, but this
19 was pretty
typical. We would get something like 3,
20 5 to 10 percent or
so. Ten percent I think is the
21 most we ever saw of
the donor mitochondrial DNA in
22 the founder
females. We got that in most females.
23 So, the expectation is
if you put in, at least in
24 the mouse model, 10-15
percent of cytoplasm you are
25 going to get out
something which is not so
175
1 dissimilar from
that. It is a little bit less.
2 Again, this is just a
real eyeball estimate. We
3 haven't measured
anything in terms of how much
4 cytoplasm we put in.
5 What we saw,
and this is a very typical
6 pedigree, is that some
animals had completely lost
7 that mitochondrial DNA
and other animals in fact
8 looked like they had
amplified it. In fact, I will
9 show you they don't
amplify it, it is just a
10 stochastic
phenomenon. So, in one single
11 generation, from a
very small amount of
12 mitochondrial DNA that
is added to this, and this
13 would be analogous to
the human situation that we
14 are talking about, you
could in the next generation
15 completely lose it or
it can become more frequent
16 in the offspring from
that mom.
17 This pretty
much parallels what we have
18 seen in terms of
transmission of pathogenic
19 mutations in human
pedigrees with disease. So, we
20 wanted to sort out
what the basis for this was, and
21 the way we did it was
using single-cell PCR. We
22 simply went back in
the female germline to find out
23 what the level of
heteroplasmy was in mature
24 oocytes versus primary
oocytes versus the
25 primordial germ cells
that were going to give rise
176
1 to the entire female
germline.
2 The
conventional wisdom was, as we knew
3 from the observations,
that there must be a
4 bottleneck here
somewhere because it looked like
5 the 100,000 copies of
mitochondrial DNA in the
6 mature oocyte were not
being transmitted to the
7 next generation, if
you will, because you couldn't
8 possibly get rapid
fixation for a mutation if the
9 sample size of every
generation was 100,000;
10 100,000 is a huge
sample size. So, if ten percent
11 of those were carrying
a particular mutation and
12 you sample the 100,000
in the next generation you
13 are going to get about
ten percent, plus or minus a
14 little bit. So, it was pretty clear you must be
15 sampling, effectively
sampling a much smaller
16 number and we wanted
to determine what that number
17 was.
18 I will just
show you two pieces of data
19 from that because it
has been published years ago.
20 Using single-cell PCR,
we measured the proportion
21 of heteroplasmy from
the donor genome. In this
22 case we have added the
BALB genome on the NZB
23 background.
Here we are comparing what we see in
24 the mature oocytes
sampled from the female that
25 produced these
offspring. So, these are offspring
177
1 and oocytes from the
same female mouse. You can
2 see that the
distributions pretty much overlap,
3 meaning that by the
time you are a mature oocyte
4 there is no
significant segregation of the sequence
5 variant that we put
in, that we donated to create
6 the founder up to the
point of the offspring being
7 born.
8 We then went
back a step further and we
9 looked at primary and
mature oocytes in the same
10 animals by doing a
little trick, and you can see
11 here that the
distributions also overlap. So, even
12 by the time the
primary oocytes are set aside,
13 which happens in fetal
life, all of the segregation
14 of the sequence
variants, of the heteroplasmy that
15 is going to happen,
that is going to be important
16 in the babies that are
born from this experiment,
17 has happened. So, if you were to measure the
18 heteroplasmy in the
primary oocyte population, it
19 would predict what it
would look like in the
20 offspring. Or, if you were to measure it in the
21 mature oocytes, it
would also predict what it would
22 look like in the
offspring.
23 I won't give
you the rest of the data, but
24 we went back and
collected primordial germ cells
25 and what we saw was
that there was not that much
178
1 variation in the
primordial germ cells, but by the
2 time they reached this
stage, the primary oocyte
3 stage, all of the segregation
has happened.
4 This is just
a summary slide of what we
5 think is the life
cycle of mitochondrial DNA in the
6 female germline. It is worth probably just
7 spending a couple of
minutes to work through it,
8 just to refresh your
memory about the things I have
9 told you.
10 The mature
oocyte, at least in the mouse,
11 contains about 105
mitochondrial DNAs. The sperm
12 brings in 100; they
are completely destroyed. So,
13 the zygote still has
105 mitochondrial DNAs and
14 then, as has been
mentioned before, there is no
15 application of
mitochondrial DNA in the early
16 stages of
embryogenesis. So, up to the blastocyst
17 stage where the
inter-cell mast cells are set
18 aside, there is a
reduction in copy number of
19 mitochondrial DNA from
about the 105 that is in the
20 oocyte, here, to about
103, 1000 per cell which is,
21 if you will, about the
somatic number of
22 mitochondrial DNAs in
your average, if you can say
23 there is an average,
cell. But it reduces it from
24 the very large number
that is in the oocyte to
25 here.
179
1 Then, when
this implants we don't really
2 know what happens, but
we suspect mitochondrial DNA
3 replication still
doesn't restart and a small
4 population of cells,
called the primordial germ
5 cells, are set
aside. If you look at pictures of
6 these cells in all
mammals where it has been done,
7 which is now in
several species, they contain about
8 10 mitochondria. So, the mature oocyte had 100,000
9 copies of
mitochondrial DNA and there are about 10
10 in these cells. So, this is where the bottleneck
11 is. It is a natural physical bottleneck in the
12 female germline. A very small number of
13 mitochondria with a
small number of mitochondrial
14 DNAs, we think
certainly less than 100 copies, are
15 transmitted outcome
the next generation.
16 These cells
then start migrating from
17 where they arise in
the embryo to the general
18 ridge, and they give
rise to the complete germline
19 population, called
primary oocytes here, and at
20 this stage, here, all
of the segregation that is
21 going to happen of the
heteroplasmic sequence
22 variants has
happened. It is not going to be
23 important further
on. In mouse this might be
24 40,000 or 50,000 cells
and six or seven million in
25 humans. Most of those die by atresia and there has
180
1 been some thought that
perhaps that cell death
2 might be related to
mitochondria, but I am going to
3 argue in a minute that
I don't think that that is
4 important.
5 That is the
state in the mouse. You can
6 actually use some
statistics to calculate the
7 effective number of
transmitting units between
8 generations, but it
depends on what model you use.
9 So, that is just a
statistic; it doesn't have any
10 physical reality.
11 What happens
in humans? Here are six
12 common mutations,
point mutations that occur in
13 humans. Patrick Chittering and his colleagues in
14 Newcastle analyzed the
transmission of these
15 mutations in all the
published pedigrees, and I
16 think this was
published in the year 2000, all the
17 pedigrees that they
could find in the literature.
18 They got rid of the
proban so that they wouldn't
19 introduce a big
ascertainment bias, and if the
20 transmission of the
pathogenic mutations were the
21 same as the neutral
polymorphic mutations that we
22 saw in the mouse, what
you would expect is a
23 symmetrical
distribution around zero, which would
24 be telling you that
mom is just as likely to give
25 more mutant
mitochondrial DNAs to her children as
181
1 less.
2 In fact,
that is more or less what you see
3 here. It is a bit difficult to analyze this. This
4 is not a random
sample. These are people who show
5 up in genetics
clinics. The proban has been
6 eliminated to get rid
of some of that ascertainment
7 bias but you can't
completely get rid of it.
8 So, the
point is that even though these
9 mutations are
pathogenic, it looks like the
10 transmission of these
mutations through the female
11 germline is
stochastic, just like it is for the
12 neutral mutations that
we studied in the mouse.
13 There is a
single good example in the
14 literature actually
looking at the distribution of
15 heteroplasmy in
oocytes from a woman carrying a
16 pathogenic mutation,
and here is what you find.
17 The mean proportion of
this particular mutation of
18 the mom in her oocytes
was something around 14
19 percent, and you can
see that a large proportion of
20 her oocytes have
completely lost it. Some had very
21 little and some had
more. I could take any of the
22 mice that we looked at
and plot the same thing
23 here, and these
distributions would absolutely
24 overlap. In fact, if you used a statistic to
25 calculate the
effective number of segregating units
182
1 that could give rise
to this distribution, it is
2 indistinguishable in
the mouse and human.
3 So, what we
find in the mouse, as far as
4 we know, looks pretty
similar to what is in the
5 human. So, the transmission of sequence variants
6 between generations
appears to be largely
7 stochastic.
8 The effective number of mitochondrial DNAs
9 in the germline is
small because of the bottleneck
10 that happens at the
primordial germ cell stage.
11 That causes rapid
segregation of sequence variants.
12 So, if an individual
were to get, from whatever
13 mechanism,
mitochondrial DNAs from a donor
14 individual, the next
generation would now rapidly
15 segregate those. So, some of her offspring may
16 contain a lot of that
particular sequence variant;
17 some may contain none.
18 I think the
evidence that pathogenic
19 mutations are largely
transmitted in a stochastic
20 fashion, which is
almost indistinguishable from
21 what we see in the
mouse, suggests that there is no
22 strong selection for
mitochondrial function during
23 this process. So, what we are talking about here,
24 one of the aspects of
what we are talking about
25 here today is whether
the additional boost, if you
183
1 will, that could be
given to a zygote from a small
2 amount of extra
mitochondria there, I don't think
3 in the disease cases
there is any reason to suspect
4 that that is true
because there are lots of babies
5 born who are perfectly
normal, who later get into
6 trouble, and they
might get into trouble even in
7 the first few months
of life but they may be
8 carrying 90 percent or
95 percent of mutant
9 mitochondrial
DNAs. If those mutants are organized
10 as one per
mitochondrion, then certainly those
11 mitochondria have very
little function. So, I
12 think the point is you
don't need much
13 mitochondrial function
either to go through
14 oogenesis or to go
through fetal life and have a
15 perfectly normal
baby. Later on things can happen,
16 and they do in
disease.
17 What about
segregation? What about after,
18 in post natal
life? Well, if you look at human
19 disease, there are any
number of patterns of
20 segregation of
pathogenic mutations. Let just
21 focus on two that I
have on this slide, two common
22 mutations in tRNAs
that are associated with
23 well-recognized
clinical phenotypes. One is the
24 point mutation in
lysine that is segregating in a
25 pedigree that I showed
you earlier on. Here, it
184
1 looks like the
affected individuals have high
2 proportions of this
mutation in their skeletal
3 muscle, always over 85
percent. There is also a
4 high proportion in the
blood which is usually about
5 ten percent less than
what is in the muscle.
6 If you
contrast that with another mutation
7 that is again a point
mutation in the tRNA that
8 produces a completely
different clinical phenotype,
9 it is all over the map
in the blood, the proportion
10 of these
mutations. There is a high proportion
in
11 rapidly dividing
epithelial cells and they can
12 collect in the
urine. We don't know what that
13 looks like for this
particular mutation, and it
14 decreases with age in
the blood, whereas here we
15 don't really have any
evidence that there is much
16 of a change in the
proportion of these mutants with
17 life.
18 So, here are
two different tRNA point
19 mutations. They have been worked on quite a lot.
20 We know that they
produce translation defects in
21 mitochondria and, yet,
the segregation of these
22 sequence variants, the
pattern of segregation is
23 very different. You wouldn't really predict that
24 if the segregation
pattern depended upon function,
25 mitochondrial
dysfunction, if you will, in some
185
1 way. The pattern of segregation we know
2 determines, in muscle
at least because this is the
3 tissue we have the
most access to, what the muscle
4 phenotype looks like.
5 Here are
muscle biopsies from two patients
6 that are carrying this
tRNA lysine mutation that is
7 associated with
MERF. One of them has this very
8 typical pathology
called ragged red fibers. These
9 are grossly abnormal
muscle fibers. If you stain
10 them for cytochrome
oxidase activity, which is one
11 of the enzymes in the
mitochondrial respiratory
12 chain, they are
completely negative. They have
13 absolutely no
activity. And, you have huge
14 proportions of these
mutants here.
15 There was
another patient who had
16 completely normal
muscle biopsy, but the
17 proportion, if you
just took a piece of muscle of
18 the mutation in both
biopsies they are virtually
19 identical. So, how they distribute in muscle and
20 presumably other
tissues determines, to a large
21 extent, what the
phenotype or how serious the
22 biochemical phenotype
is and presumably that
23 determines some of the
clinical picture.
24 If we look
again, comparing these same two
25 mutations with age,
and Here Joanne Pulsion, in
186
1 Oxford, first did this
plot and she said, well, if
2 there is no real
pattern to what is going on in the
3 blood of patients
carrying this particular 3243
4 mutation, maybe what
is happening is that it is
5 changing in the blood
and it is changing in the
6 muscle as well. So, the difference between what
7 you find in the muscle
and the blood might be
8 linear with age. In fact, that is, indeed, what
9 she saw. Subsequent studies have shown that this
10 mutation does decrease
in age with the blood and
11 probably increases
with age in the muscle. The
12 mutation they talked
about at 8344 doesn't do
13 anything with
time. It is absolutely flat. So,
14 the evidence there is
that what you get at birth
15 determines how sick
you will be, whereas things can
16 change with other
mutations.
17 So, that is
all extremely confusing. You
18 are probably confused
so here is the conclusion:
19 there is no simple
relationship between the
20 oxidative
phosphorylation dysfunction and the
21 pattern of
segregation. There are lots of
22 different patterns and
we don't understand what it
23 is. It could be some subtleties associated with
24 the mitochondrial
dysfunction that mutations
25 produce, or it could
be that some other nuclear
187
1 genes are controlling
this whole process, and that
2 is what I want to talk
about in the last little
3 bit.
4 To come back
to our mouse model of
5 segregation, when Jack
Jenuth was in the lab and we
6 had sorted out the
transmission we thought, well,
7 that is kind of
neat. We expected to see something
8 that was different and
stochastic and we didn't and
9 we thought let's look
in the tissues and see what
10 we see. We expect it would just be random there,
11 just like it was in
the female germline; there
12 wouldn't be any
particular pattern and sometimes
13 the proportion of this
sequence variant would go up
14 and sometimes it would
go down, and whatever tissue
15 we measured, it would
be all over the map.
16 In fact, we
saw something completely
17 different. If we looked at rapidly turning over
18 cells, like colonic
crypts, we found out that that
19 was the segregation
which turned over in the mouse
20 about once every 24
hours. That was completely
21 random. If we then looked later on at age, what we
22 found was that most of
those crypts had completely
23 lost the mutation but
a few were going towards
24 fixation of the
mutation.
25 So, this is
a picture like you could see
188
1 in a population in a
textbook, and this is the
2 probability of
fixation of a rare neutral mutation
3 in a randomly mating
population, and this shows it
4 par excellence. So, the proportion of crypts that
5 should be fixing the
mutation should be directly
6 proportional to the
initial frequency of the
7 genotype in that
population, which was about six
8 percent and that is
about what we saw here. About
9 six or eight percent,
I can't remember the exact
10 number were fixing but
most of them had lost it by
11 pure random genetic
drift so there was no selection
12 at all involved here.
13 If we looked
in the brain, the heart and
14 skeletal muscle we
couldn't even see any evidence
15 for that in the
lifetime of the animal. Very
16 surprisingly, if we
looked at a few other tissues
17 like the liver, the
kidney, the spleen and the
18 peripheral blood we
saw a very strange phenomenon
19 that had never been
described before, and that was
20 tissue-specific and
age-dependent selection for
21 different polymorphic
mitochondrial DNA genotypes.
22 So, the liver and the
kidney without exception
23 would select for the
NZB mitochondrial DNA and the
24 BALB in the spleen,
without exception would select
25 for the BALB, the
opposite mitochondria in the same
189
1 animal. So, we didn't know what that meant.
2 Then a
graduate student came to my lab,
3 Brendan Battersby, and
picked up on this and he
4 wondered what was
going on, what was selecting for
5 this particular
sequence variants that we would
6 have predicted would
not have functional
7 consequences, and that
were transmitted through the
8 female germline as
completely neutral variants in a
9 completely stochastic
fashion.
10 So, he did
some sing-cell PCR in the liver
11 and basically wanted
to measure what the increased
12 fitness was for the
NZB mitochondrial DNA which, as
13 I mentioned, always
selects in the liver. By 18
14 months, most of the
hepatocytes in the liver are
15 fixed for that, and it
doesn't matter where they
16 started--they could
start at one percent or two
17 percent, if you look a
year and a half later, they
18 are fixed. So, it happens at a constant rate. It
19 is independent of
genotype frequency, which is very
20 mysterious if it were
a function but it is not what
21 you would predict
because of these threshold
22 phenomena.
23 Initially we
said, well, that can't be
24 related to
function. So, we measured this relative
25 fitness simply by
comparing the initial and final
190
1 genotype frequencies
in animals. The way we got
2 the initial frequency
was to look at tissues where
3 these things weren't
segregating. So, we assumed
4 that is what the
animals were born with. By the
5 way, we have pretty
good evidence--we have data
6 actually to show that
at birth all tissues are
7 pretty much the same
in terms of the level of
8 heteroplasmy of these
patients. So, if you get two
9 percent or five
percent or ten percent, every
10 tissue has the same
amount and you would predict
11 that amniocytes would
have the same amount too.
12 There is a little bit
of data in humans to suggest
13 that that is true.
14 So, if we
measured this relative fitness
15 for this thing at two,
four and nine months of age
16 we pretty much got the
same answer. So, there is a
17 constant advantage for
this genotype in the liver.
18 Every time
mitochondrial DNA turns over this
19 particular genotype
has a 14 percent advantage.
20 So, if you wait long
enough, no matter where you
21 start, it will fix for
that mitochondrial DNA
22 genotype.
23 If you look
at oxygen consumption, a
24 fairly crude way to
look at function of
25 mitochondria, this
measures Vmax of the respiratory
191
1 chain and we couldn't
see any difference. So, we
2 put essentially a very
high proportion--we couldn't
3 get 100 percent at the
time we did these
4 experiments, NZB
mitochondrial DNA on a BALB
5 nuclear background or
100 percent BALB on a BALB
6 nuclear background,
and these are just measures of
7 mitochondrial
respiratory chain function, and there
8 was no difference,
which is what we also would have
9 predicted.
10 We then
thought maybe it is just
11 replication, although
the base substitutions in
12 these two different
molecules did not affect any of
13 the known regulatory
sites that have been defined
14 in the literature, but
we thought maybe we will
15 measure replication
and see if there is a
16 difference
anyway. So, we did an in vivo
17 experiment where we
injected with BrdU to label
18 mitochondrial
DNA. We isolated mitochondrial DNA
19 and did a Southwestern
analysis. So, the idea here
20 is that we are looking
at incorporation of BrdU in
21 the mitochondrial
DNA. We strip this and then we
22 just do a straight
Southern to look at how much
23 mitochondrial DNA is
there.
24 We have two
different sequence variants
25 that we can recognize
because there are restriction
192
1 fragments here. So, we have the NZB or the BALB
2 mitochondrial
DNA. These are five different
3 animals obviously
because we had to sacrifice them
4 at different times
during the experiment, but the
5 number to compare is
this versus this. So, if
6 there is no
replicative advantage in this
7 experiment for this
molecule, for the NZB molecule
8 over the BALB, then
the incorporation rate of BrdU
9 should reflect the
proportion of the genome that is
10 there and that is, in
fact, the case. So, we have
11 no evidence that this
is based on replication.
12 If you take
hepatocytes out of these
13 animals and culture
them you get exactly the
14 opposite effect. They now select for the BALB
15 mitochondrial DNA and
not the NZB mitochondrial
16 DNA--completely
unexpected. We don't know why this
17 happens. It turns out it is not so easy to grow
18 mouse hepatocytes so I
am not going to pursue that,
19 but you can actually
calculate the relative
20 fitness. The copy number of mitochondrial DNA
21 drops when hepatocytes
start to proliferate in
22 culture and the
relative fitness goes up by about a
23 factor of two, but for
the opposite mitochondrial
24 DNA.
25 So, this was
all very mysterious. We got
193
1 a small advantage for
the this NZB thing, not based
2 on function as near as
we can tell. It is not
3 based strictly on
replication. If we change the
4 mode of growth in the
genotype the selection can be
5 opposite. So, what we concluded from all of this
6 was that selection
must be acting at the level of
7 the genome itself. It doesn't have anything to do
8 with the
function. It is not acting at the level
9 of the cell or the
organelle; it is acting at the
10 level of the genome.
11 So, we
hadn't made any progress with the
12 biochemistry here so
we needed to do something
13 else, and my son
summed this up very well. He was
14 working with his mom
in the kitchen one day. He
15 was making a cake and
he turned around and he said,
16 "Daddy, you know,
every experiment has a wet part
17 and a dry part."
18 Here is the
dry part, the genetics. We
19 had to turn to
genetics. So, the idea now was to
20 see if we could tease
out a gene, a quantitative
21 trait locus that would
determine whether or not you
22 would select for the
BALB or the NZB mitochondrial
23 DNA in a
tissue-specific way.
24 So, here is
the breeding strategy but it
25 is a pretty typical
thing you do in genetics. In
194
1 mouse genetics you
just breed two inbred strains
2 together and look in
the F2 generation and see if
3 the phenotype, and the
phenotype here is
4 tissue-specific
directional selection of
5 mitochondrial DNA, see
if it segregates. In fact,
6 it does.
7 I will just
show you the example in the
8 liver in the interest
of time. This is a random
9 collection of about 50
animals. Actually it is a
10 little bit less, they
are not all in the liver
11 here; from muscle in
animals at 3 months or 12
12 months of age. These are F2 mice in this
13 experiment. The muscle is not doing anything
14 interesting but you
can see that at 3 months there
15 is kind of a bimodal
distribution in the liver, and
16 by 12 months they are
completely fixed to that
17 genotype. I won't show the other tissues in the
18 interest of time.
19 We then
calculated the relative fitness
20 using the same measure
that we used before in these
21 animals and it looked
to us like in the F2 animals
22 there were some that
looked like the parents that
23 were selecting, strong
selectors for the NZB
24 genotype; there were
others that were weak
25 selectors for the NZB
genotype; and then there were
195
1 some in the
middle. This kind of looked like a one
2 to one distribution to
us, which is suspiciously
3 Mendelian, and we
thought maybe there is a single
4 strong gene that
underlies this effect, a nuclear
5 gene which is
controlling segregation behavior.
6 The idea
would be that in the BALB animals
7 are behaving like a
parent, and the animals that we
8 bred them with, which
are actually a subspecies
9 called moos-moos
castenius, were homozygous for the
10 castenius. So, we tested that. We did genome
11 scans that were done
on all the tissues. I am just
12 going to summarize the
data rather than going
13 through how we did
this genetically because I don't
14 think anybody is
particularly interested in those
15 details and if you
are, you can ask me.
16 We did a
genome scan at three months in
17 the liver, and what we
found was a locus on mouse
18 chromosome 5, which a
giant LOD score which, those
19 of you who know about
LOD scores, that is pretty
20 big; I haven't seen
too many that are bigger, that
21 explained almost 40
percent of the variants of that
22 genotype. In the kidney we saw another locus on
23 chromosome 2 that
explained less. This acted in a
24 dominant way; this one
was recessive. At this
25 stage we couldn't
really score the phenotype in the
196
1 spleen accurately so
we didn't really pick up any
2 linkage.
3 If we look
at 12 months of age we don't
4 see any linkage in the
liver because all the
5 animals are fixed so
the segregation has all
6 happened. That is telling us that the BALB allele
7 is presumably a strong
allele than the castenius
8 allele but eventually
the castenius alleles catch
9 up. But we saw the same locus in chromosome 6
that
10 could account for
15-20 percent of the variants in
11 the kidney and the
spleen and it was the same one,
12 the same locus. If you remember, these are going
13 in opposite
directions. So, this is selecting the
14 NZB mitochondrial DNA;
this is selecting the BALB
15 mitochondrial DNA.
16 We ended up
with three quantitative trait
17 loci that explain a
fair proportion, especially in
18 the liver, of this
variation that seem to control
19 the selection of what
we think are neutral variants
20 of mitochondrial
DNA. How that happens is a
21 complete mystery so
far. We don't know what they
22 look like. They are probably not molecules that
23 are involved in the
replication of it because it
24 looks like the
replication is the same. So, we
25 think they may be
codes for molecules that are
197
1 involved in its
maintenance.
2 So, this is a summary of the three
3 different loci on
three different chromosomes. One
4 of them, in the liver,
is very highly significant;
5 the other ones are
significant by the normal
6 criteria used in
quantitative trait analysis.
7 I will just
conclude, just to sum this
8 whole thing up, the
transmission of these sequence
9 variants in the
germline, as I said, looks like it
10 is completely
stochastic to us. There doesn't seem
11 to be any bias one way
or another. We have now
12 actually bred animals
completely in the other
13 direction. So, we have put in a couple of percent
14 of the NZB or the
BALB--now we are just doing it
15 with NZB on a BALB
background into founder females
16 and we can get 100
percent if we just breed for a
17 few generations of NZB
on a BALB background
18 starting out with
two. So, it doesn't matter where
19 you start from. Because it rapidly segregates
20 through the germline
in a stochastic way, you can
21 just pick animals that
have a high percentage and
22 the offspring from
those mothers are going to have
23 a higher percentage,
and in about three or four
24 generations you can
get 100 percent the other way.
25 There is
tissue-specific nuclear genetic
198
1 control of this
segregation process which does not
2 seem to be based
strictly on replication of the
3 molecules or on
function of the molecules, which is
4 very surprising, and
we think, but this is not just
5 hand waving, that
perhaps the genes that code for
6 these molecules might
be involved in the
7 organization of
mitochondrial DNA in the nucleoid.
8 There could be a lot
of other things and we are now
9 trying to clone those.
10 In closing,
I just want to acknowledge the
11 two people in my lab
who have done most of this
12 work, two very
talented students, a graduate
13 student, Brendan
Battersby and a postdoc who
14 started it, Jack
Jenuth. Thanks very much.
15 DR.
SALOMON: I never know whether to clap
16 or not, but as we
didn't clap before--
17 [Laughter]
18 --that was a very nice talk. Just in
19 terms of trying to be
efficient with time, given
20 that the next talk is
also about mitochondria and
21 you are sitting on the
panel with us, unless
22 someone has a question
which just totally be out of
23 context and they just
have to ask it now--I don't
24 see anyone jumping up
because of the way I put
25 that, I guess--I would
like to go on and have Dr.
199
1 Van Blerkom give his
talk and then what I think we
2 need to do is stop and
talk a little bit about what
3 does this tell us now
about mitochondria and how
4 this specifically
relates back to safety and other
5 issues with respect to
ooplasm transfer.
6 Mitochondrial
Function and Inheritance Patterns
7 in
Early Human Embryos
8 DR. VAN
BLERKOM: Thank you very much.
9 Let's see if I can put
a number of different
10 aspects of human development
and mitochondrial
11 function, other than
necessarily respiratory
12 function, in the
context of what this is all about,
13 which has to do with
cytoplasmic transfer. So, I
14 would like to talk a
little bit about the oocyte,
15 since we are really
dealing with the human, and the
16 types of information
that we can gather from
17 available studies on
the behavior of oocytes and
18 their biology.
19 In the human
this is what we deal with
20 initially in the IVF
lab, which is the mast cells,
21 the cells surrounding
cell structure, about 100
22 microns in diameter,
which is the oocyte. You can
23 see it right
here. What we know now from a fairly
24 substantial amount of
biochemical and physiological
25 studies is that, in
large measure, the potential of
200
1 this oocyte that is
here, its developmental
2 competence is largely
determined by factors that
3 have occurred before
this egg has even been
4 ovulated. So, influences to which this oocyte is
5 exposed in the
follicle during oogenesis actually
6 largely determine its
competency after
7 fertilization. We know this from studies of
8 biochemistry on
follicles, the physiology of blood
9 flow, some of which
have been used as predictors of
10 oocyte competence in
trying to select oocytes such
11 as these from many
that may be retrieved from an
12 IVF procedure.
13 The next
slide shows a picture of an
14 oocyte, and here is
the problem. I mean, when you
15 look at this egg here,
this human oocyte it is
16 normal in
appearance. It has a polar body and
17 everything else that
it should have. Most eggs
18 look equivalent but
their potential is different.
