Tools and Technologies II
$1 829 478
Previous work has attempted to use recombinant protein to make useful pluripotent stem cells such as induced pluripotent stem cells. However, due to protein degradation in cultures or inside the cells, the efficiency of making such kind of stem cells is extremely low beyond any practical use. Our research team has recently developed a novel nanotechnology to manufacture protein capsules containing recombinant proteins including OCT4, SOX2, KLF4, and C-MYC (OSKM). These protein capsules will allow optimal intracellular delivery of capsulated proteins. Nanocapsulated recombinant proteins are much stable in culture media and much efficient for intracellular delivery. Therefore, we expect to use this novel technology to improve protein delivery into cells. With this technology, we can efficiently make useful stem cells for future clinical applications. By the same technology, we can promoter neuronal cell differentiation from human stem cells, thus achieving a large quantity of neural cells for cell transplantation and drug screening purpose. The success of our research project will allow us to efficiently make patient specific stem cells and neural cells. This is important for cell replacement therapy in neural repair and in drug discovery. Our proposal research will be an important step to translate stem cell research into future clinical applications.
Statement of Benefit to California:
Stem cell transplantation has emerged as a method that may improve recovery of many human diseases such as neurodegenerative disorders (Parkinson's disease and Alhzeimer's disease) and brain injuries. Studies of stem cell transplantation have been limited because the source of transplantable brain cells is limited. Recent technology demonstrated that induced pluripotent stem cells could be a feasible source for patient specific stem cells. However, current technology is limited in the derivation of clinically useful stem cells. We have recently found that a new method to introduce proteins into cells, thus paving the way to promote the generation of induced pluripotent stem cells. The experiments in this grant will devise a new method to promote stem cell derivation as well as enrich for the useful brain cells differentiated from pluripotent stem cells. These results are applicable to the treatment of many neurological disorders such as Parkinsons, Alzheimer’s and Huntington’s diseases. Our proposed research will accelerate the pace in the application of human embryonic stem cells and induced pluripotent stem cells for neural repair, thus benefiting the health care of the State of California and its citizens.
The goal of this proposal is to develop a novel nanocapsule-based protein delivery system to improve the efficiency of non-viral cellular reprogramming and to facilitate the differentiation of pluripotent stem cells (PSCs) and direct reprogramming of somatic cells into neural derivatives. The applicant suggests that, if successful, this delivery system will provide a safe, vector-free reprogramming system that could also be used to improve lineage specific differentiation; these goals will address major bottlenecks of reprogramming efficiency and safety in the use of induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) for patient specific treatments. To address these bottlenecks, three specific aims are proposed. First, the applicant plans to engineer degradable nanocapsules containing Oct4, Sox2, IKlf4 and C-Myc proteins (OSKM) and other reprogramming factors to achieve optimal and efficient intracellular delivery of reprogramming proteins. Next, nanocapsulated OSKM and other reprogramming factor proteins will be used to derive vector-free human iPSCs. Finally, the principal investigator (PI) proposes to engineer nanocapsules containing neural differentiation transcription factors for use in improving differentiation of human PSCs and performing direct reprogramming of human and mouse fibroblasts into neural derivatives, the functionality of which will be tested using an in vivo murine transplant model. Reviewers agreed that the lack of efficient, non-viral cellular reprogramming tools is a major bottleneck for the development of stem cell-based therapies, and advancement in this area would be of significant value to the field. Reviewers were divided as to whether the proposed research is innovative; although some reviewers felt the technology incorporated novel ideas, others questioned the novelty of the nanocapsule system for protein delivery and thought that other aspects of the proposed technology provided little advantage beyond those approaches already in development in other labs. Reviewers found the proposal to be focused and carefully designed with methodologies, strategies and analyses that are logical and appropriate to complete the specific aims. They further agreed that the applicant provided quantitative measures of success and included convincing preliminary data supporting the ability of the team to carry out the proposed research. However, reviewers were concerned that some important feasibility issues were not adequately addressed in the experimental plan and found that the proposal lacked sufficient consideration of potential pitfalls and alternative approaches. For example, the applicant did not adequately address potential difficulties related to the intracellular half-life of proteins following nanocapsule-mediated delivery; this issue could be critical, since the delivered proteins may need to remain active for days to weeks in order to drive reprogramming. Another major concern was the lack of a systematic approach to control levels of cellular protein delivery to achieve optimal reprogramming. Additionally, the applicant did not address potential toxicity or efficacy problems related to protein modification during encapsulation. Finally, reviewers found the description of the in vivo experiments proposed in Aim 3 to be superficial and lacking in important details. Reviewers described the PI and the collaborator as excellent scientists with outstanding track records who bring complementary expertise to the project. The PI is fully qualified to oversee all aspects of the project, and the collaboration is integrative and well established. The institutional support and environment were judged to be of excellent quality. In summary, while reviewers appreciated the outstanding research team and logical research plan, they were not convinced that the proposed work is highly innovative, competitive, or would have a significant impact in the field. Further, reviewers questioned the feasibility of the proposed work given the absence of critical details from the experimental plan. Therefore, the reviewers did not recommend this application for funding.
- A motion was made to move this application into Tier 1, Recommended for Funding. Although reviewers agreed there is potential merit to the proposal, they felt that on the basis of its innovativeness, the proposed technology is not sufficiently innovative to be competitive in such a rapidly evolving field. As such, there was not sufficient support to move this application from Tier 2 to Tier 1. The motion failed.