A porcine model of Becker muscular dystrophy cardiomyopathy.

Abstract

To establish a model large animal of Becker muscular dystrophy (BMD) cardiomyopathy, especially a model pig (male pig) often used for evaluation of a blood circulation system and / or a carrier pig (female pig) keeping the model pig as a strain, and to provide a method for sorting mother pigs giving birth to BMD model pigs.SOLUTION: A method for sorting mother pigs giving birth to BMD model pigs includes: administering an anesthetic to male piglets born from the mother pigs; and using decline in the cardiac function induced by the administration as an index. The sorting method of the mother pigs is established and on the basis of the sorting method, a male pig as the BMD cardiomyopathy model pig and a carrier female pig of the cardiomyopathy are constructed, both pigs characterized by having mutated dystrophin genes obtained by artificially inducing deletion of exon 21-28 regions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for selecting sows that will give birth to a pig model of Becker muscular dystrophy cardiomyopathy; a female pig that is a carrier of Becker muscular dystrophy cardiomyopathy, characterized by having a mutant dystrophin gene lacking exons 21 to 28, and eggs or fertilized eggs collected from the female pig; or a male pig that serves as a pig model of Becker muscular dystrophy cardiomyopathy, characterized by having the mutant gene, and sperm collected from the male pig. [Background technology]

[0002] Muscular dystrophy is a general term for hereditary muscle disorders in which muscle fibers undergo repeated destruction and degeneration (myo-necrosis) and regeneration, gradually leading to progressive muscle atrophy and muscle weakness. The primary pathology is skeletal muscle degeneration and necrosis, and clinically, progressive muscle weakness is observed. Muscular dystrophy is classified into various disease types based on factors such as age of onset, inheritance pattern, and clinical course, and was designated an intractable disease in July 2015. While the disease is further classified based on inheritance pattern, the most common type is caused by mutations in the dystrophin gene.

[0003] Dystrophin is a cytoplasmic protein that forms part of a protein complex known as the costamere. This complex connects the muscle fiber cytoskeleton to the surrounding extracellular matrix across the cell membrane. Dystrophin deficiency or mutation leads to abnormalities in the intracellular signaling pathway, resulting in irreversible muscle fiber necrosis, muscle weakness, and fatigue. Because the dystrophin gene is located on the X chromosome and is inherited recessively, boys develop symptoms. Severe cases are diagnosed as Duchenne muscular dystrophy (DMD), while milder cases are diagnosed as Becker muscular dystrophy (BMD).

[0004] DMD is a childhood disease caused by a complete deficiency of dystrophin. Patients become unable to walk around the age of 10, have a short life expectancy of less than 30 years, and are fatal. In contrast, BMD shares the same pathology as Duchenne, but the onset of symptoms such as muscle weakness is later, the progression of symptoms is slower, and patients become unable to walk until their late 20s. Like Duchenne, dystrophin is abnormal, but while dystrophin is barely expressed in DMD, BMD is known to produce abnormal dystrophin, such as partial deficiency, or to have reduced expression. This is thought to be the cause of the differences in symptoms between the two diseases.

[0005] Although the prognosis for BMD is far better than that for DMD, some cases of BMD have been reported in which heart failure appears earlier than limb muscle weakness, and in some cases heart failure has been reported as the initial symptom. Heart failure accounts for only 20% of deaths in DMD patients, but 50% in BMD patients, so controlling cardiac function is extremely important in the disease management of BMD patients.

[0006] In May 2020, Japan's first DMD treatment (viltolarsen) was launched (Non-Patent Document 1). DMD develops when a point mutation converts a single codon into a stop codon, terminating protein synthesis, preventing the synthesis of dystrophin protein. This treatment restores the production of mutant dystrophin, skipping exon 53, in DMD patients who lack dystrophin by converting an out-of-frame deletion in the dystrophin gene into an in-frame deletion. This is expected to slow the progression of DMD and improve symptoms. In other words, with the development of such novel therapeutic agents and treatment methods, severe DMD symptoms will become more similar to milder BMD. Therefore, in the treatment of not only BMD but also DMD, it is expected that management of cardiac dysfunction, such as cardiomyopathy, which is the greatest risk factor for sudden death in BMD, will become increasingly important in disease management after remission of DMD, which is normally fatal. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 2000-316420 [Non-patent literature]

[0008] [Non-Patent Document 1] Nippon Shinyaku Co., Ltd., May 20, 2020, News Release, https: / / www.nippon-shinyaku.co.jp / file / download.php?file_id=3109 [Non-patent document 2] Teramoto et al., 2020, Disease Models & Mechanisms, vol. 13, 9, dmm0444701 [Non-patent document 3] Rady Ho et al., 2016, World Journal of Cardiology, vol.8, 6, pp.356-361 [Non-patent document 4] Yamashita et al., 1976, Anaesthesist, vol. 25, pp. 76-79 Summary of the Invention [Problem to be solved by the invention]

[0009] As mentioned above, in both the treatment of BMD and the treatment process of DMD, it is extremely important to advance research into the control of the decline in cardiac function that is characteristic of the disease, as exemplified by cardiomyopathy, etc. In general, animal models that mimic the disease are known to be very useful tools for studying the mechanisms of the disease and methods of prevention and treatment. To date, animal models of muscular dystrophy for DMD have been created using a variety of animals, including mice, dogs, and pigs, contributing to the development of treatments. In particular, models using large animals such as dogs and pigs play a very important role in conducting pharmacological and performance tests as a preliminary step to human clinical trials after testing using small animal models such as mice. In contrast, although a rat model of BMD was reported in 2020 (Non-Patent Document 2), no animal models using large animals have been reported to date.

