CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I

Abstract

CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1^-/- mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites.

Fig. 1

Efficient GO inhibition using CRISPR/Cas9 in PH1 animals. a Schematic representation of the CRISPR/Cas9-mediated SRT strategy targeting the Hao1 locus. b Editing efficiency measured by TIDE in 12–14-week-old PH1 animals 4 weeks after treatment with saline (n = 5), Cas9 (n = 5), Hao1-g1 (n = 5) and Hao1-g2 (n = 5). c Quantification of Hao1 mRNA expression levels by RT-qPCR in animals treated as in (b). Data are presented as mean ± SEM and Kruskal–Wallis statistical test was used to evaluate differences between groups. d Western blot analysis of GO protein levels in representative PH1 animals treated with saline, Cas9, Hao1-g1, and Hao1-g2. GAPDH was used as loading control. e Representative IHC images of liver sections stained for GO, from PH1 animals treated with saline, Cas9, Hao1-g1, and Hao1-g2. Scale bar: 100 μm. *p < 0.05

Fig. 2

Characterization of CRISPR/Cas9-mediated Hao1 gene editing. Deep sequencing was performed 1 month after treatment on the DNA from livers of 12–14-week-old PH1 animals treated with Hao1-g1 (n = 5) and Hao1-g2 (n = 5), as well as Cas9 (n = 3) and saline (n = 1). a Frequency of CRISPR/Cas9 introduced variants in the Hao1 gene of individual animals. b Characterization of the variants according to their type, size, and frameshift potential of each animal treated with Hao1-g1 and Hao1-g2. a, b Each bar represents an individual mouse. c Frequency distribution of indel size in base pairs (bp) for each animal treated with Hao1-g1 (green lines) and Hao1-g2 (orange lines). Each line represents an individual mouse

Fig. 3

Therapeutic efficacy of CRISPR/Cas9-mediated STR in PH1 animals. a Schematic experimental procedure, where 8–10-week-old PH1 animals were intravenously treated with saline (n = 7), Cas9 (n = 6), Hao1-g1 (n = 6) and Hao1-g2 (n = 5). A 7-days EG challenge was performed 4 months after vector administration, and 24 h urine samples were collected before and on days 3 and 7 of challenge. b Quantification of basal urine levels of oxalate and glycolate (µmol/24 h) 4 months after treatment. c, d Quantification of urine oxalate (c) and glycolate (d) levels (µmol/24 h) before and on days 3 and 7 of EG challenge. Data are presented as mean ± SEM and Kruskal–Wallis statistical test was used to compare the groups in each day. e Weight change of the animals during a one-week EG challenge. f Representative histological analysis of CaOx accumulation in the kidneys of PH1 animals from control or treatment groups after a 10-days EG challenge. Scale bar: 200 μm. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 4

Safety of the expression of CRISPR/Cas9 system. a Representative hematoxylin-eosin staining and CD45⁺ IHC of liver sections from 8–10-week-old PH1 animals sacrificed 4 months after treatment with saline (n = 7), Cas9 (n = 6), Hao1-g1 (n = 6) and Hao1-g2 (n = 5). Scale bar: 200 μm. b Quantification of CD45⁺ areas (%) of liver sections. (c, d) Serum ALT (U/L) (c) and bilirubin (mg/dL) (d) levels measured in animals sacrificed 6 months after treatment. Mean ± SEM of data are presented. Kruskal–Wallis test revealed no significant differences between groups. Dotted lines represent the range of normal reference values for ALT and bilirubin serum levels