19 We know that some of
these eggs will be competent
20 to go on and divide
normally and implant. Others
21 that look the same
don't. This is the notion of
22 why you might want to
rescue the cytoplasm, or
23 there may be a
cytoplasmic defect of some sort in
24 these eggs that render
them incompetent.
25 One of the
things that Jacques Cohen
201
1 mentioned, of course,
is the fact that with
2 increased maternal age
the frequency of aneuploidy,
3 the frequency of
chromosomal dysfunctions, as well
4 as dysfunctions in the
organization of the spindle
5 to which these
chromosomes are attached actually
6 increases
substantially. So, a large number of
7 oocytes that exist in
women of advanced
8 reproductive age will
not be rescued by any means
9 because the
chromosomal abnormalities, and
10 spindles, structural
abnormalities exist and they
11 will not be fixable.
12 But in other
cases, especially for eggs of
13 older women, they look
entirely normal and they
14 really are
indistinguishable from oocytes of
15 younger women. But in large measure, whatever has
16 happened prior to this
egg meeting sperm, which
17 this particular egg
has not, things have happened
18 to this egg which
largely determine its competence,
19 and it is the question
of whether cytoplasm
20 transfer or other
procedures actually will be able
21 to rescue whatever
insults have been imposed on an
22 egg prior to its
meeting with the sperm.
23 This slide
just shows examples of an egg.
24 Here is a two-cell
embryo, starting off perfectly
25 normal except this has
multiple nuclei. One of the
202
1 features that might be
of interest to some in this
2 case, because the
issues of chromosomal segregation
3 and additional
chromosomal additions from
4 cytoplasmic transfer
came up, is the fact that the
5 early human embryo,
especially at the one- and
6 two-cell stage, has
unique capacity actually to
7 encapsulate individual
chromosomes in a nuclear
8 membrane. So, you can get multiple nuclei that
9 occur in these
embryos, some of which you can show
10 have one or two
chromosomes and others have more,
11 but these eggs tend to
be developmentally lethal.
12 So the ectopic
transmission of the chromosome may
13 or may not be an
important issue in cytoplasmic
14 transfer.
15 This slide
shows an example of an embryo
16 which, as you have
heard, is fragmented. You can
17 see some fragments
here. The severity differs
18 between embryos within
the same cohort so you can
19 have 12 or 15
embryos. Some of them have much more
20 extensive
fragmentation, some have none. So, it
is
21 an embryo-specific
event. Some patients have all
22 their embryos fragment
like this, which is
23 relatively rare, but
it is common to see
24 fragmentation of this
sort.
25 The problem
is, is this rescuable? Is it
203
1 a problem in terms of
the ability of the embryo to
2 implant? Here again, as Jacques mentioned and
3 others have shown, in
fact the fragmentation
4 patterns seen at a
static image such as a four-cell
5 stage can change.
6 So, embryos
that had this fragmentation
7 that looked relatively
severe early, in fact, go to
8 the blastocyst and
hatch and, in fact, implant. I
9 don't show baby
pictures but I do show embryo
10 pictures, and this is
a little girl that was born
11 about two years ago.
12 So, we have
the situation where we can see
13 dysmorphologies and
some may be of clinical
14 significance and
others are not. There is recent
15 work that shows that
some types of patterns of
16 fragmentation are
transient; that they exist in one
17 stage and later on in
development seem to
18 disappear. So, it is not clear whether subjective
19 criteria looking at
embryos is actually predictive
20 of competence. In some cases, obviously, if there
21 are no cells left that
is a problem.
22 So, you have
to look in terms of sort of
23 the molecular
mechanisms that take place in eggs
24 where their competence
may be affected by
25 influences that they
have experienced.
204
1 This slide
tells us a little something
2 about eggs in terms of
mitochondria. What you see
3 here is a pronuclear
human egg. These are the two
4 nuclei and these
little dots here are mitochondria.
5 I always grew up with
the notion that, in fact, the
6 number of mitochondria
in human eggs was about
7 150,000, although that
is not based on any
8 morphometric analysis
but that is about the right
9 number. All these dots here are, in fact, what we
10 are talking about,
mitochondria.
11 One of the
interesting things about
12 mitochondria both in
the human and in the mouse,
13 and in other systems
as well, is the fact that
14 their distribution is
not static, that during
15 different stages of
oocyte maturation, especially
16 before the egg comes
out of the follicle as well as
17 during embryogenesis,
there is a lot of spatial
18 remodeling of the
cytoplasm. These mitochondria
19 can move around and
have different locations and
20 different positions
based on what the cell is
21 doing.
22 If we look
at this slide, it gives you an
23 example of
mitochondria in human at the electron
24 microscope level. These guys here, the little dots
25 are at the surface of
the cell. It is upside down
205
1 but here are the
mitochondria. They are fairly
2 unusual when compared
to somatic cell mitochondria.
3 These are relatively
undeveloped. They are not in
4 a dormant state but
developmentally and in terms of
5 their differentiation
they are in a more primitive
6 state. But they do move around. During oogenesis
7 for example, in the
mouse and other rodents they
8 migrate around the
nucleus. You can barely see
9 that here but they do.
They form interesting
10 patterns that extend
from the plasma membrane down
11 to the nuclear
membrane, shown here, almost arrays
12 which we and others
have suggested may be important
13 in certain signal
transduction pathways. So, they
14 are unusual. Their distribution is not static, and
15 they undergo
remodeling as the embryo and oocyte
16 progress.
17 This slide
shows this a little more here,
18 mitochondria at higher
magnifications, a two-cell
19 embryo in the
human. Their spherical structure is
20 about half a micron in
diameter, and they remain
21 this way in a pretty
undeveloped state until fairly
22 late in the
pre-implantation period as the embryo
23 becomes a
blastocyst. Some of these then will
24 start to change into
the more orthodox
25 configuration that one
sees in somatic cells, but
206
1 these are really
unique structures.
2 This slide
just shows some rearrangements
3 that occur fairly
rapidly during oocyte maturation.
4 This is a mouse oocyte
that is stained, the
5 mitochondria are
stained with a mitochondrial
6 specific fluorescent
probe, and what happens is
7 that as the oocyte
matures, in this case in vitro,
8 mitochondria
translocate and move around towards
9 the center of the cell
around the nuclear region to
10 form a very compact
structure here. Then, during
11 the first miosis they
start to redistribute
12 themselves and they go
back to a more or less
13 uniform distribution
at metaphase II, which is when
14 the oocyte is
ovulated.
15 These are
dynamic structures. They are
16 dynamic in their
orientation and organization, and
17 they undergo spatial
remodeling as eggs and embryos
18 divide. This is maybe actually an important
19 feature in
determinants of the oocyte's competence
20 while the oocyte is
still in the ovary. In other
21 words, how these
organelles are located and
22 distributed may
actually be fairly important.
23 Their
distribution, shown in this slide,
24 is directed by
microtubules in many species, in
25 this case the mouse,
and you can see this is the
207
1 central region where
chromosomes are maturing,
2 oocytes are
forming. These are mitochondria that
3 have translocated from
around the cytoplasm towards
4 this rim or ring of
mitochondria around the nuclear
5 region. Here are microtubules and the
6 mitochondria, we
think, migrate as in other cells
7 and are translocated
along microtubular paths. So,
8 the organization of
the cytoplasm in terms of its
9 microtubular
organization may, in fact, be a very
10 important determinant
of how mitochondria are
11 distributed, and
whether the distribution of
12 mitochondria in space
and time, in fact, turns out
13 to be determinant of
competence.
14 This slide
shows another example of
15 presumed mitochondrial
function, and this has to do
16 with energy. We have heard, and it is true, that
17 energy may not be a
critical component of
18 competence because it
is clear that while you have
19 mutations in
respiratory mitochondria the embryos
20 develop quite
normally, otherwise they wouldn't be
21 individuals that carry
this particular respiratory
22 mutations in their
mitochondria.
23 In this type
of experiment, what we did in
24 the mouse was to knock
down mitochondrial
25 respiration
substantially and we found that you
208
1 could reduce mitochondrial
respiration by about 60
2 percent and still get
the eggs to mature normally.
3 They fertilize in
vitro, but what is interesting
4 about this particular
experiment is the fact that
5 when these embryos
reach pre-implantation stages
6 they start to die
off. This may or may not be a
7 mitochondrial
effect. It may be a downstream toxic
8 effect of this
treatment which was done days before
9 at the oocyte
level. But the point is that we were
10 able to establish here
that, in fact, there was a
11 downstream consequence
during embryogenesis, early
12 embryogenesis of
knocking down respiration at the
13 beginnings of
maturation in vitro which is, in this
14 case the germinal
vesicle stage.
15 This
experiment showed that at zero hours
16 in culture knocking
down mitochondrial respiration
17 actually had no effect
on maturation, which is what
18 would occur in the
ovary prior to ovulation,
19 fertilization cleavage
but did progressively have
20 effects on the
embryo's ability to develop to the
21 blastocyst stage and
implant. So, it was an effect
22 that was actually seen
four or five days later.
23 In the case
of the human, one of the
24 proposed effects of
mitochondria and why would
25 mitochondrial transfer
or cytoplasmic transfer if
209
1 it involves
mitochondria be beneficial? One is ATP
2 generation during
pre-compaction stages seems to be
3 respiratorily driven
rather than driven by
4 glycolysis. So, the early stages seem to be
5 requiring some level
of mitochondrial input. It is
6 not clear in the human
whether glycolysis in the
7 presence of
mitochondrial defects that affect
8 respiration can be
up-regulated to supply enough
9 ATP.
10 Of course,
mitochondrial replication
11 begins after
implantation. So the putative effects
12 of mitochondrial
dysfunctions that have been
13 suggested, not proven
yet but suggested for early
14 human development
which may be rescuable is
15 cytochrome C release
if perhaps the mitochondria
16 are damaged resulting
in apoptosis; reactive oxygen
17 species generation
which may be a toxic effect from
18 mitochondrial
dysfunction of some sort that hasn't
19 been identified; or
low ATP production from
20 metabolically
incompetent mitochondria. These have
21 been proposed but not
clearly identified.
22 This slide
suggests something that is
23 really quite
interesting. This asks the basic
24 question. As I said, I always grew up with the
25 notion that there were
about 150,000 mitochondria
210
1 and a number of years
ago we approached this
2 problem for actually
completely different reasons,
3 looking at the
question of how many mitochondrial
4 DNA copies were
present and we looked at a
5 particular
mitochondrial gene at that time using
6 PCR, and we had quite
a few oocytes from gift
7 procedures that were
left over. One of the things
8 that we saw and tried
to quantitate is that the
9 number of copies of
this participant mitochondrial
10 gene, in fact, ranged
from about 30,000 upwards
11 to--I don't remember
the actual number but
12 something like 400,000
or 500,000. We were seeing
13 variations in the
number of mitochondrial DNA
14 copies per oocyte
within the same patient.
15 In that
particular situation, what we were
16 seeing is almost an
order of magnitude difference
17 in the number of
mitochondrial DNA copies in
18 oocytes from the same
patient. We never did
19 anything with this
data because, actually, I simply
20 didn't believe
it. I didn't believe that you could
21 get that variability.
22 But recently
work has come out from a
23 number of groups,
including Jacques Cohen and
24 others, who have
looked at the number of
25 mitochondrial DNA
copies, and the number is about
211
1 20,000 to over
600,000, 700,000. Now, does that
2 mean that an oocyte
that looks the same, that you
3 cannot distinguish at
the light microscope level,
4 one from the other,
that in one case you have
5 20,000 mitochondria if
there is one mitochondrial
6 DNA copy per egg all
the way up to 800,000? Which
7 is a problem because
if that is the case, then if
8 there is one
mitochondrial DNA copy per
9 mitochondria you are
dealing with eggs that look
10 identical at the light
microscope level from the
11 same patient, whether
it is a patient or a donor,
12 where the number of
mitochondria can differ by an
13 order of magnitude?
14 If that is
the case, then going into an
15 egg with a pipet and
removing cytoplasm could be
16 problematic because
you cannot make the assumption
17 that the number of
mitochondria that are being
18 transferred are the
same. In other words, from egg
19 to egg or from patient
to patient. That is a real
20 issue and that has to
be addressed.
21 So, it looks
like the number of
22 mitochondria, in fact,
seem to vary, at least
23 mitochondrial DNA copy
number almost by an order of
24 magnitude and that is
not predictable by any
25 morphology or by any
light microscopic inspection.
212
1 So, this is a problem,
potentially. It is
2 surprising in the
human, but it may not be so
3 surprising as I will
show you in the next slide.
4 This slide basically
shows a picture of an
5 egg, and this is just
stained for mitochondria and,
6 again, here we see
some interesting differences.
7 In this particular
egg, and these eggs are from the
8 same patient, pretty
much the fluorescence is
9 uniformly distributed,
very little in this case in
10 the polar body but
pretty well uniformly
11 distributed, and we
can quantitate and do all sorts
12 of interesting
measurements about the fluorescence
13 intensity and
correlate this with mitochondrial
14 numbers, the point
being that here is one egg that
15 is stained.
16 The next egg
from that same patient shows
17 something a little bit
different. This is not an
18 artifact of the
procedure or the staining. These
19 are live eggs. What has happened here is that, in
20 fact, there are
regions of this particular
21 cytoplasm where
mitochondria are absent. This is
22 something that we
consistently see looking at eggs,
23 that you have regional
differentiation and regional
24 specialization of
mitochondrial distributions that
25 are not predicted by
any other means, other than
213
1 this. So, you cannot say that going into this
2 particular region of
the cytoplasm will produce an
3 equivalent number
going into this region of the
4 cytoplasm. So, now we have the further complexity
5 of having perhaps a
difference in an order of
6 magnitude or certainly mitochondrial DNA
numbers
7 and now we have
regional specializations in terms
8 of distribution within
the cytoplasm that is not
9 predicted by just
looking at morphology.
10 This slide
shows another example of this
11 where, in fact, the
relative fluorescence intensity
12 is quite reduced. So, there may be something to
13 correlating
fluorescence intensity by this method
14 and mitochondrial DNA
numbers, except that in order
15 to do that you have to
destroy the egg, which means
16 it is not very useful
other than for experimental
17 purposes.
18 This slide
shows something about energy
19 distributions in human
eggs. This is some old data
20 that we published a
number of years ago. It simply
21 asks a basic question,
what is the ATP content of
22 eggs in the same
cohort? A very simple-minded
23 question. Just look at the distribution. It is
24 quite remarkable. These are eggs that were gotten
25 by stimulation for IVF
in the same way we normally
214
1 do it, and the
distribution was over an order of
2 magnitude. Again, this was one of these puzzling
3 findings, except that
in terms of outcome, when we
4 had eggs that were
left over, excess donated, that
5 were in the high range
of ATP content, those tended
6 to be the women that
got pregnant from embryos that
7 were transferred in their
cycles. Those that had a
8 preponderance of low
ATP content eggs, when we
9 transferred their
embryos, even though they were
10 morphologically
identical to ones from high ATP
11 cohorts, in fact they
rarely got pregnant.
12 So, here you have a spectrum of an order
13 of magnitude
difference in ATP content, and both
14 differences within
cohorts and between cohorts of
15 patients. So, in this case we now have the
16 complexity of saying
we now know that not only do
17 we have a huge
variability in mitochondrial DNA
18 content, we may have a
mitochondrial numbers
19 variability in terms
of how actual mitochondria are
20 in an egg, which is
not detectable just by looking
21 at it, and now we have
energy differences that may
22 be related either to
mitochondria numbers or to
23 something else that is
going on in these particular
24 cells.
25 So, it is
not just simple to say that, in
215
1 fact, when you have a
mitochondrial basis for
2 certain types of
infertility that, in fact, it is
3 related strictly to
mitochondria because there are
4 too many complex,
confounding issues with
5 mitochondria alone
that are important.
6 This slide
just sort of summarizes this.
7 The size of the
mitochondrial complement, how many
8 mitochondria really
are there? We really don't
9 know how mitochondria
there are in human oocyte.
10 The variability in
mitochondrial DNA content is
11 important, but how
does it relate to the size of
12 the complement? And, is the size of the complement
13 actually
important? Differential spatial
14 distribution at the
pronuclear state, and I will
15 talk a little bit
about that, and disproportionate
16 inheritance during
cleavage, which is another issue
17 in terms of how we
understand the relationship
18 between mitochondria,
if any, and development.
19 This is
shown on this slide. I think I
20 will just pass this
one up. Here, we started
21 looking at how
mitochondria are spatial distributed
22 within the egg and in
the early embryo. This is
23 one of the earlier pictures
that we have seen from
24 looking at an analysis
of the mitochondrial
25 distribution. Here are the two pronuclei, one here
216
1 and one there. Here is the mitochondria around it.
2 What you see here is
the relative intensity of how
3 many mitochondria are
present, but what is
4 particularly
interesting about this guy is the fact
5 that the mitochondria
are asymmetrically
6 distributed in the pronuclear
stage. This is just
7 before cell division.
8 So, we
followed this along in quite a few
9 embryos from the
pronuclear stage onward. I will
10 just summarize the
results. This basically says
11 that you have symmetrical
and asymmetrical
12 distributions. Here are mitochondria around the
13 pronuclei from the
one-cell stage, in a cross
14 section. The point being that the segregation, at
15 least the inheritance
of the mitochondria at the
16 one-cell stage, the
pattern or the spatial
17 distribution at the
one-cell stage determines in
18 large measure the
proportion of mitochondria that
19 are distributed at the
first cell division in the
20 human.
21 So, we
follow this along and we see
22 embryos that have
fairly good and equivalent
23 segregation, others
where the segregation is
24 disproportionate. We can do this both by looking
25 at mitochondrial DNA
copy numbers as well as by
217
1 metabolism.
2 Just to show
you some examples of that,
3 here you have
relatively unusual segregation.
4 Again, all of these
were first examined at the
5 pronuclear stage, the
one-cell stage, and then
6 subsequently. What we found is that you can have
7 different
distributions. For example, a normal
8 appearing embryo,
absolutely normal appearing, can
9 have some cells where
you have relatively high,
10 relatively moderate
and relatively low inheritance.
11 We can, again,
quantify this in a number of ways
12 reflect the intensity
of fluorescence. At the
13 eight-cell stage
in perfectly normal embryo you
14 have some cells that have
relatively few
15 mitochondria, others
that have inherited quite a
16 few.
17 The
consequence of this is that cells that
18 have
under-representation of mitochondria tend to
19 die. They tend to divide more slowly, which may
be
20 what Jacques Cohen
described as the slowly dividing
21 embryos but,
nevertheless, if there are enough
22 cells that have
inherited a fairly reasonable
23 amount or close to
normal amounts, the embryo is
24 still competent.
25 So here,
just the organization of
218
1 mitochondria and their
distribution can have
2 profound effects on
embryo development and
3 competence, and that
is shown on this slide, where
4 you have, for example,
blastomeres of an eight-cell
5 stage, where you have
mitochondria that are
6 relatively evenly
distributed. So, here we have
7 relatively normal,
even distribution.
8 This slide
shows examples where that
9 distribution actually
is quite asymmetric, again,
10 traceable back to the
one-cell stage leaving
11 several cells that are
deficient. These cells
12 eventually lyse and
disappear. Other cells that
13 are deficient, such as
this one, simply don't
14 divide again and
remain in that position.
15 So, not only
do we have the situation
16 where we have
differences in mitochondrial number
17 initially present in
the oocyte, but now we also
18 have the complexity of
how these mitochondria are
19 distributed at cell
division, which is not
20 necessarily
uniform. It is not an equivalent
21 distribution.
22 This slide
just simply shows the basis of
23 this, and we think a
lot of this has to do with
24 microtubules. These are mitochondria that you can
25 see. Most eggs and embryos will slide along
219
1 microtubular
tracks. It is the position of the
2 microtubules and their
organization, both at the
3 one-cell level and
multi-cell level, that we think
4 determines the
proportion or uniqueness of
5 segregation whether it
is even or disproportionate
6 among blastomeres.
7 This slide
is an example of what is called
8 a central zonal
defect. What has happened here is
9 that you normally see
mitochondrial clustering
10 around
microtubules. In this case there are no
11 microtubules because
he has a central zonal defect
12 and there is no
migration of the mitochondria.
13 This comes
to another point, that I will
14 end with, and that has
to do with the notion of
15 cytoplasmic
transfer. We have talked about and
16 published work on
mitochondrial transfusions, going
17 from one oocyte to
another and I just want to show
18 you some of the
complications that come in with
19 this type of approach
to cytoplasmic transfer,
20 something that needs
also to be considered.
21 In this
method what we have done, we have
22 segregated pretty much
all the mitochondria into
23 one compartment. This was an original oocyte where
24 you can see one
compartment here. This contains
25 DNA and here are the
mitochondria.
220
1 This slide
shows a different method. Here
2 is a cytoblast. Here is the nucleoblast which is
3 very, very efficient
in mitochondria. This is very
4 heavy. So, we did a number of experiments, taking
5 by micropipet,
mitochondria from this enriched
6 fraction and asking a
very simple question, what
7 happened to it.
8 That is shown
in the next series of
9 slides. Here what you see is the case of putting
10 mitochondria that are
labeled into a germinal
11 vesicle stage oocyte,
and this cloud material,
12 here, is about five to
ten hours after mitochondria
13 were injected as a
bolus, right around here. This
14 is stained both for
mitochondria and nuclear DNA.
15 This is the nucleus of
the germinal vesicle and
16 this was, with think,
the injected mitochondria.
17 This is
shown in other slides. This slide
18 shows different
variations. Here is an oocyte
19 injected at an earlier
stage of maturation, after
20 the germinal
vesicle. These are the labeled
21 mitochondria and, in
fact, some of those
22 mitochondria have
gotten quite heavily into the
23 first polar body. So, this shows that, yes, you
24 can inject
mitochondria and many hours later you
25 can detect them and
they seem to be pretty well
221
1 segregated or at least
spatially oriented in a sort
2 of uniform manner,
except it again is egg specific.
3 So, if we
look at this slide, it just
4 shows another example
where mitochondria were
5 placed in the center
of the egg. These are stained
6 mitochondria so the
resident mitochondria are not
7 visible. In this case, here is a polar body but
8 there was virtually no
detectable segregation of
9 mitochondria into this
polar body. So, sometimes
10 they are lost;
sometimes they are not. But in most
11 cases they seem to
sort of evenly distribute when
12 injected early in the
maturation phase, that is,
13 well before the time
that we would consider doing
14 this in the human,
which is after ovulation where
15 the egg is mature.
16 Now, if we
inject mature eggs, here is the
17 issue. These are
metaphase II eggs. In this case,
18 what has been done is
to inject mitochondria in
19 different places,
here, here and here, and watch
20 what happens. In fact, in some cases the
21 mitochondria simply
stay in one position. There is
22 no spatial remodeling
or redistribution. If we
23 activate these eggs,
not by fertilization but so
24 that they divide, in
fact, the segregation is
25 entirely
asymmetric. One cell will have a fairly
222
1 substantial,
disproportionately high distribution
2 of the injected
mitochondria, others will not.
3 Here is
another example of this. Here you
4 can see three zones of
mitochondria that were
5 injected at the
metaphase II stage and they stayed
6 in place. They did not move in this particular
7 egg.
8 This shows
another example where actually
9 they did move. Here we put mitochondria in the
10 center and a little
bit later there were, in fact,
11 a lot in the center
but they had actually migrated
12 to the cortex of the
egg as well.
13 This slide
shows another pattern where
14 they were injected in
the subcortical location and
15 pretty much stayed
there. Again, when you activate
16 these eggs you get
unequal segregation. We have
17 not yet seen in any of
our eggs that we have
18 examined by this
method of injection equivalent
19 segregation. It is all asymmetrical, which is a
20 problem in terms of
how mitochondria may, in fact,
21 find their way into one-cell
lineage or placenta or
22 perhaps different
tissues in the fetus.
23 I just want
to end, if I have two more
24 minutes, and I just
want to talk about one
25 potential other
function of mitochondria early in
223
1 development that has
very little to do with
2 metabolism. This has to do with the notion of
3 involvement in calcium
signaling or in ionic
4 signaling.
5 This is a human
egg that is stained with a
6 probe that picks up
mitochondria that are high
7 polarized. These are mitochondria that have a high
8 membrane
potential. We think these are actively
9 involved in other
cells in calcium signaling. What
10 you are seeing here
are these little dots or the
11 high polarized
mitochondria. They are at the
12 cortex. What we think happens is that at
13 fertilization these
mitochondria participate in an
14 important way in
calcium modulation.
15 Shown in
this slide is that when we
16 actually activate
these eggs, in fact you get a
17 very early calcium
discharge which we have now been
18 able to show comes
from those mitochondria. We
19 think this discharge
is actually very important in
20 terms of subsequent
signal transduction pathways
21 that occur later on in
development, which are
22 required for normal
gene activation and normal
23 development.
24 This slide
shows an example--well, you
25 can't see it but there
are very few asymmetric high
224
1 polarized
mitochondria. When we activate this
egg,
2 we see the following,
which simply shows that, in
3 fact, the signaling is
restricted to one part of
4 the egg.
5 This slide
shows where, in fact, there are
6 no detectable, in the
egg, high polarized
7 mitochondria. They are only found in the polar
8 body. When we activate these eggs we get nothing.
9 So, in
addition to metabolic and in
10 addition to other
functions that these mitochondria
11 may have, they also
appear to be involved in early
12 events in calcium
signaling which we think actually
13 turn out to be
important in setting up the right
14 signaling transduction
pathways in the cytoplasm as
15 the egg and embryo
develops. It is an influence on
16 the normality of
development. So, there are a
17 number of different
functions that these organelles
18 are involved in, other
perhaps than metabolic,
19 which are important in
competence determination.
20 Thank you.
21
Question and Answer
22 DR.
SALOMON: Thank you very much for a
23 very interesting
topic. We should have a
24 discussion of sort of
mitochondria per se for a few
25 minutes. As a scientist, I have fifty questions
225
1 here that are just
about mitochondria, but you
2 don't need to waste
your valuable time answering
3 those. It is obviously a fascinating area.
4 There are a
number of questions that
5 specifically relate to
the issues on the table
6 today. So, just to kind of start, one of the
7 things I heard was
that this is pretty safe because
8 there is a very high
threshold for dysfunctional
9 mitochondria and that
would be a safety feature. I
10 am just trying to get
little key things here, but
11 that is something I
got.
12 DR.
SHOUBRIDGE: Yes, I think that is
13 true. The other safety feature in the animal
14 experiments that we
have, we really haven't seen
15 any evidence that the
animals are sick in any way.
16 We haven't done
careful studies in
17 histopathological
things, behavioral tests or
18 anything, but we have
had this colony since 1995, a
19 colony of
heteroplasmic animals, and done all these
20 kinds of different
genetic experiments and
21 different back crosses
or half a dozen other
22 nuclear backgrounds
and we have really never seen
23 anything unusual. I mean, we haven't been looking
24 for it either so we
haven't done a careful analysis
25 but the mice look
pretty normal.
226
1 DR.
SALOMON: Good.
2 DR.
CASPER: From the other point of view
3 then, it was suggested
earlier this morning that
4 you might be able to
treat mitochondrial diseases
5 by mitochondrial
transfer. In view of the
6 stochastic segregation
that happens, is that
7 possible to actually
happen from generation to
8 generation, or would
it only be feasible in the
9 actual injected
offspring?