[0010] One of the reasons why it is difficult to create an animal model of BMD is that it is not clear what mutations in the causative gene, dystrophin, are suitable for creating a BMD animal model. Since DMD is a disease caused by a complete deficiency of dystrophin, it is relatively easy to create a DMD animal model by adding some kind of mutation that results in almost no dystrophin expression. In contrast, because BMD is a disease caused by partial deficiency or reduced expression of dystrophin, there are many unknowns regarding what mutations in the dystrophin gene would enable an animal model that reflects the pathological characteristics of intrinsic BMD. BMD model rats, which have already been reported as disease models, express only truncated dystrophin protein by inducing an in-frame mutation at a specific site in the dystrophin gene. However, compared to wild-type rats, no differences in muscle function or the onset of heart failure symptoms have been confirmed, raising many questions about their feasibility as a BMD animal model. Furthermore, it is unclear whether BMD model animals can be created in large animals, similar to rats, by modifying the dystrophin gene containing the mutation. Furthermore, there have been no reports of other dystrophin gene mutations that could be used to create BMD model animals.

[0011] Furthermore, human muscular dystrophy, caused by dystrophin gene mutations, is known to be recessively inherited and primarily occurs only in males, regardless of whether it is DMD or BMD, because the dystrophin gene is localized on the X chromosome. Therefore, even in BMD model animals caused by a dystrophin mutant gene, the disease only develops in male animals carrying the mutant gene, while female animals heterozygous for the mutant gene do not generally develop the disease, and the model can be maintained for subsequent generations. Therefore, the former male animals can be used as BMD model animals for various evaluations and analyses, and the latter female animals can serve as carrier animals to maintain the BMD model animal lineage.

[0012] In view of the above circumstances, an object of the present invention is to establish a large animal model of BMD, particularly a model pig (male pig) that is often used for evaluating the blood circulatory system, and / or a carrier pig (female pig) that maintains the model pig as a lineage. Another object of the present invention is to establish a method for selecting sows that will give birth to the BMD model pigs. [Means for solving the problem]

[0013] The inventors conducted research to address the above-mentioned problem of establishing a disease model pig with characteristics of BMD, particularly a model pig with cardiomyopathy characteristic of BMD or some kind of cardiac dysfunction similar to that of the disease. As a result, they found that, rather than using a conventional method (Patent Document 1) that uses molecular genetic techniques, such as introducing a causative gene known in humans into the animal's genes, it is more effective to find a BMD model pig or a carrier pig that will give birth to such a model pig by selecting pigs that meet some indicators that characterize the disease from a large number of pigs with various genetic backgrounds, since a dystrophin gene mutation that induces a BMD model in a large animal has not yet been identified.

[0014] Many cases of BMD have been reported in which heart failure appears earlier than limb muscle weakness, and in which heart failure is the initial symptom (Non-Patent Document 3). In particular, anesthesia during surgery is known to pose a risk of sudden death due to myocardial damage and postoperative pulmonary infections (Non-Patent Document 4), making it a disease that presents difficulties in anesthesia management.

[0015] Therefore, the inventors conducted extensive research, suspecting that certain anesthesia conditions might induce some kind of decline in cardiac function as an indicator of BMD. As a result, various anesthesia procedures were administered to male litters born to sows that could potentially give birth to BMD model pigs. For example, when a triple anesthetic (medetomidine hydrochloride, midazolam, and butorphanol tartrate) was administered intramuscularly, approximately half of the pigs died of cardiac failure. However, when anesthesia was induced by intramuscular administration of alphaxalone and diazepam and maintained by inhalation administration of isoflurane, cardiac dysfunction symptoms such as cyanosis and arrhythmia were observed. Then, pathological autopsies and immunohistochemistry using anti-dystrophin antibodies were performed on male piglets that had been found to have these abnormalities. These piglets were found to have characteristics of BMD, such as degenerative lesions in skeletal and cardiac muscles and reduced dystrophin expression. Furthermore, the piglets also exhibited symptoms of cardiomyopathy, such as right ventricular dilatation. Therefore, the piglets were identified as BMD cardiomyopathy model pigs.

[0016] Genetic analysis of the BMD cardiomyopathy pig model revealed that it produced dystrophin lacking exons 21-28. A BMD animal model with this dystrophin deletion had not been reported before, and it was found to be a completely new strain. The inventors further investigated whether artificial induction of this mutation could produce a BMD cardiomyopathy pig model, leading to the completion of this invention.

