Development of ARL4C antisense oligonucleotide with reduced off-target effects and enhanced efficacy as an anti-cancer drug.

ARL4C, a member of the small GTP-binding protein superfamily, is overexpressed in the tumor lesions of various cancers. Antisense oligonucleotide (ASO) therapy targeting ARL4C (ASO-1316-A), a 15-mer ASO, has been shown to inhibit xenograft tumor formation. To enhance therapeutic efficacy and reduce off-target effects, fourteen 18-mer ARL4C-targeting ASOs were developed. Among them, ASO-2025-A/L demonstrated superior suppression of ARL4C mRNA and protein expression compared to ASO-1316-A. In vitro, ASO-2025-A/L inhibited the proliferation, migration, and adhesion abilities of PANC-1 and S2-CP8 pancreatic cancer cells as well as PC-9 lung cancer cells more effectively than ASO-1316-A. ASO-2025-A/L was predicted to reduce the expression of 26 potential off-target genes, compared to 2824 potential off-target genes affected by ASO-1316-A in PANC-1 cells. While ASO-2025-A/L downregulated 317 genes, which were confirmed by RNA-sequence analysis, the majority was involved in cell adhesion pathways downstream of ARL4C signaling, and only two genes corresponded to off-target genes. In vivo, intravenous administration of ASO-2025-A/L inhibited xenograft tumor growth induced by PC-9 and PANC-1 cells, accompanied by a reduction in ARL4C mRNA levels in tumors without elevating serum liver toxicity markers. These findings highlight the development of an improved 18-mer ARL4C-targeting ASO with enhanced therapeutic efficacy and reduced off-target effects.