Curative Ex Vivo Hepatocyte-Directed Gene Editing in a Mouse Model of Hereditary Tyrosinemia Type 1

Abstract

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). It has been previously shown that ex vivo hepatocyte-directed gene therapy using an integrating lentiviral vector to replace the defective Fah gene can cure liver disease in small- and large-animal models of HT1. This study hypothesized that ex vivo hepatocyte-directed gene editing using CRISPR/Cas9 could be used to correct a mouse model of HT1, in which a single point mutation results in loss of FAH function. To achieve high transduction efficiencies of primary hepatocytes, this study utilized a lentiviral vector (LV) to deliver both the Streptococcus pyogenes Cas9 nuclease and target guide RNA (LV-Cas9) and an adeno-associated virus (AAV) vector to deliver a 1.2 kb homology template (AAV-HT). Cells were isolated from Fah^-/- mice and cultured in the presence of LV and AAV vectors. Transduction of cells with LV-Cas9 induced significant indels at the target locus, and correction of the point mutation in Fah^-/- cells ex vivo using AAV-HT was completely dependent on LV-Cas9. Next, hepatocytes transduced ex vivo by LV-Cas9 and AAV-HT were transplanted into syngeneic Fah^-/- mice that had undergone a two-thirds partial hepatectomy or sham hepatectomy. Mice were cycled on/off the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) to stimulate expansion of corrected cells. All transplanted mice became weight stable off NTBC. However, a significant improvement was observed in weight stability off NTBC in animals that received partial hepatectomy. After 6 months, mice were euthanized, and thorough biochemical and histological examinations were performed. Biochemical markers of liver injury were significantly improved over non-transplanted controls. Histological examination of mice revealed normal tissue architecture, while immunohistochemistry showed robust repopulation of recipient animals with FAH+ cells. In summary, this is the first report of ex vivo hepatocyte-directed gene repair using CRISPR/Cas9 to demonstrate curative therapy in an animal model of liver disease.

Keywords: CRISPR/Cas9; gene therapy; hepatocytes; hereditary tyrosinemia type 1; metabolic liver disease.

Figure 1.

Schematics of Fah gene and viral vectors used. (A) Fah^−/− mouse gene locus. The hereditary tyrosinemia type 1 (HT1) mutation is on the last nucleotide of exon 8, as shown in red. The protospacer adjacent motif (PAM) sequence for the indicated guide RNA is highlighted in blue. (B) Adeno-associated virus (AAV) vector homology template with 1.2 kb homology fragment flanked by inverted terminal repeats. The corrected Fah gene sequence is shown in red, and the PAM sequence, which is modified to prevent re-cutting, is shown in blue. (C) The LV-Cas9 vector contains a single-guide RNA (gRNA) under the control of the U6 promoter. Cas9 from Streptococcus pyogenes is co-expressed with green fluorescent protein (GFP) under the control of the EFS promoter by means of a P2A cleavage site.

Figure 2.

LV-Cas9-mediated gene editing at the Fah^−/− locus in fibroblasts. (A) A 452 base pair (bp) region around the HT1 single nucleotide polymorphism was amplified using polymerase chain reaction (PCR), re-annealed, and analyzed using a T7 endonuclease assay, which cleaves DNA at mismatched sequences. The lower bands in the gel indicated that LV-Cas9 did disrupt the Fah locus. (B) Gene disruption was confirmed by next-generation sequencing of amplicons. The top line is the non-modified mutant sequence, and the lines below indicate the frequency of each modified sequence at this locus. The guide sequence is italicized in the top line. (C) Representative Tracking of Indels by DEcomposition analysis of cells treated with LV-Cas9 at a multiplicity of infection of 8,000 lentiviral (LV) particles. The Cas9 efficiency in this sample was 34.4%, with the most frequent indel being a +1 insertion (24.7%).

Figure 3.

Fah^−/− gene correction using AAV is dependent on LV-Cas9. (A) Hepatocytes were transduced with different serotypes of AAV vector expressing GFP, and the percentage of cells transduced was measured by flow cytometry. AAV-2 and AAV-DJ yielded statistically significant more GFP+ cells compared to control, AAV-8, and AAV-9 (****p < 0.001) but not from each other. AAV-DJ was used for the duration of the experiments. (B) Fah^−/− fibroblasts were transduced, as indicated with a combination of a LV vector carrying Cas9 and a guide for the Fah locus (LV-Fah sgRNA), a LV vector carrying Cas9 and a guide for the LacZ locus (LV-LacZ sgRNA), and AAV-HT for the Fah locus. Using PCR primers that selectively amplify corrected sequence, only cells that were transduced with both LV-Fah sgRNA and AAV-HT showed correction. The expected product size is 859 bp. (C) Fah^−/− fibroblasts were transduced as indicated. Using the same PCR primers as (B), only cells transduced by LV-Cas9 and AAV-HT together resulted in gene correction.

Figure 4.

Transplantation of ex vivo gene edited hepatocytes is curative. (A) Schematic depicting the ex vivo procedure. Fah^−/− hepatocytes were isolated from a donor mouse, transduced with the LV-Cas9 and AAV-HT, cultured for <24 h, and transplanted into syngeneic Fah^−/− mice via splenic injection. One cohort of mice (n = 3) received a partial hepatectomy at the time of transplant. A second cohort (n = 3) received splenic injection only. After 6 months of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) cycling, biochemical and histological data were obtained. (B) Mice were cycled on and off NTBC (gray bars) following transplant until NTBC-independence was obtained. Weight data from all mice are shown. Mice were NTBC-independent for 92 days before sacrifice. (C) Change in average weights (n = 3 for each group) from day 5 post transplant indicates that mice that received a partial hepatectomy (Hx) became weight stable off NTBC more quickly than mice that did not receive a partial hepatectomy (No hx).

Figure 5.

Biochemical analyses confirmed amelioration of metabolic disease in transplanted mice. Plasma from the time of sacrifice (6 months post transplantation) in all transplanted mice (n = 6) was compared to plasma of Fah^−/− untreated controls off NTBC (–NTBC; n = 5) and untreated Fah^−/− mice on NTBC (+NTBC; n = 5). ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; TBIL, total bilirubin. ***p < 0.001; ****p < 0.0001.

Figure 6.

(A) Histology and immunohistochemistry slides from treated and untreated control mice. Low-magnification fumarylacetoacetate hydrolase (FAH) images are on the left; higher-magnification view of dashed box is on the right. (B) Average percentage of FAH+ cells in untreated controls (n = 3), +Hx mice (n = 3), and −Hx mice (n = 3). *p < 0.05; ***p < 0.0001. (C) Combined sequencing data from the time of autopsy detailing frequency of targeted and untargeted modifications at the Fah locus.