Tools and Technologies I
Statement of Benefit to California:
This proposal focuses on the development of methods to model human neurodegenerative diseases in human embryonic stem cells (hESCs). Specifically, the Principal Investigator (PI) proposes to use homologous recombination (HR) to target and modify the hESC genome to express disease-causing genes. As proof-of-principle, the research team will focus on amyotrophic lateral sclerosis (ALS). In Aim1, they will introduce three common ALS point mutations by HR in hESCs. In Aim 2 they plan to further target these mutations in hESC reporter lines of the two important cell types in ALS: motoneurons and astrocytes. Finally, in Aim 3, they will analyze cell survival, apoptosis and SOD1 protein expression in the SOD1 mutant hESC lines they create. The availability of these SOD1 mutant hESCs and hESC reporter lines will allow researchers to obtain purified “diseased” motoneurons and astrocytes, which offer direct clues to ALS pathogenesis. The reviewers agreed that this proposal could have a significant impact on the field of stem cell biology. The disease models that exist for neurodegenerative diseases show poor correlation to the human disease state(s). If successful, this proposal would provide an unlimited source of purified “diseased” motoneurons and astrocytes: novel and valuable tools for the study of ALS. Importantly, because the cells would be generated from a normal starting population, the appropriate, genetically identical, controls will be available for comparative experiments. In addition, the HR technology developed in this proposal could easily be applied to other neurodegenerative diseases for which genetic defects have been identified, including Huntington’s and Parkinson’s disease. Finally, a reviewer noted that this approach is complementary to those generating disease models using induced pluripotent stem cells (iPSCs) from ALS patients. Overall, reviewers found the proposal very well written, carefully designed and ultimately feasible. They commented that the strategies outlined are sensible and achievable and the preliminary data strongly suggests that the team is capable of carrying out gene targeting in hESC cells. However, a few comments were raised concerning the ambitious nature of the proposal and the lack of detail provided in some cases. Reviewers expressed doubts about the timelines presented, which they thought were based on best-case scenarios. Reviewers expect most aspects of the proposal will take considerably longer than stated, particularly in cases where serial gene targeting is required. One reviewer appreciated that the applicants intend to generate independent clones derived from independent parental cell lines, originating from independent laboratories, even while noting that it makes the proposal more ambitious. A reviewer also commented that because the applicants intend to introduce these SOD mutations into the motoneuron and astrocyte reporter lines, the benefit of generating similar SOD mutant lines without reporter genes is unclear and this aspect of the first aim could be eliminated. Another reviewer was frustrated by a lack of experimental detail provided for the third aim. This reviewer questioned how the applicants will measure survival of mutant and wild-type hESCs. This reviewer also commented that the genomic and proteomic analyses are not well defined and no preliminary data for this specific project was presented. This reviewer also noted that the targeting efficiency in the preliminary data from a different knockin line (Olig2-GFP) was low (4-5%) and wondered if it was typical. While the reviewers generally praised the discussion of pitfalls and alternative approaches in the proposal, one felt that the section proposing to use a transgene strategy to generate the desired reporter lines should the HR strategy fail was poorly justified. Reviewers praised the research team’s molecular and cell biology expertise but noted a lack of experience on neurodegenerative disease. One reviewer strongly recommended including a consultant from the ALS field in this proposal. Reviewers generally found the budget appropriate, although one commented that not all of the proposed rounds of gene targeting on all of the proposed lines are absolutely necessary to demonstrate proof-of-principle (as detailed above) and if some were eliminated the budget would be smaller. Overall, reviewers found this to be a strong, well-organized proposal that clearly addresses a roadblock in stem cell biology and provides new tools for therapeutic research. They raised a few concerns about it feasibility but were ultimately convinced by the quality of the preliminary data and research team.