Background: Peptide-conjugated PMOs (PPMOs) are a next-generation chemistry platform in which a cell-penetrating peptide is conjugated to the PMO backbone, with the goal of increasing cellular uptake, exon skipping, and dystrophin production. Tissue inhibitor of metalloproteinases-1 (TIMP-1) and creatine kinase muscle type (CKM) serum levels are elevated in patients with DMD and in the mdx mouse model of DMD and correlate with disease severity.
Objective: We assess the utility of TIMP-1 and CKM as serum biomarkers of response to PPMO in mdx mice.
Methods: Mdx mice were injected intravenously every 4 weeks (Q4W) for 24 weeks with vehicle or RC-1001, a PPMO targeting the mdx mutation (exon 23), at doses of 20, 40 or 80 mg/kg (N=6 mice per group). Exon 23 skipping and dystrophin protein expression were assessed by droplet digital polymerase chain reaction and western blot respectively. Grip strength was also measured. TIMP-1 and matrix metallopeptidase 9 (MMP9) serum levels were measured by ELISA; CKM serum levels were measured by Meso Scale Discovery electrochemiluminescence assay.
Results: Single dose of RC-1001 resulted in sustained concentration-dependent exon 23 skipping and dystrophin protein increase up to 24 weeks with Q4W dosing; grip strength also improved throughout the study. Single dose RC-1001 40 mg/kg significantly decreased TIMP-1 (<2000 pg/ml; P < 0.0001) and CKM (<10 ?g/ml; P = 0.044) levels but not MMP9, demonstrating biomarker response specificity. Q4W dosing resulted in sustained concentration-dependent decrease in TIMP-1 and CKM levels, but not MMP9, throughout 24 weeks. Increased grip strength correlated in a dose-dependent manner with decreased TIMP-1 (r = -0.74; P = 0.0003) and CKM (r = -0.69; P = 0.0007) levels as early as after one administration of the PPMO.
Conclusions: These data support further investigation of TIMP-1 and CKM as potential noninvasive biomarkers of therapeutic response for exon-skipping therapies for DMD.