278 - A solution for cell therapy safety

Abstract

Safety concerns such as tumourigenicity have been limiting the implementation of cell therapies to treat degenerative diseases. Here we introduce a concept and an associated genome editing strategy that addresses this issue. To maximize the expression of an inducible suicide system in dividing cells, we transcriptionally linked a suicide gene to a cell division essential locus (CDEL). Our prototype CDEL and suicide gene is CDK1, and HSV-TK, respectively. First, we generated mouse and human embryonic stem cell (ESC) lines with the described heterozygous modification. Our results showed we could eliminate proliferating cells in vitro and in vivo with dosing the prodrug ganciclovir (GCV). Elimination of these proliferating cells stopped the growth of teratomas generated by these ESCs and rendered this tissue dormant for up to one year. Despite the teratoma dormancy post-GCV treatment, we could detect GCV “escapees” among heterozygous cells, the majority of which were due to loss-of-heterozygosity (LOH). To avoid this, we generated cell lines with a homozygous CDK1 modification. Since we were unable to identify escapees in these lines in vitro, we used mathematical modelling to quantitate the risk of generating a GCV-resistant escapee. Furthermore, we demonstrated the homozygous modification of CDK1 does not compromise the differentiation capacity of the cells by evidence of viable homozygous mice and in vitro differentiation assay of modified hESCs. We also illustrated that GCV spares quiescent therapeutic cells. Lastly, we showed that our system is able to prevent tumour formation and eliminate formed tumours in a clinically relevant in vivo experiment. We are confident that our definition of safety, and the genome editing concept presented and characterized will provide a solution for cell therapy safety.