Tools and Technologies I
Embryonic stem (ES) cells offer great promise for normal tissue repair and diseased tissue replacement. It is believed that this cell therapy someday will revolutionize medicine. However, the use of currently available human ES cell lines in patients risks rejection by the patient’s own immune system. Creating “autologous” stem cells specific to individual patients by therapeutic cloning has offered an alternative way but not without ethical concerns and technical challenges. The procedure requires human eggs, and the successful rate of cell cloning is extremely low, making it hard to be broadly applied to common research and clinical facilities. For more than ten years we have studied a process called cell reprogramming, by which stem cells are produced from a person’s skin cells. In 2005 we made a breakthrough by converting human skin into stem cells using three genes known to be involved in maintaining characteristics of ES cells, in combination with ES cell extracts. Recent breakthroughs by two groups of scientists in Japan and US demonstrate that human stem cells can be generated directly by viral introduction of four genes. These cells are named “induced pluripotent stem” (iPS) cells. The characteristics of iPS cells thus produced are indistinguishable from human ES cells. Thus, this approach heightens the hope and promise beyond the use of ES cells in clinical application, since it bypasses the ethical controversy and can be tailored for each patient for a given therapeutic purpose. Nevertheless, this technology is still in its infancy and is in need of improvement. Major hurdles include a risk of cancer from the stem cells, low efficiency, and the risk of mutation and gene instability. Studies in our current proposal aim at overcoming these hurdles by using three novel strategies. First, we will get around part of the cancer problem by replacing the tumor-causing gene c-Myc with an anti-aging gene, a most potent reprogramming factor recently identified in our lab. This gene is known to affect aging and cell differentiation. Second, instead of using a cocktail of four virus-carried genes, we will construct the new set of genes as a single “poly-gene” to simplify the process of cell transduction and reduce the copies of viral DNA being randomly inserted into the cell. Third, we will create a genetically engineered enzyme that can enter the cells to specifically remove the viral DNA, while not affecting the features of iPS cells. Through this proposed project, we hope to establish a powerful tool that generates personalized stem cells with significantly improved efficiency and safety. We believe our “genetically cleansed” stem cells will bring us one step closer to clinical use in humans. Thus, there is every reason to hope that this revolutionary new approach will result in radically improved ways to create human stem cells to treat disease.
Statement of Benefit to California:
Our proposed research aims at establishing a new approach to the production of so-called ìinduced pluripotent stemî (iPS) cells with the benefits of high efficiency, and little or no risk of genetic instability or oncogenesis. We believe this project will make the following contributions to the state of California and its citizens. 1. Improve Californiaís health service by providing a cost-effective and time-saving way of producing custom iPS cells tailored to each individual patient. These stem cells will be more economically produced and will not be rejected by the patientís immune system when transplanted into the body. Thus, they will be much safer than ES cells that are derived from an embryo when applied to clinical cell therapy. 2. Set up an iPS cell bank with the technology developed in this grant. [REDACTED] will use its own resources to create thousands of human iPS cells carrying various genetic backgrounds including HLA subtypes. It will benefit the basic research and clinical applications in California and world-wide. 3. Increase Californiaís visibility in stem cells research. With the success of proposed research, we will attract more federal funding to sponsor future research and fulfill the wish of California citizens who voted unanimously to support stem cell research. 4. Benefit the health and quality of life of California citizens. Our proposed improvements in the procedures of iPS, when translated into therapeutic interventions in regenerative medicine, will directly benefit the health and quality of life of California citizens. 5. Stimulate Californiaís economy growth by a state-wide increase in the regenerative medicine industry. The iPS technology may significantly boost the growth of biotechnology and pharmaceutical industries in California, making our state the front runner of regenerative medicine industry. The increased revenues in the state will further enhance research and development activities leading to treatment or cure of diseases.
This application focuses on the development of novel technologies for generating induced pluripotent stem cells (iPS) that are safer and more efficient than current methods. To achieve these ends, the Principal Investigator (PI) proposes to express a single polygene containing a novel reprogramming factor in concert with Oct4, Sox2 and Nanog. This viral vector construct will be transfected into fibroblast cell lines to induce expression of pluripotency factors and generate iPS cells. Next, the PI will introduce an enzyme capable of removing the viral sequences from the iPS cells. Once this “genetic cleansing” has been achieved, the resulting cell lines will be evaluated for pluripotency and compared to embryonic stem cells by genetic and epigenetic analysis. The reviewers valued the technology presented in this proposal, which was notable for its unique and elegant approach. Although several technical deficiencies were noted, the reviewers were encouraged by its potential to advance the field. The impact of the proposed technology is potentially high, as it addresses three limitations in current iPS methodologies. First, by eliminating c-Myc from the reprogramming strategy, the risk of tumor formation would be significantly reduced. Second, by combining pluripotency regulators into a single viral vector, induction of pluripotency could be achieved more efficiently. Finally, the enzymatic removal of viral sequences would minimize the risk of insertional mutagenesis. If successfully realized, the technology would facilitate development of powerful new tools for biological applications. Reviewers were confident in the experience of the applicants, but expressed some uncertainty that the proposed approach could be sufficiently effective to generate iPS cells that are free of viral integration. The proposal was well written and clearly presented, and the milestones were thoroughly and convincingly articulated. In addition to encouraging preliminary data, the PI carefully considered potential difficulties and possible solutions. The reviewers were particularly enthusiastic about two aspects of this proposal. First, the polygene approach for concerted expression of pluripotency-inducing factors was praised for its originality as an approach to limit integrations. Second, excision of viral sequences by the engineered enzyme was thought to be mostly feasible. However, reviewers were not convinced that the applicants would be able to successfully minimize viral integrations to a single copy per cell and, if not, whether the enzyme would necessarily remove multiple integrations. The PI failed to discuss how the number of viral insertions would be minimized and to justify whether one integration event would be sufficient for induction of pluripotency. More importantly, it was unclear to the reviewers that a 1:1:1:1 stoichiometry of induction factors would be effective for activating pluripotency. Reviewers also questioned whether this approach could ultimately gain the regulatory approval needed for clinical use but recognized that it would be a valuable research tool. Finally, while the reviewers emphasized the importance of using clonally derived cells, this was not addressed. The reviewers noted that the PI and assembled team of investigators have the appropriate expertise to carry out this project. They have a strong track record in stem cell biology and biotechnology development, and there is an appropriate collaborator who will perform the epigenetic analyses. In summary, the proposed technology would address multiple deficiencies in the generation of iPS cells. The proposal was found to be unique and exciting with a few technical issues that could encumber the intended outcome but overall scientifically meritorious.