FOOD AND DRUG ADMINISTRATION






                              OPEN SESSION


                              Meeting #32




                         Thursday, May 9, 2002


                               8:00 a.m.



                              Hilton Hotel

                         Gaithersburg, Maryland






                        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




      Bruce R. Blazar, M.D., Industry Representative

      Katherine A. High, M.D.

      Richard C. Mulligan, Ph.D.

      Alice H. Wolfson, J.D., Consumer Representative

      Alison F. Lawton, Consumer Representative

      Mahendra S. Rao, M.D., Ph.D.


      Temporary Voting Members:


      Lori P. Knowles, L.L.B., B.C.L., M.A., LL.M.

      Thomas F. Murray, Ph.D.

      Robert K. Naviaux, M.D., Ph.D.

      Eric A. Shoubridge, Ph.D.

      Jonathan Van Blerkom, Ph.D.

      Edward A. Sausville, M.D., Ph.D.

      Eric A. Schon, Ph.D.


      Guests and Guest Speakers:


      Robert Casper, M.D., Ph.D.

      Susan Lanzendorf, Ph.D., H.C.L.D.

      Marina O'Reilly, Ph.D.

      Jacques Cohen, Ph.D.

      Amy Patterson, M.D.

      Stephen M. Rose, Ph.D.


      FDA Participants:


      Jesse Goodman, M.D.

      Philip Noguchi, M.D.

      Scott Monroe, M.D.

      Mercedes Serabian, M.D.

      Jay B. Siegel, M.D.

      Deborah Hursh, Ph.D.

      Malcolm Moos, M.D.



                            C O N T E N T S


      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


                Lori Plasma Knowles, LL.B.                     257


      Open Public Hearing:

                Jamie Grifo, M.D., American Society

                  for Reproductive Medicine                    278

                Pamela Madsen, American Infertility

                  Association                                  283

      Questions to the Committee                               287



  1                      P R O C E E D I N G S


  2             DR. SALOMON:  Welcome this morning to the


  3   Biological Response Modifiers Advisory Committee.


  4   I have been complaining about the lack of titles


  5   but at least they had numbers but ow they don't


  6   even have a number here.  Oh yes, we do, meeting


  7   number 32.  Eventually they will get the idea and


  8   give me titles.


  9             I am Dan Salomon.  I have the pleasure of


 10   chairing the committee today.  What we are going to


 11   do this morning is have about a one-hour open


 12   session here that I guess merges into a closed


 13   session at 8:45.  Then, there will be a break at


 14   9:00 and at 9:00 we will get into the main topic of


 15   the morning.  So, a lot of things like introducing


 16   the members of the committee I will save for nine


 17   o'clock if you guys will forgive the lack of pomp


 18   and circumstance this early in the morning.  I also


 19   reserve the right to say something totally stupid


 20   for the next hour since I am from California and it


 21   is awfully early for me right now.


 22             Without any further ado, we should get


 23   going.  It is Amy getting up there, Amy Rosenberg


 24   from the Laboratory of Gene Regulation, to give us


 25   an update on research programs, and that will be



  1   followed by Ezio Bonvini, from the Laboratory of


  2   Immunobiology.


  3                     Update Research Program


  4                  Laboratory of Gene Regulation


  5             DR. ROSENBERG:  I am actually the Director


  6   of the Division of Therapeutic Proteins, and I am


  7   here to speak for Ed Max and Serge Beaucage, who


  8   are members of the Laboratory of Gene Regulation


  9   who, unfortunately, could not be here today.


 10             This is a follow-up to the site visit and


 11   I will run through the follow-up for Dr. Max first.


 12   Dr. Max works with three research scientists, as


 13   you can see here.  The non-research


 14   responsibilities of a laboratory include primary


 15   review responsibility for several cytokines and


 16   thrombolytics and anticoagulants.  They


 17   additionally provide expert consultation on issues


 18   of molecular biology, particularly quantitative PCR


 19   assays and immunoglobulin genes.  In addition, Dr.


 20   Max performs a lot of administrative functions.  He


 21   is the associate director for research in OTRR and,


 22   as well, he organizes semina series; he chairs the


 23   research coordinating committee; and he manages the


 24   CBER library.


 25             The projects that are ongoing in his



  1   laboratory, two were primarily dealt with in the


  2   site visit, mechanisms of immunoglobulin isotype


  3   switching and characterization of the human 3'


  4   immunoglobulin heavy chain enhancer complex.


  5             The mission relevance of the research is


  6   listed here.  Regarding gene regulation, FDA


  7   regulates strategies to alter gene expression.


  8   Basically, we have a lot of products being produced


  9   by knock-in technology.  Insulators are now


 10   becoming increasingly important in transgenic


 11   animals. Regarding isotype switching, there is a


 12   little more activity, in fact.  There are specific


 13   strategies to have TH2 to TH1 switches.  So,


 14   increasing IgG, decreasing IgE to protect against


 15   allergic type reactions.  Additionally, our


 16   division regulates several agents that are known to


 17   directly affect isotype switching, cytokines IL4,


 18   TGF-beta and CD40 ligand.  As we all fervently


 19   believe, good basic science enables appropriate


 20   regulation.


 21             Dealing with the first project, mechanisms


 22   of immunoglobulin isotype switching, this is just


 23   to remind you that isotype switching involves a


 24   switch recombination event which juxtaposes VDJ


 25   segments with downstream constant regions of



  1   different isotype genes.


  2             The first aspect of this project involves


  3   a study of the Ku protein complex, how does this


  4   participate in immunoglobulin gene recombination?


  5   Ku protein has been found to be key in sealing


  6   double-stranded DNA breaks, and it is found that


  7   during isotype switching this protein increases in


  8   B cells and that knockout mice that are deficient


  9   for Ku seal DNA breaks inappropriately.  Since the


 10   site visit, this laboratory has cloned additional


 11   breakpoints in tumors from Ku knockouts that they


 12   are trying to characterize to clarify the role of


 13   Ku in sealing these double-stranded breaks.


 14             The second aspect of this project involves


 15   characterization or identification of the role of


 16   the ATM proteins in switch recombination.  This is


 17   a collaboration with Dr. Hodes at NCI.  They found


 18   that the ATM knockout mice show a defect in isotype


 19   switch recombination intrinsic to B cells, and


 20   since the site visit they have basically adapted


 21   their assay to become really a quantitative assay


 22   so that they can more accurately measure the degree


 23   of switch recombination.


 24             Regarding the second project, which is the


 25   characterization of the human 3' IgH enhancer



  1   complex, there are many aspects that they are


  2   investigating, one, the genomic neighborhood.  That


  3   aspect has been completed.  The human IgH 3'


  4   enhancer complex in humans resulting from a


  5   duplication event that causes large segments to be


  6   duplicated so that downstream of C-alpha 1 and


  7   C-alpha 2 constant regions the laboratory


  8   characterized these nearly identical enhancer


  9   complexes, each composed of a strong enhancer


 10   designated HS12, which are flanked by two weaker


 11   enhancers, HS3 and HS4.  Both HS12 enhancers are


 12   flanked by inverted repeats.


 13             So, they went on to study the functional


 14   motifs in HS12 and other 3' enhancers.  The have


 15   identified functional motifs in the enhancers by


 16   sequence conservation between the human enhancers


 17   and the murine homologs.  They have performed in


 18   vivo footprinting using LM-PCR, and they have


 19   performed transient transfections with luciferase


 20   reporter constructs that are driven by enhancers


 21   mutated in putative functional motifs.


 22             Regarding this aspect, since the site


 23   visit the laboratory has used DNA swan protection


 24   as an alternative technique for in vivo


 25   footprinting.  They have extended the footprinting



  1   analysis outside the evolutionary conserved cores


  2   of the HS12 and HS4 areas, and they have


  3   constructed and tested additional reporter plasmid


  4   containing DNA outside the core enhancers.


  5             With regard to the response of this


  6   enhancer complex to IL4 and CD40 ligand, it is


  7   found that these are factors, which are TH2


  8   stimuli, actually inhibited the action of the HS12


  9   enhancer in the germinal center B cell lines.


 10   Other enhancers, an endogenous one here, were


 11   unaffected.  Since the site visit they have


 12   investigated candidate IL4 or CD40 responsive


 13   elements in the HS12 enhancer by constructing


 14   reporter plasmid driven by multimerized candidate


 15   enhancer motifs.


