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RS1-00165-1: Genome Replacement in Human Embryonic Stem Cells

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Embryonic stem cells have tremendous potential value for the treatment of many diseases and injuries, but like other transplants their use is likely to be limited because the immune system of the recipient will probably reject the transplant unless there is a good genetic match between donor and recipient. The ideal solution to this problem would be to replace the genetic material of stem cells with genetic material from the prospective patient, and to use the genetically modified stem cells in therapy. Work on experimental animals has shown that it is possible to do this by implanting a nucleus from one of the patient’s body cells into an oocyte from which the nucleus is removed. The oocyte is then allowed to grow into an embryo, and stem cells are derived from the embryo when it reached the appropriate stage. This method suffers from several drawbacks, the most significant being the need for oocyte donors, the technical difficulty of performing the nuclear replacement, and ethical objections to the production of embryos which are later to be destroyed for the benefit of the patient. We have devised an alternative strategy in which the nuclei of existing stem cell lines will be replaced by nuclei from cells of the prospective patient, by physically fusing together the stem cell with a body cell from the patient under condition preventing the survival of the stem cell nucleus. The resulting cell lines, with the genetic material from the patient in stem cell cytoplasm, will then be grown up and should be suitable for use in cellular therapy. In the proposed research we will compare several methods for incapacitating or removing the stem cell nucleus from the fused cells, and we will rigorously test the resulting cell lines for the correct genetic makeup as well as the retention of developmental properties characteristic of stem cells.

Statement of Benefit to California (provided by applicant)

California has taken the lead in the nation in providing support for stem cell research and especially in support for developing the use of human embryonic stem cells in cellular therapy for many diseases and disorders. Many laboratories are investigating methods to control the differentiation of these cells, so that they will no longer be likely to produce tumors in the patient, and they will be more suited to regenerative therapy in specific organs. But the full potential of these cells cannot be met until a solution is found to the problem of immune rejection by the patient. Claims made outside the U.S. for success in generation of stem cell lines that are genetically tailored to the patient turned out to be fraudulent, so the field still awaits a solution to this problem.The conventional approach to generating patient-specific stem cell lines would be to transplant a somatic cell nucleus into an oocyte obtained from a donor, then allow the oocyte to develop into an embryo and recover stem cells from that embryo. However, this has several drawbacks as stated elsewhere in this proposal. California could continue to lead the nation in embryonic stem cell research and in developing the use of these cells in cellular therapy if a solution cold be found to the problem of immune rejection. Here we propose a novel but feasible method of generating patient-specific embryonic stem cells from existing stem cell lines, avoiding the problems of obtaining egg donors, and of generating embryos for later destruction. If this method could be developed in California it would allow the state to continue the momentum that has built up in stem cell research, and to continue to build upon it by moving to the next stage where these cells are actually used in the clinic to treat some of the most devastating injuries and diseases affecting our people.

Review

SYNOPSIS: The proposed studies will seek to develop alternative methods to re-program somatic cell nuclei to an embryonic stem (ES) cell state using cell fusion. The experiments will be performed in the human system using human embryonic stem cells (hESC) and somatic cells lines of a specific lineage. Briefly, the nucleus of the hESC will be incapacitated by promoting nuclear degeneration, either before or after fusion, while the somatic cell cytoplasm will be incapacitated with a mitochondrial poison. Following fusion, the only cells that can survive in principle will be hybrids (nucleus from somatic cell and cytoplasm from hESC). An alternative method is to label the hESC nucleus with a vital dye, and destruction of this nucleus following fusion will also be explored. Any surviving hybrid cells will be evaluated genotypically and phenotypically. The PI will utilize somatic cells and hESC that are genotypically distinguishable using genetic markers. These markers will be used to insure the exclusion of ES cell-derived nuclear DNA and to identify the origin of the organelles in any viable hybrids. Particular emphasis will be placed on examining pluripotency markers in the somatic nucleus.

