In vivo epigenome editing reduces circulating lipids and attenuates atherosclerosis in mice

Wei Wang; Yingjie Zhang; Lin Chen; Yuanbin Lu; Yunjie He; Hui Cheng; Ting Tang; Xinyi Li; Jiayi Zhang; Yan Liu; Yang Wang; Yuxuan Kong; Huijun Yuan

doi:10.1016/j.jgg.2026.04.004

Turn off MathJax

Article Contents

Article Navigation > Journal of Genetics and Genomics > 2026 > Accepted Manuscript

PDF( 0 KB)

In vivo epigenome editing reduces circulating lipids and attenuates atherosclerosis in mice

doi: 10.1016/j.jgg.2026.04.004

Institute of Rare Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China

Funds:

This work is supported by grants from the National Natural Science Foundation of China (82030030 and 82530036) and the 1·3·5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (ZYJC20002). Sincere appreciation is also extended to the National Super-computing Center in Chengdu and the Information Center of West China Hospital for providing data analytical and storage resources, along with technical support.

Received Date: 2025-11-28
Accepted Date: 2026-04-08
Rev Recd Date: 2026-04-07

Available Online: 2026-04-15

Abstract

Abstract

Atherosclerotic cardiovascular disease remains the leading cause of global mortality, with hypercholesterolemia serving as a critical driver of atherogenesis. Although current lipid-lowering therapies substantially improve circulating lipid profiles, strategies that provide more durable, safe, and efficient control of lipid metabolism are still needed. Epigenome editing offers a promising approach for long-lasting repression of disease-modifying genes without altering the underlying DNA sequence. Here, we develop CRISPRoff platforms delivered by adeno-associated virus or lipid nanoparticle to epigenetically silence hepatic Hmgcr or Pcsk9 in vivo. In both C57BL/6J wild-type and ApoE^−/− mice, CRISPRoff mediates robust and durable repression of these targets, leading to marked reductions in circulating total cholesterol, low-density lipoprotein cholesterol, and triglycerides. In the ApoE^−/− context, epigenetic silencing of Pcsk9 confers pronounced vascular protection, including decreased lipid accumulation in the liver and aortic root, reduced necrotic core formation, diminished macrophage infiltration, and enhanced plaque stability. Together, these results provide proof of principle that CRISPRoff-based epigenome editing enables stable repression of clinically relevant targets and ameliorates key features of atherosclerotic disease. This work lays the foundation for broader therapeutic applications of epigenetic modulation in cardiovascular disorders.
- Epigenome editing,
- Hypercholesterolemia,
- Atherosclerosis,
- Pcsk9,
- Hmgcr

FullText(HTML)

References(67)

References

Amabile, A., Migliara, A., Capasso, P., Biffi, M., Cittaro, D., Naldini, L., Lombardo, A., 2016. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219-232.

Aussel, C., Cathomen, T., Fuster-Garcia, C., 2025. The hidden risks of CRISPR/Cas: structural variations and genome integrity. Nat. Commun. 16, 7208.

Banach, M., Surma, S., 2023. A look to the past - what has had the biggest impact on lipids in the last four decades? A personal perspective. Arch. Med. Sci. 19, 559-564.

Blom, D.J., Hala, T., Bolognese, M., Lillestol, M.J., Toth, P.D., Burgess, L., Ceska, R., Roth, E., Koren, M.J., Ballantyne, C.M., et al., 2014. A 52-week placebo-controlled trial of evolocumab in hyperlipidemia. N. Engl. J. Med. 370, 1809-1819.

Bolsoni, J., Liu, D., Mohabatpour, F., Ebner, R., Sadhnani, G., Tafech, B., Leung, J., Shanta, S., An, K., Morin, T., et al., 2023. Lipid nanoparticle-mediated hit-and-run approaches yield efficient and safe in situ gene editing in human skin. ACS Nano 17, 22046-22059.

Cannon, C.P., 2022. Statin intolerance: how common is it and how do we work with patients to overcome it? Eur. Heart. J. 43, 3224-3226.

Cappelluti, M.A., Mollica Poeta, V., Valsoni, S., Quarato, P., Merlin, S., Merelli, I., Lombardo, A., 2024. Durable and efficient gene silencing in vivo by hit-and-run epigenome editing. Nature 627, 416-423.

Centa, M., Prokopec, K.E., Garimella, M.G., Habir, K., Hofste, L., Stark, J.M., Dahdah, A., Tibbitt, C.A., Polyzos, K.A., Gistera, A., et al., 2018. Acute loss of apolipoprotein E triggers an autoimmune response that accelerates atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 38, e145-e158.

Chadwick, A.C., Evitt, N.H., Lv, W., Musunuru, K., 2018. Reduced blood lipid levels with in vivo CRISPR-Cas9 base editing of ANGPTL3. Circulation 137, 975-977.

