The project is focused on developing efficient strategies to modify the genome of human embryonic stem cells (hESCs). Such modifications are likely to be essential for the full therapeutic potential of hESCs to be realized because they will allow for precise changes to be made in the hESC genome (without unwanted or unanticipated effects on the genome). Therapeutic changes made to the hESC genome could include those that correct inherited diseases, or those that augment the functions of critical genes that can confer a needed therapeutic effect. hESCs carrying changes of this sort can eventually be used in transplant settings to alleviate or cure diseases. The strategies favored by this project (those that depend on homologous recombination) have been widely exploited in model systems, but have yet to be optimized for use with hESCs. Various approaches will be tested in the project to optimize homologous recombination procedures for hESC genome modification. The cell lines generated in this project, the methodological expertise acquired, and the DNA reagents should all help to facilitate future efforts to generate hESCs carrying therapeutic mutations.
Statement of Benefit to California:
The expertise we hope to gain in this project is essential for future projects that depend on generating precise changes in the human embryonic stem cell (hESC) genome. As has been the case with other experimental systems, once established, the technique of gene targeting by homologous recombination should be widely exploited for research and ultimately for therapeutic purposes. The State of California will benefit from this project because it will provide a means for realizing the full therapeutic and research potential that hESCs embody. Engineering specific changes into the hESC genome will allow for genes that cause disease to be corrected prior to the use of hESCs in transplant settings. It will also make a critical experimental tool available to researchers so that they can further understand biology of hESCs and thus make better use of the cells in therapeutic contexts.
SYNOPSIS: The aim of this proposal is to improve genetic engineering of human embryonic stem cells (hESC) by homologous recombination. Initially, effort will be devoted to developing the technology by targeting four loci (2 that are widely expressed and 2 hematopoietically expressed) by use of BACs modified in E. Coli. Subsequently the recombinase-mediated cassette exchange method (RMCE) will be employed to permit controlled targeting and predictable expression of transgenes.
INNOVATION AND SIGNIFICANCE: Improved genetic manipulation of hESC would be an important goal. Such approaches would permit creation of disease models and also facilitate introduction of markers for subsequent studies. The work would provide cell lines with "conveniently" located spots for homologous recombination to introduce the gene of interest. This would overcome the problems with using conventional methods to introduce exogenous genes using transfection or viral vectors, where there is no control over where the vectors would integrate, and the integration itself could perturb endogenous gene expression, potentially creating an artifactual situation for studying the impact of expression of the gene of interest. The use of RMCE is a logical step in this process. The significance is high, but there is little novelty in the proposed experiments.
STRENGTHS: The strength of the proposal lies principally with the prior experience of the PI in gene targeting in the context of the mouse. The availability of hESC with targeted loci that could be then employed in RMCE would be a useful step forward and helpful to the general community of researchers. The data provided on the use of the BAC in murine ESC demonstrates that they have used these approaches in another system with success, and that the screening methods they propose using (for example the quantitative PCR shown in figure 1B) are feasible.
WEAKNESSES: There is little innovation with respect to the design of targeting approaches. There is no strong reason to believe that use of BACS will enhance efficiency (as work in mESC has not shown advantages over conventional constructs). Thought might be given to other strategies that might enhance efficiency of recombination.The investigators have not considered whether there would be a problem in using two selection steps, puromycin and gancyclovir, with regards to ESC phenotype and biology. The PI does not seem to include any controls such as analysis of the hESC lines for typical ESC markers before and after each step in the process of deriving the targeted cell lines.
DISCUSSION: The PI proposes to employ a method (RMCE) that inserts transgenes at a single site. The PI proposes the use of BACs, however discussants noted that increasing the length of the targeting vector does not appear to increase targeting efficiency in other system(s). There was a general belief among the reviewers that hESC are more difficult to target than mESC. The goal of the application is laudable but there was no new 'hook' proposed.