TAT Cell-Permeable Protein Delivery of siRNAs for Epigenetic Programming of Human Pluripotent and Adult Stem Cells
Tools and Technologies I
Statement of Benefit to California:
The applicant proposes to use cell permeable peptides called Peptide Transduction Domains (PTDs) to deliver short interfering RNAs (siRNAs) into cells. These siRNAs cause RNA interference (RNAi), i.e. they selectively degrade targeted mRNA, which leads to the down regulation (knock down) of the encoded protein. This approach will be tested in human embryonic stem cells (hESCs) in vitro and on a population of adult stem cells in an in vivo model in an effort to disrupt the circuitry of pluripotency and self renewal, respectively. In Aim 1, the principal investigator (PI) will optimize the knock down protocol in hESC. The duration of the knock down as well as toxicity and off target effects will be considered. Then the PI will test in Aim 2 the effects of knocking down two specific RNAs with the goal of obtaining mesoderm differentiation in hESCs. Finally, the PI proposes to knock down two different RNAs by application of the PTD-coupled siRNA in an in vivo model and assess the effect of these siRNAs on the proliferative capacity of the targeted population of adult stem cells. Reviewers agreed that the potential impact of this work is substantial. The ability to introduce siRNAs into hESC without the use of DNA-based vectors is required for this field to move forward. The bottleneck for the development of RNAi is the delivery of these molecules intracellularly, in vivo. This proposal, if successful, will solve the problem, and remove one of the major roadblocks to developing therapies using hESC and induced pluripotent stem cells. Furthermore, the proposed method has the potential for controlling stem cells in specific ways, such as inducing their differentiation into specific cell types. The impact of this work will be on stem cell research and on stem cell-based therapies. Reviewers praised the experimental design and found the preliminary data to be very compelling. Some of the key issues, such as the durability of the RNAi effect over time and potential off-target effects, are addressed with well-designed studies, including initial proof of concept studies, analyses of dosage effects and a time course. One reviewer especially appreciated the proposed experiments to address possible off-target effects of given siRNAs. One addition could have been to perform the control siRNA experiment in mouse ESCs and then demonstrate that these cells can still give rise to a mouse, the ultimate test of pluripotency. However, since this proposal is geared towards human cells and is limited to two years, it is understandable that mouse ESC experiments were not included. The in vivo experiments in which the RNAi technology will be examined in adult stem cells were a nice addition. Overall this is an outstanding proposal. The team is uniquely positioned to undertake the proposed experiments. The PI has extensive experience with the use of PTD-mediated cell permeability approaches. The PI and co-investigator propose to commit 15 and 20% effort, respectively, and three full time laboratory personnel to this project. The budget was considered appropriate. Overall, the reviewers were very enthusiastic about this proposal, based on the expertise of the PI, the potential impact of the proposed studies on the field of stem cell biology and the high quality of the preliminary data.