From bench to bedside: Improving the clinical safety of GalNAc–siRNA conjugates using seed-pairing destabilization
Mark K. Schlegel, Maja M. Janas, Yongfeng Jiang, Joseph D. Barry, Wendell Davis, Saket Agarwal, Daniel Berman, Christopher R. Brown, Adam Castoreno, Sarah LeBlanc, Abigail Liebow, Tara Mayo, Stuart Milstein, Tuyen Nguyen, Svetlana Shulga‐Morskaya, Sarah Hyde, Sally Schofield, John Szeto, Lauren Blair Woods, Vedat O. Yilmaz, Muthiah Manoharan, Martin Egli, Klaus Charissé, Laura Sepp‐Lorenzino, Patrick Haslett, Kevin Fitzgerald, Vasant Jadhav, Martin A. Maier
Abstract
Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc-siRNA conjugates. Here, we demonstrate that a single glycol nucleic acid or 2'-5'-RNA modification can substantially reduce small interfering RNA (siRNA) seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established hepatotoxicity driven by off-target effects, these novel designs with seed-pairing destabilization, termed enhanced stabilization chemistry plus (ESC+), demonstrated a substantially improved therapeutic window in rats. In contrast, siRNAs thermally destabilized to a similar extent by the incorporation of multiple DNA nucleotides in the seed region showed little to no improvement in rat safety suggesting that factors in addition to global thermodynamics play a role in off-target mitigation. We utilized the ESC+ strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient and asymptomatic alanine aminotransferase elevations in healthy volunteers. The redesigned ALN-HBV02 (VIR-2218) showed improved specificity with comparable on-target activity and the program was reintroduced into clinical development.