Pluripotent stem cells, by virtue of their un-differentiated, un-committed state, have been a major focus for regenerative medicine, as they offer the potential of being able to replace diseased tissue with healthy, functional tissue. Despite the ability of embryonic stem cells (ESCs) to contribute to any cell type of the body, their derivation raises ethical issues over harvesting cells from embryos for research or clinical treatment purposes. In addition, as with any transplant, their use raises concerns over rejection.
The recent development of methods for reprogramming adult cells into induced pluripotent stem cells (iPSCs) presents a tremendous opportunity for regenerative medicine. iPSCs possess most, if not all, of the capacities that make ESCs promising for novel human therapies. iPSCs are free from the ethical concerns surrounding ESC production from embryonic tissue, and can avoid complications of rejection by deriving the iPSCs from cells of the patients themselves. Unfortunately, the methods for generating iPSCs are not very efficient and utilize viruses to introduce the genes needed to reprogram the cells, both of which are major bottlenecks.
The first protocols to generate iPSCs employed viral vectors to express four reprogramming genes. Clinical experience with viral vectors suggests that such iPSCs will be unsafe for use in humans. Viruses that integrate into the genome can result in mutagenesis and can lead to cancer. Although alternative approaches involving drug treatment permit iPSCs to be generated with as few as two expressed factors, they still employ integrating viral vectors. Even viral vectors that do not integrate can evoke a robust immune reaction by the host, raising concerns such as the destruction of the engrafted cells. In order for IPS cells to be safe for use in humans, it is essential to develop methods for their generation without the use of viral vectors.
A second bottleneck is presented by the inefficiency of the current techniques, as the methods employing viruses induce cells at a rate less than 1%. What is needed are methods to express the needed gene products, to suppress unwanted gene products, and to follow the reprogramming process as it takes place so that it can be optimized.
The proposed research answers both of these bottlenecks by developing a set of non-viral vectors optimized for iPSC production. The non-viral vectors are nanoparticles that assemble around and protect RNA so that adult cells can be preprogrammed without fear of cancer-inducing mutations. The nanoparticles are designed to introduce mRNA for the reprogramming factors and to introduce siRNA for suppressing unwanted factors, in order to increase the efficiency of the reprogramming. In parallel, the research will provide the tools to follow the reprogramming as it takes place, so that protocols for iPSCs can be optimized for a given source of adult cell and desired clinical use.
Statement of Benefit to California:
The proposed work involves the design and use of new reagents that can be used to reprogram adult cells into induced pluripotent stem cells (iPSCs). iPSCs offer many of the same advantages as embryonic stem (ES) cells in that they can be induced to differentiate into diverse cell types, making them an important resource for regenerative medicine. Unlike ES cells, iPSCs can be derived from the patients themselves, eliminating ethical issues surrounding the use of embryonic cells, and avoiding the complication of tissue rejection. However, the generation of iPSCs at present involves the use of viruses to accomplish the reprogramming, and hence raise concerns of cancer if they were to be used in patients.
The proposed works will have several major benefits to the state of California and its citizens:
The technologies to be refined in this proposal will allow the creation of iPSCs without the use of viruses, which will allow regenerative medicine with iPSCs to advance to clinical utility in a rapid fashion. Instead, chemically defined nanoparticles will be utilized to deliver the factors in a fashion that cannot create mutation, and are hence unable to create cancer. Tests of the nanoparticles in animal and human studies suggest strongly that the reagents to be developed will be safe for use in humans. This should impact the citizens by making a major avenue of regenerative medicine available for clinical use.
The technology will be simple to deploy, as test experiments show that the compounds can be supplied in a form so that nanoparticles can be created by non-specialists without the need of specialized techniques or equipment. This will allow the reagents to be deployed broadly in both research and clinical settings.
Parallel technologies will be developed to follow the reprogramming of the cells. These tools will be useful to all stem cell researchers, and will allow more directed test experiments to be performed. Because of these tools, researchers will be able to more effectively program stem cells into the progenitor types that are desired, and will be able to follow the cells more accurately. Thus, it will be possible for research and development money from CIRM to be used more efficiently.
Finally, the reagents to be generated by the proposed work offer an excellent opportunity for businesses in California to generate a new and important reagent stream. The optimized targeting of the nanoparticles involves technologies aligned with the capabilities of current industry in the state and also offers opportunities for start-up companies.
The goal of this project is to develop more effective and safer technologies for somatic cell reprogramming using non-viral transfection methodologies, and to understand the biological mechanisms involved in generating induced pluripotent stem cells (iPSCs). Two bottlenecks to the development of personalized cell-based therapies are referenced: the issue of the use of viral vectors to deliver reprogramming factors to human somatic cells, and the low efficiency with which human somatic cells are reprogrammed. The applicant proposes to use nanoparticles to deliver mRNAs of the different reprogramming factors into adult mouse somatic cells, which avoids conventional viral transfection. The applicant also proposes to increase the efficiency of iPSC generation utilizing this technology to deliver the optimal set of reprogramming molecules. Once the new iPSCs are generated, they will be characterized using multiple in vitro and in vivo assays and novel imaging approaches. These technologies will also be applied to generating and characterizing iPSC from human somatic cells
Reviewers appreciated the innovative nanotechnology and imaging tools that were being used to resolve issues regarding viral vector contamination of iPSCs. The technology was considered exciting and the work was rated as first class. However, the proposal drew criticism for not having a clear disease-specific target in mind and the development of this tool was not considered appropriate for a translational research award. There was no clear biological target identified and only a brief discussion of the therapeutic personalized cell lines that could be derived. Overall, reviewers were unconvinced that this proposal would lead to the development of novel tools to enable progression of stem cell therapies towards clinical applications.
In addition to the lack of responsiveness of this application, reviewers felt that the experimental design was overly descriptive and lacking in experimental details. The application lacked preliminary data and the research group did not have experience generating human or mouse iPSCs by conventional methods (that is, using retroviral vectors). In some cases (for instance, the use of imaging during embryogenesis) there was little biological rationale for the proposed experiments, and at times experiments did not seem relevant to the overall aims of the project. The budget was extremely high, and the proposed personnel budget seemed inappropriate for the project. One reviewer commented that there was too much time allotted for technicians dedicated to general tasks, such as computer and animal work, while the unique, creative work was left to two graduate students that had yet to be appointed.
Reviewers commented that the strength of this proposal was the experience and skill of the PI and co-PI. The principal investigator was described as outstanding, very highly regarded in his/her area of expertise with exceptional knowledge in related fields, and reviewers were confident that the PI would be capable of directing a large research project. The co-investigators listed were also highly experienced researchers in technical fields related to nanotechnology and developmental biology. However, the research group lacked investigators with expertise in translational research. The research environment was described as appropriate for a project of this type. No external collaborations were identified in the application.
In summary, this is a proposal to create a novel tool for generating iPS cells through nanopolymer delivery of RNA. The strengths of this application include the outstanding team of applicants and the innovative approach to an important bottleneck in stem cell science. However, reviewers noted that this was a technology development project, and the rationale and experimental design did not address a bottleneck to the development of stem cell-based therapies. The application was therefore deemed unresponsive and not competitive.