GENETIC DESIGN OF PIGS AS EXPERIMENTAL MODELS IN THE COMBAT BETWEEN CHRONIC DISEASES AND HEALTHY AGING

Abstract

Chronic, dysregulatory disease processes are becoming a major medical problem in the aging populations of our world. Metabolic syndrome (obesity, diabetes, atherosclerosis, and hypertension), cardiovascular diseases, neurodegeneration (e.g. Alzheimer syndrome), inflammatory diseases, and cancers affect a rapidly increasing number of people. The sequencing of the human genome should help in disease prevention by allowing mapping and characterisation of “illness” and “wellness” gene variants that convey susceptibility or resistance to the dysregulatory and degenerative disease processes. Common chronic disorders are genetically very heterogeneous with some subtypes showing Mendelian inheritance with quite high penetrance. Finding such monogenetic causes will allow truly personalized prevention and treatment. However, genetics is just the first step – functional studies in model systems will be necessary. The pig is an excellent model for medical research as well as for testing of new methods and drugs for disease prevention and treatment. Its size and longevity makes it especially useful for the study of chronic disease processes that can be monitored and repeatedly biopsied for long periods with and without intervention. The genome of different pig breeds have been sequenced, revealing that the pig is genetically more similar to man than conventional laboratory animals – in agreement with the similarities in organ development, physiology and metabolism. Genetically designed minipigs (Göttingen and Yucatan) are obtained by genetic engineering of somatic cells and animal cloning by somatic cell nuclear transfer. Primary minipig fibroblasts are genetically modified in culture by transposon-based transgenesis and/or homologous recombination with AAV-transduced constructs. The designed pig cells are electro-fused with enucleated oocytes (from normal slaughtered pigs) and the reconstructed oocytes develop in vitro into blastocysts that are transferred to surrogate production sows giving birth to clones of the designed pigs. Our HMC (hand made cloning) technology is very cost-efficient and allows large-scale production, without a need for micromanipulation. Thus, minipigs have been produced that should be prone to develop disease processes such as neurodegeneration (dominant negative human AAP and/or PS1 expression), atherosclerosis (human gain-of-function PCSK9 expression, ApoE knockout, LDL-R knockout), inflammation (ectopic expression of human α2 and β1 integrins in suprabasal epidermis), and cancer (BRCA1 knockout). Interesting phenotypes are observed in many of these minipigs. The Yucatan pigs with liver-specific expression of gain-of-function PCSK9, for example, display reduced hepatic LDL-R levels, impaired LDL clearance, severe hypercholesterolemia with accumulation of ApoB100-containing lipoproteins, and spontaneous development of progressive atherosclerotic lesions in multiple vascular beds. The genetic load can be further increased or modulated by breeding or cross-breeding of the different model pigs. We can also produce clones of pigs, some disease prone and some fluorescing, to perform experiments in regenerative medicine where the fate of healthy fluorescent cells can be followed in the, basically identical, disease prone animals. It is also our hope that our pig models can contribute to the digital revolution in medicine, combining detailed genomic sequencing and analysis with the introduction of wireless biosensors and advanced imaging methods. “Digitalized” pigs should teach us how to apply these fantastic new possibilities clinically. We envisage that this will become one of the biggest shake-ups in the history of medicine.