Funding opportunities

Mechanisms of small RNA regulation in early embryonic development

Funding Type: 
New Faculty II
Grant Number: 
RN2-00906
Principle Investigator: 
Funds requested: 
$3 029 897
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
The promise of embryonic stem cells in regenerative medicine is based on their potential to make every cell in the body, a property coined pluripotency. With rapid recent advances in technology, it is becoming relative straightforward to make embryonic stem cell-like lines from adult tissues. In the near future, the generation of these lines will become increasingly common practice including the production of patient-specific lines that can be used to evaluate the disease and even replace damaged tissue in these patients. However, we still do not fully understand the molecules that underlie pluripotency. It is essential for us to do so, in order to improve on the generation and quality control testing of the embryonic stem cells. Exciting recent work has shown that modifications to the genome that do not change the actual DNA sequence, but do change how that sequence is presented, is a central component of pluripotency. These modifications have been coined epigenetic modifications because they are not altering the underlying genetic code. Specifically, it was recently shown that these epigenetic modifications maintain the stem cell’s capacity to proliferate while poising them to differentiate into all tissues of body. They do so by keeping the programs required for differentiation into adult tissues off, but still accessible to activation. Failure in establishment and/or maintenance of the correct epigenetic program leads to diminished pluripotency and even tumor risk. Unfortunately, very little is known about how the epigenetic program of embryonic stem cells is established and maintained. This grant proposes that a novel class of regulators called small RNAs is essential for the establishment and maintenance of epigenetic program underlying pluripotency. To test this proposition, the investigators intend to use tools that enable them to remove different subtypes of small RNAs. They will test the effects of the removal of these RNAs on the establishment and maintenance of the epigenetic program underlying pluripotency. Furthermore, they will identify the molecular nature of these small RNAs and evaluate how individual small RNAs influence these epigenetic modifications. Once the critical small RNAs are identified and their specific functions uncovered it should be possible to use these small RNAs both to improve the efficiency of derivation of quality embryonic stem cell lines as well as potentially even fix damaged embryonic stem cell lines, in particular lines that have lost some aspects of their potential to differentiate into adult tissues.
Statement of Benefit to California: 
This grant proposes to uncover mechanisms that are central to providing embryonic stem cells with their amazing potential to produce all the cell types of the body. This potential makes embryonic stem cells a powerful tool for both evaluating and treating disease. For example, it is now becoming increasingly clear that soon it will be possible to produce patient specific embryonic stem cells that can then be differentiated into any tissue(s) of choice including those responsible for a patient’s specific ailment. The resulting tissue can then be analyzed for the unique causes of the patient’s disease and even evaluated for responsiveness to a panel of drugs. This personalized treatment of disease will have broad positive impacts in how health care is delivered as it will take in account the differences between every patient’s specific ailment and response to treatment. However, before this approach can be put into clinical practice in the most efficient way, there remains a lot to be learned about embryonic stem cells themselves. That is, what provides them with their amazing potential? This is the question addressed by this grant proposal. In particular, the grant proposes to uncover mechanisms central to determining and maintaining the potential of embryonic stem cells. Uncovering such mechanisms will allow the medical and pharmaceutical community to improve the efficiency at which they can produce embryonic stem cell lines as well as quality control the resulting cells. It could even enable the correction of defects in a particular stem cell line’s potential. The little we already have learned about the mechanisms underlying a embryonic stem cell’s developmental potential has lead to major practical advances including the recent success of producing human embryonic stem cells from adult skin cells. This will be the tip of iceberg. The more mechanisms we uncover, the more sophisticated the manipulations we will be able to perform and the more profound the therapeutic possibilities. Therefore, completion of this proposal should have positive health and financial impacts for the state of California.
Review Summary: 
