Funding opportunities

Regulation of Alternative Splicing During Embryonic Stem Cell Differentiation

Funding Type: 
Basic Biology III
Grant Number: 
RB3-02260
Funds requested: 
$1 425 600
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
To generate the variety of cell types found throughout our bodies, progenitor cells undergo a specification process termed differentiation. Appropriate cellular differentiation is essential for both embryonic development and adult tissue homeostasis, whereas misregulation of these pathways is commonly observed in many human diseases. Recent work suggests that progenitor cells and their differentiated counterparts express variations of the same protein through a process called alternative splicing. By characterizing the regulation of alternative splicing during differentiation, the proposed research will enhance our understanding of the regulatory networks governing normal cellular biology and may uncover novel therapeutic opportunities for a variety of human diseases.
Statement of Benefit to California: 
This project has the potential to benefit both the economy of the state of California as well as the health care provided to residents. In the successful study of stem cells and how they differentiate, we hope the results will lead to new therapeutic strategies, diagnostics, and cures for diseases (specifically in the area of neurodegenerative diseases). These new therapies would significantly impact the care given to California residents with these diseases. Clinicians will better understand the disease of their patients and be able to tailor care for them. As there is much room for growth in the area genetic therapeutics, we believe new discoveries in this area would be a catalyst for the creation of biotechs in [REDACTED]. Also, we are requesting funds from CIRM which will provide salaries for several full-time employees to a number of people who reside in California as well as purchasing the majority of our supplies from area companies.
Review Summary: 
Project Synopsis: The applicant proposes to investigate the hypothesis that the factors that regulate RNA processing change during the differentiation of human pluripotent stem cells (hPSCs) to a specific cell lineage. The principal investigator (PI) hypothesizes that the number of RNA variants becomes more simplified as cells differentiate to a more specialized fate. The process being studied, known as alternative splicing, results in the RNA produced from a gene being reconnected in multiple ways. Thus, a single gene may encode for multiple proteins. The PI seeks to identify and understand the role of factors that regulate alternative splicing during human embryonic stem cell (hESC) differentiation. In the first aim, the investigators propose to determine the degree to which transcript complexity decreases as hESCs differentiate. In Aim 2, they propose to use bioinformatics methods to identify RNA binding proteins (RBPs) and other RNA modulation proteins involved in cell differentiation and then inhibit them to assess their function. Finally, in Aim 3, the PI plans to characterize RBPs that mediate cell differentiation to identify targets in conjunction with RBP overexpression studies. Significance and Innovation: - The role of RNA splicing in differentiation could be significant and is under studied. While the project has the potential to uncover a new mechanism for human pluripotent cell differentiation, reviewers considered this a high-risk project due to concerns with the feasibility and experimental design, and therefore were not sure that the proposal would have a major impact on the field of regenerative medicine. - The approaches and techniques proposed are not particularly creative or novel and the proposal is primarily descriptive and discovery in nature. - Reviewers suggested that Aim 1 could be expanded to determine if induced pluripotent stem cells (iPSC)-derived cultures displayed similar levels of post-differentiation complexity. This would help incorporate a broader view of regenerative medicine into the proposal and more clearly define the impact of the proposed research. Feasibility and Experimental Design: - The experimental design was not logical. Key experiments are proposed, but the sequence for the experiments is poorly justified. For example, it was suggested that gain-of-function over-expression studies should be conducted earlier within the experimental plan. - The proposal lacked sufficient detail and alternative plans. -The preliminary data do not offer particularly compelling evidence that the proposed studies will be successful. As one example, the applicant did not include data demonstrating the ability to differentiate hESCs into the relevant cell types. - The entire effort hinges on a small number of RBPs playing critical roles in hESC differentiation; yet, it is impossible to discern from the preliminary data whether any of these candidates would be important factors in differentiation. - A more thorough proteomics strategy in Aim 2 would identify more candidates. - Including time points along the differentiation pathway in Aim 1 would be instructive. - The proposal is underdeveloped and not well written. Although blank space remained in the application, key experimental details were absent and sufficient supportive preliminary data was not provided. Principal Investigator (PI) and Research Team: - The PI has an outstanding track record and is well suited for this project. - The research team has the appropriate expertise to conduct this project and was a strength of the proposal. Responsiveness to RFA: - The proposal is responsive to the RFA as it is focused on examining the molecular mechanism of lineage specification from hPSCs.
Conflicts: 

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