Conti, A., Di Micco, R., 2018. p53 activation: a checkpoint for precision genome editing? Genome Med. 10, 66.

Crisby, M., Nordin-Fredriksson, G., Shah, P.K., Yano, J., Zhu, J., Nilsson, J., 2001. Pravastatin treatment increases collagen content and decreases lipid content, inflammation, metalloproteinases, and cell death in human carotid plaques: implications for plaque stabilization. Circulation 103, 926-933.

Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., Gingeras, T.R., 2013. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21.

Duan, Y., Gong, K., Xu, S., Zhang, F., Meng, X., Han, J., 2022. Regulation of cholesterol homeostasis in health and diseases: from mechanisms to targeted therapeutics. Signal Transduct. Target. Ther. 7, 265.

Emini Veseli, B., Perrotta, P., De Meyer, G.R.A., Roth, L., Van der Donckt, C., Martinet, W., De Meyer, G.R.Y., 2017. Animal models of atherosclerosis. Eur. J. Pharmacol. 816, 3-13.

Eryilmaz, E., Shah, N.H., Muir, T.W., Cowburn, D., 2014. Structural and dynamical features of inteins and implications on protein splicing. J. Biol. Chem. 289, 14506-14511.

Fang, Q., Lu, X., Zhu, Y., Lv, X., Yu, F., Ma, X., Liu, B., Zhang, H., 2024. Development of a PCSK9-targeted nanoparticle vaccine to effectively decrease the hypercholesterolemia. Cell Rep. Med. 5, 101614.

Ference, B.A., Ginsberg, H.N., Graham, I., Ray, K.K., Packard, C.J., Bruckert, E., Hegele, R.A., Krauss, R.M., Raal, F.J., Schunkert, H., et al., 2017. Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel. Eur. Heart. J. 38, 2459-2472.

German, C.A., Shapiro, M.D., 2020. Small interfering RNA therapeutic inclisiran: a new approach to targeting PCSK9. BioDrugs 34, 1-9.

He, B., Peng, W., Huang, J., Zhang, H., Zhou, Y., Yang, X., Liu, J., Li, Z., Xu, C., Xue, M., et al., 2020. Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver. Protein Cell. 11, 518-524.

Hernandez, I., 2017. Revisiting outcomes-based pricing propositions for the PCSK9 inhibitor evolocumab. JAMA Intern. Med. 177, 1388-1390.

Herrington, W., Lacey, B., Sherliker, P., Armitage, J., Lewington, S., 2016. Epidemiology of atherosclerosis and the potential to reduce the global burden of atherothrombotic disease. Circ. Res. 118, 535-546.

Hooper, A.J., Tang, X.L., Burnett, J.R., 2024. VERVE-101, a CRISPR base-editing therapy designed to permanently inactivate hepatic PCSK9 and reduce LDL-cholesterol. Expert Opin. Investig. Drugs 33, 753-756.

Ilyas, I., Little, P.J., Liu., Z., Xu, Y., Kamato, D., Berk, B.C., Weng, J., Xu, S., 2022. Mouse models of atherosclerosis in translational research. Trends Pharmacol. Sci. 43, 920-939.

Issa, S.S., Shaimardanova, A.A., Solovyeva, V.V., Rizvanov, A.A., 2023. Various AAV serotypes and their applications in gene therapy: an overview. Cells 12, 785.

Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A., Liu, D.R., 2016. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424.

Kong, N., Xu, Q., Cui, W., Feng, X., Gao, H., 2022. PCSK9 inhibitor inclisiran for treating atherosclerosis via regulation of endothelial cell pyroptosis. Ann. Transl. Med. 10, 1205.

Kramer, A., Green, J., Pollard, J., Tugendreich, S., 2014. Causal analysis approaches in Ingenuity Pathway Analysis. Bioinformatics 30, 523-530.

Laffin, L.J., Nicholls, S.J., Scott, R.S., Clifton, P.M., Baker, J., Sarraju, A., Singh, S., Wang, Q., Wolski, K., Xu, H., et al., 2025. Phase 1 trial of CRISPR-Cas9 gene editing targeting ANGPTL3. N. Engl. J. Med. 393, 2119-2130.

Levy, J.M., Yeh, W.H., Pendse, N., Davis, J.R., Hennessey, E., Butcher, R., Koblan, L.W., Comander, J., Liu, Q., Liu, D.R., 2020. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nat. Biomed. Eng. 4, 97-110.

Li, B., Dewey, C.N., 2011. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323.

Li, H., Yang, Y., Hong, W., Huang, M., Wu, M., Zhao, X., 2020. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Signal Transduct. Target. Ther. 5, 1.