10 DR.
SHOUBRIDGE: I am not quite sure I
11 understand the
question. The transmission is
12 stochastic so if you
look, for instance, at the
13 distribution of mutant
mitochondrial DNA in the
14 ovary of the woman
where about 50 or 60 oocytes are
15 available, some of
them have no mitochondrial DNA
16 mutations at all. So, in that case, I think if you
17 were going to treat
the disease, the best option
18 would be to look for
an oocyte that didn't have any
19 mitochondrial DNA
mutations at all. If you were to
20 completely remove that
cytoplasm and then put in
21 donor cytoplasm, the
prediction would be that to
22 the extent that you
left the recipient cytoplasm in
23 there you would get
the same kind of stochastic
24 transmission to the
next generation. But having
25 taken out most of it,
the chances are that the
227
1 child, if it developed
in that egg, would have
2 mostly donor
mitochondrial DNA and not very many
3 from mom, but in the
next generation in the female
4 that would segregate.
5 DR.
CASPER: In other words, if you had an
6 embryo that would be
heteroplasmic with a mutation
7 as well as normal
mitochondrial DNA, you could
8 change the threshold
by putting in more normal
9 mitochondria. Is that right?
10 DR.
SHOUBRIDGE: Probably, yes. It is
11 simply stochastic; it
is a numbers game so you can
12 treat this as a bowl
of marbles, black and white
13 marbles. The sample size with determine the rate
14 of segregation. So, if you actually do the
15 statistic, you can
calculate in the mice under a
16 particular model the
effective number of
17 segregating units as
about 200 in the next
18 generation, and you
can figure out, given the
19 sample size of 200,
that you would get
20 distributions like I
showed you.
21 DR.
MULLIGAN: On that point, if you had
22 diseased mitochondria
that behaves like whatever
23 the mouse strain,
wouldn't that be a way of
24 actually promoting
disease because you would
25 actually over a time
course--I mean, if you were so
228
1 unlucky that the
diseased mitochondria also had the
2 same property that is
the property that allows you
3 to selectively reconstitute
cells, wouldn't that be
4 a way to amplify that?
5 DR.
SHOUBRIDGE: Absolutely, and that is
6 probably what happens
in many of the diseases,
7 probably not all, but
there is pretty good evidence
8 for directional increases
in the mutant
9 mitochondrial DNAs in
many diseases, in muscle
10 tissue for
instance. The idea there is that the
11 muscle cell is
continuously reading out the
12 oxidative
phosphorylation capacity. So, if you
13 decided you wanted to
be a marathon runner
14 tomorrow--maybe you
are today, I don't know--but if
15 you wanted to be, you
can up-regulate the number of
16 mitochondria in your
post-mitotic muscle cells. We
17 don't really
understand the nature of the signals
18 that are involved in
that pathway. Then, if you
19 decide you don't want
to run a race it will go
20 down.
21 The thinking
is, at least my thinking on
22 this is that the
selection of that occurs at the
23 level of
organelles. So, some signals are given
at
24 the organelles and
that somehow feeds back to the
25 nucleus. Factors are produced to give you more
229
1 mitochondria. If you now have an organelle that
2 has mutant
mitochondrial DNA the same signals go
3 back. It looks like overworked mitochondria. It
4 looks like it is
running a marathon. In fact, it
5 is just the
mutation. The nucleus doesn't know
6 that. What it does is make more of those guys and
7 so it makes more of
the bad ones. So, there is
8 kind of a positive
feedback loop. It doesn't seem
9 to happen in the
context of every mutation so it
10 isn't a completely general
phenomenon, but it
11 certainly could be a
problem.
12 DR.
MULLIGAN: In this concept of loading
13 with an excess of
certain type, what is the role of
14 the decay of the
existing mitochondria? That is,
15 in principle, there is
a competition and there is a
16 fixed number of
mitochondria that should be in this
17 particular kind of
cell, then whatever determines
18 that number presumably
influences the decay
19 characteristics of the
mitochondria. So, if the
20 cell only usually has
X number and you put in 10 X,
21 presumably for it to
refix itself there has to be
22 loss of some
mitochondria.
23 DR.
SHOUBRIDGE: Nothing, virtually
24 nothing is known about
mitochondrial turnover.
25 DR.
SCHON: Maybe the definition of the
230
1 word mitochondria
needs to be expanded a little.
2 What we do know is
that cells control the mass of
3 mitochondrial
DNA. That is what is being
4 regulated, and it is
controlled rather well. The
5 number of organelles
that enclose those DNAs is
6 what we don't
know. But since it is a completely
7 dynamic system, at two
o'clock in the afternoon you
8 can have a thousand
organelles and at 3:30 you
9 could have two hundred
merely because they are
10 fusing and then they
are repartitioning. So, it
11 may not be that useful
for this discussion to talk
12 about organelles per
se, although I agree 100
13 percent, I think
selection is at the level of the
14 organelle, not at the
level of the DNA.
15 I would like
to amplify a little bit about
16 the tissue
specificity. Bioenergetics probably
17 play some role in the distribution and the
18 amplification but it
can't be everything, and I
19 will give you two
examples.
20 There is a
disease caused by deletions of
21 mitochondrial
DNA. Invariably the deletions pile
22 up, among other places besides muscle, in the
23 choroid plexus of the
brain and in the dentate
24 nucleus of the
cerebellum more than they do in,
25 let's say, in the
epithelium of the ventricles, and
231
1 we have no idea why
that is but there is
2 predilection. There is some signal that is going
3 back and forth that is
operating. It is hard to
4 see how it is
operating at the level of the genome
5 but it is a
genome-specific effect.
6 DR.
SHOUBRIDGE: There is one thing to
7 add. Even though it is true in cells in culture
8 that the regulating
mitochondrial DNA mass seems to
9 be the signal, but
obviously it is not happening in
10 pathology because
there is dysregulation in muscle
11 cells. There can be 50 or 100 times more
12 mitochondrial DNAs in
a segment of a muscle fiber
13 than normal. So, there is some feedback that is
14 due to the presence of
the mutation, presumably,
15 which dysregulates
that.
16 DR.
MULLIGAN: When the DNA replicates,
17 what is the
organelle's status?
18 DR.
SCHON: I am not understanding the
19 question really.
20 DR.
MULLIGAN: Does the DNA replicate
21 within an otherwise
intact organelle? Or, is it
22 compromised or changed
in shape in some fashion?
23 DR.
SCHON: We don't know anything about
24 it. It just happens.
25 DR. SALOMON: Remember, what I want to
232
1 focus you guys on is
what about all of this relates
2 back to the safety and
to the kinds of biological
3 questions that these
guys in the IVF field are
4 going to face in
developing an IND? I think they
5 will be happy to say
that they will screen patient
6 donors for
mitochondrial disease, which they have
7 admitted they haven't
done up until now, but if
8 they do that, then one
is assuming we are
9 transferring normal
mitochondria and, therefore, if
10 they add that one
little piece it seems like they
11 will substantially
remove this as a safety issue,
12 and the fact that
there is this high threshold
13 anyway would seem to
even enhance that.
14 So, that is
all good news for them in
15 terms of safety
issues. What I want to make sure
16 though is, as we go
around here, that there aren't
17 other issues that they
need to address.
18 DR. VAN
BLERKOM: So, maybe the first
19 question is, from what
we have heard so far, is
20 there any evidence
that mitochondria are rescuing
21 these eggs to begin
with?
22 DR.
SALOMON: Right. I was listening to
23 you and the one
question I wrote down, and I am
24 going to put it to you
now--I wrote down first any
25 specific measure of
oocyte mitochondrial function
233
1 that compares good or
normal oocytes to those from
2 infertile
females. You then launched into an ATP
3 content slide and made
one comment, which I thought
4 was at least partially
addressing this, and that is
5 that there seems to be
some correlation. Now, how
6 much of that was hand
waving and how much of that
7 was stuff that would
really stand up to statistical
8 analysis?
9 DR. VAN
BLERKOM: First of all, that was
10 published stuff and it
was actually statistically
11 analyzed so it wasn't
hand waving. But the point
12 is that at the time it
was done there was a
13 relatively limited
number of patients. We had 30
14 or 40 in that
group. But there was no explanation
15 for why those
differences existed because, again,
16 these were analyzed at
the same time, from the same
17 patients, so there was
no culture artifact or
18 anything of that sort.
19 Now, with
the notion that you have
20 differences in
mitochondrial DNA copy numbers that
21 can be an order of
magnitude, the question then
22 comes are the ones
that are the low ATP producers
23 low ATP because they
had, for some reason,
24 inherited a low number
of mitochondria? What the
25 metabolic experiment
showed was that, in fact, to
234
1 make an egg and to
make an embryo you don't need a
2 lot of mitochondria,
functional mitochondria. It
3 may be that at later
stages at some point you do,
4 but the number of
mitochondria that are being
5 injected is so small,
and since they are not
6 replicating, it is
hard to imagine that if you are
7 starting out with an
egg that is below a certain
8 threshold to get a
normal embryo through the first
9 four or five days of
development you need 150,000
10 and you put an extra
10,000 in, it is hard to
11 imagine that that is
going to make a difference.
12 So, numerically it doesn't make sense. I
13 think there are eggs
that fall away in terms of
14 natural developmental
failure, perhaps their
15 inheritance of
mitochondria is very low for
16 whatever reason. But, you see, those are gone
17 anyway. They are not going to be rescued.
18 DR.
SALOMON: Can I follow-up on that?
19 Actually, another
question I wrote down was just
20 what you said. I am still confused here. So, the
21 question I wrote down
is why are there so many
22 mitochondria in an
oocyte, 100,000, as compared to
23 a somatic cell--
24 DR. VAN
BLERKOM: Because they are
25 replicating until
after implantation.
235
1 DR.
SALOMON: So you think they need all
2 these in order to
survive?
3 DR. VAN
BLERKOM: I mean, that is it. I
4 mean, all mitochondria
come from that. All the
5 mitochondria that are
present as the cell divides
6 and parceled out come
from that initial population.
7 DR.
SALOMON: So, you think there has to
8 be this big reservoir
of mitochondria and then, as
9 you go to eight and
twelve and so many cells, you
10 start distributing
around and you get down to what
11 a normal somatic cell
has. So, that is a real
12 simple explanation
like that. Does anyone know
13 what the function of
the 100,000 mitochondria--it
14 is obviously not
100,000 times the ATP reservoir of
15 a somatic cell. Is that right?
16 DR. VAN
BLERKOM: Well, they are involved
17 in APTP product. They seem also to be involved in
18 calcium signaling in
the cell. They also seem to
19 be involved in other
functions that are not
20 necessarily
metabolic. They redistribute
21 themselves, as I
showed, in terms of spatial
22 remodeling, presumably
for ionic purposes or energy
23 purposes, early in the
division. But you are
24 dealing with a very
big cell. I mean, this is a
25 100 micron cell. So, I don't know why whoever put
236
1 in 100,000 or whatever
mitochondria decided that
2 was an important
number, but it probably was an
3 important number in
terms of the reservoir that
4 exists for later
on. It is probably an
5 over-capacity or
redundancy in terms of development
6 because you can knock
out function for a fairly
7 substantial proportion
of those mitochondria and
8 the egg still divides.
9 DR.
SCHON: We shouldn't have tunnel
10 vision here. The mitochondria is not synonymous
11 for ATP
production. There are TCA cycles,
steroid
12 oogenesis, beta
oxidation, amino acid synthesis,
13 and on and on and on,
especially steroids for
14 oocytes. You might need 100,000 just to partition
15 out little molecules
that are important for this
16 egg, and that could be
the end of it, and the ATP
17 goes to sleep because
you don't need it until down
18 the road, and that is
the simple answer.
19 DR.
SALOMON: I guess you guys see where I
20 am going with
this. I am asking the question how
21 can you construct a
rational series of experiments
22 even to test the
hypothesis that injecting the
23 extra mitochondria
from the good eggs into the bad
24 eggs, if you will
allow me to be that simplistic,
25 is doing anything
here? You are injecting 10,000
237
1 to 20,000, but the
point is that if you don't know
2 what it is about the
function of 100,000, what do
3 you measure? So, can we even think of a way to
4 compare these, or is
this really possible right
5 now?
6 DR.
SCHON: I don't think this is the
7 venue for experimental
design. Having said that,
8 if you want to test
whether ATP production had an
9 impact, there is a
line of cells that make no ATP;
10 they are otherwise
normal and you can inject those.
11 It is not an easy
experiment but it can be done. I
12 am not sure what you
would learn from such a thing
13 however, to be
honest. It goes back to the issue
14 of what I said
before. This is a multi-level
15 interacting system and
checking one at a time may
16 or may not give an
answer, and I don't know how to
17 interpret it.
18 DR.
SHOUBRIDGE: There is an experiment
19 you could do but it is
an inhibitor experiment, and
20 they are all
inherently dirty, but there are some
21 dyes that irreversibly
knock out mitochondria, like
22 rhodamine 6G for
instance, so you could treat your
23 extract with rhodamine
6G, kill the mito's and
24 inject the ooplasm and
see if you got the same
25 rescue. So, I mean, it can be approached this way.
238
1 I prefer to do things
genetically because I think
2 it is a little tidier,
but there are ways to do
3 that genetically--not
ways to do that experiment
4 but I can think of a
lot of genetic experiments
5 that would test the
notion that you need that many.
6 I personally
think, and this may be an
7 extreme view, that you
just need them to parcel
8 them out. So, if you look at the mitochondria at
9 the egg level,
morphologically the look like
10 mitochondria in the
rozero cells that Eric was
11 talking about that
have no mitochondrial DNA. They
12 look like inactive or
dead mitochondria and I think
13 it is just a mechanism
to hand them out to the
14 descendants in a
system, for whatever reason, where
15 there is no
mitochondrial replication.
16 DR.
MOOS: A couple of things, just a
17 quick, offhand comment
although we are not going to
18 get into details of
experimental design, if we
19 generate some good
ideas for experiments that we
20 should all be thinking
about, that is a great
21 outcome for this
meeting.
22 I too was
struck by the ATP slide, not
23 necessarily because it
might all by itself be
24 definitive but there
is a hint there perhaps of
25 something that we can
use. So, I am curious
239
1 whether what was done
was simply to measure total
2 ATP content, or
whether P31 NMR to look at energy
3 charge, or techniques
to look at metabolism either
4 have been or might be
considered because the other
5 thing that needs to be
kept in mind is that the
6 oocyte is not a bag of
stuff that is mixed
7 isotopically and,
indeed, there might be extremely
8 rapid turnover of nucleotides
tightly localized in
9 particular regions
that, you know, some
10 high-powered
analytical biochemistry might be used
11 to address. That would then give us the beginnings
12 of something that we
can use to look at the process
13 and keep it
characterized and controlled.
14 DR. VAN
BLERKOM: Can I answer that? That
15 was total ATP
measurements, but you are right about
16
micro-compartmentalization of ATP.
It turns out to
17 be really important in
terms of cell function, and
18 I don't know how you
would actually study that--oh,
19 he does; he is
smarter!
20 The issue is
that you want to keep these
21 things alive and
actually do something to them
22 functionally afterwards
rather than just looking at
23 them in static.
24 DR.
MOOS: Sure. There are two tiers.
25 There is the
investigative tier and that is
240
1 separate from a QA sort
of tier.
2 DR. VAN
BLERKOM: Right.
3 DR.
SALOMON: Dr. Casper?
4 DR.
CASPER: Coming back to the point of
5 why maybe just
injecting 10,000 mitochondria would
6 be helpful, from the
clinical point of view, we
7 have been discussing
patients who make fragmented
8 embryos and trying to
rescue those fragmented
9 embryos, there is some
data that embryo
10 fragmentation may be
related to apoptosis or
11 programmed cell death
sort of issue. We have
12 actually shown that
cell death gene transcription
13 does increase with
increasing embryo fragmentation.
14 Nobody has
mentioned so far that
15 mitochondria actually
have Bcl-2 family member
16 proteins associated
with them. So, one of the
17 issues may well be
that we are injecting enough
18 mitochondria that we
are adding some cell death
19 suppressors, enough to
sort of inhibit or
20 antagonize cell death
genes that could be turned on
21 abnormally in some of
these embryos.
22 DR.
SALOMON: That is really interesting.
23 The problem with that
is that at least our current
24 understanding of this
is that these are occurring
25 at the mitochondrial
cell surface itself. It would
241
1 be an interesting
concept to set up competition
2 with controlling
caspase activation at the native
3 mitochondria by
injecting new mitochondria because
4 these proteins are not
necessarily translocating to
5 new mitochondria in
the process.
6 DR.
CASPER: No, but they wouldn't
7 translocate. You are putting them in right at the
8 time of fertilization,
so very early on in the
9 process. It could be controlled by the nucleus of
10 the cell. You may just have to get the embryo past
11 a certain stage so
mitochondria can replicate and
12 make more of its own
protective proteins.
13 DR. SALOMON: Dr. Naviaux and then Dr.
14 Rao.
15 DR.
NAVIAUX: There is a dynamic interplay
16 in bioenergetics. There are two ways that the cell
17 can produce ATP and,
because of the interplay where
18 we started to get some
understanding of that,
19 actually in the last
century when Pasteur, you
20 know, defined the
suppression of glycolysis by
21 oxygen and later,
around 1927 a biochemist,
22 Crabtree, defined the
suppression of oxidase
23 phosphorylation by
glucose. Traditionally, when
24 you try to measure the
contributions of glycolytic
25 and ox phos pathways
to overall ATP synthesis, you
242
1 do it under laboratory
conditions of ambient
2 oxygen, let's say, at
20 percent. But the female
3 reproductive tract, of
course, is one of the most
4 anaerobic environments
in the human body and low
5 oxygen tension
actually does alter the relative
6 contributions of
bioenergetics available to the
7 egg, particularly
before implantation and the blood
8 supply is established.
9 There are
some early experiments that look
10 at radiolabeled
glucose and its oxidation to either
11 lactate of 14-labeled
CO2, and in early embryos a
12 very large proportion,
exceeding 80 percent of the
13 carbon, can come out
as 14C-labeled lactate as
14 opposed to 14C-labeled
CO2, emphasizing the
15 importance of
glycolysis in bioenergetics of
16 embryos at least at an
early stage.
17 DR.
SALOMON: Dr. Rao and then Dr. Murray.
18 DR.
RAO: I want to try and take off from
19 what you just said
about rather than looking at
20 experiments to see
what we can take home from here
21 in terms of
application, and there are two issues
22 that struck me from
the points you made. Does this
23 tell us anything about
the reproducibility of
24 taking ooplasm at any
site? Should one suggest a
25 particular site, or does it tell you that
there is
243
1 going to be so much
variability that you have no
2 predictive power at
all?
3 The second
thing is does this tell you
4 about selection of the
donor oocyte or the
5 recipient oocyte in
any fashion in terms of doing
6 this?
7 Lastly, if
one assumes that mitochondria
8 can play an important
role in signaling, then does
9 this tell us that even
the small number that you
10 place, because of
patterns of signaling which are
11 critical in terms of
dynamism in this thing, that
12 small number can be
quite critical and, therefore,
13 where you place them
might be very important as
14 well? If anybody can comment on the
15 specifications?
16 DR.
WILLADSEN: I am Steen Willadsen, from
17 St. Barnabas. First of all, I think I should tell
18 you a little bit about
the historical start of
19 this. We weren't concerned about mitochondria
20 specifically, and I
think that in a way we are now
21 barking up the wrong
tree with the wrong dog.
22 Obviously,
this committee is concerned
23 because there is DNA
being transferred. That was
24 not our primary
concern. It would be very easy, I
25 think, to design
experiments where no mitochondria
244
1 were transferred. In fact, we don't even know that
2 the mitochondria that
are in the egg have any
3 particular function at
the time. As was pointed
4 out by one of the
speakers, they are probably
5 useful for making the
egg, which is a very
6 specialized cell. So, I think the real issue with
7 the mitochondria in
this context is are they
8 dangerous and how the
egg otherwise gets along. I
9 think it is wrong to
focus so completely on the
10 mitochondria because
they can very easily be
11 brought out of the
picture. Then, where would the
12 FDA be?
13 The second
thing is that obviously when
14 you look at these
risks, and I think I will say at
15 this point if you look
at the risks, I can only
16 speak from the basis
of the evidence that I have
17 some insight into, the
major risk if you enter as a
18 patient into this
program is that you could get
19 pregnant. That is the major risk. Whether you
20 would like to say that
this because it is a
21 treatment or whether
you say it is because of the
22 place, it is a big
risk if you go into the program
23 because 40 percent of
the patients got pregnant.
24 Thank you.
25 DR.
RAO: Can I respond to that?
245
1 DR.
SALOMON: Okay, but I think what we
2 have to realize here
is that what we are doing
3 right this second is
focusing on the mitochondria.
4 It doesn't mean that
we will end the day focusing
5 on it, it is just that
we are following a
6 discussion of two
very, you know, high level
7 professors telling us
about mitochondria. So, I
8 think it is very
appropriate right this minute to
9 be focusing on the
mitochondria. But I think that
10 to think of this in
context, to be reminded that we
11 have to put it in
context is perfectly fair, and I
12 think we will have to
come back to it because you
13 articulated some of
the issues we are going to have
14 to deal with in about
half an hour. But in that
15 context, it is
okay. I just don't think we have to
16 defend why we are
talking about mitochondria right
17 now. I think that is what we are supposed to be
18 doing.
19 DR. SCHON: This is not really in the
20 realm of safety but I
would just like to bring it
21 to the floor. The transfer of ooplasm means the
22 transfer of
mitochondria right now, unless the
23 protocol is
changed. So, I would like to spend
24 just a couple of
minutes talking about the
25 evolutionary
implications of this, not safety, not
246
1 viability.
2 It comes to
the heart of why nature
3 invented maternal
inheritance in the first place.
4 So, why is that? In fact, nobody really knows but
5 the most reasonable
answer is the same reason why
6 nature invented sex,
and it comes down to something
7 Muller's ratchet which
in economics would be called
8 Gresham's law--all
things being equal, things go
9 from bad to
worse. I think that would be the best
10 way to describe
Muller's ratchet.
11 So, if you
had clonal expansions of DNAs
12 that were going to
their progeny, eventually they
13 would call up
mutations and wipe out that organism
14 in evolutionary
time. So, sex was invented to
15 erase that--well, that
is a little bald statement
16 there. That is part of the reason I think sex was
17 invented, to help
accommodate, to deal with those
18 kinds of mutations.
19 Now, when
you have an organelle that is
20 present not at one or
two copies per cell but at
21 thousands, it is very
difficult to deal with that
22 kind of a problem of
Muller's ratchet where, if a
23 mutation arises, it
just naturally will spread
24 through the
population, as you saw so dramatically.
25 So, what appears to
have happened is that maternal
247
1 inheritance came
around so that when mutations
2 arose you shut them
down. In fact, when we look at
3 pedigrees with real
diseases, first of all, the
4 pedigrees are short,
meaning they go from
5 great-grandmother to
proband and might go one more
6 generation and then,
like a light going out, that
7 pedigree is
extinguished carrying that mutation.
8 That is what is really
going on.
9 That
mutation only passes through the
10 maternal line and goes
nowhere else. So, all
11 mitochondrial
mutations that we study are really
12 only a few hundred
years old, if you will, or less
13 in time. They come on and they go out.
14 So, what does this have to do with
15 ooplasmic
transfer? So, now we are taking
oocytes,
16 ooplasm containing
mitochondrial haplotype A and
17 sticking it into a
recipient cell with
18 mitochondrial
haplotype B. This is lateral genetic
19 transfer. All right?
We haven't eliminated
20 Muller's ratchet but
we haven't made things that
21 much better either
because now you are putting in a
22 new genotype from this
pedigree into a new
23 pedigree. If you do this with one person, two
24 people, ten people, a
hundred people it is probably
25 irrelevant. But if you start doing this with tens
248
1 of thousands of
people--I don't expect this ever to
2 happen at that scale
but it is something just to
3 think about--y are now
transferring mitochondrial
4 genotypes horizontally
through the population that
5 otherwise would never
have been transferred because
6 they all pass
vertically. That is the only point I
7 am trying to
make. I can't quantitate the impact
8 of this, it is just a
fact.
9 DR.
MURRAY: This will be a question for
10 Dr. Van Blerkom. Thanks to both speakers.
11 Fascinating, I have
learned a lot from both
12 presentations. I am going to focus on one thing
13 which we may actually
be able to put aside, but one
14 of the striking things
in your presentation was the
15 information about the
dynamic patterning and
16 remodeling of the
location of mitochondria in the
17 egg. You showed us some slides of how that might
18 affect calcium ion
transport, and the like. Is
19 there any reason to
think that the injection of
20 another 10,000, a
bolus of cytoplasm with 10,000
21 mitochondria in some
particular site in the egg
22 would be either
readily integrated and made to
23 dance the same way as
the native ones, or might
24 there be some
disruption of, say, fine structure of
25 transport structures,
the architecture within the
249
1 cell that might make
it more difficult? One, is
2 this important enough
to worry about? Two, are
3 there ways to sort of
answer that question?
4 DR. VAN
BLERKOM: I don't think I have an
5 answer for that,
except to say that the work we
6 have done with regard
to mitochondrial transfer
7 indicates that you
can't predict how they we dance.
8 In some eggs they will
remain where you place them
9 as the cells divide;
in others there is a more
10 pronounced
distribution. So, that is the level of
11 predictability, which
is a problem.
12 As far as
interrupting, I don't get the
13 sense that the amount
of cytoplasm that is put in
14 and the number of
mitochondria that are transferred
15 is actually
significant in terms of disrupting any
16 of the normal cell
functions or even contributing
17 to them, for that matter.
18 DR.
MURRAY: You don't think it makes a
19 difference?
20 DR. VAN
BLERKOM: I don't think it makes a
21 difference.
22 DR.
MULLIGAN: Is there anything that
23 aggregates the
mitochondria or keeps them in any
24 constrained fashion
that, upon transfer--this is
25 kind of a similar
question to what Tom was asking,
250
1 that is, some
cytoskeletal structure that you
2 transfer like a
precipitative mitochondria?
3 DR. VAN
BLERKOM: I think Jacques actually
4 alluded to this when
he spoke about differences in
5 the cytoplasmic
texture, and we have to think in
6 terms of the human and
our experience, those of us
7 who have experience in
working with human eggs, is
8 that even with the
standard ICSI procedure eggs
9 differ substantially
in how they receive sperm, how
10 the cytoplasm is
withdrawn, the viscosity of the
11 cytoplasm, and you can
actually see this as you do
12 it. I have seen this many times. I think the
13 situation that Jacques
has described, where you
14 have different
cytoplasmic textures and you can
15 actually see in his
cytoplasmic transfer studies
16 the cytoplasm that is
injected in some eggs but not
17 in others, I think
indicates why in some cases when
18 you put in a bolus of
mitochondria or a bolus or
19 cytoplasm they remain
fixed in position and in
20 other cases they are
more diffuse. I think you
21 cannot predict
that. I don't think you want to
22 relax the cytoplasm by
treating it with drugs so
23 that you have some
sort of uniform distribution or
24 some controllable
distribution.
25 DR.
MULLIGAN: Can you alter the viscosity
251
1 or whatever you want
to call it--
2 DR. VAN
BLERKOM: In a sense you can relax
3 the cytoplasm. It usually requires treatment with
4 some relaxant drugs
that will relax
5 cytoarchitectural
components. I don't think you
6 want to do that in
clinical IVF. The problem in
7 the cytoplasm
injection is that you have already
8 injected the cytoplasm
and now you discover that,
9 in fact, the recipient
egg has, let's say, a
10 particular viscosity
where the cytoplasm remains
11 intact in one
position. Maybe those type of
12 studies will be useful
to determine whether or not
13 the mitochondria
remain fixed or not as a prelude
14 to a clinical
trial. But they are differences that
15 are egg specific. They are hard to predict and
16 what I tried to
emphasize is that just by looking
17 at an egg you really
can't tell.
