Genetically blocking HPD via CRISPR-Cas9 protects against lethal liver injury in a pig model of tyrosinemia type I

Abstract

Hereditary tyrosinemia type I (HT1) results from the loss of fumarylacetoacetate hydrolase (FAH) activity and can lead to lethal liver injury (LLI). Therapeutic options for HT1 remain limited. The FAH ^-/- pig, a well-characterized animal model of HT1, represents a promising candidate for testing novel therapeutic approaches to treat this condition. Here, we report an improved single-step method to establish a biallelic (FAH ^-/- ) mutant porcine model using CRISPR-Cas9 and cytoplasmic microinjection. We also tested the feasibility of rescuing HT1 pigs through inactivating the 4-hydroxyphenylpyruvic acid dioxygenase (HPD) gene, which functions upstream of the pathogenic pathway, rather than by directly correcting the disease-causing gene as occurs with traditional gene therapy. Direct intracytoplasmic delivery of CRISPR-Cas9 targeting HPD before intrauterine death reprogrammed the tyrosine metabolism pathway and protected pigs against FAH deficiency-induced LLI. Characterization of the F1 generation revealed consistent liver-protective features that were germline transmissible. Furthermore, HPD ablation ameliorated oxidative stress and inflammatory responses and restored the gene profile relating to liver metabolism homeostasis. Collectively, this study not only provided a novel large animal model for exploring the pathogenesis of HT1, but also demonstrated that CRISPR-Cas9-mediated HPD ablation alleviated LLI in HT1 pigs and represents a potential therapeutic option for the treatment of HT1.

Keywords: CRISPR/Cas9; HPD ablation; lethal liver injury; liver hemeostasis; metabolic reprogramming; oxidative stress; pig; tyrosinemia type I.

Graphical abstract

Figure 1

The generation of FAH^−/− pigs via direct zygote injection (A) FAH mRNA levels were determined in the liver, heart, spleen, lung, kidney, brain, and intestine of healthy pigs (n = 6). (B) Schematic diagram of the target sequence at the FAH locus. The sgRNA sequences are underlined and highlight in red and the protospacer adjacent motif (PAM) sequences are highlighted in blue. (C) Schematic overview of the single-step generation of FAH mutant pigs. (D) Top panel: representative photograph of microinjection into the cytoplasm of one-cell-stage zygotes; bottom panel, representative photograph of newborn FAH^−/− piglets. (E) Sanger sequencing of mutant sequences from four newborn piglets. The targeted sequences are underlined and shown at the top. The mutations are shown in red. Deletions and insertions are denoted as “Δ” and “+” plus the number of base pairs, respectively. (F) Kaplan-Meier survival curve for FAH^−/− and FAH^+/− pigs. (G) Western blotting and quantification of FAH and GAPDH protein levels in liver tissue of FAH^−/− founders (#1, #3, #4). Data are presented as means ± SD. ∗∗∗p < 0.001. (H) Immunohistochemical staining for FAH in liver sections derived from FAH^−/− pigs showing the absence of FAH expression. Scale bar, 200 μm (left) and 50 μm (right).

Figure 2

The generation of FAH^−/−/HPD^−/− double-knockout pigs (A) Schematic diagram of the target sequence at the HPD locus. The sgRNA sequences are underlined and highlighted in red and the PAM sequences are highlighted in blue. (B) Representative photographs of FAH^−/−/HPD^−/− founder piglets (2 months old). (C) Sanger sequencing results of the target regions at the FAH and HPD loci in FAH^−/−/HPD^−/− founders. The targeted sequences (FAH and HPD) are underlined and shown at the top. The mutations are shown in red. Deletions and insertions are denoted as “Δ” and “+” plus the number of base pairs, respectively. (D–F) FAH and HPD protein expression as detected by western blotting (D), immunohistochemistry (scale bar, 200 μm [left panel] and 50 μm [right panel]; E) and immunofluorescence (scale bar, 50 μm; F). The results show that the FAH^−/−/HPD^−/− founders were negative for FAH and HPD protein expression in liver tissue, whereas FAH^−/− pigs expressed a reduced amount of HPD compared with that in wild-type (WT) pigs. Data are presented as means ± SD. ∗∗∗p < 0.001.

