Mdx mice dosed with antisense to CD49d and dystrophin exon skip morpholino; reduced force loss and affected gene expression pathways support ATL1102 combination therapy in DMD patients

Background: VLA-4 is a disease progression biomarker in patients with Duchenne Muscular Dystrophy (DMD) including those treated with corticosteroids. ATL1102 an antisense oligonucleotide (ASO) inhibitor of human CD49d chain of adhesion molecule VLA-4 shows promising phase-2 results in non-ambulant DMD stabilizing upper limb function (PUL2.0), grip strength, and MRI muscle fat fraction, compared to worsening with corticosteroids. Methods: ASO to mouse CD49d (ISIS348574) was used in combination with a known mdx dystrophin morpholino exon-23 skipping restoration drug (PMO), dose-time chosen for low dystrophin restoration. Mdx mice were treated once weekly for 8 weeks with ASO, control mismatch oligonucleotide, saline, or PMO alone for 4 weeks, and PMO in combination with ASO or mismatch. Results: ASO+PMO demonstrated increased specific maximum force and eccentric muscle force retention in the exterior digitorum longus (EDL) muscle after 1-7 muscle eccentric contractions relative to saline. Area-under-curve force remaining after 8-10 contractions was higher for ASO+PMO versus PMO monotherapy and ASO monotherapy, PMO monotherapy versus saline, and the ASO monotherapy versus saline and mismatch. RNA-sequencing of quadriceps muscle evaluated gene expression changes, with false discovery rate (FDR) adjusted p-values up to <0.1. ASO monotherapy at FDR<0.05 showed 2 unique genes, Gm2a in immune neutrophil function and Trdn with Ryr2 muscle calcium channel function, versus mdx saline and none with MM. The lowest FDR PMO gene was another calcium channel Cacna1s, with PMO+MM FDR<0.05. ASO+PMO treatment at FDR<0.05 modulated 55 genes, 53 unique to the combination and 2* also in ASO FDR<0.06. Affected genes were involved in immune response (Btla*, Ppml1, Tnfsm13), lipolysis (Fabp4*, G0s2), fibrosis (Igfbp-7, Calu), muscle cell (Asb15) and muscle stem cell function (Adam10, Mt-Tp, Myom1). Conclusion: ASO+PMO EDL muscle function protection in mdx mice, and gene expression pathways affected, support the development of ATL1102 in combination therapy with conditionally approved morpholino dystrophin restoration drugs in DMD patients.