 16             Regarding the last project, looking at


 17   locus control region function in chromatin, they


 18   found that there is a CPG island within a cluster


 19   of DNA swan hypersensitivity sites that showed the


 20   activity of gene insulators.  So, the level of


 21   transcription in the normal situation is here.  If


 22   you have gene insulators it cuts down dramatically,


 23   and these CPG islands as well cut down dramatically


 24   on transcription.  So, since the site visit they


 25   have constructed additional plasmid to define the



  1   active insulator element.  They are also searching


  2   for a possible homologous insulator downstream of


  3   the murine enhancers.


  4             Additional studies in progress involve


  5   chromatin immunoprecipitation studies to identify


  6   transcription factors found to be enhancers in


  7   vivo, and they are using single cell assays for the


  8   3' enhancer function using stable transfectants of


  9   GFP constructs.  That is the follow-up on the Max


 10   lab.


 11             DR. SALOMON:  Thank you, Amy.  I feel bad


 12   for Alice since she is an attorney and she came in


 13   a little late, she is going to have trouble with


 14   the test questions on enhancer.


 15             [Laughter]


 16             We will try and help you through it.  The


 17   next is from the representing the laboratory of


 18   immunobiology.


 19             DR. ROSENBERG:  No, I have to give


 20   follow-up on Dr. Beaucage.  I am sorry.  So, the


 21   laboratory of Dr. Beaucage, he works with five


 22   postdoctoral fellows.  His regulatory


 23   responsibilities include primary review of


 24   hematologic products, enzyme replacement therapies,


 25   anti-cancer enzymes and thrombolytics.  He provides



  1   expert consultation on all of the nucleotide


  2   diagnostic kits with the Center's Office of Blood.


  3   He has large responsibility for helping to draft


  4   the guidance for industry on submission of CMC


  5   information for synthetic oligonucleotides.  He has


  6   also performed some inspections regarding


  7   hematologic products and thrombolytics.


  8             Overview of his program--as you know, he


  9   is an oligonucleotide chemist, and he is


 10   responsible in large part for development of the


 11   phosphoramidite method so he has three major


 12   efforts.  The first is effects in development of


 13   deoxyribonucleotide cyclic anacylphosphoramidetes


 14   and stereo-controlled synthesis of oligonucleotide


 15   phosphorofioates for potential therapeutic


 16   applications.


 17             Essentially, since the site visit the


 18   group has optimized the coupling efficiency of


 19   deoxynucleoside cyclic anacylphosphoramidites to


 20   enable synthesis of nuclease-resistant P


 21   stereo-defined oligonucleotides containing all four


 22   nucleotides.  They found that pryrrolidin and DBU


 23   are the preferred bases for efficient coupling of


 24   deoxyribonucleotide acylphosphoramidites


 25   uncontrolled for GLAS, which is important for



  1   potential applications for microarray.  They


  2   published a paper in the Journal of the American


  3   Chemical Society, describing the development of a


  4   simple NMR method to determine the absolute


  5   configuration of deoxyribonucleotide


  6   phosphoramidites at phosphorus, and the findings,


  7   again, have appeared in the Journal.  They are also


  8   working to improve the resistance of CPG


  9   oligonucleotides to nuclease activities by using


 10   P-stereo defined oligos.


 11             The second effort involves efforts towards


 12   the discovery of phosphodiester protecting groups


 13   for potential applications to large-scale


 14   production of alphalation free therapeutic


 15   oligonucleotides and to the synthesis of


 16   oligonucleotides on microarrays.  They found that


 17   the 3-NN-dimethyl carboxymedopropryl group--this


 18   group right here, is a novel phosphate


 19   thiophosphate protecting group for solid phase


 20   synthesis that has recently been developed.  The


 21   monomers which are required are easily prepared


 22   from inexpensive raw materials.  The protecting


 23   group can be removed from the oligonucleotides


 24   under the basic conditions that are used


 25   standardly, and, thus, it is actually a very



  1   convenient protecting group.  But, most


  2   importantly, the thermolytic properties of the


  3   protecting group are particularly attractive to the


  4   synthesis of DNA oligonucleotides on microarrays


  5   because it minimizes exposure of the arrays to the


  6   harsh nucleophilic conditions used for


  7   oligonucleotide protection.  So, these conditions


  8   are actually quite mild and favorable.


  9             The third effort is involved in the


 10   development of thermophilic 5'hydoxyl protecting


 11   groups for nucleoside or nucleotides for synthesis


 12   of, again, DNA oligos on microarrays.  The


 13   thermolytic phosphate protecting groups described


 14   in the site visit report have been applied to the


 15   protecting group in the 5'hydroxyl of nucleosides


 16   as carbonates, but this was found to be quite


 17   impractical.  Recently the laboratory has


 18   discovered that the 5'O and methyl, 1 phenylmethyl


 19   oxycarbinol protecting group can be thermolytically


 20   cleaved from nucleosides in aqueous ethanol within


 21   10 minutes at 90 degrees.  Here is the loss of this


 22   protecting group.


 23             Interestingly enough, this forms a


 24   fluorescent byproduct and it permits the accurate


 25   determination of the D-protection deficiency.  The



  1   protecting group appears to be stable in organic


  2   solvents at ambient temperature, which also again


  3   makes it increasingly attractive to the synthesis


  4   of oligonucleotides on microarrays.  That is the


  5   follow-up for the Beaucage lab.


  6             DR. SALOMON:  I think someone should get


  7   the message back to them that you have represented


  8   them really remarkably well.  That was a beautiful


  9   presentation of not your own laboratory efforts.  I


 10   think anybody who didn't know that would have had a


 11   clue that this wasn't your own work.


 12             DR. ROSENBERG:  That is because they


 13   didn't ask questions.


 14             [Laughter]


 15             Thank you very much, Dan, I do appreciate


 16   it.


 17             DR. SALOMON:  It is also a representation


 18   of the kind of quality work going on at the FDA.


 19   My only regret is there aren't enough people in the


 20   audience that should hear that kind of thing


 21   because that is something that we should have saved


 22   for the end of day when there are a lot of people


 23   here.  The next presentation is from Ezio Bonvini,


 24   the Laboratory of Immunobiology, Division of


 25   Monoclonal Antibodies.



  1                   Laboratory of Immunobiology


  2             DR. BONVINI:  Thank you very much.  I


  3   would like to thank Dr. Salomon and the members of


  4   the advisory committee.


  5             My duty today is to summarize the work


  6   that we have done, and the focus of my laboratory


  7   is on the regulation of phospholipase C-gamma


  8   activation in immune cells.  The laboratory is


  9   operationally divided into two inter-related units,


 10   one focusing on the coupling of C-gamma-1 to the


 11   antigen receptor TMB cells.  The second, which is


 12   headed by Dr. Rellahan, looks at the control of


 13   phospholipase C activation, and in particular the


 14   control mediated by a complex molecule called


 15   C-Cbl.


 16             Recapitulating the functional division, we


 17   have two interacting units, one that I coordinate


 18   which is currently made up of a research assistant,


 19   Karen DeBell, and a postdoctoral fellow, Carmen


 20   Serrano.  I would also like to acknowledge past


 21   postdoctoral members of the laboratory that, in one


 22   way or another, have contributed to this project,


 23   and they have actually all left and found


 24   employment elsewhere.


 25             Dr. Rellahan has one permanent staff



  1   member, Dr. Laurie Graham, a lab associate, and she


  2   also enjoys the benefit of a number of students who


  3   have actually contributed during the summer to her


  4   project.


  5             Now, we do what we do for a number of


  6   reasons.  The laboratory has the regulatory


  7   responsibility for monoclonal antibodies and


  8   protein directed against T-cells for the purpose of


  9   immune suppression or immunomodulation.  More and


 10   more so, these antibodies interact with surface


 11   receptors that interfere either in signalling


 12   blockade or signalling manipulation with the


 13   purpose of immunomodulation.  Furthermore, signal


 14   transvection targeting can be used as surrogate for


 15   potency of biologics.  A number of biologics and a


 16   number of monoclonal antibodies, also trigger a


 17   number of adverse events to undesired signaling.


 18   Another fundamental reason is the familiarity with


 19   the knowledge base and technology.


