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

Jicong Cao, Eva Maria Novoa, Zhizhuo Zhang, William C. W. Chen, Dianbo Liu, Gigi C. G. Choi, Alan S. L. Wong, Claudia Wehrspaun, Manolis Kellis () and Timothy K. Lu ()
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Jicong Cao: Massachusetts Institute of Technology
Eva Maria Novoa: Broad Institute of MIT and Harvard
Zhizhuo Zhang: Broad Institute of MIT and Harvard
William C. W. Chen: Massachusetts Institute of Technology
Dianbo Liu: Massachusetts Institute of Technology
Gigi C. G. Choi: Massachusetts Institute of Technology
Alan S. L. Wong: Massachusetts Institute of Technology
Claudia Wehrspaun: Massachusetts Institute of Technology
Manolis Kellis: Broad Institute of MIT and Harvard
Timothy K. Lu: Massachusetts Institute of Technology

Nature Communications, 2021, vol. 12, issue 1, 1-10

Abstract: 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.

Date: 2021
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DOI: 10.1038/s41467-021-24436-7

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