Dysregulated metabolic pathways implicated in coronary artery disease (CAD) and diabetes mellitus represent tractable therapeutic targets whose modulation has the potential to reduce disease progression and associated global health burden. By integrating human genetic discovery with tissue-specific in vivo perturbation, we aimed to distinguish causal disease drivers from proteins that are circulating biomarkers. To address this, we employed a multi-ancestry genetic meta-analysis of plasma proteins associated with CAD or DM risk in participants from the UK Biobank and All of Us cohorts. This analysis identified established causal mediators, including APOE and PCSK9, alongside novel candidate plasma proteins significantly associated with cardiometabolic disease risk. One such candidate was ITIH4, a liver-enriched secreted factor associated with an increased risk of CAD.
To investigate the functional role of ITIH4, we harnessed CRISPR gene-editing technology delivered using lipid nanoparticles (LNPs) to the liver of mice. Cas9 transgenic mice were injected with LNP-encapsulated guide RNAs targeting eGFP (control), the low-density lipoprotein receptor (LDLR), or LDLR and ITIH4 in combination. Targeted silencing of the LDLR in combination with western diet feeding for 20 weeks was used to induce hypercholesterolaemia and atherosclerotic phenotypes. At the study endpoint, liver and plasma were collected for molecular and metabolic analyses.
CRISPR editing produced a 60% reduction in hepatic LDLR protein expression compared with controls (p<0.0001). Dual targeting reduced hepatic LDLR and ITIH4 expression by 55% (p<0.001) and 85% (p<0.0001) respectively and a 90% (p<0.01) decrease in circulating ITIH4. Reduced hepatic LDLR expression increased plasma cholesterol concentrations (7.70, 9.82 and 8.20 mmol/L in control, LDLR-targeted and dual-targeted mice, respectively; p<0.01). Importantly, concurrent ITIH4 depletion attenuated this increase and reduced multiple disease-associated plasma lipid species, including cholesterol.
Building on this liver-targeted proof of concept, we evaluated new LNP formulations for delivering CRISPR machinery to metabolically relevant extrahepatic tissues, including the pancreas. As an initial step in targeting the pancreas, we selected the murine insulin genes INS1 and INS2. These genes serve as positive controls, with successful editing expected to disrupt insulin production and alter blood glucose, thereby providing a direct functional readout of pancreatic CRISPR activity.
Collectively, these findings suggest that CRISPR-mediated depletion of ITIH4 may protect against hypercholesterolaemia that promotes CAD. More broadly, our platform provides a feasible and cost-effective strategy for validating candidate disease genes in vivo and expanding tissue-directed CRISPR editing across the liver and pancreas to uncover new mechanisms and therapeutic targets for cardiometabolic disease.