 20             The focus on PLC-gamma, PLC-gamma


 21   regulates calcium mobilization in a variety of


 22   cells, including immune cells, and I don't think I


 23   need to go any further for this audience but


 24   calcium is a critical component in control for


 25   transcriptional activation through a number of



  1   elements, one of which is an important element,


  2   calcineurin phosphatase as a target for a number of


  3   drugs; the other path being calcium dependent


  4   proteinases.  The duration of the effects of the


  5   flux of calcium controls a number of cellular


  6   responses with a prolonged calcium flux being a


  7   requirement for immunocompetence.  As I said


  8   earlier, a number of calcium-dependent pathways are


  9   a target of immunosuppressive structures which


 10   include cyclosporin A, among others.


 11             Again, I don't think I can go through the


 12   data in detail, but what I would like to give you


 13   is a flavor for how complex PLC-gamma is.  This is


 14   the molecule which is a cytoplasmic molecule which


 15   contains a number of separate domains.  The


 16   molecules need to be recruited to the surface where


 17   the substrate where PTdinsP, a lipid, resides, and


 18   needs to undergo presumably a confirmation or


 19   modification to bridge together the X and Y domains


 20   of the catalytic subdomain.


 21             Our focus has been largely on the


 22   cytochromology 2 domain, which are individual


 23   domains which are known to interact with calcium


 24   and phosphorolytic protein and the cytochromology 3


 25   domains which are known to interact with the



  1   protein rich region.  When we started these


  2   investigations, the mechanism of activation of


  3   PLC-gamma was largely unknown or misinterpreted, I


  4   should say, so we focused on this largely because


  5   by their own nature we thought they were


  6   responsible for targeting phospholipase C-gamma


  7   with a number of regulatory proteins.  So, we


  8   pursued this by mutational analysis of the enzyme,


  9   and recently we obviously focused on a number of


 10   other domains but I will not go into any of this.


 11             This enzyme is regulated by


 12   phosphorylation, and there are at least four known


 13   targets in phosphorylation, here in yellow, and


 14   that is also another focus of our investigation but


 15   we use studies of phosphorylation somewhat as a


 16   surrogate marker for activation.


 17             So, I will briefly summarize the results


 18   of our studies, which have all been published, and


 19   I will split them vertically into the different


 20   domains.  The cytochromology of amino-2 terminal


 21   domain is the most critical domain in the


 22   activation of PLC-gamma-1 in T and B cells.  This


 23   domain is required in sufficient phosphorylation.


 24   It is required for membrane translocation and this


 25   requirement, we think, is required for activation



  1   because its activation correlates with the degree


  2   of phosphorylation.  What this domain does is bind


  3   a number of adapters which were recently


  4   discovered.  One is Lat which we identified in


  5   collaboration with Larry Samuelson.  The other is


  6   BLnk which we identified in collaboration with Tom


  7   Korozaky, who actually cloned it.  The


  8   cytochromology to the C domain appeared to be


  9   dispensable for phosphorylation of membrane


 10   translocation, although it is required for


 11   activation in vivo, and the function of this domain


 12   is largely unknown, but since the site visit report


 13   we have gained quite a number of insights and this


 14   is a very critical domain to investigate as it


 15   pertains to the ability of PLC-gamma to couple to a


 16   number of different pathways, including


 17   co-stimulatory pathways, and to a function of


 18   PLC-gamma that is independent of this catalytic


 19   activity.


 20             The cytochromology 3 domain appears to be


 21   dispensable phosphorylation, however, enhances


 22   membrane translocation, and I will provide a


 23   summary at the end of how it does that, and by


 24   virtue of its announcement of membrane


 25   translocation, enhanced activation of the enzyme in



  1   vivo.  Its function, we have identified binding to


  2   the protocol gene C-Cbl and Art Wizer's group, one


  3   of the leaders in the field, has shown that the


  4   domain binds with Lp-76, another adaptive molecule.


  5             Of course, I don't have the time to go


  6   through all the details but I just want to


  7   summarize again some of the milestones that we have


  8   achieved since we started this project.  With


  9   respect to PLC coupling to the receptor, we


 10   reported initially that PLC-gamma-1 SS-2 domain was


 11   critical for coupling it to the T-cell receptor.


 12   Then, we explored the role of cytochromology domain


 13   of PLC-gamma coupling to the B cell receptor.


 14   Recently we have focused on the ability of membrane


 15   raft, which are a microdomain, to function at the


 16   microdomain that segregates PLC-gamma and other


 17   molecules for their regulators, and we have shown


 18   that recompartmentalization of PLC-gamma to this


 19   microdomain is, in itself, sufficient to lead to


 20   PLC-gamma activation, activation of the cells and


 21   IL-2 separation.


 22             With respect to the negative regulation of


 23   PLC-gamma, which is the focus of Dr. Rellahan's


 24   research, we have shown that C-Cbl inhibits


 25   TCR-induced 81 activation, a reporter gene whose



  1   activation depends on raft and isoglycerol, and


  2   isoglycerol is under the control of PLC-gamma.


  3   PLC-gamma-1 binds C-Cbl in its HS-3 domain and


  4   C-Cbl exerts inhibitory function, however, it


  5   transforms a counterpart of C-Cbl-70Z-3 Cbl which


  6   lacks the ability of C-Cbl molecule to ubiquinate


  7   the target protein.  This molecule, 76-C-Cbl,


  8   activates PLC-gamma and does so through a


  9   differential pathway, a pathway which is not shared


 10   completely by the T cell receptors, suggesting the


 11   possibility of regulation of PLC-gamma through an


 12   alternate mechanism of activation.


 13             Rather than going through data, I would


 14   like to give you a model that will try to summarize


 15   our findings with those of other laboratories and


 16   put everything together.


 17             This is a schematic TCR receptor.  The TCR


 18   receptor interacts with the antigen it encounters


 19   of antigen presenting cells.  Now, in the membrane


 20   of many cells, including T cells, it is


 21   homogeneous.  Depicted here in red are rats which


 22   contain a number of different molecules, including


 23   the Lck which is brought together through the


 24   T-cell receptor by the action of the antigen into


 25   the raft.  The rafts contain an adaptor molecule,



  1   called raft, which we have shown to interact with


  2   phospholipase C.  This occurs subsequent to


  3   phosphorylation of Lck of the CD3 molecules which


  4   are associated with the alpha and beta chain of the


  5   T cell receptor.  Following phosphorylation, a


  6   cytoplasmic kinase called Zap 70 is recruited, and


  7   it is the Zap 70 that phosphorylates these other


  8   transmembrane adapters into the rat.


  9             This is the signal that tells PLC-gamma,


 10   which is a cytoplasmic enzyme which is


 11   constitutively bound to the Lck-76 through the


 12   SSS-3 domain.  That is the signal to recruit


 13   PLC-gamma through the amino termini cytochromology


 14   to this adaptor.  This interaction is further


 15   stabilized by the presence of Gads, a second


 16   adaptor molecule, which interacts with Lck-76 and,


 17   in turn, interacts with the cytochromology-2


 18   domain.  That explains the contribution of the


 19   cytochromology-3 domain to stabilize the


 20   interaction of PLC-gamma to the membrane.


 21             PLC-gamma in the raft compartment can be


 22   phosphorylated by a number of kinases which are


 23   either present in the raft compartment, such as


 24   RLK, or recruited to the raft compartment via the


 25   action of another specialized phosphorylated lipid



  1   PIP-3, such as ITK.  These are a member of the TAK


  2   family of kinase which are a member of the


  3   subfamily of kinase, although their mechanism of


  4   regulation is different.  The contribution of Lck


  5   and RLK in our hands shows that it leads to


  6   phosphorylation of PLC-gamma-1 which presumably


  7   induces a confirmation of modification of PLC-gamma


  8   and the ability of PLC-gamma to activate and


  9   mobilize calcium.


 10             Our data showed that if we artificially


 11   target PLC-gamma through the lipid raft we


 12   basically bypass this entire initial phase,


 13   although Lck and RLK are still required, presumably


 14   because of their contribution to the


 15   phosphorylation.  Artificially targeted PLC-gamma


 16   to the raft compartment is phosphorylated and is


 17   active bypassing the receptor entirely.  So, this


 18   is a dominant, positive variant of the PLC-gamma.


