Thymidylate synthase disruption to limit cell proliferation in cell therapies

• DOI: 10.1016/j.ymthe.2024.06.014
• Publication Date: 2024-06-12T22:57:30Z
• Authors: Rocio Sartori-Mal...; Hossam Montaser; Inkeri Soppa; Solja Eurola; Juhana Juutila; Melanie Balaz; Henri Puttonen; Timo Otonkoski; Jonna Saarimäki-Vire; Kirmo Wartiovaara
• Keywords: 11832 Microbiology and virology; Pluripotent Stem Cells; Medical biotechnology; Cell- and Tissue-Based Therapy; Cell Differentiation; Thymidylate Synthase; Cell Line; Mice; Insulin-Secreting Cells; Humans; Animals; Original Article; CRISPR-Cas Systems; Cell Proliferation; Thymidine

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.