Recently, researchers have shown that they can genetically alter adult cells so that they display the same properties as embryonic stem cells. These genetically altered adult cells are called induced pluripotent stem cells (iPS). Like embryonic stem cells, iPS cells can renew themselves indefinitely, and are capable of becoming any of the cell types in the body. Even better, this form of stem cell would be compatible with a person’s immune system, and could be used to develop tissues and organs that would not be rejected.
Unfortunately, the methods used to genetically alter these cells involve viruses. The involvement of viruses raises safety concerns about the use of iPS cells in future medical treatments. Thus, we propose to develop a virus-free strategy to make iPS cells. Our approach involves the use of proteins that have the capacity to enter the nucleus of the cells (where the DNA resides) and turn on stem cell genes. The proteins that we will use are identical to native proteins in the body that reprogram genes (reprogramming factors), except that we will add a small protein (R9) to them that will permit the proteins to enter the cell. Thus, the proteins that we will use are a fusion of the reprogramming factors and R9. The small R9 protein is composed of a natural amino acid (L-arginine). Nine of the L-arginines are strung together to form R9. It is a small protein that the body can metabolize, and has already been used safely in humans in FDA trials. We have previously shown that R9 is very effective at entering the cell nucleus.
Thus our approach is likely to be effective, and to be much safer, than the current viral approaches to making iPS cells.
With your support, we intend to a) synthesize the fusion proteins (containing R9 and the reprogramming factors), b) confirm that the fusion proteins can enter the nucleus of human cells and induce reprogramming, eg. expression of stem cell genes and c) confirm that a mixture of the fusion proteins can produce iPS cells. To create the reprogramming factor protein products we will use a relatively new cell-free protein synthesis system. After assessing the ability of the proteins to bind, enter into cells, and function normally, human cells will be exposed to the proteins and we will characterize the cells for the appearance of embryonic stem cell-like qualities. These qualities include the presence of certain genes and markers found only in stem cells.
We anticipate that this virus-free technology will become an indispensable tool for clinician-investigators in regenerative medicine, and will accelerate the insights regarding iPS cells into regenerative therapies for a host of diseases due to aging and other degenerative disorders of the heart, brain and other organs.
Statement of Benefit to California:
Human embryonic stem cells have an unparalleled ability to regenerate, and can potentially develop into any tissue in the body. There is great promise in using these cells for regenerative medicine, that is, to help replace diseased or damaged tissue. This type of cell therapy could be used to treat multiple diseases including; diabetes, heart disease, Parkinson’s disease and Alzheimer’s disease. However, the use of human embryonic stem cells for treatment has raised ethical issues. Furthermore, the use of embryonic stem cells is complicated by the possibility of rejection of these cells by the patient’s immune system. Accordingly new approaches to cell therapy are necessary.
In a remarkable development last year, a research group in Japan and one in America showed that human skin cells could be turned into stem cells. These cells are called induced pluripotent stem (iPS) cells. They are currently made using viruses that deliver factors capable of switching on and off genes associated with normal embryonic stem cells.
iPS cell technology is exciting because it doesn’t require the destruction of embryos and provides the opportunity to produce patient-specific stem cells. However, a major obstacle for clinical application of this technology is that viruses are used to make iPS cells. The use of viruses raises many safety concerns. Therefore, we aim to develop a virus-free approach for generating pluripotent (iPS) cells on a large scale. We then want to use these cells to make cells capable of forming new blood vessels.
In this CIRM-funded project, we will make and characterize iPS cells using a virus-free approach to deliver the reprogramming factors. Our approach, if successful, could make stem cells for individuals from their own skin cells. These cells could be used to replace damaged or diseased tissues in that individual.
Our virus-free approach will employ engineered proteins comprising the reprogramming factors fused to a sequence that allows the proteins to enter into cells. In other words, these proteins will be able to cross the cell’s membranes and activate the genes required to induce an iPS state. This novel approach is superior to the current technique of using viruses, in that it avoids the potential to create cancerous cells, associated with viral strategies. As a result, this project has a great potential to directly lead to new cell-based therapies for conditions where there is insufficient blood supply, as described in the proposal, but is also likely to lead to other applications in regenerative medicine. Successful execution of this project will produce an indispensable tool for researchers in regenerative medicine, will lead to new insights regarding the induction of pluripotentiality and will provide for a novel therapeutic strategy in the state of California and its citizens. Especially, California patients who are waiting transplantation will benefit from the availability of a curative stem cell therapy option.
This proposal focuses on the development of a novel, non-viral technology for the generation of induced pluripotent stem cells (iPS cells). The applicant proposes to create fusion peptides of four proven reprogramming factors (Oct3/4, Sox2, Nanog, and Lin28) designed to translocate into the cytoplasm and nucleus. First, these peptides will be generated using novel cell-free protein synthetic technology, and their nuclear localization and transcriptional transactivation capabilities when applied to cells will be confirmed. Then each fusion peptide’s reprogramming capacity will be tested sequentially by attempting to generate iPS cells from fibroblasts virally transduced with the other three reprogramming factors. After optimizing each fusion peptide individually, the researchers will attempt to combine all four to generate iPSCs. Finally the pluripotent phenotype of these iPSCs will be characterized using a range of approaches, including microarray comparison with human embryonic stem cells and iPSCs generated by viral transduction; genomic sequencing and teratoma formation assays.
The reviewers felt that this proposal could have broad impact on the field. Recent pioneering studies in the laboratories of Yamanaka, Thomson and Jaenisch have resulted in the successful reprogramming of somatic cells to a pluripotent phenotype. However, the use of viral delivery systems to transduce somatic cell targets is problematic for clinical applications, since these viral systems have a number of biosafety concerns including the potential for oncogenesis and disruption or silencing of endogenous genes. The proposed approach could avoid these problems and considerably advance the potential for therapeutic application of iPSCs.
The reviewers had major concerns about feasibility of the proposed study. There was a unanimous opinion by reviewers that the proposal did not adequately address the issue of peptide stability. The applicant proposes to test a number of experimental variables, but it is unclear whether he/she will adequately examine peptide stability following introduction into target cells. Short protein half-lives and intracellular protein degradation were not discussed. Additionally, important experimental details such as the necessity of treating cells repeatedly with peptides and possible secondary effects of cell exposure to high levels of proteins were not addressed. Another issue raised by reviewers was whether the fusion proteins would activate genes with the same specificity and kinetics as the transcription factors when delivered by viral vectors. Finally, reviewers agreed that the fourth and final aim, to design and test novel fusion proteins as new candidates emerge in the literature, is unlikely to be feasible, given the magnitude of the experimental task facing these investigators in their first three aims.
Reviewers agreed that the assembled research team is excellent and well qualified to carry out the work described in the proposal. They noted the impressive track records of the PI and co-investigators and found their experience to be a major strength of the proposal. The budget was found to be generally appropriate, although there was some concern that the requested funding for three post-docs with 100% effort was beyond the needs of the proposal.
Overall, this is an innovative proposal from an excellent team that addresses a significant roadblock to stem cell research. However, the reviewers described it as high-risk and raised serious doubts about its feasibility.