Funding opportunities

The APOBEC3 Gene Family as Guardians of Genome Stability in Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
Principle Investigator: 
Funds requested: 
$777 467
Funding Recommendations: 
Grant approved: 
Public Abstract: 
Statement of Benefit to California: 
Review Summary: 
SYNOPSIS: This is a well developed proposal from a highly experienced investigator to evaluate endogenous mobile genetic elements in human embryonic stem cells. Such elements are highly methylated in somatic cells and it is believed that such methylation prevents their retrotransposition. However, methylation levels are markedly reduced in hESC. The investigator speculates that these reduced levels of methylation could enhance the frequency of retrotransposition of mobile genetic elements. The project is focused around evaluating the role of APOBEC3 genes in modulating mobility of genetic elements in hESC. Three specific aims are proposed: Specific Aim 1: To determine the profile of expression of each of the 8 known A3 genes and the expression of LINE and SINE retroelement RNAs in multiple NIH-approved and newly established non-NIH-approved hESC lines cultured under GMP standards in the absence of feeder cells and xenogenic components; RT-PCR will be used to estimate the concentration of RNAs encoding the APOBEC family members of cytidine deaminases and the concentration of various retrotransposon RNA species and Western Blot analysis will be used to confirm estimated concentrations. Specific Aim 2: to assess whether specific A3 proteins oppose LINE1 or Alu retrotransposition in hESCs. Knockdown of A3 protein expression will be achieved by shRNA delivered by lentiviral vectors. Retroposition will be scored by activation of a drug resistance gene through intron excision during the retrotransposition process. Specific Aim 3: to analyze whether knock-down of an individual A3 RNAs in hESC increases the frequency of malignant transformation of hESCs or perturbs their successful differentiation into various cell types. Assays will be performed to determine whether cells expressing A3-specific shRNA display distinct growth properties including accelerated doubling time, proliferation in the absence of added bFGF, clonal growth in soft agar or proliferation independent of extracellular matrix proteins. SIGNIFICANCE AND INNOVATION: The investigator has focused on a highly important issue related to the potential stability of the ESC phenotype in the context of a possibly enhanced potential for retrotransposition. The methodologies proposed are all state of the art and, in combination, render the proposal highly innovative. The investigator has focused on an important biological process that may be profoundly significant with regard to the stability of a stem cell phenotype during ex vivo propagation. The studies proposed by Dr. Greene are innovative. While much work has been done on the role of APOBEC3 (A3) family members in the context of viral infection, little is known about their role in the absence of exogenous viral infection. Some work has begun to evaluate their role in inhibiting retrotransposition events of endogenous elements (some of which has been done by Dr. Greene), but not in embryonic stem cells. Given the fact that retrotransposons are more active in embryonic and germ cells than somatic cells, the question posed by the investigator is of direct relevance and importance to understanding not only the biology of embryonic stem cells, but also the genetic stability. While genetic stability of ES cells has been looked at on the "gross" level (i.e., looking at diploid stability, telomere length, or chromosomal rearrangements as markers for genetic instability), it has not been looked at on a more detailed level. Indeed, understanding the mechanisms that regulate the transposition events may be critical to ultimately develop genetically stable cell lines that could be used for clinical indications. STRENGTHS: The investigator is highly experienced, among the top in the field working on APOBEC3 and therefore is well-positioned to do the proposed studies. They have numerous publications in this area, and therefore, a proven track record of productivity and high quality science. The Aims of the grant as defined by the investigator are very feasible (again in particular for an investigator with his experience in this area) -- Aim 1 is easily achievable -- the investigator has all the reagents and tools set up to analyze the expression profile of A3 genes and proteins, as well as the expression of the Line element L1 and Alu. Aim 2 again is easily achievable -- the investigator has already used knockdown of A3G technology effectively in other contexts (for the purpose of studying the impact on viral infection). In addition, preliminary data provided in Figure 3 demonstrates that the investigator has successfully established an assay to measure retrotransposition events and has demonstrated that expression of APOBEC3G inhibits these events, further underscoring the investigator's ability to carry out the proposed studies. Aim 3: again the knock-down technology has already been used by the investigators successfully. The methods that they plan to use to assess the phenotypic impact of A3 knockdown are all established methods. Collaboration with the Gladstone Stem Cell Core brings in additional investigators who have worked with embryonic stem cells and with the assays that are proposed to evaluate the phenotypic impact of the knockdown experiment. The research plan is well-developed, well-described and acknowledges specific issues and potential technical complexities. There is extensive preliminary data. WEAKNESSES: The only "weakness" of the proposal, and this is relatively minor, is that the investigators need to delineate what cell passages or time points would be used for comparison over time. The time points that will be used to analyze the phenotypic and genotypic impact of A3 gene knockdown on ES cell function need to be clarified. DISCUSSION: This was regarded as the best application read by the primary reviewer. The investigator is highly experienced and new to the field. He has proposed a novel assay for scoring retroposition using intron excision to activate a drug resistance gene. Reviewer 2 commented favorably upon the amount of preliminary data submitted to support the application; reviewer 2 also felt that the proposed assay was feasible.

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