Abstract
Recent advances in gapmer antisense oligonucleotides (ASOs) have led to the regulatory approval of ASO therapeutics that target the liver and central nervous system. Efficient delivery of gapmer ASOs to muscle tissue will further broaden their therapeutic applications. Here, we evaluated the knockdown activity of lipid-conjugated DNA/RNA heteroduplex oligonucleotides (HDOs) in muscle tissue. Cholesterol-conjugated HDO (Chol-HDO) exhibited effective gene suppression in both cardiac and skeletal muscles, outperforming unconjugated ASO and cholesterol-conjugated ASO (Chol-ASO). Chol-HDO and Chol-ASO showed greater binding to low-density lipoprotein (LDL); however, while Chol-ASO also exhibited enhanced binding to other serum proteins, Chol-HDO displayed weaker nonspecific interactions. Mechanistically, Chol-HDO enhanced cellular uptake through an LDL receptor-mediated mechanism. Moreover, subcellular distribution analysis revealed that the duplex structure of Chol-HDO promoted efficient nuclear delivery. Regarding safety, intravenous injection of Chol-ASO induced thrombocytopenia, whereas Chol-HDO mitigated this effect at the same dose. Ex vivo platelet activation assays confirmed that platelet activation was significantly more suppressed by Chol-HDO treatment than by Chol-ASO. Taken together, these results underscore the therapeutic potential of HDOs for safe and effective knockdown in muscle tissue, and provide a novel platform for the clinical development of gapmer ASO therapy for muscular disorders.