An Investigational RNAi Therapeutic Targeting Glycolate Oxidase Reduces Oxalate Production in Models of Primary Hyperoxaluria

Abstract

Primary hyperoxaluria type 1 (PH1), an inherited rare disease of glyoxylate metabolism, arises from mutations in the enzyme alanine-glyoxylate aminotransferase. The resulting deficiency in this enzyme leads to abnormally high oxalate production resulting in calcium oxalate crystal formation and deposition in the kidney and many other tissues, with systemic oxalosis and ESRD being a common outcome. Although a small subset of patients manages the disease with vitamin B6 treatments, the only effective treatment for most is a combined liver-kidney transplant, which requires life-long immune suppression and carries significant mortality risk. In this report, we discuss the development of ALN-GO1, an investigational RNA interference (RNAi) therapeutic targeting glycolate oxidase, to deplete the substrate for oxalate synthesis. Subcutaneous administration of ALN-GO1 resulted in potent, dose-dependent, and durable silencing of the mRNA encoding glycolate oxidase and increased serum glycolate concentrations in wild-type mice, rats, and nonhuman primates. ALN-GO1 also increased urinary glycolate concentrations in normal nonhuman primates and in a genetic mouse model of PH1. Notably, ALN-GO1 reduced urinary oxalate concentration up to 50% after a single dose in the genetic mouse model of PH1, and up to 98% after multiple doses in a rat model of hyperoxaluria. These data demonstrate the ability of ALN-GO1 to reduce oxalate production in preclinical models of PH1 across multiple species and provide a clear rationale for clinical trials with this compound.

Keywords: RNAi therapeutics; end-stage renal disease; oxalate; primary hyperoxaluria type I; siRNA.

Figure 1.

Hepatic oxalate synthesis pathway. ALN-GO1 targets hepatic GO. Copyright clearance center license number 3863190482947.

Figure 2.

Silencing liver HAO1 mRNA results in increased serum glycolate in healthy rodents. Levels of (A) liver HAO1 mRNA and (B) serum glycolate 10 days after a single subcutaneous dose of ALN-GO1 in C57BL/6 mice. Levels of (C) liver HAO1 mRNA and (D) serum glycolate 10 days after a single subcutaneous dose of ALN-GO1 in Sprague–Dawley rats. Bars represent the mean of three or four animals and error bars depict the SD. Stars indicate significance of Dunnett multiple comparison tests of each dose level versus PBS (after one-way ANOVA; all treatment effects significant at P<0.001): **P<0.01; ***P<0.001.

Figure 3.

A single dose of ALN-GO1 results in sustained liver HAO1 mRNA silencing. Liver HAO1 mRNA at various time points following a single subcutaneous dose of PBS or 3 mg/kg ALN-GO1 in C57BL/6 mice. Points represent the mean of three animals and error bars depict the SD. Overall difference in HAO1 mRNA over time between PBS and 3 mg/kg groups was assessed via repeated ANOVA measures; treatment and treatment×time interactions were both significant at P<0.001 and P<0.001, respectively. Stars indicate significance level of Tukey post hoc comparisons of mean HAO1 mRNA in PBS versus 3 mg/kg groups at each day: *P<0.05; ***P<0.001. Bracket below stars indicates that all points in span share the noted significance level.

Figure 4.

Silencing liver HAO1 mRNA leads to increased serum glycolate in cynomolgus monkeys. Levels of (A) liver HAO1 mRNA and (B) serum glycolate in healthy nonhuman primates treated monthly with PBS or ALN-GO1 at 1, 2, or 4 mg/kg. Points represent individual animals (mRNA) or group averages (glycolate) of n=3 animals/group and error bars depict the SD. Arrows indicate dosing days. For HAO1 mRNA (A), stars indicate significance of Tukey post hoc tests of each group daily mean versus corresponding PBS daily mean (after repeated ANOVA measures; treatment, time, and treatment×time interaction all significant at P<0.001): *P<0.05; **P<0.01; ***P<0.001; ****P<0.001. For glycolate (B), stars indicate significance of Tukey post hoc tests after repeated ANOVA measures (treatment, time, and treatment×time interaction all significant at P<0.001): the stars above the data points indicate pairwise comparisons of each group daily mean versus corresponding PBS daily mean; the stars below the data points indicate pairwise comparisons of each group daily mean versus baseline levels (average of predose values). Colors indicating 4, 2, and 1 mg/kg as in legend: *P<0.05; **P<0.01; ***P<0.001. Bracket above/below stars indicates that all points in span share the noted significance level(s).

Figure 5.

Urinary oxalate lowering and serum glycolate increases following ALN-GO1 dosing in PH1 mice. (A) Twenty-four-hour urine oxalate and (B) 24-hour urine glycolate levels in male Agxt1^−/− mice after a single subcutaneous dose of ALN-GO1 at the concentrations shown. Points represent the mean of three animals, error bars depict the SD. Differences among treatments over time were assessed via repeated ANOVA measures (one model for each analyte). For both oxalate and glycolate, treatment, time, and treatment×time interactions were significant at P<0.01, P<0.001, and P<0.001, respectively. Stars indicate significance of Tukey post hoc tests of each weekly mean versus week 0 mean, with colors indicating 3, 1, and 0.3 mg/kg according to legend: *P<0.05; **P<0.01; ***P<0.001.

Figure 6.

Silencing liver HAO1 mRNA reduces urinary oxalate in a rat model of hyperoxaluria. Levels of (A) liver HAO1 mRNA on day 14 and (B) 24-hour urinary oxalate levels at multiple time points after a single subcutaneous dose of ALN-GO1 in hyperoxaluric rats that received 1 mg/kg AGXT targeting siRNA and 1% ethylene glycol in drinking water. Points and bars represent the mean of three animals and error bars depict the SD. For HAO1 mRNA (A), stars indicate significance of Dunnett multiple comparison tests of each dose level versus PBS (after one-way ANOVA; overall treatment effect significant at P<0.001): *P<0.05; **P<0.01; ***P<0.001. For oxalate (B), stars indicate significance of Tukey post hoc tests of each group daily mean versus corresponding PBS daily mean (after repeated measures ANOVA; treatment, time, and treatment×time interaction all significant at P<0.001), with colors indicating 0.3, 0.1, 0.03, and 0.01 mg/kg as in legend: *P<0.05; **P<0.01; ***P<0.001.

Figure 7.

Linear relationship between HAO1 mRNA silencing and urinary oxalate lowering in hyperoxaluric rats. Percent liver HAO1 mRNA remaining versus percent urinary oxalate remaining on day 14 or 28 in hyperoxaluric rats treated with PBS or doses of ALN-GO1 ranging from 0.01 to 3 mg/kg. Points represent individual hyperoxaluric rats normalized to vehicle control. R², equation, and P value from linear regression.