| Autor: |
Ferreira MV; iBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal., Fernandes S; iBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal., Almeida AI; iBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal., Neto S; iBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal., Mendes JP; iBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal., Silva RJS; iBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal., Peixoto C; iBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal., Coroadinha AS; iBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal. |
| Abstrakt: |
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. |
