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Recombinant adeno-associated virus (rAAV) stands as a crucial in vivo gene therapy platform, valued for its high transduction efficiency and established safety. However, challenges such as immunogenicity and the transient nature of transgene expression impede its widespread application. A key area of research focuses on AAV capsid development, employing strategies such as isolating natural serotypes, rational design, directed evolution, and in silico design. 2-9 Engineered AAV capsids often incorporate modified AAV capsid genes with canonical amino acids or additional chemical decorations. A study recently published in “Molecular Therapy: Methods & Clinical Development” from Dr. Guangping Gao and Dr. Dan Wang’s team at the University of Massachusetts Chan Medical School explores genetic code expansion, incorporating non-canonical amino acids (ncAAs), for AAV5 vectors. 1 Some NAEK-AAV5 vectors exhibit enhanced transduction without ligand conjugation, particularly 374NAEK, suggesting promising AAV capsid engineering through genetic code expansion.

In this investigation, genetic code expansion was applied to introduce the non-canonical amino acid NAEK into specific sites of the AAV5 capsid during vector production. Twenty NAEK-AAV5 vector designs were generated, selecting ten amino acid residues for NAEK engineering across variable regions III to VIII. While NAEK modification generally compromised packaging yield, five top candidates were identified. Structural analysis confirmed solvent-facing incorporation of NAEK into AAV5 capsids. In vitro studies demonstrated that 374NAEK-AAV5 exhibited significantly higher transduction efficiency in various cell lines compared to rAAV5. In vivo experiments in mice revealed enhanced lung-specific transduction of 374NAEK-AAV5, both following systemic delivery and intranasal instillation. Also, immunostaining showed increased transduction of alveolar type 2 cells in the lung. Importantly, NAEK-AAV vectors, particularly 374NAEK-AAV5, were well-tolerated without triggering inflammatory responses or histological changes in the lung. Mechanistic studies suggested that the enhanced transduction of 374NAEK-AAV5 may be attributed to its unique cellular trafficking pattern and potential interactions between NAEK at residue 374 and nearby amino acids, inducing conformational changes near the 3-fold axis.

Various non-canonical amino acids (ncAAs) have been utilized to modify recombinant proteins for research purposes.10-17 In gene therapy vector development, the incorporation of ncAAs into the proteinaceous AAV capsid has primarily relied on NAEK, followed by ligand conjugation via click reaction. Unlike other methods involving chemical modifications, the study emphasizes a one-step ncAA incorporation approach, making it comparable to standard rAAV production. This method, based on genetic code expansion at a predefined termination codon, ensures precision and consistency in generating homogeneous batches of vectors, distinguishing it from two-step capsid modification methods that may be more complex and costly. This streamlined one-step method for non-canonical amino acid incorporation suggests a new approach for Capsid Engineering, yet still considers manufacturability critically, offering a much more simplified vector manufacturing process compared to other chemical modification approaches.

1. Chang H, Du A, Jiang J, Ren L, Liu N, Zhou X, Liang J, Gao G, Wang D, Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice, Molecular Therapy: Methods & Clinical Development (2023), [Link: https://doi.org/10.1016/j.omtm.2023.101129]
2. Li, C., and Samulski, R.J. (2020). Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet 21, 255-272. Journal Pre-proof 22
3. Gao, G.P., Alvira, M.R., Wang, L., Calcedo, R., Johnston, J., and Wilson, J.M. (2002). Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U S A 99, 11854-11859.
4. Yao, Y., Wang, J., Liu, Y., Qu, Y., Wang, K., Zhang, Y., Chang, Y., Yang, Z., Wan, J., Liu, J., et al. (2022). Variants of the adeno-associated virus serotype 9 with enhanced penetration of the blood-brain barrier in rodents and primates. Nat Biomed Eng 6, 1257-1271.
5. Zhang, C., Yao, T., Zheng, Y., Li, Z., Zhang, Q., Zhang, L., and Zhou, D. (2016). Development of next generation adeno-associated viral vectors capable of selective tropism and efficient gene delivery. Biomaterials 80, 134-145.
6. Yang, L., Jiang, J., Drouin, L.M., Agbandje-McKenna, M., Chen, C., Qiao, C., Pu, D., Hu, X., Wang, D.Z., Li, J., et al. (2009). A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection. Proc Natl Acad Sci U S A 106, 3946-3951.
7. Deverman, B.E., Pravdo, P.L., Simpson, B.P., Kumar, S.R., Chan, K.Y., Banerjee, A., Wu, W.L., Yang, B., Huber, N., Pasca, S.P., et al. (2016). Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol 34, 204-209.
8. Nonnenmacher, M., Wang, W., Child, M.A., Ren, X.Q., Huang, C., Ren, A.Z., Tocci, J., Chen, Q., Bittner, K., Tyson, K., et al. (2021). Rapid evolution of blood-brain-barrier-penetrating AAV capsids by RNA-driven biopanning. Mol Ther Methods Clin Dev 20, 366-378.
9. Tabebordbar, M., Lagerborg, K.A., Stanton, A., King, E.M., Ye, S., Tellez, L., Krunnfusz, A., Tavakoli, S., Widrick, J.J., Messemer, K.A., et al. (2021). Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Cell 184, 4919-4938 e4922
10. Zhang, C., Yao, T., Zheng, Y., Li, Z., Zhang, Q., Zhang, L., and Zhou, D. (2016). Development of next generation adeno-associated viral vectors capable of selective tropism and efficient gene delivery. Biomaterials 80, 134-145.
11. Kelemen, R.E., Mukherjee, R., Cao, X., Erickson, S.B., Zheng, Y., and Chatterjee, A. (2016). A Precise Chemical Strategy To Alter the Receptor Specificity of the Adeno-Associated Virus. Angew Chem Int Ed Engl 55, 10645-10649.
12. Katrekar, D., Moreno, A.M., Chen, G., Worlikar, A., and Mali, P. (2018). Oligonucleotide conjugated multi-functional adeno-associated viruses. Sci Rep 8, 3589.
13. Puzzo, F., Zhang, C., Powell Gray, B., Zhang, F., Sullenger, B.A., and Kay, M.A. (2023). Aptamer-programmable adeno-associated viral vectors as a novel platform for cell-specific gene transfer. Mol Ther Nucleic Acids 31, 383-397.
14. Seidel, L., Zarzycka, B., Zaidi, S.A., Katritch, V., and Coin, I. (2017). Structural insight into the activation of a class B G-protein-coupled receptor by peptide hormones in live human cells. Elife 6.
15. Horowitz, E.D., Weinberg, M.S., and Asokan, A. (2011). Glycated AAV vectors: chemical redirection of viral tissue tropism. Bioconjug Chem 22, 529-532.
16. Mevel, M., Bouzelha, M., Leray, A., Pacouret, S., Guilbaud, M., Penaud-Budloo, M., AlvarezDorta, D., Dubreil, L., Gouin, S.G., Combal, J.P., et al. (2019). Chemical modification of the adeno-associated virus capsid to improve gene delivery. Chem Sci 11, 1122-1131.
17. Liu, Y., Fang, Y., Zhou, Y., Zandi, E., Lee, C.L., Joo, K.I., and Wang, P. (2013). Site-specific modification of adeno-associated viruses via a genetically engineered aldehyde tag. Small 9, 421-429.

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