Potential of Cell-Penetrating Peptide-Conjugated Antisense Oligonucleotides for the Treatment of SMA

Abstract

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder that is caused by mutations in the survival motor neuron 1 (SMN1) gene, hindering the production of functional survival motor neuron (SMN) proteins. Antisense oligonucleotides (ASOs), a versatile DNA-like drug, are adept at binding to target RNA to prevent translation or promote alternative splicing. Nusinersen is an FDA-approved ASO for the treatment of SMA. It effectively promotes alternative splicing in pre-mRNA transcribed from the SMN2 gene, an analog of the SMN1 gene, to produce a greater amount of full-length SMN protein, to compensate for the loss of functional protein translated from SMN1. Despite its efficacy in ameliorating SMA symptoms, the cellular uptake of these ASOs is suboptimal, and their inability to penetrate the CNS necessitates invasive lumbar punctures. Cell-penetrating peptides (CPPs), which can be conjugated to ASOs, represent a promising approach to improve the efficiency of these treatments for SMA and have the potential to transverse the blood-brain barrier to circumvent the need for intrusive intrathecal injections and their associated adverse effects. This review provides a comprehensive analysis of ASO therapies, their application for the treatment of SMA, and the encouraging potential of CPPs as delivery systems to improve ASO uptake and overall efficiency.

Keywords: DG9; antisense oligonucleotides (ASOs); cell-penetrating peptides (CPPs); delivery; phosphorodiamidate morpholino oligomers (PMOs); spinal muscular atrophy (SMA).

Figure 1

Mechanism of action of ASOs through either RNase H-mediated degradation (a) or steric blockage (b). (a) ASOs bind to target RNA, and the RNA-ASO complex is recognized by RNase H, which subsequently degrades the target RNA. (b) ASOs bind to target RNA and, based on the region targeted, block translation or splicing machinery. Blocking splicing machinery results in alternative splicing of the pre-mRNA transcript. Blocking translation machinery prevents translation of the target RNA.

Figure 2

Nusinersen mechanism of action for the treatment of SMA. In healthy cells, SMN1 is processed primarily into full-length SMN mRNA, containing exon 7. Exon 7 is predominantly skipped in SMN2 pre-mRNA processing, resulting in a dysfunctional SMN protein. In SMA, no functional SMN protein is translated from SMN1. Nusinersen, an ASO targeting the ISS-N1 element, promotes the inclusion of exon 7 in SMN2 pre-mRNA processing to increase full-length SMN proteins.

Figure 3

Schematic representation of the mechanisms reported to facilitate the uptake of CPPs into cells. Energy-dependent pathways include receptor-mediated endocytosis and micropinocytosis, which result in the CPP being internalized in an endocytic vesicle for subsequent release into the cytoplasm. Energy-independent mechanisms include pore formation, inverted micelle formation, and the carpetlike model, which allow the CPP to enter the cell directly.