Autologous Gene and Cell Therapy Provides Safe and Long-Term Curative Therapy in A Large Pig Model of Hereditary Tyrosinemia Type 1

Abstract

Orthotopic liver transplantation remains the only curative therapy for inborn errors of metabolism. Given the tremendous success for primary immunodeficiencies using ex-vivo gene therapy with lentiviral vectors, there is great interest in developing similar curative therapies for metabolic liver diseases. We have previously generated a pig model of hereditary tyrosinemia type 1 (HT1), an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). Using this model, we have demonstrated curative ex-vivo gene and cell therapy using a lentiviral vector to express FAH in autologous hepatocytes. To further evaluate the long-term clinical outcomes of this therapeutic approach, we continued to monitor one of these pigs over the course of three years. The animal continued to thrive off the protective drug NTBC, gaining weight appropriately, and maintaining sexual fecundity for the course of his life. The animal was euthanized 31 months after transplantation to perform a thorough biochemical and histological analysis. Biochemically, liver enzymes and alpha-fetoprotein levels remained normal and abhorrent metabolites specific to HT1 remained corrected. Liver histology showed no evidence of tumorigenicity and Masson's trichrome staining revealed minimal fibrosis and no evidence of cirrhosis. FAH-immunohistochemistry revealed complete repopulation of the liver by transplanted FAH-positive cells. A complete histopathological report on other organs, including kidney, revealed no abnormalities. This study is the first to demonstrate long-term safety and efficacy of hepatocyte-directed gene therapy in a large animal model. We conclude that hepatocyte-directed ex-vivo gene therapy is a rational choice for further exploration as an alternative therapeutic approach to whole organ transplantation for metabolic liver disease, including HT1.

Keywords: gene therapy; fumarylacetoacetate hydrolase; hepatocyte transplantation; hereditary tyrosinemia type 1; porcine model.

Fig. 1.

Biochemical analyses confirmed amelioration of metabolic disease. (A) Long-term weight data on Y842 from birth (day 0) though 532 days of age. Time on NTBC is represented by shaded bars. (B–E) Plasma or serum from the time of sacrifice was compared to historical wildtype (WT; n = 4) and Fah^-/- controls that received no cell transplant (No Cell; n = 5). (F) Urine succinylacetone (SUAC) from the time of sacrifice was compared to historical wildtype (n = 3) and Fah^-/- untreated controls (n = 4).

Fig. 2.

(A) Complete liver of Y842 at time of euthanasia. (B) Representative cross-sections of liver showing no significant pathology.

Fig. 3.

Histological analyses revealed no abnormal liver pathology. (A–B) Representative low (left) and high (right) magnification images from Y842 at time of euthanasia after FAH IHC. (C–D) Representative low (left) and high (right) magnification images from Y842 at time of euthanasia after H&E staining. (E) Masson’s Trichrome (MT) staining in a representative liver section at time of euthanasia. (F) Quantification of collagen staining was compared in the same animal from time of transplant (Pre), 1-year post-transplantation biopsy (12 mo), and at time of euthanasia (31 mo). (G) Ki-67 staining in a representative liver section at time of euthanasia in Y842. (H) Quantification of Ki-67-positive nuclei staining was compared in Y842 and a 42-month-old control pig.

Fig. 4.

(A) Whole kidneys of Y842 at time of euthanasia. (B) Cross-sections of both kidneys showing no significant pathology.

Fig. 5.

Representative low (left) and high (right) magnification images of the kidney of Y842 at time of euthanasia after FAH IHC. (Top) Representative low (left) and high (right) magnification images from kidney of Y842 at time of euthanasia after H&E staining (Bottom).

Fig. 6.

Bioinformatics demonstrated a benign integration profile for lentiviral vector into treated hepatocytes. (A) Chromosomal map of the pig genome showing regions of integration (red peaks) for each chromosome based on frequency. (B) Relative integration of lentiviral vector in genomic features, including exons, introns, intergenic regions and regions in and out of transcription units (TU), note 1 = random distribution (dashed line). (C) Integration of lentivirus relative to the transcription start sites (TSS) showing preference for downstream integration. (D) Lentiviral vector integrations into genes categorized by increasing transcriptional activity normalized by million base pairs (MBP) of each. (E) Lentiviral vector integrations into tumor and non-tumor associated genes normalized by million base pairs (MBP) of each. (F) Relative integration of lentiviral vector in CpG islands normalized to the percent of the pig genome represented by each, note 1 = random distribution (dashed line).