Human embryonic stem cells (hESc) represent a potentially unlimited supply of tissues for regenerative therapies. However, current differentiation methods result in heterogeneous populations of cells in which the desired product is, at best, a fraction of the total. Moreover, carry-over undifferentiated cells, as well as cells that de-differentiate upon transplantation, have been shown to form teratomas in animal models. The risk of teratomas is arguably the foremost obstacle towards the clinical implementation of hESc-based therapies, and a major cause of concern for regulatory agencies. In order to address this problem, we set out to engineer hESc with control mechanisms allowing for the selective ablation of (a) tumorigenic cells; and (b) cells differentiated along non-desired fates. Our preliminary data indicate that a construct of our invention, stably integrated in the genome of the H1 hESc line, imparts such selectivity both in vitro and upon transplantation in vivo. We have chosen pancreatic ?-cells as a model to test our hypothesis, as hESc-derived ?-like cells are now entering clinical trials for type 1 diabetes. However, the principle behind our strategy could be adapted to any cell type of choice. Our control mechanisms are based on (a) the activation of a suicide gene (HSV-TK) in cells that resume/continue tumorigenic proliferation; and (b) the irreversible inactivation of a second suicide gene (NTR) in cells that express insulin. Our central hypothesis is that hESc lines where these constructs are targeted to a ?safe harbor? genomic location (the ROSA26 locus) will work as effectively as they do when their integration is random, as per our preliminary data. We plan to test this hypothesis by pursuing the following specific aims: (1) To integrate the above constructs into the ROSA26 locus of a human hESc line by CRISPR/Cas9 gene editing techniques; and (2) To perform in vivo tests of function in rodents. The proposed research is innovative because it provides a degree of selectivity that is lacking in conventional suicide gene-based strategies, in which activation of the transgene typically brings about the destruction of the entire graft. We anticipate a positive impact of our research on the field, because such cell lines would be ideally suited for the development and safe clinical implementation of differentiation protocols. Our research, therefore, is designed to break barriers that stand in the way of the widespread clinical use of hESc and will pave the ground for a subsequent Phase II proposal aimed at the generation of cGMP-grade hESc with this targeted modification for safe use in the clinical practice.
In order to realize the clinical potential of human embryonic stem cells (hESc), methods to overcome both the relatively high incidence of tumors (teratomas) and the low efficiency of differentiation must be devised. Here we propose a double fail-safe mechanism to address these two problems. hESc engineered with a construct of our invention will activate a suicide gene in tumorigenic cells and de-activate a second in cells of the desired characteristics. Our proposal thus responds to the general mission of the NIH, as a positive outcome will speed up the safe and efficacious translation of hESc research into clinical therapies.
