Skip main navigation
×

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

  • Huan Zhao
  • , PhD
  • Yan Li
  • , PhD
  • Lingjuan He
  • , PhD
  • Wenjuan Pu
  • , PhD
  • Wei Yu
  • , PhD
  • Yi Li
  • , PhD
  • Yan-Ting Wu
  • , MD, PhD
  • Chenming Xu
  • , PhD
  • Yuda Wei
  • , PhD
  • Qiurong Ding
  • , PhD
  • Bao-Liang Song
  • , PhD
  • Hefeng Huang
  • , MD
  • Bin Zhou
  • MD, PhDState 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.).International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, China (Y.-T.W., C.X., H.H.).Shanghai Key Laboratory of Embryo Original Diseases, China (Y.-T.W., C.X., H.H.).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.).Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Institute for Advanced Studies, Wuhan University, China (B.-L.S.).School of Life Science and Technology, ShanghaiTech University, China (B.Z.).Key Laboratory of Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China (B.Z.).Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, China (B.Z.).Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing (B.Z.).
Originally publishedhttps://doi.org/10.1161/CIRCULATIONAHA.119.042476Circulation. 2020;141:67–79

    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.

    Footnotes

    Sources of Funding, see page 77

    The online-only Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/circulationaha.119.042476.

    https://www.ahajournals.org/journal/circ

    Hefeng Huang, MD, 910 Hengshan Rd, Shanghai, China 200031; or Bin Zhou, MD, PhD, 320 Yueyang Rd, A2112, Shanghai, China 200031. Email or

