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.


 

                                                                 2

 

                        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.

                                                                 3

 

                            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.

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  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

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  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

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  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.