New Cell Lines
Embryonic stem cells open up exciting new prospects for medicine, because they can differentiate into any tissue in the body. Therefore, they have the potential to be used to repair faulty tissues in diseases like diabetes, heart disease, and neural disorders. Furthermore, stem cells can be corrected by gene therapy and transplanted, in order to treat a wide variety of genetic diseases, such as sickle cell anemia. However, embryonic stem cell research has been difficult because of the technical and ethical problems involved in obtaining these cells from human embryos, as well as the need to transplant cells that are immune-matched to the recipient patient. The solution to these challenging biological and ethical problems may emerge from recent findings that show that cells that behave like embryonic stem cells can be derived from ordinary cells easily obtained from a patient, such as skin cells. This process is known as reprogramming. This breakthrough means that embryos may no longer be required to generate the stem cells needed for exciting new therapies. However, the methodology that is currently used to create reprogrammed cells involves introducing many viruses into patient cells. This procedure is itself dangerous and can lead to tumors and other abnormalities in the cells. As a result, the reprogrammed cells created to date are not suitable for use in the clinic. This proposal seeks to solve this problem by creating a novel method for introducing the reprogramming genes into one safe place in the chromosomes that will have no adverse effects on the cells. In these experiments, a simple, safe way to make reprogrammed cells without viruses will be developed. The reprogrammed cells made by this method will be thoroughly tested to ensure that they have all the beneficial properties of embryonic stem cells and are safe to use. The emphasis will be on generation of human reprogrammed cells that are safe and effective in therapies. Reprogrammed mouse cells will also be generated, for use in testing in mice before human clinical trials. The reprogrammed cells will be evaluated for their ability to differentiate in culture into tissues such as nerve, heart, and blood cells. The cells will then be tested for their capacity to cure a genetic disorder in a mouse model. Success in these experiments will provide a simple and safe method to generate reprogrammed stem cells and will speed the use of these cells in a wide variety of clinical applications.
Statement of Benefit to California:
Human pluripotent stem cells derived from ordinary adult cells are a scientific breakthrough that could speed medical advances to the public. These “reprogrammed” stem cells, made from ordinary cells, could remove the technical and ethical impasses that have delayed advances with stem cells that are derived from human embryos. However, current methods to make reprogrammed cells utilize viruses that are themselves dangerous. This proposal will apply new, California-invented technology, in the form of a novel gene addition system, to create an easy and safe method to make reprogrammed stem cells without the use of viruses. By replacing the current ~20 random viral integration sites with one safe, defined integration site, the resulting stem cells are likely to be suitable for clinical use, without the fear of tumors or other abnormalities. This project is feasible in all its elements, highly relevant to the goals of CIRM, and will result in a variety of new lines of pluripotent human stem cells. The availability of high-quality stem cells, made from ordinary patient tissue, will allow researchers to move more quickly to develop safe, effective, stem cell therapies for the people of California. This highly innovative project could create a leap forward for the entire stem cell field and greatly speed clinical applications. Therefore, this application is of great importance to California.
Executive Summary This is a proposal to develop a new approach for the generation of induced pluripotent stem (iPS) cells that avoids the use of retroviral vectors and random integration. Human and mouse iPS cells will be generated by using an innovative molecular tool to create a single integration site for gene insertion. Genes encoding canonical transcription factors for iPS cell generation will be targeted to this site. Resulting iPS cells will be characterized and differentiated in vitro, and differentiated cells will be tested in mice for their ability to cure a genetic blood disorder. This proposal outlines a highly significant approach that can improve the safety of iPS cell generation. It could provide a key advantage in avoiding the use of retroviral vectors and their potential to induce random insertional mutations throughout the genome of transfected cells. The proposed method is innovative and may allow the integration of activating genes at a single, defined, genomic site. The procedure also may prove to be more efficient than previous approaches. The proposed approach is highly feasible. Although reviewers had some concerns about potential difficulties in adjusting gene expression levels and a lack of specific experimental detail, the principal investigator is recognized as an expert in the use of the abovementioned molecular tool and should be successful in optimizing its use for iPS cell generation. The overall experimental plan is appropriate and thoughtful. The proof-of-principle test in the correction of a specific blood disorder in mice using cells generated by this technique was viewed as a particularly worthwhile component of the study. The proposal is responsive to the RFA. It should yield a new and highly improved approach for the generation of a wide variety of iPS cells. Reviewer Synopsis The overall goal of this proposal is to develop a new, improved approach to creation of iPS cells that is safer, simpler, and more efficient than the current protocols. The proposed approach will eliminate the use of retroviruses and associated random integration, and instead, use the efficient, sequence-specific integrase from phage phiC31 to create one safe integration site for gene insertion. The first specific aim will be to optimize a protocol for generation of human and mouse iPS cells using phiC31 integrase. The second specific aim is to characterize these cells in vitro and analyze their potential for differentiation. In a third specific aim, differentiated iPS cells will be used to cure a genetic blood disorder, SCID-ADA, in mice. Reviewer One Comments Significance: Highly significant if one can improve safety of iPS cells, in this case by targeting integration of all elements to one genomic site. Feasibility: Pluses: 1. PI is expert in integrase system 2. Plasmids would be delivered by transfection and integrate in one site 3. good plans for testing quality of iPS cells and also for doing proof of principle experiment with SCID-ADA Negatives: 1. Amount of expression may be hard to adjust in this system. 2. It will be necessary to shut off exogenous factor expression. PI alludes to this briefly but doesn’t describe the approach in any detail. 3. The proposal is very sketchy as to the nature of the promoter(s) used to drive the cDNAs. The proposal says “strong” promoters, but the nature of these will be important. Are they silenced on conversion to iPS state? Responsiveness to RFA: The proposal is appropriately responsive. Reviewer Two Comments Significance: The proposal is about using the integrase from phage phiC31 for delivering genes necessary for the production of iPS cells. This would have the advantage that the integration event is more defined. They will first optimize the protocol for the generation of iPS cells with phiC31 integrase, in a second step characterize the resulting iPS cells and finally use this approach to correct one specific defect, SCID-ADA, as a gene therapy approach. Feasibility: This is a feasible plan, likely to work if optimized. It is a very nice idea, and there is a very clear need to find specific means to deliver the reprogramming genes. This system is very promising and the investigator has significant experience with the system. The “proof-of-concept” SCID-ADA experiment is a nice add-on to the study. There are several gaps in describing experimental detail.