TAT Cell-Permeable Protein Delivery of siRNAs for Epigenetic Programming of Human Pluripotent and Adult Stem Cells

Public Summary of Scientific Progress (3/10). Grant Number: RT1-01063-1; PI Name: Dowdy, Steven F.; Project Title: TAT Cell-Permeated Delivery of siRNAs for Epigenetic Programming of Human Pluripotent and Adult Stem Cells. This summary is for a 9 month funding period. Use of induced-pluripotent (iPS) cell, embryonic stem cell, and adult stem cell research opens the door to promising new medical cell therapy applications and discoveries. However, one of the major obstacles in translating these basic science discoveries into safe cell based therapies for patients is the risk of acquiring mutations from viral and DNA vectors that are used to manipulate pluripotent cells into the specific cell types required to treat human diseases. Current approaches to manipulate stems cells into specific cell types or lineages requires exposure of stem cells to DNA vectors can result in integration of the DNA element into the chromosome of the stem cell and thereby potentially induce a non-desirable effect, including malignant mutation. In addition, use of small molecule inhibitors that cause global changes to stem cells and their cell lineage progeny for therapeutic benefit may also result in undesirable cell stress and cytotoxicities that can permanently alter the cell’s physiology in an unintended manner. Consequently, to clinically develop stem cell therapies, there is a great need to develop reagents and protocols that can gently alter stem biology, but do not result in either use of DNA vectors or stress the cells, both of which can lead to mutagenic events. Our labs have developed small domains from proteins called cell-permeable peptides or peptide transduction domains (PTDs) that enter cells, including embryonic stem cells and non-dividing adult stem cells, in a non-cytotoxic manner that is independent of exposing the stem cells to DNA vectors. We have generated over 50 transducible proteins that enter the entire population of all cell types tested, including human embryonic stem cells and adult stem cells. Importantly, PTD-mediated delivery of peptide and protein cargo has now been tested in over 1,000 patients in multiple phase I and II clinical trials for heart disease, pain, and cancer. None of these clinical trials have reported any ill effects or consequences of PTD-mediated delivery. While still quite early, these clinical trial results are very promising that the approach we are using will likely not have any unintended consequences to the cells. Our approach utilizes PTD-mediated delivery of small double stranded RNA molecules, called siRNAs, that induce a RNA interference or RNAi response that selectively eliminates expression of specific gene products. However, siRNAs are difficult to deliver into cells and current delivery approaches result in cytotoxicity, poor percentage of cells, changes in the overall transcription and biology of the cells, and even DNA damage. So siRNA induced RNAi responses have great potential to manipulate stem cells and iPS cells into specific types or lineages; however, their delivery into cells is the problem. To solve this problem, our labs developed an approach to combine the advances in cell-permeable PTD peptides with siRNA delivery. Over the last 9 months of funding, we have rapidly advanced this approach, called PTD-DRBD mediated siRNA delivery, and find that it is non-cytotoxic, delivers siRNAs into the entire population of all cell types, including stem cells and iPS cells, and has a minimal effect on the overall cell biology or other non-targeted gene expression profiles. During the last 9 months, we have been able to make a second-generation version of PTD-DRBD that further refines the technology and will make it more applicable to other laboratories working on stem cell manipulation. Over the coming year, we anticipate that these technological advances will increase our productivity to gently manipulate stem cells and iPS cells into specific cell types.

Embryonic stem cell, adult stem cell and induced-pluripotent (iPS) cell research opens the door to promising new medical cell therapeutic applications to treat human diseases that would likely not be accessible by traditional small molecule therapeutics. However, one of the major obstacles in translating these basic science discoveries into safe cell based therapies for patients is the risk of acquiring mutations from viral and DNA vectors that are used to manipulate pluripotent cells into the specific cell types required to treat human diseases. Many of the current approaches to manipulate stems cells into specific cell types or lineages requires exposure of stem cells to DNA vectors that can result in integration of the DNA element into the chromosome and potentially induce a non-desirable effect, including malignant mutation. In addition, use of small molecule inhibitors that cause global changes to stem cells and their cell lineage progeny for therapeutic benefit may also result in undesirable cell stress and cytotoxicities that can permanently alter the cell’s physiology and induce genetic mutations. Consequently, to clinically develop stem cell therapies, there is a great need to develop reagents and protocols that gently alter stem biology, avoid use of DNA vectors or stress the cells that could lead to mutagenic chromosomal events that would negate the overall approach. Over the last 15 years, our labs have developed small domains from proteins called cell-permeable peptides or peptide transduction domains (PTDs) that enter cells, including embryonic stem cells and non-dividing adult stem cells, in a non-cytotoxic manner that is independent of exposing the stem cells to DNA vectors. PTDs have the potential to deliver macromolecules, including peptides, proteins, siRNAs, mRNA that otherwise have no bioavailability, into the entire population of cells. We have generated over 50 transducible proteins that enter the entire population of all cell types tested, including human embryonic stem cells and adult stem cells. Importantly, PTD-mediated delivery of peptide and protein cargo has now been tested in over 2,000 patients in multiple phase I and II clinical trials for heart disease, pain, and cancer. None of these clinical trials have reported any ill effects or consequences of PTD-mediated delivery. While still quite early, these clinical trial results are very promising in that they begin to show that PTD-mediated delivery of macromolecules likely does not result in any detrimental effects to the cells. Our overarching approach to manipulate the phenotypes of stem cells has been focused on PTD-mediated delivery of small double stranded RNA molecules, called siRNAs, that induce RNA interference (RNAi) responses to selectively eliminates expression of specific gene product. In addition, we have developed a non-cytotoxic approach to efficiently deliver mRNA encoding transcription factors into stem cells. siRNAs and mRNAs have great potential to manipulate stem cells and iPS cells into specific types or lineages, but cellular delivery was problematic. To solve the siRNA delivery problem, our labs developed an approach to combine the advances in cell-permeable PTD peptides with a dsRNA Binding Domain fuse protein (PTD-DRBD) that delivers efficiently delivers siRNAs into stem cells and iPS cells, and has a minimal effect on the overall cell biology or other non-targeted gene expression profiles. We have also spent significant effort and resources to develop a non-cytotoxic approach to deliver mRNAs into stem cells and iPS cells. To do so, we developed a small peptide that self polymerizes into long polyplexes that bind and neutralize the mRNA into a nanoparticle. We then coat the outside of the mRNA-peptide nanoparticle with a PTD delivery peptide. Overall, both of these technological advances will further allow for the epigenetic manipulation of stem cells and iPS cells into specific cell lineages for application in the clinics.