 19             What happened with the negative


 20   regulation, initial phase is the same and PLC-gamma


 21   is interacting with the Lck-76.  C-Cbl binds to the


 22   SU-3 domain of PLC-gamma very much in the manner


 23   seen with Lck-76.  So, there is probably


 24   competition by a mechanism which we still don't


 25   understand.  C-Cbl is also phosphorylated in



  1   response to activation of the T cell receptor and


  2   that leads to inhibition of PLC-gamma presumably


  3   via a mechanism of ubiquitilation.  We are still


  4   investigating this, however, data that confirm that


  5   this may be the case is that the variant to 73-Z


  6   C-Cbl, and we now have data with another variant


  7   that is Ub-ligase deficient, which results in the


  8   dephosphorylation of PLC-gamma by a mechanism that


  9   we still do not know but that does not require


 10   Lck-76, and that leads to the activation of


 11   PLC-gamma by a mechanism that is independent of the


 12   T-cell receptor.  So, we believe that C-Cbl and


 13   Lck-76 and the equilibrium between the two


 14   coordinate the assembly of the complex that in one


 15   case is activatory and in the other case is


 16   inhibitory.


 17             As far as our future plan, we will


 18   continue to investigate the role of PLC-gamma-1 and


 19   gamma-2 as a second isozyme present preferentially


 20   in B-cells and in other hematopoietic cells where


 21   gamma-1 is ubiquitously present in all cells.  We


 22   will focus further between these two enzymes and


 23   other pathways in the co-stimulatory activation of


 24   T cells.


 25             I mentioned earlier the function that the



  1   function of the SS2 domain is still unknown and we


  2   have obtained quite a bit of new exciting results


  3   on the function of this domain and its coupling to


  4   a number of different molecules, but the bottom


  5   line that I want to give you is that domain


  6   regulates the intrinsic activity of PLC-gamma by


  7   intermediate intermolecular interaction which


  8   regulates its opening up and the availability of


  9   the other subdomains.  So, it is a fundamental


 10   mechanism of regulation.


 11             We will continue, of course, to


 12   investigate the role and mechanism of


 13   phosphorylation of PLC-gamma.  What the enzymes are


 14   that phosphorylate the PLC-gamma are largely


 15   unknown.  We have a candidates are, as I mentioned


 16   earlier, but what the different candidates do in


 17   terms of individual residues, and there are at


 18   least four and mostly likely five residues, and


 19   what is the role of the individual residue is still


 20   quite unclear.


 21             Because we have made a dominant positive,


 22   we have now also developed a dominant negative


 23   PLC-gamma, and we will certainly ask the question


 24   of the role of PLC-gamma development by using


 25   transgenic technology.  Finally, and I am not going



  1   to dwell on this, but we are using technology to


  2   recompartmentelize PLC-gamma intracellularly by a


  3   condition of mechanism.


  4             With respect to the role of C-Cbl again,


  5   C-Cbl is probably a threshold for activation, and


  6   the impact of C-Cbl on the co-stimulatory signal is


  7   the ability of the cell to behave as naive or


  8   memory will be investigated.  We are going to


  9   generate some C-Cbl-deficient lines and we are


 10   going to try to do that by a number of different


 11   strategies.  As I said, we have some new data on


 12   the C-Cbl-mediated with the delineation of


 13   PLC-gamma-1.  I can tell you that it is


 14   ubiquitilated.  The role of C-Cbl in this remains


 15   to be determined but we have evidence that by using


 16   Ub-ligase to inhibit the C-Cbl negative cells is,


 17   in fact, the case.


 18             Finally, we will try, as I said earlier,


 19   to generate some C-Cbl deficient cell line using


 20   interferon RNA and that will help us in the study


 21   of kinetics in mice for PLC-gamma activation.


 22             I just want to leave you with the number


 23   of individuals who have contributed in one way or


 24   another with particular reagents and a number of


 25   collaborators that we have worked with whom I would



  1   like to acknowledge for their help in this.  And, I


  2   will be glad to take any questions.


  3             DR. SALOMON:  That was a very nice


  4   presentation and good work, and also my same


  5   comments, that I wish more people could see the


  6   kind of quality work that is going on in the FDA,


  7   oftentimes, with a lot less support not because of


  8   your fault or the FDA support but just because of


  9   the budget constraints than we are used to in


 10   academia.  It is excellent.


 11             The part that is confusing me here,


 12   besides the fact that I really am still asleep, is


 13   that we now have to switch officially to a closed


 14   session to vote on accepting the report.  Gail will


 15   make sure that the right people have to leave.


 16   Anyway, we will see you again very shortly.


 17             [Whereupon, the open session was recessed


 18   to continue in closed session and reconvene in open


 19   session at 9:15 a.m.]



  1                      P R O C E E D I N G S


  2                Welcome and Administrative Remarks


  3             DR. SALOMON:  If we can get everybody to


  4   sit down we will start the main show, I guess we


  5   should say.  For the larger group here now, this is


  6   meeting number 32 of the Biological Response


  7   Modifiers Advisory Committee.  My name is Dan


  8   Salomon.  I have the pleasure to chair the meeting


  9   this morning.  What we usually do at the start, as


 10   in many big committee meetings where a lot of us


 11   don't know each other initially--we will certainly


 12   get to know each other as the day goes on, is just


 13   to go around the table and introduce yourself, and


 14   make a couple of quick sentences about what your


 15   interests are and your scientific expertise.  We


 16   can start at that end of the table.  Dr. Casper?


 17             DR. CASPER:  Hi.  I am Bob Casper, am a


 18   professor of obstetrics and gynecology and


 19   physiology at the University of Toronto, and I am


 20   head of the Division of the Reproductive Sciences.


 21   I have clinically been involved st in vitro


 22   fertilization for several years, and our laboratory


 23   at the present time has an interest in


 24   mitochondrial research involving aging of human


 25   oocytes.  We have also been doing some work with



  1   mitochondrial transfer experiments in mice.


  2             DR. SALOMON:  There is a button here that


  3   you push and then you have to remember to turn it


  4   off, otherwise there will be feedback.


  5             DR. KNOWLES:  Thank you.  I am Lori


  6   Knowles.  I am from the Hastings Center.  I have a


  7   background in international law and policy, and I


  8   am principal investigator right now of an


  9   international project on reprogenetic regulation


 10   and affects, and also do work in international stem


 11   cell policy.


 12             DR. NAVIAUX:  I am Bob Naviaux, from the


 13   Mitochondrial Metabolic Disease Center at the


 14   University of California, San Diego.  My basic work


 15   is in mitochondrial DNA replication, and we also


 16   have interest in inborn errors of metabolism and


 17   adult and childhood mitochondrial disorders.


 18             DR. SHOUBRIDGE:  I am Eric Shoubridge.  I


 19   am a professor at McGill University in the


 20   Departments of Human Genetics and Neurology and


 21   Neurosurgery.  I have a research lab at the


 22   Montreal Neurological Institute and our laboratory


 23   is interested in the basis of mitochondrial


 24   disease, the molecular basis, and we are interested


 25   in basic, fundamental aspects of mitochondrial



  1   genetics.


  2             DR. SCHON:  My name is Eric Schon.  I am a


  3   professor of genetics and development in the


  4   Department of Neurology at Columbia University, and


  5   I do everything that Eric Shoubridge does.


  6             [Laughter]


  7             DR. VAN BLERKOM:  Jon Van Blerkom.  I am


  8   from the University of Colorado, Molecular Biology


  9   Department, and I am also in clinical practice in


 10   in vitro fertilization, for about twenty years.


 11             DR. MURRAY:  I am Tom Murray.  I am from


 12   the Hastings Center these days, after fifteen years


 13   of medical schools, most recently Case Western


 14   Reserve University.  My research has been broadly


 15   in the field of ethics and medicine and the life


 16   sciences, and I have done a lot of work on


 17   reproductive technologies, genetics and parents and


 18   children.


 19             DR. RAO:  My name is Mahendra Rao, and I


 20   am a section chief in stem cell biology at the


 21   National Institute of Aging, and I am a member of


 22   this committee.  My interests are in embryonic stem


 23   cells and adult stem cells.


 24             DR. MULLIGAN:  I am Richard Mulligan.  I


 25   am from the Harvard Medical School, Children's



  1   Hospital.  I am a stem cell person and a gene


  2   transfer person, and a member of BRMAC.


  3             DR. SALOMON:  I am Dan Salomon.  I am from


  4   the Scripps Research Institute and my lab is doing


  5   cell transplantation, tissue engineering,


  6   angiogenesis and therapeutic gene delivery.


  7             MS. DAPOLITO:  Gail Dapolito, Center for


  8   Biologics, executive secretary.


  9             DR. SAUSVILLE:  Ed Sausville.  I am the


 10   associate director of NCI's Division of Cancer


 11   Treatment and Diagnosis, with responsibility for


 12   the development of our therapeutics program, and


 13   our interest is in the preclinical studies leading


 14   to the approval for INDs for drugs and biologics.


 15             MS. WOLFSON:  Alice Wolfson.  I am the


 16   consumer representative on the committee.  I am an


 17   attorney specializing in policy holder


 18   representation, with particular emphasis on


 19   disability policy holders and their struggles with


 20   their insurance companies.  I have a strong


 21   interest in health.  I am a founder of the National


 22   Women's Health Network, and I am particularly


 23   interested in the social effects of postponing


 24   fertility as well as the social effects of not


 25   postponing fertility and I think it may have, along



  1   with the scientific elements in it, the beginnings


  2   of a possibility of a resurgence of another wing of


  3   the women's movement.


  4             DR. ROSE:  I am Stephen Rose.  I am from


  5   the National Institute of Health, Office of


  6   Biotechnology Activities, deputy director for the


  7   recombinant DNA program.


  8             DR. MONROE:  I am Scott Monroe.  I am from


  9   the Division of Reproductive and Neurologic Drug


 10   Products at CDER.  I am an


 11   obstetrician/gynecologist and a reproductive


 12   endocrinologist.


 13             DR. SERABIAN:  I am Mercedes Serabian.  I


 14   am an expert toxicologist with the Office of


 15   Therapeutics in the Division of Clinical Trials,


 16   and I will be part of the review team at CBER that


 17   will be reviewing these INDs when they come in.


 18             DR. MOOS:  I am Malcolm Moos, from the


 19   Division of Cellular Gene Therapy at the FDA.  My


 20   research interests are cell and tissue


 21   specification and patterning, and I am also


 22   concerned with review of cellular products,


 23   primarily that have to do with that general


 24   biological area.


 25             DR. HURSH:  I am Deborah Hursh.  I am also



  1   a cellular product reviewer in the Division of Cell


  2   and Gene Therapy, and I have a research lab


  3   studying developmental biology and signal


  4   transduction.


  5             DR. NOGUCHI:  I am Phil Noguchi.  I am the


  6   director of the Division of Cell and Gene Therapy,


  7   where we see these and other novel technologies and


  8   continually struggle with doing the right thing.


  9             [Laughter]


 10             DR. SIEGEL:  I am Jay Siegel.  I direct


 11   the Office of Therapeutics Research and Review at


 12   the Center for Biologics, FDA.


 13             DR. SALOMON:  I welcome all of you.  I


 14   think one of the privileges of being on the


 15   committee and certainly chairing it is the chance


 16   to interact with experts at each of these sessions


 17   that take me into areas that are often new to me,


 18   and today is definitely one of those areas.  It is


 19   a fantastically important discussion that we are


 20   going to have that has a lot of implications on


 21   what is going to happen over the next several


 22   years.  So, I specifically feel a lot of


 23   responsibility to this particular session and how


 24   we go forward.


 25             There will be some more comments later on



  1   that, just simple administrative things.  My job,


  2   obviously, is to stay on time and also to get the


  3   questions the FDA answered and keep everybody on


  4   track.  So, if you will forgive me sometimes


  5   playing my administrative role which sometimes


  6   includes being rude.  I apologize in advance.


  7             The button thing, we have all been through


  8   it.  It gets to be a real problem with feedback and


  9   also with the transcriber.  So, if I ever sort of


 10   look at you and kind of point to the button, it is


 11   just to let you know.  I think that is the major


 12   thing.  I want to try and keep track of sort of


 13   what we are going to do next so you will sort of


 14   know where we are going.


 15             What we will do now is a presentation of


 16   the certificate of appreciation to Dr. Ed


 17   Sausville, with some more comments to follow that.


 18   Then Gail Dapolito has some official things to read


 19   into the record and then we will start the full


 20   session with Dr. Hursh.


 21           Presentation of Certificate of Appreciation


 22             DR. SIEGEL:  It is indeed an honor, tinged


 23   with regret at his departure but an honor to speak


 24   of the many services that Dr. Sausville has


 25   provided to us through his participation in BRMAC



  1   in recent years, and to thank you for them.  Those


  2   of you on the committee, of course, are aware of


  3   his many thoughtful contributions to the


  4   deliberations to this committee.  Some of you may


  5   be somewhat less aware of his many contributions as


  6   a representative of BRMAC to the Oncological Drugs


  7   Advisory Committee and other FDA committees to


  8   which we have taken products for consideration of


  9   approval, as well as contributions to our lab


 10   evaluation and site visiting program.


 11             We ask a lot, as you know, of BRMAC


 12   members.  It ranges from discussion of the issues


 13   regarding manufacturing a product, viral purity,


 14   protein stability, immunogenicity, and so forth,


 15   and how we should focus on safety.  The issues of


 16   clinical testing of a product; what is the


 17   appropriate trial design to get the answers we need


 18   and what to make of the answers when those trials


 19   are done; and, of course, as you heard this morning


 20   the issues of evaluating our research programs and


 21   how to make sure that they are tied in intimately


 22   to our mission and our goals and are of the highest


 23   quality.


 24             We choose experts in each and all of these


 25   areas to help us in our functions, but it is rare



  1   that we have an expert--rare both inside the agency


  2   and outside but very much appreciated when we have


  3   someone such as Dr. Sausville who really is the


  4   regulatory expert triple threat, who integrates an


  5   understanding of the clinical evaluation of the


  6   basic science, of the research needed to support


  7   that, and can participate in an integrated


  8   assessment in any of those areas, understanding the


  9   implications for the others.  That is what you have


 10   done for us for these several years and it is very


 11   much appreciated.  Thank you very much.


 12             [Applause]


 13             DR. GOODMAN:  I know Dr. Zoon and I really


 14   second that and appreciate the tremendous breadth


 15   of expertise Dr. Sausville has brought.  I was


 16   going to stress the same thing.  From what I have


 17   understood and seen, this translational ability


 18   between the laboratory and the clinical setting,


 19   and an understanding of product development, those


 20   things are just extremely important and we really


 21   appreciate it.  We look forward to continuing to


 22   call on you and get your input and help.  Thanks


 23   very, very much.  So, we have a nice certificate


 24   and plaque.


 25             [Applause]



  1             DR. SALOMON:  I can't not make my own


  2   personal comments, having been together with Ed on


  3   this committee for four years.  I don't know how


  4   many of you have seen the movie "The Scorpion


  5   King."  I guess is depends on how old your kids


  6   are, but the actor in it is called "The Rock"


  7   because I suppose he is a professional wrestler as


  8   well.  But I really think that he is competing with


  9   the real "rock" who is Ed Sausville.  On any


 10   committee like this you have to have a rock.  I


 11   mean, you have to have the one guy who you can


 12   always turn to, even though everything has gone to


 13   shreds, and he just hits it right on the head.  You


 14   have to shut up and listen to him whenever he says


 15   anything.  Really, whenever there has been any kind


 16   of issue here, he is one of the people that I come


 17   to at the break and say, "you know, Ed, what the


 18   heck do we do now?"  And, he always has good


 19   advice.  This is not good at all, to have Ed


 20   leaving and all I can do is say I will always be


 21   dragging you back here, and he is really, really


 22   going to be a loss to the committee.  Thank you.


 23   Gail?


 24             MS. DAPOLITO:  I would like to read the


 25   meeting statement.  This announcement is part of



  1   the public record for the May 9, 2002 Biological


  2   Response Modifiers Advisory Committee meeting.


  3             Pursuant to the authority granted under


  4   the Committee Charter, the director of FDA Center


  5   for Biologies Evaluation and Research has appointed


  6   Ms. Lori Knowles and Drs. Thomas Murray, Robert


  7   Naviaux, Eric Schon, Eric Shoubridge, Daniel


  8   Salomon and Jonathan Van Blerkom as temporary


  9   voting members for the discussions on issues


 10   related to ooplasm transfer in assistive


 11   reproduction.  In addition, Dr. Salomon serves as


 12   the acting chair for this meeting.


 13             To determine if any conflicts of interest


 14   existed, the agency reviewed the submitted agenda


 15   and all financial interests reported by the meeting


 16   participants.  In regards to FDA's invited guests,


 17   the agency has determined that the services of


 18   these guests are essential.  The following


 19   interests are being made public to allow meeting


 20   participants to objectively evaluate any


 21   presentation and/or comments made by the guests


 22   related to the discussions and issues related to


 23   ooplasm transfer in assisted reproduction.


