Tools and Technologies II
The use of stem cells or stem cell-derived cells to treat disease is one important goal of stem cell research. Major human illnesses, such as amyotrophic lateral sclerosis (Lou Gehrig’s disease), Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, diabetes, and macular degeneration represent opportunities where stem cell-based therapies may help patients. A second, important use for stem cells is the creation of cellular models of human development and disease, critical for uncovering the molecular and cellular roots of illness and testing new drugs. However, a major limitation in achieving these goals is the difficulty in manipulating the genes of human stem cells. Existing means of generating genetically modified stem cells are not ideal, as they do not preserve the normal gene regulation, are inefficient, and insert foreign genetic sequences. For the promise of human stem cells to be realized, we must develop the tools to shape their properties. We have developed and extensively tested a method of genetically modifying mouse embryonic stem cells that is much more efficient than traditional methods. We propose to adapt this approach for use with human embryonic stem cells so that these cells can be better understood and harnessed for modeling, or even treating, human diseases. This approach seeks to overcome existing barriers to both basic and translational research using stem cells, and will improve and accelerate diverse aspects of biomedical research and fundamentally enhance the research enterprise. Drawing upon our previous experience generating and genetically modifying mouse stem cells, we will create a resource of easily modified human stem cells, will integrate our technology with other resources, and will provide the research community with open access to these reagents and techniques. These stem cells will thus represent a large, unique public domain source of genetically modifiable human stem cells accessible to all researchers.
Statement of Benefit to California:
Although human stem cells hold the potential to generate new understanding about human biology and new approaches to important human diseases, the inability to efficiently and specifically modify stem cells limits the pace of current research. Presently, there is no safe means of changing human stem cell genes compatible with the use of the stem cells in human therapies. This proposal seeks to develop new genetic tools to allow for the tractable manipulation of human stem cells. By accelerating diverse other stem cell research projects, these tools will enhance the scientific, therapeutic, and economic development of California. The ability to engineer specific genetic changes in human stem cells will be useful for widespread applications including the creation of models of early human development, replacement cells for personalized therapies, reporter lines for stem cell-based drug screens, and disease-associated mutations to explore the cellular bases of human illness. Additionally, the creation of a resource of manipulable stem cells will provide the larger research community with the tools to tailor stem cell genes for purposes beyond those envisioned here. Thus, the technology developed in this project will spur the research endeavors of the stem cell community in both the public and private arenas, contributing to economic growth and new product development. This research will increase biological and medical understanding and may promote the development of safer and more effective means of controlling embryonic stem cells. This project will also train students and postdoctoral scholars in human stem cell biology, who will contribute to the economic and research prowess of California.
This proposal is focused on the development of an efficient technology for the rapid and targeted modification of genes in human embryonic stem cells (hESCs) to overcome the inefficiency of current gene modification methods. This would enable more extensive implementation of hESC-based applications such as generation of cellular models of human disease or reporter lines for differentiation assays or drug screens. Using a random integration approach, the applicant intends to generate a large collection of hESC lines, with each line easily modifiable at specific gene loci. The applicant proposes to adapt and improve this system originally developed for genetic modification of mouse ESCs (mESCs) for use in hESCs and to generate a resource of over a thousand genetically modified hESC lines for use by the research community. The reviewers agreed that this proposal addresses a significant translational bottleneck and is innovative and clever in its approach. However, they were not convinced that it would have a major impact on the field of regenerative medicine. Reviewers noted that in this approach, the selection of modified genes occurs randomly and while a thousand lines is a good start, these will only represent a few hundred distinct genes, i.e. a small fraction of all human genes. Therefore it is possible that few of these loci will be of interest to the research community. The power of this approach depends on the availability of a much larger collection of gene tagged hESC lines representing a substantial proportion of all human genes, a stated long-term goal beyond the scope of this proposal. In addition, reviewers were concerned that insufficient consideration had been given to the choice of hESC line. They noted that hESC lines vary considerably and no single line may be optimal for a master collection of genetic modifications. Given the intent to generate a widely used resource, reviewers would have appreciated a more detailed justification of the applicant’s choice of hESC line. The reviewers found the research plan to be logical and detailed with a sound scientific rationale. They appreciated the strong preliminary data presented in mESCs, but were concerned that feasibility has not yet been demonstrated in hESCs. They cautioned that targeting large numbers of genes could be much more difficult in hESCs than mESCs and were concerned that alternative approaches are discussed only sparingly. In general, reviewers had some doubts that one thousand genetically modified hESC lines could be generated in the context of this proposal. They also noted that while milestones are defined there is a lack of meaningful quantitative measures to determine if the aims and objectives of the project have been achieved. The reviewers praised the principal investigator (PI) as a talented and creative young scientist with an excellent track record. They noted that the PI has made innovative discoveries and published in several high-impact journals. Reviewers found the research team well-suited to perform the proposed experiments. They appreciated the letters of support from accomplished colleagues at the applicant institution but noted that these do not identify specific collaborations that would enable rapid translational use of the hESC lines likely to be generated with the proposed funding. Overall, while the reviewers appreciated that this proposal from an excellent PI addresses a significant translational bottleneck, they raised some concerns about the feasibility of transferring the technology developed for mESC to hESC and were not convinced this study would have a major impact on the field.
- 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.