Safety evaluation of 2'-deoxy-2'-fluoro nucleotides in GalNAc-siRNA conjugates

  • Nucleic Acids Res. 2019 Apr 23;47(7):3306-3320. doi: 10.1093/nar/gkz140.
Maja M Janas  1 ,  Ivan Zlatev  1 ,  Ju Liu  1 ,  Yongfeng Jiang  1 ,  Scott A Barros  1 ,  Jessica E Sutherland  1 ,  Wendell P Davis  1 ,  Jingxuan Liu  1 ,  Christopher R Brown  1 ,  Xiumin Liu  1 ,  Mark K Schlegel  1 ,  Lauren Blair  1 ,  Xuemei Zhang  1 ,  Biplab Das  1 ,  Chris Tran  1 ,  Krishna Aluri  1 ,  Jing Li  1 ,  Saket Agarwal  1 ,  Ramesh Indrakanti  1 ,  Klaus Charisse  1 ,  Jayaprakash Nair  1 ,  Shigeo Matsuda  1 ,  Kallanthottathil G Rajeev  1 ,  Tracy Zimmermann  1 ,  Laura Sepp-Lorenzino  1 ,  Yuanxin Xu  1 ,  Akin Akinc  1 ,  Kevin Fitzgerald  1 ,  Akshay K Vaishnaw  1 ,  Peter F Smith  1 ,  Muthiah Manoharan  1 ,  Vasant Jadhav  1 ,  Jing-Tao Wu  1 ,  Martin A Maier  1
Affiliations
  • 1. Alnylam Pharmaceuticals, Inc., Cambridge, MA 02142, USA.
PMID: 30820542 DOI: 10.1093/nar/gkz140
Abstract

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2'-F-monomer metabolites was low and transient in rats and humans. In vitro, 2'-F-nucleoside 5'-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2'-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2'-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2'-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.

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