Ex vivo gene editing and cell therapy for hereditary tyrosinemia type 1

Abstract

Background: We previously demonstrated the successful use of in vivo CRISPR gene editing to delete 4-hydroxyphenylpyruvate dioxygenase (HPD) to rescue mice deficient in fumarylacetoacetate hydrolase (FAH), a disorder known as hereditary tyrosinemia type 1 (HT1). The aim of this study was to develop an ex vivo gene-editing protocol and apply it as a cell therapy for HT1.

Methods: We isolated hepatocytes from wild-type (C57BL/6J) and Fah-/- mice and then used an optimized electroporation protocol to deliver Hpd-targeting CRISPR-Cas9 ribonucleoproteins into hepatocytes. Next, hepatocytes were transiently incubated in cytokine recovery media formulated to block apoptosis, followed by splenic injection into recipient Fah-/- mice.

Results: We observed robust engraftment and expansion of transplanted gene-edited hepatocytes from wild-type donors in the livers of recipient mice when transient incubation with our cytokine recovery media was used after electroporation and negligible engraftment without the media (mean: 46.8% and 0.83%, respectively; p=0.0025). Thus, the cytokine recovery medium was critical to our electroporation protocol. When hepatocytes from Fah-/- mice were used as donors for transplantation, we observed 35% and 28% engraftment for Hpd-Cas9 ribonucleoproteins and Cas9 mRNA, respectively. Tyrosine, phenylalanine, and biochemical markers of liver injury normalized in both Hpd-targeting Cas9 ribonucleoprotein and mRNA groups independent of induced inhibition of Hpd through nitisinone, indicating correction of disease indicators in Fah-/- mice.

Conclusions: The successful liver cell therapy for HT1 validates our protocol and, despite the known growth advantage of HT1, showcases ex vivo gene editing using electroporation in combination with liver cell therapy to cure a disease model. These advancements underscore the potential impacts of electroporation combined with transplantation as a cell therapy.

Graphical abstract

FIGURE 1

Transplantation of cytokine-treated hepatocytes electroporated with Hpd-targeting CRISPR-Cas9 RNPs. (A) Schematic of the experimental setup. (B) Mean normalized weight data for recipient Fah^−/− mice (n=5) after transplantation with wild-type hepatocytes EP with Hpd-Cas9 RNPs and incubated with cytokine recovery media or plain HMX media. The dotted lines indicate the NTBC-on periods and the solid lines represent the NTBC-off periods. (C) Percent liver engraftment estimated using IHC staining against Fah (lines inside the box plot represent mean levels, and the lower and upper bars represent the minimum and the maximum values). Levels of significance *p<0.05, **p<0.01 (one-way ANOVA with Tukey multiple comparison). Abbreviations: EP, electroporated; NTBC, 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3 cyclohexanedione; RNP, ribonucleoprotein.

FIGURE 2

Electroporated hepatocytes isolated from wild-type donor mice protect against liver failure in Fah^−/− recipient mice. (A) Levels of liver biomarkers in serum: ALP, ALT, AST, and TBIL, respectively, for recipients transplanted with untransfected wild-type hepatocytes or EP and incubated with cytokine recovery media. Untransplanted mice maintained on NTBC or kept off NTBC were used as controls. Bars represent the mean (n=5), and error bars represent SEM. Levels of significance *p<0.05, **p<0.01, and ****p<0.0001 (one-way ANOVA with Tukey multiple comparison). (B) Representative H&E-stained histological images of the liver for recipient mice transplanted with Cas9 RNP and cytokine-treated hepatocytes compared with Fah^−/− controls kept off NTBC. The scale bar represents 50 μm. Abbreviations: ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase; EP, electroporated; NTBC, 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3 cyclohexanedione; RNP, ribonucleoprotein; TBIL, total bilirubin.

FIGURE 3

Diseased hepatocytes electroporated with Hpd-Cas9 mRNA and RNPs engraft in Fah^−/− recipient mice. (A) Quantitative data for Hpd protein levels measured by ELISA assay (n=5, error bars represent SEM, and each dot represents a different sample). Level of significance ***p<0.001 (one-way ANOVA with Tukey multiple comparison). (B) Percent engraftment in transplanted recipients (n=5) estimated by immunohistochemical staining against Hpd (lines inside the box plot represent mean levels, and the lower and upper bars represent the minimum and the maximum values). (C) Representative H&E-stained histology liver images from recipient mice transplanted with hepatocytes electroporated with Hpd-Cas9 RNP and mRNA. The scale bar represents 50 μm. Abbreviations: H&E, hematoxylin and eosin; RNP, ribonucleoprotein.

FIGURE 4

Correction of HT1 phenotype in Fah^−/− recipient mice transplanted with diseased hepatocytes electroporated with Hpd-Cas9 mRNA and RNPs. (A) Progressive weight data in transplanted Fah^−/− recipients on-NTBC and off-NTBC (n=5). The dotted lines indicate the NTBC-on periods and the solid lines represent the NTBC-off periods. (B) Mean phenylalanine and tyrosine levels (n=5) with error bars representing the SEM. (C) Biochemical markers of liver function were measured in serum for transplanted recipients and control mice (n=5) with error bars representing the SEM. Levels of significance *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 (one-way ANOVA with Tukey multiple comparison). Abbreviations: ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase; NTBC, 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3 cyclohexanedione; RNP, ribonucleoprotein.

FIGURE 5

Establishing the dose by the number of viable hepatocytes after electroporation improves engraftment. (A) Percent engraftment estimated by IHC staining against Hpd (error bars represent SEM, and each dot represents a different mouse) in mice transplanted with 350,000 or 500,000 viable cells after electroporating Hpd-Cas9 RNP. (B) The percent editing efficiency estimated by TIDE analysis of on-target indels in gDNA isolated from homogenized liver from recipients transplanted with 500,000 viable hepatocytes after electroporation. (C) Phenylalanine and tyrosine levels and (D) biochemical markers in serum from recipients transplanted with 500,000 viable cells. The controls consisted of untreated wild-type C57BL/6J mice and Fah ^−/− mice kept off NTBC. Horizontal lines or bars represent mean levels (n=5), and error bars represent the SEM. Levels of significance *p<0.05, ***p<0.001, and ****p<0.0001 (one-way ANOVA with Tukey multiple comparison). Abbreviations: ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase; IHC, immunohistochemistry; NTBC, 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3 cyclohexanedione; RNP, ribonucleoprotein.