Thymidylate synthase disruption to limit cell proliferation in cell therapies.
Rocio Sartori-MaldonadoHossam MontaserInkeri SoppaSolja EurolaJuhana JuutilaMelanie BalazHenri PuttonenTimo OtonkoskiJonna Saarimäki-VireKirmo WartiovaaraPublished in: Molecular therapy : the journal of the American Society of Gene Therapy (2024)
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.
Keyphrases
- induced apoptosis
- cell proliferation
- cell cycle arrest
- crispr cas
- cell therapy
- living cells
- single cell
- endoplasmic reticulum stress
- type diabetes
- cell cycle
- gene therapy
- pluripotent stem cells
- pi k akt
- endothelial cells
- oxidative stress
- genome wide
- metabolic syndrome
- gene expression
- machine learning
- fluorescent probe
- copy number
- deep learning
- big data
- high fat diet induced
- artificial intelligence