Thymidylate synthase disruption to limit cell proliferation in cell therapies.

Abstract

Stem and progenitor cells hold great promise for regenerative medicine and gene therapy approaches. However, transplantation of living cells entails a fundamental risk of unwanted growth, potentially exacerbated by CRISPR-Cas9 or other genetic manipulations. Here, we describe a safety system to control cell proliferation while allowing robust and efficient cell manufacture, without any added genetic elements. Inactivating TYMS, a key nucleotide metabolism enzyme, in several cell lines resulted in cells that proliferate only when supplemented with exogenous thymidine. Under supplementation, TYMS^-/--pluripotent stem cells proliferate, produce teratomas, and successfully differentiate into potentially therapeutic cell types such as pancreatic β cells. Our results suggest that supplementation with exogenous thymidine affects stem cell proliferation, but not the function of stem cell-derived cells. After differentiation, postmitotic cells do not require thymidine in vitro or in vivo, as shown by the production of functional human insulin in mice up to 5 months after implantation of stem cell-derived pancreatic tissue.