[0017] Based on the above findings, the present invention has been completed. That is, the present invention relates to the following (1) to (23). (1) A method for selecting a sow that will give birth to a Becker muscular dystrophy cardiomyopathy model pig, characterized in that the method uses the induction of cardiac dysfunction as an indicator by administering an anesthetic to the male piglets given birth to by the sow. (2) The method for selecting a sow according to (1), wherein the sow carries a mutated dystrophin gene. (3) The selection method according to (2), wherein the mutation is a mutation that deletes the exon 21 to 28 region of the dystrophin gene. (4) A selection method described in any one of (1) to (3), characterized in that the administration of the anesthetic is intramuscular administration of a triple anesthetic (medetomidine hydrochloride, midazolam, and butorphanol tartrate), and the decline in cardiac function is heart failure. (5) A selection method described in any of (1) to (3), characterized in that the administration of anesthetic agents involves inducing anesthesia by intramuscular administration of alphaxalone and diazepam, followed by maintaining the anesthesia state by inhalation administration of isoflurane, and the cardiac dysfunction is cyanosis and / or arrhythmia. (6) A selection method described in any one of (2) to (5), characterized in that before administering the anesthetic to the male piglet, dystrophin expression analysis in the muscle and / or pathological examination of the muscle are performed on the male piglet, and only male piglet with reduced dystrophin expression and / or muscle degenerative lesions are pre-selected, and the anesthetic is administered only to the selected male piglet. (7) A method for selecting sows carrying Becker muscular dystrophy cardiomyopathy, comprising: conducting dystrophin expression analysis in the muscles and / or histopathological examination of the muscles of sows born from sows selected by the selection method described in any one of (2) to (6); and selecting sows with reduced dystrophin expression and / or degenerative lesions in the muscles as sows carrying Becker muscular dystrophy cardiomyopathy. (8) A method for selecting male pigs to become model pigs for Becker muscular dystrophy cardiomyopathy, comprising: conducting dystrophin expression analysis in the muscles and / or histopathological examination of the muscles of male piglets born from sows selected by the selection method described in any one of (2) to (6); and selecting male piglets with reduced dystrophin expression and / or degenerative lesions in the muscles as male pigs to become model pigs for Becker muscular dystrophy cardiomyopathy. (9) Oocytes or fertilized eggs selected by the method according to any one of (1) to (6) above, which have been artificially collected, stored and / or frozen from a sow that gives birth to a Becker muscular dystrophy cardiomyopathy model pig. (10) A female pig carrier of the cardiomyopathy, which is the offspring of a sow that gives birth to a Becker muscular dystrophy cardiomyopathy model pig selected by the method according to any one of (1) to (6). (11) Oocytes or fertilized eggs artificially collected, stored and / or frozen from a female pig carrying Becker muscular dystrophy cardiomyopathy described in (10). (12) A boar that will become a Becker muscular dystrophy cardiomyopathy model pig, which is an offspring of a sow that gives birth to the cardiomyopathy model pig selected by the method according to any one of (1) to (6). (13) Sperm artificially collected, stored and / or frozen from a boar that will be used as a Becker muscular dystrophy cardiomyopathy model pig according to (12). (14) A female pig carrier of Becker muscular dystrophy cardiomyopathy according to (10), characterized in that it has a mutant dystrophin gene lacking exons 21 to 28. (15) Oocytes or fertilized eggs artificially collected, stored and / or frozen from a sow carrying Becker muscular dystrophy cardiomyopathy according to (14). (16) A male pig serving as a Becker muscular dystrophy cardiomyopathy model pig according to (12), characterized by having a mutant dystrophin gene lacking exons 21 to 28. (17) Sperm artificially collected, stored and / or frozen from a boar that will be used as a Becker muscular dystrophy cardiomyopathy model pig according to (16). (18) A method for screening or evaluating therapeutic agents for Becker muscular dystrophy cardiomyopathy, comprising administering a drug to a cardiomyopathy model pig that is the offspring of a sow that gives birth to a Becker muscular dystrophy cardiomyopathy model pig selected by the method described in any one of (1) to (6), and evaluating the drug using an anesthetic to induce cardiac dysfunction and / or a cardiac injury marker in the serum as an indicator. (19) A female pig carrier of Becker muscular dystrophy cardiomyopathy, characterized in that the female pig is a carrier of the cardiomyopathy and has a mutant dystrophin gene in which a deletion in the exon 21 to 28 region has been artificially induced. (20) Eggs or fertilized eggs collected, managed, and / or frozen from a sow carrier of Becker muscular dystrophy cardiomyopathy according to (19). (21) A male pig that serves as a Becker muscular dystrophy cardiomyopathy model pig, characterized in that the male pig has a mutant dystrophin gene in which a deletion in the exon 21 to 28 region has been artificially induced. (22) Sperm collected, managed, and / or frozen from a boar that will be used as a Becker muscular dystrophy cardiomyopathy model pig according to (21). (23) A method for screening or evaluating therapeutic agents for Becker muscular dystrophy cardiomyopathy, comprising administering a drug to a pig model of Becker muscular dystrophy cardiomyopathy described in (16) or (21) and evaluating the drug using as an indicator the induction of cardiac dysfunction by administering an anesthetic and / or cardiac damage markers in serum. [Effects of the Invention]

[0018] According to the present invention, it is possible to select sows that give birth to BMD cardiomyopathy pig models, and to secure novel BMD cardiomyopathy boar model pigs and / or BMD cardiomyopathy carrier sows that give birth to such model boars, which have been difficult to find in the past. Furthermore, by using the BMD cardiomyopathy pig model caused by dystrophin lacking the exons 21-28 region according to the present invention as a pathological model of the disease, it can contribute to the development of effective and safe therapeutic agents for muscular dystrophy. [Brief explanation of the drawings]

[0019] [Figure 1] A male piglet and its heart that died of cardiac failure due to anesthesia stress. Left: A male piglet that died with cyanosis. Right: A severely dilated right ventricle. [Figure 2] Pathological images of the skeletal muscle (left) and cardiac muscle (right) of a male piglet that died of cardiac failure due to anesthesia stress. Degeneration (arrows) and regeneration (arrowheads) are observed in the skeletal muscle. The cardiac muscle is severely atrophied and tortuous. [Figure 3] Immunostaining of dystrophin in the myocardium of a normal male piglet (left), a sow (center), and a dead male piglet (right). Compared to the normal male piglet, dystrophin expression is significantly reduced in the hearts of the sow and the dead male piglet. DETAILED DESCRIPTION OF THE INVENTION

[0020] A first aspect of the present invention is a method for selecting a sow that will give birth to a BMD cardiomyopathy model pig, characterized in that the method uses as an indicator the induction of cardiac dysfunction by administering an anesthetic to the male piglets given birth to by the sow.

[0021] In the present invention, "cardiomyopathies" refers to myocardial diseases accompanied by cardiac dysfunction. Generally, cardiomyopathy is classified into hypertrophic, dilated, restrictive, and arrhythmogenic right ventricular cardiomyopathy, as well as unclassifiable cardiomyopathy. The cardiomyopathy of the present invention is not particularly limited to any of these types. However, since many cases of human BMD cardiomyopathy are of the dilated type, the cardiomyopathy of the present invention is preferred. Becker muscular dystrophy (BMD) cardiomyopathy is cardiomyopathy caused by a disease known as BMD. It may also be, but is not limited to, cardiomyopathy that develops due to impaired myocardial function caused by progressive necrosis of muscle (muscle fibers), a symptom of muscular dystrophy.

[0022] The BMD cardiomyopathy pig model is a pig model that mimics cardiomyopathy caused by a disease called BMD. Originally, BMD is caused by a mutation in the dystrophin gene, and since the dystrophin gene is located on the X chromosome, it is recessively inherited and is generally known to occur only in males. Therefore, the BMD cardiomyopathy pig model that mimics this not only has the characteristics of BMD cardiomyopathy described in

[0021] , but also has a characteristic that is generally only observed in male pigs. This means that the characteristics of the model pig are induced by a dystrophin mutant gene and / or a recessively inherited causative gene that is similarly located on the X chromosome. This also means that the disease does not generally occur in sows that heterozygously carry the causative gene, and that the disease can be maintained for generations using such sows. The "BMD cardiomyopathy carrier sow" described below according to the present invention is a sow that heterozygously carries the causative gene, including the dystrophin mutant gene, and the "sow that gives birth to a BMD cardiomyopathy pig model" according to the present invention also corresponds to such a sow.