 24             Dr. Robert Casper is employed by the


 25   University of Toronto in the Division of



  1   Reproductive Science at Mt. Sinai Hospital in


  2   Toronto.  Dr. Jacques Cohen is employed by the St.


  3   Barnabas Medical Center.  Dr. Susan Lanzendorf is


  4   employed by the Eastern Virginia Medical School at


  5   the Jones Institute of Reproductive Medicine.  Drs.


  6   Amy Patterson, Marina O'Reilly and Stephen Rose are


  7   employed by the Office of Biotechnology Activities,


  8   NIH.


  9             In the event that the discussions involve


 10   other products or firms not already on the agenda


 11   for which FDA participants have a financial


 12   interest, the participants are aware of the need to


 13   exclude themselves from such involvement and their


 14   exclusion will be noted for the public record.


 15             With respect to all other meeting


 16   participants, we ask in the interest of fairness


 17   that you state your name, affiliation, and address


 18   any current or previous financial involvement with


 19   any firm whose product you wish to comment upon.


 20   Thank you.


 21             DR. SALOMON:  Thank you, Gail.  Before we


 22   officially get started, let me just make a couple


 23   of quick comments.  That is, the task we have here


 24   is to begin now, through about four o'clock this


 25   afternoon at which point we will have gone through



  1   a series of presentations on this issue of ooplasm


  2   transfer that clearly touch on some absolutely


  3   major areas, we encourage you to ask questions and


  4   to set the stage for critical discussions which I


  5   will try to keep on time, but also it is so


  6   important that these critical discussions develop


  7   that we will have to be a little flexible about how


  8   that goes, leading up to a discussion at 4:00 of


  9   specific questions that have been put together by


 10   the FDA that will frame issues the FDA wants input


 11   from us on regarding developing an IND process for


 12   this field.


 13             The only other comment I want to make to


 14   all of you is get your thoughts out on the table.


 15   There is no need to force an agreement on anybody.


 16   You are more than welcome to articulate and defend


 17   a minority opinion.  I don't believe my job here is


 18   to come up with some absolute consensus.  My job is


 19   to identify where consensus can be reached,


 20   however, as well as to have you help us figure out


 21   where there isn't consensus and perhaps other


 22   additional efforts in those areas are coming.


 23             We have to make sure that when we are


 24   done--I feel very strongly--that we can say to the


 25   public that this was an open, balanced discussion



  1   of the issues.  That is a major responsibility.


  2   If, in the middle of discussions, somebody goes,


  3   you know, we are really missing this piece and we


  4   just don't have it here today, then that should go


  5   into the record as well because I think that is


  6   part of being fair to the whole field.


  7             With respect to the audience, I feel you


  8   are as much part of this discussion as we are.  It


  9   is a little harder to control you so you will have


 10   to forgive that, but you are certainly not just


 11   welcome but encouraged to step up at key points of


 12   the discussion and bring your expertise and your


 13   viewpoints to it.  The rules are simply to keep it


 14   brief and identify yourself, and realize that with


 15   the competition to try to keep everything on time I


 16   will also have to manage that.  But very much,


 17   please feel part of the discussion that will take


 18   place today.


 19             That is basically it and I am really


 20   looking forward to any discussion that follows and


 21   the diversity of expertise we have here.  With that


 22   introduction, Dr. Hursh?


 23            Ooplasm Transfer in Assisted Reproduction


 24                         FDA Introduction


 25             DR. HURSH:  I would also like to welcome



  1   the participants and the audience to this meeting


  2   of the Biological Response Modifiers Advisory


  3   Committee.


  4             This is day one of a two-day meeting of


  5   the Biological Response Modifiers Advisory


  6   Committee.  On this first day we will discuss


  7   ooplasm transfer in the treatment of female


  8   infertility.  On the second day the topic will be


  9   potential germline transmission during gene


 10   therapy.  We have chosen to link these two topics


 11   as both of them deal with the transfer of genetic


 12   material go gametes, sperm and eggs.


 13             This has occurred in the case of ooplasm


 14   transfer and is a potential inadvertent risk of


 15   gene therapy.  In both cases heritable genetic


 16   modifications will be produced.  While the FDA and


 17   the Recombinant DNA Advisory Committee have


 18   discussed some of these issues previously, FDA felt


 19   it was timely to have further open public


 20   discussion on the subject of gene transfer in


 21   gametes in light of the evidence of new mechanisms,


 22   such as the manipulation of oocytes by which germ


 23   cells can be genetically modified.


 24             Since today's discussion is focused on


 25   ooplasm transfer, I will limit the rest of my



  1   remarks to that topic.  We will hear about this in


  2   much greater detail from our first two speakers


  3   but, in brief, in ooplasm transfer 5 percent to 15


  4   percent of an unfertilized egg cytoplasm, which is


  5   called ooplasm, is transferred from a donor into a


  6   recipient, and is then fertilized in vitro.


  7   Recipients are women who have been unable to


  8   conceive through conventional in vitro


  9   fertilization.  The cytoplasm of an oocyte is


 10   considered specialized and it contains proteins,


 11   messenger RNAs, small molecules and organelles.  It


 12   is not clear which of these components is the


 13   putative active component of ooplasm, but it is


 14   with one of these organelles, the mitochondria,


 15   that we will be primarily concerned with.


 16             Most of you are probably aware that


 17   mitochondria are the powerhouse of a cell, the site


 18   where aerobic respiration, the production of energy


 19   using oxygen occurs.  But they have other


 20   functions.  They are involved in fatty acid


 21   metabolism, intracellular ion balance and


 22   programmed cell death.


 23             As you can see on the schematic diagram


 24   here, they are a very specialized subcellular


 25   structure, membrane bound, and each cell has many,



  1   many mitochondria to support the energy


  2   requirements of that cell.  I would like to draw


  3   your attention to the little squiggle in the middle


  4   because that is one of the issues about


  5   mitochondria that concerns us here.  Perhaps the


  6   most important feature for our purposes is that,


  7   due to their supposed evolution from primitive


  8   bacteria, mitochondria contain their own genome.


  9             The mitochondrial genome is very small.


 10   It is only about 17,000 base pairs as opposed to


 11   several billion for the human genome.  However, it


 12   has 37 distinct genes.  Unrelated individuals have


 13   distinct genotypes of mitochondria, so distinct


 14   that they can be used by forensic biologists to


 15   establish relatedness among human beings.  The


 16   mitochondrial DNA, while small, is very important


 17   because mutations associated with mitochondrial DNA


 18   result in human disease.  While I realize you


 19   cannot read what is in the balloons, the point of


 20   the schematic diagram here is this is the circular


 21   mitochondrial genome and each one of these balloons


 22   represents positions of mapped mitochondrial


 23   mutations that result in human disease.


 24             Mitochondria obey unusual rules of


 25   inheritance.  In mammals, after fertilization, the



  1   mitochondria contributed by the sperm are


  2   apparently destroyed.  Therefore, the only


  3   population of mitochondria in a developing embryo


  4   and in the resultant progeny come from the pool


  5   existing in the oocyte prior to fertilization.


  6             In general, oocytes therefore get all of


  7   their mitochondria from the mother and that


  8   mitochondria is a homogeneous pool of a single


  9   genetic type.  This is a condition that is called


 10   homoplasmy.  This is the more common situation in


 11   human oocytes.  Having two distinct genetic forms,


 12   two distinct pools of mitochondria is less common


 13   and this is referred to as heteroplasmy.  While


 14   heteroplasmy is unusual with wild type


 15   mitochondria, it is actually seen in people who


 16   have mitochondrial disease where you can have a


 17   population of mutant and a population of wild type


 18   mitochondria co-existing in the same cell.


 19             In studies of heteroplasmy it has been


 20   observed that mitochondrial genotypes can be


 21   partitioned unequally among tissues, and I believe


 22   we will hear a great deal more about this from one


 23   of our speakers this morning, Dr. Eric Shoubridge.


 24             So, what happens after ooplasm transfer?


 25   If there are mitochondria transferred during



  1   ooplasm transfer, what is the result?  In March of


  2   2001, a laboratory of Dr. Jacques Cohen reported


  3   that two children born after the ooplasm transfer


  4   protocol were heteroplasmic, which means the


  5   genotypes of both the ooplasm donor and the mother


  6   could be detected in their tissues.  These children


  7   were approximately one year old at the time of this


  8   analysis, so this was a persistent heteroplasmy


  9   that had been maintained.