Li, W., Liu, H., Liang, J., Wang, T., Liu, J., Pi, X., Zou, W., Qu, L., 2023. Effects of atorvastatin on bile acid metabolism in high-fat diet-fed ApoE−/− mice. J. Cardiovasc. Pharmacol. 81, 454-462.

Libby, P., 2021. The changing landscape of atherosclerosis. Nature 592, 524-533.

Lin, K., Zou, C., Hubbard, A., Sengelmann, S., Goudy, L., Wang, I.C., Sharma, R., Pak, J., Foster, K., Ozawa, T., et al., 2025. Multiplexed epigenetic memory editing using CRISPRoff sensitizes glioblastoma to chemotherapy. Neuro. Oncol. 27, 1443-1457.

Liu, C., Chen, J., Chen, H., Zhang, T., He, D., Luo, Q., Chi, J., Hong, Z., Liao, Y., Zhang, S., et al., 2022. PCSK9 inhibition: from current advances to evolving future. Cells 11, 2972.

Liu, T., Zhao, D., Qi, Y., 2022. Global trends in the epidemiology and management of dyslipidemia. J. Clin. Med. 11, 6377.

Liu, X.S., Wu, H., Ji, X., Stelzer, Y., Wu, X., Czauderna, S., Shu, J., Dadon, D., Young, R.A., Jaenisch, R., 2016. Editing DNA methylation in the mammalian genome. Cell 167, 233-247.

Lu, Y., Qu, H., Qi, D., Xu, W., Liu, S., Jin, X., Song, P., Guo, Y., Jia, Y., Wang, X., et al., 2019. OCT4 maintains self-renewal and reverses senescence in human hair follicle mesenchymal stem cells through the downregulation of p21 by DNA methyltransferases. Stem Cell Res. Ther. 10, 28.

Mehta, J.L., Zhou, S., Fan, Y., Deng, X., Theus, S., Wang, X., Liu, S., Ding, Z., 2018. PCSK9 regulates expression of scavenger receptors and ox-LDL uptake in macrophages. Cardiovasc. Res. 114, 1145-1153.

Neumann, E.N., Bertozzi, T.M., Wu, E., Serack, F., Harvey, J.W., Brauer, P.P., Pirtle, C.P., Coffey, A., Howard, M., Kamath, N., et al., 2024. Brainwide silencing of prion protein by AAV-mediated delivery of an engineered compact epigenetic editor. Science 384, 7082.

Nicholls, S.J., Ballantyne, C.M., Barter, P.J., Chapman, M.J., Erbel, R.M., Libby, P., Raichlen, J.S., Uno, K., Borgman, M., Wolski, K., et al., 2011. Effect of two intensive statin regimens on progression of coronary disease. N. Engl. J. Med. 365, 2078-2087.

Nunez, J.K., Chen, J., Pommier, G.C., Cogan, J.Z., Replogle, J.M., Adriaens, C., Ramadoss, G.N., Shi, Q., Hung, K.L., Samelson, A.J., et al., 2021. Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell 184, 2503-2519.

Olmastroni, E., Scotti, S., Galimberti, F., Xie, S., Casula, M., 2024. Ezetimibe: integrating established use with new evidence - a comprehensive review. Curr. Atheroscler. Rep. 27, 10.

Pan, R., Ren, J., Chen, X., Flores, L.F., Gonzalez, R.V.L., Adonnino, A.A., Lofts, B., Waldo, J., Halmai, J., Devinsky, O., et al., 2025. Editing DNA methylation in vivo. Nat. Commun. 17, 527.

Reiner, Z., 2014. Resistance and intolerance to statins. Nutr. Metab. Cardiovasc. Dis. 24, 1057-1066.

Robinson, M.D., McCarthy, D.J., Smyth, G.K., 2010. EdgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139-140.

Robinson, M.D., Oshlack, A., 2010. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 11, R25.

Roth, E.M., Davidson, M.H., 2018. PCSK9 inhibitors: mechanism of action, efficacy, and safety. Rev. Cardiovasc. Med. 19, S31-S46.

Rothgangl, T., Dennis, M.K., Lin, P.J.C., Oka, R., Witzigmann, D., Villiger, L., Qi, W., Hruzova, M., Kissling, L., Lenggenhager, D., et al., 2021. In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels. Nat. Biotechnol. 39, 949-957.

Schafer, E., Irizarry, R., Negi, S., McIntyre, E., Small, D., Figueroa, M.E., Melnick, A., Brown, P., 2010. Promoter hypermethylation in MLL-r infant acute lymphoblastic leukemia: biology and therapeutic targeting. Blood 115, 4798-4809.

