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Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice.

ABSTRACT: Gene therapy using recombinant adeno-associated virus (rAAV) relies on safe, efficient, and precise in vivo gene delivery that is largely dependent on the AAV capsid. The proteinaceous capsid is highly amenable to engineering using a variety of approaches, and most resulting capsids carry substitutions or insertions comprised of natural amino acids. Here, we incorporated a non-canonical amino acid (ncAA), Nε-2-azideoethyloxycarbonyl-L-lysine (also known as NAEK), into the AAV5 capsid using genetic code expansion, and serendipitously found that several NAEK-AAV5 vectors transduced various cell lines more efficiently than the parental rAAV5. Furthermore, one NAEK-AAV5 vector showed lung-specific transduction enhancement following systemic or intranasal delivery in mice. Structural modeling suggests that the long side chain of NAEK may impact on the 3-fold protrusion on the capsid surface that plays a key role in tropism, thereby modulating vector transduction. Recent advances in genetic code expansion have generated synthetic proteins carrying an increasing number of ncAAs that possess diverse biological properties. Our study suggests that ncAA incorporation into the AAV capsid may confer novel vector properties, opening a new and complementary avenue to gene therapy vector discovery.

SUBMITTER: Chang H

PROVIDER: S-EPMC10597788 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice.

Chang Hao H Du Ailing A Jiang Jun J Ren Lingzhi L Liu Nan N Zhou Xuntao X Liang Jialing J Gao Guangping G Wang Dan D

Molecular therapy. Methods & clinical development 20231007

Gene therapy using recombinant adeno-associated virus (rAAV) relies on safe, efficient, and precise <i>in vivo</i> gene delivery that is largely dependent on the AAV capsid. The proteinaceous capsid is highly amenable to engineering using a variety of approaches, and most resulting capsids carry substitutions or insertions comprised of natural amino acids. Here, we incorporated a non-canonical amino acid (ncAA), Nε-2-azideoethyloxycarbonyl-L-lysine (also known as NAEK), into the AAV5 capsid usin ...[more]

PMID: 37886602

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Project description:Synthetic biology holds promise to revolutionize the life sciences and biomedicine via expansion of macromolecular diversity outside the natural chemical space. Use of non-canonical amino acids (ncAAs) via codon reassignment has found diverse applications in protein structure and interaction analysis, introduction of post-translational modifications, production of constrained peptides, antibody-drug conjugates, and novel enzymes. However, simultaneously encoding multiple ncAAs in vivo requires complex engineering and is sometimes restricted by the cell's poor uptake of ncAAs. In contrast the open nature of cell-free protein synthesis systems offers much greater freedom for manipulation and repurposing of the biosynthetic machinery by controlling the level and identity of translational components and reagents, and allows simultaneous incorporation of multiple ncAAs with non-canonical side chains and even backbones (N-methyl, D-, β-amino acids, α-hydroxy acids etc.). This review focuses on the two most used Escherichia coli-based cell-free protein synthesis systems; cell extract- and PURE-based systems. The former is a biological mixture with >500 proteins, while the latter consists of 38 individually purified biomolecules. We delineate compositions of these two systems and discuss their respective advantages and applications. Also, we dissect the translational components required for ncAA incorporation and compile lists of ncAAs that can be incorporated into polypeptides via different acylation approaches. We highlight the recent progress in using unnatural nucleobase pairs to increase the repertoire of orthogonal codons, as well as using tRNA-specific ribozymes for in situ acylation. We summarize advances in engineering of translational machinery such as tRNAs, aminoacyl-tRNA synthetases, elongation factors, and ribosomes to achieve efficient incorporation of structurally challenging ncAAs. We note that, many engineered components of biosynthetic machinery are developed for the use in vivo but are equally applicable to the in vitro systems. These are included in the review to provide a comprehensive overview for ncAA incorporation and offer new insights for the future development in cell-free systems. Finally, we highlight the exciting progress in the genomic engineering, resulting in E. coli strains free of amber and some redundant sense codons. These strains can be used for preparation of cell extracts offering multiple reassignment options.

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Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice.

Publications

Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice.

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