TY - JOUR
T1 - Extending AAV Packaging Cargo through Dual Co-Transduction
T2 - Efficient Protein Trans-Splicing at Low Vector Doses
AU - Ferreira, Mariana V.
AU - Fernandes, Sofia
AU - Almeida, Ana Isabel
AU - Neto, Salomé
AU - Mendes, João P.
AU - Silva, Ricardo J.S.
AU - Peixoto, Cristina
AU - Coroadinha, Ana Sofia
N1 - Funding Information:
Author Mariana V. Ferreira acknowledges Fundação para a Ciência e Tecnologia for PH.D. fellowship UI/BD/151256/2021 within the scope of the Ph.D. Program in Bioengineering—Cell Therapies and Regenerative Medicine. This work was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associate Laboratory LS4FUTURE (LA/P/0087/2020).
Publisher Copyright:
© 2023 by the authors.
PY - 2023/7
Y1 - 2023/7
N2 - 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.
AB - 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.
KW - AAV co-transduction
KW - dual AAV vector
KW - dual AAV-intein mediated systems
KW - full capsid enrichment
KW - split-inteins
UR - http://www.scopus.com/inward/record.url?scp=85164843969&partnerID=8YFLogxK
U2 - 10.3390/ijms241310524
DO - 10.3390/ijms241310524
M3 - Article
C2 - 37445701
AN - SCOPUS:85164843969
SN - 1661-6596
VL - 24
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 13
M1 - 10524
ER -