Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity

Abstract

Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity can be associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing using a thermally destabilizing chemical modification, which significantly improves the safety profile of a GalNAc-siRNA in rat and may minimize the occurrence of hepatotoxic siRNAs across species.

Fig. 1

Blocking RISC loading mitigates hepatotoxicity. a Structures of nucleotide analogs used at 5′-ends of siRNAs to prevent 5′-phosphorylation thus reducing RISC loading. b Liver exposures for parent (RNAi-active) and capped (RNAi-inactive) GalNAc-siRNAs in rat and mouse toxicity studies as assessed by stem-loop RT-qPCR for the antisense strand (AS) at necropsy (nx). Dashed vertical lines demarcate studies conducted separately. c Serum alanine aminotransferase (ALT) levels measured at necropsy. Differences between group means were evaluated for statistical significance using one way ANOVA with post hoc corrections (for multiple siRNAs) in GraphPad Prism 7. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars represent standard deviation of the mean. d H&E staining of liver sections collected at necropsy. In the rat, hepatotoxic siRNAs (siRNA-1 shown here) had hepatocellular degeneration (bracketed area), increased sinusoidal cells due to Kupffer cell hyperplasia and/or leukocyte infiltration (#), single cell necrosis (*), increased mitoses (^), and hepatocellular vacuolation (arrow). In the mouse, hepatotoxic siRNAs (siRNA-7 shown here) were associated with single-cell necrosis and lower incidence and severity of the other findings commonly seen in the rat. Capped RNAi-inactive siRNAs had minimal vacuolation or no histologic findings in both species. Cytoplasmic clearing present in the mice was consistent with glycogen due to incomplete fasting and was not considered test article-related. Microscopic liver findings for all tested siRNAs are tabulated in Supplementary Table 1. N = 3 males (6–8 weeks old) per group; qw, weekly dosing; q2d, every other day dosing

Fig. 2

Changing siRNA chemical modifications does not mitigate hepatotoxicity. a Chemical modification patterns of the high 2′F and low 2′F GalNAc-siRNAs with the same PS content and sequence. b Liver exposures in rat and mouse toxicity studies as assessed by stem-loop RT-qPCR for the antisense strand (AS) at necropsy (nx). c Liver RISC loading as assessed by stem-loop RT-qPCR for the antisense at necropsy. d Serum alanine aminotransferase (ALT) levels measured at necropsy. Differences between group means were evaluated for statistical significance using one way ANOVA with post hoc corrections (for multiple siRNAs) in GraphPad Prism 7. ns, not significant. Error bars represent standard deviation of the mean. e H&E staining of liver sections collected at necropsy. In the rat, both high 2′F and low 2′F siRNA-6 compounds were associated with hepatocellular degeneration (bracket), single cell necrosis (*), increased sinusoidal cells consistent with Kupffer cell hyperplasia and/or infiltrating leukocytes (#), and hepatocellular vacuolation (arrow). In the mouse, findings consisted of single-cell necrosis for both chemical modification patterns. All microscopic liver findings are tabulated in Supplementary Table 4. N = 3 males (6–8 weeks old) per group; qw weekly dosing

Fig. 3

Blocking antisense strand-loaded RISC activity mitigates hepatotoxicity. a Study design depicting prevention and treatment of rat toxicity by GalNAc-siRNAs using REVERSIR^TM. b Liver exposures for GalNAc-siRNAs in rat prevention (siRNA-1 and siRNA-4) or treatment (siRNA-5) toxicity studies as assessed by stem-loop RT-qPCR for the antisense strand (AS) at necropsy (nx). c Liver RISC loading with or without REVERSIR^TM treatment as assessed by stem-loop RT-qPCR for the antisense strand at necropsy. d Serum glutamate dehydrogenase (GLDH) levels measured at necropsy. Differences between group means were evaluated for statistical significance using one way ANOVA with post hoc corrections (for multiple siRNAs) in GraphPad Prism 7. ns. not significant; *p < 0.05; ****p < 0.0001. Error bars represent standard deviation of the mean. e H&E staining of liver sections collected at necropsy. Known toxic siRNAs administered alone or with a scrambled, control (Ctr) REVERSIR^TM were associated with hepatocellular degeneration (bracket), single cell necrosis (*), increased sinusoidal cells consistent with Kupffer cell hyperplasia and/or infiltrating leukocytes (#), increased mitoses (^), bile duct hyperplasia with fibrosis (+), and hepatocellular vacuolation (arrow). Co-administration of a complementary REVERSIR^TM decreased the severity of these findings and often limited their distribution. All microscopic liver findings are tabulated in Supplementary Table 5. N = 3 males (6–8 weeks old) per group; qw, weekly dosing; q2d, every other day dosing