[0023] As described above, the sow that gives birth to the BMD cardiomyopathy pig model of the present invention need only have a heterozygous recessive causative gene that is localized on the X chromosome and causes BMD cardiomyopathy. The causative gene is not particularly limited, but may be a mutant dystrophin gene, preferably a deletion in the exon 21-28 region, or a deletion, duplication, or point mutation / microdeletion of a gene in-frame with no deviation in the mRNA reading frame (in-frame), and particularly preferably a mutant dystrophin gene lacking the exon 21-28 region.

[0024] In addition, the "mutated dystrophin gene" according to the present invention is not limited to a mutation in the dystrophin gene itself encoded on the genome, but also includes all dystrophin proteins that have some kind of mutation (e.g., the structure or expression level of the dystrophin protein) due to a defect in the transcription stage from the dystrophin gene to mRNA and / or the translation stage from mRNA to protein. For example, a mutant dystrophin gene lacking the exons 21 to 28 region may be a dystrophin gene encoded on the genome that lacks the exons 21 to 28 region (in this case, it may include all dystrophin genes with differences in the remaining introns), but it may also include cases where a dystrophin protein is expressed that lacks the portion of the protein corresponding to the exons 21 to 28 region due to some defect in the process of transcription and translation from the dystrophin gene to the dystrophin protein.

[0025] In the present invention, anesthesia refers to the artificial elimination of sensations, including pain, by means of drugs or the like, and an anesthetic is a medicine used for anesthesia. Generally, anesthetics are classified into general anesthetics and local anesthetics, but the present invention does not particularly limit the scope of the present invention. The primary purpose of the anesthetic according to the present invention is to induce some kind of cardiac dysfunction in the administered animal by administering the anesthetic. Therefore, a preferred anesthetic in the present invention is an anesthetic that has a sedative effect to such an extent that some cardiac function can be measured in the administered animal, and that can induce some kind of cardiac dysfunction in the administered animal. The inventors administered various anesthetics under various conditions to male piglets born from various sows they owned, and as a result, they found several preferred anesthetics and administration conditions. These include, but are not limited to, intramuscular administration of a triple anesthetic mixture consisting of medetomidine hydrochloride, midazolam, and butorphanol tartrate, intramuscular administration of alphaxalone and diazepam followed by inhalation of isoflurane, intravenous administration of propofol followed by intramuscular administration of medetomidine hydrochloride and midazolam, and intramuscular administration of maflopan followed by inhalation of isoflurane. In particular, with regard to intramuscular administration of a triple anesthetic mixture consisting of medetomidine hydrochloride, midazolam, and butorphanol tartrate, the mixing ratio of each anesthetic may be, for example, at least 10% of each anesthetic, preferably with the amount of medetomidine hydrochloride being equal to or greater than that of the other two anesthetics. For example, a preferred administration ratio is medetomidine hydrochloride:midazolam:butorphanol tartrate = 3:2:2 (ml), and the dosage is, for example, 0.05 to 5 mg / kg, preferably 0.05 to 0.5 mg / kg, and for example, a preferred dosage is 0.1 ml / kg. Furthermore, with regard to intramuscular administration of alphaxalone and diazepam, for example, diazepam may be present in 5 to 50% of the total dosage, preferably 5 to 20%, and preferred dosages are alphaxalone 5 mg / kg and diazepam 0.5 mg / kg, but are not limited thereto. There is no particular limitation on the frequency of administration, and the entire amount may be administered in a single dose or in multiple divided doses, with the entire amount preferably being administered in a single dose.

[0026] The cardiac function of the present invention refers to the function of the heart, a muscular organ, to circulate blood to various parts of the body, like a pump, through rhythmic contractions of the cardiac muscle (myocardium). Therefore, the cardiac dysfunction of the present invention refers to a state in which, for some reason, the heart is unable to fully exert its pumping function. While not particularly limited, a suitable example is cardiac failure, a state in which the heart is unable to pump enough blood to the body. Cardiac failure can be divided into acute and chronic heart failure. In the former case, symptoms such as severe dyspnea appear within a short period of time, and in severe cases can be fatal, while in the latter case, symptoms such as palpitations and shortness of breath are observed. Although not particularly limited, cardiac failure of the present invention is preferably acute heart failure. Furthermore, organic disorders such as myocardial infarction, in which an insufficient blood supply causes the heart to become undernourished and partially die, and cardiac hypertrophy, in which the heart muscle enlarges and its weight increases; phenomenal disorders such as cyanosis (including both central cyanosis and peripheral cyanosis), in which the skin and mucous membranes turn blue-purple when the oxygen concentration in the blood decreases; arrhythmia, in which the heart rate and / or rhythm are irregular and / or there are abnormalities in the electrocardiogram; abnormalities detected by echocardiography, such as decreased cardiac contractility and blood reflux within the heart, and a significant increase in serum cardiac damage markers, may also be included, but are not limited to these.

