Tools and Technologies I
The ability to modify the genes that are expressed in pluripotent stem cells or the mature cells produced from the stem cells will be important for many applications to regenerative medicine and for experimental research studies to understand better the biology of stem cells. For examples, inserting genes into stem cells may lead to cells with improved survival or ability to develop into specific mature cell types, may allow the developmental pathways to be studied and may be used to correct genetic diseases. Gene transfer into pluripotent stem cells is often limited by the transferred gene getting “turned off” as the stem cells grow into the desired type of adult cells. The primary goal of this project will be to develop improved gene delivery tools that will keep genes transferred into stem cells working and active. Studies will be performed to determine how the genes are turned off or kept on, to guide future development of optimal gene deliver tools. Diverse hESC and iPS lines will be studied to verify the generality of results; some of the hESC lines to be used cannot be studied with federal funds.
Statement of Benefit to California:
Development of methods for regenerative medicine using pluripotent stem cells will have wide-spread applications to improve the health and to provide novel, effective therapies for millions of Californians and tens of millions of people world-wide. Regenerative medicine may provide new treatments for diseases including diabetes mellitus, Parkinson’s disease, organ failure and injuries, inherited diseases and cancer and leukemia. The major challenge facing the field of regenerative medicine is to increase knowledge of the processes by which the mature cells of tissues (pancreas, brain, bone marrow, etc.) develop from stem cells, so that clinical approaches can be developed to produce cells suitable for transplantation. This Project will produce and apply novel tools for experimental studies of pluripotent stem cells and for the development of clinical therapies using hESC. The central focus is on the development of improved and optimal gene delivery vectors that can transfer and express new genes in stem cells, to influence their properties for research or for clinical purposes. These studies will help to advance the capacities for regenerative medicine. All scientific findings and biomedical materials produced from our studies will be publicly available to non-profit and academic organizations in California, and any intellectual property developed by this Project will be developed under the guidelines of CIRM to benefit the State of California.
This proposal aims to identify promoters that will facilitate reliable expression of transgenes in undifferentiated human embryonic stem cells (hESCs) and their differentiated progeny. The applicant proposes to systematically test a series of lentiviral vectors with different constitutive and lineage-specific promoters, as well as different genetic boundary elements, for the ability to stably express a green fluorescent protein (GFP) reporter gene in a number of hESC and induced pluripotent cell (iPSC) lines. The goal of this systematic approach is to identify promoters and elements that avoid epigenetic silencing and lineage-inappropriate expression, two common problems with lentivirus-mediated transgene expression techniques. The reviewers agreed that the overall goals of this proposal are worthy and important for the field. However, they questioned whether the specific approach would ever be feasible in a clinical setting. They also found the proposal overly ambitious and raised several concerns about the research design. Finally, the reviewers praised the applicant and research team but were worried about overlap with other work in the applicant’s portfolio. All of the reviewers felt that this proposal addresses a major roadblock in the field. Effective methods for the introduction of genetic material into hESCs will be needed when these cells are ready for clinical treatment of genetic disorders. Different approaches have been considered, including viral vector mediated gene transfer, in situ repair via homologous recombination, or episomally retained self-replicating vectors. The advantage of lentivirus-mediated gene delivery is that it appears to be relatively safe; one of the current disadvantages is that gene expression is often affected by epigenetic silencing. Therefore, identifying promoters that are stably expressed could potentially increase the practical use of lentiviruses, although reviewers expressed doubts as to whether an integrating lentiviral approach would ultimately be clinically relevant. Because conventional lentiviruses integrate randomly, they raise the possibility of insertional mutagenesis and inadvertent activation of endogenous genes. One reviewer suggested that methods such as in situ repair via homologous recombination combined with “recombineering” or addition of zinc finger nucleases to enhance efficiency, or non-integrating self-replicating vector approaches may hold more promise for generating clinically suitable stem cell therapies. Other reviewers noted that the proposal would be strengthened by a broader discussion of the disease states in which enforced exogenous gene expression might be beneficial. Reviewers specifically wondered what diseases might be ameliorated in erythroid progenitors or differentiated epithelial cells, two of the differentiated cell types chosen in the application. Reviewers described this proposal as very ambitious and raised questions about its feasibility. Although some reviewers described the research plan as well thought out and praised its systematic approach, another reviewer was concerned that identifying promoters for stable transgene expression in lentivirus-transduced stem cells, which is the principal objective of the proposal, is not feasible using this method. Since the applicant will analyze cell populations rather than individual cell clones, the average level of transgene expression will be identified in cell populations comprising multiple clones. Unless there are relatively dramatic differences in the strength of the various promoters in undifferentiated or differentiated stem cells, the applicant may not be able to distinguish the relative capacity of the various lentivirus vectors to affect transgene expression in the appropriate cells. As another example of the ambitious nature of the project, reviewers noted that nearly a dozen lentivirus constructs would be tested on at least 3 different pluripotent cell lines and presumably several more, as the total number of iPS cell lines to be tested is not mentioned. The rationale for using each of the promoters was limited to the observation that each possesses some measure of activity in undifferentiated hESCs. One reviewer questioned whether various constitutive promoters are likely to vary much in their activity, or whether knowing their precise comparative activity is an important goal. Finally, reviewers commented that there was a lot of work in the application that was redundant – for instance, assessment of transgene expression will be carried out by quantitative fluorescent activated cell sorting (FACS), global gene expression (RNA) profiling, and monitoring of the “epigenomic”status of DNA and histones comprising chromatin. The ambitious research plan raised concerns that the research project was not feasible. The reviewers agreed that the applicant is well qualified to carry out this research and has assembled an excellent team. The applicant is an expert in lentiviral design and stem cell biology and has an outstanding track record. The reviewers raised concerns about the number of grants the principal investigator holds with similar objectives to this proposal, and were worried about the possibility of double funding. Overall, while this proposal addresses an important area of stem cell research the reviewers raised several serious questions about its feasibility and clinical relevance, especially given the applicant’s current research activities.