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Cited by 159Virus-based gene therapy researchRNA and protein synthesis mechanismsRNA Interference and Gene Delivery

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

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Cao, Jicong, Novoa, Eva Maria, Zhang, Zhizhuo, Chen, William C. W., Liu, Dianbo, Choi, Gigi C. G., Wong, Alan S. L., Wehrspaun, Claudia +2 more·2021·Nature Communications
DOI 10.1038/s41467-021-24436-7PMC PMC8260622

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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.

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  • Neo-2 has previously manifested high translational efficiency.

    “In addition, we used synthetic sequence Neo-2, which has previously manifested high translational efficiency too [35].”

    From Effects of Combinations of Untranslated-Region Sequences on Translation of mRNA (2023) · cites this paper as [35]

    The Results section ('Synthetic 5′ UTRs enhance protein expression levels in non-viral DNA therapies'; page unavailable) reports that NeoUTR2 increased GFP abundance and VEGF production. It does not report a measured translational efficiency for NeoUTR2; the paper distinguishes translational efficiency from protein expression, so the specific efficiency claim is not established. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • The synthetic 5′ UTR neoUTR3 shows enhanced protein production.

    “the synthetic 5′ UTR neoUTR3 showing enhanced protein production (UTR3).9”

    From Evaluation of synthetic mRNA with selected UTR sequences and alternative poly(A) tail, in vitro and in vivo (2025) · cites this paper as [9]

  • The number of studies assessing the contribution of certain UTRs to the expression of heterologous RNAs is rather small.

    “Of note, despite active research into the properties of UTRs, the number of studies assessing the contribution of certain UTRs to the expression of heterologous RNAs is rather small [34,35].”

    From Effects of Combinations of Untranslated-Region Sequences on Translation of mRNA (2023) · cites this paper as [35]

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Cited by

Sorted by citations

Authors
Cao, J., Novoa, E. M., Zhang, Z., Chen, W. C. W., Liu, D., Choi, G. C. G., Wong, A. S. L., Wehrspaun, C., Kellis, M., & Lu, T. K.
Affiliations
  • Broad Institute (US)
  • Massachusetts Institute of Technology (US)
Corresponding author
  • Kellis, Manolis
  • Lu, Timothy K.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2021-07-06
Volume
12
Issue
1
Pages
4138-4138
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-021-24436-7
PMC
PMC8260622
OpenAlex
W3180908419
Cited by
159
References
6
Citations by year
2026: 31 · 2025: 44 · 2024: 37 · 2023: 30 · 2022: 13 · 2021: 4
FWCI
12.85
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
  • Repositori digital de la UPF (Universitat Pompeu Fabra) (submittedVersion, cc-by)
Funders
  • U.S. Department of Health & Human Services | National Institutes of Health
  • United States Department of Defense | United States Army | U.S. Army Research, Development and Engineering Command | Army Research Office
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
protein expression optimization, human cytomegalovirus promoter, high-throughput screening, genetic algorithm, non-viral gene therapy, lentiviral vectors, translation efficiency
MeSH terms
Algorithms, Cell Line, Genetic Engineering, Humans, Plasmids, Promoter Regions, Genetic, Genetic Therapy, Gene Expression, 5' Untranslated Regions, Recombinases, High-Throughput Screening Assays, HEK293 Cells

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