Development of a novel technology for precise, efficient, and safe genetic modification of stem cells
Tools and Technologies I
$1 146 312
Statement of Benefit to California:
This is a proposal to develop a new, non-viral approach for targeted and efficient gene transfer into human embryonic stem cells (hESC). The overall aim is to develop zinc finger recombinase technology for mediating site-specific integration of foreign DNA (transgenes) in hESCs, with the ultimate goal of introducing reporters and therapeutic genes into desired loci. In Aim 1, two specific loci, chosen because they are either dispensable (and therefore disrupting them will have no deleterious effects) or because they are believed to maintain an open chromatin configuration (thus allowing inserted transgenes to be expressed), will be targeted in a human somatic cell line. Optimization of the technology involves the in vitro selection of zinc finger recombinases with enhanced preference for targeting integration into these loci. In Aim 2, building on the optimization in Aim 1, reporter genes for monitoring pluripotency and neural differentiation will be targeted into the same two loci in hESCs. The purpose is to develop a system that will enable the easy monitoring of the transition between the undifferentiated hESC phenotype and differentiated neural progeny. Reviewers agreed that this is a goal-driven proposal that is worth pursuing. The proposed technique would allow the introduction of genetic material into hESCs in a safe and efficient manner, thereby enabling the dissection of the genetic basis of disease, the replacement of defective genes, and the safe and precise introduction of disease-fighting genes into hESCs, potentially enabling treatment of many conditions. Furthermore, reviewers lauded potential incidental by-products of this work, such as its application to the manipulation of the genomes of other stem cell populations. They also appreciated that one of the hESC lines to be generated could prove effective as a cell-based treatment of HIV. In support of the feasibility of the proposed project, reviewers commended the PI’s strong expertise with the design of zinc finger recombinases and they found the preliminary data to be compelling. One reviewer questioned whether the somatic cell line used in Aim 1 will enable identification of adverse consequences of the overall approach. With regard to Aim 2, the reviewer identified several potential pitfalls related to reporter design. Overlapping emission spectra of the chosen fluorescent proteins requires sophisticated microscopy capabilities, when emission spectra of other reporter combinations are more easily separable and hence more widely applicable. Furthermore, the reviewer expressed concern that the promoters chosen to report pluripotency and neural differentiation may not be optimal, and that due to their proximity in the construct they may interfere with each other. However, if the system works as proposed, the PI will have created a very interesting reporter cell line that will facilitate analysis of hESC differentiation. Furthermore, reviewers appreciated that the PI is aware of the difficulties of some of the proposed experiments and proposes alternative approaches. Clear milestones are proposed and plans to share reagents are mentioned. Reviewers considered the applicant team to be strong, the PI is clearly a world leader in the engineering of zinc finger recombinases, has consistently published in highly rated journals, is well funded and is committing 15% effort to this project. A collaborator provides expertise in hESC culture, differentiation and characterization, but one reviewer felt that the expertise of a developmental biologist to address promoter choices would strengthen the proposal. In conclusion, reviewers were very enthusiastic about this proposal, which, if successful, will lead to the development of a valuable tool. Although some concerns were raised about a few technical issues, they were not considered critical, and taking the strength of the applicant team into account, reviewers felt that success of the project was likely.