The overlapping approach was developed recently to expand the adeno‐associated viral (AAV) packaging capacity. In this approach, a gene is split into two partially overlapping fragments and separately packaged into an upstream and a downstream vector, respectively. Transgene expression is achieved in co‐infected cells after homologous recombination. Despite the promising proof‐of‐principle results in the lung, the efficiency has been very disappointing in skeletal muscle. Here we examined two potential rate‐limiting factors including AAV serotype and the transgene sequence.

To study serotype effect, we delivered AAV‐2, ‐5 and ‐6 overlapping vectors (5 × 10⁸ vg particles of the upstream and the downstream vectors, respectively) and 5 × 10⁸ vg particles of the intact gene vector to the tibialis anterior muscles of 7‐week‐old C57Bl/6 mice, respectively. To determine the effect of transgene sequence, we compared LacZ and alkaline phosphatase (AP) overlapping vectors. Transduction efficiency was quantified 6 weeks later by scoring the percentage of transgene‐positive myofibers.

AAV‐2 overlapping vectors barely resulted in detectable transduction. Transduction efficiency was significantly improved in AAV‐5 and AAV‐6. The highest level was achieved in AAV‐6 that reached 42% and 96% of that of the intact gene vector for the LacZ gene and the AP gene, respectively. Surprisingly, AAV‐6 overlapping vector resulted in higher transduction than did AAV‐2 and AAV‐5 intact gene vectors.

Our findings suggest that AAV serotype and the transgene sequence play critical roles in the overlapping approach. AAV‐6 holds great promise for overlapping vector‐mediated muscle gene therapy. Copyright © 2005 John Wiley & Sons, Ltd.