Fig. 4

Swapping seed regions mitigates hepatotoxicity. a Chemical structures of seed swapping between a hepatotoxic and a non-hepatotoxic GalNAc-siRNA. b Liver exposures for parent and seed-swapped GalNAc-siRNAs in rat toxicity study as assessed by stem-loop RT-qPCR for the antisense strand (AS) at necropsy (nx). c Liver RISC loading as assessed by stem-loop RT-qPCR for the antisense strand at necropsy. d Serum alanine aminotransferase (ALT) levels measured at necropsy. Differences between group means were evaluated for statistical significance using one way ANOVA with post hoc corrections (for multiple siRNAs) in GraphPad Prism 7. ns, not significant; **p < 0.01, ***p < 0.001. Error bars represent standard deviation of the mean. e H&E staining of liver sections collected at necropsy. The toxic siRNA had hepatocellular degeneration (bracket), single cell necrosis (*), increased sinusoidal cells consistent with Kupffer cell hyperplasia and/or infiltrating leukocytes (#), and hepatocellular vacuolation (arrow), while the non-toxic siRNA had only minimal vacuolation. The non-toxic seed in the toxic backbone was comparable to the full non-toxic siRNA, and the toxic seed in the non-toxic backbone had single cell necrosis, increased sinusoidal cells and vacuolation but at a lower severity grade than the full-length toxic compound. Microscopic liver findings for all tested siRNAs are tabulated in Supplementary Table 6. N = 3 males (6–8 weeks old) per group; q2d, every other day dosing

Fig. 5

siRNA off-targets are enriched for seed complementarity in vitro and in vivo. a Volcano plots depicting global gene expression changes in rat hepatocytes at 24 h after transfection with 10 nM of GalNAc-siRNAs of four different sequences. Enrichment of the seed region complementarity in 3′UTRs is tabulated. N = 3 technical replicates. b Volcano plots depicting global gene expression changes in rat liver at 24 h after subcutaneous administration of GalNAc-siRNAs at 50 mg/kg. Two parent GalNAc-siRNAs and their RNAi-inactive versions blocked with inverted abasic (iB) caps are shown. Enrichment of the seed region complementarity in 3′UTRs is tabulated. Blue points, adjusted p ≤ 0.05; red points, adjusted p > 0.05; N = 3 males (6–8 weeks old) per group. The adjusted p-value for fold change was calculate in DESeq2 using the Wald test with multiple test correction. Seed enrichment p-value was calculated using the Fisher’s exact test. The variance was similar between groups that were statistically compared. iB, inverted abasic; NA, not applicable (p-values could not be calculated due to absence of downregulated genes)

Fig. 6

Destabilizing seed-mediated base-pairing minimizes off-target effects and mitigates hepatotoxicity. a Bad actor siRNA-5 containing a single thermally destabilizing glycol nucleic acid (GNA) nucleotide at position seven of the antisense strand. b Volcano plots depicting global gene expression changes in rat hepatocytes at 24 h after transfection with 10 nM of parent or GNA-modified GalNAc-siRNAs. N = 3 technical replicates. c Liver exposures for parent and seed-modified siRNA-5 in rat toxicity study as assessed by stem-loop reverse transcription-quantitative PCR (RT-qPCR) for the antisense strand (AS) at necropsy (nx). d Liver RISC loading as assessed by stem-loop RT-qPCR for the antisense strand at necropsy. e Serum glutamate dehydrogenase (GLDH) levels measured at necropsy. Differences between group means were evaluated for statistical significance using one way ANOVA with post hoc corrections (for multiple siRNAs) in GraphPad Prism 7. ns not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent standard deviation of the mean. f H&E staining of liver sections collected at necropsy. The toxic parent siRNA-5 had fibrosis (circle), hepatocellular degeneration (bracket), single cell necrosis (*), increased mitoses (^), increased sinusoidal cells consistent with Kupffer cell hyperplasia and/or infiltrating leukocytes (#), and hepatocellular vacuolation (arrow), while the non-toxic siRNA had only minimal vacuolation. The GNA-modified siRNA-5 had degeneration, single cell necrosis, increased mitoses, and vacuolation but at a lower incidence and severity grade than the parent siRNA-5. Microscopic liver findings for all tested siRNAs are tabulated in Supplementary Table 7. N = 4 males (6–8 weeks old) per group; qw weekly dosing, GNA glycol nucleic acid