    References

    • 1. Goldstein JL, Schrott HG, Hazzard WR, Bierman EL, Motulsky AG. Hyperlipidemia in coronary heart disease, II: genetic analysis of lipid levels in 176 families and delineation of a new inherited disorder, combined hyperlipidemia.J Clin Invest. 1973; 52:1544–1568. doi: 10.1172/JCI107332CrossrefMedlineGoogle Scholar
    • 2. Defesche JC, Gidding SS, Harada-Shiba M, Hegele RA, Santos RD, Wierzbicki AS. Familial hypercholesterolaemia.Nat Rev Dis Primers. 2017; 3:17093. doi: 10.1038/nrdp.2017.93CrossrefMedlineGoogle Scholar
    • 3. Hobbs HH, Brown MS, Goldstein JL. Molecular genetics of the LDL receptor gene in familial hypercholesterolemia.Hum Mutat. 1992; 1:445–466. doi: 10.1002/humu.1380010602CrossrefMedlineGoogle Scholar
    • 4. Nordestgaard BG, Chapman MJ, Humphries SE, Ginsberg HN, Masana L, Descamps OS, Wiklund O, Hegele RA, Raal FJ, Defesche JC, et al.; European Atherosclerosis Society Consensus Panel. Familial hypercholesterolaemia is underdiagnosed and undertreated in the general population: guidance for clinicians to prevent coronary heart disease: consensus statement of the European Atherosclerosis Society.Eur Heart J. 2013; 34:3478–3490a. doi: 10.1093/eurheartj/eht273CrossrefMedlineGoogle Scholar
    • 5. Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.J Clin Invest. 1993; 92:883–893. doi: 10.1172/JCI116663CrossrefMedlineGoogle Scholar
    • 6. Rader DJ, Cohen J, Hobbs HH. Monogenic hypercholesterolemia: new insights in pathogenesis and treatment.J Clin Invest. 2003; 111:1795–1803. doi: 10.1172/JCI18925CrossrefMedlineGoogle Scholar
    • 7. Oka K, Pastore L, Kim IH, Merched A, Nomura S, Lee HJ, Merched-Sauvage M, Arden-Riley C, Lee B, Finegold M, et al.. Long-term stable correction of low-density lipoprotein receptor-deficient mice with a helper-dependent adenoviral vector expressing the very low-density lipoprotein receptor.Circulation. 2001; 103:1274–1281. doi: 10.1161/01.cir.103.9.1274LinkGoogle Scholar
    • 8. Sun XM, Patel DD, Webb JC, Knight BL, Fan LM, Cai HJ, Soutar AK. Familial hypercholesterolemia in China: identification of mutations in the LDL-receptor gene that result in a receptor-negative phenotype.Arterioscler Thromb. 1994; 14:85–94. doi: 10.1161/01.atv.14.1.85LinkGoogle Scholar
    • 9. Raal F, Panz V, Immelman A, Pilcher G. Elevated PCSK9 levels in untreated patients with heterozygous or homozygous familial hypercholesterolemia and the response to high-dose statin therapy.J Am Heart Assoc. 2013; 2:e000028. doi: 10.1161/JAHA.112.000028LinkGoogle Scholar
    • 10. Soutar AK, Naoumova RP. Mechanisms of disease: genetic causes of familial hypercholesterolemia.Nat Clin Pract Cardiovasc Med. 2007; 4:214–225. doi: 10.1038/ncpcardio0836CrossrefMedlineGoogle Scholar
    • 11. Vallejo-Vaz AJ, Kondapally Seshasai SR, Cole D, Hovingh GK, Kastelein JJ, Mata P, Raal FJ, Santos RD, Soran H, Watts GF, et al.. Familial hypercholesterolaemia: a global call to arms.Atherosclerosis. 2015; 243:257–259. doi: 10.1016/j.atherosclerosis.2015.09.021CrossrefMedlineGoogle Scholar
    • 12. Bilheimer DW, Goldstein JL, Grundy SM, Starzl TE, Brown MS. Liver transplantation to provide low-density-lipoprotein receptors and lower plasma cholesterol in a child with homozygous familial hypercholesterolemia.N Engl J Med. 1984; 311:1658–1664. doi: 10.1056/NEJM198412273112603CrossrefMedlineGoogle Scholar
    • 13. Revell SP, Noble-Jamieson G, Johnston P, Rasmussen A, Jamieson N, Barnes ND. Liver transplantation for homozygous familial hypercholesterolaemia.Arch Dis Child. 1995; 73:456–458. doi: 10.1136/adc.73.5.456CrossrefMedlineGoogle Scholar
