Right Column

RL1-00660-1: Induction of pluripotent stem cells by small RNA-guided transcriptional activation

Recommendation: Recommended for funding
Scientific Score: 71

First Year Funds Requested: $453,288
Total Funds Requested: $1,375,144

Public Abstract (provided by applicant)

Embryonic stem cells have great potential in therapeutic use to replace diseased or damaged tissues because they have the unique capability of giving rise to any cell type of the body while perpetuating their own identity, even after repeated cell divisions. Recent advances in this area have resulted in a new way to generate stem cells from specialized adult cells by introducing 3 to 4 genes encoding proteins called stem cell factors, which are highly active in natural stem cells, into these adult cells using viruses as the carrier. These derivatives are called induced pluripotent stem (iPS) cells and have properties that are very similar to those of embryonic stem cells. Because iPS cells can be generated from the patient’s own tissues, problems associated with immune rejection are avoided. Furthermore, this process does not use embryos, so there are no ethical concerns. Unfortunately, the use of viruses to generate these cells is problematic because the virus may also activate harmful genes in the cells, such as those that cause cancer. We recently developed a way to switch on inactive genes in human cells using small RNA molecules instead of viruses, and coined the technique ”RNAa” for RNA-induced gene activation. We have shown that RNAa can induce robust and prolonged activation of a variety of genes. RNAa therefore seems well suited to replace virus-mediated reprogramming as a means to generate iPS cells. The main goal of this application is to develop a novel method of transforming adult cells into stem cells without using viruses. Accomplishment of our study will bring iPS cells one step closer to the clinical application of stem cell therapy.

Statement of Benefit to California (provided by applicant)

The aim of this application is to develop new approaches for the generation of pluripotent stem cell lines without using virus as the gene expression vector. Stem cells so generated can be used to replace diseased or damaged tissues without the concern of virus-related adverse effects such as insertional mutagenesis. Success of these approaches will benefit the health of the population and the economy of the State of California. Californians suffer many diseases and injuries that are treatable by using stem cells, such as Parkinson’s and Alzheimer’s disease, diabetes and cancer. The new stem cell lines and reagents we generate will likely be commercialized by California-based biotechnology companies and thus generate revenue and new job opportunities for the state.

Review

The proposal focuses on the development of a novel method to generate induced pluripotent stem (iPS) cells from adult human somatic cells. The approach will involve reprogramming through the use of small activating RNAs (saRNAs) to activate expression of endogenous genes encoding the known reprogramming factors. Specific saRNAs that target the promoter region of these genes will be identified. saRNAs found to confer gene activation will be used to replace virally-driven reprogramming factors in a standard iPS cell protocol, and conditions for the function of these saRNAs will be optimized. Finally, iPS cells will be derived from somatic cells by combining the various saRNAs and optimizing the reprogramming process.

This is a proposal of high significance in that it seeks to overcome a major obstacle to the utilization of iPS cells. The proposed approach could eliminate the need for viral vectors that can cause insertional mutagenesis and other safety concerns hindering the clinical application of iPS cells. Additionally, the saRNA procedure would act on endogenous genes, negating problems of gene copy number. Cell lines developed with the proposed approach would be a significant improvement over those that have been derived to date via existing iPS cell techniques.

The proposal is clear, logical and innovative. Convincing preliminary data showing that saRNAs can activate one of the reprogramming genes supports the likelihood for success of the approach. Plans for the systematic step-wise testing of individual factors are well developed and straight-forward. Reviewers had some concern that the level and duration of gene expression may be difficult to optimize and that some genes may not be good candidates for saRNA regulation. However, the advantages and potential of the approach heightened reviewers’ enthusiasm for the application.

The PI is a leader in the study of RNA–mediated gene activation. He/she is highly qualified to carry out the proposed research. The co-investigator is an accomplished expert in stem cell and developmental biology.

The proposed study is highly responsive to the RFA. New cell lines derived with the proposed approach are likely to be superior to those generated using viral vectors. Plans for distributing cell lines created in the project are adequate.

Overall, the project was viewed as risky but with very high potential to make significant contributions toward the clinical utilization of stem cell-based therapies.

Programmatic Discussion
A motion was made to recommend that this application be moved to Tier 1 – Recommended for Funding. Reviewer enthusiasm was raised by the convincing preliminary data and the potential of the approach relative to other technologies for stem cell generation. The motion to move this application to Tier 1 carried.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• None