To date, most basic and applied research on human disease is performed with animal models, the majority being mice. Unfortunately, most of what is learned from mouse models is not associated with human disease. Moreover, even with current advanced genetic technology, it is nearly impossible to generate models of rare diseases such as Amyotrophic Lateral Scelerosis (ALS), also known as Lou Gehrig’s disease. “Therapeutic Cloning” will provide a novel opportunity to study patient specific stem cells allowing one to understand the genetic causes of disease. It is a process known as somatic cell nuclear transfer (SCNT) in which the nucleus of a donor cell is transferred into an egg that has been emptied of its chromosomes. That egg now contains an exact duplicate of the donor's genome, and if all goes well when it is cultured in incubator for five to seven days, an SCNT-embryo will develop into a stage in the incubator for derivation of embryonic stem cells (ESC) which can differentiate into any cell type a patient my need for possible therapy. However, although the potential of combining SCNT technology with ESC research for the future therapies in animals has been demonstrated; there is little data demonstrating its feasibility in humans. While we understand and respect those who oppose SCNT-ESC research, we are equally sincere in our belief that the life-and-death medical needs of suffering children and adults will be ameliorated by SCNT-ESC work. This proposal marks the beginning of the effort to use human embryonic stem cells in a series of experiments whose principle has already been proven in animals. In our proposed research, we initially aim at understanding ALS following establishment of SCNT-ESC lines. However, there have been no reports of successfully generating SCNT-embryos from cultured adult cells in the human. Therefore, initial development and validation of the lab procedures for embryo development following cloning procedures needs to be accomplished. In our proposed research, we will use depository eggs donated by infertile couples undergoing in vitro fertilization who have had successful therapy and do not wish to keep frozen eggs for future replacement, which will be discarded. Frozen eggs as well as somatic cells from ALS patients will be obtained with informed consent in a controlled and scientific manner. Based on our proven record with successful SCNT in animal studies, we have reasoned that ESC lines can be established from SCNT-embryos which will be genetically identical to ALS patients’ own cells using frozen human eggs. Upon completion of this project, we will be able to prove the concept of SCNT-ESC research for the development of novel therapies for the treatment of human diseases. Furthermore producing such stem cell lines will provide a novel resource to the biomedical research community to study and understand how genes correlate with the development of disease.
Statement of Benefit to California:
The proposed research involves three cutting edge technologies: embryonic stem cell research, somatic cell nuclear transfer and gene modification. These technologies which are complementary to each other, have yet to be developed and optimized in humans, and when combined, will offer great potential in medicine. The ability to stay close to emerging technologies is paramount to keeping California business at the leading technological edge. In addition to the likely economic benefits to the State of such a commercial program, it is critical to point to the paramount medical benefits to the public of such collaboration in terms of the development of milestone medical therapies for future cell therapy. Upon completion of this project, we will be able to prove the concept of SCNT-ESC research as a future therapy for numerous diseases. In addition, stem cells derived from SCNT-embryos will likely be a conduit to drug-development, eventually leading to new pharmaceuticals. Furthermore, producing such stem cell lines would establish a novel resource to study the role of individual genes in disease development. With the improvement of cloning methodologies as described in our proposal, SCNT-ES cells could be used as a cellular transplantation resource. Cell therapy technologies overlap with those of gene therapy, cancer vaccines, drug delivery, tissue engineering and regenerative medicine. Collectively, the value of the cell-based markets is estimated to be $26.6 billion in 2005, $56.2 billion in 2010, and $96.3 billion in 2015. The largest expansion will be in diseases of the nervous system and cancer. Skin and soft tissue repair as well as diabetes mellitus will be other major markets. The number of companies involved in cell therapy has increased remarkably during the past few years. In 2006, more than 500 companies have been identified to be involved in cell therapy. Of these, 104 are involved in stem cell therapy. If the proposed research is successful as anticipated in contributing to the development of cell therapy products, there are likely to be substantial benefits to two critical sectors of the California economy, health care and pharmaceuticals. Additionally, our proposed research will benefit through further refinement and expansion of its SCNT-ESC technology and through collaboration in other areas of biotechnology, including molecular biology and immunobiology. Furthermore, the advancement of ESC research together with SCNT technique will enhance partnership developing programs for start-up as well as established biotechnology companies. Support for this project will help maintain our leadership in embryonic stem cell and cloning technologies and will help a California business to be better equipped to compete for a multi-billion dollar market.
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.