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

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 approximate to 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.