 10             At the time of Dr. Cohen's publication the


 11   FDA was already considering action in the area of


 12   ooplasm transfer.  The report of heteroplasmy


 13   raised our concerns, as did information in two


 14   pregnancies occurring after ooplasm transfer


 15   resulted in fetuses with Turner's syndrome, a


 16   condition where there is only one X chromosome.


 17             In addition, despite the fact that Dr.


 18   Cohen refers to this as an experimental protocol


 19   that should not be widely used, we felt that it was


 20   beginning to spread rapidly into clinical practice


 21   in the United States by 2001.  There were at least


 22   23 children born in the United States after using


 23   ooplasm transfer.  Three United States clinics had


 24   published on this procedure and we, at FDA, were


 25   able to find five additional clinics that were



  1   advertising this procedure on the internet.


  2             FDA had concerns about whether we


  3   understood all the ramifications of this procedure


  4   and whether we understood its safety in particular,


  5   and reacted by sending letters to practitioners who


  6   were identified by publications on ooplasm transfer


  7   or by advertisements offering the procedure.  We


  8   advised practitioners that we would now require the


  9   submission of an investigational new drug


 10   application, or IND, to the agency and its


 11   subsequent review to continue to treat new


 12   patients.  After the letter was issued we had


 13   telephone conversations with several practitioners


 14   who wanted to know more about the IND submissions


 15   procedure.


 16             After these conversations FDA felt this


 17   topic would be well served by open public


 18   transparent discussion of the ooplasm transfer


 19   procedure and the data behind it, hence this


 20   meeting.  The major issue we, at FDA, are trying to


 21   achieve consensus on at this advisory committee


 22   meeting is are preclinical and clinical data


 23   supporting the safety and efficacy of ooplasm


 24   transfer sufficient to justify the risks of


 25   clinical trials?  If additional data are needed,



  1   what types of data would be the most informative,


  2   what model systems, what size studies?


  3             FDA's tasks in regulating new therapies is


  4   to weigh risks and benefits and to determine what


  5   safeguards need to be in place to ensure the safety


  6   of human subjects.  That is what we will do with


  7   ooplasm transfer.  While the FDA welcomes


  8   discussion with all interested parties, our topic


  9   today is very limited.  We will, therefore, limit


 10   today's discussion to the science behind ooplasm


 11   transfer and not extend that discussion to FDA's


 12   jurisdiction in general, FDA's proposed rules for


 13   the regulation of human cells and tissues and other


 14   assisted reproductive technologies.  Thank you very


 15   much.


 16             DR. SALOMON:  Thank you, Deborah.  Unless


 17   there are any pressing questions, I think the


 18   purpose of that was clearly just to set the stage


 19   for what is to follow.  What I would like to do is


 20   invite Dr. Susan Lanzendorf to present cytoplasmic


 21   transfer in the human oocyte.   She is from the


 22   Jones Institute of Reproductive Medicine.


 23                Cytoplasmic Transfer in the Human


 24             DR. LANZENDORF:  I have come here today to


 25   share some of the experiences that we have



  1   encountered at the Jones Institute with the


  2   procedure of cytoplasm transfer in the human.


  3             Cytoplasmic transfer was first considered


  4   at the Jones Institute back in 1990 when an


  5   investigator, a clinical fellow, Flood et al.,


  6   reported that the developmental potential of


  7   oocytes to mature in vitro can be increased by


  8   injecting with the cytoplasm of oocytes matured in


  9   vivo.  This was performed in the monkey model.


 10             This study found that 13 percent of the


 11   injected oocytes resulted in pregnancies while none


 12   of the sham-injected or non-surgical controls


 13   resulted in a pregnancy.  The investigators felt


 14   that this suggested that factors may be present


 15   within the cytoplasm that control genetic,


 16   maturational and/or developmental properties.


 17             Then, in 1997, Cohen and coworkers


 18   reported the first human pregnancy from the


 19   transfer of cytoplasm from donor eggs.  They


 20   reported that the goal of the procedure was to


 21   provide healthy cytoplasmic factors to the eggs of


 22   the patients who repeatedly produce embryos of poor


 23   quality.


 24             We were very interested in this report.


 25   We see a lot of patients who come through in vitro



  1   fertilization who repeatedly fail to achieve a


  2   pregnancy and many times we are at a loss on how to


  3   continue treatment in these patients who just don't


  4   seem to get pregnant.  So, we approached our


  5   institutional review board to see if we could


  6   investigate this procedure.


  7             We decided to look at two groups of


  8   patients, in one of which the wife is 40 years of


  9   age or older, or in couples who have had at least


 10   two previous IVF attempts which resulted in only


 11   poor quality embryos.  In in vitro fertilization we


 12   have found that when you transfer embryos that have


 13   an ideal morphology they result in a higher


 14   pregnancy rate than those who have less than an


 15   ideal morphology.  So, this was an attempt to try


 16   to improve this and, hopefully, increase the


 17   pregnancy rate.


 18             Again, we put this to the institutional


 19   review board and we requested permission to do this


 20   with 15 consenting patients.  We worked very hard


 21   on our consent form, being that this was a


 22   procedure where very, very little was known.  So,


 23   of course, we tried to emphasize to the patients


 24   the risks that they might encounter, including that


 25   the effect of the procedure on the couple's eggs or



  1   their ability to establish a pregnancy totally


  2   unknown.  What is also unknown is if the procedure


  3   would increase the risk of obstetric complications,


  4   or if the thawed donor eggs would even survive.  I


  5   should point out here that we used frozen and


  6   thawed donor eggs for our procedure.  So, we


  7   emphasized to the patient that if the thawed eggs


  8   didn't survive the procedure would not be performed


  9   and they may not get a transfer.  In addition, the


 10   patient's eggs may not survive the procedure or


 11   they may fail to fertilize and develop normally and


 12   they would not obtain a transfer.


 13             We also emphasized the risk to the


 14   offspring.  It is not known if the procedure would


 15   increase risk of obstetric complications or fetal


 16   abnormalities.  The eggs could be damaged in some


 17   way that could affect the offspring.  And, there


 18   was the possibility that genetic material could be


 19   transferred from the egg donor to the patient's


 20   eggs and it is unknown if this could adversely


 21   affect the offspring.


 22             In our consent form we did break this out


 23   into talking and making clear to the patient that


 24   there are two types of genetic material, DNA from


 25   the nucleus of the egg and the DNA from the



  1   mitochondria.  So, we were careful to make them


  2   understand that the two different possibilities of


  3   genetic material could be transferred.


  4             The consent form also stressed that


  5   because the procedure is so new there is no way to


  6   determine what the exact risks are, or at what rate


  7   the risks occur.  In our other consent forms we try


  8   to say, you know, we have seen a 50 percent


  9   survival rate, or we have seen a 60 percent


 10   pregnancy rate but we couldn't even do this with


 11   this procedure because it is so new so we


 12   emphasized this to them.


 13             It was also recommended that all of the


 14   patients who achieve a pregnancy have an


 15   amniocentesis regardless of their age.  Then, of


 16   course, the boiler plate other risks that cannot be


 17   identified at that time.


 18             This is just to show you quickly how we


 19   perform the procedure.  Again, we used


 20   frozen-thawed donor eggs so the donor eggs that


 21   contributed the cytoplasm were collected and


 22   cryopreserved at a previous state.  Then, when the


 23   patient came through on the day of their aspiration


 24   and cytoplasm transfer, the donor eggs were thawed.


 25             So, before we get here what we will have



  1   done is--this is the pipet here that we also use to


  2   do the donation.  This is the egg-holding pipet


  3   which just holds the egg in place.  This is the


  4   egg.  So, prior to getting here we would have got a


  5   drop of sperm and picked up a sperm from the


  6   patient's husband and loaded it in the pipet.  We


  7   then take this pipet with the sperm and insert it


  8   into the donor egg.  Then, once in the donor egg,


  9   we draw up cytoplasm that will be transferred.


 10             We then move to the recipient's egg, the


 11   patient in this scenario, and then put that pipet


 12   into the egg, inject that cytoplasm into the egg,


 13   along with the husband's sperm.  Actually, what


 14   occurs is the cytoplasm transfer and the


 15   utilization of the egg at the same time.


