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

Abstract

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.