18 DR.
SALOMON: Dr. Hursh and then Dr.
19 Sausville. Then what I would like to do is move on
20 to Dr. Knowles, only
because I am just trying to
21 have some time at the
end.
22 DR.
MALTER: Very brief?
23 DR.
SALOMON: Yes, sure.
24 DR.
MALTER: I am Henry Malter, from St.
25 Barnabas. Jonathan, the experience you showed,
252
1 what exactly did you
do? Was that where you were
2 isolating essentially
mitochondria in part of the
3 cytoplasm and taking
it from there?
4 DR. VAN
BLERKOM: The experiments I showed
5 were not cytoplasmic
injections. These were
6 procedure where we
have actually compartmentalized
7 the mitochondria and
then took mitochondria in
8 relatively small
drops, smaller than you would
9 actually use in a
cytoplasmic transfer, and
10 actually deposited it
into the egg. So, those were
11 enriched mitochondrial
fractions.
12 DR.
MALTER: I just wanted to remind of
13 some images that
actually Jacques showed because we
14 have done this as
well. In fact, we have done it
15 with spare human
material and it is essentially
16 duplicating exactly
what is done during the
17 clinical cytoplasmic
transfer material, loading an
18 egg with labeled
mitochondria and injecting them.
19 Those were not
extensive experiments but we never
20 saw that just sitting
in one place. Basically, you
21 showed right after
injection you can see this
22 bolus, this red image
in part of the cytoplasm and
23 then, as development
proceeded, they just
24 essentially seemed to
disperse and it was just
25 variable. You would see it in some blastomeres.
253
1 DR. VAN
BLERKOM: So, these were
2 fertilized eggs after
injection?
3 DR.
MALTER: Yes.
4 DR.
HURSH: This question is for Dr.
5 Shoubridge. You don't feel that heteroplasmy
6 itself is a problem,
but if there was a situation
7 where the mitochondria
became asymmetrically
8 distributed so you had
one, say, organ that was
9 primarily donor
mitochondria could you foresee any
10 problems with that
mitochondria with a disconnect
11 with the nucleus in
any way? Would that be a
12 safety consideration
that we need to be
13 considering?
14 DR.
SHOUBRIDGE: Our data would suggest
15 that it is not a big
problem, but I don't think you
16 can rule it out
because, I mean, what happens
17 biologically is that
every time you have a child,
18 of course, the
father's nuclear DNA is introduced.
19 So, now that nuclear
DNA is introduced to
20 mitochondrial DNA that
it has never seen and the
21 mother's genome has
seen that mitochondrial DNA.
22 So, it is a natural
process for new nuclear genes
23 to be introduced into
mitochondrial DNA genes to
24 dance with them and
they have never danced with
25 them before, to follow
the dancing analogy. But in
254
1 the case of our mice,
of course, that is exactly
2 what we have, we have
complete fixation of a donor
3 genotype in the
liver. In that case it doesn't
4 seem to produce any
particular phenotype that we
5 can recognize but we
haven't done any liver
6 function tests. The mice seem to be pretty normal,
7 but I don't think you
can rule it out.
8 DR.
SAUSVILLE: So, this question's last
9 comment sort of
follows along on that. First of
10 all, I want to thank
both of the speakers this
11 afternoon because I
think they have put, at least
12 for me, a lot of the
biological issues somewhat in
13 greater perspective.
14 But, I
guess, addressing one of the other
15 major concerns that
goes into the IND and, again,
16 this is somewhat to
what Dr. Hursh's question
17 alludes to, is the
issue of safety. I seem to be
18 hearing that if one
looks to safety either from the
19 implications for the
recipient, the organism who
20 receives it, the mouse
experiments don't suggest
21 that there is a
tremendously great effect for
22 having radically
different mitochondrial genomes
23 and, moreover, do
suggest that if there were to be
24 a bad different you
would have to have an enormous
25 amount of penetration
in one participant organ.
255
1 Then, the
comment that you made
2 subsequently is that
if one looks at safety from
3 the standpoint of
evolutionary safety, at one level
4 you could construe
that as an argument that the
5 mechanism is designed
to keep itself safe because
6 it is going to
extinguish itself within a very few
7 generations and you
would have to posit that if
8 this were a threat to
our collective genomes you
9 would have to have a
succession of almost continued
10 maintenance through
some sort of artificial system.
11 So, I guess
quite apart from the issue of
12 whether mitochondria
really do anything for you or
13 whether, indeed, the
cytoplasm does anything for
14 you, my initial
reaction to this is that it is hard
15 to make the case that
the procedure appears unsafe,
16 at least from the standpoint
of mitochondrial
17 related matters.
18 DR.
SALOMON: Yes, I think I was earlier
19 saying the same thing
in another way, that it seems
20 like with the
threshold issue there is a lot of
21 safety.
22 DR. SHOUBRIDGE: I guess the only thing I
23 would add there is
that the slight caution is that
24 because we know there
are mechanisms that increase
25 the proportion of bad
guys in cells from patients
256
1 who have disease, if
you unwittingly put in
2 something from an
individual that is below the
3 threshold you could
select for it in a
4 tissue-specific
way. I think that may be a very
5 small risk but I don't
think it is zero.
6 DR.
MULLIGAN: I think that one issue
7 about mechanism that
is important is that if you
8 really did think that
mitochondria weren't
9 important, by not
having mitochondria in your
10 ooplasm you could,
obviously, reduce whatever risk
11 you otherwise would be
concerned about. So, it is
12 a relevant issue just
because you have wiped out
13 that risk completely
if you didn't have any.
14 DR.
SAUSVILLE: But then that becomes
15 impossible to
investigate in the conventionally
16 clinically oriented
situation since what we have
17 heard is that while,
in an ideal sense, you would
18 parse out precisely
which part of this works, I
19 inferred from the discussion
earlier that that is
20 going to be very
difficult from a practical point
21 of view to ever do in
a meaningful sense
22 clinically.
23 DR.
MULLIGAN: There might be people who
24 don't feel that that
is an important part of the
25 method and would
choose to go down the regulatory
257
1 pathway that wouldn't
make use of mitochondria.
2 DR.
SAUSVILLE: I don't think there is
3 anything that would
prevent that from a regulatory
4 standpoint, but the
issue is whether or not the
5 user community would
actually go down that path. I
6 think that is
uncertain to me from what I have
7 heard.
8 DR. SALOMON: We certainly haven't gotten
9 to where we need to be
by the end of the day, but I
10 think we have made
some progress along that line.
11 Ms. Knowles is going
to talk to us about ethical
12 issues and then, just
to give you the lay of the
13 land, we are going to
do the public comment
14 section, take a break
and come back and really get
15 into the key
questions, and that is when we will
16 have to have it all in
perspective, mitochondrial
17 safety, ooplasm, other
components of the ooplasm
18 and its impact on this
group of scientists and
19 physicians.
20 Ethical
Issues in Human Ooplasm
21
Transfer Experimentation
22 MS.
KNOWLES: Thank you for inviting me to
23 be a part of
this. I have been charged with
24 elucidating the
ethical issues in human ooplasm
25 transfer
experimentation. So, we are going to
step
258
1 back a little bit from
all the mitochondrial data
2 we have been talking
about, and stepping back from
3 the animal models, and
we are looking now at the
4 issue that we started
with today, looking at the
5 experimentation of
ooplasm transfer, that I am
6 going to call OT just
for shorthand, in humans, and
7 looking at some of the
ethical issues.
8 In terms of
context, I just want to
9 highlight that all
medical experimentation takes
10 place in the context of
some risk and some
11 uncertainty. The question, therefore, is what is
12 the threshold of risk
and uncertainty that is
13 acceptable? One way that we can better understand
14 the risk and
uncertainty of OT experimentation is
15 by looking at what I
am calling the knowns and
16 unknowns.
17 So, in terms
of elucidating safety and
18 efficacy concerns, we
are going to say to ourselves
19 what threshold of risk
and uncertainty exists in
20 this context and so
what are the knowns and
21 unknowns. I am going to look at the implications
22 this has not only for
whether it is ethical to
23 proceed with this
technique in women and to create
24 children, but also the
implications for informed
25 consent.
259
1 Considerable
amount of thought, discussion
2 and work has been
devoted to the question of the
3 ethics and science of
both therapies and
4 experiments that
result in inheritable genetic
5 modifications, and I
adopt that term from the AAAS
6 report of 2000. In the time that is allotted to
7 me, I can't do justice
to that work but what I can
8 do is nod to some of
the work and some of the
9 issues that are on the
table when we are talking
10 about inheritable
genetic modifications.
11 Similarly, I don't
actually have time to address
12 the depth of the issue
of what I call the invisible
13 woman, the other woman
who is involved in all of
14 these procedures, the
egg provider.
15 So, it is
extremely important to realize
16 that the implications
of proceeding with OT both in
17 experiments and as a
clinical technique have larger
18 ripple effects which implicate the safety of
the
19 women who undergo the
egg provision, the egg
20 donation as it is
called, to enable this technique
21 to go forward. So, whereas ooplasm transfer is
22 primarily concerned
with transplanting genetic
23 material that is
believed, although we don't know
24 certainly at all, to
not have an impact on
25 phenotypic development
of the embryo, there is a
260
1 likelihood then that
the market for oocytes will be
2 increased and will
pull on women who have not
3 typically been pulled
on for provision of eggs
4 based on their
phenotypic characteristics which
5 aren't going to be, we
assume, as important in this
6 market. So, that has some larger social ripples
7 and ramifications that
we should be thinking about
8 as well.
9 That leads
me to my last area of concern
10 that I am actually not
going to touch on. Given
11 FDA's mandate, I am
not going to address the social
12 and legal
ramifications of this technique but I
13 think it is necessary
to underline the importance
14 these issues have, the
uncertainty that exists
15 where genetic
parenthood is tripartite and the
16 ethical imperative now
to have a broad and
17 multidisciplinary
review of the ethical and
18 scientific
issues. So, somebody needs to be free
19 to deliberate about
these larger ethical issues as
20 well, and I think it
is my responsibility to just
21 outline that.
22 Turning then
to safety and efficacy, we
23 are asking ourselves
what are the unknowns. There
24 are clearly more
unknowns than I have on this list
25 so I am just going to
highlight what I think some
261
1 of the most important
unknowns are. The first is
2 it is not known, and
we have heard this many times
3 so a lot of what I am
going to say is going to be
4 sort of
summarizing--what is not known are the
5 defects that ooplasm
transfer is trying to correct.
6 It is not
known what is doing the work in
7 OT. Although we have concentrated on
mitochondria
8 recently, we have to
remember that we don't
9 actually know what is
doing the work. We don't
10 know whether OT
techniques have an adverse effect
11 on transferred
material. We don't know that. We
12 don't know whether OT
helps actualize abnormal
13 embryos that would not
otherwise be actualized.
14 And, we don't know the
effects on embryos, infants
15 and
toddlers--humans--with heteroplasmy.
Would
16 don't know what its
effects are.
17 So, let's
delve a little bit into that.
18 Our scientific
understanding of why an embryo does
19 not develop is still
incomplete. We heard that a
20 number of different
ways today. We know there are
21 a number of different
factors that may be
22 implicated including
maternal age and including ATP
23 deficiencies. So, let's look at what some of the
24 other factors may be.
25 This is a
partial quotation from The New
262
1 England Journal of
Medicine, March 7, 2002, many
2 factors can lead to
poor embryonic development,
3 including chromosomal
abnormalities, genetic
4 defects, and cellular
abnormalities. Impaired
5 embryonic development
may also be consequence of
6 other problems within
the embryo or in its
7 immediate environment.
8 In the Huang
experiment, in fertility and
9 sterility, October,
1999 it was stated, the reasons
10 for previous
implantation, and this is in
11 describing the failure
of the nine patients in that
12 study, the reasons for
previous implantation
13 failure in these nine
patients are not clear
14 because their oocytes
appeared morphologically
15 normal and the embryo
transferred were of fair
16 quality.
17 This is
complicated by a great variation
18 in the women in each
of the studies, incomplete
19 histories of the
techniques each woman underwent
20 prior to OT, the
number of attempts, the techniques
21 tried after OT and
inclusion and exclusion criteria
22 for the women in each
group. This is complicated
23 by what Dr. Lanzendorf
and her colleagues refer to
24 as the subjective
grading of embryos in vitro
25 performed by various
embryologists, which renders a
263
1 comparison between
patients' previous IVF cycles
2 and treatment cycles
unavailable. So, we know that
3 that information in terms
of comparison is not
4 available to us in
many circumstances.
5 Continuing
with the unknowns, what is
6 doing the work? We don't know this. Since we
7 don't know what is
doing the work and whether in
8 all cases it is the
same beneficial factor, which
9 we can't assume it
necessarily is and we don't even
10 know if the same
beneficial factors are
11 transferred, it is not
actually possible to know
12 whether OT is
clinically indicated in a particular
13 case.
14 I have
shorthanded the citations because I
15 have so many words on
these slides, but I have the
16 citations if you would
like them. The mechanisms
17 involved are still
enigmatic. It remains unclear
18 as to which cellular
components are transferred in
19 the donor
ooplasm. Exact mechanisms and factors
20 that help to rescue
the function of the defective
21 oocytes remain
unknown. It is not yet clear how
22 ooplasm transfer
works. Specialized proteins or
23 messenger RNAs may
direct subsequent cell cycle
24 events. it is also possible that donor
25 mitochondria is
providing the benefit.
264
1 So, can
transfer techniques have an
2 adverse effect on
material that is transferred,
3 transferred
material? Here, of course, we are
4 concerned with the
risks that are implicit in this
5 technique. Interestingly, it seems that all the
6 research of the
clinicians in the protocols that we
7 were provided express
some concern with the source
8 of ooplasm or
cytoplasm used either in their own
9 experiment outcome
used by other in the other
10 experiments. These concerns include the effects of
11 cryopreservation of
the material transferred since
12 that has been studied
and shows that
13 cryopreservation can
have negative impact on
14 oocytes and
embryos. That, obviously, has to be
15 considered.
16 So, let's
look at what they said, because
17 it is still not known
what is being transferred to
18 recipient oocytes, it
cannot be determined if
19 cryopreservation may
have an averse effect on these
20 factors.
21 This is the 3PN protocol and they are
22 commenting on the use
of metaphase II oocytes, one
23 concern we have is the
risk of transferring donor
24 chromosomes, and we
heard about this earlier, from
25 metaphase II oocytes
of donors into the recipient's
265
1 oocytes.
2 We feel
validation is still required to
3 provide absolute proof
that donor nuclear DNA has
4 not been accidentally
transferred. That is
5 referring to the 3PN
protocol.
6 What are the
effects on embryos? Well,
7 the bottom line is we
don't actually know. Let's
8 take a look at what
they said. Even though the use
9 of cytoplasmic
transfer has been employed in
10 several IVF
clinics--this is from the abstract, by
11 the way, of this
report--and pregnancies have
12 resulted, it is not
known definitively whether the
13 physiology of the
early embryo is affected.
14 There may be an improved developmental
15 potential of hybrid
cytoplasm in chromosomally
16 normals as well as
abnormal embryos. So, here we
17 know the following
risk exists with respect to the
18 effect that OT may
have on embryos and that
19 abnormal embryos may
be actualized as well as
20 normal embryos getting
the boost that we talked
21 about.
22 We do know
at this point that ooplasmic
23 transfer can alter the
normal inheritance of
24 mitochondrial DNA
resulting in sustained
25 heteroplasmy
representing both donor and recipient
266
1 mitochondrial
DNA. That is also a quotation.
2 What are the
effects on the embryos,
3 infants and toddlers
with heteroplasmy? And, we
4 are talking about
humans. Well, because little is
5 understood about the
maintenance of mitochondrial
6 heteroplasmy and its
nuclear regulation during
7 human development, the
effects of potentially
8 mixing of two
mitochondrial populations are still
9 being debated. In other words, we don't know.
10 We do know
that mitochondrial heteroplasmy
11 may result in embryos,
approximately 50 percent
12 from my reading that
particular study, of
13 non-viable embryos
used in Barritt's study
14 exhibited this
trait. We also know that two
15 children now exhibit
mitochondrial heteroplasmy,
16 but we don't know what
this means and it is unclear
17 whether all the
children created from ooplasm
18 transfer have been
tested for mitochondrial
19 heteroplasmy. It sounds like, from the first
20 speaker's
presentation, that we know that, in fact,
21 not all the children
that have been created this
22 way have been tested.
23 So, let's
look at what we do know. Well,
24 we know that the
incidence of chromosomal anomalies
25 is higher in this
population than the rate of major
267
1 congenital
abnormalities observed in the natural
2 population. This is a quotation from page 430 of
3 Barritt et al. in the
European Society of Human
4 Reproduction and
Embryology journal.
5 We know that
one 18-month old boy, as Dr.
6 Cohen was mentioning
this morning, has been
7 diagnosed with
PDD. And, we know that the
8 mitochondrial DNA
inheritance is changed in some
9 children resulting in
an inheritable genetic
10 modification.
11 Let's talk
about inheritable genetic
12 modification. I want to say first of all that
13 there has been kind of
an interesting discussion
14 going on in the
literature about whether this is,
15 in fact, a case of
germline genetic modification.
16 I think that is, in
fact, interesting in and of
17 itself, the fact that
there is a lot of energy
18 being spent to make
sure that we are not labeling
19 this a germline
genetic modification. That should
20 be telling us
something. I have seen some very
21 interesting arguments
about why it is not a case of
22 germline genetic
modification, including one that I
23 have mentioned to
several people before, that it
24 can't be considered a
germline genetic modification
25 because is doesn't
pass through males. Well, I am
268
1 not actually going to
discuss that particular
2 argument but the
energy that is being expended
3 should be telling us
something about whether, in
4 fact, it is an
inheritable genetic modification.
5 Well, why
are we concerned about IGMs?
6 Here I have to be
really very concise. This term
7 IGM, inheritable
genetic modification, as I
8 mentioned, I am taking
from the AAAS, the American
9 Association for the
Advancement of Science, their
10 2000 report which
brought together a group of
11 eminent scientists
including gene therapists,
12 ethicists and policy
analysts, and they say the
13 following, they say
essentially due to the
14 transmission of
inheritable genetic modifications,
15 there would need to be
compelling scientific
16 evidence that these
procedures are safe and
17 effective, compelling
scientific evidence. For
18 those techniques that
have foreign material, their
19 stability across
generations would need to be
20 determined based
initially on molecular and animal
21 studies before
proceeding with germline
22 interventions in
humans. It is not yet possible to
23 meet these standards,
nor is it possible to predict
24 when we will be able
to do so. One footnote I
25 should add that was
correctly mentioned earlier, we
269
1 don't know whether,
because the blood only of these
2 children has been
tested, the germ cells have also
3 inherited this
mitochondrial heteroplasmy. But we
4 haven't tested for
that yet because we can't at
5 this point. So, it is important to recognize that
6 that, in fact, is true
but it doesn't mean that
7 this is not
inheritable genetic modification. That
8 is important.
9 They also go on and say the
possibility of
10 genetic problems
occurring as a result of the
11 unintended germline
side effects seems at least as
12 great or greater than
those that might arise from
13 intentional
inheritable genetic modifications which
14 at this time we don't
permit in many, many
15 countries. Why?
Because knowing you were creating
16 an IGM assumes that
you would have safeguards and
17 rigorous monitoring in
place and we know that in
18 this case that is
actually not true because they
19 allegedly didn't think
that they were going to be
20 transmitting genetic
modification.
21 So, those
are the AAAS conclusions.
22 Clearly, we have a
duty to future
23 generations--there is
a lot of theoretical work on
24 this, but we can
intuit that we do have a duty to
25 future generations to
be thinking about what we
270
1 are, in fact, passing on
to them, to be doing it
2 carefully if we are
going to do it.
3 I would note
that there is almost never
4 consensus in the
international community, but there
5 is pretty close to a
consensus in the international
6 community that we
should not be doing research that
7 results in inheritable
genetic modifications. I
8 just want to
highlight, in terms of the
9 international work,
that this would not be
10 permitted in most
countries, this kind of protocol,
11 and in the U.K., which
is arguably the most liberal
12 with respect to embryo
research, they are going to
13 allow some stem cell
protocols that we are not in
14 this country, they
prohibit germline modification,
15 and the House of Lords
stem cell report noted
16 that--they didn't
discuss OT in the context of
17 fertility treatments
at all, but discussed what we
18 were discussing, the
use of a similar procedure
19 with respect to
screening out mitochondrial disease
20 and they said that
very little research has been
21 carried out on this
procedure and it would need
22 extensive testing in
animal models and in human
23 eggs before it could
be used therapeutically in
24 humans. Remember that they are talking about a
25 therapy in a disease,
not fertility, in that
271
1 context.
2 What does
heteroplasmy of this type, the
3 type that we have been
discussing in humans in the
4 two children that we
have been talking about, what
5 does it mean? Well, the bottom line is we don't
6 know. We do know that there are diseases
7 associated with
mitochondrial heteroplasmy. We
8 know that. Yet, there is no reason to consider
9 this mitochondrial DNA
heteroplasmy from this OT
10 protocol as harmful
because it is known to occur
11 naturally in normal
individuals.
12 Well, to be
fair, we don't know if this
13 type of heteroplasmy
resulting from these
14 experiments results in
mitochondrial disease
15 because it doesn't
occur naturally. So, we haven't
16 been able yet to
determine that it is benign. We
17 simply know that this
other type of heteroplasmy
18 can occur in normal
individuals and it can occur
19 and be associated with
disease states as well. So,
20 we cannot say that it
is benign because we don't
21 know. We don't have the information at hand to
22 know. We haven't done the experiments yet to know
23 or the follow-up to
know.
24 We do know
that one child has PDD but we
25 don't know whether
that child actually is
272
1 heteroplasmic or
not. I would like to know that if
2 we have that
information available. I don't think
3 we know that.
4 What
else? Well, since mitochondrial
5 diseases are
associated with heteroplasmy that can
6 be early or late
onset, we cannot know whether this
7 heteroplasmy is benign
until these children grow
8 up. That is a basic conclusion from logic.
9 Limitations
of clinical data, well we
10 heard very candidly
from our speakers, and it is
11 much appreciated, some
limitations of the clinical
12 data. It is very helpful. Small sample sizes;
13 incomplete information
on the women in the
14 experiment for a
number of very legitimate reasons.
15 We don't know
necessarily whether previous
16 procedures are the
reasons for their failure.
17 Incomplete testing of
the children who have been
18 born; and the lack of
long-term follow-up.
19 This is
particularly troubling. There is
20 clearly a need for
long-term monitoring of the
21 children that are born
with a heteroplasmic
22 condition and those
that aren't born with a
23 heteroplasmic
condition. In addition, there is
24 likely going to need
to be extensive follow-up of
25 these children until they have children to
273
1 determine whether, in
fact, we have an inheritable
2 genetic modification
and what happens to it through
3 the generations.
4 This follow-up can, and will
likely be
5 very intrusive
because, as we were hearing on the
6 mouse models, the
mitochondrial segregation is
7 tissue specific and
differs. So, if you are going
8 to do proper follow-up
you would need to take
9 tissue biopsies from
different tissues to
10 understand how the
mitochondria has been
11 differentially
segregated. This, of course, could
12 be extremely
intrusive. Whether one could
13 ethically consent to
this kind of long-term
14 monitoring and
invasive follow-up for a child that
15 is not yet conceived
has to be added to the ethical
16 picture when we are
looking at this.
17 So, what do
the knowns and unknowns tell
18 us? Well, this has pretty profound implications
19 for informed
consent. How you get meaningful
20 informed consent in
this environment is a real
21 question and a real
challenge, not only because of
22 all the information
that we don't know but also
23 because of the
specific environment which we are
24 dealing with. We are dealing with the environment
25 of reproductive
medicine which has a reputation for
274
1 having a tremendous
overlap between clinical
2 innovation and human
experimentation. This
3 environment has to be
factored into the whole
4 question of the
meaningfulness of informed consent.
5 Added on to
that is the fact that patients
6 that come into
fertility clinics are desperate,
7 truly desperate for
real reasons to get pregnant.
8 We heard very candidly
that they will pressure
9 concentrations,
researchers, to provide techniques
10 for them even when
they are not necessarily
11 indicated. We have clinicians who are very
12 thoughtful people but
who have developed their
13 practice as clinician
researchers where much of
14 their practice is the
practice of experimentation
15 because they can. This is an interesting area
16 where they can
actually do a lot of clinical
17 innovation and human
experimentation.
18 So, what
does that mean? It means that
19 perhaps this is not
the best environment for basic
20 research to be
conducted when down the road the
21 risks could be much
more than society or even the
22 individuals are
actually willing to bear despite
23 what they say in this
context. There is near
24 consensus in the
literature, in the briefing
25 package, the protocols
that we have been
275
1 discussing, that this
is not ready for widespread
2 clinical
applications. Pretty much all the
3 protocols we read or
people who have spoken to us
4 earlier today indicate
in their work that they do
5 not believe it is
appropriate to conduct this
6 experiment in a
widespread fashion in fertility
7 clinics in this
country. They are very candid
8 about that.
9 So, should
there be more animal testing?
10 Yes. At the very least, one of the things I was
11 struck by was when Dr.
Shoubridge was talking is
12 that at the very least
we could be doing the tests
13 on his animals,
tissue-specific tests to find out
14 whether they are, in
fact, normal. He says they
15 appear normal, very
candidly, but he doesn't know.
16 They haven't tested
for that. So, we could be
17 doing that work.
18 Given the level of uncertainty of the
19 risk, I think the
answer is quite clearly yes. All
20 the studies that we
look at rely on animal studies.
21 So little is known
about the function of
22 mitochondria, about
heteroplasmy, about the
23 bottleneck, about
mitochondrial diseases that
24 animal experimentation
of various kinds, mice,
25 primates, can surely
help elucidate these
276
1 underlying uncertainties.
2 Finally,
must there be further human
3 embryo experimentation
before embryos are implanted
4 and children are
born? Yes. There must be more
5 human embryo
experimentation before implantation.
6 This is a lovely quote
from The New England Journal
7 of Medicine, the use
of novel reproductive
8 techniques must be
based on more than their mere
9 availability. There has to be clear clinical
10 indication for using
such techniques, evidence of
11 their efficacy and
consideration of the risks to
12 the mother and
society.
13 This is
difficult. We make decisions
14 about bringing
techniques to human trials by
15 looking at the risks
and uncertainties, the
16 potential harm to the
patients, offspring and other
17 individuals
involved. But we have to also factor
18 in the nature of the
condition that is the focus of
19 these experiments in
examining the risk to the
20 patients. Here we are talking about how quickly we
21 move forward. How imperative is it that this
22 results in human
experimentation in the clinics
23 tomorrow? So, this is a factor in our
24 deliberations.
25 In this
case, although infertility can be
277
1 a very serious
condition with serious and real
2 emotional impacts and
personal side effects, this
3 is not always the case
with infertility. More
4 importantly, we are talking
about the ability to
5 have a genetically
related child. Let's make it
6 even more of a finer
point here. The inability to
7 have a genetically
related child is not a
8 life-threatening or
fatal condition.
9 So, my point
is simply that when we
10 discuss how quickly we
move forward, the necessity
11 of making this happen
quickly in fertility clinics,
12 we have to keep this
in mind as well. Finally and
13 very importantly, we
have a duty to the children
14 that we help to be
born to do our utmost to see
15 that they are born
free from disease or impairment,
16 and we are not there
yet.
