Advances in pig models of human diseases

Abstract Animal models of human diseases play a critical role in medical research. Pigs are anatomically and physiologically more like humans than are small rodents such as mice, making pigs an attractive option for modeling human diseases. Advances in recent years in genetic engineering have facilitated the rapid rise of pig models for use in studies of human disease. In the present review, we summarize the current status of pig models for human cardiovascular, metabolic, neurodegenerative, and various genetic diseases. We also discuss areas that need to be improved. Animal models of human diseases play a critical role in medical research. Advances in recent years in genetic engineering have facilitated the rapid rise of pig models for use in studies of human disease. In the present review, we summarize the current status of pig models for human cardiovascular, metabolic, neurodegenerative, various genetic diseases and xenotransplantation.


| INTRODUC TI ON
Research on human disease pathogenesis is critical for progress in therapeutic medicine. Insufficient sample acquisition, environmental conditions, and ethics often impede studies to examine human disease directly, and therefore animal models are crucial for gaining in vivo insight into disease etiology and pathogenesis. Mice and other small rodents have long been important model animals for basic research, and have contributed greatly to our understanding of human disease pathogenesis. However, the limitations of rodent models are many. For example, metabolic rate is influenced by body size, and their small size leads to difficulties in performing surgery and using organs ( Table 1). Considerable differences exist between rodents and humans in the regulatory networks controlling the activity of the immune system, metabolic functions, and responses to stress. 1,2 For example, age-associated fasting blood glucose exhibits differential trends between mice and monkeys/humans. 3 Importantly, more than 80% of potential therapeutics fail in human trials despite showing safety and efficacy in mice. 4 Pigs are one of the most common domestic animals in the world.
Compared to other livestock and primates, pigs have a rapid growth rate, short generation intervals, large litter sizes, and standardized breeding techniques. These advantages, combined with comparable human and pig body sizes, anatomical and physiological characteristics, diets, and genome (Table 1), 5 have driven a gradual rise in the use of pigs as animal models for human diseases.
Similar body and organ sizes between pigs and humans will likely hasten the translation of pig studies (in comparison to mouse studies) to the clinic. Even before the advent of transgenic and gene-editing technology, pig models enabled important advances in human heart, bone, metabolism, and even genetic diseases, to name a few. For example, pig models of acute myocardial infarction (MI) were generated by permanently ligating the trunk near one-third of the apex after the first branch or by inflating an angioplasty balloon in the mid-left anterior descending artery, [12][13][14][15][16] facilitating testing, and development of MI therapies for use in humans. Similarly, bone and cartilage models have been generated through surgically-induced lesions in pigs for the development of biomaterials. 17,18 As both humans and pigs are monogastric omnivores, diet modification has been a fruitful approach for creating pig models of human metabolic disease. A highfat diet (HFD) induces obesity and metabolic syndrome and has been used in pigs to research the renal disease and nonalcoholic fatty liver disease. 19,20 To obtain genetic disease models, ENU chemical mutagenesis has been used to induce a set of point mutations that frequently mimic the subtlety and heterogeneity of human genetic lesions. 21 For example, microphthalmia-associated transcription factor (MITF +/l247s ) mutants mimic Waardenburg's syndrome type II, dual oxidase 2 (DUOX 2 D409G/D409G ) mutants mimic congenital hypothyroidism, SRY-box transcription factor 10 (SOX 10 +/R109W ) mutants mimic Mondini dysplasia, and mutants with a 2 bp CC insertion in the melanocortin receptor 1 (MC1R) mimic albinism. [22][23][24][25] These genetic models are heritable and require no special diet or surgical intervention to obtain experimental animals ( (HNF-1α), which has been reported to cause type III maturity-onset diabetes of the young (MODY3). 33 Although the majority of cloned MODY3 pigs died two weeks after birth, the viable pigs, showed high blood glucose levels and proved useful for studying the disease.

| Atherosclerosis
Atherosclerosis promotes cardiovascular disease, and lipid metabolism disorder is the pathological basis of atherosclerosis. Therefore, understanding abnormal lipid metabolism, such as high blood lipid, high cholesterol, and obesity, is vital. 36,37 Atherosclerosis is usually characterized by the deposition of lipids, cholesterol, and sugar complexes beginning from the intima and histiocytosis, leading to calcification. 38 Low-density lipoprotein and apolipoprotein are closely In addition to abnormal lipid metabolism, atherosclerosis can be caused by abnormal glucose metabolism. 45 In 2017, Yang et al. 46 used zinc finger nuclease technology to create PPARγ mono-allelic knockout pigs, which proved to be a good model for both atherosclerosis and type 2 diabetes. These pig models provide new research opportunities for early asymptomatic human atherosclerosis and other cardiovascular diseases that are difficult to study and treat.

