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Induction of iPS Cells Using Transient Episomal Expression Vectors: Validation of Toxicity Testing in iPS-Derived Hepatocytes.

Funding Type: 
New Cell Lines
Grant Number: 
Funds requested: 
$1 386 261
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
One of the biggest scientific breakthroughs of the past decade has been the isolation of human embryonic stem (hES) cells, which hold vast potential to treat conditions including diabetes, Parkinson’s, spinal cord injury, hearing loss, and cardiac disease. However, the ethical and political issues surrounding the use of human embryos have hindered development of these therapies. Recently, innovative publications have demonstrated that human skin cells can be reprogrammed into cells that are very similar to hES cells. These “induced pluripotent stem cells,” or iPSC, hold similar potential to hES cells but avoid the use of human embryos. Furthermore, since skin cells are obtained more easily than embryos, iPSC facilitate the generation of patient- or disease-specific cell lines. Currently, viruses are used to transmit the genes necessary for reprogramming the skin cells, and these viruses permanently integrate into the DNA of the cells. Although the genes may be shut off after iPSC are induced, at least one of these genes, c-Myc, is capable of causing tumors when reactivated in cells derived from the iPSC. Here we propose a novel method of generating iPSC that does not use viruses and, therefore, avoids insertion of the reprogramming genes into the cells. These genes will only exist temporarily, long enough to allow the skin cells to change into iPSC. Once iPSC have formed, the reprogramming genes will be "kicked out" of the cells, eliminating the potential of tumor formation from reactivation of c-Myc. Furthermore, this method allows finer control over the amount of each reprogramming gene delivered to the cells than does the viral method, which means this method should be more efficient at producing iPS cells. Genetic differences between individuals, particularly differences in certain liver genes, lead to differences in how patients respond or react to drugs. Currently, few good cellular models of liver are available, especially those that model the genetic diversity of human populations. Normal liver cells isolated from the body last in culture only a very short time, during which they gradually lose appropriate drug processing activity. Transformed liver cell lines can be cultured indefinitely due to activation of a gene normally involved in cancer, but because of this gene, it is unclear whether these cells respond to drugs in the same way that normal liver cells do. We plan to use our new iPSC method to derive three patient-specific lines that represent variation in some of the important drug metabolizing liver enzymes. The advantage of using iPSC is that they are a renewable source of normal, non-embryo-derived cells that can be turned into liver cells, thus allowing earlier identification of drug-induced liver toxicity, decreasing the number of animal studies needed for drug development, and permitting study of the effects of genetic variation on drug metabolism. This should lead to the development of safer and more effective drugs.
Statement of Benefit to California: 
Drug-induced liver toxicity is the leading cause of liver failure in California, as is the case in the rest of the U.S. as well (Goldkind and Laine 2006). This includes drugs that were approved by the U.S. Food and Drug Administration (FDA) but subsequently removed from the market. In fact, the FDA estimates that increasing efficiency of early detection of toxicity by only 10% would save the pharmaceutical industry $100 million per drug, on average, of R&D development costs. In addition to the generalized liver toxicity, a significant reason for these adverse events results from the genetic differences (“polymorphisms”) between individuals with respect to certain drug metabolizing genes found in the liver. What the industry needs is a more robust clinically predictive drug screening system for the early detection of generalized and polymorphism-specific liver toxicity. Our iPSC-derived liver cells will be the basis of both types of screens by allowing the generation of polymorphism-specific cell lines. Eventually a panel of these donor-specific cell lines could be generated that represent most of the variation seen in humans. This should result in earlier identification of drug-induced liver toxicity, keeping many of the more dangerous clinical drugs from ever reaching the market and leading to the development of safer and more effective drugs for all Californians. This proposal has been submitted by a California company whose mission is to develop and commercialize ES cell-based assays for the pharmaceutical industry. The Company has many years of experience working with human and mouse embryonic stem cells as tools for drug discovery and development, and has a successful pharmaceutical partnership using ES cells for diabetes drug screening. Partnering and licensing of the technology developed in this proposal, as well commercialization of new and safer drugs, would bring revenue and additional jobs to California. Intellectual property gains from patents arising from these technologies are also potentially significant for California, due to the licensing revenue fees that would go back to the state. The cell lines developed would also be the basis of additional academic and corporate collaborations, increasing California’s leadership role in stem cell applications. Goldkind, L. and Laine, L. (2006). A systematic review of NSAIDs withdrawn from the market due to hepatotoxicity: lessons learned from the bromfenac experience. Pharmacoepidemiology and Drug Safety. 15:213-220.
