Hypertriglyceridemia and atherosclerosis in apolipoprotein A5 mutant rabbits generated using CRISPR/Cas9

Apolipoprotein A5 (APOA5) plays an important role in regulating plasma triglycerides (TGs). Most in vivo data, however, derive from murine models whose lipoprotein metabolism diverges substantially from humans, and no rabbit APOA5 loss-of-function model has previously been described. Microinjection of Cas9 mRNA and two exon-2-targeting single-guide RNAs into rabbit zygotes yielded 10 founders carrying biallelic loss-of-function alleles, and with an approximately 90% reduction in plasma APOA5 protein. On chow, APOA5 mutant rabbits developed a stable atherogenic dyslipidemia with selectively elevated plasma TGs and reduced HDL-C, both more severe in male than in female mutants. The dyslipidemia was paralleled by accumulation of apolipoprotein B-containing lipoproteins, an enlarged VLDL distribution by fast-protein liquid chromatography, and reduced pre-heparin plasma activities of lipoprotein lipase and hepatic lipase together with correspondingly lower plasma FFAs. Mutants also exhibited elevated plasma IL-1β, TNF-α and CCL2 with a selective expansion of circulating monocytes, granulocytes, and platelets, indicating a low-grade systemic inflammatory state already under chow conditions. After 8 weeks on a high-fat, cholesterol-enriched diet, mutants developed severe combined dyslipidemia, extensive aortic atherosclerosis with macrophage- and smooth-muscle-rich plaques, and marked hepatic steatosis. In conclusion, biallelic disruption of rabbit APOA5 produces a translationally relevant phenotype that recapitulates principal features of human APOA5 deficiency. The APOA5 mutant rabbit thus provides a tractable large-animal platform for studying residual cardiovascular risk and for evaluating emerging TG-lowering therapeutics.