High-throughput 5' UTR engineering for enhanced protein production in non-viral gene therapies

Cao, Jicong; Novoa, Eva Maria; Zhang, Zhizhuo; Chen, William C. W.; Liu, Dianbo; Choi, Gigi C. G.; Wong, Alan S. L.; Wehrspaun, Claudia; Kellis, Manolis; Lu, Timothy K.

doi:http://dx.doi.org/10.1038/s41467-021-24436-7

High-throughput 5' UTR engineering for enhanced protein production in non-viral gene therapies

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Cao 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/s41467-021-24436-7

Abstract

Despite 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.