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

Abstract

Mutations in low-density lipoprotein (LDL) receptor (<i>LDLR</i>) 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. 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 <i>Ldlr</i> mutant in a mouse model. We generated a nonsense point mutation mouse line, <i>Ldlr</i>^<i>E208X</i>, based on a relevant familial hypercholesterolemia-related gene mutation. The AAV-CRISPR/Cas9 was designed to correct the point mutation in the <i>Ldlr</i> gene in hepatocytes and was delivered subcutaneously into <i>Ldlr</i>^<i>E208X</i> mice. We found that homogeneous <i>Ldlr</i>^<i>E208X</i> mice (n=6) exhibited severe atherosclerotic phenotypes after a high-fat diet regimen and that the <i>Ldlr</i> 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. Our work shows that in vivo AAV-CRISPR/Cas9-mediated <i>Ldlr</i> gene correction can partially rescue LDLR expression and effectively ameliorate atherosclerosis phenotypes in <i>Ldlr</i> mutants, providing a potential therapeutic approach for the treatment of patients with familial hypercholesterolemia.