Funding opportunities

Development of non integrating gene delivery system for reprogramming adult cells and identify molecules to enhance reprogramming efficiency

Funding Type: 
Tools and Technologies I
Grant Number: 
Funds requested: 
$846 332
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
Embryonic stem cells have great potentials in the future of medicine and research because they can develop into virtually any kind of tissue type and potentially become an unlimited resource for cell therapy. However, the ethical issue surrounding the use of embryonic stem cells comes from the fact that they are derived from human embryos in a process that causes the death of the embryos. In 2006, scientists reported that stem cells can be created without eggs or embryos by using viruses to deliver four regulator genes, a process called induced pluripotent stem cells (iPSC). However, before iPS cells can be used in the clinic, many technical problems need to be overcome, such as eliminating the use of viruses or inserting regulator genes into the genome that potentially can cause cancer. Here we propose to develop a non integrating system to deliver the regulator genes which can be turned off after cells have been reprogrammed. This delivery system will enable researchers to introduce genes into the cells, switch them on during the reprogramming period, and turn off once cells have been reprogrammed. This method does not integrate genes into the cell's genome and thus, preventing additional changes in the host's genome. In addition, another technical difficulty to reprogram cells is that the process is very inefficient. Scientists have reported the success rate of 0.1-5% of obtaining iPS cells. We will be screening for molecules that will enhance the reprogramming process. Reprogramming adult stem cells into induced pluripotent stem cells could generate a potentially limitless source of immune-compatible cells for tissue engineering and transplantation medicine. However, before reprogrammed cells can be used in the clinic, they must be grown in conditions are free of any animal origin. We will perform and adapt reprogramming of cells in defined serum free medium containing enhancing molecules identified in this study to generate iPS cell lines that will be suitable for clinical studies.
Statement of Benefit to California: 
Stem cells have potential in many areas of medical research, such as they offer the possibility of renewable sources of replacement cells and new tissues to treat many kinds of diseases, conditions, and disabilities. However, the use of stem cells has been very controversial due to the fact that they are derived from human embryos. The recent discovery of transforming adult cells back to the equivalent of embryonic state without the use of eggs or embryos will open a new era of research in cell-based therapy and tissue engineering. Before reprogrammed cells can be used in the clinic, there are some technical hurdles need to be overcome, such as the use of viral vectors to deliver genes and introducing regulator genes that can cause cancer. We propose to develop a novel delivery method that does not insert genes into the cell's genome and enables genes to be turned on and off, thus preventing genes to be expressed and potentially cause cancer after cells have been reprogrammed. In addition, in order to obtain large number of reprogrammed cells for cell therapy, the reprogramming process needs to be more robust than the current state of obtaining between 0.1-5% reprogramming efficiency. We will screen for molecules and examine their ability to enhance reprogramming efficiency. Finally, we will generate iPS cells using techniques developed in this study and adapt them to animal origin free medium which will be applicable for clinical studies. Results from our research will increase the statue of California's research and benefit its citizens by enhancing the process of creating patient-specific pluripotent stem cells that can be used for disease research and cell replacement therapies.
Review Summary: 
The Principal Investigator (PI) proposes to develop a new method for generating human induced pluripotent stem (iPS) cells that could be safer and more efficient than the current, retroviral-based strategies. First, a non-integrating system will be developed using an inducible episomal vector. This system will enable the transient introduction of the reprogramming genes into adult somatic cells. Because episomal vectors do not easily integrate into the host genome, the potential for insertional mutagenesis is minimized. In the second objective, the applicants propose to improve the efficiency of iPS cell generation by evaluating the effects of various molecules on the induction of pluripotency. Finally, the PI proposes to develop serum-free culturing conditions for deriving clinical grade iPS cells. If successful, this proposal would have a clear impact on the future success of stem cell therapy. Human iPS cells are a desirable resource for a wide variety of applications at the basic and clinical level. Their potential for use is limited, though, by their potential for oncogenic transformation and the inefficiency of their production. The PI addresses both of these drawbacks by proposing a new method for iPS generation. One reviewer made the point that low reprogramming efficiency does not pose a limitation to the applicability of iPS cells, since sufficient numbers of cells are reprogrammed from most cell sources, thus calling the impact of the second objective into question. The proposal was poorly written and the rationale for many important experimental details was not provided. The feasibility of the overall proposal was unclear but varied considerably by aim. The reviewers were most enthusiastic about the first objective and felt that the strategy for creating the non-integrating vector system was sound. The reviewers were divided in their assessment of the preliminary data, as they support the PI’s expertise in vector construction and expression but not his/her ability to reprogram somatic cells. The feasibility of the remaining aims was difficult to ascertain due to a lack of critical details in the application. For example, the applicants propose to evaluate 10 of a specific class of molecules for their ability to improve reprogramming. However, they do not provide the identity of the molecules, the reasons they were chosen, or the justification for their use. Similar lack of rationale was pervasive throughout the proposal and led the reviewers to conclude that the experiments were poorly conceived. The reviewers found the research team to be well qualified for conducting the proposed studies. The PI is young but has relevant experience in molecular virology. The collaborating investigators provide strong support in the other areas of the proposal such as stem cell biology. Finally, the research environment is very well suited for this work and provides unique resources and advanced vector tools. In conclusion, this proposal describes a promising new technology of potentially high impact, but the applicants failed to provide sufficient explanations and rationale to convince the reviewers of its feasibility.

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