Antisense oligonucleotide (ASO) has emerged as a promising therapeutic approach for treating CNS disorders by modulating gene expression with high selectivity and specificity. However, the poor permeability of ASO across the blood-brain barrier (BBB) diminishes its therapeutic success. Here, we designed and synthesized a series of BBB-penetrating peptides (BPPs) derived from the receptor-binding domain of apolipoprotein E (ApoE) or transferrin receptor-binding peptide (THR). The BPPs were conjugated to a phosphorodiamidate morpholino oligomers (PMO), an FDA-approved ASO for the treatment of spinal muscular atrophy (SMA). The BPP-PMO conjugates significantly increased full-length SMN2 levels in patient-derived SMA fibroblasts in a concentration-dependent manner with minimal toxicity. Furthermore, the BPP-PMO conjugates were determined to increase the expression of full-length SMN2 in the brain and spinal cord of SMN2 transgenic adult mice. Notably, a retro-inverso (RI) ApoE-PMO conjugate (BPP8-PMO) showed a 1.25-fold increase in the expression of full-length functional SMN2 in mice brains, as confirmed by fluorescence imaging studies that demonstrated biodistribution throughout the CNS. Additionally, the P-PMO conjugates containing RI D-BPPs were found to possess extended half-lives compared to their L counterparts, indicating increased stability against protease degradation while preserving bioactivity. This delivery platform based on BPPs enhances the CNS bioavailability of PMO targeting the SMN2 gene, paving the way for the development of systemically administered neurotherapeutics for CNS disorders.
Keywords: BBB-penetrating peptides, endosomal escape, blood-brain barrier, antisense oligonucleotides, antisense therapy.