    • 14. Al-Ashwal A, Alnouri F, Sabbour H, Al-Mahfouz A, Al-Sayed N, Razzaghy-Azar M, Al-Allaf F, Al-Waili K, Banerjee Y, Genest J, et al.. Identification and treatment of patients with homozygous familial hypercholesterolaemia: information and recommendations from a Middle East advisory panel.Curr Vasc Pharmacol. 2015; 13:759–770. doi: 10.2174/1570161113666150827125040CrossrefMedlineGoogle Scholar
    • 15. Daya S, Berns KI. Gene therapy using adeno-associated virus vectors.Clin Microbiol Rev. 2008; 21:583–593. doi: 10.1128/CMR.00008-08CrossrefMedlineGoogle Scholar
    • 16. Mueller C, Flotte TR. Clinical gene therapy using recombinant adeno-associated virus vectors.Gene Ther. 2008; 15:858–863. doi: 10.1038/gt.2008.68CrossrefMedlineGoogle Scholar
    • 17. Tontonoz P, Wu X, Jones M, Zhang Z, Salisbury D, Sallam T. Long noncoding RNA facilitated gene therapy reduces atherosclerosis in a murine model of familial hypercholesterolemia.Circulation. 2017; 136:776–778. doi: 10.1161/CIRCULATIONAHA.117.029002LinkGoogle Scholar
    • 18. Muramatsu S. Gene therapy using adeno-associated virus vectors.Cancer Sci. 2018; 109:1200–1200.Google Scholar
    • 19. Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.Cell. 2013; 154:1370–1379. doi: 10.1016/j.cell.2013.08.022CrossrefMedlineGoogle Scholar
    • 20. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, et al.. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339:819–823. doi: 10.1126/science.1231143CrossrefMedlineGoogle Scholar
    • 21. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell. 2013; 153:910–918. doi: 10.1016/j.cell.2013.04.025CrossrefMedlineGoogle Scholar
    • 22. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9.Science. 2013; 339:823–826. doi: 10.1126/science.1232033CrossrefMedlineGoogle Scholar
    • 23. Panier S, Durocher D. Push back to respond better: regulatory inhibition of the DNA double-strand break response.Nat Rev Mol Cell Biol. 2013; 14:661–672. doi: 10.1038/nrm3659CrossrefMedlineGoogle Scholar
    • 24. Yang Y, Wang L, Bell P, McMenamin D, He Z, White J, Yu H, Xu C, Morizono H, Musunuru K, et al.. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice.Nat Biotechnol. 2016; 34:334–338. doi: 10.1038/nbt.3469CrossrefMedlineGoogle Scholar
    • 25. Tabebordbar M, Zhu K, Cheng JKW, Chew WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, et al.. In vivo gene editing in dystrophic mouse muscle and muscle stem cells.Science. 2016; 351:407–411. doi: 10.1126/science.aad5177CrossrefMedlineGoogle Scholar
    • 26. Long C, Amoasii L, Mireault AA, McAnally JR, Li H, Sanchez-Ortiz E, Bhattacharyya S, Shelton JM, Bassel-Duby R, Olson EN. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy.Science. 2016; 351:400–403. doi: 10.1126/science.aad5725CrossrefMedlineGoogle Scholar
    • 27. Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb EA, Castellanos Rivera RM, Madhavan S, Pan X, Ran FA, et al.. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy.Science. 2016; 351:403–407. doi: 10.1126/science.aad5143CrossrefMedlineGoogle Scholar
    • 28. Traxler EA, Yao Y, Wang YD, Woodard KJ, Kurita R, Nakamura Y, Hughes JR, Hardison RC, Blobel GA, Li C, et al.. A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition.Nat Med. 2016; 22:987–990. doi: 10.1038/nm.4170CrossrefMedlineGoogle Scholar
    • 29. Amoasii L, Long C, Li H, Mireault AA, Shelton JM, Sanchez-Ortiz E, McAnally JR, Bhattacharyya S, Schmidt F, Grimm D, et al.. Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy.Sci Transl Med. 2017; 9:eaan8081. doi: 10.1126/scitranslmed.aan8081CrossrefMedlineGoogle Scholar
    • 30. Amoasii L, Hildyard JCW, Li H, Sanchez-Ortiz E, Mireault A, Caballero D, Harron R, Stathopoulou TR, Massey C, Shelton JM, et al.. Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy.Science. 2018; 362:86–91. doi: 10.1126/science.aau1549CrossrefMedlineGoogle Scholar
