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Overcoming GNA/RNA Base-Pairing Limitations Using Isonucleotides Improves the Pharmacodynamic Activity of ESC+ GalNAc-siRNAs

ABSTRACT: We report the impact of two pairs of GNA nucleotides, the native GNA-C and -G compared with the isomeric GNA-isoC and -isoG, on the on- and off-target activity of chemically modified siRNAs in primary mouse hepatocytes by RNA sequencing. We found that both GNA-C and -isoC mitigate the suppression of off-targets in a similar fashion, however, GNA-isoG was less efficacious in the mitigation of off-targets as the native GNA-G. This study informs the further design of more potent and specific chemically modified siRNAs for potential application in RNAi therapeutics.

ORGANISM(S): Mus musculus

PROVIDER: GSE183164 | GEO | 2021/09/24

REPOSITORIES: GEO

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

Project description: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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Overcoming GNA/RNA Base-Pairing Limitations Using Isonucleotides Improves the Pharmacodynamic Activity of ESC+ GalNAc-siRNAs

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Bisulphite-sequencing of chromatin immunoprecipitated DNA directly informs methylation status of histone-modified DNA

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