[0027] Various combinations can be envisioned for the types of anesthetics and their administration conditions, and the items that indicate a decline in cardiac function that serve as indicators during the administration of the anesthesia. A person skilled in the art can determine an appropriate combination by conducting preliminary experiments to examine the many items that indicate a decline in cardiac function under various anesthetic administration conditions. For example, in this study, a triple-anesthesia mixture (medetomidine hydrochloride, midazolam, and butorphanol tartrate) was administered intramuscularly to male piglets from a litter born to a sow that had the potential to give birth to a BMD cardiomyopathy model pig. Approximately half of the piglets showed cyanosis after waking up from anesthesia, and pathological autopsies revealed signs of heart failure. In addition, male piglets born to the same mother were anesthetized by intramuscular administration of alphaxalone and diazepam, and then given isoflurane by inhalation.The inhaled concentration of the agent was gradually increased to 4% and maintained at that level.All pigs became anesthetized without any abnormalities, but approximately half of them showed symptoms such as cyanosis and arrhythmia. Based on the above, suitable examples of combinations of anesthetic administration conditions and cardiac function items in the present invention include, but are not limited to, 1) a combination of intramuscular administration of a triple-anesthesia (medetomidine hydrochloride, midazolam, butorphanol tartrate) and heart failure and / or a significant increase in serum cardiac damage markers, and 2) a combination of induction of anesthesia by intramuscular administration of alphaxalone and diazepam, followed by maintenance of the anesthetic state by inhalation administration of isoflurane, and cyanosis and / or arrhythmia and / or heart failure and / or a significant increase in serum cardiac damage markers. In addition to or instead of the cardiac dysfunction, cardiac injury markers in serum can be used as an indicator in the method for selecting sows according to the present invention. Here, cardiac injury markers are factors released into the blood from the myocardium when cardiomyocytes are damaged. These factors are collected in the serum after blood collection, and the extent of cardiac damage can be measured by quantifying the factors. Specific examples of cardiac injury markers include, but are not limited to, cardiac fatty acid-binding protein (H-FABP), cardiac troponin I, troponin T, myoglobin (Mb), creatine kinase enzyme MB (CK-MB), and myosin light chain. Cardiac troponin I and troponin T are preferred, and cardiac troponin I is particularly preferred. In

[0049] , the inventors confirmed that, after administering a specific anesthesia treatment to a BMD cardiomyopathy model pig, symptoms indicative of cardiac dysfunction, such as cyanosis and arrhythmia, appeared simultaneously with an increase in serum cardiac troponin I concentration.

[0028] The characteristics of the BMD cardiomyopathy pig model according to the present invention are induced by a mutant dystrophin gene and / or a recessively inherited causative gene that, like the gene, is localized on the X chromosome, and therefore the disease only develops in male pigs that have the gene on their X chromosome and does not generally develop in female pigs that have a heterozygous version of the causative gene, making it possible to maintain the disease for subsequent generations using such female pigs (

[0022] ). That is, particularly when the causative gene is a mutant dystrophin gene, only about half of the male piglets and female piglets born to a sow carrying the mutant dystrophin gene will stochastically carry the mutant gene, and the remaining half will not become BMD cardiomyopathy model male pigs or female pigs carrying the mutant dystrophin gene, respectively.

[0029] Therefore, in order to select a sow that will give birth to a BMD cardiomyopathy pig model caused by a mutant dystrophin gene, if it is possible to exclude in advance male piglets that do not have the mutant gene, i.e., male piglets that express normal dystrophin, when administering an anesthetic to the male piglets given birth to by the sow, the sow can be selected more efficiently.

[0030] Various methods can be considered to determine whether or not normal dystrophin expression is present in the male piglets. For example, but not limited to, dystrophin expression analysis at the gene, mRNA, and protein levels in muscle may be used to determine whether normal dystrophin expression is attenuated and / or abnormal dystrophin expression or augmented, or whether muscle degeneration, necrosis, atrophy, and / or a decrease in anti-dystrophin antigen are detected by histopathological examination of muscle or immunohistochemical staining using an anti-dystrophin antibody.

[0031] Therefore, in the present invention, for the purpose of selecting a sow that will give birth to a BMD cardiomyopathy pig model caused by a mutant dystrophin gene, some method for determining whether or not normal dystrophin expression is present in the male piglets given birth to by the sow can be carried out before administering an anesthetic to the male piglets, and only male piglets with reduced dystrophin expression and / or muscle degenerative lesions can be selected in advance, and the anesthetic can be administered to only these selected male piglets.Although not particularly limited, dystrophin expression analysis in muscle and / or muscle histopathological examination are preferred methods for determining whether or not normal dystrophin expression is present.

[0032] In addition, the sow selected by the method of selecting a sow that will give birth to a BMD cardiomyopathy model pig according to the first embodiment of the present invention, and the offspring of the sow, a female pig carrying BMD cardiomyopathy and a male pig that will become a BMD cardiomyopathy model pig, are all included as one embodiment of the present invention. Here, the term "offspring" refers to pigs born from a mother pig that inherit some of the mother pig's genes, and includes both males and females. The inherited genes include the causative genes for the BMD cardiomyopathy pig model described in

[0022] . Although not particularly limited, the dystrophin gene with some mutation is preferred, and a mutant dystrophin gene (described below) lacking exons 21 to 28 is particularly preferred.

[0033] When a sow that gives birth to a BMD cardiomyopathy model pig with a dystrophin mutant gene as the causative gene is selected using the selection method of the present invention, the male piglet born from the sow will be a BMD cardiomyopathy model pig, and the female piglet will be a carrier sow for BMD cardiomyopathy. However, as described in

[0028] , approximately half of both the male and female piglets will be BMD cardiomyopathy model male piglets and carrier sows.

[0034] Therefore, the inventors have devised a method for more efficiently selecting BMD cardiomyopathy model male pigs / carrier sows from the above-mentioned selected sows, by carrying out some method described in

[0030] and

[0031] to determine whether or not normal dystrophin expression is present in the sows selected as those that will give birth to BMD cardiomyopathy model pigs whose causative gene is a dystrophin mutant gene, and have completed the invention.

[0035] That is, the second aspect of the present invention is a selection method in which a method for determining whether or not normal dystrophin expression is present in piglets born from a sow that will give birth to a BMD cardiomyopathy pig model caused by a mutant dystrophin gene and that has been selected by the selection method according to the first aspect of the present invention, and male piglets and female piglets that have reduced dystrophin expression and / or muscle degenerative lesions are selected as BMD cardiomyopathy model male pigs and BMD cardiomyopathy carrier sows, respectively. Although not particularly limited, the above-mentioned method for determining whether or not normal dystrophin expression is present is preferably dystrophin expression analysis in muscle and / or muscle histopathological examination.

