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 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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Jul 06, 2021
- Source ID
- 10.1038/s41467-021-24436-7
Entities
People
- Alan S. L. Wong
- Claudia Wehrspaun
- Dianbo Liu
- Eva Maria Novoa Pardo
- Gigi C.G. Choi
- Jicong Cao
- Manolis Kellis
- Timothy K. Lu
- William C. W. Chen
- Zhizhuo Zhang
Organizations
- Army Research Office
- National Institutes of Health