The rapid progress of embryonic stem cell, induced-pluripotent cell, and adult stem cell research opens the door to thousands of promising, new medical applications and discoveries. However, one of the major obstacles in translating these basic science discoveries into safe therapies for patients is the risk of acquiring mutations from viral and DNA vectors. Exposure of stem cells to DNA vector can result in integration of the DNA element into the chromosome of the stem cell and thereby potentially induce a malignant mutation. Consequently, to clinically develop stem cell therapies, there is a great need to develop reagents and protocols that do not expose the stem cells to DNA vectors.
Over the last 10+ years our lab has 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. We have also used this approach to introduce tumor suppressor proteins into pre-clinical mouse models of cancer. Moreover, PTDs are currently being tested in multiple clinical trials in the United States for heart disease and cancer.
One non-genetic (no DNA vector) approach with great potential to manipulate stem cells in specific cell types, such as heart muscle or neurons for spinal cord injury, is by RNA Interference (RNAi). RNAi, which was won the Nobel prize in 2006, allows for the selective degradation of mRNA and hence their protein product by introduction of short interfering dsRNAs (siRNA). 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. Recently, our labs have developed an approach to combine the advances in cell-permeable PTD peptides with siRNA delivery. Pilot experiments show that we can efficiently deliver siRNAs into human embryonic stem cells and adult stem cells and induce specific RNAi responses. In this proposal, we will determine the overall efficiency of siRNA delivery using cell-permeable proteins in pluripotent and adult stem cells and will test for possible negative effects of this approach on their pluripotency and regenerative capacity. We will further test the feasibility of this approach to stimulate pluripotent stem cells to produce clinically relevant cells such as nerve, heart muscle and blood. If successful, the use of cell-permeable peptides to deliver siRNA into cells has great potential for scientists and biotech companies to remove a gap between stem cell discoveries and safe, medical treatments.
Our California universities and biomedical industries are poised for the development of an entirely new spectrum of potential patient therapies based on discoveries in the stem cell field. However, a major obstacle in bringing these discoveries to the clinics is the absence of safe, non-DNA based approaches. Consequently, an alternative approach to manipulate stem cells that avoids the use of DNA vectors is critical to advance stem cell therapies into the clinics.
Over the past 10 years, our labs have pioneered alternative epigenetic, non-DNA based approach that allows for the introduction of proteins into embryonic and adult stem cells. We have used this approach to introduce more than 50 active proteins into a broad spectrum of human and animal cell types. In this proposal, we will develop this non-genetic approach to manipulate embryonic and adult stem cells by introducing RNA Interference (RNAi) inducing short interfering dsRNAs (siRNAs) in differentiate these pluripotent cells and adult stem cells into specific cell lineages such as heart muscle and neurons. Proof-of-Concept experiments have demonstrated the potential of approach to efficiently deliver siRNAs into stem cells in a non-cytotoxic fashion to induce RNAi response in the absence of DNA vectors (No DNA). Here we propose to expand this approach and ascertain its potential to manipulate stem cells to differentiate into therapeutically important cell types and understand the consequences of RNAi on stem cells therapeutics.
Any breakthrough from this proposal could have an immediate impact on biomedical field. Furthermore, these technologies will have an immense economic benefit to California by removing a major obstacle for biotechnology and pharmaceutical companies in California and reducing the barriers between new discoveries and new treatments.
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.