Lentiviral rescue of UMPS auxotrophy enables drug-free selection and stable vector expression

Retroviral gene transfer remains a cornerstone of biomedical research and gene therapy, yet its effectiveness is occasionally compromised by low transduction efficiency, transgene silencing, and the reduced fitness of engineered cells. Although antibiotic-based selection systems can enrich transduced populations in vitro, their use in vivo is limited by safety and regulatory constraints. To overcome these challenges, we present a drug-free selection strategy based on uridine-5'-monophosphate synthase (UMPS) auxotrophy. Efficient UMPS disruption via CRISPR-Cas9 rendered cells dependent on uridine supplementation for survival and concomitantly enabled the selection of knockout cells based on their acquired resistance to the chemotherapeutic prodrug 5-fluoroorotic acid. Lentiviral reconstitution of UMPS restored uridine independence, and enabled the formation of >90% gene-marked cells within 7-14 days upon uridine removal. Likewise, splitting UMPS into its two functional domains-orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxylase (ODC)-for expression from separate vectors promoted the effective selection of double-transduced cells under auxotrophic conditions. Apart from robust cell enrichment, long-term stabilization of transgene-expressing cells without pharmacological intervention could be demonstrated and was superior to antibiotic selection. Our work thus establishes UMPS auxotrophy-rescue as a versatile platform for both in vitro and possibly in vivo applications, offering a safe and scalable alternative to conventional drug-based selection methods.