Maximizing the Safety of Induced Pluripotent Stem Cells as an Infusion Therapy: Limiting the Mutagenic Threat of Retroelement Retrotransposition during iPSC Generation, Expansion and Differentiation
Early Translational I
$1 327 973
Statement of Benefit to California:
This proposal addresses the safety of transplanted (patient-specific) induced pluripotent stem (iPS) cells. It is based on the hypothesis that genomic integrity may be jeopardized during reprogramming through activation of retrotransposition, a process in which mobile genetic retroelements, present in the human genome, replicate and move to new chromosomal locations. This process leads to insertions and/or genomic rearrangements that can produce disease- or cancer-causing mutations. The applicant hypothesizes that it might also contribute to the relatively low efficiency of the reprogramming process. Since DNA methylation is one of the main mechanisms employed by somatic cells to limit retrotransposition, and since demethylation of the genome is widespread during reprogramming, the applicant suggests that reprogramming may lead to enhanced levels of retrotransposition with potentially detrimental downstream consequences. The applicant intends to assess the levels of retrotransposition during reprogramming of human skin cells mediated by lentiviral transduction of required transcription factor genes (aim 1), to compare the levels of retrotransposition occurring in iPS cells and hESCs during their differentiation (aim 2) and to evaluate the role of various cellular defenses against retrotransposition in pluripotent stem cells (aim 3). Ultimately, the goal is to develop potentially safer ways to generate and maintain iPS cells in culture by blocking retrotransposition, using an FDA-approved compound, or by developing methods to enhance the relevant endogenous anti-retrotransposition defenses. Reviewers agreed that the clinical safety of iPS cells or their derivatives is a fundamental unanswered question, and that this proposal thus addresses a critical bottleneck. They felt that is the applicant presented a strong scientific rationale for the project, and that iPS cells may be particularly prone to retrotransposition, which could lead to detrimental genomic alterations even if non-viral approaches will be developed for reprogramming. Although there is currently no evidence that retrotransposition represents a safety issue, reviewers argued that the answer to this question, even if it was negative, would provide a valuable contribution to the field. Alternatively, if retrotransposition turns out to be activated in iPS cells, it needs to be addressed. The reviewers did not agree that the hypothesized effect of retrotransposition on reprogramming efficiency represents a significant bottleneck, since that efficiency, although low, is sufficient for generating lines. Reviewers also warned that unless non-viral, non-oncogene driven iPS approaches are used; it will be difficult to sort out the myriad potential causes of any tumor formation. Reviewers felt that the proposed studies were well designed and feasible; the experimental methods were sound, the study can be completed within the proposed timelines, and is supported by convincing preliminary data. A reviewer expressed concern that reporter lines will need to be generated for each of the hESC and iPS cell lines to be analyzed, which complicates comparisons between lines. Reviewers agreed that aim 3 addresses an interesting scientific question, and will lead to the identification of the cellular defenses against retrotransposition that are operating in pluripotent stem cells, but it falls short of addressing the translational goals of the project, the improvement of the clinical safety of iPS cells, and emphasizes instead a basic biological question. The applicant is a very accomplished scientist and has successful worked in related areas. One existing post-doc, who is accomplished, although not in the stem cell biology field, and a research associate are assigned to the study, raising concern that not enough personnel have been committed to this project. Reviewers appreciated that the budget was appropriate for the proposed studies. The environment for stem cell research at the applicant institution is outstanding and resources are available for carrying out this project. There are no collaborators that would provide needed expertise in iPS cell generation or stem cell differentiation listed in this proposal, although core facilities and neighboring groups can presumably help if necessary. In summary, reviewers agreed that this proposal addresses an important translational bottleneck, the clinical safety of iPS cells, although some of the proposed experiments were judged to address more basic scientific questions. Despite being based on a currently unproven hypothesis, reviewers felt that the scientific rationale for this project is compelling, and that this proposal has the potential to answer important questions related to translatability of iPS cells. Reviewers were enthusiastic about this proposal because of the quality of the experimental plan, the applicant’s qualifications and the excellent research environment.