The applicant proposes to study mechanisms of epigenetic control of early development. The premise is that an understanding of these mechanisms will guide manipulations of embryonic stem cells (ESCs) to allow ESCs to reach their full promise as developmental model systems and their full therapeutic potential. The specific focus of the research plan is on epigenetic regulation by classes of small non-coding RNAs distinguishable using transgenic mouse models which differentially affect the generation of these RNAs. In the first aim, the applicant will look at the complement of small non-coding RNAs during pre-implantation development by activating conditional knockouts of genes differentially affecting small RNA generation at different time points, then phenotypically characterizing these early embryos; isolating and analyzing certain small RNAs that may underlie interesting phenotypes; and finally, for promising small RNAs, looking for potential human homologs and performing subsequent expression analysis. In the second aim, the applicant will use wild type and the above-described transgenic mice to address the relationship of small non-coding RNAs to histone modifications. The reviewers uniformly noted that the proposed research is in an exciting and critically important area of basic research. Although there is no immediate or short-term therapeutic benefit, the proposed studies are likely to unravel mechanisms of development and provide a basis for manipulating ESCs to specific cell types, thus there is a high degree of significance for many disease applications. The reviewers found the proposal to be very well-written and considered the experimental plan to be a well-laid out. One reviewer found the research proposal to be ambitious, another commented that it was not overreaching in either the aims or the claims. All reviewers noted that the PI has already been working in the field and that this proposal is an extension of ongoing, funded work. The PI has demonstrated significant expertise in the techniques for each of the specific aims, and some preliminary data speaks to feasibility. The PI provided good detail in describing potential pitfalls and possible solutions. Several weaknesses in the proposal were discussed. Reviewers expressed concern over the difficulty of drawing conclusions relevant to normal embryonic development through the use of conditional knockouts. They noted that correlating the effects of such knockouts with precise points in development would be very difficult since the knockouts affect multiple components with varying kinetics. A reviewer did note that alternative methods for knockdown of the respective genes are likely to have similar problem in terms of kinetics and timing. Reviewers also noted the tremendous amount of work involved in isolating and analyzing, from thousands of murine embryos, the small RNAs that may underlie interesting phenotypes. Other concerns highlighted by the reviewers include the heterogeneity of the cell populations which could make it difficult to distinguish direct versus indirect effects of the small non-coding RNAs, and the lack of detail for applying findings from the mouse system to the human system. One reviewer noted that the only part of the proposal potentially not fundable by NIH is the transition to studies in hESC, and questioned the rationale for CIRM funding for this proposal. The PI has already demonstrated an ability to obtain NIH grants in exactly this area of research, and the proposed work is just an extension of his/her preliminary data. Despite these considerations, overall, the reviewers found the research plan to be very strong and interesting, albeit somewhat ambitious, and expected it to lead to interesting and interpretable results in this exciting and important area of stem cell research. The reviewers found the PI to be an exceptionally talented young investigator who has already made important contributions to the field of stem cells and is on a trajectory to have a stellar scientific career. S/he is a well-trained M.D./Ph.D. who completed a postdoctoral fellowship studying the epigenetics of development in an internationally known laboratory. S/he has been an independent investigator for about 3 years at the applicant institution and has significant extramural funding, including 3 grants from the NIH. The applicant has over 10 publications, including recent publications in high quality journals. The reviewers were mixed about the career development plan, one describing it as “modest” another as “very good”. It describes what the PI has already achieved and how s/he hopes to improve in three key areas: research, education, and leadership. A highly structured formalized mentoring plan is in place for the candidate, that includes mentorship from internationally recognized senior faculty, each of whom are experienced in mentoring and training. The institutional commitment to this spectacular young investigator is extraordinary in all aspects. There was a very strong letter of commitment from the institution, with significant financial support, along with an endowed chair for salary support. The PI has been given ample lab space, along with additional office areas and has access to considerable shared resources. The institution has a growing number of talented young scientists populating its stem cell program, and the environment is rich for collaboration. The institution has a very strong track record for developing leading scientists. In short, the institutional environment and commitment is superb. Overall, this is an exceptionally strong and well-written application from a talented young investigator working in a rich and nurturing environment.
Conflicts: 

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