Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats

Abstract

Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutation of fumarylacetoacetate hydrolase (FAH). Because of the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. HTI is an ideal model for gene therapy, and several strategies have been shown to ameliorate HTI symptoms in animal models. Although CRISPR/Cas9-mediated genome editing is able to correct the Fah mutation in mouse models, WT Cas9 induces numerous undesired mutations that have raised safety concerns for clinical applications. To develop a new method for gene correction with high fidelity, we generated a Fah mutant rat model to investigate whether Cas9 nickase (Cas9n)-mediated genome editing can efficiently correct the Fah First, we confirmed that Cas9n rarely induces indels in both on-target and off-target sites in cell lines. Using WT Cas9 as a positive control, we delivered Cas9n and the repair donor template/single guide (sg)RNA through adenoviral vectors into HTI rats. Analyses of the initial genome editing efficiency indicated that only WT Cas9 but not Cas9n causes indels at the on-target site in the liver tissue. After receiving either Cas9n or WT Cas9-mediated gene correction therapy, HTI rats gained weight steadily and survived. Fah-expressing hepatocytes occupied over 95% of the liver tissue 9 months after the treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. These results strongly suggest that Cas9n-mediated genome editing is a valuable and safe gene therapy strategy for this genetic disease.

Keywords: CRISPR/Cas; Cas9 nickase; fibrosis; gene editing; gene therapy; genetic disease; genome editing; hereditary tyrosinemia; liver.

Figure 1.

Comparison of indel frequencies between Cas9 and Cas9n in HEK293T cells. A, on-target and off-target sites in the EXM1 and VEGFA loci. Target sequences are labeled in red and protospacer adjacent motifs (PAMs) are in blue. Mismatches in the off-target sites are labeled in green. B, indel frequencies of Cas9 and Cas9n in EMX1 and VEGFA on-target sites. C, indel rate of Cas9 and Cas9n in the off-target sites of EMX1 and VEGFA. OT, off-target.

Figure 2.

Generation of the Fah^Δ10/Δ10 at and phenotype identification. A, the Fah^Δ10/Δ10 rat was generated by the CRISPR/Cas9 technology. sgRNA targeting the wildtype (WT) Fah sequence is indicated by the red line and the PAM sequence is labeled in blue. The reading frames of the WT and mutant Fah sequence are listed below. The 10-bp deletion in Fah results in an early STOP codon. B, sequencing results of the targeted Fah sites of WT and Fah^Δ10/Δ10 rats. The red box indicates the 10 missing bp in the Fah^Δ10/Δ10 rat. C, IHC staining to check Fah expression in WT, Fah^Δ10/+ and Fah^Δ10/Δ10 rat liver tissues. I, 100 μm. D and E, knockout of the Fah gene in rat caused severe liver damage including liver fibrosis/cirrhosis indicated by Masson's trichrome staining (D) and α-SMA (α-smooth muscle actin) (E) staining. F, WT and Fah rat serum AST, ALT, ALB, and TBIL levels were tested in WT and Fah^Δ10/Δ10 rats. *, p < 0.01 (n = 3) using two tailed unpaired Student's t test. Data are presented as mean ± S.D.

Figure 3.

In vivo delivery of the CRISPR/Cas9 system into the rat HTI model via recombinant adenoviral vectors. A, left, schematic views of the recombinant adenoviral vector design and the strategy to repair the Fah gene in Fah^Δ10/Δ10 rats. The sgRNA target site is listed under the Fah^Δ10/Δ10 allele. The dashed line in the target site sequence represents the 10-bp deletion in the Fah^Δ10/Δ10 rat. The theoretically repaired sequence is listed under the HDnR allele. The red-dashed rectangle indicates the fully repaired Fah sequence. The PAM sequence is labeled in blue. Silent mutations are labeled in green. Right, an overview of the NTBC withdrawal process; the black lines indicate the days under NTBC treatment and the gray lines indicate NTBC withdrawal periods. PHT, partial hepatectomy. B, co-infection of the AdV–Cas9/AdV–Cas9n (red) and the AdV-HDR (GFP). Scale bar, 200 μm. C, indel frequencies caused by AdV–Cas9 or AdV–Cas9n in the rat liver on day 8. Data were obtained after sequencing 98 clones. D, immunohistochemistry staining of rat Fah protein in liver tissues from AdV–Cas9 and AdV–Cas9n groups on day 8. Fah^Δ10/Δ10 and WT rats served as negative and positive controls, respectively.

Figure 4.

Delivery of AdV–Cas9/AdV–Cas9n with AdV–HDR cures HTI in Fah^Δ10/Δ10 rats. A, delivery of AdV–Cas9/AdV–Cas9n with AdV–HDR rescued the body weight loss following NTBC withdraw. Arrow indicates the partial hepatectomy (PHT) on day 8. B, liver damage marker levels (AST, ALT, and TBIL) of the AdV–Cas9- or AdV–Cas9n-treated groups decreased steadily over time. C, IHC staining of liver tissues to detect Fah expression 3 and 9 months after treatment. Fah^Δ10/Δ10 rat liver slide served as a negative control. Scale bar, 100 μm. PHT, partial hepatectomy.

Figure 5.

Detection of homology directed repair in rat Fah locus. A, rats treated with Cas9/Cas9n had comparable levels of serum AST, ALT, and TBIL (liver damage markers) to WT healthy rat, 3 months after treatment. *, p < 0.05; **, p < 0.01 (n = 3) using the two-tailed unpaired Student's t test. Data are presented as mean ± S.D. B, precise repair of the Fah locus was detected 3 months after treatment. The 10-bp lost in the Fah^Δ10/Δ10 HTI model were precisely repaired after the treatment as indicated by the red-dashed rectangle. C, detection of collagen deposition and α-SMA expression in rat liver tissues, 3 and 9 months after the treatment. HTI rats with NTBC on and with NTBC off for 1 month served negative and positive controls, respectively.

Figure 6.

Low dose adenovirus treatment elicited a mild immune response in rats. On day 96, rat liver mRNA levels of inflammatory cytokines from each group were determined by real-time PCR. Data presented as mean ± S.D.