Combined viral and nonviral delivery of CRISPR components corrects liver disease in adult rodents. Homo sapiens

Abstract: The ability to correct defective genes in vivo has broad potential utility for both therapy and the study of genetics. The combination of Cas9, guide RNA, and repair template DNA can induce gene editing, and the correction of genetic disease in adult mammals. However, implementation of this promising technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. By combining viral and nonviral nucleic acid delivery we report the first therapeutically relevant formulations capable of inducing repair of a disease gene in an adult animal. In vivo repair was mediated by lipid and lipid-like formulation of Cas9 mRNA in combination with adeno-associated virus encoding sgRNA and a ssDNA repair template to an adult mouse model of the human hereditary tyrosinemia. This treatment fully rescued body weight loss, alleviated liver damage-associated serum markers and generated Fah- positive hepatocytes. The Fah splicing mutation was corrected in the liver, which restored fully-spliced Fah mRNA. Importantly, the efficiency of correction was more than 4% of hepatocytes after a single application, suggesting potential utility for a range of diseases. Our study indicates that systemic CRISPR-mediated genome editing is possible in vivo, and provides proof-of-principle for therapeutic correction of genetic diseases in adult animals.