Tools and Technologies II
Cellular therapies represent the most advanced technologies in medicine and have a myriad of potential applications. To date, the chromosome and gene composition of cell therapeutics have undergone only minimal evaluation, an oversight that could be of significant detriment to the long-term development of these products. Two major issues need to be addressed during the earliest stages of developing cells for therapy. First, the overall chromosomal integrity of cells must be assured. There are many examples in biology where alterations and mutations in genes can have disastrous effects, most significantly in the development of cancer. Furthermore, an increasing number of studies are documenting large scale alterations in the chromosomes of cell culture populations that mirror several of the changes seen in tumor cells. Secondly, the complete DNA sequence of cell therapy products should be assessed. The number of documented genetic disease with clearly defined gene lesions is over 300, while the number with presently undefined molecular lesion is over 1600. An analogous reasoning holds for disease propensity alleles; shouldn’t we know the satus of genes that are substantially implicated in disease processes? ApoE is one of dozens of genes that can dramatically affect overall health. The above considerations weigh even more heavily on transgenic and genetically modified cell therapeutics. Most cellular therapeutics are intended as permanent grafts and as our discovery and understanding of genetic disease and disease propensity progresses, it will be of considerable importance to be able to review archived genomic data on the products transplanted. Our proposal will provide important new platforms for ensuring both manufacturing consistency and the long-term safety of cellular therapeutics. We will apply both low (cytogenetic) and high-resolution (complete genome sequencing) methods to thoroughly analyze the DNA content of cell therapeutics and will do this using already available service providers. Our goal is to provide standardized protocols, quality measurements and reports to improve quality assessment and safety of cellular therapeutics.
Statement of Benefit to California:
California is unquestionably the leading state in the nation for funding and supporting regenerative medicine. Since much of the support provided by CIRM targets the development of expensive and complex cell-based therapeutics, making efficient use of these dollars is crucial. Cell therapeutics represent, in most applications, permanent transplants, and thus it is critical that the overall safety of these products is assessed. DNA-based assessment of cell therapy candidates is a under-utilized quality control step and one where the consequences of unintended mutations or alterations of the genome could have disastrous effect. Our studies will apply the most modern and high resolution methods to thoroughly analyze and document the DNA content of cell therapeutics. In California, we are home to most of the major service companies that provide complete genome sequencing and our study will capitalize on these private industry resources. In addition, applying these methods to the earliest stages of research and development can help optimize the selection of cell therapy candidates and prevent the expenditure of dollars on candidates with unsafe genetic profiles.
This proposal is focused on the development and application of a standardized set of tools to assess the genomic integrity of human embryonic stem cells (hESC), human embryonic progenitor cell lines (hEPC), induced pluripotent stem cells (iPSCs) and CIRM-funded cell therapy candidates. The applicant cited evidence that many hESC lines accumulate genetic abnormalities and emphasized that even the most commonly used lines have not been fully sequenced for mutations or genetic polymorphisms associated with human disease. The applicant argued that this lack of genomic integrity data represents a critical safety issue and a bottleneck to the translation of stem cell-based therapies. The specific goals of the proposal include: (1) development of standard protocols and procedures for calibration of molecular and cytogenetic assays; (2) implementation of higher sensitivity probe arrays to detect chromosomal abnormalities; and (3) complete genome sequencing of hESC lines, iPSC lines and CIRM-funded clinical cell therapy candidates. While the reviewers agreed that in-depth genomic analyses of stem cell lines would provide valuable information, they noted that the lack of this information does not currently represent a translational bottleneck to stem cell therapy. Instead, this proposal looks forward to a potential safety and regulatory bottleneck. Reviewers appreciated the scientific rationale for the proposal, as genetic abnormalities in transplanted stem cells could pose significant risks to patients and current detection methods are costly and of low-resolution. However, they concluded that this proposal would fall short of having a major impact on the field. They noted that the interpretation of genome sequencing data is not yet advanced enough to be of benefit in determining the safety of cell therapy. The significance of the proposal is also weakened by the lack of a clear plan to follow up on evidence of genomic changes, such as correlating these changes with specific phenotypes. Finally, reviewers did not find the proposal to be particularly innovative, noting that it does not entail the creation of new technologies, but rather the implementation of existing, cutting-edge tools. The reviewers agreed that the research plan is focused and scientifically sound. They appreciated the strong preliminary data demonstrating that many hESC lines have undergone some degree of selective genomic rearrangement during expansion in culture. The data also demonstrate that the research team has the fundamental tools in place for examining the genomic stability of pluripotent cell lines as well as for sequencing their entire genomes. However, reviewers noted that the applicant proposes to collect a lot of data while doing little to improve the analysis of that data. For example, measurement of SNP array sensitivity to abnormal subpopulations is proposed, but not experiments to improve this critical limitation. The applicant also does not propose to investigate the time course over which genetic abnormalities accumulate, or the significance of pre-existing polymorphisms and mutations in the hESC or iPS cell lines. There is no discussion on whether particular methods of cell line establishment, propagation or cryopreservation correlate with specific genomic rearrangements. While reviewers found the research plan to be well-organized and generally feasible, they noted that potential difficulties and alternative approaches are not described or addressed. Finally, reviewers described the lack of experiments designed to tie specific changes in genetic stability to biological outcomes as a major shortcoming of the research plan. Reviewers found the principal investigator (PI) and assembled research team to be strengths of the proposal. They praised the PI’s considerable expertise in stem cell research and the logical collaborations established with companies specializing in cytogenetic analysis and whole genome sequencing. One minor reviewer criticism was that the proposed SNP analysis would be performed by a service provider rather than a collaborator, likely precluding the sort of experimentation that would improve molecular cytogenetics as a tool for stem cell analysis. In terms of the budget, one reviewer noted that the bulk of the cost for each cell line would go into sequencing, the analysis that currently provides the least amount of information about safety for cell transplantation. A reviewer also suggested that full genomic sequencing only be performed on a few lines, those which do not display genetic abnormalities in other, lower-cost assays. Overall, while reviewers appreciated that useful data that would result from this proposal, they were not convinced that it addresses a translational bottleneck. They noted that the applicant does not propose technical improvements or biological followup, limiting the project’s potential impact.
- A motion was made to move this application into Tier 1, Recommended for Funding. The programmatic reason cited was a lack of bioinformatics proposals in Tier 1. Reviewers recapitulated the scientific review. Some GWG members argued that this would be a unique grant in CIRM’s portfolio and could have a broad impact on CIRM’s development pipeline, while others seriously doubted the potential impact of the proposed study. The motion failed.
- Minority Report: The motion was opposed by a majority of the GWG. Because the motion was supported by more than 35% of members, supporters have exercised their right to have that position reported to the ICOC. Three elements were cited by the minority group in support of moving the application up to Tier 1: 1) the proposal was submitted by a “for profit” applicant; 2) the project uses bioinformatics approaches; 3) the proposed research expands a global capacity to assess safety of cell therapy products derived from embryonic stem cells that have been expanded in culture. Also, the proposal supports efforts to characterize 10 cell lines that are being derived as part of a previously funded CIRM award.