Funding opportunities

Funding Type: 
New Cell Lines
Grant Number: 
Principle Investigator: 
Funds requested: 
$1 375 144
Funding Recommendations: 
Grant approved: 
Public Abstract: 

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: 

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 Summary: 

Executive Summary

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.

Reviewer Synopsis

The goal of this proposal is to develop methods to reprogram adult human somatic cells into pluripotent stem cells by activating specific genes with double-stranded RNA (dsRNA). The first specific aim will be to use screens to identify small activating RNAs (saRNAs) that stimulate expression of reprogramming factors. A second specific aim will be to optimize RNA activation for each of the relevant genes. The third specific aim will be to reprogram somatic cells into iPS cells. This study should lead to the development of a virus-free approach for the generation of iPS cells.

Reviewer One Comments

The proposed use of a non-viral approach to create iPS cells would be very significant. In addition, this proposal will yield additional data on RNAa, a new and potentially powerful method to activate gene expression.

There are two major drawbacks to this proposal. The first is that Aims 2 and 3 entirely depend on the successful completion of Aim 1. In Aim 1, the PI proposes experiments using RNAa to activate genes known to be involved in reprogramming. RNAa is a very new technique that has been shown to be capable of activating gene expression in vitro. However, to date there has been <4 papers published using this technique. In addition, only a handful of genes (and none of the ones in this proposal) have been activated using RNAa. It is currently unknown what the rules are that govern why some dsRNAs activate gene expression while others do not. It is also very unclear if this technique can be used in all cell types (the PI’s own publication on this topic suggests that RNAa will not work well in some cell lines) and if all genes can be activated using RNAa. If NANOG, SOX2, C-MYC, KLF4 or LIN28 cannot be activated using RNAa then Aims 2 and 3 cannot be attempted. Activation of OCT4 by RNAa in HeLa and IMR-90 cells is promising. However, there is no evidence that the other genes listed above can be expressed using RNAa.

The PI assumes that if RNAa can induce gene expression that it will be at the same level as the above genes are normally expressed in hES cells. This is impossible to know until the experiment has been successfully performed. There are no alternative experiments proposed to address the possibility that RNAa will not drive high enough levels of gene expression.

The proposal to add a DNA demethylating agent to cells in which “genes prove refractory to RNAa” is a risky strategy. Even a slight alteration in the methylation state could cause severe problems when using hES or iPS cells for future studies. The ability of RNAa to activate gene expression needs to be demonstrated prior to using hES or iPS cells.

The PI proposes an estimated cost of $ 40,000 per year for dsRNAs. Twenty dsRNAs have been proposed for each gene to be tested, since it is unclear why some dsRNAs work while others do not. I would suggest ordering a much smaller number (~5) and investigate if these work prior to ordering all 20. It would be waste of money if the first dsRNA ordered induced a 15-fold increase in gene expression.

While using viruses to create iPS lines is clearly not desired, I do not think that using a technique that apparently modifies chromatin structure through an unknown mechanism is a productive area of research at this time for creating iPS lines. RNAa is a promising technique but should be investigated further using traditional cell lines prior to use in iPS or hES cells. Once it is clear how this technique functions, how long induced gene expression lasts and what makes a good dsRNA molecule it could be used to create iPS cell lines. There is no need to perform these experiments in hES or create iPS cells at this time using RNAa.

Responsiveness to RFA:
The successful completion of this proposal would create new iPS cell lines that would have not been exposed to viruses. These cells lines would be very useful and there is an adequate plan for distributing cell lines created during this project.

Reviewer Two Comments

A major obstacle to the use of induced pluripotent stem (iPS) cells for cell replacement therapies is the reliance on viral vectors to accomplish reprogramming. Moreover, the integration of these vectors randomly into the genome of iPS cells may complicate the interpretation of the results. Li and colleagues seek to overcome these problems by creating iPS cells via small RNA-guided transcriptional activation of OCT4, NANOG, SOX2, C-MYC, KLF-4, and LIN28. These cell lines would be a significant improvement over the current state-of-the-art techniques employed to generate iPS cells.

Overall, the proposal is clear, logical and fairly straightforward. Li and colleagues propose to first find small activating RNAs (saRNAs) that activate the endogenous expression of OCT4, NANOG, SOX2, C-MYC, KLF-4, and LIN28. Given that this group was among the pioneers of the technique of using RNA to induce gene activation, they are likely to succeed in this aim. Further, their preliminary data suggests they have already discovered saRNAs capable of activating endogenous OCT4. As this technology is still very new, it is not clear how broadly it can be applied across the genome and whether it would work to activate the requisite genes necessary for creating iPS cells. Second, they propose to replace one lentiviral driven reprogramming factor at a time with saRNAs that activate the corresponding endogenous gene. To aid in identifying properly reprogrammed cells they plan to use human fibroblast derived from human embryonic stem cells that carry a transgene that drives expression of green fluorescent protein and neomycin from an Oct4 promoter. By proceeding step-wise to replace individual factors in a system that allows selection and reliable monitoring of reprogramming they increase the chances of this aim in succeeding. Third, they plan to reprogram somatic cells with optimized saRNAs. The major weakness of both aim two and three is that it may be necessary to repeatedly transfect cells with the saRNAs to ensure expression of reprogramming factors for the time required to generate iPS cells. Preliminary data demonstrating long-term activation of endogenous genes by repeated transfection would have gone a long way to assuage these concerns.

Responsiveness to RFA:
The proposal is very responsive to the RFA