Genome Editing With Adv-Cas9 In Adult Rats Corrects Mutation In FAH Causing Tyrosinemia Type 1 And Phenotype

Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use.CRISPR/Cas9-mediated in vivo genome editing has been applied to correct disease-causing mutations in adult animals.In this study,we used a rat model of hereditary tyrosinemia type I(HTI),an autosomal recessive error of metabolism genetic disease caused by mutation of fumarylacetoacetate hydrolase(FAH),the terminal enzyme in the tyrosine catabolic pathway.Compared to traditional animal models,such as pigs and mice,the Fah-deficient rats recapitulate all the typical features of clinical symptoms,such as liver fibrosis and cirrhosis,more close to human disease.First,we applied the widely used CRISPR/Cas9 technique to construct the Fah-/-rat model and confirmed that it was well represent the phenotypic characterizations of liver function damage.Subsequently,delivering adenovirus,Cas9 by tail-vein injection was used to reverse liver damage and rescue hepatocytes function in adult rats.Experimental group treated with adenovirus injections could be free of NTBC and survival for a long term more than 3 months.Although the initial repair efficiency in hepatocytes was low,which was sufficient to rescue the Fah-/-rats from death.Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype.Serum examination showed that liver injury parameters alanine aminotransferase(ALT),asparate aminotransferase(AST)and total bilirubin(TBIL)were decreased,albumin(ALB)was improved.Further,Immunohistochemical staining of liver sections revealed that the percentage of Fah positive hepatocytes got up to 90%.Moreover,the corrected hepatocytes significantly resolved liver fibrosis and cirrhosis.In conclusion,these results revealed that based-adenovirus gene therapy had been successfully rescue liver function.This proof-of-principle study indicates that Cas9-mediated in situ genome editing is possible in adult animals and a potential therapeutic approach of human genetic diseases.