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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.

SUBMITTER: Cao J

PROVIDER: S-EPMC8260622 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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

Cao Jicong J Novoa Eva Maria EM Zhang Zhizhuo Z Chen William C W WCW Liu Dianbo D Choi Gigi C G GCG Wong Alan S L ASL Wehrspaun Claudia C Kellis Manolis M Lu Timothy K TK

Nature communications 20210706 1

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. ...[more]

PMID: 34230498

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Project description:High-density mesenchymal stem cell (MSC) aggregates can be guided to form bone-like tissue via endochondral ossification in vitro when culture media is supplemented with proteins, such as growth factors (GFs), to first guide the formation of a cartilage template, followed by culture with hypertrophic factors. Recent reports have recapitulated these results through the controlled spatiotemporal delivery of chondrogenic transforming growth factor-β1 (TGF-β1) and chondrogenic and osteogenic bone morphogenetic protein-2 (BMP-2) from microparticles embedded within human MSC aggregates to avoid diffusion limitations and the lengthy, costly in vitro culture necessary with repeat exogenous supplementation. However, since GFs have limited stability, localized gene delivery is a promising alternative to the use of proteins. Here, mineral-coated hydroxyapatite microparticles (MCM) capable of localized delivery of Lipofectamine-plasmid DNA (pDNA) nanocomplexes encoding for TGF-β1 (pTGF-β1) and BMP-2 (pBMP-2) were incorporated, alone or in combination, within MSC aggregates from three healthy porcine donors to induce sustained production of these transgenes. Three donor populations were investigated in this work due to the noted MSC donor-to-donor variability in differentiation capacity documented in the literature. Delivery of pBMP-2 within Donor 1 aggregates promoted chondrogenesis at week 2, followed by an enhanced osteogenic phenotype at week 4. Donor 2 and 3 aggregates did not promote robust glycosaminoglycan (GAG) production at week 2, but by week 4, Donor 2 aggregates with pTGF-β1/pBMP-2 and Donor 3 aggregates with both unloaded MCM and pBMP-2 enhanced osteogenesis compared to controls. These results demonstrate the ability to promote osteogenesis in stem cell aggregates through controlled, non-viral gene delivery within the cell masses. These findings also indicate the need to screen donor MSC regenerative potential in response to gene transfer prior to clinical application. Taken together, this work demonstrates a promising gene therapy approach to control stem cell fate in biomimetic 3D condensations for treatment of bone defects.

Dataset Information

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

Publications

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

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