[0036] Furthermore, the inventors conducted extensive selection from pigs of various lineages using the method for selecting sows that will give birth to BMD cardiomyopathy model pigs, which is the first embodiment of the present invention, and found that a female pig heterozygously carrying a dystrophin mutant gene lacking exons 21 to 28 can become a sow that will give birth to a BMD cardiomyopathy model pig. Furthermore, they analyzed the piglets selected by the selection method according to the second embodiment of the present invention, and confirmed that male piglets carrying the defective gene became BMD cardiomyopathy model pigs, and female piglets carrying the defective gene became sows carrying BMD cardiomyopathy.

[0037] Based on the above, a third aspect of the present invention is a male pig that serves as a BMD cardiomyopathy model pig, characterized in that it has a mutant dystrophin gene in which a deletion in the exon 21-28 region has been artificially induced, and a female pig that is a carrier of BMD cardiomyopathy, characterized in that it has a mutant dystrophin gene in which a deletion in the exon 21-28 region has been artificially induced. The mutant dystrophin gene in which the deletion of exons 21 to 28 region has been artificially induced according to the present invention refers to an artificially induced "mutant dystrophin gene in which the deletion of exons 21 to 28 region" as defined in

[0024] . The inventors have now discovered for the first time a mutant dystrophin gene lacking exons 21-28. They have confirmed that this genetic mutation generates a BMD cardiomyopathy pig model. Therefore, it is possible to generate this cardiomyopathy pig model using existing techniques known to those skilled in the art. That is, it is possible to artificially generate pigs carrying a mutant dystrophin gene lacking exons 21-28 by, but not limited to, introducing the Cas9 protein and guide RNA constituting the CRISPR / Cas9 system into fertilized eggs at the fertilized egg stage, or by introducing a foreign gene into fertilized eggs using microinjection, electroporation, transfection, lipofection, or other methods. This means that a method for generating a BMD cardiomyopathy pig model using this mutant gene has been completed.

[0038] Furthermore, sperm collected, stored and / or frozen from a male pig that will be the BMD cardiomyopathy model pig described in

[0032] and

[0037] , and / or eggs or fertilized eggs collected, stored and / or frozen from a female pig that is a carrier of BMD cardiomyopathy described in

[0032] and

[0037] , constitute a fourth aspect of the present invention. Here, collection means physically separating from the animal body, and is not particularly limited. For example, sperm may be ejaculated manually after conventional rearing until sexual maturity, or may be collected as epididymal sperm from the cauda epididymis. Eggs and fertilized eggs can also be surgically collected from carrier sows. The method for storing sperm, eggs, and fertilized eggs is not particularly limited, but it is desirable to immerse them in a predetermined preservative solution or the like and maintain them at an ultra-low temperature.

[0039] The fifth aspect of the present invention is a method for screening or evaluating therapeutic agents for cardiomyopathy, comprising administering a drug to a cardiomyopathy model pig that is the offspring of a sow that gives birth to a BMD cardiomyopathy model pig selected by the method according to the first aspect of the present invention, and evaluating the drug using as an indicator the induction of cardiac dysfunction by administering an anesthetic and / or cardiac injury markers in the serum. The BMD cardiomyopathy pig model used in this method is not particularly limited, but is preferably a male pig having a dystrophin gene with some mutation, and particularly preferably a male pig having a mutant dystrophin gene lacking the exon 21 to 28 region. The fifth aspect also includes a method for screening or evaluating a therapeutic agent for cardiomyopathy, which comprises administering a drug to a BMD cardiomyopathy pig model consisting of a male pig having a mutant dystrophin gene in which deletion of exons 21 to 28 has been artificially induced, and evaluating the drug using an anesthetic to induce cardiac dysfunction and / or a cardiac injury marker in the serum as an index, according to the third aspect of the present invention. It is to be noted that there is no particular problem if the indicator for the screening or evaluation method of the present invention is only the induction of cardiac dysfunction. However, since rapid judgment is required, it is desirable to use, without limitation, either a serum cardiac damage marker alone or both a cardiac dysfunction marker and the induction of cardiac dysfunction as the indicator.

[0040] The drug of this embodiment is not particularly limited, and any substance can be used. Examples include, but are not limited to, naturally occurring compounds, artificially synthesized compounds, peptides, proteins, lipids, and nucleic acids (ribonucleic acid, deoxyribonucleic acid, etc.). Food-derived substances may also be used as the drug. The drug may be a single compound or a composition such as an extract.

[0041] In administering the drug according to this embodiment, the administration route and / or administration interval of the drug to the model pig are not particularly limited. Examples of administration routes include, but are not limited to, oral administration such as buccal administration and sublingual administration, and parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, nasal administration, and pulmonary administration. Furthermore, examples of administration intervals include, but are not limited to, a single administration, multiple consecutive administrations, and continuous administration for a certain period of time, such as intravenous drip administration.

[0042] The pigs of the present invention are mammals of the order Cetacean, family Suidae, and may belong to any species, although this is not limitative. Pigs are closer to humans than apes in terms of weight, skin condition, and organ size, and are similar to humans in terms of physiology and anatomy. Domestic pigs and miniature pigs are particularly used for experiments in the fields of medicine and pharmacy. Major breeds of domestic pigs include the Large Yorkshire, Landrace, Duroc, Berkshire, Hampshire, and crossbreeds thereof, but this is not particularly limited. Crossbreeds of Landrace, Large Yorkshire, and Duroc are preferred. Miniature pigs include the Potbelly and Göttingen breeds, and crossbreeds of these with domestic pigs. Furthermore, miniature pigs and micropigs, which are further miniaturized miniature pigs, are also included, but this is not particularly limited.

[0043] The timing of administering an anesthetic to a male piglet according to the first aspect of the present invention is not particularly limited, but is preferably during the piglet rearing period, and particularly preferably when the piglet is three months old. Furthermore, there are no particular limitations on the timing of performing some method to determine whether or not normal dystrophin expression is present in the piglets according to the second aspect and / or before administering the anesthetic according to the first aspect, but this is preferably performed during the piglet rearing period after weaning, and particularly preferably at 70 days of age. [Example]

[0044] Examples are shown below. These are merely illustrative examples and do not limit the scope of the present invention, and various improvements and design changes may be made without departing from the spirit of the present invention.

