Tools and Technologies II
This proposal is directed at RFA 10-02: CIRM Tools and Technology Awards II. This call was developed to support the inception, early stage development and evaluation for stem cell research applications of innovative tools and technologies that will overcome current roadblocks in translational stem cell research. The proposed research is aimed at developing an efficient and safe tool for reprogramming human fibroblasts and blood cells into induced pluripotent stem cells (iPSC) without the use of viruses or viral DNAs, thus avoiding integration of foreign DNA into the genome. The basis of this tool will be a nano-scale particle, called a vault. Vaults are naturally occurring cellular structures found in nearly every cell of the human body. Because of their broad distribution and highly conserved structure, they are quite benign and do not trigger an immune response. Although they are a nano-scale structure, vaults have a sizable hollow interior cavity able to enclose a large payload. Furthermore, they can self-assemble from multiple copies of a single protein in the laboratory and they can be engineered to allow the particle to be utilized for delivery of proteins and/or nucleic acids into cells without toxicity. The research proposed will work out the conditions and properties needed to target vault particles to differentiated (somatic) cells, to package reprogramming proteins or nucleic acids encoding these proteins into the vault particles, and to monitor the functional quality of human iPSCs that are generated by treating cells with engineered vault particles. The roadblocks addressed in this proposal are the safety concerns that have surrounded the use of iPSC since the early report that these cells could be generated. First of all the reprogramming factors are themselves proto-oncogenes, normal genes that can become oncogenes (tumor causing genes) due to mutations or increased expression. Second, the introduction of these genes into somatic cells often requires viral delivery systems, whose use may result in insertion into a cell’s DNA causing mutations and tumor formation. As a consequence stem cell therapeutics awaits the development of a reproducible, reliable and safe non-viral approach for the generation of human iPSCs.
Statement of Benefit to California:
Over the past few years, reprogramming technologies have been developed which allow pluripotent stem cells to be induced from differentiated cells. These induced pluripotent stem cells (iPSC) have enormous potential to radically alter the practice of medicine, as they can be derived without the use of embryos, and from a patient’s own skin or blood cells. In theory, scientists could differentiate iPSCs into any desired cell type in the laboratory and use them to replace any cell type that is defective or dysfunctional in the patient. To realize the full potential of this exciting technology, we will need a better understanding of how iPSCs are formed, maintained and differentiated. This proposal addresses the safety concerns that have surrounded the use of iPSCs since the early report that these cells could be generated. First of all the reprogramming factors are themselves proto-oncogenes, normal genes that can become oncogenes (tumor causing genes) due to mutations or increased expression. Second, the introduction of these genes into somatic cells often requires viral delivery systems, whose use may result in insertion into a cell’s DNA causing mutations and tumor formation. As a consequence stem cell therapeutics awaits the development of a reproducible, reliable and safe non-viral approach for the generation of human iPSCs. This proposal presents a plan to develop an entirely new tool for generating iPSC that will overcome these limitations. An entirely novel, non-toxic, non-viral vehicle for introducing transcription factor proteins, and/or mRNAs will be engineered, based on the naturally-occurring, non-immunogenic vault nanoparticle. The proposal is a collaboration between three scientists who each bring unique skills and strong research records to the problem, including proven abilities to translate basic science into clinical therapeutics, execute somatic cell reprogramming, and engineer therapeutic delivery systems. The investigators have productive experience working with California industry, and understand the importance of developing tools that will benefit both the health and the economy of California. In addition, this collaborative project will focus diverse research groups with many trainees on an important interdisciplinary project at the interface of science and engineering, thereby training future employees and contributing to the technological and economic development of California. Finally, these novel tools will have future use in regenerative medicine, since technologies perfected here will result in novel, effective therapies that have the potential to improve the health of millions of Californians and of people world-wide. All scientific findings and biomedical materials produced from these studies will be publicly available to non-profit and academic organizations in California, and any intellectual property developed by this Project will be developed under the guidelines of CIRM to benefit the State of California.
This proposal is focused on the development of a novel vector, called a vault, to deliver reprogramming factors for induced pluripotent stem cell (iPSC) generation without viruses, thus avoiding integration of foreign DNA into the genome. The novel vector is based on naturally occurring, non-immunogenic nano-scale cellular structures with a hollow interior cavity that can deliver a protein or nucleic acid payload. These structures naturally occur in almost every cell of the human body. The applicant identifies the lack of efficient, non-integrating reprogramming methods as a roadblock to clinical translation. There are three Specific Aims: (1) to optimize vault targeting and uptake in different somatic cell types; (2) to engineer vaults containing iPSC reprogramming factors, in both protein and mRNA form; and (3) to optimize vault-mediated reprogramming using the vaults developed in Aims 1 and 2. The reviewers agreed that this proposal addresses a significant translational bottleneck. They described the idea of using vault proteins as reprogramming vectors as highly innovative and appreciated that it takes advantage of a natural cellular structure. However, the reviewers noted that the significance and potential impact of the proposal are limited by a recently published study demonstrating virus-free reprogramming with synthetic mRNA. It wasn’t clear that the vault technology would be significantly better for iPSC reprogramming than synthetic mRNA, or other non-viral delivery systems currently under investigation. Reviewers described the research plan as complex and challenging, but generally feasible. They noted that the preliminary data are strong, including demonstration of the ability to produce the required modified vault proteins. Reviewers did suggest that the project would benefit from more data addressing the specific problems at hand. For example, it is not clear that vaults have the capability to package and deliver sufficient quantities of protein or mRNA to induce reprogramming. Reviewers appreciated the applicant’s approach in Aim 1, but would have liked more detail about the attachment of ligands to vaults as well as discussion of potential pitfalls and alternative approaches for this Aim. Experimental design for Aims 2 and 3 were considered straight forward with adequate considerations for alternate approaches. One reviewer suggested that it could benefit from testing one vaulted reprogramming factor at a time, with the remainder delivered virally. In addition, a reporter system to assess the activity of various components in transduced cells could be useful. It was noted that although vault technology has been around for some time it has not been applied as a delivery device in any clinical applications so far. The reviewers described the Principal Investigator (PI) as an expert in the field of vaults and highly qualified to oversee every aspect of the project. They appreciated the contributions of two collaborating senior investigators, who provide critical expertise in gene transfer and cellular reprogramming. Reviewers found the research team to be integrative and complementary, with a combined track record that suggests a high probability of success. Overall, reviewers appreciated this innovative proposal from a strong research team. However, they were not convinced that the complex reprogramming system proposed would provide significant advantages over currently available technologies.
- This application scored below the initial scientific merit funding line, no programmatic reason to fund the application was suggested, and the GWG voted to place the application in Tier 3, Not Recommended for Funding.