High-Throughput 5’ UTR Engineering for Enhanced Protein Production in Non-Viral Gene Therapies

Abstract

Despite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major challenge. One approach to increase protein expression is by optimizing translation through the engineering of 5’ untranslated regions (5’ UTRs). Here, we developed a high-throughput strategy to design, screen, and optimize novel 5’UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identified naturally occurring 5’ UTRs with high translation efficiencies and used this information with in silico genetic algorithms to generate synthetic 5’ UTRs. A total of ∼12,000 5’ UTRs were 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 identified three synthetic 5’ UTRs that outperformed commonly used non-viral gene therapy plasmids in expressing protein payloads. Furthermore, combinatorial assembly of these 5’ UTRs enabled even higher protein expression than obtained with each individual 5’ UTR. 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.

mRNA vaccines for COVID-19: what, why and how

“…New and improved methodologies will continue to be explored to optimize the stability and translation efficiency of mRNA and the delivery of LNP-mRNA complexes. Novel approaches, including deep learning and genome-wide screening method to identify the optimal codon usage and UTR design of mRNA are already being tested empirically [72,115]. Recent studies have screened a library of the total mRNA containing 5'-UTR using computational and empirical analyses and determined the optimal 5'-UTR for the maximum RNA stability and translation efficiency in vitro and in vivo [116,117].…”