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A hybrid dual-vector system was developed recently as a universal platform to double the packaging capacity of recombinant adeno-associated virus (AAV). In this system, the expression cassette is split into two independent AAV vectors. A highly recombinogenic bridging DNA sequence is engineered in both vectors to mediate target gene-independent homologous recombination between the split vector genomes. In the prototype hybrid vectors, a 0.87-kb DNA fragment from the middle portion of the human placental alkaline phosphatase (AP) gene was used as the bridging sequence. Here we report the development of the minimized bridging sequences. Five independent bridging sequences (0.26 to 0.44 kb) were evaluated in MO59K cells and/or murine skeletal muscle in the context of the AP overlapping vectors and/or the β-galactosidase (LacZ) hybrid vectors. Robust reconstitution comparable to that of the …