Cao, JicongNovoa, Eva MariaZhang, ZhizhuoChen, William C. W.Liu, DianboChoi, Gigi C. G.Wong, Alan S. L.Wehrspaun, ClaudiaKellis, ManolisLu, Timothy K.2022-06-072022-06-072021Cao J, Novoa EM, Zhang Z, Chen WCW, Liu D, Choi GCG et al. High-throughput 5' UTR engineering for enhanced protein production in non-viral gene therapies. Nat Commun. 2021 Jul 6;12(1):4138. DOI:10.1038/s41467-021-24436-72041-1723http://hdl.handle.net/10230/53394Despite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major technical challenge. Here, we develop a high-throughput strategy to design, screen, and optimize 5' UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identify naturally occurring 5' UTRs with high translation efficiencies and use this information with in silico genetic algorithms to generate synthetic 5' UTRs. A total of ~12,000 5' UTRs are then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identify three synthetic 5' UTRs that outperform commonly used non-viral gene therapy plasmids in expressing protein payloads. In summary, we demonstrate that high-throughput screening of 5' UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.application/pdfeng© Jicong Cao et al. 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were madeGenèticaTeràpia genèticaProteïnesinfo:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s41467-021-24436-7info:eu-repo/semantics/openAccess