    • 31. Long C, Li H, Tiburcy M, Rodriguez-Caycedo C, Kyrychenko V, Zhou H, Zhang Y, Min YL, Shelton JM, Mammen PPA, et al.. Correction of diverse muscular dystrophy mutations in human engineered heart muscle by single-site genome editing.Sci Adv. 2018; 4:eaap9004. doi: 10.1126/sciadv.aap9004CrossrefMedlineGoogle Scholar
    • 32. Yin H, Song CQ, Dorkin JR, Zhu LJ, Li Y, Wu Q, Park A, Yang J, Suresh S, Bizhanova A, et al.. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo.Nat Biotechnol. 2016; 34:328–333. doi: 10.1038/nbt.3471CrossrefMedlineGoogle Scholar
    • 33. Austin MA, Hutter CM, Zimmern RL, Humphries SE. Genetic causes of monogenic heterozygous familial hypercholesterolemia: a HuGE prevalence review.Am J Epidemiol. 2004; 160:407–420. doi: 10.1093/aje/kwh236CrossrefMedlineGoogle Scholar
    • 34. Kapourchali FR, Surendiran G, Chen L, Uitz E, Bahadori B, Moghadasian MH. Animal models of atherosclerosis.World J Clin Cases. 2014; 2:126–132. doi: 10.12998/wjcc.v2.i5.126CrossrefMedlineGoogle Scholar
    • 35. Nykjaer A, Willnow TE. The low-density lipoprotein receptor gene family: a cellular Swiss army knife?Trends Cell Biol. 2002; 12:273–280. doi: 10.1016/s0962-8924(02)02282-1CrossrefMedlineGoogle Scholar
    • 36. Allahverdian S, Chaabane C, Boukais K, Francis GA, Bochaton-Piallat ML. Smooth muscle cell fate and plasticity in atherosclerosis.Cardiovasc Res. 2018; 114:540–550. doi: 10.1093/cvr/cvy022CrossrefMedlineGoogle Scholar
    • 37. Wang L, Yang Y, Breton CA, White J, Zhang J, Che Y, Saveliev A, McMenamin D, He Z, Latshaw C, et al.. CRISPR/Cas9-mediated in vivo gene targeting corrects hemostasis in newborn and adult factor IX-knockout mice.Blood. 2019; 133:2745–2752. doi: 10.1182/blood.2019000790CrossrefMedlineGoogle Scholar
    • 38. Yu W, Huang X, Tian X, Zhang H, He L, Wang Y, Nie Y, Hu S, Lin Z, Zhou B, et al.. GATA4 regulates Fgf16 to promote heart repair after injury.Development. 2016; 143:936–949. doi: 10.1242/dev.130971CrossrefMedlineGoogle Scholar
    • 39. Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.Physiol Rev. 2008; 88:125–172. doi: 10.1152/physrev.00013.2007CrossrefMedlineGoogle Scholar
    • 40. Ganem NJ, Pellman D. Limiting the proliferation of polyploid cells.Cell. 2007; 131:437–440. doi: 10.1016/j.cell.2007.10.024CrossrefMedlineGoogle Scholar
    • 41. Guidotti JE, Brégerie O, Robert A, Debey P, Brechot C, Desdouets C. Liver cell polyploidization: a pivotal role for binuclear hepatocytes.J Biol Chem. 2003; 278:19095–19101. doi: 10.1074/jbc.M300982200CrossrefMedlineGoogle Scholar
    • 42. Kim K, Ryu SM, Kim ST, Baek G, Kim D, Lim K, Chung E, Kim S, Kim JS. Highly efficient RNA-guided base editing in mouse embryos.Nat Biotechnol. 2017; 35:435–437. doi: 10.1038/nbt.3816CrossrefMedlineGoogle Scholar
    • 43. Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions.Nat Biotechnol. 2017; 35:371–376. doi: 10.1038/nbt.3803CrossrefMedlineGoogle Scholar
    • 44. Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H, Teramura H, Yamamoto T, Komatsu H, Miura K, et al.. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion.Nat Biotechnol. 2017; 35:441–443. doi: 10.1038/nbt.3833CrossrefMedlineGoogle Scholar
    • 45. Zong Y, Wang Y, Li C, Zhang R, Chen K, Ran Y, Qiu JL, Wang D, Gao C. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion.Nat Biotechnol. 2017; 35:438–440. doi: 10.1038/nbt.3811CrossrefMedlineGoogle Scholar
    • 46. Jin S, Zong Y, Gao Q, Zhu Z, Wang Y, Qin P, Liang C, Wang D, Qiu JL, Zhang F, et al.. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice.Science. 2019; 364:292–295. doi: 10.1126/science.aaw7166MedlineGoogle Scholar
    • 47. Zuo E, Sun Y, Wei W, Yuan T, Ying W, Sun H, Yuan L, Steinmetz LM, Li Y, Yang H. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos.Science. 2019; 364:289–292. doi: 10.1126/science.aav9973MedlineGoogle Scholar