Glycolate Oxidase Is a Safe and Efficient Target for Substrate Reduction Therapy in a Mouse Model of Primary Hyperoxaluria Type I  Cristina Martin-Higueras, Sergio Luis-Lima, Eduardo Salido  Molecular Therapy  Volume 24, Issue 4, Pages 719-725 (April 2016) DOI: 10.1038/mt.2015.224 Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Targeted mutagenesis of the mouse Hao1 locus. (a) Design of Hao1 gene exon 3 deletion by homologous recombination in ES cells. (b) Upper Western blot of 50-µg liver protein from Hao1+/+, Hao1+/−, and Hao1−/− mice probed with affinity-purified rabbit antibody raised against recombinant mouse glycolate oxidase (GO) shows lack of expression of the targeted allele and reduced levels in the heterozygous sample. Lower Reprobing of the blot with anti-glyceraldehyde-3-phosphate dehydrogenase detects even loading of the gel. (c). Left Western blot of wild-type (wt) mouse tissues (B: brain, H: heart, L: liver, K: kidney, T: testis) shows liver-specific expression of glycolate oxidase. Right No differences were found in GO expression between male and female mice. GAPDH, antiglyceraldehyde-3-phosphate dehydrogenase. Molecular Therapy 2016 24, 719-725DOI: (10.1038/mt.2015.224) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 A 24-h urine glycolate and oxalate excretion by different mouse genotypes. Data is represented as mean ± SD (n = 6 per group). ANOVA statistical signification: ***P<0.001, NS = nonsignificative. Molecular Therapy 2016 24, 719-725DOI: (10.1038/mt.2015.224) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Kinetics of the mouse glycolate oxidase (GO) inhibition by 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1,2,3-thiadiazole (CCPST). (a) Cornish-Bowden plot for the inhibition of mouse glycolate oxidase by CCPST. Increased inhibitor concentrations were tested at every glycolate (substrate) concentration and represented against glycolate/velocity (v). CCPST behaves as a noncompetitive inhibitor as all lines intersect on the x axis at the point X = −Ki = −91.2 µM. (b) Dose–response curve of mouse glycolate oxidase activity against CCPST concentration. Data are represented as mean ± SD. Discontinue lines represent 95% confidence interval; nonlinear regression analysis. Molecular Therapy 2016 24, 719-725DOI: (10.1038/mt.2015.224) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 In vitro response of mouse Agxt1−/− primary hepatocytes. (a) Oxalate excretion in Agxt1−/− hepatocytes treated with 5 mM glycolate compared with nontreated controls at 24, 48, and 72 hours. (b) Agxt1−/− hepatocytes viability after treatment with increased concentrations of 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1,2,3-thiadiazole (CCPST) in presence of 5 mM glycolate, measured by methyl thiazol tetrazolium reduction assay. (c) Graphic representation of the positive relationship between CCPST added to the medium and that detected in the intracellular extract. Simple linear regression analysis (r = 0.952, P < 0.001; R2 = 0.906, P < 0.001). (d) Relative amount of excreted oxalate by Agxt1−/− hepatocytes measured at 24, 48, and 72 hours post-treatment with increased concentrations of CCPST, and compared to the corresponding nontreated control. Data are represented as mean ± SD. ANOVA statistical signification: *P < 0.05, **P < 0.01, ***P < 0.001, NS = nonsignificative, relative to control at each time point. Molecular Therapy 2016 24, 719-725DOI: (10.1038/mt.2015.224) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 In vivo effects of 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1,2,3-thiadiazole (CCPST) treatment in PH1 mice. 24-h urine oxalate (a) and glycolate (b) excretion in Agxt1−/− mice after daily oral administration of CCPST in complex with β-cyclodextrin during 11 days. Significant reduction in oxalate levels from the first dose compared to basal oxaluria (P< 0.05). At the 10th and 11th dose, the oxalate levels significantly decrease with respect to the first dose (P< 0.01). Mean ± SD of three independent assays of six male mice. Paired t-test statistical analysis. (c) Glycolate oxidase (GO) specific activity measured in perfused liver after the 11th oral dose of CCPST-β-cyclodextrin (CD) in Agxt1−/− mice. Hao1-deficient mice (Hao1−/−) was used as control of nonenzymatic GO activity. Significant reduction in GO specific activity compared to nontreated Agxt1−/− mice (P< 0.001). Molecular Therapy 2016 24, 719-725DOI: (10.1038/mt.2015.224) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions