{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,23]],"date-time":"2026-01-23T20:35:38Z","timestamp":1769200538064,"version":"3.49.0"},"reference-count":43,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2023,6,23]],"date-time":"2023-06-23T00:00:00Z","timestamp":1687478400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia\/Minist\u00e9rio da Ci\u00eancia, Tecnologia e Ensino Superior (FCT\/MCTES, Portugal)","award":["UIDB\/04462\/2020"],"award-info":[{"award-number":["UIDB\/04462\/2020"]}]},{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia\/Minist\u00e9rio da Ci\u00eancia, Tecnologia e Ensino Superior (FCT\/MCTES, Portugal)","award":["UIDP\/04462\/2020"],"award-info":[{"award-number":["UIDP\/04462\/2020"]}]},{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia\/Minist\u00e9rio da Ci\u00eancia, Tecnologia e Ensino Superior (FCT\/MCTES, Portugal)","award":["LA\/P\/0087\/2020"],"award-info":[{"award-number":["LA\/P\/0087\/2020"]}]},{"name":"the Associate Laboratory LS4FUTURE","award":["UIDB\/04462\/2020"],"award-info":[{"award-number":["UIDB\/04462\/2020"]}]},{"name":"the Associate Laboratory LS4FUTURE","award":["UIDP\/04462\/2020"],"award-info":[{"award-number":["UIDP\/04462\/2020"]}]},{"name":"the Associate Laboratory LS4FUTURE","award":["LA\/P\/0087\/2020"],"award-info":[{"award-number":["LA\/P\/0087\/2020"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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\u2019 performances by three-fold when compared to low-quality preparations (60\u201375% vs. 20\u201330% 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.<\/jats:p>","DOI":"10.3390\/ijms241310524","type":"journal-article","created":{"date-parts":[[2023,6,26]],"date-time":"2023-06-26T01:42:32Z","timestamp":1687743752000},"page":"10524","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8676-827X","authenticated-orcid":false,"given":"Mariana V.","family":"Ferreira","sequence":"first","affiliation":[{"name":"iBET-Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2781-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Sofia","family":"Fernandes","sequence":"additional","affiliation":[{"name":"iBET-Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2781-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Ana Isabel","family":"Almeida","sequence":"additional","affiliation":[{"name":"iBET-Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2781-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Salom\u00e9","family":"Neto","sequence":"additional","affiliation":[{"name":"iBET-Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2781-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Jo\u00e3o P.","family":"Mendes","sequence":"additional","affiliation":[{"name":"iBET-Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2781-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2959-102X","authenticated-orcid":false,"given":"Ricardo J. S.","family":"Silva","sequence":"additional","affiliation":[{"name":"iBET-Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2781-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Cristina","family":"Peixoto","sequence":"additional","affiliation":[{"name":"iBET-Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2781-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9728-6890","authenticated-orcid":false,"given":"Ana Sofia","family":"Coroadinha","sequence":"additional","affiliation":[{"name":"iBET-Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2781-901 Oeiras, Portugal"},{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2023,6,23]]},"reference":[{"key":"ref_1","unstructured":"US Food and Drug Administration (2023, April 08). 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