17 The
combination of these factors quite
18 clearly, in my mind,
mandates that further trials
19 not be conducted on
human embryos that will be
20 implanted in women
with the hope of creating more
21 children at this
time. If the FDA decides
22 otherwise, there are,
in fact, all kinds of factors
23 that should be introduced,
that I don't have time
24 to go
through--informed consent procedures,
25 rigorous screening,
etc. that we can discuss at
278
1 another time. That is the end of my remarks.
2 DR.
SALOMON: Thank you for a really
3 superb presentation
and actually an excellent
4 transition. What I would like to do now, before
5 the break, is to
invite three people who are on the
6 official docket for public
comment. We have
7 allotted seven minutes
each for these people. Then
8 we will take a break
and then come back and face
9 the set of questions,
many of which we have set
10 groundwork for and
some of which we will have to
11 try and put in a
proper context.
12 The first
person I would call for the
13 public hearing is Dr.
Jamie Grifo, representing the
14 American Society for
Reproductive Technology.
15 Welcome, Dr. Grifo.
16 Open
Public Hearing
17 DR.
GRIFO: Thank you. I appreciate the
18 opportunity to
speak. My name is Jamie Grifo. I
19 am a clinician
researcher. I am a reproductive
20 endocrinologist. I am the division director at
21 MIU, University School
of Medicine for Reproductive
22 Endocrinology. I oversee our laboratory; I oversee
23 our research. I run the fellowship and I am a
24 practicing clinician.
25 In my spare
time I am the president of
279
1 SARD. SARD is an
organization of the American
2 Society of
Reproductive Medicine. It has been in
3 existence since
1988. We are composed of
4 physicians,
scientists, researchers, embryologists,
5 nurses, mental health
providers and patient
6 advocates. We set the standard for the practice of
7 our medicine.
8 You have
never heard a story about this
9 organization because
we are not sensational and
10 there is no journalist
that will tell our story.
11 We have effectively
set the standard for our field;
12 we have self-regulated
and no one knows this story.
13 We are the only group
of physicians in the world
14 who collect data,
validate data, publish data in
15 collaboration with the
CDC about clinic specific
16 and national birth
rates. We have strict
17 membership
guidelines. We have strict criteria for
18 lab and medical
directors of programs. We validate
19 data by random site
visits. We have ethical
20 guidelines and
practice guidelines that are
21 required to be
followed in order to maintain
22 membership. We have teeth. We have eliminated 30
23 people from our
membership for failure to adhere to
24 our guidelines.
25 More
recently, we now require performance
280
1 standards and if they
are not met we offer remedial
2 services to these clinics
to assure quality of
3 care. We have also issued a statement saying that
4 we do not think
reproductive cloning should be done
5 at the current time
until it is proven to be safe
6 and effective.
7 So, we have
set the standard for our
8 field. We do regulate our field, and we have done
9 a very good job. Unfortunately, the media prefers
10 to talk about people
who are not our members and
11 who are not doing
things that people say they are
12 doing.
13 We are very
pleased that the FDA has taken
14 an active role in
regulating the medicines and the
15 devices that we use to
assure safety for our
16 patients. Our goal is that our patients have
17 healthy outcomes.
18 I do not
believe, and we do not believe
19 that ooplasmic
transfer is a food or a drug. It is
20 a research
protocol. Research protocols
21 traditionally have
been regulated by a very fine
22 situation that has
withstood the test of time. It
23 is called informed
consent and institutional review
24 board. That method has worked. Human research has
25 been done
ethically. Results have been good.
281
1 Safety has been
assured.
2 One must
realize that you can never assure
3 safety in any new
technique. The safest thing that
4 we can do is stop all
research in our field.
5 Unfortunately, the
series of letters sent out from
6 FDA has just done that
in our field. That has
7 assured that our work
will be done in other
8 countries by people
who perhaps do not have the
9 skills or the support
to do what we, Americans, can
10 do. We have been the best in our field.
11 Unfortunately, we have
had that privilege taken
12 away from us.
13 Through
informed consent and IRB we have
14 introduced in our
specialty, in very rapid
15 sequence, techniques
that did not exist. We have
16 made the practice of
IVF better. We have helped
17 more patients. Techniques such as ICSI, assisted
18 hatching, embryo
biopsy in co-culture have been in
19 existence and have
helped many patients. Embryo
20 biopsy was done
initially in England. It took me
21 four years to get
institutional review board
22 approval to do embryo
biopsy. In collaboration
23 with Jacques, we had
the first baby in the United
24 States. We were the second group in the world.
25 There have been
hundreds of thousands of babies
282
1 born free of genetic
disease by this technique. If
2 we attempted to
institute this practice into our
3 field today in this
environment, we would not be
4 able to do that.
5 I applaud
the FDA in wanting to assure
6 safety, but human
research will always have
7 inherent risks. You cannot get rid of risk. With
8 informed consent patients
are educated about what
9 those risks may be and
they make a decision whether
10 or not to undergo
those risks.
11 The FDA must
add value to the practice of
12 research in this
field. I hope that there is a
13 better mechanism,
other than stopping us from doing
14 our research, that can
exist. Thank you for the
15 opportunity to speak.
16 DR.
SALOMON: Thank you. The next speaker
17 is Dr. Sean Tipton,
also from the American Society
18 for Reproductive
Medicine. Does anyone know, is
19 Dr. Tipton here? Mr. Tipton, sorry. Maybe I could
20 invite the third
speaker since there wasn't any
21 particular order or
priority here, Pamela Madson,
22 from the American
Infertility Association.
23 MS.
MADSEN: It is an honor to be with all
24 of you here
today. It is an encouraging and
25 auspicious start that
so many members of the
283
1 medical and scientific
research and government
2 communities have come
together.
3 For the
millions of us who are locked
4 together in the
wrenching battles against
5 infertility, this
meeting embodies the hope of
6 achieving increasingly
effective and safe
7 treatments as quickly
as possible because we have
8 no time to waste.
9 The
population of the infertile is
10 growing, with one in
six couples actively
11 experiencing
problems. Let's be clear, we are raw.
12 Recent headlines made
public what most of us
13 already know, that our
collective ignorance about
14 fertility is
extracting an enormous toll. That
15 women who delayed
childbearing, either by choice or
16 force of circumstance,
feel duped out of their shot
17 at genetic
motherhood. That their partners, who
18 also long for the
children that are uniquely
19 theirs, are just as
saddened and infuriated by the
20 loss. That the individual and societal costs of
21 infertility are
intolerable. Let me respond to
22 you, no, it is not
life-threatening; it is
23 life-stopping.
24 What do we
do about it? Certainly we
25 raise public awareness
about infertility, its
284
1 prevalence, its causes
and prevention. We make a
2 concerted effort to
educate everyone about the
3 human reproductive
life cycle. But we must also
4 rededicate ourselves
to refining the infertility
5 treatments we have and
to discovering new ones.
6 Like any other
ruthless disease, infertility
7 ravages not just the
immediate sufferers but their
8 families and friends,
employers, peers and
9 employees. With age, a genetic inheritance, a
10 physiological fluke or
a medical condition is to
11 blame, all those
affected by infertility have one
12 thing in common, an
urgent need for reliable paths
13 to biological
parenthood.
14 As patients, we understand, to a large
15 extent, that the fees
we pay for services propel
16 developments in
reproductive technology. It is
17 worth noting, however,
that we are here when our
18 government does not
provide any funding for
19 research. Yes, we need more embryo research. No,
20 it is not funded by
our government. We are
21 cognizant of the risks
we voluntarily take as the
22 subjects of clinical
experimentation that are
23 required to move the
research expeditiously. We
24 know that we are
treading on uncharted territory.
25 To date,
ooplasm transfer research offers
285
1 the greatest potential
to help women with oocyte
2 problems. It is potential. We need research, we
3 need it to move
forward. It is the avenue that
4 seems to be leading to
many different technologies
5 that may deal with the
multiple forms of
6 egg-related
infertility. We want to do everything
7 we can to facilitate
this work because right now,
8 as far as we know,
there is nothing else.
9 Of course,
we are concerned that
10 researchers adhere to
the highest standards
11 possible. It is not only our health at stake, but
12 the health of future
generations as well. We have
13 always relied on the
twin mechanisms of IRBs and
14 informed patient
consent, and it is our
15 understanding that the
system has worked reasonably
16 well.
17 As willing
participants in experimental
18 procedures, patients
have the right to honest and
19 forthright information
before giving consent. That
20 includes anticipated
outcomes and possible
21 pitfalls; what is
known and best guesses about what
22 isn't. We wonder why IRBs can't be overhauled to
23 include a broader
array of interests--patient
24 advocates and possibly
government representatives
25 among them. We wonder why we don't have uniform
286
1 IRB standards. This is likely to be far less
2 intrusive and
economically onerous than the
3 creation of an
entirely new system.
4 If, however,
the government is committed
5 in its current plans,
we do urge restraint. We
6 would like to know
that government federal
7 guidelines will not be
so cumbersome and expensive
8 that they inhibit
researchers from pursuing
9 promising leads. We want to know that the costs of
10 regulation which are
passed down to consumers will
11 be reasonable and
contained. Remember, most of the
12 infertile around this
country are paying out of
13 pocket. We don't have coverage.
14 Otherwise,
we jeopardize the access to
15 treatment for all but
a very wealthy few. As it
16 is, the financial
burden of largely uninsured
17 reproductive
technology puts an enormous strain on
18 the infertile. We are asking that we build on the
19 cooperation and open
communication that we have
20 witnessed here today,
and we would urge, if we are
21 going to work
together, that whatever body it is,
22 whether it is through
overhauling of systems that
23 are in place or a new
body, that it be composed of
24 regulators,
researchers, reproductive clinicians
25 and patient advocates
to ensure that politics do
287
1 not interfere with the
community's need for
2 scientific
breakthroughs. We are depending on a
3 true collaborative
process. The infertile cannot
4 afford, and do not
deserve any less. Thank you.
5 DR.
SALOMON: Very nicely spoken. As I
6 said, we are going to
take basically a ten-minute
7 break. It is 4:15 right now. We will start again
8 at 4:25 regardless of
anyone who isn't here, just
9 so you take me
seriously this time. I want to make
10 sure we have enough time. Thanks.
11 [Brief
recess]
12
Questions to the Committee
13 DR.
SALOMON: To initiate the final phase
14 of this afternoon and
where things have to come
15 together, all the
different pieces that we have
16 explored all day, is
in dealing with a series of
17 specific FDA
questions. These will be briefly
18 reviewed by Dr. Moos.
19 DR.
MOOS: I am just going to try and tie
20 together a few things
that we have heard today by
21 way of introducing our
list of questions. I am not
22 going to subject you
to a detailed reiteration of
23 this list; it is in
the briefing package.
24 The first
thing I want to say is directed
25 to the folks whom we
consider really the most
288
1 important people in
the room, who are the patient
2 interest
advocates. I think that if you have a
3 look at the kinds of
questions we have been asking
4 and discussing,
implicit in the entire format of
5 the meeting and the
discussion is that we have no
6 intention of stopping
any kind of research. Our
7 intention is to
balance carefully the avoidable
8 risks and the benefits
in a way that we optimize
9 the balance between
the two.
10 To do that,
we need to make use of the
11 best scientific and
medical evidence and analysis.
12 I think the presenters
have done an excellent job
13 of laying out much of
the critical information that
14 we will need to make
use of to synthesize how we go
15 ahead with this.
16 Many of our
judgments will depend on some
17 kind of treatment of
numerical data. We have seen
18 a great many mentions
of how small the numbers are
19 and what the
statistics are like. And, one of the
20 things which, over in
the FDA corner, we found very
21 striking is just this
fact. We heard some very
22 useful information
suggesting that experiments
23 might be quite
feasible and relatively
24 straightforward to
design that would satisfy us
25 that heteroplasmy per
se represents a manageable
289
1 risk.
2 But there is
a fly in the ointment,
3 particularly with
respect to the incidence of
4 Turner syndrome that
has been reported in some of
5 the data. We know that it is very common. The
6 best information that
we can get out of the
7 literature suggests
that the incidence of Turner
8 syndrome in the
general population is perhaps 1/100
9 conceptions, not live
births but conceptions. If
10 someone wants to weigh
in with a better number, we
11 are all ears. In contrast, the series that has
12 been reported has an
incidence of 23 percent, more
13 than 20-fold
higher. If you factor in the
14 biochemical
pregnancies, which were very likely
15 aneuploid, the figure
becomes higher.
16 We
acknowledge that the confidence
17 interval around 3/13
is very, very large, but this
18 is something that
can't be ignored. There are a
19 couple of
scenarios. We can reduce this with
20 respect to the
efficacy question either to a
21 situation in which
ooplasm transfer has no
22 beneficial effect on
fertility, in which case the
23 additional risks of
instrumentation, of
24 superovulation and so
forth are not reasonable, or
25 that it does give a
boost, in which case the
290
1 potential to bring
marginal embryos that perhaps
2 should not come to
term to a point where something
3 bad might happen
actually exists. So, this is an
4 issue that we have not
heard sufficient discussion
5 on and that I would
like for the committee to keep
6 in mind as we tackle
the question.
7 If we can
address the salient safety
8 issues, I just want to
say one or two words about
9 product
characterization. There has been I
think a
10 very interesting
discussion about what it is that
11 is doing
something. I would like to point out
that
12 the better we
characterize the material that is
13 being transferred, the
better we will be able to
14 manage those risks
from a number of standpoints.
15 There will be
questions that we will need to
16 consider both to
initiate experiments in what we
17 call Phase I or safety
studies, and there will be
18 questions that we will
need to confront at the time
19 of the licensure which
will, indeed, require much
20 more detailed
information about what is in the
21 product that is making
it work and definitive proof
22 that the product, in
fact, is working.
23 With that
brief introduction to the
24 questions, I will
yield the floor to our chairman,
25 with thanks for his
able service, and to all the
291
1 members of the
committee and panelists for the
2 discussion today. Thank you.
3 DR.
SALOMON: Thank you very much. So,
4 there are two pages of
questions, but some of them
5 are more important
than others and I will do my
6 best to prioritize
them.
7 As stated
here, to me, there are a couple
8 of principal
goals. The first is to determine
9 whether there are data
available right now that
10 support the safety or
support the rationale for
11 ooplasm transfer that
is sufficient to justify any
12 perceived risk
involved in the clinical trial. We
13 need to deal with
that.
14 We also need
to determine a separate
15 issue, what additional
data are needed prior to
16 initiation of a
broader use of this technology or
17 clinical trials if the
first discussion should come
18 to the conclusion that
clinical trials shouldn't go
19 forward.
20 So, I think
there are a couple of
21 different options that
the committee can now
22 consider. You can consider that, no, there is not
23 enough data; no
clinical trials. But you can't
24 just say that. You have to say what exactly has to
25 be done. We have to come to some grips with the
292
1 concept of where is
the bar going to be set for
2 this. We can also say, no, there is sufficient
3 data; go forward with
clinical trials but, in
4 parallel, we need
additional data. You know, you
5 need to be show us
evidence that the field is
6 working on these
additional data but we can also
7 then go forward and
talk about what is a good
8 clinical trial. So, I think that is a major issue.
9 We can't leave without
really trying to come to
10 grips with it.
11 A second
major issue to me is regardless
12 of the answer to
either of those, even though they
13 have such important
immediate implications, another
14 issue here is to begin
at least a dialogue with the
15 community regarding
what you will need to
16 characterize this
product. I mean, that is going
17 to be something that
you can't change. Whether we
18 are talking about
islet transplantation,
19 therapeutic gene
transfer in any number of cells,
20 stem cells of any
sort, you have to have a sense of
21 product. We are not talking about, "hey, trust
me
22 with this wonderfully
ethical group of scientists,"
23 it has to be,
"trust me, we are going to do this in
24 40 centers, in 50
states and charge money for it."
25 I mean, that is
okay. That is fine; that is the
293
1 American way. But in the process of doing that,
2 the direction that the
FDA has to have from us is
3 how you are going to
make sure that in 50 states
4 and 50 places or 100
places, or whatever, there is
5 a sense of objective
measurements for the quality
6 of the product, what
we call lot release criteria.
7 Those things are much
more difficult to do in a
8 biologic. I know that. We all know that. But
9 they are not
impossible.
10 So, with
that background let's start kind
11 of with the first
concept. I am getting off the
12 strict question order
a little bit but I am going
13 to do that on
purpose. So, the first question here
14 is we have heard the
clinical presentations and I
15 have to start with a
discussion of is there enough
16 data, preclinical or
clinical, right now to do a
17 human clinical
trial? Let's assume that that is a
18 really good clinical
trial that is going to answer
19 a question, just are
we comfortable doing a
20 clinical trial or
should we say, no, we are not
21 comfortable; it should
be put on hold and then we
22 have to set a bar?
23 MS.
WOLFSON: Well, as one of the few
24 non-scientists here,
first of all, I would like to
25 say I really thought
that Lori posed very
294
1 interesting questions
and I don't think you can
2 answer your question
without kind of addressing all
3 of those questions.
4 From what I
have heard and what I have
5 read up to this point,
I do not think we have
6 enough clinical data
to allow human studies in any
7 form. I think that there are so many things that
8 have to be answered
that haven't been answered.
9 When Lori spoke about
informed consent, I thought
10 to myself, well, it is
one thing for a couple to
11 give informed consent
for any dangers that they
12 might encounter, but
how can they give informed
13 consent for future
generations? I would even
14 wonder if they could
really give informed consent
15 for their own possible
child if there is a risk
16 that, for instance,
there is a 23 percent chance
17 that that child would
have Turner syndrome?
18 I think
these questions have to be
19 addressed. I don't think we got enough information
20 here today to say that
there is enough clinical
21 data out there at all.
22 DR.
SALOMON: Okay, that is clear. What
23 are other thoughts
here?
24 DR.
NAVIAUX: An alternative to that would
25 be a limited number of
expert centers, one, two--a
295
1 small number that
would be guided by the
2 recommendations of
this body in obtaining some of
3 the human data that is
necessary in the process of
4 offering the
technique. I will leave it at that
5 for now.
6 MS.
WOLFSON: Just a point of
7 clarification, do you
mean human data as in
8 pregnancies, or are
you talking about
9 experimentation with
human embryos?
10 DR.
NAVIAUX: I think there are practical
11 difficulties. We definitely need the embryo
12 research but we have
kind of left the human
13 reproductive
technology people out on a limb
14 without any support
because there is no mechanism
15 for funding human
gamete research. So, yes, I we
16 need that data but, you
know, in the U.S. there may
17 not be a mechanism,
and someone can correct me
18 perhaps.
19 DR.
SALOMON: Dr. Sausville?
20 DR.
SAUSVILLE: I think there returns a
21 little bit to the
importance of the animal
22 experiments that were
discussed previously.
23 Recognizing that the
lack of support for human
24 gamete related
research and subsequent production
25 of zygotes is an issue
that is ultimately one that
296
1 this committee does
not have the purview to, shall
2 we say, change, I do
think that the scientific
3 rationale that might
emerge from a considerably
4 larger body of
research that can be funded on
5 animal-related matters would increase my
enthusiasm
6 for the possibility,
and possibly the fact, that
7 there is something
actually happening here. We
8 heard that we don't
know what components of this
9 process convey a
salubrious outcome. Maybe the
10 whole combination of
things is necessary, but then
11 that gets to the
product issue that was raised. I
12 mean, do you define
this product as having a lot of
13 ATP? Do you define it as having a certain minimum
14 level of ATP or
calcium, or whatever you favorite
15 component is?
16 So, to me,
while I actually want the field
17 to move ahead and
potentially give what benefit it
18 can within the context
of its limitations, I just
19 feel that in
comparison to many other therapies
20 that have come to this
committee before, some of
21 which are very
specialized, in each case the
22 proponents were able
to make the scientific case
23 preclinically for the
ones that went forward; that
24 there was a basis for
actually regarding this as an
25 ultimately successful
outcome and I don't actually
297
1 see that here.
2 DR.
SALOMON: Lori?
3 MS.
KNOWLES: I just want to make the
4 point that it is true,
and we are obviously not
5 going to discuss it at
any length, that there is a
6 lack of publicly
funded human embryo research, but
7 there is private money
for human embryo research
8 and the fact that
there isn't public money for it
9 doesn't, to me, say
that then you skip that stage
10 and do the
experimentation in humans, live humans.
11 So, if you
actually want to be able to
12 offer this technique
and make money from it, you
13 have to do the
experimentation that shows that it
14 is safe. It is just part of the equation, the way
15 that I see it.
16 DR.
SALOMON: I just want to point out
17 that here is where it gets kind of
complicated
18 because we have to be
very careful. One is talking
19 about efficacy and one
is talking about safety. I
20 am not saying that we
don't have to discuss both
21 but we need to be
careful. Ed is talking about
22 efficacy and I was
talking about efficacy, and now
23 you kind of throw in
safety, that is okay but we
24 need to be sure that
we stay intellectually clear
25 that the domains of
safety and efficacy are
298
1 different.
2 MS.
KNOWLES: Right, and I actually agree.
3 My feeling is exactly
what you were saying, that
4 there are all kinds of
information that we can get
5 from animal models--it
sounds like, about the
6 efficacy.
7 DR.
SAUSVILLE: And I would go so far as
8 to say that both
safety and efficacy are uncertain
9 to me.
10 DR. VAN
BLERKOM: As far as efficacy, we
11 are dealing with
long-standing infertile couples,
12 women whether have
been through lots of treatments
13 unsuccessfully. What animal model do you propose
14 that will be
relevant? I mean, as far as a mouse,
15 put in cytoplasm and
get mice. Would you use a
16 primate model. I don't know if there are any
17 long-standing
infertile Macaques. Maybe there are.
18 So, I am not sure
about the relevancy specifically
19 of animal models.
20 I think the
basic question is, is this
21 effective? If you look at all the publications on
22 cytoplasm transfer,
they all say we don't know that
23 this is
effective. We don't know what is
causing,
24 if anything, a boost
in efficacy. So, I think in
25 reality what it is
going to come down to is that
299
1 the only system that
is really suitable for a test
2 of efficacy is going
to be the human. I just don't
3 see an animal system
providing the types of
4 information that you
would like to see.
5 DR.
SAUSVILLE: I would respectfully
6 suggest that while I
can understand the ultimate
7 human relevance of
both the use of the procedure
8 and the judgment of
its value, what we are talking
9 about here is the
setting up of some boundary
10 conditions which would
begin to be able to be
11 applied to that which
is used in this critical
12 human experiment. I mean, the very presentation
13 that I believe came
from you showed that there is a
14 great deal of
variability in terms of where you
15 stick the needle, the
different types of eggs--I
16 mean, this becomes
very problematic, therefore, for
17 deciding how we would
set up the human experiments,
18 at least to me it
does.
19 DR. VAN
BLERKOM: That is the whole point.
20 I think the human
experiment is unique, unique in
21 the sense that I think
there are confounding issues
22 that happen in human
eggs that you are not going to
23 find in other species.
24 DR.
SIEGEL: May I interrupt? We really
25 need to focus this in
a context that will be more
300
1 useful to us if we are
trying to deal with the
2 questions. The question you asked is whether there
3 is enough data to do
clinical research but then you
4 are focusing on the
efficacy side. We worded our
5 question somewhat
differently, and for a reason,
6 and that has to do
with what our regulatory
7 authorities are. I would like to have this
8 discussion within the
context of what our
9 regulatory authorities
are.
10 So, your
question bears some significant
11 similarity to question
number three, which I would
12 like to take just a
moment to read and explain the
13 context of why it is
worded that way. Are these
14 data, referring to the
clinical and preclinical
15 data currently
availability, sufficient to
16 determine that ooplasm
transfer does not present an
17 unreasonable and
significant risk to offspring and
18 mother, and to support
further clinical
19 investigations?
20 The determination
we need to make
21 specifically is
whether there is an unreasonable
22 and significant
risk. That is largely a safety
23 determination, but
what risks are reasonably and
24 what risks are not
reasonable is clearly linked to
25 the issues of what disease is being treated,
what
301
1 the prospective
outcome is and how strong is the
2 rationale. So, efficacy does figure in but we are
3 not going to decide
simply that because we don't
4 think that this is
going to work; you shouldn't
5 study it in humans to
find that out. So, the
6 question is a little
more safety oriented in the
7 context.
8 DR.
SALOMON: Right. We don't always
9 agree on how I get
there but I am trying to get
10 there.
11 [Laughter]
12 If you will
indulge me just a little
13 longer, not too much
longer--
14 DR.
SIEGEL: Now that I am on record, you
15 go where you want to
go but I hope we will get to
16 where we need to get.
17 DR.
SALOMON: Fair enough. I don't want
18 to delve too deep, I
just want to stay on the
19 surface here but I
still want to just get a sense
20 of the committee along
the lines of where we are
21 starting here. We have been doing a pretty good
22 job of that and we
have identified this sort of
23 knife-edge balance
between efficacy and safety and,
24 in that case, what Dr.
Siegel just said is
25 absolutely true
because what we are going to do
302
1 then is dive into the
safety side. But I would
2 just like to hear a
few more minutes of the
3 gut-level feeling at
this point of should this
4 discussion go more
toward--we need to deal with the
5 safety issues and then
step in and say, okay, what
6 is the good clinical
design because we are going to
7 go forward with
clinical design, or we are going to
8 say, no, this
committee does not feel that a
9 clinical design is
appropriate now so we had better
10 set a bar in
preclinical studies for safety. I am
11 trying to decide where
we are going to go as a way
12 of guiding
myself. So, Dr. Murray and then Dr.
13 Rao.
14 DR.
MURRAY: I think I want to ask what
15 for me, at least, is a
prior question, one that I
16 have to get an answer
to before I can answer the
17 one you gave me. There is an expression in my
18 field, bioethics,
which is that ethics begin with
19 the facts and I don't
know all the facts I need to
20 know at this
point. I have heard a lot of raw
21 information. I would really like to hear the
22 considered judgments
of a number of the scientists
23 around here about what
we actually know about
24 safety and, if not
efficacy, about the plausibility
25 of the mechanisms by
which this intervention is
303
1 presume to have its
positive effect.
2 I certainly
have to defer to Jonathan
3 about animal models
and what is an adequate animal
4 model, but it seems to
me we were getting answers
5 to some of those
questions from animal data. They
6 may not be animal
models in some very cosmically
7 broad sense but I feel
a lot better about the risks
8 for heteroplasmy now
having heard the discussion
9 that took place after
lunch here. I am much less
10 worried about it than
I was when I first read the
11 papers.
12 So, I think
there is a lot of wisdom that
13 has come in front of
us today. It would be nice to
14 see that digested, get
kind of a best read on it,
15 and then I would be
ready to talk about the human
16 trials.
17 DR.
SALOMON: My response to you is you
18 will be one of our
bench marks. I will look to you
19 to tell us you have
heard enough information. That
20 is important. Dr. Rao?
21 DR.
RAO: As you said, I don't want to
22 dive too deep into
this but say that even though we
23 may not have data for
efficacy, maybe we have some
24 data from the mouse
models for a rationale for why
25 one might want to do
ooplasm transfer, and maybe
304
1 that may best be
addressed by the doctor, I don't
2 know which one;
someone right at the end, where
3 they had the mouse
model which showed that if you
4 have mitochondrial
deficit you actually see
5 degeneration which
looks similar, and if you
6 replace those
mitochondria you actually see much
7 less
degeneration. So, there is a rationale
in
8 some sense that, yes,
if you transfer something
9 which is present in
the cytoplasm you might see
10 some improvement. That certainly doesn't address
11 what happens in human
but it does give you a
12 rationale for why you
may want to try and address
13 that therapy.
