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Article

Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses

by
Mariana V. Ferreira
1,2,
Sofia Fernandes
1,2,
Ana Isabel Almeida
1,2,
Salomé Neto
1,2,
João P. Mendes
1,2,
Ricardo J. S. Silva
1,2,
Cristina Peixoto
1,2 and
Ana Sofia Coroadinha
1,2,*
1
iBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal
2
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(13), 10524; https://doi.org/10.3390/ijms241310524
Submission received: 19 May 2023 / Revised: 16 June 2023 / Accepted: 19 June 2023 / Published: 23 June 2023
(This article belongs to the Special Issue Virus Engineering and Applications)
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Abstract

Adeno-associated viral (AAV) vectors represent one of the leading platforms for gene delivery. Nevertheless, their small packaging capacity restricts their use for diseases requiring large-gene delivery. To overcome this, dual-AAV vector systems that rely on protein trans-splicing were developed, with the split-intein Npu DnaE among the most-used. However, the reconstitution efficiency of Npu DnaE is still insufficient, requiring higher vector doses. In this work, two split-inteins, Cfa and Gp41-1, with reportedly superior trans-splicing were evaluated in comparison with Npu DnaE by transient transfections and dual-AAV in vitro co-transductions. Both Cfa and Gp41-1 split-inteins enabled reconstitution rates that were over two-fold higher than Npu DnaE and 100% of protein reconstitution. The impact of different vector preparation qualities in split-intein performances was also evaluated in co-transduction assays. Higher-quality preparations increased split-inteins’ performances by three-fold when compared to low-quality preparations (60–75% vs. 20–30% full particles, respectively). Low-quality vector preparations were observed to limit split-gene reconstitutions by inhibiting co-transduction. We show that combining superior split-inteins with higher-quality vector preparations allowed vector doses to be decreased while maintaining high trans-splicing rates. These results show the potential of more-efficient protein-trans-splicing strategies in dual-AAV vector co-transduction, allowing the extension of its use to the delivery of larger therapeutic genes.
Keywords: dual AAV vector; split-inteins; full capsid enrichment; AAV co-transduction; dual AAV-intein mediated systems dual AAV vector; split-inteins; full capsid enrichment; AAV co-transduction; dual AAV-intein mediated systems
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MDPI and ACS Style

Ferreira, M.V.; Fernandes, S.; Almeida, A.I.; Neto, S.; Mendes, J.P.; Silva, R.J.S.; Peixoto, C.; Coroadinha, A.S. Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses. Int. J. Mol. Sci. 2023, 24, 10524. https://doi.org/10.3390/ijms241310524

AMA Style

Ferreira MV, Fernandes S, Almeida AI, Neto S, Mendes JP, Silva RJS, Peixoto C, Coroadinha AS. Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses. International Journal of Molecular Sciences. 2023; 24(13):10524. https://doi.org/10.3390/ijms241310524

Chicago/Turabian Style

Ferreira, Mariana V., Sofia Fernandes, Ana Isabel Almeida, Salomé Neto, João P. Mendes, Ricardo J. S. Silva, Cristina Peixoto, and Ana Sofia Coroadinha. 2023. "Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses" International Journal of Molecular Sciences 24, no. 13: 10524. https://doi.org/10.3390/ijms241310524

APA Style

Ferreira, M. V., Fernandes, S., Almeida, A. I., Neto, S., Mendes, J. P., Silva, R. J. S., Peixoto, C., & Coroadinha, A. S. (2023). Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses. International Journal of Molecular Sciences, 24(13), 10524. https://doi.org/10.3390/ijms241310524

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