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

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• dc.contributor.author Cao, Jicong
• dc.contributor.author Novoa, Eva Maria
• dc.contributor.author Zhang, Zhizhuo
• dc.contributor.author Chen, William C. W.
• dc.contributor.author Liu, Dianbo
• dc.contributor.author Choi, Gigi C. G.
• dc.contributor.author Wong, Alan S. L.
• dc.contributor.author Wehrspaun, Claudia
• dc.contributor.author Kellis, Manolis
• dc.contributor.author Lu, Timothy K.
• dc.date.accessioned 2022-06-07T09:05:44Z
• dc.date.available 2022-06-07T09:05:44Z
• dc.date.issued 2021
• dc.description.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.
• dc.description.sponsorship The work was financially supported by Army Research Office (funding under the OSP account 6924758), Boston University (funding under the OSP account 6924758), HFSP to T.K.L., and NIH (R01 GM113708, R01 HG004037) to M.K.. E.M.N. thanks Human Frontier Science Program (LT000307/2013-L) for their financial support
• dc.format.mimetype application/pdf
• dc.identifier.citation 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
• dc.identifier.doi http://dx.doi.org/10.1038/s41467-021-24436-7
• dc.identifier.issn 2041-1723
• dc.identifier.uri http://hdl.handle.net/10230/53394
• dc.language.iso eng
• dc.publisher Nature Research
• dc.rights © 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 made
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri https://creativecommons.org/licenses/by/4.0/
• dc.subject.other Genètica
• dc.subject.other Teràpia genètica
• dc.subject.other Proteïnes
• dc.title High-throughput 5' UTR engineering for enhanced protein production in non-viral gene therapies
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion