Perfluoroaryl Bicyclic Cell-Penetrating Peptides for Delivery of Antisense Oligonucleotides

Exon-skipping antisense oligonucleotides are effective treatments for genetic diseases, yet exon-skipping activity requires that these macromolecules reach the nucleus. While cell-penetrating peptides can improve delivery, proteolytic instability often limits efficacy. We hypothesized that bicyclization of arginine-rich peptides would improve their stability and their ability to deliver oligonucleotides into the nucleus. Here, we introduce two methods for the synthesis of arginine-rich bicyclic peptides using cysteine perfluoroarylation chemistry. Then, the bicyclic peptides are covalently linked to a phosphorodiamidate morpholino oligonucleotide (PMO) and assayed for exon skipping activity. The perfluoroaryl cyclic and bicyclic peptides improve PMO activity roughly 14-fold over the unconjugated PMO. The bicyclic peptides exhibited increased proteolytic stability relative to the monocycle, demonstrating that perfluoroaryl bicyclic peptides are potent and stable delivery agents.