Overcoming GNA/RNA base-pairing limitations using isonucleotides improves the pharmacodynamic activity of ESC+ GalNAc-siRNAs

• Published in: Nucleic acids research Vol. 49; no. 19; pp. 10851 - 10867
• Main Authors: Schlegel, Mark K, Matsuda, Shigeo, Brown, Christopher R, Harp, Joel M, Barry, Joseph D, Berman, Daniel, Castoreno, Adam, Schofield, Sally, Szeto, John, Manoharan, Muthiah, Charissé, Klaus, Egli, Martin, Maier, Martin A
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 08.11.2021
• Subjects: Acetylgalactosamine; Adenosine - chemistry; Alcohol Oxidoreductases - antagonists & inhibitors; Alcohol Oxidoreductases - genetics; Alcohol Oxidoreductases - metabolism; Animals; Base Pairing; Chemical Biology and Nucleic Acid Chemistry; Chlorocebus aethiops; COS Cells; Cytidine - chemistry; Dimethylformamide - analogs & derivatives; Dimethylformamide - chemistry; Ethylamines - chemistry; Female; Glycols - chemistry; Guanosine - chemistry; Hepatocytes - cytology; Hepatocytes - metabolism; Hydrogen Bonding; Mice; Mice, Inbred C57BL; Oligoribonucleotides - chemistry; Oligoribonucleotides - genetics; Oligoribonucleotides - metabolism; Organophosphorus Compounds - chemistry; Prealbumin - antagonists & inhibitors; Prealbumin - genetics; Prealbumin - metabolism; Primary Cell Culture; RNA Stability; RNA, Double-Stranded - chemistry; RNA, Double-Stranded - genetics; RNA, Double-Stranded - metabolism; RNA, Small Interfering - chemistry; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkab916

Abstract We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc–siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity.