Funding opportunities

Genetically modified human ES cells as novel tools for developing human cell replacement therapy

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01032
Funds requested: 
$925 200
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Human ES cells (hESCs) are capable of unlimited duplication (self-renewal) and retain the ability to differentiation into all cell types in the body. Therefore, hESCs hold great promise for the human cell replacement therapy. While significant progress has been made to differentiate hESCs into various cell types, major obstacles remain that hinder clinic trial in human patients. First, during the cell type-specific differentiation of hESCs, a small fraction of undifferentiated hESCs frequently remain mixed with the differentiated cells. These undifferentiated hESCs pose serious cancer risk by forming tumors after transplanted into patients. Second, the cell type-specific differentiation of hESCs is usually consisted of multiple steps, resulting in a mixture of cells of various lineages and developmental stages. Approach to purify the cells of interest from this mixture of differentiated cells, a prerequisite for human therapy, is generally not established. Our overall goal is to develop novel tools and strategies to overcome these major obstacles by genetic manipulation of hESCs through efficient homologous recombination, a technology already established in my lab. Since the federally approved hESCs cannot be used for efficient homologous recombination due to their growth requirement as cluster of cells, our proposed research must use non-federally approved hESCs and thus cannot be supported by any federal agencies. To eliminate the undifferentiated hESCs during cell type-specific differentiation, I propose to employ homologous recombination to genetically modify hESCs so that the undifferentiated hESCs but not their differentiated derivatives can be effectively killed by existing FDA-approved drugs. One of a few clinically proven human cell replacement therapies is the transplantation of β cells harvested from human donors into diabetic patients. Due to the limited source of human donors, hESCs have become a very promising source for β cells. Recent studies have provided definite evidence that hESCs can be differentiated into β cell precursors, which differentiate into functional β cells in mice. However, the lack of purification steps leads to tumor formation and highly mixed cell populations in the transplants. To solve these problems, I propose a strategy to easily purify the hESC-derived β cell precursors by genetically modifying hESCs through homologous recombination so that these β cell precursors are marked with fluorescence. These genetic modifications will introduce genes into the specific sites of the hESC genome that should have no impact on the expression of the endogenous genes. Products of these genes should have little toxicity on hESCs and their differentiated derivatives. Therefore, once validated, these tools and strategies could be directly applied to the therapeutic grade hESCs and improve their safety and efficacy for human therapy.
Statement of Benefit to California: 
Human ES cell (hESC)-based therapy holds great promise for curing many life-long devastating diseases such as the Type 1 diabetes. Type 1 diabetes is one of the most common chronic diseases in children and young adults in California. This life-long disease, caused by the autoimmune destruction of the insulin-secreting pancreatic β cells, is the major cause of blindness and kidney failure despite existing insulin replacement therapy. Recent success in restoring long-term insulin independence by transplanting β cells harvested from human donors into diabetic patients provides the proof-of-concept evidence that human cell replacement therapy could cure the disease. Due to the very limited supply of β cells from human donors, hESCs have become a very promising source of unlimited supply of β cells. Recent studies have provide definite evidence that hESCs can be differentiated into functional β cells. However, the cancer risk associated with the undifferentiated hESCs mixed with the differentiated cells and the lack of an approach to purify the β cell precursors differentiated from hESCs pose serious obstacles for the clinic trials in human patients. Our proposed research is aimed to overcome these major obstacles so that clinic trial of this highly promising human ES cell based therapy becomes feasible in the very near future. If successful, our proposed tools and strategies should be applicable to eliminate cancer risk associated with all other hESC-based cell replacement therapies of major human diseases.
Review Summary: 
This application focuses on the development of tools for eliminating undifferentiated human embryonic stem cells (hESC) from differentiating populations as well as improved methods for purification of endocrine beta cell progenitors differentiated from hESC. The applicants will use homologous recombination to engineer hESC lines that express a specific marker only when undifferentiated, and therefore will be selectively eliminated from differentiating populations by use of known drugs that specifically target the marker. The applicants also propose a similar strategy to engineer stem cells that express fluorescent markers under the control of two pancreatic lineage promoters. The dual expression of these markers will subsequently be used as a tool to purify endocrine progenitors from differentiating hESC populations that may eventually produce insulin when transplanted into mice. The reviewers agreed that in theory, the proposed technology addresses critical roadblocks that must be overcome in order to advance stem cell science. First, the ability to safely remove undifferentiated stem cells from differentating populations is an essential goal for preventing teratomas. Second, a reporter cell line such as the one described would be valuable for developing improved methods for preparing safer and more authentic tissues for regenerative medicine. Despite these implications, it was not clear how the proposed methods would offer advantages over the many similar approaches that are currently in use or under development. Thus, the reviewers were not convinced that the overall impact would be significant. The reviewers were also uncertain of the feasibility of this effort for a variety of technical and practical reasons. While the applicants were very qualified to develop techniques for homologous recombination, the level of cell biology expertise on the team seemed insufficient. The applicants did not have in hand many of the tools and reagents that would be necessary for this endeavor, and it was not clear that the path to obtaining them would be straightforward. For example, it is not known whether or how well the proposed hESC line can be driven to the pancreatic lineage, preliminary differentiation data were presented using a different hESC line. Furthermore, the applicants did not have the proposed vectors in hand that would be used for the described genetic modifications. Another significant concern was the perceived lack of rigor and ambition in the proposed validation experiments. For example, the applicants proposed only PCR as a means to evaluate differentiating pancreatic lineages rather than assessing their functionality before transplanting into animal models. Furthermore, the choice of promoters for the endocrine cell selection suggested, based on known expression patterns of the corresponding genes during pancreatic differentiation, that the proportion of double labeled cells would be extremely low and perhaps very challenging to obtain in quantity. Beyond these potential hurdles, the reviewers expressed concern that the technical challenges of achieving 2 sequential rounds of homologous recombination in hESC, and simultaneously retaining ESC properties, would preclude this effort from being realized in two years. Finally, although some reviewers felt that the experiments were well described, others found experimental detail lacking in some parts of the proposal. The reviewers agreed that the principal investigator and much of the team are highly qualified in molecular techniques. In addition to their strong track record, the preliminary data spoke to their capabilities with homologous recombination in hESC. While the co-investigator is a highly qualified expert in beta cell biology, and thus provides needed expertise, there were concerns that the 3% level of commitment would be insufficient. This observation was largely evidenced by a perceived naiveté in the applicants’ assumptions that achieving pancreatic lineage differentiation would be straightforward despite a decade of precedent suggesting otherwise. Reviewers indicated that the budget was highly excessive for an endeavor of this magnitude. In conclusion, the reviewers acknowledged that the proposed technology addresses important problems but they were unconvinced of its feasibility based on technical issues and the team’s overall expertise and commitment. Thus, in general, enthusiasm for this proposal was only moderate.
Conflicts: 

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