Basic Biology I
The use of embryonic and other stem cells is providing Medical science with the opportunity to develop a deep and entirely new understanding of the causes of many human diseases and to develop completely new approaches to their treatment. Particularly important will be a combination of cells and gene therapy techniques to arrive finally at therapies that produce definitive cures rather than mere symptomatic control of terrible and poorly-treated diseases such as many forms of cancer, neurological and psychiatric diseases, muscle-wasting disorders and many others. While the promise and the allure of stem cell treatments is enormous, it would be hazardous to this wonderful goal to underestimate the scientific, ethical and policy difficulties that still stand in the way of safe and effective treatments. We currently know only the most superficial aspects of stem cell biology and understand little of the technical and medical difficulties that must still be overcome before wide-spread application to clinical trials can be undertaken effectively and safely and with ethical confidence that we do more good than harm in clinical applications. For instance, the excitement surrounding the production of stem cells from non-embryonic sources such as skin cells (induced pluripotent stem cells – iPSC) opens the door to solutions for both scientific and ethical/policy dilemmas involved in the use of embryonic stem cells. However, the mechanisms involved in making and using such non-embryonic stem-like cells are virtually completely unknown. In our studies of the terrible and untreatable childhood counterpart of Parkinson’s disease known as Lesch Nyhan Disease (LND), we have discovered that there seems to be a powerful obstacle preventing the production of iPSC from skin cells from these patients. We wish to understand how this block works so that we can eventually produce normal brain neurons that reveal the reasons why cells from LND patients function incorrectly and so that we can design methods to replace or supplement those defective cells with functional neurons in this disorder. Learning more about the mechanisms of iPSC production will open vast new opportunities to iPSC- and embryonic stem cell-based understanding of the causes and treatment of many additional diseases that are appropriate targets for stem cell-based regenerative cellular and genetic therapy. Furthermore, we hope that success with this rare model disease can teach us a great deal about similar and much more common and societally burdensome diseases of the brain, especially the related disorder Parkinson’s disease.
Statement of Benefit to California:
With the exploding world-wide interest in regenerative medicine, California is in an excellent position to solidify its current role as one of the world’s most important centers for stem cell research and for the search for stem cell-based regenerative cellular and genetic therapies. Some of this work will center around the preparation and use of disease-specific and even patient-specific induced pluripotent stem cells (iPSC) from non-embryonic sources. However, a major obstacle to some of these non-embryonic advances involves the difficulty by current reprogramming methods to produce large amounts of well-characterized patient-specific iPSC to deepen our understanding of diseases and potentially to treat them by grafting and other cellular regenerative and genetic methods. To reach such a goal, we must understand the basic mechanisms of reprogramming and to our knowledge this HPRT blockade model provides the first genetic system to indentify the genes and their actions in driving the programming process. The work of CIRM is already spearheading all aspects of stem cell and iPSC-based research, and the impending relaxation of federal guidelines for stem cells research and the increasing pace of cellular and genetic research and their resulting advances will surely lead to important scientific and medical discoveries with enormous payoff in the clinical and in the biotechnology and pharmaceutical industries that support this work. Regenerative medicine is almost certain to become a cornerstone for much of Medicine in the future, and that evolution will come about through collaboration between academia and the private sector in many centers and parts of the world. But California will again lead the way toward many of those advances, as it is already beginning to do through the CIRM program. The power and depth of research and the current momentum in this area in California is unmatched anywhere in the world. Furthermore, much of the medical payoff will benefit tens or hundreds of thousands of patients in California who are suffering from intractable and untreatable disease and who will eventually benefit from improved health. Furthermore, the State will benefit from a healthier and more productive citizenry and from reduced health care costs and an enhanced tax base. California-based industry will benefit from the creation of very large and lucrative new forms of regenerative medical industries discovering and delivering these new therapies. Biomedicine in California is uniquely on the edge of developments reminiscent in earlier times of the discovery of antibiotics, cancer therapeutic agents, and transplantation methods and anti-rejection drugs. Thanks partly to the unique power of California academic institutions, biotechnology and pharmaceutical industries and to CIRM, scientists in California are poised to play a pace-setting role in this epochal advance in Science and Medicine.
This proposal is based on the applicant’s observation that fibroblasts from Lesch-Nyhan disease (LND) patients, lacking the function of the gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT), cannot be reprogrammed into induced pluripotent stem cells (iPSCs). The applicant proposes to explore the mechanism responsible for this reprogramming blockade in five specific aims: 1) to determine if overexpression of HPRT in deficient fibroblasts restores iPSC generation and if HPRT knockdown blocks iPSC generation in cells known to reprogram; 2) to determine whether the reprogramming blockade in HPRT deficient cells is specific to method of reprogramming; 3) to identify effectors downstream of HPRT that may be responsible for the reprogramming blockade; 4) to perform epigenetic analysis of HPRT deficient cells; and 5) to determine whether fibroblasts from patients with other cell cycle disorders also demonstrate reprogramming blockades or inefficiencies. Reviewers were not convinced that this proposal addresses a major unsolved problem in stem cell biology or is likely to have a significant impact. They were not sure that studying the mechanism underlying the reprogramming blockade in HPRT deficient cells would necessarily uncover something fundamental about reprogramming with the potential for broad application. The proposal’s rationale was considered potentially flawed, because a negative result (the applicant’s inability to reprogram HPRT deficient cells) was used to support a positive conclusion (that HPRT deficiency is blocking reprogramming). Reviewers raised significant doubts about the feasibility of the research plan and found it lacking detail and alternative strategies. They observed that the preliminary results section contains no direct evidence that the research team has generated or characterized any iPSC lines. The reprogramming data consists solely of a table listing a few cell lines and the applicant’s success or failure in generating iPSCs from them. There is also no preliminary data demonstrating that the described cell cycle defects in HPRT deficient fibroblasts isolated from LND patients can be rescued by introduction of a wild-type HPRT gene. Without this data it remains unclear whether HPRT rescue of the reprogramming blockade, as proposed in Aim 1, is feasible. Further, the entire project depends on showing in Aim1a correlation of HRPT expression with the ability to produce iPSC; failure to demonstrate that correlation will result on the end of the project as there are not clear alternatives. A reviewer found the suggestion that cell cycle aberrations could affect the efficiency of reprogramming to be quite interesting and suggested building a proposal around this observation. However, the concern was raised that the proposed experiments would only address how HPRT deficiency affects cellular processes and wouldn’t answer broader questions about reprogramming and pluripotency. Finally, reviewers felt that the focus of Aim 2 on the alternative reprogramming method(s) proposed was not well justified and poorly connected with the rest of the proposal. Reviewers had high praise for the applicant, whom they described as a world-renowned expert with a long and successful track record. They were enthusiastic that that the applicant is extending his/her research into potential therapeutic avenues for LND using reprogrammed cells. One reviewer did note that the research team could use additional expertise in iPSC generation. Overall, while reviewers appreciated the quality of the applicant, they raised significant doubts about the feasibility and potential impact of the proposal.