| Myocardial infarction
Myocardial infarction (MI) is a major cause of morbidity and mortal- ing MI. 47 MicroRNAs have proven to be another rewarding avenue for MI research. MiR-590-3p was shown to suppress proliferation, migration, and differentiation of cardiac fibroblasts, whereas 13 MiR-144-3p and microRNA-199a appear to induce these cardiac fibroblast programs. 12,48 At present, most research models of myocardial infarction are disposable models prepared by surgery, which have limitations for long-term use. If a stable genetic model can be developed in the future, the research on myocardial infarction will be greatly accelerated (Table 3).   and hPS1 (M146V and L286P) were generated using the polycistronic vector system. These pigs were similarly found to accumulate Aβ-40

| Neurodegenerative diseases
and Aβ-42 in their brain, 54 a significant phenotype of AD patients.

| Parkinson's disease
Parkinson's disease, also known as paralysis tremors, is a neurode-

| Huntington's disease
Huntington's disease is a rare autosomal dominant genetic disorder.
Due to variations in Huntington protein (HTT), patients typically develop motor symptoms, cognitive dysfunction, and mental disorders.
In 2010, Yang et al. 61 generated HD pigs with HTT mutations that suffered significant involuntary movements. In 2018, Yan et al. 62 found that endogenous expression of full-length HTT mutants in pigs elicited significant neuronal degeneration, which effectively mimics human Huntington's disease. This single gene mutation has resulted in the current pig models that simulate Huntington's disease well. Future use of these models to search for effective treatments will be an important application of these pig models (Table 4).

| Genetic diseases
Genetic diseases generally refer to diseases caused by changes in genetic material or disease genes. In addition to the metabolic diseases and neurodegenerative diseases discussed above, pig models of cystic fibrosis, Duchenne muscular dystrophy, hemophilia, and various cancers have also been developed for medical research.

| Cystic fibrosis
Cystic fibrosis (CF), a recessive genetic disease with a single gene mutation, is caused by dysfunction of the CF transmembrane conductance regulator (CFTR). The disease starts in early childhood and affects many tissues and organs, including the respiratory tract, lungs, gastrointestinal tract, pancreas, liver, reproductive tract,  Pig DMD models hold great promise in the development of drugs and treatments for DMD. Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that often causes premature aging and cardiovascular complications. Introducing heterozygous mutations of the LMNA gene into pigs induced growth retardation, lipodystrophy, skin and bone changes, cardiovascular disease, and death in adolescence. 86 The mean lifespan of these pigs is just about 6 months, making them good models for longevity studies in clinics.

| Cancer
Loss-of-function mutations in the COL2A1 gene are the etiology of type II collagenopathy. COL2A1 mutant pigs exhibit bone dysplasia and tracheal collapse, modeling aspects of human spondyloepiphyseal dysplasia and stickler syndrome type I. 87 Waardenburg's disease is a syndrome of deafness, white hair, and eye disease. Wang et al. 88

| Xenotransplantation
One and has provided important materials for the advancement of xenotransplantation. [112][113][114][115][116][117] Solid organ xenotransplantation between pig and non-human primates is also a key research priority before human clinical trials. In recent years, with the development of xenotransplantation, several types of solid organ xenotransplantation have been tested in non-human primates with some success, including heart, 118,119 kidney, 120 lung, 121 and liver. 122 Even more exciting, the world's first gene-edited pig heart transplant into a human was carried out in January 2022. Although the patient died unfortunately after two months, this is still a milestone in the search for a solution to the shortage of human organs. Almost at the same time, the world's first pig kidney transplant into a human was reported. 123 We expect that in the future, gene-modified pigs will certainly provide new opportunities for the shortage of human organs (Table 6). Inactivation of porcine endogenous retroviruses PERV Knockout 115 in pig models for studying genetic background and for testing drugs, therapeutics, and methods of delivery, safety, and ethical issues cannot be ignored. On the one hand, humans and pigs are different in many ways, and drugs and treatments developed in pig models must be determined to be safe before clinical tests. On the other hand, because of the existence of zoonosis, care must be taken at every stage of the experiment to avoid cross-contamination and the spread of disease. Apart from safety and ethical issues, animal welfare also affects society's willingness to condone animal research. The health of the animal used as a model is not only critical to obtaining reliable results but is also a responsibility for every researcher. Improving the nutrition, physical environment, health, behavioral interactions, and mental state of pigs will promote the development and social acceptance of pig models. 125 By addressing the importance of these issues, pig models will continue to be an important source of support for the advancement of human medicine in the future.

ACK N OWLED G M ENT
We thank Dr. Lara Carroll (University of Utah) for the careful read-

CO N FLI C T O F I NTE R E S T
The authors declared no conflicts of interest.