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

  • Nucleic Acids Res. 2021 Nov 8;49(19):10851-10867. doi: 10.1093/nar/gkab916.
Mark K Schlegel  1 ,  Shigeo Matsuda  1 ,  Christopher R Brown  1 ,  Joel M Harp  2 ,  Joseph D Barry  1 ,  Daniel Berman  1 ,  Adam Castoreno  1 ,  Sally Schofield  1 ,  John Szeto  1 ,  Muthiah Manoharan  1 ,  Klaus Charissé  1 ,  Martin Egli  2 ,  Martin A Maier  1
Affiliations
  • 1. Alnylam Pharmaceuticals, Inc., Cambridge, MA 02142, USA.
  • 2. Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN 37232, USA.
PMID: 34648028 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.

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