SIGNIFICANCE AND INNOVATION: If successful, the studies in this proposal may alleviate 2 major problems that will arise in re-programming of somatic nuclei to generate patient-specific hESC lines. First, a need for oocytes (and oocyte donors) will be eliminated, and second there will be no need for sophisticated nuclear transfer technologies. The heart of the proposal lies in a complementation approach where a hESC with an incapacitated nucleus, and a somatic cell with incapacitated cytoplasm are fused to obtain hybrids. These are then cultured in conditions that require both mitochondrial and nuclear functions for survival. It is hoped that any resultant viable hybrid will have the DNA content (genotype) of the somatic nuclear “donor” cell and the cytoplasm and mitochondrial component originating from the starting hESC. It is further hoped that the hESC cytoplasm will re-program the somatic nucleus to an ES cell fate. Reviewers had differing opinions regarding the significance and innovation of the proposed studies. Nonetheless, reviewers clearly felt that the ability to re-program somatic nuclei without a need for oocytes would be a major advance. Whether this will be possible or not remains an open question; however recent results suggest that a limited number of factors may be sufficient at least to approximate effective oocyte-mediated re-programming. The approaches in this proposal are novel, although with significant potential complications. Overall, these studies are of high risk, but potentially have very high significance. The PI proposes to use non-federally approved hESC; thus, these studies are not eligible for NIH funding, but there is no compelling reason why federally-approved hES cells could not equally be utilized for the proposed studies.

STRENGTHS: The strengths of the proposal include its collaborative nature and the attempt to establish a system for creating patient specific hESC that would not involve new embryos or oocytes. In addition, the proposed back-up plan of specifically labeling the donor nucleus and ablating it after fusion using a newly engineered device is interesting. While it has been shown that fusion of ES cells with somatic cells often re-reprograms the latter to an ES cell state (at least to a very good first approximation), the resultant heterokaryons have fused nuclei, and are therefore tetraploid. Even if methods could be developed to reproducibly reduce ploidy to a diploid state, the preferential exclusion of the genetic complement originally derived from the ES cell is difficult to envision. Clearly, this is not a general problem when using oocyte donors. The key to this proposal is to develop ways to incapacitate the nucleus of the hESC and the cytoplasm of the somatic cell, respectively, and prior to (or simultaneously with) cell-cell fusion. The PI presents some preliminary data to indicate that the poisons to be used are effective in somatic cell lines. The proposed studies are contingent on obtaining viable and genotypically verified hybrid cell lines; thereafter, the remaining studies to evaluate the extent of re-programming would be straightforward.

WEAKNESSES: The proposed studies are very risky, given the lack of evidence that viable, growing hybrids can be obtained and that these can proliferate for prolonged periods using the proposed methodologies. This is a major drawback to the proposed studies. Early observations indicate that growing cells were shown to be contaminant poison “escapees”, and given the rate at which they observed “escape” from the nuclear and mitochondrial poisons it is possible the hybrid cells are tetraploid or even not the product of fusion. Thus, there is little confidence that the proposed fusion studies will be successful. Even if permanently growing cell lines are obtained, it is not at all clear whether the hybrid cells generated via this method actually contain the correct nuclear composition. Are the genomic complements original-cell-type homogeneous or mixtures originating from the two donor genomes? There is also concern that the cytoplasm of ES cells may not be adequate to re-program somatic cell nuclei. There does not appear to be any direct proof of this, at least to the knowledge of reviewers, and there is no evidence that reprogramming can occur without nuclear factors. The most well established cell for reprogramming is the metaphase II oocyte, which has no nuclear envelope and free access of nuclear factors to the cytoplasm. In fact, recent studies have suggested that 4 nuclear proteins are sufficient for re-programming at least to an approximate ES-like state. These are very real considerations that substantially temper the enthusiasm for this proposal.

DISCUSSION: One reviewer recommends that, should future funding opportunities arise, this proposal be re-written to emphasize the unique ablation technique that is both highly innovative and avoids many of the reviewers’ concerns.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• Lansing, Sherry