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

Kanae Kawai1, Shinji Matsumoto2,3, Akikazu Harada2,4

  • 1Department of Physiology, Graduate School of Medicine, The University of Osaka, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

In Vitro Cellular & Developmental Biology. Animal
|March 6, 2026
PubMed

Insights

A new antisense oligonucleotide (ASO), ASO-2025-A/L, effectively targets ARL4C in cancer cells, inhibiting tumor growth with fewer off-target effects than previous therapies. This improved ARL4C ASO therapy shows promise for enhanced cancer treatment.

Area of Science:

  • Oncology
  • Molecular Biology
  • Pharmacology

Background:

  • ARL4C, a small GTP-binding protein, is frequently overexpressed in various cancer types.
  • Antisense oligonucleotide (ASO) therapy, such as the 15-mer ASO-1316-A, has shown potential in inhibiting tumor growth by targeting ARL4C.
  • Enhancing therapeutic efficacy and minimizing off-target effects are crucial for developing effective ASO treatments.

Purpose of the Study:

  • To develop and evaluate novel 18-mer ARL4C-targeting ASOs with improved efficacy and reduced off-target effects compared to ASO-1316-A.
  • To assess the in vitro and in vivo performance of the lead candidate ASO, ASO-2025-A/L, in relevant cancer models.
  • To investigate the impact of ASO-2025-A/L on ARL4C expression, cancer cell behavior, and potential off-target gene regulation.

Main Methods:

  • Development of fourteen 18-mer ARL4C-targeting ASOs.
  • In vitro assessment of ASO efficacy on cancer cell proliferation, migration, and adhesion (PANC-1, S2-CP8, PC-9 cells).
  • RNA-sequencing analysis to confirm gene expression changes and identify off-target effects.
  • In vivo evaluation of ASO-2025-A/L in xenograft tumor models (PC-9 and PANC-1 cells) and assessment of liver toxicity markers.

Main Results:

  • ASO-2025-A/L demonstrated superior suppression of ARL4C mRNA and protein compared to ASO-1316-A.
  • In vitro, ASO-2025-A/L significantly inhibited proliferation, migration, and adhesion of pancreatic and lung cancer cells.
  • ASO-2025-A/L showed a substantial reduction in predicted off-target genes (26 vs. 2824) and confirmed downregulation of cell adhesion pathway genes.
  • In vivo, ASO-2025-A/L effectively inhibited xenograft tumor growth without inducing liver toxicity.

Conclusions:

  • ASO-2025-A/L represents an improved 18-mer ARL4C-targeting ASO with enhanced therapeutic potential.
  • This novel ASO exhibits superior efficacy in inhibiting cancer cell functions and tumor growth.
  • ASO-2025-A/L offers a promising therapeutic strategy with a significantly reduced off-target effect profile.
Keywords:
ARL4CAnti-cancer drugAntisense oligonucleotideOff-target effect,

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