487. Therapeutic Genome Editing by Combined Viral and Non-Viral Delivery of CRISPR System Components to the Mouse Liver

Abstract

The CRISPR(clustered regularly interspersed short palindromic repeats)/Cas9 system has emerged as a transforming genome editing tool. Cas9/sgRNA recognizes the protospacer-adjacent motif (PAM) sequence and a complementary 20 nucleotide genomic sequence and induces double stranded DNA breaks (DSBs), which are repaired by error-prone non-homologous end-joining (NHEJ) or precise homology-directed repair (HDR). CRISPR/Cas9 genome editing has been applied to correct disease-causing mutations in mouse zygotes and human cell lines, but delivery to adult mammalian organs to correct genetic disease genes has not been reported prior to our study. The liver disease hereditary tyrosinemia type I is a particularly suitable model for gene repair-based therapy because the repaired hepatocytes will expand and repopulate the liver. In tyrosinemia patients, mutation of fumarylacetoacetate hydrolase (FAH), the last enzyme catalyzing the tyrosine catabolic pathway, leads to accumulation of toxic metabolites and severe liver damage. The Fahmut/mut mouse model is caused by a G->A point mutation in the last nucleotide of exon 8. This causes splicing skipping of exon 8 and truncated Fah messenger RNA (mRNA). These mice can be treated with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), an inhibitor of an enzyme upstream of FAH, to prevent toxin accumulation in hepatocytes. In our first proof-of-concept study, we demonstrate CRISPR/Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR/Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ~1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that for the first time CRISPR/Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases. The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here in our second study, we combined lipid nanoparticle—mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated Fah-positive hepatocytes by correcting the causative Fah splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.