Comparison of U7snRNA-induced dystrophin expression following systemic delivery with AAV9 and AAVrh74 capsids

We have developed a U7snRNA vector that targets DMD exon 2 to result in exclusion of this exon from the mature mRNA. Preclinical studies resulted in significant expression of full-length dystrophin in our duplication of exon 2 (Dup2) mouse model, and the scAAV9.U7.ACCA vector is now in a clinical trial (NCT04240314). Here we assess efficiency of AAV9 and AAVrh74 capsids by delivery of this transgene at clinically relevant doses, as judged by RT-PCR and by dystrophin expression. Both vectors were intravenously delivered in a blinded fashion at a dose of 8E13 vg/kg to Dup2 mice at 10-12 weeks of age (n=6 mice per group), mice were euthanized 4 weeks later for analysis of mRNA splicing by RT-PCR, dystrophin expression by both quantitative immunofluorescence (IF) and western blot (WB) analyses, and vector genome biodistribution by qPCR. Biodistribution by transgene-specific qPCR showed no significant difference between capsids. In tibialis anterior (TA), Quadriceps (Quad) and Triceps Brachii (Tri) muscles, mRNA splicing efficiency by RT-PCR showed that AAV9-encapsidated vector outperformed AAVrh74-encapsidated vector, with AAV9 resulting in 74-85% total therapeutic transcript and AAVrh74 resulting in 43-48%. Protein expression in skeletal muscle was consistently better in Dup2 mice treated with AAV9.U7.ACCA. In TA, percent dystrophin-positive fibers (PDPF) by IF analysis were 57.8% for AAV9 and 35.3% for AAVrh74, while in diaphragm AAV9 showed 68.2% PDPF vs. AAVrh74 with 49.4%. In cardiac tissue, AAVrh74 appeared superior, with PDPF of 91.8% versus 79.4% for AAV9. These results suggest that robust target engagement for alteration of splicing can occur with either serotype, presenting an additional clinical avenue for therapeutic delivery. However, the AAV9 capsid ultimately results in greater skeletal muscle dystrophin expression. We have developed a U7snRNA vector that targets DMD exon 2 to result in exclusion of this exon from the mature mRNA. Preclinical studies resulted in significant expression of full-length dystrophin in our duplication of exon 2 (Dup2) mouse model, and the scAAV9.U7.ACCA vector is now in a clinical trial (NCT04240314). Here we assess efficiency of AAV9 and AAVrh74 capsids by delivery of this transgene at clinically relevant doses, as judged by RT-PCR and by dystrophin expression. Both vectors were intravenously delivered in a blinded fashion at a dose of 8E13 vg/kg to Dup2 mice at 10-12 weeks of age (n=6 mice per group), mice were euthanized 4 weeks later for analysis of mRNA splicing by RT-PCR, dystrophin expression by both quantitative immunofluorescence (IF) and western blot (WB) analyses, and vector genome biodistribution by qPCR. Biodistribution by transgene-specific qPCR showed no significant difference between capsids. In tibialis anterior (TA), Quadriceps (Quad) and Triceps Brachii (Tri) muscles, mRNA splicing efficiency by RT-PCR showed that AAV9-encapsidated vector outperformed AAVrh74-encapsidated vector, with AAV9 resulting in 74-85% total therapeutic transcript and AAVrh74 resulting in 43-48%. Protein expression in skeletal muscle was consistently better in Dup2 mice treated with AAV9.U7.ACCA. In TA, percent dystrophin-positive fibers (PDPF) by IF analysis were 57.8% for AAV9 and 35.3% for AAVrh74, while in diaphragm AAV9 showed 68.2% PDPF vs. AAVrh74 with 49.4%. In cardiac tissue, AAVrh74 appeared superior, with PDPF of 91.8% versus 79.4% for AAV9. These results suggest that robust target engagement for alteration of splicing can occur with either serotype, presenting an additional clinical avenue for therapeutic delivery. However, the AAV9 capsid ultimately results in greater skeletal muscle dystrophin expression.