Huan Zhao1, Yan Li1, Lingjuan He1, Wenjuan Pu1, Wei Yu1, Yi Li1, Yan-Ting Wu2,3, Chenming Xu2,3, Yuda Wei4, Qiurong Ding4, Bao-Liang Song5, Hefeng Huang2,3, Bin Zhou1,4,6,7,8,9. 1. State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences (H.Z., Y.L., L.H., W.P., W.Y., Y.L., B.Z.). 2. International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, China (Y.-T.W., C.X., H.H.). 3. Shanghai Key Laboratory of Embryo Original Diseases, China (Y.-T.W., C.X., H.H.). 4. CAS Key Laboratory of Nutrition, Metabolism, and Food Safety, Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences (Y.W., Q.D., B.Z.). 5. Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Institute for Advanced Studies, Wuhan University, China (B.-L.S.). 6. School of Life Science and Technology, ShanghaiTech University, China (B.Z.). 7. Key Laboratory of Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China (B.Z.). 8. Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, China (B.Z.). 9. Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing (B.Z.).
Abstract
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, LdlrE208X, 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 LdlrE208X mice. RESULTS: We found that homogeneous LdlrE208X 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.
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, LdlrE208X, 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 LdlrE208X mice. RESULTS: We found that homogeneous LdlrE208X 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.
Entities:
Keywords:
CRISPR-associated protein 9; atherosclerosis; gene editing; hypercholesterolemia; receptors, LDL
Authors: Tanner Rathbone; Ilayda Ates; Lawrence Fernando; Ethan Addlestone; Ciaran M Lee; Vincent P Richards; Renee N Cottle Journal: CRISPR J Date: 2022-03-02
Authors: Marco De Giorgi; Ang Li; Ayrea Hurley; Mercedes Barzi; Alexandria M Doerfler; Nikitha A Cherayil; Harrison E Smith; Jonathan D Brown; Charles Y Lin; Karl-Dimiter Bissig; Gang Bao; William R Lagor Journal: Mol Ther Methods Clin Dev Date: 2021-04-24 Impact factor: 6.698