793. Efficient Expression of the 6 kb Mini-Dystrophin Gene by Trans-Splicing Adeno-Associated Viral (AAV) Vector Restores the Entire Dystrophin-Associated Glycoprotein Complex and Reduces Contraction-Induced Damage in the Mdx Mouse Model of Duchenne Muscular Dystrophy
Authors: Yi Lai, Yongping Yue, Mingju Liu, Arka Ghosh, John F. Engelhardt, Jeffrey S. Chamberlain, Dongsheng Duan
Journal: Molecular Therapy (2005)

Duchenne muscular dystrophy (DMD) is the most common lethal muscle disease caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) has been used to express the 3.8 kb C-terminal deleted micro-dystrophin gene for DMD gene therapy. Despite amelioration of some disease-associated changes, the microgene is not fully functional. Trans-splicing AAV vectors have been developed recently to express the 6 kb H2-R19 mini-dystrophin gene, the most competent gene besides the full-length gene. Our recent studies suggest that the RNA processing represents a critical barrier in trans-splicing AAV vector. In this study, we first screened a series of endogenous exon/intron/exon junctions by the RNase protection assay (RPA). The 56/56/57, 60/60/61 and 63/63/64 junctions hold the highest theoretic splicing values and may represent the most favorable sites to split the dystrophin gene. We generated synthetic mini-constructs carrying the splicing signals from each of above junctions. We also inserted the inverted terminal repeat junction in the middle of the intron. As a control, we included the 53/53/54 junction that has a low splicing value. The most efficient splicing was achieved in the 63/63/64 junction where the ratio of the spliced to unspliced RNA (S/U ratio) reached 7.1 0.9. The 60/60/61 and the 53/53/54 junctions yielded medium S/U ratios of 3.0 0.4 and 2.5 0.3, respectively. The 56/56/57 junction resulted in the lowest S/U ratio of 1.0 0.6. We also quantified the level of accumulated mRNA. Surprisingly, the highest mRNA was obtained from the 60/60/61 junction (255.1 20.6, relative unit), followed by the 63/63/64 (139.5 6.4), the 53/53/54 (48.8 20.6) and the 56/56/57 (0.8 0.2) junctions. We next generated two sets of AAV-6 trans-splicing vectors based on the 60/60/61 and the 63/63/64 junctions, respectively. 2 1010 particles of each set of vectors were delivered to the anterior tibialis (TA) muscle of 2-m-old mdx mice. Mini-dystrophin expression was quantified at 1 m post-infection by imunofluorescence staining. Consistent with the RPA results, trans-splicing vectors based on the 60/60/61 junction yielded 34 fold higher expression. To further evaluate the therapeutic potential, we delivered the 60/60/61 trans-splicing vectors (donor/acceptor co-infection, or donor or acceptor single infection) to both the TA and the extensor digitorium longus (EDL) muscles of the 2-m-old mdx mice. At 3 m post-infection, transduction efficiency in co-infected TA muscle reached 60-80%. Importantly, the entire dystrophin-associated glycoprotein complex (including dystroglycan, sarcoglycan, dystrobrevin and syntrophin) was restored in co-infected muscle. Furthermore, trans-splicing vector mediated mini-dystrophin expression provided significant protection against eccentric contraction induced injury in the EDL muscle.