[0045] 1. Selection of sows that will give birth to BMD cardiomyopathy model pigs (1) Whether or not the administration of anesthetics to male piglets born from candidate sows causes heart failure Overview From the various pedigrees of pigs owned by the inventors, we attempted to select a sow that would give birth to a BMD cardiomyopathy model pig. Because there have been cases in which anesthesia treatment has induced sudden death due to myocardial damage in human BMD patients, we suspected that administering anesthesia under certain conditions to a male sow candidate to give birth to the model pig might induce some kind of decline in cardiac function, and so we conducted the following experiment.

[0046] 1-2. Method In selecting sows that would give birth to BMD cardiomyopathy pig models, four male piglets from the same litter born to a sow from one particular family that was the subject of the selection test were anesthetized at three weeks of age by intramuscular administration of a triple anesthesia mixture (medetomidine hydrochloride, midazolam, butorphanol tartrate (medetomidine hydrochloride: midazolam: butorphanol tartrate = 3:2:2 (ml), 0.1 ml / kg). Histopathological examination of skeletal and cardiac muscles of male piglets administered the above anesthetics, and immunohistochemical staining using anti-dystrophin antibodies were performed as follows: histopathological examination of skeletal and cardiac muscles (skeletal and cardiac muscles were fixed in 10% neutral buffered formalin and embedded in paraffin blocks. They were then sectioned at 4 μm using a microtome, stained with hematoxylin and eosin (HE staining), and examined for lesions under an optical microscope), and immunostaining using anti-dystrophin antibodies (paraffin sections (4 μm) of skeletal and cardiac muscles were used. Two types of anti-dystrophin primary antibodies were used: Abcam's ab3149 (dilution: 100) and ab15277 (dilution: 500, C-terminal recognition antibody). Antigen retrieval was performed using immunosaver (Nissin EM Co., Ltd.) at 100°C for 45 minutes. Alexa fluor 488-labeled anti-mouse IgG antibody (dilution: 1000) and anti-rabbit IgG antibody (dilution: 1000) were used. The sections were mounted and nuclear stained using VECTASED with DAPI (water-soluble mounting medium), and observed under a fluorescence microscope FSX100 (Olympus).

[0047] 1-3.Results Four male piglets from the same litter were anesthetized with the above-mentioned anesthetic. Three of the four piglets developed cyanosis after awakening from anesthesia, and two of them died by the next day (Fig. 1, left). Autopsies of these four piglets revealed signs of heart failure in the three pigs that showed abnormalities after anesthesia, with severe dilation of the right ventricle (Fig. 1, right). The other pig that showed no abnormalities during anesthesia showed no significant changes. Histopathological analysis, immunostaining with anti-dystrophin antibodies, and dystrophin expression analysis revealed degenerative lesions in the skeletal and cardiac muscles of the former three piglets (Fig. 2) and reduced dystrophin expression (Fig. 3, right). The other pig that showed no abnormalities during anesthesia showed no abnormalities in the skeletal and cardiac muscles, and normal dystrophin expression was observed (Fig. 3, left). From the above, it became clear that the anesthetic administration conditions used on the male piglets born from the sows evaluated in this study are conditions that can induce a decline in cardiac function known as heart failure when selecting sows that will give birth to BMD cardiomyopathy model pigs. Furthermore, the male piglets born to the sows evaluated in this study fulfilled the requirements for a BMD cardiomyopathy pig model, and in particular, degenerative lesions of muscle structure and reduced dystrophin expression were observed in skeletal and cardiac muscles, strongly suggesting that the model pigs may have been formed due to the inheritance of some kind of dystrophin mutant gene. For this reason, further studies were conducted using the sows evaluated in this study and / or the piglets born to these sows.

[0048] 2. Identification of novel dystrophin mutations as the causative gene for the BMD cardiomyopathy pig model 2-1. Overview In order to clarify the identity of the dystrophin mutant gene found in item 1 above as the causative gene for the formation of the BMD cardiomyopathy pig model, mRNA was extracted from the muscle of the model pig and the expressed mutant dystrophin was identified.

[0049] 2-2. Method Dystrophin gene mRNA analysis was performed using muscle collected and cryopreserved from male piglets, a pig model of BMD cardiomyopathy (see section 1 above). mRNA extraction was performed by powdering the sample with a mortar and pestle chilled on dry ice, followed by RNeasy Plus Micro Kit (QIAGEN) and DNase treatment using RQ1 RNase-Free DNase (Promega). Reverse transcription was performed using ReverTra-Plus (Toyobo) and according to the manufacturer's instructions. The resulting cDNA was diluted and PCR was performed using KODFX neo (Toyobo) as the template. Primers were designed based on the sequence of Sus scrofa dystrophin (DMD) mRNA (NM_001012408.1). PCR products were electrophoresed on a 1% agarose gel, stained with Midori Green Advance (NIPPON Genetics EUROPE GmbH), and observed in a UV cabinet. The identified band of interest was excised and purified using NucleoSpin Gel and PCR Clean-up (Takara Bio Inc.), and the purified PCR product was subjected to DNA sequence analysis (Eurofins Genomics).

[0050] 2-3.Results Analysis of the mutant dystrophin gene found in Item 1 above as the causative gene for forming the BMD cardiomyopathy pig model revealed that the dystrophin gene lacked 1041 bp (NM_001012408.1 Sus scrofa dystrophin (DMD) mRNA, 2966-4006) in the region of exons 21 to 28. It was suggested that the presence of this mutant dystrophin gene reduces dystrophin expression in cardiac and skeletal muscles, resulting in male piglets becoming model pigs exhibiting symptoms of BMD cardiomyopathy, and female piglets heterozygous for the mutant gene becoming carrier pigs for BMD cardiomyopathy.

[0051] 3. Selection of sows that will give birth to BMD cardiomyopathy model pigs (2) Further investigation of anesthetic administration conditions and cardiac dysfunction using male piglets born to the sows that gave birth to the model pigs. Overview Using a sow that gave birth to the BMD cardiomyopathy model pig discovered in Item 1 above, we investigated a case in which a different cardiac dysfunction phenomenon from that embodied in Item 1 above was induced by subjecting the male piglets that the sow gave birth to to anesthesia treatment different from that described in Item 1 above.