14 On the
safety side too, I think if one
15 defines the problem
and says that, well, what you
16 are doing is a
procedure which is very similar to
17 what you are already
doing in ICSI where you have a
18 lot of expertise, then
you have a lot of data,
19 clinical data with
humans in the appropriate model
20 on safety. What you don't have in those models is
21 safety in terms of the
issues that were raised here
22 in terms of
heteroplasmy and in terms of what Dr.
23 Mulligan raised in the
sense of what happens with
24 naked DNA transfer or
what happens with chromosomal
25 damage.
305
1 So, maybe we
should compartmentalize it a
2 little bit and say
that there is a rationale. We
3 don't have any data on
efficacy maybe, and we have
4 some data on safety,
except in sort of critical
5 issues.
6 DR. MULLIGAN: Yes, I think the data issue
7 is very key to think
about what would you consider
8 the definition of data
versus a rationale. I think
9 that is the mystery we
are having here. I think
10 there is no data. I think that every scientist has
11 to figure out where he
wants to set the bar. Even
12 if you set the bar
really low, there is no data.
13 Yet, there is some
rationale, and the rationale,
14 probably my bright
ten-year old could come up with
15 listening to me talk
about how injecting things
16 into cells can change
their function. While we
17 dance around all the
embryo work, and whatever,
18 yes, there is a
rationale. It is a pretty simple
19 rationale that, of
course, you can profoundly
20 affect the way a cell
functions by introducing
21 things into it. So, I think there is no data and I
22 would like to have
some controversy stirred up
23 about that.
24 From the
safety point of view, I think
25 this is so clearly a
gene transfer issue that the
306
1 safety issues ought to
be focused on essentially
2 what is an unwanted
substance in the product that
3 could have a safety
effect. I can tell you from a
4 background in gene
transfer, and I am an expert in
5 that little narrow
part of things, and you can get
6 very, very different
efficiencies of gene transfer
7 by doing the method in
different ways, things that
8 are typically
efficiencies that are one tenth or
9 fifth can be 40
percent if you do it differently.
10 So, I see this no
different than the whole
11 regulatory process
with gene therapy vectors where
12 having someone say,
well, that isn't going to
13 happen, or there isn't
enough DNA there, or we do
14 this all the time is
and it just can't happen.
15 These guys are
laughing. They have heard that
16 before.
17 So, I would
say that my concern, based on
18 the whole process in
the gene therapy field, is
19 that this is an
analogous case where setting the
20 bar as low as you want
for efficacy, there is still
21 no data. But maybe there are some things that can
22 be done. There is clearly some rationale but it
23 ought to be focused on
essentially what are you
24 essentially
doing? What are you injecting? And,
25 what toxic substances
or things that can cause some
307
1 risk are in it? I would think that trying to
2 document what kind of
tests, checking for whether
3 or not there is
chromosomal DNA or naked
4 mitochondrial DNA are
things that are supportable.
5 They are not embryo
types of things. And, those
6 would be very
important, as well as to characterize
7 the consistency, as
best you can, of what you are
8 going to use, like
count the mitochondria or
9 measure the amount of
DNA, just so that in the
10 future you may be able
to draw some correlations
11 between some of the
most obvious types of things.
12 DR.
SALOMON: I think we will continue.
13 That is a nice
beginning to dive into where I
14 promised Jay I would
go in a few minutes, the
15 safety issues, because
I think that takes us there.
16 Dr. Shoubridge and
then Dr. Casper.
17 DR.
SHOUBRIDGE: I don't think the problem
18 with mitochondrial DNA
is a real safety issue here.
19 I think the chance of
getting naked mitochondrial
20 DNA to do anything
real bad, or even getting it, is
21 zero essentially in
this kind of a procedure. When
22 you can do subcellular
fractionation, and you don't
23 get much more severe
methods than this, you just
24 don't get naked
mitochondrial DNA unless you
25 isolate DNA. So, certainly the nuclear genomes is
308
1 another issue.
2 For me, the
safety issue that revolves
3 around
heteroplasmy--it is almost impossible to get
4 that information in
humans because if we take our
5 mice as an example and
look at the tissue that had
6 the strongest effect
for selecting for one
7 genotype, it took
basically the mouse's lifetime to
8 do that. It is quite a slow process. So, if we
9 just extrapolate to
the human it could take decades
10 to find out whether
that is ever going to happen.
11 So, I don't think realistically
we are ever going
12 to have that
information to go on.
13 But coming
back to something, Dr.
14 Mulligan, that you
said earlier on, to me it is
15 crucial to establish,
and it would change the whole
16 nature of the
enterprise whether mitochondria are
17 important here at
all. There, I think Dr. Casper's
18 mouse model, even
though it may not be perfect, he
19 has injected
mitochondria and shown some effects
20 there. And, I can think of a list of what I think
21 would be pretty decent
experiments, some of them
22 genetic and some of
them not, that would tell you
23 whether mitochondria
or at least the energy
24 metabolism part of
mitochondria are at all
25 important in this process. If you could come to
309
1 the conclusion that
they weren't, then we wouldn't
2 even be having a lot
of this discussion because the
3 heteroplasmy issue
would be a non-issue. It would
4 be another factor and
then maybe we would be
5 interested in the
biological effects of putting in
6 pieces of spindles, or
having a centriole, or
7 having an RNA
population, or something like that.
8 So, to me,
it would be critically
9 important to establish
whether or not mitochondria
10 are in fact important
in human embryos in a
11 research
situation. I don't know if you would
call
12 that clinical research
because the endpoint here
13 wouldn't be
pregnancies. You would have to have
14 some other endpoint,
like morphology objectively
15 determined or some
biochemical endpoint in an
16 embryo. And you would have to use the mouse
17 models. As imperfect as they are, it is the best
18 we have.
19 DR.
SALOMON: Dr. Casper and then I will
20 take us into dealing
with the first question on the
21 safety issue.
22 DR.
CASPER: You asked earlier about gut
23 feeling responses also. I can tell you just from
24 doing clinical IVF for
many years and dealing with
25 patients who have
repeated fragmented of rested
310
1 embryos, it is my
impression that it is not a
2 condition that
corrects spontaneously. So, I think
3 the fact that there
have been pregnancies produced
4 in that group of
patients with this procedure
5 suggests to me that
there is probably something
6 that is working,
although we don't have the numbers
7 to actually support
that.
8 So, I think
what we have essentially at
9 this point is the
equivalent of a pilot study that
10 demonstrates potential
efficacy, and I think it is
11 worthwhile to move on
to some more significant
12 research studies.
13 I think the
most important thing, however,
14 is to find out what it
is that actually makes this
15 work. I think it is also important to do away with
16 ooplasm transfer
because, first of all, we don't
17 really want to have to
subject women to egg
18 donation in order to
make this work. If we could
19 figure out what the
actual component is we could
20 use that component
perhaps without having to get
21 donor eggs. Secondly, the cytoplasm injections
22 also have that small
but inherent risk of
23 transferring genomic
DNA as well.
24 So, I think
there probably is some
25 efficacy to this
procedure. I think it probably
311
1 does warrant going
ahead with clinical and animal
2 trials, but on a more
specific level to try to find
3 out what it is that is
actually working in the
4 transfer.
5 DR.
SALOMON: That is good. You touched
6 on something for
me. You know, I have been trying
7 to decide for my own
self, independent of my job as
8 chair, when I say,
well, we should do some clinical
9 research at the same
time we are advancing our
10 understanding in the
basic models. I am kind of
11 leaning in that
direction. Then I think of things
12 like, well, if you
really don't know whether it is
13 the mitochondria or
some sort of soluble element,
14 maybe you ought to
know that before you do the
15 clinical studies and
that has all kinds of safety
16 implications, and we
will come back to that.
17 The other
thing is if you don't need to
18 use an oocyte donor if
you, for example, could do
19 it from a human
embryonic stem cell, you know, if
20 you could do that then
wouldn't that be an ethical
21 step in the right
direction in the sense that now
22 you wouldn't be
involving the invisible woman? I
23 thought that was an
interesting visual. Or, you
24 could use somatic
cells from the mother even.
25 So, there
are some other questions here
312
1 that could have really
profound implications as to
2 how the procedure was
done without saying that this
3 procedure actually
would work and, yet, get the
4 benefits for the
infertile mothers which I think
5 was well articulated
in the public comment period.
6 So, that is a dynamic
I guess we will have to deal
7 with for the rest of
the next hour or so.
8 Speaking in
terms of risks to the
9 offspring then, the
FDA proposes four specific
10 issues that directly
affect risks to the offspring,
11 all dancing around the
concept of how the procedure
12 might damage or alter
the oocyte--mechanical
13 damage, inadvertent
transfer of chromosomes and
14 chromosome fragments
or cellular constituents,
15 enhanced survival of
abnormal embryos and risks
16 with
heteroplasmy. We don't have to do an
hour
17 discussion of this
because we have already touched
18 on a lot of aspects of
this, but let's deal with
19 these four specific
issues of safety.
20 Number one,
mechanical damage to oocyte
21 architecture. What do you guys think? Dr. Rao?
22 DR.
RAO: I just want to reiterate that
23 there is a lot of data
for ICSI and there is no
24 difference in the procedure,
except for additional
25 volume injections, in
terms of mechanical damage.
313
1 So, I would say, from
what I have heard, that it
2 seems that the amount
of mechanical damage should
3 be the same and there
is data from lots of
4 successful births.
5 DR.
SALOMON: So, is that true? I have no
6 clue. I mean, is it true that the amount of
7 physical puncturing of
the recipient cells is
8 identical for ICSI as
for that? That is a fair
9 point from everything
I have heard today. There
10 are issues that you
are injecting cytoplasm,
11 whereas before you
were injecting the sperm in some
12 sort of natural
buffer. Right?
13 AUDIENCE
PARTICIPANT: [Not at microphone;
14 inaudible.]
15 DR.
SALOMON: So, would you say there is
16 an incrementally,
albeit incrementally small,
17 difference with the
ooplasm injection because of
18 the volume issue? Fair enough.
19 DR.
MURRAY: There are people here more
20 qualified than I am to
recite all the data on
21 ICSI's impact on
children but, as I recall it,
22 there is some increase
in various abnormalities
23 over the natural
background rate, although it is
24 not an outrageous
increase, and there is I think
25 roughly a doubling of
low birth rate among the
314
1 children, and low
birth weight is a predictor of a
2 lot of later
problems. But, again, so far at least
3 those have been deemed
to be acceptable I guess by
4 the people who employ
them.
5 DR.
SALOMON: So, the point here now is
6 that ICSI is
essentially close to, maybe slightly
7 incrementally
different but I think we can live
8 with that incremental
difference for safety. Now
9 the question is what
increase in risk does ICSI
10 cause versus
age-matched infertile women?
11 DR.
SABLE: Just to address the ICSI
12 questions, once one
factors out the couples who
13 conceive who would
never conceive on their own
14 because there is no
sperm in the ejaculate, and
15 these are couples
where the sperm has to be
16 literally surgically
removed from the testicle,
17 once you factor those
couples out--and these are
18 not people to be doing
cytoplasmic transfer--the
19 risks drop down to the
background risk.
20 Regarding
the low birth weight, that is a
21 study that actually
included all IVF patients,
22 including the ICSI
patients. There did not seem to
23 be an incremental
increase in risk of low birth
24 weight versus the
background IVF population, just
25 to clarify that.
315
1 DR.
SALOMON: So, for question number one
2 I assume that there is
a fairly high level of
3 comfort here, comfort
as defined by mechanical
4 damage to the oocyte
cytoarchitecture induced by
5 this procedure is
incrementally small over the
6 overall risk of these
procedures that are already
7 ongoing.
8 DR.
SAUSVILLE: Right, I would say numbers
9 one and four under the bullet "risks to
offspring"
10 are obviously there
and are things that are
11 reasonably tolerable
or at least known, recognizing
12 the long-term risks
associated with heteroplasmy
13 have been extensively
discussed that are at one
14 level unknowable but
that are intrinsic to the
15 procedure.
16 I guess I am
more concerned with numbers
17 two and three. As Dr. Mulligan articulated, the
18 procedures that are
currently in place do seem to
19 be somewhat
uncontrolled on whether or not matters
20 of technique or
instrumentation can minimize the
21 likelihood of
chromosomal fragments being an issue.
22 Lastly, we
heard the figure cited by Dr.
23 Moos about if one just
does the crude calculation,
24 there is approximately
20-some odd incidence of
25 major abnormalities in
the series that have been
316
1 reported so far. So, I am a little concerned that
2 that is a higher level
of abnormality than I at
3 least would feel
comfortable with.
4 MS.
KNOWLES: I don't want to get off
5 topic if we want to
follow this up but since you
6 were taking about number
one and four, my feeling
7 about number four, and
this may in fact be just a
8 question of my
ignorance of the animal models, what
9 I have heard is that
we have some limited work in
10 mice that shows that
this is not a problem. Yet, I
11 have also heard a
discussion that the mouse models
12 are, in fact, not
something that we can really use
13 to translate for other
questions to the humans.
14 So, I am not a hundred
percent convinced that that
15 does away with all of
the questions about
16 heteroplasmy. So, I also wonder if there isn't
17 some kind of closer
animal model, like a non-human
18 primate, that we could
do a study in heteroplasmy
19 that might be quite
useful. Perhaps I just don't
20 understand.
21 DR.
SAUSVILLE: I could respond to that, I
22 think we agree that
the actual risk or the
23 dimensions in which
heteroplasmy would enter being
24 something that could
be considered an adverse event
25 are actually
unknown. I agree entirely with your
317
1 analysis. I guess to me, from the standpoint of
2 writing an informed
consent, it becomes at one
3 level something that
could be state, look, we don't
4 know anything about
this and I could imagine
5 scenarios where, if
donors were properly screened
6 for the known
mitochondrial issues etc., that one
7 might reasonably take
the risk of tolerating that
8 statement, recognizing
that it is an unknown.
9 My issues
with respect to number two, that
10 is very much, in my
mind, a matter of how the
11 technique would
actually be practiced on an
12 individual sense and,
therefore, is a potential
13 basis of extraordinary
variability.
14 With respect
to number three, I am
15 concerned that the
incidence of 20-some odd
16 percent recognizing,
if that is true and the issue
17 of how broad the error
bars are, ultimately society
18 is going to be asked
to, at one level, take care of
19 these children in some
way or fashion. So, to
20 countenance a
technique that has that level of
21 abnormality
generation, if that is truly the
22 number, I think is a
matter of concern.
23 DR.
MULLIGAN: On that point, if you drop
24 statistics for the
efficacy part of things, that is
25 a gut feeling that
maybe there is something to
318
1 this, not evoking
statistics, then we might as well
2 not evoke statistics
for the potential toxic effect
3 too. Since there is not statistically significant
4 info, I think it is
important to weigh the data
5 comparably. That is, on one side it looks like
6 there may be
difficulty; on the other side there
7 may be some efficacy.
8 DR.
SALOMON: I am happy for this
9 discussion. So, we are still focused now maybe
10 more on questions two
and three, the inadvertent
11 transfer of
chromosomes or the enhanced survival of
12 abnormal embryos, with
the emphasis in the last few
13 minutes on the
abnormal embryos. What is the
14 feeling of the panel
on that?
15 DR. RAO:
I would just like to second what
16 Dr. Sausville said,
that it is really a big issue
17 and what Dr. Mulligan
said, that in a system where
18 you don't know, and
where you have a spindle and
19 you have DNA, there is
a chance of incorporation of
20 extra chromosomal into
nucleus is much higher. So,
21 one cannot extrapolate
from low amounts and make
22 conclusions, and that
we be a really important
23 concern. Likewise, I think the issue of enhanced
24 survival and the
society responsibility are really
25 major concerns.
319
1 DR.
MULLIGAN: Also, I think there are
2 always more or less
competent people. You know,
3 for this sort of thing
I am sure it makes a big
4 difference and you are
going to have people that
5 are going to do this
that, I am positive, are going
6 to be much less
competent than the experts that we
7 heard. Therefore, you have to have in place some
8 characterization of
what damage can occur, what DNA
9 you can get and so
forth.
10 DR.
SALOMON: Now speaking for myself, I
11 absolutely agree with
that. That is why I said
12 earlier on that no matter
how we end up, the field
13 has to accept the
mantle toward understanding what
14 it is their product
is, what they are injecting.
15 Even if that is not
absolutely settled in the first
16 trials, that is fine
but that is the direction this
17 has to go for all
those reasons. It is not just to
18 do it in three or four
really wonderful
19 laboratories, which is
where it has been done up to
20 now, but it is doing
it in 40 or 50.
21 DR.
CASPER: I think we have to be a bit
22 careful because the
numbers are so small in terms
23 of looking at
chromosomal abnormalities, and so on.
24 Just as an analogy,
there was a paper published
25 concerning sex
chromosome abnormalities in ICSI
320
1 offspring that showed
a 33 percent incidence of sex
2 chromosome
abnormalities but it was based on 15
3 pregnancies, and here
we are talking about less
4 than 20
pregnancies. Whether that 20 percent
5 figure is going to
hold up or not, I very much
6 doubt it. I think it will be very much lower,
7 probably close to
baseline if you got to the
8 position where you had
enough pregnancies to
9 actually look at. I understand that we are talking
10 about small numbers
but that can just magnify a
11 problem out of
proportion.
12 DR.
SCHON: Could you elaborate on why you
13 believe that is a
tenable position?
14 DR. CASPER: Only based on the previous
15 experience with ICSI
which really didn't hold up at
16 all. The initial paper that came out, suggesting
17 that there was a 33
percent abnormality rate turned
18 out not to be correct
at all when people started to
19 examine hundreds of
ICSI pregnancies.
20 DR.
MURRAY: I am definitely not a
21 statistician but this
is the classic case of why
22 take that point of
view. I mean, it could be a
23 statistical
abnormality in either direction. I
24 don't understand why
it is that in this particular
25 case this will turn
out to be in the wrong
321
1 direction. I just don't get the logic behind why
2 that would be the case. You are saying that in one
3 other case there is a
side effect that turned out
4 not to prove to be
statistically significant. I
5 mean, how many
hundreds of examples of that sort of
6 thing are the
case? But there are also cases where
7 the data set shows you
a certain percentage and
8 then the next data set
shows twice that percentage.
9 I just don't
understand it. I don't get it.
10 DR.
CASPER: It just seems to me that that
11 is a very high
number. It is out of proportion to
12 the sorts of
chromosomal abnormalities that we see
13 with most assisted
reproductive technology type
14 procedures. That is all. I am just saying that I
15 think we have to be careful
in interpreting the
16 numbers because the
numbers are so small at this
17 point.
18 DR.
SALOMON: Dr. Moos?
19 DR.
MOOS: It is worth stirring into the
20 pot the consideration
that we don't know the
21 prevalence of
chromosomal abnormalities in the
22 population of women
presenting these procedures.
23 It may be
significantly higher than in the normal,
24 healthy
population. So, we don't know the
25 denominator. It is, however, impossible to ignore
322
1 this even if, given
the sample size, it is a
2 statistically
improbably event, not likely to be
3 repeated. Dr. Mulligan's point that the coin could
4 come up heads or tails
I think is perfectly well
5 taken.
6 DR.
SAUSVILLE: But to me that is all the
7 more cause for some of
the product characteristic
8 issues that we just
talked about previously. After
9 some sort of modeling
process and after figuring
10 out whether
mitochondria are necessary, and whether
11 it is the RNA that is
doing it, we come forward
12 with a pristine, let's
say, product and there still
13 may be evidence of
this occurring, then that would
14 become a more obvious
conclusion. As the issue
15 stands now, if this
outcome were to occur we would
16 not know whether any
of those other things, plus
17 the intrinsic
susceptibility of the recipient egg
18 to this sort of thing
would be relevant.
19 DR.
MURRAY: I am more focused on the
20 second worry, the
worry about chromosomal DNA or
21 the cellular
fragments, and I cannot disentangle my
22 thinking about that
from exactly the point Dr.
23 Sausville was
raising. What is it that is
24 operating here? I mean, we are injecting a soup
25 or, maybe even better,
a stew into the egg and it
323
1 is full of lots of things,
and we sort of roughly
2 know what is in the
stew but we have no idea what
3 component or
components of the stew are making a
4 difference, if they
are making a difference,
5 including the DNA
fragments and the other cellular
6 components. Until we have a clear idea, we have a
7 plausible notion of a
mechanism and some evidence,
8 and I think it would
not be impossible to create
9 some experiments in
both animal cells and human
10 embryos that would
take us toward answers, it is
11 difficult to justify
doing a human trial with the
12 risk of transfer or
chromosomal elements until we
13 have a sense of
whether they are, in fact, at all
14 necessary in that
stew.
15 DR.
SAUSVILLE: To be clear, the issue is
16 not only the transfer
of chromosomal elements, but
17 multiple experiments,
extending back to some of the
18 classical experiments
in bacterial genetics, is
19 that DNA is
mutagenic. So, it is not only a
20 question of passively
adding something, it is
21 something actively
altering something.
22 DR.
SALOMON: I think the other thing that
23 just came out in last
weeks is studies on the
24 nature of the
algorithms used to call the number of
25 genes in the human
genome. Just to explain that
324
1 for those of you who
didn't catch the last issue of
2 Nature Biotechnology,
the call was that there were
3 30,000 to 40,000 human genes, which upset a
lot of
4 humans--
5 [LAUGHTER]
6 --because
there didn't seem to be enough
7 genes to make us
different than mice and everybody
8 was uncomfortable with
that concept. It comes down
9 to the fact that when
they really began looking at
10 different ways of
calling genes that there may be a
11 lot of RNA transcripts
in cytoplasm that encode
12 for--
13 [Laughter]
14 --see, I
told you you would like this
15 stuff! There would be a lot of RNA transcripts
16 that are clearly not
called formal genes in the
17 original genome
project algorithm. What that also
18 raised was the
possibility that a lot of these RNAs
19 wouldn't necessarily
have to encode proteins but
20 would encode RNA
molecules, like ribosomes for
21 example, that have
enzymatic activities that alter
22 different cell
functionalities. So, I just bring
23 up to you that one thing
that we haven't talked
24 about that is
certainly reasonable to put on the
25 table here is that
another uncertainty in the
325
1 safety issue is RNAs
that are not transcriptionally
2 active for proteins
but, rather, are important
3 perhaps in other
cellular functions. I mean, maybe
4 one of the reasons you
are getting these XO
5 chromosome
abnormalities is some sort of imprinting
6 phenomenon. That is just a wild speculation, but I
7 think it is more than
just mitochondrial DNA that
8 is getting transferred
that has a genetic lineage.
9 That is just to make
it a little more complicated.
10 I am told
the other mike is now fixed.
11 You will be the
experiment on this.
12 DR.
SABLE: I am David Sable, medical
13 director for the
Institute for Reproductive
14 Medicine at St.
Barnabas. I really want to clarify
15 the very excellent
point Dr. Moos made regarding
16 the baseline
chromosomal abnormality issue, and I
17 really want to make
sure that are assumptions for a
18 control group are
appropriate. The pregnancy loss
19 rate in an IVF
population at our center, and that
20 is what we are
comparing this particular subset to,
21 with a mean age of 37
is 22 percent, and the
22 overwhelming majority
of these are chromosomally
23 abnormal, and the
single most common chromosomal
24 abnormality in a
pregnancy loss is 45 XO. So,
25 these numbers together
suggest that we are actually
326
1 very close to the
middle of the bell curve. The
2 direction of the
conversation seems to keep veering
3 to where we have this
assumption that there is this
4 huge discrepancy
behind the background population
5 and I don't believe
the data supports that.
6 DR.
SALOMON: That is an excellent point.
7 Before you sit down,
the question then would be if
8 we have a population
of infertile women, many of
9 whom are older but not
all of whom are older, and
10 we now are capable,
with this technique or a
11 technique that we are
discussing a few months from
12 now, of rescuing a
higher percentage of those
13 oocytes, is it not
reasonable then to be concerned
14 about all the
implications of rescuing embryos with
15 potential genetic
abnormalities?
16 DR.
SABLE: That is an excellent point,
17 however, let's make
sure we are not reading too
18 much into a single
case. One of the XOs aborted
19 spontaneously.
20 DR.
SALOMON: We will stipulate that your
21 point on the XOs was
well taken--
22 DR.
SABLE: No, theoretically I agree
23 completely. I just don't want to imply or allow us
24 to infer that the data
supports that that is
25 actually
happening. I think in theory, yes, it
is
327
1 the same point that we
would be concerned about
2 ourselves, however, I
don't want to take that
3 additional step and
say that the data so far,
4 including the losses
we have had, really deviates
5 significantly from
what the background control
6 should be.
7 DR.
SIEGEL: In that same population
8 though, what is the
proportion of 45 XO in the
9 successful live birth
pregnancies?
10 DR.
SABLE: I am sorry, repeat the
11 question.
12 DR.
SIEGEL: You said that 27 percent--I
13 don't want to re-quote
your numbers but that 45 XO
14 was a common cause in
spontaneously aborted
15 pregnancies, many of
which were chromosomal
16 abnormalities. What about in successful
17 pregnancies, what has
been your incidence of 45 XO?
18 DR.
SABLE: I don't think we have had a
19 report of 45 XO, but
we have had pregnancies
20 terminated after
second trimester genetic testing.
21 Thank you.
22 DR.
SALOMON: I think that in general here
23 there is consensus on
the part of the committee
24 that there are real
safety issues potentially that
25 play in this field,
and that the amount of data
328
1 that we have right now
in animal models, which we
2 will talk about a
little more a little later but
3 for right now the
amount of data in the animal
4 models doesn't really
settle the issue adequately,
5 albeit they contribute
in some ways positively, and
6 the data in the human
system is just really not
7 adequate to make any
statements at all about,
8 neither safety or
efficacy. That is my attempt to
9 summarize this first
part of the discussion. Does
10 anyone disagree? I told you from the beginning you
11 are welcome to
disagree. I am just trying to make
12 sure I am giving you a
good summary.
5:30
DR. NOGUCHI: Dan, is it true
that there
13
14 are a few safety
issues that seem to have been at
15 least allayed to a
certain extent? When you are
16 speaking of the human
experience I think it is with
17 that caveat that in
terms of some of the mechanical
18 parts of ICSI that may
be helpful. But you are
19 talking about two and
three specifically.
20 DR.
SALOMON: I think two, three and four.
21 I think number one, I
think everybody kind of
22 agreed, you are right
and thanks for pointing that
23 out, we sort of agreed
that that didn't seem to be
24 a big deal in that
they have a lot of experience
25 doing ICSI and this is
an incrementally small
329
1 increase. I think we said that, if everybody
2 agrees with that.
3 But for two
and three there is clearly
4 some real risk there
and the clinical data doesn't
5 address it. For four, I don't think we really
6 know. I think it is correct to point out that at
7 least the animals are
reproductively active and are
8 overtly healthy, but
we are not very good mouse
9 veterinarians when it
comes to really know what
10 their kidney, heart,
liver and other functions are,
11 and living in little
sterilized boxes, being
12 perfect food is not
really a measure of health
13 either as judged by
SKID animals, fine, but look at
14 SKID children. So, the heteroplasmy thing I think
15 still remains an
unclear issue.
16 DR.
MURRAY: Just to follow-up on that
17 point, Lori Knowles
observed, and I believe this is
18 correct, that many of
the human manifestations of
19 mitochondrial disease
are late onset. So, we would
20 have an issue of would
we have an ability to
21 follow-up with such
children to see if there are
22 early signs of these
later onset diseases. That is
23 not, to me, an
absolute barrier to doing it; it is
24 a challenge for us.
25 DR.
SALOMON: I think it is an interesting
330
1 similarity to all
these other fields that we have
2 dealt with in biology,
in gene therapy, cell
3 transplantation and
stem cells that there is going
4 to be this demand or
strong pressure for long-term
5 follow-up of the
recipients.
