Familial Hypercholesterolemia and Atherosclerosis in Cloned Minipigs Created by DNA Transposition of a Human PCSK9 Gain-of-Function Mutant

Abstract

Lack of animal models with human-like size and pathology hampers translational research in atherosclerosis. Mouse models are missing central features of human atherosclerosis and are too small for intravascular procedures and imaging. Modeling the disease in minipigs may overcome these limitations, but it has proven difficult to induce rapid atherosclerosis in normal pigs by high-fat feeding alone, and genetically modified models similar to those created in mice are not available. D374Y gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene cause severe autosomal dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low-density lipoprotein (LDL) receptor levels, impaired LDL clearance, severe hypercholesterolemia, and spontaneous development of progressive atherosclerotic lesions that could be visualized by noninvasive imaging. This model should prove useful for several types of translational research in atherosclerosis.

Recommendation on Design, Execution, and Reporting of Animal Atherosclerosis Studies: A Scientific Statement From the American Heart Association

“…74,75 These pig models develop extensive atherosclerosis in several arterial regions, including the coronary. The nature of the lesions extends from primarily lipid-laden macrophages to more advanced stages of evaluation that include necrotic core formation, extensive fibrosis, calcification, angiogenesis, and intralesion hemorrhage.…”

Pcsk9

“…A liver-specific expression of this mutant in minipigs resulted in markedly reduced levels of hepatic LDLR, impaired LDL-C clearance, severe hypercholesterolemia, and eventually these minipigs developed spontaneous progressive atherosclerotic lesions that could be visualized by noninvasive imaging. 104 Two conclusions can be drawn from these observations: first, inhibition of PCSK9 would be a potential therapeutic modality; second, the use of large species models will better advance the translational research aspect of treating atherosclerosis from animals to human.…”

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

“…As an alternative to the direct embryo injection described here, both the SB [50][51][52] and the piggyBac 53 transposon systems have been used to genetically modify porcine cells in vitro, which were subsequently used as donors in SCNT for pig transgenesis.…”