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RL1-00633-1: High-fidelity derivation of new stem cell lines

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Statement of Benefit to California (provided by applicant)

Review

In this proposal, the applicant proposes to improve methods of generating human induced pluripotent stem (iPS) cells by using artificial transcription factors (designed regulatory proteins, DRPs) designed to activate lineage reprogramming genes. These DRPs will be delivered by protein transduction, which will circumvent issues of viral integration, and will be optimized for dose and mix of the various activators. The applicant then proposes to generate disease-specific iPS cells by reprogramming fibroblasts taken from patients suffering from Alzheimer’s Disease (AD) and Chronic Lymphocytic Leukemia (CLL), in order to create human cell-based models of these diseases.

AD is the most common neurodegenerative disorder. Accurate cell-based models will represent a significant step towards a greater understanding of disease pathology, and may even yield new therapies. And while CLL is less prevalent, a reliable cell-based model would represent a unique opportunity to identify new treatments for this debilitating disorder. In addition, the described goal of improving iPS cell generation, and particularly of avoiding the use of integrating viruses, is highly significant. However, reviewers felt that the alternative iPS methodology was at too preliminary a stage to determine its feasibility. In addition, although one reviewer stated that the experiments were clear and concise, most felt that the application was not cohesive and was poorly focused.

The derivation of iPS cells from AD patients is feasible based on the published work of others. The molecular pathology at the core of AD is complex and poorly understood, and research into the underlying mechanisms would greatly benefit from the derivation of iPS cell lines from patients afflicted with AD. These experiments, described in Specific Aim 1, were considered significant.

The second aim of the proposal seeks to develop a method for generating clinical grade iPS cells. To accomplish this aim they propose to use a new technology, namely DRPs, to achieve reprogramming and generate iPS cells. The idea that artificial DNA binding domains can be targeted selectively to specific promoters is a relatively recent discovery and the proposed use of this technology by the applicant and colleagues to achieve reprogramming is intriguing. As there are no preliminary data shown for DRPs that activate Oct4, Sox2, Klf4 or cMyc it remains to be seen whether this technique will truly be feasible given the levels and duration of gene expression likely required to achieve reprogramming. Overall, reviewers were concerned that the technology was at too preliminary a stage to judge its feasibility.

Aim 3 of the proposal received a high level of criticism from reviewers. In this aim, the applicant proposes to derive clinical-grade iPS cells for AD and CLL, which like AD is currently incurable, remains poorly understood in terms of the genetic lesion responsible, and lacks an appropriate disease model. These experiments depend upon the success of the DRP technology, which as discussed above might not be feasible. Moreover, the experimental rationale for examining CLL cell-based models is lacking, and by combining AD and CLL cell-based models this aim weakens the entire proposal by undermining a coherent experimental rationale.

The applicant trained as a plant molecular geneticist, and has an outstanding track record in terms of publications and a long history of innovative research. S/he developed the DRP technology to be applied in the reprogramming experiments proposed in the current project. The applicant proposes very strong collaborations.

Programmatic Discussion
A motion was made to recommend that this application be moved to Tier 3 – Not Recommended for Funding. Reviewers commented that the DRP technique was clever, but felt that the chance of it working was pretty low. They also commented that the grant was unfocused and poorly written. The doubts about feasibility were cited as reasons to move this application to tier 3. The motion to move this application to Tier 3 carried.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• None