Gene Editing Targeting Angptl3 By SlugCas9 Ameliorates Lipid Metabolism And Atherosclerosis In Familial Hypercholesterolemia

Background: Familial Hypercholesterolemia(FH) is a genetic disease causing severe hyperlipidemia and atherosclerosis at early age. ANGPTL3, as an important factor for regulating lipid metabolism, has been proved as a promising target for gene therapies. The SlugCas9 system, as a newly-developed gene-editing tool with advantages of small size and wide targeting site, can be wrapped into AAV vectors and facilitate gene therapy. The application of SlugCas9 system towards FH has not been previously reported. Methods: We screened two guide RNA with the highest targeted angptl3 editing efficiency labeled g2 and g3 and built the AAV8-SlugCas9 system. Ldlr-/- mice with 4-week-old were divided into AAV-SlugCas9 group (including g2, g3 and g2+g3), AAV-control group(empty AAV vectors) and normal diet group. AAV-SlugCas9 and AAV-control were injected through tail vein after two weeks of western-diet feeding. Two weeks later, expression of protein ANGPTL3 in both serum and liver were detected and editing efficiency was assessed. We also evaluated serum lipid levels and atherosclerotic level by oil-red staining as well as immunofluorescence labeling of inflammatory markers in aorta. Adverse effect was evaluated by liver histology. Results: Two weeks after western-diet feeding, serum lipid and inflammatory markers increased significantly in ldlr-/- mice. We found dramatically decreased ANGPTL3 protein expression in both serum and liver after AAV-SlugCas9 treatment, with no off-target condition found. Our results also showed that compared with the AAV-control group, the AAV-SlugCas9 group had significant reductions in serum lipid levels and that we found improved atherosclerosis and decreased inflammatory marker expression in serum and aorta. No obvious damage in liver histology was found. Conclusions: In this study, we used AAV as a delivery vector to evaluate the editing efficiency of SlugCas9 for the angptl3 gene in liver for the first time and found out by reducing its expression by gene editing of angptl3 in vivo can reduce blood lipid levels and delay the progression of atherosclerosis with no obvious adverse effect in mice. It proves that SlugCas9 is a useful tool in gene therapies targeting FH with potential clinical application.