Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses
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, wi...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-06-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/13/10524 |
| _version_ | 1797591613582606336 |
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| author | Mariana V. Ferreira Sofia Fernandes Ana Isabel Almeida Salomé Neto João P. Mendes Ricardo J. S. Silva Cristina Peixoto Ana Sofia Coroadinha |
| author_facet | Mariana V. Ferreira Sofia Fernandes Ana Isabel Almeida Salomé Neto João P. Mendes Ricardo J. S. Silva Cristina Peixoto Ana Sofia Coroadinha |
| author_sort | Mariana V. Ferreira |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-11T01:39:50Z |
| format | Article |
| id | doaj.art-36e289fa08f7437a8e82a2bfb54094fa |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T01:39:50Z |
| publishDate | 2023-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-36e289fa08f7437a8e82a2bfb54094fa2023-11-18T16:39:28ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-06-0124131052410.3390/ijms241310524Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector DosesMariana V. Ferreira0Sofia Fernandes1Ana Isabel Almeida2Salomé Neto3João P. Mendes4Ricardo J. S. Silva5Cristina Peixoto6Ana Sofia Coroadinha7iBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, PortugaliBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, PortugaliBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, PortugaliBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, PortugaliBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, PortugaliBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, PortugaliBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, PortugaliBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, PortugalAdeno-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.https://www.mdpi.com/1422-0067/24/13/10524dual AAV vectorsplit-inteinsfull capsid enrichmentAAV co-transductiondual AAV-intein mediated systems |
| spellingShingle | Mariana V. Ferreira Sofia Fernandes Ana Isabel Almeida Salomé Neto João P. Mendes Ricardo J. S. Silva Cristina Peixoto Ana Sofia Coroadinha Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses International Journal of Molecular Sciences dual AAV vector split-inteins full capsid enrichment AAV co-transduction dual AAV-intein mediated systems |
| title | Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses |
| title_full | Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses |
| title_fullStr | Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses |
| title_full_unstemmed | Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses |
| title_short | Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses |
| title_sort | extending aav packaging cargo through dual co transduction efficient protein trans splicing at low vector doses |
| topic | dual AAV vector split-inteins full capsid enrichment AAV co-transduction dual AAV-intein mediated systems |
| url | https://www.mdpi.com/1422-0067/24/13/10524 |
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