Atherosclerosis Gene-Edited Cell Models for Drug Discovery

Atherosclerosis is the primary cause of cardiovascular diseases (CVD), which remain the leading cause of death globally. According to the World Health Organization (WHO), an estimated 17.9 million people died from CVDs in 2019, representing 32% of all global deaths. Of these, 85% were due to heart attack and stroke, both driven by atherosclerosis. Key risk factors include hyperlipidemia, hypertension, smoking, diabetes, and obesity. The disease progresses silently over decades, with clinical events often occurring suddenly. Five-year survival after a major cardiovascular event varies significantly by age and comorbidities; for example, post-myocardial infarction, the 5-year survival rate is approximately 70-80% in high-income countries (NCI SEER data for heart disease). The chronic, progressive nature of atherosclerosis makes it an ideal target for mechanistic studies and drug development.

Atherosclerosis is a complex, multi-factorial disease involving endothelial dysfunction, lipid accumulation, inflammation, and smooth muscle cell proliferation. Its pathogenesis is driven by both genetic and environmental factors. Public datasets such as the Genome-Wide Association Studies (GWAS) Catalog and the NCBI Gene database have identified numerous risk loci (e.g., 9p21, PCSK9, LDLR, APOE). Open questions include the exact mechanisms of plaque rupture, the role of clonal hematopoiesis, and the interplay between immune cells and vascular cells. Gene-edited cell models are essential for dissecting these pathways in a controlled, human-relevant system.

Atherosclerosis development involves several interconnected pathways:

• • Lipoprotein Retention and Modification:
• • Low-density lipoprotein (LDL) particles infiltrate the arterial intima.
• • LDL undergoes oxidation (oxLDL) by reactive oxygen species.
• • oxLDL is taken up by macrophages via scavenger receptors (e.g., SR-A, CD36), forming foam cells.
• • Endothelial Dysfunction:
• • Reduced nitric oxide (NO) bioavailability.
• • Increased expression of adhesion molecules (VCAM-1, ICAM-1).
• • Enhanced permeability to lipoproteins and leukocytes.
• • Inflammatory Cascade:
• • Monocyte recruitment and differentiation into macrophages.
• • Macrophage activation via toll-like receptors (TLRs) and inflammasomes (NLRP3).
• • Secretion of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6).
• • Smooth Muscle Cell (SMC) Phenotypic Switching:
• • SMCs migrate from media to intima.
• • They transition from contractile to synthetic phenotype.
• • SMCs contribute to fibrous cap formation and plaque stability.

While atherosclerosis is not a monogenic disease, several genes harbor common variants that significantly influence risk. Key alterations include:

Data sources: ClinVar, NCBI Gene, GWAS Catalog.

Atherosclerosis involves the dysregulation of several key signaling networks:

• • NF-κB Pathway:
• • Central mediator of inflammation.
• • Activated by oxLDL, cytokines, and shear stress.
• • Upregulates adhesion molecules, chemokines, and pro-inflammatory genes.
• • PI3K/AKT/mTOR Pathway:
• • Regulates cell survival, proliferation, and metabolism.
• • Dysregulated in SMCs and macrophages.
• • Promotes foam cell formation and plaque progression.
• • MAPK/ERK Pathway:
• • Involved in SMC proliferation and migration.
• • Activated by growth factors (PDGF, FGF) and mechanical stress.
• • NLRP3 Inflammasome:
• • Activated by cholesterol crystals and oxLDL.
• • Leads to IL-1β and IL-18 secretion.
• • Key driver of vascular inflammation.

Commonly used cell lines for atherosclerosis research include:

Organoids: 3D vascular organoids derived from iPSCs are emerging as advanced models that recapitulate vessel structure and cell-cell interactions, enabling more physiologically relevant studies of atherosclerosis.

Animal models are critical for in vivo atherosclerosis research:

• • Genetically Engineered Mouse Models (GEMMs):
• • ApoE-/- mice: Spontaneously develop atherosclerotic lesions on a chow diet.
• • LDLR-/- mice: Develop lesions on a high-fat diet.
• • PCSK9-/- mice: Reduced LDL-C and protection from atherosclerosis.
• • Induced Models:
• • Partial carotid artery ligation: Induces flow disturbance and rapid plaque formation.
• • Angioplasty balloon injury: Used for restenosis studies.
• • Patient-Derived Xenograft (PDX) Models:
• • Limited use in atherosclerosis due to the systemic nature of the disease.
• • Human artery segments can be implanted into immunodeficient mice for vascular biology studies.

CRISPR-Cas9 technology enables the creation of isogenic cell lines with precise genetic modifications, providing powerful tools for studying atherosclerosis. Examples include:

• • LDLR Knockout (LDLR-/-) in HepG2 cells: Models familial hypercholesterolemia; used to study LDL uptake and cholesterol metabolism.
• • PCSK9 Gain-of-Function (e.g., D374Y) in HepG2 cells: Models hypercholesterolemia; used for drug screening of PCSK9 inhibitors.
• • APOE Knockout (APOE-/-) in THP-1 macrophages: Models impaired lipoprotein clearance; used to study foam cell formation.
• • IL-1β Knockout in THP-1 cells: Used to study the role of inflammation in atherosclerosis.

Commercially available, sequence-verified gene-edited cell models accelerate research by providing ready-to-use, validated tools. These models are generated using CRISPR-Cas9 ribonucleoprotein (RNP) complexes and are confirmed by Sanger sequencing and functional assays. They enable reproducible, high-throughput studies without the need for time-consuming in-house gene editing.

Gene-edited cell lines are essential for validating the role of candidate genes identified from GWAS and other genomic studies. For example:

• • LDLR knockout in HepG2 cells confirmed its essential role in LDL uptake and cholesterol homeostasis.
• • PCSK9 knockout in HepG2 cells demonstrated increased LDLR expression and enhanced LDL clearance.
• • APOE knockout in THP-1 macrophages showed increased foam cell formation and altered cytokine secretion.

These models allow researchers to directly assess the impact of specific genetic variants on cellular phenotypes relevant to atherosclerosis.

Isogenic cell line pairs (e.g., wild-type vs. PCSK9 gain-of-function) are powerful tools for drug screening:

• • PCSK9 D374Y knock-in HepG2 cells are used to screen for small molecule inhibitors of PCSK9.
• • LDLR-/- HepG2 cells are used to test LDLR-independent cholesterol-lowering strategies.
• • Resistance modeling: Gene-edited cells can be used to study mechanisms of resistance to statins or PCSK9 inhibitors by introducing specific mutations in target genes.

CRISPR-based screens in relevant cell types can identify novel biomarkers and therapeutic targets:

• • Synthetic lethality screens: For example, identifying genes that become essential in the context of LDLR deficiency, revealing potential drug targets for familial hypercholesterolemia.
• • Secretome analysis: Conditioned media from gene-edited macrophages (e.g., NLRP3 knockout) can be analyzed to identify novel inflammatory biomarkers.
• • Epigenetic editing: CRISPR-dCas9 fusion proteins can be used to modulate gene expression and identify epigenetic regulators of atherosclerosis.