New Cell Lines
$1 406 875
Statement of Benefit to California:
Executive Summary This is a proposal to develop a new approach for the generation of induced pluripotent stem (iPS) cells that avoids the use of retroviral vectors and random integration. Human and mouse iPS cells will be generated by using an innovative molecular tool to create a single integration site for gene insertion. Genes encoding canonical transcription factors for iPS cell generation will be targeted to this site. Resulting iPS cells will be characterized and differentiated in vitro, and differentiated cells will be tested in mice for their ability to cure a genetic blood disorder. This proposal outlines a highly significant approach that can improve the safety of iPS cell generation. It could provide a key advantage in avoiding the use of retroviral vectors and their potential to induce random insertional mutations throughout the genome of transfected cells. The proposed method is innovative and may allow the integration of activating genes at a single, defined, genomic site. The procedure also may prove to be more efficient than previous approaches. The proposed approach is highly feasible. Although reviewers had some concerns about potential difficulties in adjusting gene expression levels and a lack of specific experimental detail, the principal investigator is recognized as an expert in the use of the abovementioned molecular tool and should be successful in optimizing its use for iPS cell generation. The overall experimental plan is appropriate and thoughtful. The proof-of-principle test in the correction of a specific blood disorder in mice using cells generated by this technique was viewed as a particularly worthwhile component of the study. The proposal is responsive to the RFA. It should yield a new and highly improved approach for the generation of a wide variety of iPS cells. Reviewer Synopsis The overall goal of this proposal is to develop a new, improved approach to creation of iPS cells that is safer, simpler, and more efficient than the current protocols. The proposed approach will eliminate the use of retroviruses and associated random integration, and instead, use the efficient, sequence-specific integrase from phage phiC31 to create one safe integration site for gene insertion. The first specific aim will be to optimize a protocol for generation of human and mouse iPS cells using phiC31 integrase. The second specific aim is to characterize these cells in vitro and analyze their potential for differentiation. In a third specific aim, differentiated iPS cells will be used to cure a genetic blood disorder, SCID-ADA, in mice. Reviewer One Comments Significance: Highly significant if one can improve safety of iPS cells, in this case by targeting integration of all elements to one genomic site. Feasibility: Pluses: 1. PI is expert in integrase system 2. Plasmids would be delivered by transfection and integrate in one site 3. good plans for testing quality of iPS cells and also for doing proof of principle experiment with SCID-ADA Negatives: 1. Amount of expression may be hard to adjust in this system. 2. It will be necessary to shut off exogenous factor expression. PI alludes to this briefly but doesn’t describe the approach in any detail. 3. The proposal is very sketchy as to the nature of the promoter(s) used to drive the cDNAs. The proposal says “strong” promoters, but the nature of these will be important. Are they silenced on conversion to iPS state? Responsiveness to RFA: The proposal is appropriately responsive. Reviewer Two Comments Significance: The proposal is about using the integrase from phage phiC31 for delivering genes necessary for the production of iPS cells. This would have the advantage that the integration event is more defined. They will first optimize the protocol for the generation of iPS cells with phiC31 integrase, in a second step characterize the resulting iPS cells and finally use this approach to correct one specific defect, SCID-ADA, as a gene therapy approach. Feasibility: This is a feasible plan, likely to work if optimized. It is a very nice idea, and there is a very clear need to find specific means to deliver the reprogramming genes. This system is very promising and the investigator has significant experience with the system. The “proof-of-concept” SCID-ADA experiment is a nice add-on to the study. There are several gaps in describing experimental detail.