 16             Our results, we had eight patients in


 17   eight cycles who were 40 years of age or over, with


 18   an average age of 44.  The procedure did not appear


 19   to have an effect on embryo quality.  I say "did


 20   not appear" because there are too few numbers of


 21   actual embryos to compare with other embryos to


 22   make a significant conclusion.  No pregnancies were


 23   established in any of these eight patients.


 24             In the same 40 years or older group, 39


 25   eggs were retrieved, with a mean of 3.2 eggs per



  1   patient.  This is low but is normal in patients in


  2   this age group.  We had a 54 percent fertilization


  3   rate, and this would be with the cytoplasm transfer


  4   occurring at the same time.  To do these


  5   procedures, we had to use cytoplasm from nine donor


  6   eggs, and these donors ranged in age from 25 to 29.


  7   Of the donor eggs, 62 percent survived the thaw


  8   procedure and were used.


  9             We had three patients who came through who


 10   had a history of poor quality embryos.  Actually,


 11   this is the group of patients that we thought we


 12   could really help with this procedure.  We did not


 13   go into it thinking that the older patients would


 14   be the ones that would benefit mostly, and I think


 15   the other investigators who performed this


 16   procedure would probably agree that it is not


 17   helping the older aged couples.


 18             So, these were three patients who had


 19   significant history of poor quality embryos in the


 20   past.  The age of these patients was 35, 35 and 38.


 21   The procedure did appear to have an effect on


 22   embryo quality.  To us, the embryos looked much


 23   better than those that we had seen from these same


 24   patients previously.  Of those three patients, one


 25   achieved a pregnancy.  It was a twin pregnancy that



  1   was established.  That particular patient had


  2   undergone six previous IVF attempts with fresh


  3   transfer and three attempts with cryotransfer and


  4   never achieved a pregnancy.


  5             In these three patients 42 eggs were


  6   retrieved, a mean of 14.3 which, as you can see, is


  7   much higher than in the older patients; 62 percent


  8   fertilization rate with the cytoplasm transfer.


  9   This is the information on the donors that provided


 10   the eggs, and they had a 66 percent survival, those


 11   three donors.


 12             These are the twins.  I have been told


 13   that the medical director has spoken with the


 14   couple about having their twins evaluated


 15   genetically for all the questions that we are here


 16   about today.  The couple is not interested.  They


 17   feel their children, who are now three or four


 18   years old, are very healthy and very normal and


 19   they don't want anything else done with that.


 20             We were also looking at other things when


 21   we were doing these studies and before we received


 22   our letter to stop doing them.  One of the things


 23   that we were interested in was the inadvertent


 24   transfer of the nuclear material, the chromosomes


 25   from the donor egg into the recipient egg.  I



  1   should point out here that would had actually met


  2   with a mitochondrial geneticist at our institution


  3   to find out--you know, we posed this problem of


  4   transferred mitochondria, and ask him did he think


  5   we would have a problem there; did he think that


  6   these mitochondria that we transferred we be passed


  7   on.  He assured us no, it was too few mitochondria


  8   and it couldn't happen.  So, we really didn't go


  9   into it thinking that that would be the problem.


 10   We were more concerned with accidentally


 11   transferring the nuclear material.


 12             So, we looked at some of the eggs that we


 13   had taken cytoplasm out of using staining.  We can


 14   actually see the spindle of the egg, and with this


 15   stain we can see the chromosomes on the spindle.


 16   So, we looked at these eggs that provided the


 17   cytoplasm, and this was published just recently,


 18   last year, and the oocytes that we evaluated


 19   resulted from either clinical cases I just


 20   described to you or research procedures which we


 21   are doing.


 22             In this case 12 oocytes were thawed but


 23   were not used for the transfer.  They weren't


 24   needed to provide cytoplasm so we used those as


 25   controls.  We had 23 eggs that we thawed which



  1   survived the donation procedure.  These are the


  2   ones that served as tests.


  3             When we did the staining procedure on


  4   these eggs, the control eggs all demonstrated


  5   normal meiotic spindle but when we looked at the


  6   test eggs we found that 2/23 eggs that provided


  7   cytoplasm demonstrated total dispersion of the


  8   chromosomes from the metaphase plate, and complete


  9   disorganization of the spindles.


 10             Of course, the numbers are very small but


 11   there was no significant difference between the two


 12   groups.  So, we wondered if this was something to


 13   do with the drawing out of the cytoplasm that


 14   potentially disrupts the spindle.  We wondered,


 15   since it is a procedure that is very similar to


 16   ICSI, if this would be the same rate of meiotic


 17   spindle damage that you would see in ICSI oocytes.


 18             Because we were worried about this we


 19   looked at ways to see if there were some way we


 20   could prevent this.  So, we looked at a new


 21   microscope that was on the market, the PolScope.


 22   Having this attached to your microscope actually


 23   lets you visualize, while you are doing a


 24   procedure, the actual spindle so that you can see


 25   the spindle and you can stay clear of it.



  1             Here is the egg, just a small part of the


  2   egg, the polar body and the spindle here.  So,


  3   while you are doing the procedure, you are sticking


  4   something into the egg and you can see the spindle


  5   and stay clear of it.  This is equipment that is


  6   currently used in many laboratories, including ours


  7   now, in which clinical ICSI cases are performed, or


  8   research involving enucleation where they want to


  9   see where the spindle is so they can take out the


 10   nuclear material.


 11             We also did a little work with looking at


 12   this from a research aspect.  We had a clinical


 13   fellow, Sam Brown, who wanted to see if the


 14   original work of Flood in 1990, where we used


 15   immature eggs, would have the same ft, cytoplasmic


 16   transfer.  The idea with it is the developmental


 17   failure of human embryos derived from oocytes


 18   matured in vitro may be due to the deficiency of


 19   cytoplasmic factors.  In in vitro fertilization we


 20   have found that when patients get a lot of immature


 21   eggs, eggs that need more time maturing before they


 22   can be inseminated, these eggs do not do as well.


 23   So, the idea was to see if human prophase I oocytes


 24   became developmentally competent after


 25   microinjecting them with the ooplasm of eggs



  1   matured in vivo within the body.


  2             Sam hypothesized that such an injection


  3   would improve fertilization and blastocyst


  4   development of these immature eggs.  This was just


  5   a research project.  None of these eggs were


  6   transferred back to patients.  It was with the hope


  7   of salvaging immature eggs.  For example a patient


  8   who gets all immature eggs after a retrieval could


  9   have this procedure done and improve her chances of


 10   achieving a pregnancy.


 11             In the first part of the experiment looked


 12   at the effect of cytoplasmic transfer from in vivo


 13   matured eggs into PI eggs.  So, we had three


 14   groups, control eggs which were put on a stage of


 15   the microscope but not actually injected.  We found


 16   that 74 percent of these matured to metaphase II


 17   after continued culture.  Sham eggs were eggs that


 18   were injected with an equal amount of media only,


 19   not cytoplasm, and we found that only 50 percent


 20   matured to metaphase II.  Cytoplasm transfer eggs


 21   that actually had the procedure, 58 percent matured


 22   to metaphase II.  So, these findings suggested that


 23   injecting a substance into an egg may have a


 24   negative impact on maturation.


 25             We also inseminated these eggs to see if



  1   they could be fertilized, and in the control the 14


  2   eggs that matured to metaphase II we had a 50


  3   percent fertilization rate.  Shame injected, we


  4   only had 38 percent fertilization rate.  With


  5   plasmic transfer four of the eight fertilized,


  6   which was 50 percent.  The development after


  7   culture was not remarkable between the three


  8   groups.  The numbers were very low and similar to


  9   what we always see with immature eggs.


 10             We also looked at the effect of


 11   cytoplasmic transfer on eggs that matured in vitro.


 12   They were first allowed to mature in vitro and then


 13   they were given the cytoplasm of an egg 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



  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



  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



  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



  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



  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



  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,



  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



  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



  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.



  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



  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



  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



  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



  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.



  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



  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



  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



  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



  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



  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



  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



  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



  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



  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



  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.



  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



  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



  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



  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?



  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



  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



  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



  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



  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



  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



  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



  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.



  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



  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



  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



  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



  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



  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



  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



  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.



  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.



  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



  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



  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



  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.



  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



  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



  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



  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,



  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.



  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



  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



  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



  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.