6 DR.
SCHON: I am not that worried about
7 item four, and on the
particular case the worry
8 that is being
mentioned, let me remind you that
9 this invisible woman
is of age 25, 30, 35. She
10 carries the same
genotype presumably as whatever is
11 being donated to this
child, to this oocyte. The
12 woman donating the
cytoplasm is apparently normal.
13 That is why she is
donating it. The presumption is
14 that her mitochondria
are okay and, therefore, what
15 is being transferred
presumably is okay unless
16 there were some random
mutation, and these things
17 happen and, in fact,
that is what mitochondrial
18 diseases are. So, from that score, I am not all
19 that worried.
20 DR.
SIEGEL: Then that is predicated on
21 the assumption that
the donor women are screened
22 for mitochondrial
disease.
23 DR.
SCHON: No, no, the presumption is
24 that the donor woman
looks normal when she walks
25 into the clinic.
331
1 DR. SIEGEL: Is that what you would
2 recommend as
screening, that she looks normal? Is
3 that what you are
saying?
4 DR.
SCHON: I will rephrase it. This is
5 serious. Everybody in this room is different.
6 Everybody in this room
had different mitochondrial
7 genotype. We all have a sort of societal consensus
8 presumably--physicians
will disagree--that we are
9 fundamentally normal
unless proven otherwise. And,
10 for me to, let's say,
sequence somebody's genome
11 where there are 16,000
factorial possibilities of
12 genotype, and for me
to then say that this genotype
13 is good and this one
is not good is just not going
14 to happen. You have to have some kind of rule of
15 thumb. To me, if the physician says she passes my
16 criteria for donation,
I have no way of saying at a
17 molecular level,
except the most rough molecular
18 level, that she is not
a candidate.
19 DR.
SALOMON: That is a key point,
20 particularly as one of
the duties we have to this
21 field, to this group
of people here is that we
22 don't demand
unnecessary testing that is not
23 efficacious or doesn't
answer the issue.
24 DR.
SCHON: We certainly could test for
25 the 150 known
mutations. Fine.
332
1 DR.
MURRAY: I am wondering if a pedigree
2 would be useful for
the cytoplasm provider.
3 DR.
SHOUBRIDGE: If you look at the
4 pedigree that I showed
in five generations, there
5 was one affected
individual that happened in the
6 fifth generation. But I think the number that
7 might be important
here is the prevalence of these
8 mutations that we know
about in the population. No
9 epidemiological
studies have been done in North
10 America, but those
that have been done in Europe,
11 in Continental Europe
and in the United Kingdom,
12 suggest that it is
about one in 8,000 or so, one in
13 8,500. So, the chances of having somebody who
14 looks, to use your
words, normal walking into the
15 clinic as a carrier of
one of these is pretty slim,
16 and many of these
people will manifest some aspect
17 of these disorders
which a physician could pick up.
18 So, you have to
balance testing the whole genome
19 looking for mutations
against the chances that
20 somebody will come in
off the street who is a
21 carrier of a
pathogenic mutation.
22 DR.
SCHON: This returns to the point that
23 I tried to make
before, that I think heteroplasmy
24 is not without risk
for the reasons that you cited.
25 I see the risk of an
active mitochondrial disease
333
1 of being significant
is relatively low. What you
2 get into is the
unknown of having some sort of
3 interaction between a
paternal genome with some
4 maternal mitochondrial
genome that would not have
5 gone to fruition
otherwise now being in an abnormal
6 context. Again, that is the sort of thing that, in
7 my mind, reflects an
unknown procedure and could
8 probably put in some
way into an informed consent
9 that could lay that
out, not satisfactorily in an
10 absolute sense but in
a way that certainly is no
11 different than we
attempt to address when we bring
12 an unknown drug to a
population for the first time.
13 DR.
SHOUBRIDGE: Just to make it clear,
14 the paternal genome
sees a new mitochondrial DNA
15 every generation.
16 DR.
SCHON: But it is a contextual thing.
17 It is mitochondria in
the context of a given
18 maternal gene.
19 DR. MURRAY: I think that your work is so
20 interesting and
important to hear because it says
21 that, depending upon
the combination of the two,
22 different things can
happen. You showed exactly
23 that. Right?
So, if you put in something and have
24 a certain maternal
copy, it may well behave
25 differently than it
had behaved before because
334
1 there is some sort of
complicated competition or
2 genetic background in
the recipient that will maybe
3 accept that.
4 DR.
SCHON: In this case, of course, what
5 we are showing is that
there is nuclear genetic
6 control which could
just as easily come from mom or
7 dad. You are right. So, I accept the point.
8 DR.
MURRAY: I would just say that on the
9 testing I think you
would certainly want to test
10 for whatever it is,
the 150 known things even
11 though they are
infrequent. That is the least you
12 could do.
13 DR.
SCHON: It is easy to do.
14 DR.
SALOMON: It is easy to do?
15 DR.
SCHON: Yes. You would take a sample
16 from the mother and
just sequence her genome.
17 DR.
SALOMON: Sequence her mitochondrial
18 genome which is, what?
7,000 to 8,000 kb?
19 DR.
SCHON: Yes, not kb, 16 kb.
20 DR.
SALOMON: Whatever, right. I don't
21 know how easy that is.
22 DR.
SHOUBRIDGE: No, because you are
23 looking for
heteroplasmy and sequencing is the
24 absolute worst way to
look for heteroplasmy so it
25 is not a trivial
matter.
335
1 DR.
SALOMON: This is probably a little
2 too technical. This is something the FDA is going
3 to have to deal with
but, again, I feel that one of
4 the things you should
hear from us is that I don't
5 believe anyone wants
to put an unreasonable demand
6 on these people. If it is easy to sequence and
7 find these, then it is
easy. Those are the things
8 I hope you will do
internally and be fair about it.
9 DR.
HURSH: I just want to get out the
10 point that egg donors in
the United States are not
11 tested for
mitochondrial disease. There is a lot
12 of egg donation going
on. If this was a serious
13 problem I think we
would have seen it by now.
14 DR.
SALOMON: That is another good point.
15 I would like to keep
going here because time is
16 getting short.
17 DR. VAN
BLERKOM: Just one point, I guess
18 I am not concerned so
much about heteroplasmy per
19 se, but I think maybe
one issue that needs to be
20 addressed is the
extent of heteroplasmy. Is the
21 finding of 50 percent,
or 30 percent or 40 percent
22 of donated
mitochondria an issue to be concerned
23 with, number one.
24 I guess the
other issue, and maybe Dr.
25 Cohen can answer is,
is whether or not in
336
1 successful cytoplasmic
transfers there have been
2 cases where there are
no detectable donated
3 mitochondria, so there
is no issue of heteroplasmy
4 at all.
5 DR.
COHEN: I think I said that 10/13
6 tested are
homoplasmic. So, one could argue that
7 the tests are maybe
not sensitive enough and that
8 it changes over time
and next year it is better
9 again. The samples are stored and we will check
10 them again when the
technology becomes available.
11 DR. VAN
BLERKOM: But using the same
12 methodology you were
detecting high frequencies, in
13 fact there were ten
cases where there was no
14 heteroplasmy.
15 DR.
COHEN: That is right.
16 DR.
SALOMON: The only other issue I would
17 add to that is that
you are testing peripheral
18 blood. One of the problems with peripheral blood
19 testing of something
as complex as heteroplasmy--
20 DR.
COHEN: Yes, I would like to biopsy
21 all their vital organs
twice a year but it is hard.
22 DR.
SALOMON: I wasn't trying to be
23 facetious.
24 DR. COHEN:
What we try to do is go with
25 pediatric care and
when they go to the pediatrician
337
1 we come along. That is sort of what we do. I hear
2 from bioethicists that
we have to follow them for
3 life, well, that is a
stigma and we have no
4 intention at all to do
that.
5 DR.
SALOMON: That is good to know.
6 DR.
MURRAY: Don't over-interpret what has
7 been said here. I think you are taking that way
8 too far. What I heard Dr. Salomon saying was
9 weighing the
pertinence of the data that in
10 peripheral blood you
are not finding heteroplasmy,
11 one must take into
account that one could find it
12 in other tissues
because we know there is
13 differential
expression, nor were the ethicists
14 that you have heard
from today saying that these
15 children must be
hounded for life. That is not the
16 point. The point is we have to think about the
17 issue of late onset
and how we are going to deal
18 with it. One way to do it is to say it is just
19 impossible; it would
be an unreasonable burden.
20 Another way is to try
to at least persuade the
21 parents and eventually
they will be young people,
22 not children, that it
would be very helpful for the
23 future of this
procedure for them to make
24 themselves available
voluntarily. There are a lot
25 of approaches.
338
1 DR.
SALOMON: I would like to go on.
2 DR.
SHOUBRIDGE: One small point, all the
3 data we have on
humans, which is very limited, and
4 on mice, which is
quite a lot, suggests that if you
5 sample one fetal
tissue you have sampled them all.
6 So, if you really
wanted to determine whether or
7 not a fetus was
heteroplasmic you should be able to
8 do it from embryocytes
and then you would know.
9 So, the issue of what
to sample after birth to
10 determine heteroplasmy
is a thorny one and you
11 won't solve it. You are not going to biopsy
12 perfectly health
children; there is no way. But
13 you could determine it
from either a CVS sample or
14 amniocytes.
15 DR.
SALOMON: The next big section is the
16 risks to the
mother. Might risks to the mother be
17 different from those
incurred with established ART
18 procedures? For example, the possibility exists
19 that the ooplasm might
enhance the survival of
20 abnormal embryos to
incur additional medical risks
21 to the mother, for
example late term abortion. Any
22 comments?
23 DR.
RAO: I would say we just don't know.
24 There is just not
enough data; the sample size is
25 too small.
339
1 DR.
SALOMON: In the clinical experience
2 we heard today--I am
looking to Dr. Cohen and
3 others for
confirmation--it seems like there was
4 one abortion in the
group of three that Dr.
5 Lanzendorf
presented. Is that correct? There was
6 one in three. One was a miscarriage and one
7 delivered twins. Is that correct?
8 DR. COHEN:
There were a total of 15
9 pregnancies and two
were just confirmation of
10 chemical rise in
ACG. That was a biochemical
11 pregnancy. There was one who miscarried before.
12 It was after
confirmation of the fetal sac but
13 before fetal heart
beat.
14 DR.
SALOMON: That is early, right.
15 DR.
COHEN: That is early, six weeks, five
16 weeks, four
weeks. Then there is the one twin that
17 was sustained until
amnio.
18 DR.
SALOMON: What I was saying there is
19 not an overwhelming
amount of evidence yet, albeit
20 the experience is
extremely small, that there is a
21 whole bunch of late
abortions due to chromosomal
22 abnormalities.
23 DR. COHEN: Not yet.
24 DR.
SALOMON: Are the risks to the
25 mother's future
fertility or ability to engage in
340
1 subsequent ART
procedures? Actually, Dr. Cohen,
2 you addressed that specifically, or Dr.
Lanzendorf.
3 I remember at least
one or two mothers who had
4 failed this and went
on to a second procedure and
5 delivered a normal
pregnancy, or at least became
6 pregnant. I am not certain they said it was a
7 normal pregnancy. Is that fair?
8 So, I would
say here the only way the
9 risks to the mother
are going to get established
10 would be a formal
clinical trial. I don't think
11 this is an issue that
is going to get settled by
12 any further discussion
here, unless someone
13 disagrees.
14 I would like
to go to question number
15 three or four. Three was kind of where I started
16 the afternoon. Are these data sufficient to
17 determine that ooplasm
transfer does not present an
18 unreasonable and
significant risk to offspring
19 and/or mother, and to
support further clinical
20 investigations?
21 We began
with our gut-level feelings on
22 it, went into the
safety as I promised, and we are
23 sort of back here
again. Is there more discussion
24 or do we all feel
pretty comfortable with the
25 discussion we have
already had?
341
1 DR.
SIEGEL: Well, there has been
2 discussion but of a
somewhat different and related
3 question. I would like to know the advice of the
4 committee on this
question. I would on that point
5 clarify further that,
because I gave a partial
6 clarification but I
left an important piece out
7 when I said that we
put trials on clinical hold
8 based on unreasonable
and significant risks. We
9 also put trials on
clinical hold based on
10 inadequate information
to determine whether there
11 are unreasonable and
significant risks. That is
12 what we will do, for
example, if we believe that
13 there are important or
critical preclinical studies
14 that could be done
that would lead to a better
15 assessment of the
risks, a better design of the
16 trial, a better
informed consent, and so forth,
17 that need to be done
before the trials are done.
18 That is sort of where
we are going with this
19 question in asking are
there sufficient data to
20 make that
determination and, if so, is there a
21 determination that
there is not unreasonable--
22 DR.
SALOMON: So, let me make sure that we
23 pose this just right
because, as I told you at the
24 beginning, I think
this is a very key issue that
25 formed my thinking
around the discussion we have
342
1 had. If we determined that there is no
2 insufficient data to
determine efficacy, regardless
3 of the discussion we
have already had about the
4 amount of data
sufficient to establish safety, just
5 on the efficacy issue
could we advise, or would the
6 FDA agree to put a
hold on a set of studies on that
7 basis?
8 DR.
SIEGEL: If you were to determine or
9 advise that the
rationale for any benefit is so
10 slim as to not justify
the perceived risks, then we
11 could do that. So, we do consider risks in the
12 context of rationale
but we are not, in general,
13 terribly aggressive on
the rationale piece if the
14 hold is based on the
risks, and I think where there
15 is scientific
disagreement or where there is
16 scientific consensus,
or pretty close to consensus
17 or pretty solid
evidence that is one thing, but
18 where there is
disagreement we are, I think
19 appropriately,
reluctant to assess that our
20 assessment of the
rationale is better than somebody
21 else's who is also
appropriately assessing.
22 DR.
SALOMON: So, we are back to what I
23 described earlier as a
sort of knife's edge here.
24 We have some safety
issues. There are some
25 efficacy issues, and
we need to think again now in
343
1 terms of the
discussions we have already had how we
2 are going to balance
because that is really an
3 important circle that
we have to complete. So, Dr.
4 Murray?
5 DR.
MURRAY: I may jot be formulating in a
6 way that the FDA will
find useful but it is the way
7 I am formulating
it. I think we have had a good
8 discussion about a
number of risks to the offspring
9 and to the woman, to
the point where we can say
10 that for most of them,
and not all of them and that
11 is a big
"but" there is reasonable either
12 combination of
evidence or evidence sometimes by
13 analogy that they
don't seem to be outrageous
14 risks.
15 The one
piece that remains for me of
16 significant concern is
the possible transfer of
17 cellular components,
DNA of various forms, etc. I
18 would refer to that as
a very poorly characterized
19 risk. We really don't know what we are getting.
20 The problem is the
stew problem.
21 The way I am
formulating it that may not
22 be helpful is I feel
like we need to know more
23 about what the active
ingredient or ingredients are
24 in this stew because
at this point we may be
25 exposing offspring to
risks that are utterly
344
1 unrelated to the
therapeutic component of the
2 ooplasm transfer. It is longer than I meant it to
3 be.
4 DR.
SIEGEL: And that is pertinent because
5 risks that are
unrelated to a therapeutic are
6 probably less
reasonable from the perspective of
7 our regulatory
authority than risks that have to be
8 accepted in order to
have a chance of achieving the
9 benefit.
10 DR.
MURRAY: And we just don't know.
11 DR.
SIEGEL: No, definitely from
12 contaminants of active
ingredients in terms of
13 whether they need to
be removed, and if you don't
14 know which is which
you are at a disadvantage.
15 DR.
SCHON: I would like to raise
16 something to be sure
that we don't lose sight of at
17 least one part of this
picture. My lab and a lot
18 of the labs of my
colleagues work on mitochondrial
19 diseases because there
are women who have children
20 who are destined to
die, and some of them die very,
21 very early, and we
work on treatment of various
22 kinds. I hope one of these days one of those
23 treatments will be
debated in front of you guys.
24 But until that happens
the risk to benefit for
25 helping such a woman
and using a procedure like OT
345
1 is enormous. In the case of a woman who carries a
2 pathogenic mutation we
actually know what the
3 beneficial principle
is. It happens to be good
4 mitochondria, which is
a slightly different way of
5 looking at it but, no
matter how the FDA rules or
6 whatever you suggest,
I would like you to take into
7 account the enormous
benefit that might accrue to
8 those people who
really have cytoplasmic transfer,
9 if you will, would
really help even knowing that
10 there are these
problems of potential chromosomal
11 transfer, and so
forth.
12 DR.
MOOS: You are proposing that perhaps
13 pursuing an indication
where the rationale is
14 sufficiently strong
that we are not on the knife's
15 edge anymore, but the
balance is tipped strongly
16 gives us an entree
into a human trial that can
17 examine in some kind
of a safety series these
18 questions, and then
that can be extended to future
19 trials in infertility.
20 DR.
SCHON: As the other Eric pointed out,
21 there are other ways
to help these women that do
22 not necessarily
require OT but I don't want to
23 eliminate it as a
possibility, and some of these
24 other issues might
piggyback on that.
25 DR.
SALOMON: Drs. Rao, Mulligan and then
346
1 Casper.
2 DR.
RAO: I have one clarification I need
3 about the
question. When you say to support
4 further clinical
investigations, this is distinct
5 from clinical
research. Does clinical
6 investigation mean you
are thinking about
7 pregnancies in
follow-up and clinical research
8 means you are using
human blastocysts and looking
9 at those, or is there
no distinction?
10 DR.
SIEGEL: I am not sure we intended a
11 specific distinction,
but in this question what we
12 are asking is are
there enough data to do clinical
13 research that would
involve pregnancies? I am not
14 sure we have
consistently made a distinction in the
15 use of those terms but
I will tell you that in the
16 context of this, we
have IND proposals to do those
17 studies but we have
said they can only be done
18 under IND and we are
seeking advice as to whether
19 there is more that
needs to be done either in terms
20 of human egg research
that doesn't lead to
21 pregnancies or in
animal models prior to doing
22 that, or whether in
fact there are sufficient data
23 to make a judgment
that those studies with
24 pregnancies can
proceed.
25 DR.
SALOMON: Dr. Mulligan and then Dr.
347
1 Casper.
2 DR.
MULLIGAN: I was just going to propose
3 that we will never
come to consensus on any animal
4 experiment to find the
active ingredient because we
5 are not even at the
point really of finding the
6 active ingredient. We are at the point of whether
7 or not there is
anything to this. I mean, we are
8 all talking about
finding the thing, and I don't
9 think we would ever
agree, this group would ever
10 agree on anything that
would be compelling, that
11 would definitively
document that it is mitochondria
12 that is important or
that some other thing is
13 important. So, I would opt just to see if we could
14 get a consensus that
that is not an appropriate
15 avenue to pursue--well,
it is an appropriate avenue
16 to pursue but it is
not something that should limit
17 this going ahead and,
rather, focus on what
18 preclinical things do
we think really would have to
19 be accomplished before
we would want to see the
20 clinical work go back.
21 DR.
SALOMON: So, the question, Richard,
22 that you are getting
is, that I want to get to here
23 before it gets too
late, is it seems to me, and
24 correct me if my
thinking is not straight, that
25 there is this fork in
the road and we are not
348
1 getting past this fork
in the road. Depending
2 where we go on this
fork, it seems to me at least,
3 is telling us
everything that we have to discuss
4 then.
5 So, the
first fork is there is not
6 sufficient data. The trial designs weren't good;
7 there weren't enough
patients, whatever, in the
8 human studies to say
anything definitive. I think
9 we have all agreed on
that.
10 Now the
question is do we think that we
11 should go ahead and do
a study in humans, going all
12 the way to pregnancy,
using this field's sense of
13 which are appropriate
patients. Or, do we say, no,
14 there are too many
unknowns. We are not going down
15 that fork and then we
really have to define the bar
16 for preclinical
studies. Right? Because they are
17 going to want it and
they deserve that. We have to
18 go down one fork or
the other, or we ought to agree
19 that we can't agree
and we are stuck. That is okay
20 too, I guess.
21 DR.
MULLIGAN: I am saying we could say
22 there is a limited
number of things that could be
23 tested that would
impact upon the most easily
24 assessable risk.
25 DR.
SALOMON: So, are you saying that we
349
1 shouldn't do any human
clinical trials until we do
2 that?
3 DR.
MULLIGAN: Yes, but what I am saying
4 that might be is to
have people look at the
5 contaminated nuclear
DNA content or--
6 DR.
SALOMON: That is what I am saying, if
7 we take that fork,
then we can set the bar.
8 DR.
MULLIGAN: I think that we ought to
9 have a consensus on
this issue of is there
10 sufficient rationale,
and I agree that this
11 probably meets that
criteria, that there is some
12 rationale for this and
no data.
13 DR.
SALOMON: That is exactly what I
14 trying to get
that. Dr. Casper?
15 DR.
CASPER: I hope I can express this
16 properly, but I think
one logical thing that
17 follows from Dr.
Schon's comments that there could
18 be a huge upside from
treating mitochondrial
19 diseases is why not
think about mitochondrial
20 transfer, not ooplasm
transfer but mitochondrial
21 transfer? That avoids the nuclear DNA issue and
22 you are looking at one
specific component. So, if
23 it works, that would
help you to determine whether
24 or not that is the
right ingredient. If it doesn't
25 work, then you can
look at other components of the
350
1 cytoplasm but you
still might have some information
2 that may help people
with mitochondrial problems is
3 because what you are
really looking for is a good
4 source of mitochondria
for them.
5 DR.
SALOMON: I was thinking about that
6 but it doesn't really
address this fork in the road
7 issue, the reason
being that a woman with
8 mitochondrial disease
may be a candidate for
9 mitochondrial
transfer--these guys could go in that
10 direction and maybe
they have heard that today and
11 will do that. It might actually be a wonderful
12 thing to be doing, but
it won't address this issue
13 because the idea of
finding someone with
14 mitochondrial disease
is also an infertile couple
15 that would benefit
from this.
16 DR.
CASPER: I wasn't suggesting that we
17 go right to healing
mitochondrial disease, I was
18 thinking that if you
had somebody with fragmented
19 embryos and you do
mitochondrial transfer, either
20 it will work or won't
work. If it works, then
21 first of all, you have
found the active ingredient
22 for ooplasm transfer,
and also you have the upside
23 on mitochondrial
disease. If it doesn't work, then
24 you have to look in
another direction but you may
25 still have some
information that will help you in
351
1 terms of treating
mitochondrial disease.
2 DR.
SALOMON: I am sorry, I misunderstood
3 you. So, your idea is take the fork in the road
4 that takes you to
doing some limited clinical
5 trials now and do it
with mitochondria. You went
6 another step, and I don't
want to go there yet,
7 about what the
clinical trial design should be.
8 DR.
MOOS: With respect to the one issue
9 that I think many
agree is significant, the DNA
10 transfer, mention was
made of analyzing the donor
11 egg after transfer for
cytogenetics and that this
12 was very
insensitive. Is there any input that we
13 can get about how we
can satisfy ourselves, because
14 Lori Knowles certainly
made plain it was important
15 that we are not doing that,
using animal model to
16 validate our assay for
appropriate sensitivity.
17 You know 10-5 of the
human genome is still how many
18 base pairs?
19 DR.
SALOMON: I don't know anymore.
20 DR.
MURRAY: You could do something like Y
21 chromosome, some sort
of PCR, to look for whether
22 or not any inoculum
that you are going to inject
23 has Y chromosome
positively.
24 DR.
SALOMON: You could do genotyping on
25 the transfer and look
for genotypes that would be
352
1 unique to the
donor. You could take the ooplasm
2 and instead of
injecting it in an egg just do
3 genotyping on that to
see if there is chromosomal
4 DNA that was
detectable. You would actually do
5 then just DNA PCR.
6 DR.
SHOUBRIDGE: I just want to make a
7 couple of comments on
what Dr. Casper said. One is
8 there is no evidence
at all that women who carry
9 mitochondrial DNA
mutations have a fertility
10 problem that is
different than in the general
11 population.
12 DR.
SALOMON: That is where I was heading
13 before.
14 DR.
SHOUBRIDGE: Yes. The other thing is
15 that I think what you
said sort of presupposes that
16 there is a magic
bullet here, that all women have
17 the same problem and
that by doing one set of
18 experiments you are
going to identify it and I
19 would be pretty
surprised if that were true.
20 DR.
SALOMON: We have kind of danced up to
21 this fork in the road
a couple of different times.
22 A couple of people
have walked down it a little bit
23 but it is not like we
have rushed down it. Are
24 there some comments
from the community? Are you
25 guys satisfied? You have heard our discussion.
353
1 You have participated.
2 DR.
WILLADSEN: Well, it is not for us to
3 be satisfied or
dissatisfied at this point. We are
4 happy to be here, I
guess. But I should say--
5 DR.
SALOMON: No, it is for you to be
6 satisfied.
7 DR.
WILLADSEN: No, the committee is doing
8 its work. One speaker was saying that this type of
9 procedure would not be
permitted in Britain, but it
10 is actually
interesting that in Britain they left
11 an opening for
oocytoplasm transfer in the
12 legislation, I guess
on scientific advice. Now, we
13 know those people have
been wrong before in the
14 decisions that the
government makes there but,
15 nevertheless, they
have been thinking about that
16 and this particular
procedure has been kept open.
17 One of the
reasons why we have tried to
18 minimize the
intervention is that obviously at a
19 certain point if you
transfer too much cytoplasm it
20 is no longer a
cytoplasm transfer, it becomes a
21 nuclear transfer and
nuclear transfer, as we know,
22 has some big problems
that are special to itself.
23 Finally, on
the technical side, I think
24 that the chances of
getting little bits of DNA,
25 nuclear DNA transfer
with this procedure are
354
1 virtually non-existent
because the chromosomes are
2 aligned in one
bundle. You would have to transfer
3 a whole chromosome
virtually. I think it would be
4 impossible to tear off
a bit of DNA from a
5 chromosome. I am not saying it couldn't happen but
6 I don't think that is
a major concern.
7 Also, what
one can do is to check, as we
8 have done, that the
donor chromosomes are actually
9 in the remains of the
egg. That is not a
10 particularly difficult
thing to do. But the
11 concern is not nearly
as grave as we may have been
12 led to believe.
13 I should
also say that the possibility
14 that the mitochondrial
DNA that is being
15 transferred might
somehow interact unfavorably, be
16 it ever so rarely,
with the nuclear genome, well
17 the sperm provides
disintegrating mitochondria
18 every time you have
fertilization in the human.
19 Thank you.
20 MS.
KNOWLES: Can I just clarify the
21 situation in the
U.K.? I just want to be clear
22 that they have left
open the possibility for
23 mitochondrial
disease. The discussion is in the
24 context of
mitochondrial disease. In addition,
25 they are not allowing
clinical trials. They are
355
1 quite expressly not
allowing clinical trials until
2 there is more animal
and preclinical work.
3 DR.
WILLADSEN: I don't disagree about the
4 purpose of it, but you
have to understand that the
5 technique whereby they
are going to do it is going
6 to have to be this one
or not at all.
7 DR.
SALOMON: Anyone else? Dr. Cohen, at
8 this point you have
participated in this
9 discussion--I don't
think Dr. Lanzendorf is
10 here--and Dr. Grifo,
do you think that you should
11 go forward with a
limited clinical trial right now?
12 DR.
COHEN: I think we should consider it.
