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

THIS TRANSCRIPT HAS NOT 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.

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.

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

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

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

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

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

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

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

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

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

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

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

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.

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

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

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

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

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

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

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,

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

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

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

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

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

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.]

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

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

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

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

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

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

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

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

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]

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

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

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

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

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

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

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,

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

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

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

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,

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

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

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

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

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

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

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

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

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

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.

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

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

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