15. Trans-Splicing Adeno-Associated Viral Vector-Mediated Gene Therapy Is Limited by Transcription across the ITR Junction but Not by Dual Vector Co-Infection Efficiency

Abstract

Adeno-associated virus (AAV) is one of the smallest DNA viruses. Therapeutic application of recombinant AAV has been limited by its small carrying capacity. Many large genes, such as the 6 kb mini-dystrophin gene for Duchenne muscular dystrophy (DMD), are traditionally excluded from rAAV gene therapy. To overcome the size limitation, we have recently developed trans-splicing AAV vectors. This approach takes advantage of AAV inverted terminal repeat (ITR)-mediated intermolecular recombination and the eukaryotic splicing machinery. A large gene is split into two parts and engineered with splicing signals. Following packaging and co-infection, the transgene is expressed from the reconstituted genome. Despite the success of the proof-of-principle studies, the overall transduction efficiency of trans-splicing vectors is considerably lower than that of a single intact vector in skeletal muscle. To improve trans-splicing vectors for DMD gene therapy, we examined whether co-infection efficiency is a rate-limiting factor for skeletal muscle in a mouse model for DMD (mdx mouse). Two different AAV viruses carrying the gene either for alkaline phosphatase or nuclear-localized LacZ were delivered to mdx anterior tibialis muscle. Consistent with previous reports in normal muscle, co-infection efficiency reached 89.2±2.0% in the diseased muscle. This result suggests that co-infection efficiency is not a hurdle for trans-splicing vector-mediated gene therapy in dystrophic muscle.