Schuster, S., Rubil, S., Endres, M., Princen, H.M.G., Boeckel, J.N., Winter, K., Werner, C., Laufs, U., 2019. Anti-PCSK9 antibodies inhibit pro-atherogenic mechanisms in APOE*3Leiden.CETP mice. Sci. Rep. 9, 11079.

Smith, A., Johnson, D., Banks, J., Keith, S.W., Karalis, D.G., 2021. Trends in PCSK9 inhibitor prescriptions before and after the price reduction in patients with atherosclerotic cardiovascular disease. J. Clin. Med. 10, 3828.

Stelzer, Y., Shivalila, Chikdu S., Soldner, F., Markoulaki, S., Jaenisch, R., 2015. Tracing dynamic changes of DNA methylation at single-cell resolution. Cell 163, 218-229.

Strandberg, T.E., Kovanen, P.T., Lloyd-Jones, D.M., Raal, F.J., Santos, R.D., Watts, G.F., 2024. Drugs for dyslipidaemia: the legacy effect of the Scandinavian Simvastatin Survival Study (4S). Lancet 404, 2462-2475.

Tang, H., Wang, H., Wang, S., Hu, S.W., Lv, J., Xun, M., Gao, K., Wang, F., Chen, Y., Wang, D., et al., 2022. Hearing of Otof-deficient mice restored by trans-splicing of N- and C-terminal otoferlin. Hum. Genet. 142, 289-304.

Tremblay, F., Xiong, Q., Shah, S.S., Ko, C.W., Kelly, K., Morrison, M.S., Giancarlo, C., Ramirez, R.N., Hildebrand, E.M., Voytek, S.B., et al., 2025. A potent epigenetic editor targeting human PCSK9 for durable reduction of low-density lipoprotein cholesterol levels. Nat. Med. 31, 1329-1338.

Tsai, S.Q., Joung, J.K., 2016. Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat. Rev. Genet. 17, 300-312.

Wang, W.Z., Liu, C., Luo, J.Q., Lei, L.J., Chen, M.H., Zhang, Y.Y., Sheng, R., Li, Y.N., Wang, L., Jiang, X.H., et al., 2024. A novel small-molecule PCSK9 inhibitor E28362 ameliorates hyperlipidemia and atherosclerosis. Acta Pharmacol. Sin. 45, 2119-2133.

Wang, X., Liu, S., Sun, Y., Yu, X., Lee, S.M., Cheng, Q., Wei, T., Gong, J., Robinson, J., Zhang, D., et al., 2022. Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery. Nat. Protoc. 18, 265-291.

Ward, N.C., Watts, G.F., Eckel, R.H., 2019. Statin toxicity. Circ. Res. 124, 328-350.

Warden, B.A., Fazio, S., Shapiro, M.D., 2020. The PCSK9 revolution: current status, controversies, and future directions. Trends Cardiovasc. Med. 30, 179-185.

Xu, D., Besselink, S., Ramadoss, G.N., Dierks, P.H., Lubin, J.P., Pattali, R.K., Brim, J.I., Christenson, A.E., Colias, P.J., Ornelas, I.J., et al., 2025. Programmable epigenome editing by transient delivery of CRISPR epigenome editor ribonucleoproteins. Nat. Commun. 16, 7948.

Xu, S., Hu, E., Cai, Y., Xie, Z., Luo, X., Zhan, L., Tang, W., Wang, Q., Liu, B., Wang, R., et al., 2024. Using clusterProfiler to characterize multiomics data. Nat. Protoc. 19, 3292-3320.

Yang, P., Khoshandam, M., Bhia, I., Raji, S., Soltaninejad, H., Hosseinkhani, S., Sani, M., Hamidieh, A.A., Sheykhhasan, M., 2025. Integrating CRISPR/Cas technology with clinical trials: principles, progress, and challenges. Asian J. Pharm. Sci. 20, 101068.

Yin, C., Ackermann, S., Ma, Z., Mohanta, S.K., Zhang, C., Li, Y., Nietzsche, S., Westermann, M., Peng, L., Hu, D., et al., 2019. ApoE attenuates unresolvable inflammation by complex formation with activated C1q. Nat. Med. 25, 496-506.

Zhao, H., Li, Y., He, L., Pu, W., Yu, W., Li, Y., Wu, Y.T., Xu, C., Wei, Y., Ding, Q., et al., 2020. In vivo AAV-CRISPR/Cas9-mediated gene editing ameliorates atherosclerosis in familial hypercholesterolemia. Circulation 141, 67-79.

Zhou, C., Zhu, S., Luo, C., Wang, W., Fan, H., Gao, Y., Xu, X., Wang, Q., You, Y., Xie, T., et al., 2026. From IscB to Cas9: engineering and advances in the next generation of miniature gene editing tools. Biotechnol. Adv. 86, 108743.

Relative Articles

Supplements(0)

Cited By

Proportional views