The nucleobase guanine at the 3'-terminus of oligonucleotide RGLS4326 drives off-target AMPAR inhibition and CNS toxicity

  • Nat Commun. 2025 Nov 28;16(1):10762. doi: 10.1038/s41467-025-65799-5.
Tania Valencia  #  1 ,  Laura Y Yen  #  2  3 ,  Cindy Berman  4 ,  Thomas Vincent  1 ,  Scott Davis  1 ,  Francesca Varrone  1 ,  Jianfeng Huang  1 ,  Jessica Mastroianni  1 ,  Morgan Carlson  1 ,  Tate Owen  1 ,  Amin Kamel  1 ,  Denis Drygin  1 ,  Garth A Kinberger  1 ,  Shanti Pal Gangwar  3 ,  Maria V Yelshanskaya  3 ,  John Ridley  5 ,  Robert Kirby  5 ,  Jesus Alvarez  6 ,  Ronak Lakhia  6 ,  Vishal Patel  6 ,  Alexander I Sobolevsky  7 ,  Edmund C Lee  8
Affiliations
  • 1. Regulus Therapeutics Inc., San Diego, CA, USA.
  • 2. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
  • 3. Cellular and Molecular Physiology and Biophysics Graduate Program, Columbia University Irving Medical Center, New York, NY, USA.
  • 4. Berman Consulting, Wayland, MA, USA.
  • 5. Metrion Biosciences Ltd, Granta Park, Cambridge, UK.
  • 6. Department of Internal Medicine and Division of Nephrology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
  • 7. Cellular and Molecular Physiology and Biophysics Graduate Program, Columbia University Irving Medical Center, New York, NY, USA. [email protected].
  • 8. Regulus Therapeutics Inc., San Diego, CA, USA. [email protected].
  • # Contributed equally.
PMID: 41315228 DOI: 10.1038/s41467-025-65799-5
Abstract

Designing safe and effective oligonucleotide (ON) therapeutics requires thorough understanding of structural-activity relationship (SAR) with the intended on-target(s) as well as the unintended off-target(s). Despite encouraging pharmacodynamic activity in a Phase 1b study, development of the first-generation anti-miR-17 ON RGLS4326 for the treatment of autosomal dominant polycystic kidney disease was discontinued due to dose-limiting central nervous system (CNS)-related toxicity observed in nonclinical chronic toxicity studies. Here, we provide SAR evidence that the nucleobase guanine at the 3'-terminus of RGLS4326 drives an unexpected off-target aptamer-like direct interaction with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), thereby causing CNS toxicity. By replacing the 3'-terminal guanine with adenine, we discover the next-generation anti-miR-17 RGLS8429 that is devoid of off-target AMPAR interaction and CNS toxicity while preserving the potency against the on-target miR-17. Here, we show a way to avoid off-target CNS effects and, more importantly, data that support the clinical development of RGLS8429.

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