Viral serotype and the transgene sequence influence overlapping adeno-associated viral (AAV) vector-mediated gene transfer in skeletal muscle.
BACKGROUND
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.


METHODS
To study serotype effect, we delivered AAV-2, -5 and -6 overlapping vectors (5 x 10(8) vg particles of the upstream and the downstream vectors, respectively) and 5 x 10(8) 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.


RESULTS
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.


CONCLUSIONS
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.
Ghosh
Arkasubhra
A
Department of Molecular Microbiology and Immunology, The University of Missouri, School of Medicine, Columbia, MO 65212, USA.
Yue
Yongping
Y
Duan
Dongsheng
D
D000256:Adenoviridae / Q000145:classification / Q000235:genetics*; D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D018014:Gene Transfer Techniques; D005807:Genes, Overlapping; D005822:Genetic Vectors; D066298:In Vitro Techniques; D007763:Lac Operon; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000502:physiology*; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000628:therapy; D010957:Plasmids; D012703:Serotyping; D014161:Transduction, Genetic; D019076:Transgenes
D000469:Alkaline Phosphatase