More than 100,000 patients await for organ transplants nationwide this year. The ground-breaking discovery of new pluripotent human stem cell lines (iPS) derived from skin fibroblasts using a core of 3-5 transcription factors opens the door to patient-derived pluripotent stem cells and new approaches to organ and tissue replacement. Patient-derived stem cells could have an immediate impact on hundreds of other medical applications and discoveries. A major bottleneck in translating these breakthroughs into the clinic is that pre-existing mutations in patients and mutations acquired from viral and DNA vectors pose a potential risk for cancer. To overcome this obstacle, we propose an alternative approach to generating patient-derived stem cell lines using cell-permeable, pluripotent-inducing transcription factors. Introducing active proteins into cells avoids the long-term risk of genetic mutations. Over the past 10 years, our labs have pioneered this technology to introduce over 50 functional proteins in a wide spectrum of human and animal cell types. First, we will use this technology to determine the optimal number of cell-permeable proteins necessary to induce pluripotency in human cells. Second, to reduce the risk of pre-existing cancer mutations, we will identify which cells in the human body are the best source to generate mutation-free patient stem cells. These two advances will help us reach our goal of developing new medical therapies for an endless list of diseases using patient stem cells.
Almost 20,000 Californians await organ transplants. The possibility of translating pluripotent stem cell technology to patient-derived stem cells could drastically improve the outlook for organ and tissue replacement therapy in the future. Hundreds of potential medical therapies and inventions using induced-pluripotent stem cells are limited because current approaches rely on DNA technology and cause mutations. Over the past 10 years, our labs have pioneered alternative non-DNA approach, which could introduce pluripotent-inducing transcription factors into cells without causing mutations. 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 use this alternative non-genetic approach to induce pluripotent stem cells from human fibroblasts and to identify which human cell types are the best candidates for generating mutation-free patient-derived stem cells. These breakthroughs could have an immediate impact on medical treatments for Californians who are suffering from end-organ damage or have endocrine, neurological, and cardiac disease. Furthermore, these technologies will have an immense economic benefit to California by removing a major obstacle for biotechnology and pharmaceutical companies in California and giving California a leading edge in developing new treatments.
This proposal aims to study the generation of human induced pluripotent stem (iPS) cells through a technique that avoids the use of viral vectors, which have the potential to integrate into DNA and pose a risk of cancer formation. The proposed technique involves the production of cell-permeable fusion proteins of relevant transcription factors that are introduced to target cells. The applicants propose to use human cells as a model and explore which cell types are most optimal for generating pluripotent cell lines. The goal is to generate mutation-free, patient-derived pluripotent stem cells that demonstrate a potential for clinical use.
Reviewers felt that this proposal addresses a very difficult problem associated with the generation of iPS cells and provides an interesting approach to solve it. If successful, the proposed approach would lead to a significant advance in iPS cell technology. One reviewer felt that evidence provided by the applicant and by others in the field offers some support for the feasibility of the proposed approach. However, reviewers agreed that the activity and stability of proteins generated using this technique were not addressed adequately in the application. The project was viewed as being risky without evidence of how long the proteins remain in cells and the quantity of transcription factor required to achieve the desired effect.
The principal investigator (PI) was noted to be a leader in the field of fusion proteins and has been successful in applying the proposed technique to other systems. However, the PI does not have a background or experience with human embryonic stem cells and reviewers felt that collaboration with such an expert would have strengthened the proposal, since isolation and propagation of pluripotent cells is a learned art. The application presented adequate plans to share the cell lines generated.
Overall, based on the limited evidence of feasibility on the one hand, and the potential to move the iPS cell field forward on the other hand, the reviewers judged this proposal to be high risk / high gain.
Programmatic Discussion: A motion was made to recommend that this application be moved to Tier 1 – Recommended for Funding. Reviewers felt that although the project has some deficits, the overall approach is good and the PI is better poised than most to make it work. Reviewers reiterated that this represents a high risk, but potentially high gain, proposal. The motion to move this application to Tier 1 carried.
This application addresses the generation of iPS cells from human tissue using cell-permeable pluripotent-inducing transcription factors. The overall goal of this application is to generate mutation-free, patient-derived pluripotent stem cells. There are two specific aims. The first aim is to use TAT-fusion proteins to deliver Oct4, Nanog, Sox2, Klf4, Myc, and Lin28 to induce pluripotency in primary human fibroblasts. The second aim is to use this technology to identify the most efficient human cell type in the skin for generating pluripotent stem cell lines.
Reviewer One Comments
Aim 1 consists of five subaims (tasks as defined in the application). 1) Generate/purify TAT-fusion proteins. 2) Test transactivity of TAT-transcription factor fusions. 3) Test individual ability of TAT-transcription factor fusions to drive iPS cells. 4) Test complete set of TAT-transcription factor fusions to drive iPS cells. 5) Characterize iPS cells by molecular, cellular and phenotypic assays.
Aim 2 has three subaims. 1) Tissue procurement and isolation of human bulge, keratinocyte and fibroblasts. 2) Test complete set of TAT-transcription factor fusions to drive iPS cells. 3) Compare iPS cells derived from bulge, keratinocyte, and fibroblasts by molecular, cellular, and phenotypic assays.
Dr. Dowdy has an adequate track record of publications. There will be collaboration with Dr. Benjamin Yu (30%-UCSD Dermatology) and Dr. Tissa Hata MD (Associate Professor and Director of UCSD Dermatology Clinical Trials Unit).
The facilities at UCSD are adequate for accomplishing the goals of this proposal, including the Dowdy and Yu labs, the UCSD Stem Cell Core facility, and a dedicated mouse room.
Preliminary results for this proposal include PTD-dsRNA Binding Domain mediated siRNA delivery into hESCs and the generation and activity of transducible TAT-Sox2 fusion protein.
Responsiveness to RFA:
This proposal will generate induced pluripotent stem cells from TAT-Transcription factor fusion proteins.
The plan to share any generated iPS cell lines is adequate.
Reviewer Two Comments
Highly significant if the PI is able to use protein transduction for iPS cell generation. This would eliminate need for viruses with associated potential for insertional mutagenesis. If successful, this approach would be adopted for iPS cell production.
The PI is an expert in the use of TAT-protein fusions for protein transduction. As such, he is an ideal leader of the project. He shows preliminary data that a TAT-sox2 fusion functions. In poster form, other groups have shown TAT-nanog proteins. Hence, in principle, the approach may work. Concerns relate to the level of proteins achieved intracellularly by this method, the duration of time the proteins remain and how often transduction will need to be repeated, and the empirical work needed to get the right “dose” of the factors for iPS cell generation. Lacking in the proposal are details on characterization of iPS cells and their handling. The PI may need guidance from those familiar with hES cells in order to identify human iPS cells. It is non-trivial. The PI has little demonstrated experience in this area.