Jicong Cao, Eva Maria Novoa, Zhizhuo Zhang, William C. W. Chen, Dianbo Liu et al.
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.
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, Timothy K. Lu. High-throughput 5′ UTR engineering for enhanced protein production in non-viral gene therapies. 2021-07-06. https://doi.org/10.1038/s41467-021-24436-7
A 12,000-member library of 100-bp natural and computationally designed 5′ UTRs was cloned upstream of a GFP reporter and integrated as single copies into Bxb1 landing-pad HEK 293T cells. Two independently screened landing-pad cell lines were sorted into GFP-expression bins, and genomic amplicon sequencing with DESeq2 quantified UTR enrichment in the top 0–2.5%, 2.5–5%, and 5–10% bins relative to unsorted cells.
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