Review Summary: 
Executive Summary The principal investigator (PI) for this application proposes to generate hepatocytes by iPSC (induced pluripotent stem cells) technology and directed differentiation protocols. He/she proposes to develop a non-viral, non-integrating approach for delivering reprogramming genes to adult fibroblasts in order to overcome the disadvantage of the current viral vector strategy for iPSC induction. Viral vector-mediated gene integration has a potential for oncogenic transformation. As an alternative to viral vectors, the PI proposes to introduce a mixture of inducible, episomal plasmids that encode reprogramming genes. Since the reprogramming genes would not likely be required long-term they could be lost as episomes over time without harm. The newly derived pluripotent stem cells would be characterized, induced to differentiate to hepatocytes and tested for liver functions. The final aim would be to generate new iPSC lines with specific genetic characteristics useful in drug development, metabolism and toxicity screening. Although the reviewers agreed that a non-viral, non-integrating strategy for the production of iPSC could be valuable, there was concern that the episomal expression system described by the PI is quite complex and may require considerable development. Preliminary data provided by the PI was inadequate to evaluate if regulated expression will be possible. It was pointed out that since the proposed inducible vectors frequently loose their regulation over time in culture even the initial concept of regulated expression will likely be problematic. Moreover, reviewers were not convinced that the preliminary data provided by the PI demonstrates successful differentiation of embryonic stem cells into mature hepatocytes. Therefore there was some concern if the investigators will be able to produce mature hepatocytes from iPSC in sufficient numbers for the drug screening assays and for other purposes described in the proposal. Reviewers also criticized the lack of hepatocyte and iPSC research background of the PI or other personnel on the team. Reviewer One Comments Significance: Significance and Innovation Human hepatocytes have a proven value for the investigation of drug metabolism, and toxicology testing of compounds. The demand for hepatocytes is greater than the supply. Significance. If the studies worked as planned, the cell lines created would have significant value for basic science and the pharmaceutical industry. The PI proposes to generate hepatocytes by iPS technology and directed differentiation protocols. She proposes to develop a SV40 based episomal system to transiently transfect reprogramming genes. She will, thus, prevent the problems associated with genetic integration and to introduce the reprogramming genes (Oct3/4, SOX2, KLF4, and MYC, (OSKM) and a temperature sensitive mutant SV40 Large T antigen (TAg). She proposes that the episomes can be expressed at the permissive temperature (33-35) and will inactivate and shut off when cells are incubated at 39. Since the programming genes would not likely be required long-term they could be lost as episomes over time without harm. The pluripotent cells would be characterized, induced to differentiate to hepatocytes and tested for liver functions. The final aim would be to generate designer cell lines by iPS of cells with specific genetic characteristics favorable for drug metabolism. Design and Feasibility of the Research Plan. The project is theoretically simple, yet every step will require significant development. The author even presents some of the steps in development that will be required including generating the plasmids for each gene to be used, optimizing the transient transfection of the 4 different vectors, optimizing the gene dosage of each vector of the mix, optimizing the generation and characterization of the iPS, optimizing the differentiation of iPS to hepatocytes and then characterizing the hepatocytes for mature liver function. Preliminary data presented here demonstrates that the authors can transfect genes, but the regulated expression of TAg or TAg driven genes were not demonstrated. Since these temperature sensitive vectors frequently loose their regulation over time and culture even the initial concept of regulated expression of the TAg will likely be problematic. It is possible, but not assured that pluripotent cell lines would be created by the technology proposed. The only alternative method suggested in the application was to use a Tet-inducible system if tsTAg transfection did not work. Clearly space is limited in the application, however iPS generated in more traditional ways could have been considered as the primary cell type for the application with the episomal method as one alternative. The author also shows data to demonstrate the ES cells cultured in specific ways begin to express endodermal markers. The markers are early endodermal markers and not representative of mature hepatocytes. It is not clear if the differentiation is anything more than spontaneous differentiation. Feasibility: PI and Key Personnel Dr. Bonham received her Ph.D in 1997 in developmental biology from Northwestern University. She did additional work at Geron from 2002-2003 and moved to VistaGen in 2004. She is currently a senior scientist in Cell Biology. She shows 5 selected publications in developmental biology research. There is no background with liver or hepatocyte research listed for her or any of the personnel listed. There is no record of success with other projects. There is no indication in the application that cell lines, if generated, would be shared. Given the for-profit nature of the company and their clear plans to use the cell lines for drug metabolism and toxicology testing sharing might pose a problem. Responsiveness to RFA: Not high. The plan is not likely to succeed and useful cell lines would not be expected. There is no indication in the application that cell lines, if generated, would be shared. Given the for-profit nature of the company and their clear plans to use the cell lines for drug metabolism and toxicology testing sharing might pose a problem. Reviewer Two Comments Significance: Proposed is an episomal system to deliver genes for reprogramming, this is significant since it would bypass the need for retrovirus, however it appears very difficult to accomplish this even though it may be feasible since there is some experience in delivering genes using this episomal system. Feasibility: Goal in Specific Aim 1 is to develop the non-viral inducible system, it is realistic and it is possible that the system, at least with the individual genes will be possible. - In Specific Aim 2 PI plans to characterize the cells, which appears good and described in some detail - Specific Aim 3 to differentiate and characterize iPSC -derived hepatocytes: Given the established protocols it is difficult to see how they will get a significant number of hepatocytes.

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