Advancing antisense oligonucleotide delivery through click chemistry based chemical conjugation with designed short...

Duchenne muscular dystrophy (DMD) is a fatal X-linked neuromuscular disease caused by frame shift mutations in the gene encoding dystrophin. 2́-O-methyl phosphorothioate (2'-OMePS) serves as an antisense RNA platform clinically used in DMD patients to facilitate exon skipping and production of an internally truncated, yet functional dystrophin protein. Effective delivery and uptake of antisense oligonucleotides (ASOs) by target cells are crucial for their efficacy. Peptide-conjugated ASOs offer a promising next-generation platform, where a cell-penetrating peptide (CPP) is linked to the 2'-OMePS backbone to enhance cellular uptake. Herein, we designed and synthesized a new non-cationic short CPP sequence that can be efficiently conjugated with the negatively charged 2'-OMePS ASO backbone using click chemistry. Conjugation of the lead peptide ETWWK to 2'-OMePS ASO resulted in significant cellular internalization with precise nuclear localization of the ASO cargo. Cellular uptake was assessed in C2C12 and human DMD patient-derived myoblast cells via fluorescence microscopy and flow cytometry. Additionally, the synthesized ETWWK-ASO conjugate exhibits a significant 1.94 fold upregulation of dystrophin protein in the clinically relevant DMD patient-derived cell line. Our findings suggest that the identified peptide holds promise for facilitating ASO delivery at the site of splicing. This study highlights the efficient conjugation of CPPs to negatively charged 2'-OMePS ASO through tailored conjugation strategies, and will eventually be a therapeutic avenue for future ASO-based DMD treatments.