A Hybrid Vector System Expands Adeno-associated Viral Vector Packaging Capacity in a Transgene-independent Manner

The trans-splicing (ts) and overlapping (ov) vectors expand the packaging capacity of adeno-associated virus (AAV). But their application depends on the inherent properties of the target gene. The ts vectors require an optimal gene-splitting site and the ov vectors require a highly recombinogenic domain. In order to overcome these limitations, we developed a hybrid dual (hd) vector system. In the hd vectors, we inserted a highly recombinogenic alkaline phosphatase (AP) sequence in the ts vectors to allow for transgene-independent reconstitution through homologous recombination of the AP sequences. We first tested the hybrid system with the LacZ gene. Both in the cell line (in vitro) and in the mouse muscle (in vivo), the hd vectors significantly outperformed the ts and ov vectors. In muscle, the transduction efficiency of the hybrid vectors reached 80% of that from the single intact vector. Southern blot confirmed AP sequence-mediated transgene reconstitution. In order to validate the hybrid system, we split the 6 kilobase (kb) mini-dystrophin gene at the exon 55/56 junction, a predicted poor site for the ts approach. In dystrophic mdx mouse muscle, the hd vectors yielded 5.6-fold higher transduction than the ts vectors did. Taken together, these data suggest that the hybrid system efficiently expresses large therapeutic genes that are poor candidates for the ts and ov approaches.