13 We did a pilot
experiment that has been a five-year
14 long pilot
experiment. The clinical demand is
15 enormous. There are many patients who have this
16 particular profile
have become successful. We
17 didn't do a randomized
study but these patients
18 were at the end of
their rope and considered egg
19 donation or
nothing. And, there are other groups
20 of patients that are
similarly interesting. There
21 is, for instance, one
group of patients that has
22 recurrent implantation
failure but has apparently
23 normal looking embryos
and they still don't become
24 pregnant again, again
and again. So, this is just
25 one small part of the
population but the population
356
1 is larger. I think I said in my presentation there
2 is a whole slew of
techniques that are waiting at
3 the sideline that has
just studied in animal models
4 that has tremendous
potential. There are ways of
5 doing egg freezing
using cytoplasmic transfer. I
6 won't go into
details. It is not just
7 mitochondrial disease
treatment that is a
8 potential. There are ways of duplicating sperm
9 genomes so that you
can do a genetic test on one
10 duplication and use
the other one, once you have
11 tested it, for
fertilization. All these
12 technologies,
aneuploidy correction, aneuploidy
13 avoidance, all these
technologies at this point in
14 time involve, in one
way or another, some
15 cytoplasmic transfer.
16 So, this is
a very important decision we
17 are taking, and the
biggest concern we have had,
18 and I think you are
sharing this, is the safety
19 concern. These are the biggest concerns. The
20 rationale, you can
only find out when you do the
21 clinical work, when
you do the trials. You can't
22 base it on animal
models. And, the safety concerns
23 have been highlighted
appropriately today. I get a
24 lot of questions when
I give presentations about
25 cytoplasm transfer,
but the concern of little
357
1 pieces of DNA being
slashed off chromosomes that
2 are now being
transferred is a concern I haven't
3 heard about in the
six, seven years of my
4 presentations. So, I must say I am not well
5 prepared. It is an original concern. The concerns
6 about the incidence of
aneuploidy or the issue of
7 heteroplasmy I think
were well highlighted today.
8 DR.
SALOMON: As I said at the beginning
9 of the day, our
purpose is to make sure that we
10 have adequately
presented the whole discussion, and
11 when we get to the end
of today, that is what I
12 hope people feel we
have done.
13 How about a
few minutes on what would be
14 an appropriate
clinical trial? Similarly, what
15 would be the key
animal experiments to do to bring
16 the whole group
forward to the point where we would
17 all naturally go down
the curve in the road that
18 says a clinical trial?
19 DR.
SIEGEL: Before we move on to that,
20 and I know we don't
want to be here all night but
21 given that we are
going to have to make some
22 difficult decisions,
often when there is a
23 consensus of the
committee you try to sum up. I
24 haven't heard you do
that on this question.
25 Because you started
asking the question differently
358
1 from the way it is
posed, I am not sure I have an
2 appreciation of the
consensus. If we move on, I
3 assume the best advice
is that we are just supposed
4 to kind of put it all
together, but I wonder if it
5 might be helpful--
6 DR.
SALOMON: Well, I put it one way and
7 tried to get at it,
and then I put it the other way
8 with your help, and I
don't know that we got at it.
9 DR.
SIEGEL: It might be useful to poll
10 the committee members
as to whether they think
11 before doing trials in
human during pregnancy there
12 is additional animal
work to be done. If so, what?
13 That is sort of
question number four and I think
14 Dr. Mulligan pointed
out correctly that it is hard
15 to ask one question
without the other because, in
16 fact, if there is no
useful animal work, even if
17 you would like to have
more data from animals if
18 there is nothing that
is going to be relevant--
19 DR.
SALOMON: Let me just try to get a
20 consensus here, what I
have heard from everyone is
21 that this is the fork
in the road. That probably
22 based on everything we have heard, most of us
would
23 probably be okay with
a well-designed, very limited
24 clinical trial going
forward, but we haven't talked
25 enough about what a
well-designed clinical trial
359
1 would be. The rest of us would be much happier if
2 they would put
themselves on hold and do the animal
3 work and come back in,
you know, six months to a
4 year and reassure us
on some of what we have
5 articulated as safety
issues. But I think we can
6 certainly poll the
committee on that, but that is
7 my thinking. Let's go around. Dr. Casper?
8 MS.
CASPER: I am not sure I am ready to
9 decide yet. I think it would be nice to do some
10 animal work. I am just not sure there is an
11 appropriate model
available.
12 MS.
KNOWLES: I think you probably know
13 what I am going to
say. I think we should be doing
14 some animal work and
some human embryo work before
15 a clinical trial.
16 DR.
NAVIAUX: From what we have heard,
17 there doesn't seem to
be a defect in an animal
18 model to try to
correct so we would never be able
19 to get an inactive principle
in animal studies,
20 which is justification
for well-designed basic work
21 in human studies.
22 DR.
SHOUBRIDGE: I think we should be
23 doing all of the above
because I don't think there
24 is a right or wrong
answer here. As Dr. Mulligan
25 said, no one will
agree on an animal model. We
360
1 don't know what the
principles are, and the only
2 way to move a little
inch forward is to do some
3 limited, really good
trial in humans I think.
4 DR. VAN
BLERKOM: I would agree also with
5 that. I think the trial should be designed to
6 address the
fundamental issue of what defect is
7 being addressed. So, if you are transferring this
8 stew or soup, the
point is what are you really
9 addressing? What is the defect? I think if you
10 couple the cytoplasmic
transfer with the notion of
11 trying to identify
defects, whether it is
12 mitochondrial
fragmentation of whatever, I mean, I
13 think that is what is
important and I think you
14 could design it in
that way so you can get a handle
15 on the problem, if
there is one. It is a unique
16 situation because you
are not quite sure what is
17 wrong and you are not
quite sure if you are fixing
18 it.
19 DR.
MURRAY: I am actually very close to
20 Jonathan Van Blerkom
on this. We have questions
21 five and six, what
defects are being addressed, and
22 I agree, we don't
know. And number six, do
23 existing clinical data
from humans support a
24 rationale? The as is no. So, I would be unwilling
25 to favor any trial in
humans that did not have as a
361
1 main focus to identify
what it is that is actually
2 being addressed by
this therapy. In fact, I am in
3 no position to
challenge the basic scientists here
4 but it seems to me one
could do useful studies,
5 both in animals and in
human embryos. Just trot
6 out a few hypotheses,
it is the mitochondria. What
7 evidence would we have
the mitochondria are working
8 through the mechanism
of increased ATP, or calcium
9 ion transport? What sort of surrogate endpoints
10 could we study in
either humans or animals to see
11 if, in fact, what in
the cytoplasm transfer had
12 these effects? So, I think actually one could have
13 a number of
hypotheses, generate a number of
14 interesting research
questions. You know, it
15 wouldn't give you the
final answer but it would
16 indicate whether the
mechanisms we postulated are
17 plausible or not, and
I would like to see that
18 happening preferably
before we do it in humans, but
19 I wouldn't go to the
mat and say that we shouldn't
20 do a human trial to
elaborate those questions.
21 DR.
RAO: I looked through the risks with
22 the procedure that is
there and I tried to see if
23 there was any real
animal model in which one could
24 test this, and it is
very clear that if you think
25 there are going to be
late pregnancy problems or
362
1 childhood defects of
chromosomal abnormalities,
2 there is no real
clear-cut animal model which would
3 be appropriate. The best animal models are for
4 mitochondrial
defects. For those, I think it is
5 worthwhile doing
experiments in animal models.
6 But, on the other
hand, there seemed to be a
7 consensus that while
there might be a finite
8 unknowable risk in
terms of heteroplasmy, it is not
9 clear that we should
be stopping all experiments
10 because of that data.
11 So, what one
is left with then is to day,
12 yes, you have to do
this experiment. We need to
13 get more information,
and that information can only
14 come from human
testing. So, it seems that the
15 choice was between
doing human clinical work and
16 doing human clinical
investigations, and it seems
17 that both would be
necessary and it is not clear to
18 me that one can do
them one after the other or
19 whether one should do
them in parallel.
20 DR. MULLIGAN: I think I concur with that
21 point of view. I would want to see first just
22 better
characterization of whatever is being
23 injected, not only the
DNA thing but just
24 characterize the
consistency, if possible, of DNA
25 content or something
like that. Then, I like the
363
1 mouse model. I was intrigued by the mouse model
2 and I would encourage
people to look at that in
3 more detail. You know, with the history of all the
4 mouse knockouts, if
you look hard enough you may
5 well find
something. So, that is really worth
6 looking at. But I wouldn't say that you need that
7 information to go
ahead.
8 Scientifically, I think if you could
get
9 the people who are
going to do the clinical trial
10 to actually perhaps
look at--I don't know if this
11 is technically
possible--ooplasm without
12 mitochondria, or
highly decreased in it by
13 depending on where you
poke, or whatever, versus
14 things that are high,
it seems to me like that
15 would be interesting
too.
16 DR.
SALOMON: I try to be practical about
17 it. So, I see two sides to this coin. On one
18 side, I see some of
the most competent clinical
19 investigators out
there. This is a field that has
20 moved forward through
doing this kind of clinical
21 research up until
now. In general, I think
22 everyone respects the fact
that it has been done
23 well and done
ethically. There really are very few
24 smoking guns in this
field. So, I think that the
25 first part of the coin
is that I respect that, and
364
1 that gives me some
sense that a clinical trial
2 could be done, managed
properly under FDA
3 guidelines, that would
be well designed enough to
4 address the questions,
and that would be a step in
5 the direction of the
clinical trial.
6 The other
part of me sees the other side
7 of the coin, and that
is the reality that I am
8 looking out on a group
that are some of the best
9 clinical investigators
in the country, and the fact
10 is that I work in mice
and I work in non-human
11 primates as well as
humans and I think the truth is
12 that when I look at my
mouse breeders, at a certain
13 point they start
dropping off and I find that very
14 reasonable to
document, and I am not at all
15 convinced sitting here
that you couldn't find
16 quickly a mouse model
of older, less functioning
17 breeder pairs and it
wouldn't be that difficult,
18 and you would have
your mouse model.
19 Similarly, I
work at UC Davis primate
20 center where they have
3000 rhesus and over 1500
21 cinos, all of which
have got very detailed breeding
22 records and, again, I
am not certain that you
23 couldn't find--I don't
think this community is
24 really set to look in
those directions and that is
25 the other side of the
coin.
365
1 So with that
said, I think that I agree
2 with my
colleagues. At this point the people in
3 this field are willing
to do these clinical trials
4 and the mothers and
fathers that are coming to them
5 are clearly willing,
under the right umbrella of
6 consent and well-done
trials, to participate in it.
7 So, you know, I think
that is an argument for
8 taking that path. But I hope I have put it in some
9 perspective.
10 I certainly
think that we have to do
11 things to insist that
animal model work and safety
12 issues--I want to look
at messenger RNA transcripts
13 too and how this is
affecting the RNA
14 transcriptosome with
the oocyte, and I think it is
15 pretty ridiculous how
little data there is to
16 support any of this
and that worries me because it
17 is kind of a slippery
slope that I go through every
18 time, you know,
whether it is xenotransplantation
19 and, "oh come on,
leave us alone; we are just going
20 to do a little gene
therapy", or "you don't know
21 what you are doing; we
can just throw some genes
22 in." So, I am just saying I think as an
overriding
23 principle if we are
eventually going to go down
24 this clinical path, I
hope that there is a
25 consensus that there
is a real underpinning of
366
1 science.
2 DR. VAN
BLERKOM: Just to make a point, I
3 am not aware of mice
having menopause or
4 perimenppausal
conditions.
5 DR.
SALOMON: In our breeding colony, and
6 we now maintain
several different strains which we
7 have maintained for
generations, there is no doubt
8 that not only are
there better and worse breeding
9 pairs and we cull
these out because we are always
10 selecting for good
breeding pairs, but also after
11 some certain number of
generations the number of
12 pups they have per
delivery will decrease, and it
13 is very easy to
document. So, I am just suggesting
14 that that might be
when you step in and do the
15 ooplasm transfer from
a young mother.
16 MS.
WOLFSON: I am not convinced that
17 there are animal
studies that need to be done
18 before we go into
human pregnancies. I am not a
19 scientist so I can't
really go into those, but the
20 paucity of that
information frightens me when we
21 look at such a huge
outcome.
22 DR.
SALOMON: So, clinical studies or
23 animal studies?
24 MS.
WOLFSON: Animal and human embryo if
25 possible.
367
1 MS.
SERABIAN: I guess one thing I am
2 concerned with as a
toxicologist is what I call
3 worst case
scenario. I mean, here we have the best
4 of the best basically
that are performing these
5 studies in humans, and
when it gets to expanded
6 other sites, again, I
am thinking worst case, you
7 know, just going a
little too far, etc., that is
8 the kind of thing we
would want to look at in
9 animals, assume a
worst case scenario maybe not for
10 this initial phase
that we are talking about but,
11 for sure, as it
expands.
12 DR.
SALOMON: At a minimum also, if they
13 do a clinical trial
that they should do it with
14 very specific outcome
parameters for the different
15 steps, many of which
have been discussed.
16 MS.
SERABIAN: Right. Then, one other
17 comment with respect
to the animal studies, it
18 sounds like there is a
wealth of data that has been
19 published, maybe a bit
of it not published. It
20 would be kind of an
interesting idea if there are
21 certain organizations
or groups to somehow put this
22 in a document, master
files, a certain way to
23 submit to FDA that
everyone could refer to in terms
24 of the animal data.
25 DR.
MURRAY: There is one more complexity
368
1 that has come up
sporadically here but that we need
2 to bear in mind is
that I realize that, number one,
3 this isn't the kind of
thing people had in mind
4 when they wrote about
inheritable genetic
5 modifications but this
is plausibly, it will be at
6 least in some children
if they have offspring, if
7 they are females if
they have offspring, in a
8 stochastic fashion
some of the transplanted
9 mitochondrial DNA does
in fact end up in eggs that
10 become fertilized and
have children later, and I
11 don't know what to do
with that but I think it
12 would be a mistake to
simply forget that that is on
13 the table.
14 DR.
SALOMON: Dr. Schon, I realize that
15 you were out of the
room. What we did was go
16 around and just basically gave some final
thoughts
17 about which fork in
the road would you be
18 comfortable taking, to
clinical trials or no
19 clinical trials,
animal or go down both in a
20 parallel way?
21 DR.
SCHON: I have to think about this.
22 Maybe the one comment
I would like to make is that
23 it seemed to me that
there was--is everybody like
24 me? You don't answer the question, you sort of
25 make up your own
question and answer that one?
369
1 DR.
SALOMON: There have been eight
2 variations of that so
far.
3 DR.
SCHON: I have detected sort of a
4 merging of two issues,
which are the safety and the
5 efficacy, and I will
answer the question. Safety
6 means you have a level
of performance which suffers
7 no diminution when you
do something. So you are
8 here and you go
down. Efficacy is the reverse.
9 You are here and you
want to go up. One of the
10 confusions is that
when we discuss the analogy to
11 mitochondrial diseases
the bar is actually down
12 here because kids are
in bad shape, the eggs are in
13 bad shape genetically;
they are actually not in
14 such bad shape
physiologically. Now, anything you
15 do brings you up. So, to answer the question, for
16 issues of safety
clearly I think animal models are
17 the way to go. I mean, the question answers
18 itself. For issues of efficacy what I am hearing,
19 and I am no expert, is
that animal models are not
20 the way to go because
it is so hard to do. So,
21 some kind of clinical
trial for efficacy that
22 followed a preliminary
question on safety--you can
23 ask these things about
DNA fragments and so forth,
24 although you may not
be able to answer questions
25 about aneuploidy, and
maybe they can even go on
370
1 almost in parallel if
you did some of the questions
2 on human embryos,
fertilized human embryos without
3 implantation. I don't know of you are allowed to
4 do those kinds of
things, but if you were, that is
5 the way I would do it.
6 DR. SALOMON: I think we have certainly
7 answered almost all
the questions. I think the one
8 thing, sitting back
here, that we didn't really get
9 to--I mean, we have
talked about the preclinical
10 models. I don't know that there would be a lot
11 more. We have discussed the mouse model, talked
12 about the non-human
primate models. I don't think
13 that this community
has the tools to go into the
14 non-human primate and
mouse model, so we would have
15 to interest other
investigators around to come into
16 that area, and that is
the kind of thing that could
17 be done potentially
but those are unknowns.
18 The only
thing that I think we just may
19 have fallen a little
short of was exactly what
20 would be the clinical
trial. That is not a minor
21 gap. I am sure I will be reminded of this year
and
22 years from now about
how I failed the FDA on this
23 one. But we have talked a lot about the aspects
of
24 what the clinical
trial ought to be. I am going to
25 try and get some
consensus on that in a minute or
371
1 two. One thing I think we are all convinced of,
2 again correct me if I
am wrong but I think we are
3 all convinced that
there is a population of couples
4 who are not implanting
and are not being able to
5 have successful
pregnancies. I am not saying that
6 we all agree that
there is one problem for all
7 those women, and there
may not be, but there is
8 definitely an
identifiable population that is the
9 target of this.
10 I think Dr.
Cohen made the very good point
11 that there are a
number of other variations that
12 are behind this that
are relevant. So, the
13 population is outcome
there. I think population
14 choice--I think these
guys have that pretty well
15 nailed down. I don't think they have been picking
16 the wrong women to do
it in.
17 We want to
know efficacy. We have talked
18 about what the safety
issues are. So, whatever
19 that clinical trial
design is that you do, it has
20 to give us safety and
it has to give us some
21 insight into the
nature of the product, what is in
22 that ooplasm--DNA
fragments, RNA transcripts? How
23 many mitochondria are
in there? Does mitochondria
24 have anything to do
with this? What kind of
25 measures would give
you mitochondrial function? We
372
1 heard ATP and then we
heard, come on, there are 50
2 other things that
mitochondria can do; get a grip.
3 We heard about
apoptosis testing, all of which is
4 commercially
available, etc. So, I think that is
5 the kind of thing that
would come relatively easy
6 is you sat down and
said what are the aspects of a
7 clinical trial.
8 Actually, I
have just talked myself into
9 the fact that we did
answer all of the questions
10 and I don't want any
grief later.
11 [Laughter]
12 DR.
SIEGEL; Well, I could come back years
13 later or now, I
guess--
14 [Laughter]
15 I don't want
to keep the committee forever
16 and, obviously, there
are a lot of unanswered
17 questions and we are
not going to answer all of
18 them. One or two that stand out in my mind is that
19 we did hear a comment,
I think from Dr. Cohen, that
20 there is no intent for
long-term follow-up of these
21 children. I guess it would be useful to know from
22 the committee whether
they think that is an
23 acceptable way to move
forward, and if we allow
24 trials to be done
without long-term follow-up, then
25 in the long term we
still won't know what the
373
1 long-term effects are.
2 DR.
SALOMON: We fought and died over this
3 one in gene therapy in
xenotransplantation so I
4 can't believe I am
back again discussing this
5 problem. Fro Dr. Cohen's sake, xenotransplantation
6 now is follow-up
forever, and we are really not
7 interested in whether
the investigators want to do
8 that or not. That is what has been said. In gene
9 transfer studies it is
a movable target depending
10 on some of the issues
of an integrating vector,
11 non-integrating vector
etc., but it is as long as
12 15 years in some vector
classes. But the good news
13 is that in these
trials, just to give you the
14 background here so you
guys don't faint, a lot of
15 the long-term
follow-up came down to sending a
16 postcard once a year
kind of thing: "are you
17 alive?" That sort of thing. So, you guys might
18 ask "are you
alive? Do you have mitochondrial
19 defect."
20 DR.
SABLE: Just to give an idea how
21 seriously we do take
it, we had one of our
22 investigators in the
delivery room, breach
23 delivery, and the
investigator has gone to the
24 pediatrician's
appointments. So, we don't mean to
25 imply that we are not
serious about follow-up, I
374
1 think it is just a matter of degree.
2 DR.
SALOMON: With that background, I also
3 wanted to educate
those of you who are not privy to
4 these other long
discussions at multiple BRMAC
5 meetings of long-term
follow-up. What do you guys
6 think? Again, we can just get some quick opinions.
7 Why don't we just go
around? Dr. Casper, long-term
8 follow-up?
9 DR.
CASPER: Yes, I think it is
10 reasonable.
11 MS.
KNOWLES: Yes, I think obviously there
12 should be a very rich
informed consent procedure
13 about what long-term
follow-up would look like up,
14 particularly when we
are talking about inheritable
15 genetic modifications,
how long that might have to
16 be.
17 DR.
NAVIAUX: Yes, I think long-term
18 follow-up is going to
be required, and there should
19 be a default
pathway. After doing the routine
20 monitoring, if
anything abnormal comes out in
21 development, if there
is abnormal growth of the
22 child or abnormal
cognitive development, then there
23 should be an
intensified examination to look for
24 why.
25 DR.
SHOUBRIDGE: I think so too. If you
375
1 could demonstrate that
you haven't actually
2 transferred DNA, then
that would, of course, change
3 how long might want to
follow-up.
4 DR.
SALOMON: I just want to add that that
5 is one of the concepts
that came out very clearly
6 in the gene transfer
experiments as well.
7 DR.
SCHON: I don't think I am competent
8 to answer the
question. It seems to me that
9 whoever designs the
clinical trial, it is incumbent
10 on them to figure out
what the nature of the
11 follow-up is. I can't do it.
12 DR. VAN
BLERKOM: It would be nice to have
13 long-term trials, but
I just would put in a caveat
14 that in this field, in
IVF in particular,
15 compliance is an issue
because, believe it or not,
16 patients disappear,
regardless of what they signed
17 in their informed
consent, they leave their embryos
18 in storage
behind. So, it is a complicated issue
19 to get the type of
follow-up. Yes, you can put it
20 there in writing but
whether you actually get that
21 on the other end is a
different story.
22 DR.
SALOMON: I don't know that this group
23 is any less likely or
more likely to disappear than
24 our gene transfer
patients or the patients who
25 eventually will be
candidates for
376
1
xenotransplantation. But there
certainly is, on
2 the other hand, a
precedent for really
3 extraordinarily
successful long-term trials and, as
4 a principle, it is
quite possible to do, and I
5 don't think we should
approach it by saying, you
6 know, all these
patients disappear; there is no way
7 to do it.
8 DR. VAN
BLERKOM: It is not what I meant,
9 but it may be a
different category because it may
10 not be perceived on
the part of the couples that
11 this is a pressing
issue.
12 DR.
SALOMON: They won't be able to put it
13 on the income tax
return either.
14 DR.
MURRAY: No, but we can use the
15 internet. Years later it is eerily possible to
16 find you or anybody
else if you know how to look
17 and you are
determined. So, I would say, yes,
18 there should be
long-term follow-up. It should not
19 be onerous on either
the investigators or the
20 families, but
reasonable thought needs to be given
21 to what would be an
effective program of long-term
22 follow-up and I think
that is all one can
23 reasonably ask of
either party.
24 DR.
RAO: I can only second that. I just
25 wanted to add one more
thing. There were some
377
1 issues raised by Dr. Lanzendorf about
selection
2 criteria and controls,
and I think those are going
3 to be important
issues. Given that we don't think
4 there is a great
amount of data on actual benefit
5 or efficacy, that
means you have to select your
6 patient criteria for
any kind of trial and you have
7 to really define it
very carefully, along with
8 appropriate
controls. That is going to be
9 something that needs
to be factored in.
10 DR. MULLIGAN: Yes, and with your point, I
11 think the consent
form--I don't know if we are
12 going to get to that
but I think it really ought to
13 deal with this issue
of the data that does exist.
14 I am interested in
whether or not patients and
15 families would
actually find anything interesting
16 about the issue that I
think you raised about the
17 evolutionary
uncertainty. I think there ought to
18 be something about the
evolutionary things that
19 could occur.
20 DR.
SALOMON: I certainly agree with
21 long-term
follow-up. As I said, I have been
chased
22 around and around on
that already and I just accept
23 it as being a part of
the responsibility I think we
24 have. I don't mean to be facetious about it. I
25 think that in the end
the arguments for long-term
378
1 follow-up, when done
in a way that is not onerous
2 on the patients, don't
provide stigma, that carry
3 then anywhere from
school to insurance etc., if it
4 is done right I think
long-term follow-up is
5 important to the
community at large for these sort
6 of cutting edge gene
transfer experiments.
7 In terms of
a clinical trial, the only
8 other thing that I
would add to the picture is if
9 we go ahead with a
clinical trial in this area, I
10 really hope that when
you say, for example, that
11 here is a patient with
repeated failures to
12 implantation and then
we did the oocyte transfer
13 and we got such and
such a result, that those
14 patients are really
much better controlled than the
15 data we have seen so
far. I want to make sure that
16 it is all done at your
center under optimal
17 conditions and then at
your center you do it.
18 I was also
very concerned that 9 of your
19 28 patients in your
study, Dr. Cohen, were patients
20 who supposedly had
male infertility problems. I
21 wouldn't understand
why you were doing oocyte
22 transfer. Now, I may have misunderstood that
23 slide, but that is an
example of something I hope
24 you will design out of
a clinical trial.
25 DR.
COHEN: Thank you for mentioning that.
379
1 It is a very good
point. This was discovered after
2 the fact.
3 eggs were treated with
ooplasmic donation and the
4 remaining eggs from
the donor oocytes were injected
5 with the husband's
sperm. So, it is like a control
6 with the purpose of
freezing those embryos for
7 years clinically
later. But what we found is that
8 in nine cases the
embryos of those controls
9 developed as badly as
the embryos of the patient,
10 and I think that is
what I was trying to say. So,
11 it is sort of after
the fact. Looking at it
12 closer, some of these
were borderline male factors
13 and we could have
probably figured it out before
14 but that is a very
grey area.
15 DR.
SALOMON: Again, that would be
16 something that you
presumably could exclude on the
17 way to deciding this
is a repeat implantation
18 failure and won't
benefit from ICSI.
19 DR. COHEN:
Yes, you can do that but then
20 you have to do a
really big experiment, which is
21 get an egg donor and
test the sperm, yes.
22 MS.
WOLFSON: I think there should be
23 long-term follow-up in
whatever way is possible,
24 and insofar as there
could, in fact, be a DNA
25 transfer that is
involved, I think the follow-up
380
1 should go into the
second generation.
2 DR. SALOMON: Anyone else?
3 DR.
NOGUCHI: What I do want to say is
4 that I think this has
been an extraordinarily open
5 and frank meeting, and
is exactly the kind of
6 discussion and
interplay back and forth with the
7 community, the
practitioners and our colleagues to
8 really obtain advice
that we need, because these
9 are the questions that
my colleagues face daily and
10 actually are going to
have to do the reviews, and
11 this has been just an
invaluable experience. So, I
12 personally want to
thank all of you, all the
13 participants from the
public as well. This was
14 great. Thank you very much.
15 DR.
MOOS: One quick extension on a
16 comment Mercedes made
a bit ago, it seems as though
17 there are a couple of
issues that could be
18 addressed in
preclinical models, like validation of
19 DNA and so forth, that
could be done once
20 definitively in a sort
of platform mode and people
21 in the field could, in
fact, work together to
22 present us with some
useful approaches to
23 validating this. The quicker that some of these
24 safety issues, which
can be addressed in animal
25 models, can be laid to
rest, and it sounds like it
381
1 might be fairly easy
to do the DNA one for example,
2 the better for all of
us. Then we can begin with a
3 kind of staged
approach in clinical models that we
4 have all talked about,
and we have heard a lot of
5 discussion that it can
only be evaluated there.
6 So, think about it and
come talk to us.
7 DR.
SALOMON: Are there any last comments
8 from anyone that have
to be made before we adjourn?
9 If not, I would like
to thank everyone who came,
10 both the expert panel,
my committee, the FDA staff,
11 particularly staffers
like Gail and her group who
12 put all this together,
and everybody else. Thank
13 you very much for a successful
meeting. That group
14 of you who will be
here tomorrow, we will see you
15 tomorrow. Otherwise, everyone travel safely and
16 good health.
17 [Whereupon,
at 6:45 p.m., the proceedings
18 were recessed, to
reconvene on Friday, May 10,
19 2002.]
20
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