SYNOPSIS: This proposal aims at generating Amyotrophic Lateral Sclerosis (ALS) patient-specific human embryonic stem cells (hESCs) by somatic cell nuclear transfer (SCNT). In their previous experience in cloning pigs, the investigators have demonstrated that the modification of two criteria in the traditional SCNT protocol can increase the ease and efficiency of SCNT-derived porcine embryos. One is the use of frozen oocytes and the second is a modification of preexisting SCNT technique. The investigator proposes to apply this newly developed technology directly to humans. There are three specific aims. The first specific aim is to create SCNT-embryos using frozen oocytes and fibroblast cells from ALS patients. Human skin fibroblasts from ALS patients will be used for SCNT donor cells. The second specific aim is to establish reliable systems to derive stem cell lines of clinical grade from SCNT embryos. SCNT embryos at the blastocyst stage will be used to derive ESC lines. The third specific aim is to investigate future clinical application of ALS disease. IMPACT AND SIGNIFICANCE: As no ALS embryonic stem cells are currently available, the isolation and characterization of such a diseased line will not only provide the beginning of a proof of concept for this technology, but more importantly will establish a realistic platform to study the molecular basis of ALS, a devastating disease which remains incurable. The other main significance of this work is the use of frozen oocytes instead of fresh oocytes, which alleviates a number of ethical issues regarding payments for donations to patients. This should not be underestimated, as this simple fact is one of the major limitations of SCNT approaches. The use of novel SCNT techniques that have been proven useful in pigs may advance the technology in humans. Optimization of oocyte activation and SCNT techniques for the derivation of hESCs would be of use to the hESC research community by making disease model cell lines available, and is not currently fundable by the NIH. However, one reviewer felt that the lack of experience in hESC derivation, preliminary data and a complete research plan render this proposal very high risk. QUALITY OF THE RESEARCH PLAN: Each specific aim has been nicely defined and in each individual experiment the outcome analysis is done to satisfaction, according to one reviewer. The use of the proposed SCNT techniques established in the Yang lab is an exciting prospect for hESC technology. However, although the proposal relies on preliminary data from the Yang lab, it does not seem to follow the plan used by the Yang lab. There is no indication of a plan to enucleate the oocytes either in the text or the summary figure, although the plan seems otherwise to be based on work in pig oocytes where enucleation was actually done. Further, enucleation likely will be the step with the biggest loss of oocytes, and has not been tried with frozen oocytes in any animals. No plan for derivation, including no description of how this has been, or will be done in the lab was included or referenced. Further, while hESC derivations have been performed at CHA, no one on this project has experience with this crucial step in the program. In fact, rather than having personnel at CHA train someone or perform the derivations themselves the person responsible for the derivation will be recruited. Persons with this expertise are rare and in high demand. The use of frozen oocytes for SCNT has not been established. The fragility of the oocyte and the stiffness of the membrane increases with age of the oocyte, and this may also be true of frozen/thawed oocytes. This poses a technical problem both for enucleation and whole cell injection. From a scientific and ethical perspective, attempting SCNT with frozen oocytes of another species, such as mouse or pig should be done before using clinically useful human oocytes for research purposes. It is not clear why the sections on ACE-SCNT-ESC are needed or add to the project. STRENGTHS: The proposal is well-written and includes preliminary data in pigs and a novel fusion method. The research plan is nicely developed and the PI has the appropriate expertise, at least in animal cloning (less with hESCs), to be successful in this endeavor. Success of the PI in the porcine model adds strength to the plan. A large collection of letters of support provides evidence of enthusiastic collaboration with the PI that will add critically needed expertise to the project. The plan to differentiate and transplant hESC-derived neural cells in a well-established mouse model with experts in the field strengthens the lack of experience with hESC culture (but not derivation) by the rest of the group. WEAKNESSES: This is a proposal that can be easily qualified as overly ambitious. The author provides a shopping list of all the experiments that will happen after the ALS SCNT embryos have successfully been established and characterized. This seems premature. The proposal would be successful if specific aim one is first done accurately and convincingly to generate a handful of lines that will be available for the community. Preliminary data on enucleation, SCNT and hESC derivation in an animal model should be done before proposing these studies. Specifically, SCNT on frozen oocytes in an animal model should be done before using completely viable, clinically useful human oocytes. The use of frozen oocytes for SCNT has not been established, and is likely to be a significant technical problem for enucleation and whole cell injection. There is no indication of a plan to enucleate the oocytes in the proposal and a clear rationale for using one or both of the methods used by the Yang lab is required. A plan for the derivation of hESCs is also needed along with a rationale for the use of ALS cells for tranplantation studies, rather than normal cells. It also appears that no one on this project has experience with this hESC derivation, or the derivation of any ESC lines. The section on ACE-ESC is not necessary for the proposal and should be removed. The ACE-ESCs are not stable lines that have been shown to be maintained in vitro. In fact, they appear by the literature and preliminary data to be a mixture of hESCs and hESC-derived differentiated populations. The plans to differentiate hESCs to neural cells for transplantation do not require this intermediate step. It is unfortunate, because the application of novel SCNT techniques is a reasonable way to move the field of SCNT and hESC biology forward. If the rest of the proposal was as well-designed as the pig studies, the score would be very high. DISCUSSION: One reviewer felt that, although SCNT is very important and exciting, this application is weak compared to others. The proposal is over-ambitious. The proposal is significant, however, in that there are no ALS hESCs currently available. There are strong collaborators who are perfectly placed to get the SCNT work under control. The use of frozen embryos is becoming more frequent, and the thaw rate success is very high, in the 60-70% range. One reviewer felt that the series of experiments should be laid out better. Another reviewer felt that the proposal lacks preliminary data. This reviewer is also enthusiastic about encouraging work in SCNT, but a main reason that this work might not succeed is the freezing of oocytes. Freezing causes them to become brittle, and there are no preliminary studies presented from oocytes in pigs. The prediction is that it will be more difficult here, and the reviewer would have been less worried if the PI hadn’t left out enucleation as a key step in the procedure. This speaks to the naivete of the PI, according to the reviewer. Of the relevant references from the Yang lab, the applicant did not choose one of the two methods of enucleation to focus on for preliminary data preparation. This oversight is significant since no animal data were provided to support frozen oocyte SCNT. Furthermore, no one on the project has ever derived ESCs and there is no mention of how they’ll do it. Another reviewer disagreed that they should be penalized for forgetting to mention enucleation since the group is familiar with the procedure. A different reviewer thought that the frozen oocyte concept is very attractive, and it seems that this group was successful in pig. The applicants show that their cell fusion method works in pigs for cloning, and it seems that here they want to study frozen oocytes. The group has good access to an oocyte supply. There was some question about the CHA Fertility Center, including whether it is a for-profit organization, but other panel members noted that this is the biggest IVF clinic in the world with a significant history in using frozen oocytes and success in using this technique to make babies. In fact, this group holds the patent on the use of frozen oocytes, and one of the presidential lines came from CHA.