Abstract
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, we combine 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 fumarylacetoacetate hydrolase (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.
Highlights
Lipid-nanoparticle delivery of siRNA to the liver has been reported, the systemic delivery of mRNA has only recently been developed[25]
The size of β-gal mRNA is 3.3 kb whereas Cas[9] mRNA is 4.5 kb, and the activity of β-gal protein can be detected by an enzymatic reaction. β-gal protein was detected in the mouse liver by immunoblot at 14 h after administration of a single dose (1 mg/kg or 2 mg/kg), and the amount of protein expressed correlated with the dose of mRNA (Supplementary Fig. 2b)
Up to 6.2% ± 1.0% hepatocytes stained positive for the fumarylacetoacetate hydrolase (Fah) protein by immunohistochemistry in nano.Cas[9] plus associated virus (AAV)-homologydirected repair (HDR)–treated animals (Fig. 3b,c; n = 4 mice; see Supplementary Table 4 for percentage of Fah-positive cells, age and gender of each mouse)
Summary
Lipid-nanoparticle delivery of siRNA to the liver has been reported, the systemic delivery of mRNA has only recently been developed[25]. We report that systemic delivery of Cas[9] mRNA by lipid nanoparticles and sgRNA/HDR template by AAV can efficiently cure Fahmut/mut mice. 77.1 ± 2.6% of cells (n = 3) became GFP-negative after nano.Cas[9] treatment, suggesting that nanoparticle delivery of Cas[9] mRNA can mediate genome editing in cells (Fig. 1b).
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