In Vivo AAV-CRISPR/Cas9–Mediated Gene Editing Ameliorates Atherosclerosis in Familial Hypercholesterolemia

Background: Mutations in low-density lipoprotein (LDL) receptor ( LDLR ) are one of the main causes of familial hypercholesterolemia, which induces atherosclerosis and has a high lifetime risk of cardiovascular disease. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an effective tool for gene editing to correct gene mutations and thus to ameliorate disease. Methods: The goal of this work was to determine whether in vivo somatic cell gene editing through the CRISPR/Cas9 system delivered by adeno-associated virus (AAV) could treat familial hypercholesterolemia caused by the Ldlr mutant in a mouse model. We generated a nonsense point mutation mouse line, Ldlr E208X , based on a relevant familial hypercholesterolemia–related gene mutation. The AAV-CRISPR/Cas9 was designed to correct the point mutation in the Ldlr gene in hepatocytes and was delivered subcutaneously into Ldlr E208X mice. Results: We found that homogeneous Ldlr E208X mice (n=6) exhibited severe atherosclerotic phenotypes after a high-fat diet regimen and that the Ldlr mutation was corrected in a subset of hepatocytes after AAV-CRISPR/Cas9 treatment, with LDLR protein expression partially restored (n=6). Compared with the control groups (n=6 each group), the AAV-CRISPR/Cas9 with targeted single guide RNA group (n=6) had significant reductions in total cholesterol, total triglycerides, and LDL cholesterol in the serum, whereas the aorta had smaller atherosclerotic plaques and a lower degree of macrophage infiltration. Conclusions: Our work shows that in vivo AAV-CRISPR/Cas9–mediated Ldlr gene correction can partially rescue LDLR expression and effectively ameliorate atherosclerosis phenotypes in Ldlr mutants, providing a potential therapeutic approach for the treatment of patients with familial hypercholesterolemia.