Figure 3

HPD ablation ameliorated liver injury and rescued the lethality associated with HT1 (A) Kaplan-Meier survival curve for FAH^−/− pigs (n = 3), FAH^−/−/HPD^−/− pigs (n = 5), and WT pigs (n = 5). (B) The body weight of FAH^−/−/HPD^−/− pigs and WT pigs (n = 4 per group). (C–E) The serum levels of ALT (C), AST (D), and ALP (E) (n = 3 per group). (F and G) Representative images of livers (F) and hematoxylin and eosin (H&E) staining in liver samples (G) of WT (left), FAH^−/− (middle), and FAH^−/−/HPD^−/− pigs (right). Scale bar, 100 μm (top) and 50 μm (bottom). (H) Quantitative PCR analysis of hepatic TNF-α, IL1β, and IL-6 gene expression. (I–K) Representative western blot results for TNF-α, IL-6, and the NF-κB p-p65 subunit in liver tissue lysates and the semi-quantification results. Beta-actin served as loading control. Data are reported as means ± SD; n.s., not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 4

HPD deletion corrected the tyrosine metabolism profile of FAH^−/− pigs (A–C) Comparison of serum tyrosine levels (A), phenylalanine levels (B), and the Phe/Tyr ratio (C) between FAH^−/−/HPD^−/− (n = 5) and FAH^−/− pigs (n = 3). WT pigs (n = 5) served as control. (D) Western blotting assay to assess the expression levels of FAH and HPD protein in in the liver tissue of each group. (E) The mRNA levels of the tyrosine metabolism-related genes TAT, HGD, and GSTZ1 in liver tissues. (F–H) Representative western blotting results for TAT (F), HGD (G), and GSTZ1 (H); semi-quantification results are also shown. Data are reported as means ± SD; n.s., not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 5

HPD ablation reduced oxidative stress and apoptosis in the liver of HT1 pigs (A and B) The lipid peroxidation byproduct 4HNE was detected by western blot (A) and immunohistochemistry (B) to assess the oxidative stress level in each group. (C) Protein-protein interaction (PPI) network diagram of proteins related to NRF2. (D) The relative mRNA expression of KEAP1, NRF2, and NRF2 target genes (such as NQO1, HO-1, GCLM, and GCLC) in the livers of FAH^−/− and FAH^−/−/HPD^−/− pigs. WT pigs served as control. (E) Western blotting assay to assess the expression levels of NRF2-target proteins in total cell extracts; beta-actin served as the reference. (F) Western blotting analysis of the expression levels of KEAP1, total NRF2, nuclear (N)-NRF2, and cytoplasmic (C)-NRF2 in the liver tissue of each group. (G and H) TUNEL staining (G) and TUNEL-positive cell analysis (H) to assess the effect of HPD ablation on FAH deficiency-induced hepatocyte apoptosis. (I and J) Representative immunoblot (I) and immunohistochemical analysis (J) of the AFP protein expression level in individual pigs from each group and the quantification results. Scale bar, 50 μm. Data are reported as means ± SD; n.s., not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 6

Liver homeostasis was improved in FAH^−/−/HPD^−/− pigs compared with that in FAH^−/− pigs (A–C) mRNA levels of the liver-enriched transcriptional factors HNF1 (A), HNF4 (B), and C/EBPα (C). (D) Representative western blot results for HNF1β and the semi-quantification results. (E) Blood glucose concentrations of newborn piglets from each group. (F and G) The mRNA levels of the glucose regulation-related genes PCK1 (F) and G6PC (G) in the liver. (H and I) The mRNA levels of the amino acid turnover regulation-related genes SDHA (H) and MAT1A (I) in the liver. (J) Representative image and semiquantitative analysis of the expression of the liver homeostasis-associated proteins PCK1, G6PC, and SDHA in WT, FAH^−/−, and FAH^−/−/HPD^−/− pigs. Data are reported as means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 7

Characterization of the F1 double-mutant generation (A) Pedigree showing the intercrossing of the F0 FAH^−/−/HPD^−/− pigs to produce the F1 generation. Filled squares indicate FAH^−/−/HPD^−/− males, filled circles indicate FAH^−/−/HPD^−/− females. (B) Sanger sequencing chromatograms for sperm DNA from WT, F1- #1, and F1- #7 pigs. The blue arrow indicates the overlapping peaks in target sites. (C) Western blotting analysis of the FAH and HPD protein expression in liver tissues of WT, FAH^−/−, and FAH^−/−/HPD^−/− F1 pigs. (D) Representative images and semiquantitative analysis of the protein expression levels of TAT, HGD, and GSTZ1 in the liver tissues of each group as detected by western blotting. (E–G) Serum ALT, AST, and ALP levels were determined in WT, FAH^−/−/HPD^−/− founders (F0), and FAH^−/−/HPD^−/− F1 generation pigs. (H) H&E and picrosirius red staining showing normal liver tissue architecture, without obvious hepatic fibrosis, of the FAH^−/−/HPD^−/− founders (F0) and F1 generations. Scale bar, 100 μm. (I) A schematic diagram depicting the mechanism involved in genetically blocking HPD to protect against liver injury in HT1. Data are reported as means ± SD; n.s., not significant; ∗∗∗p < 0.001.