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

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.

doi:10.1093/nar/gkab916

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

Journal Article · 14 October 2021 · Nucleic Acids Research

DOI: https://doi.org/10.1093/nar/gkab916 · OSTI ID:1826226

^[1]; Matsuda, Shigeo ^[1];

^[1]; Harp, Joel M. ^[2]; Barry, Joseph D. ^[1]; Berman, Daniel ^[1]; Castoreno, Adam ^[1]; Schofield, Sally ^[1]; Szeto, John ^[1];

^[1]; Charissé, Klaus ^[1];

^[2]; Maier, Martin A. ^[1]

Alnylam Pharmaceuticals, Inc., Cambridge, MA 02142, USA
Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN 37232, USA

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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Sponsoring Organization:: USDOE

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1826226

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 19 Vol. 49; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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