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

Background & Aims 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 goal 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/6) and Fah -/- mice and then used an optimized electroporation protocol to deliver Hpd -targeting CRISPR-Cas9 ribonucleoproteins (RNP) into hepatocytes. Next, hepatocytes were transiently incubated in cytokine recovery media that we 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 liver 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 media was a critical component of our electroporation protocol. When hepatocytes from Fah -/- mice were used as donors for transplantation, we observed 35% and 28% engraftment for Hpd -Cas9 RNPs and Cas9 mRNA, respectively. Tyrosine, phenylalanine, and biochemical markers of liver injury normalized in both Hpd -targeting Cas9 RNP and mRNA groups independent of drug 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, showcase ex vivo gene editing using electroporation in combination with liver cell therapy to cure a disease model. These advancements showcase the impacts of electroporation combined with transplantation as a cell therapy. ### Competing Interest Statement The authors have declared no competing interest. * AAV : Adeno-associated viral vector ALK : Activin receptor-like kinase ALP : Alkaline phosphatase ALT : Alanine transaminase AST : Aspartate transaminase BSA : Bovine serum albumin DMSO : Dimethyl sulfoxide EGF : Epidermal growth factor EP : Electroporation FAH : Fumarylacetoacetate hydrolase FBS : fetal bovine serum GSK : Glycogen synthase kinase HGF : hepatocyte growth factor HT1 : Hereditary tyrosinemia type 1 NAC : N-acetyl cysteine NTBC : 2-nitro-4-trifluoromethylbenzoyl-1,3-cyclohexanedione IHC : Immunohistochemistry IMD : Inherited metabolic disease PBS : Phosphate-buffered saline Phe : Phenylalanine ROCK : Rho-associated, coiled-coil containing protein kinase RNP : Ribonucleoprotein TBIL : Total bilirubin TGFβ : Transforming growth factor-β