Funding opportunities

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

Funding Type: 
Early Translational I
Grant Number: 
Principle Investigator: 
Funds requested: 
$1 327 973
Funding Recommendations: 
Grant approved: 
Public Abstract: 
The ability to convert human skin cells to induced pluripotent stem cells (IPSCs) represents a seminal break-through in stem cell biology. This advance effectively circumvents the problem of immune rejection because the patient’s own skin cells can be used to produce iPSCs. This exciting technology could accelerate treatments for a number of presently incurable diseases. However, a paramount unanswered question is whether these cells or their derivatives are truly safe for administration. Specifically, it is unknown whether the integrity of the iPSC genome is maintained during the tissue culture steps required to generate, maintain, expand and differentiate iPSCs. Every cell contains roughly 3 million “jumping genes” or mobile genetic retroelements that comprise up to 45% of the human genome. This contrasts with the fact that the roughly 21,000 human genes occupy only 1.5% of genome. While many of these retroelements have been permanently silenced during evolution, many others remain active and capable of replicating and moving to new chromosomal locations potentially producing disease-causing mutations or cancer. Somatic cells limit the jumping of these mobile genetic elements (retrotransposition) chiefly by methylating the DNA in and around these elements. Strikingly, the process of converting a skin cell to an iPSC results in a profound loss of DNA methylation potentially opening the door for high level retroelement activity that could corrupt genomic integrity. These insertions can disrupt key genes, create double strand DNA breaks or lead later to loss of large sections of DNA. Whether retroelement activity contributes to the fact that only 0.01% of skin cells are successfully reprogrammed to iPSCs is unknown. Thus, key questions regarding the safety of these cells remains. We now propose to determine the level of retroelement retrotransposition occurring in iPSCs and hESCs and to develop potentially safer ways to generate and maintain iPSCs in culture by blocking a key retroelement enzyme. Further, we will assess whether differentiation of these cells triggers retroelement activity. Finally, we will explore potential additional cellular defenses brought into action to oppose these retroelements with the goal of further enhancing these defenses.
Statement of Benefit to California: 
The use of pluripotent stem cells derived either from the inner cell mass of developing blastocysts or by reprogramming of skin cells holds great therapeutic promise. These cells could provide exciting new approaches for a number of incurable human diseases like Parkinson’s and Alzheimer’s disease, type 1 diabetes, and cardiac failure. However, a paramount unanswered question in the field is whether these cells can be used in a completely safe manner. One major threat that could undermine these exciting stem cell therapies is the appearance of genetic mutations during their generation, expansion or differentiation. Such mutations could be induced by mobile genetic elements. Every cell contains roughly 3 million mobile genetic retroelements that comprise up to 45% of the genome. Active retroelements are capable of reproducing themselves and then jumping to a new chromosomal location potentially causing devastating disease-causing mutations or cancer-promoting changes. In normal differentiated cells, the jumping of these retroelements is highly constrained by DNA methylation. However, when skin cells are reprogrammed to become induced pluriopotent stem cells (iPSCs), DNA methylation is essentially erased. Human embryonic stem cells (hESCs) also exhibit dynamic changes in DNA methylation characterized by rapid losses and gains. These events open the door for repeated waves of retroelement retrotransposition that could greatly undermine the genome integrity of these cells. A real gap in our understanding of iPSC biology is that the potential activity and damaging effects of these retroelements has not been explored. To determine if hESCs and iPSCs and their cellular progeny can be safely used in patients, we propose to study the expression of retroelement RNA, the frequency of physical jumping events, and the impact of potential cellular defensive mechanisms opposing these retroelements. Since stem cells will be differentiated in vitro prior to their use in patients, we will also study levels of retroelement jumping in cells induced to differentiate into the three germ cell layers, endoderm, mesoderm and ectoderm. Additionally, we propose to explore potentially safer ways to generate and maintain iPSCs in culture where the retrotransposition process is interrupted using an FDA-approved HIV antiviral drug. Such an approach could protect the genome of these cells during their culture and manipulation in the laboratory prior to infusion into patients. The results of these studies will have both important scientific and practical value for the future therapeutic use of stems cells. As such, we believe these studies will benefit the citizens of California certainly at a societal level and potentially at a personal level.
Review Summary: 
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.

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