Funding opportunities

High-fidelity derivation of new stem cell lines

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00633
Funds requested: 
$1 308 281
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Statement of Benefit to California: 
Review Summary: 
Executive Summary 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. Reviewer One Comments Significance: The goal of improving iPS generation, and particularly avoiding the use of integrating viruses, is laudable. The PI proposes a novel method, that is the use of synthetic DRPs to activate the endogenous reprogramming genes. The derivation of AD iPS cells could be important in probing the pathobiology of AD. Feasibility: The proposal is interesting in its use of a non-viral approach to activating the endogenous reprogramming genes. The PI has experience with the DRP methodology, so in principle the experiments are feasible. I have concern about this approach, however, as it is uncertain that appropriately high levels of the important reprogramming factors will be achieved. There will be considerable trial and error presumably in identifying the most active DRPs and then they will have to be combined at various doses. Preliminary data demonstrating high level expression of one of the reprogramming factors in response to a DRP would help assuage this concern. The derivation of iPS AD cells is feasible, based on published work of others. In Aim 3 the applicant proposes to make clinical grade iPS cells from patients suffering from AD and CLL. The rationale for the CLL experiments is not well laid out in the proposal. In this respect, the proposal is not coherent and focused. Responsiveness to RFA: This proposal is very responsive to the RFA. Reviewer Two Comments Significance: • The molecular pathology at the core of Alzheimer’s disease 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. The same argument applies for CLL which like AD is currently incurable, remains poorly understood in terms of the genetic lesion responsible and lacks an appropriate disease model • The use of retrovirus mediated delivery of transcription factors to reprogram somatic cells to a pluripotent fate is acknowledged as a key proof in principle approach but has significant limitations in regard to the high potential for tumor formation that currently preclude clinical application of iPS lines derived in this manner. The proposal to use DRPs by the applicant has the potential to obviate this concern and may result in a significant advance in the therapeutic application of iPS cells. Feasibility: • The PI Dr Briggs trained as a plant molecular geneticist has an outstanding track record in terms of publication and a long history of innovative research. Developed the DRP technology to be applied in the reprogramming experiments proposed in the current project • An excellent consortium of collaborators: Larry Goldstein : Truly outstanding track record, highly productive and consistently innovative. Will generate iPS cells by retroviral delivery; Colin Jamora: will supply murine keratinocytes and expertise; Catriona Jamieson and Tom Kipps: collaboration regarding CLL • Experimental design clear, concise, systematic approach. The feasibility of much of what is being proposed appears high. • Value of comparison of approaches to reprogramming is high (Briggs : DRPs; Goldstein : retrovirus delivery). Strong likelihood that pluripotent cells will be derived from one or both approaches. • Preliminary data demonstrate feasibility of generating iPS cells by retroviral methodology in applicant’s lab using Yamanaka combination of factors. Data attest to efficiency of delivery of DRPs to target cells (keratinocytes and B-cells). • Much emphasis on methodology for quantitative proteomic analysis to characterize cells and examine cell identity using iTRAQ / 2D-LC-MS/MS analysis. This is a strength of proposal. • Some concerns include: • How pure a population of primitive endodermal cells (PEL) can be obtained? What was the rational for their choice for reprogramming? • Absence of methodology to derive B-cells from reprogrammed cells. • No evidence the applicant has experience in derivation of keratinocytes from mES cells Responsiveness to RFA: This proposal will likely yield iPS cell lines by one and/or both proposed methodologies. Stocks of characterized iPS lines will be established for distribution to the scientific community upon request. Reviewer Three Comments Significance: Briggs and colleagues propose to generate human cell-based models of Alzheimer’s Disease and Chronic Lymphocytic Leukemia by reprogramming fibroblasts taken from patients suffering from these diseases. Alzheimer’s Disease is the most common neurodegenerative disorder and thus accurate cell-based models will represent a significant step towards a greater understanding of disease pathology and may even yield new therapies. And while Chronic Lymphocytic Leukemia is less prevalent, a reliable cell-based model would represent a unique opportunity to identify new treatments for this debilitating disorder. Feasibility: 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 designed regulatory proteins (DRPs) to achieve reprogramming and generate iPS cells. The idea that artificial zinc finger DNA binding domains can be targeted selectively to specific promoters is a relatively recent discovery and the use of this technology in the form of designed regulatory proteins (DRPs) by Briggs 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 the experimental plan in this aim is not elucidated well. The third aim of the proposal attempts to derive clinical grade iPS cells from patients with Chronic Lymphocytic Leukemia (CLL) and AD. These experiments have all of the pitfalls of the second aim as it is dependent upon the success of the DRP technology. Moreover, the experimental rational for examining CLL cell-based models is lacking. Further, by combining AD and CLL cell-based models the proposal lacks a coherent experimental rationale. Responsiveness to RFA: The proposal is responsive to the RFA
Conflicts: 

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