[0052] 3-2. Method Six male piglets (3 months old) from the same sibling dam as the sow in section 1 above were anesthetized by intramuscular administration of alfaxalone and diazepam (5 mg / kg alfaxalone, 0.5 mg / kg diazepam), and maintained anesthesia by inhalation of isoflurane (1.5–2%). The isoflurane concentration was gradually increased to 4%, and changes in condition were observed by changes in body color, visible mucous membrane color, and electrocardiogram. Subsequently, the animals were autopsied and examined for the presence of lesions in skeletal muscle and heart. Furthermore, histopathological specimens of skeletal muscle and heart muscle were prepared and examined as follows: Skeletal muscle and heart muscle were fixed in 10% neutral buffered formalin, embedded in paraffin blocks, and sectioned at 4 μm using a microtome. They were stained with hematoxylin and eosin (HE) and examined for the presence of lesions under a light microscope. Immunostaining using an anti-dystrophin antibody was performed as follows. Specifically, skeletal and cardiac paraffin sections (4 μm) were used. Two anti-dystrophin primary antibodies were used: Abcam's ab3149 (dilution: 1:100) and ab15277 (dilution: 1:500, C-terminal antibody). Antigen retrieval was performed using immunosaver (Nissin EM Co., Ltd.) at 100°C for 45 minutes. Alexa fluor 488-labeled anti-mouse IgG antibody (dilution: 1:1000) and anti-rabbit IgG antibody (dilution: 1:1000) were used as secondary antibodies. The sections were mounted and nuclear stained with VECTASED with DAPI (water-soluble mounting medium), and then observed under a fluorescence microscope FSX100 (Olympus). Dystrophin gene expression analysis was performed using cryopreserved muscle samples for dystrophin gene mRNA analysis. After powdering the samples with a mortar and pestle chilled on dry ice, mRNA extraction was performed using the RNeasy Plus Micro Kit (QIAGEN) according to the accompanying instructions. DNase treatment was performed using RQ1 RNase-Free DNase (Promega). Reverse transcription was performed using ReverTra Plus (Toyobo) according to the manufacturer's instructions. The resulting cDNA was diluted and PCR was performed using KODFX neo (Toyobo) as a template.Primers were designed based on the sequence of Sus scrofa dystrophin (DMD) mRNA (NM_001012408.1). PCR products were electrophoresed on a 1% agarose gel, stained with Midori Green Advance (NIPPON Genetics EUROPE GmbH), and observed under a UV cabinet. The identified bands of interest were excised and purified using NucleoSpin Gel and PCR Clean-up (Takara Bio Inc.). DNA sequence analysis of the purified PCR products was performed (Eurofins Genomics, Inc.). Serum samples were collected before and after anesthesia, and the concentration of cardiac troponin I, a marker of cardiac injury, was measured using an ELISA kit (High sensitivity pig cardiac troponin-I ELISA, Life Diagnostics, Inc.).

[0053] 3-3.Results Six male piglets were anesthetized under the above conditions, and all were anesthetized without any abnormalities. Three of the six piglets developed symptoms of cardiac dysfunction, such as cyanosis and arrhythmia, as the isoflurane concentration was increased, suggesting that they were model pigs for BMD cardiomyopathy. Autopsy revealed findings such as right ventricular dilation, increased pericardial effusion, and petechiae in four of the six male piglets, including the three that developed symptoms under anesthesia. Histopathological examination confirmed degenerative lesions in the cardiac and skeletal muscles of four of the six male piglets with abnormalities at autopsy. Furthermore, analysis of dystrophin expression revealed decreased dystrophin expression in the same four of the six male piglets. Serum cardiac troponin I concentrations increased after treatment in four of the six male piglets with abnormalities at autopsy. From the above, it has been revealed that when male piglets born from sows of the same lineage as the sow in item 1 above are given anesthetic administration conditions different from those in item 1 above, it is possible to evaluate cardiac dysfunction phenomena different from heart failure symptoms that lead to death when selecting sows that will give birth to BMD cardiomyopathy model pigs, i.e., cardiac dysfunction (mild cyanosis, arrhythmia, etc.) without killing the model pig, and that the cardiac dysfunction is accompanied by an increase in serum cardiac troponin I concentration, a cardiac injury marker.

[0054] 4. Establishment of an efficient method for selecting sows carrying BMD cardiomyopathy Overview We attempted to establish a selection method for efficiently selecting female piglets carrying BMD cardiomyopathy from female piglets born to sows that give birth to the BMD cardiomyopathy pig model described in the above item 1. Specifically, to find female piglets with reduced normal dystrophin expression, we collected muscle samples from all sows born to the sows and performed analysis of normal dystrophin expression and histopathological examination of the muscle samples.

[0055] 4-2. Method Eight female piglets from the same litter, born to the same sow as in item 1 above, were anesthetized by inhalation of isoflurane (1.5-2%) at 70 days of age, and 1-cm pieces of muscle from the inner thigh were excised. The excised muscles were divided in half, and one half was fixed in formalin, embedded in paraffin, and used to prepare histopathological specimens and for immunostaining using anti-dystrophin antibodies. Specifically, the muscles were fixed in 10% neutral buffered formalin, embedded in paraffin blocks, and then sectioned at 4 μm using a microtome. HE staining and immunostaining were performed using anti-dystrophin primary antibody (Abcam ab3149, 1:100 dilution). mRNA and DNA were extracted from the other muscle fragment for genetic testing. Specifically, the muscle fragment was powdered using a mortar and pestle chilled on dry ice, and mRNA was extracted using an RNeasy Plus Micro Kit (QIAGEN). DNase treatment was performed using RQ1 RNase-Free DNase (Promega). Reverse transcription was performed using ReverTra-Plus- (Toyobo). The resulting cDNA was diluted and PCR was performed using KODFX neo (Toyobo) as a template. The PCR product was electrophoresed on a 1% agarose gel. The identified band of interest was excised and purified using NucleoSpin Gel and PCR Clean-up (Takara Bio Inc.). DNA sequence analysis of the purified PCR product was performed (Eurofins Genomics).

[0056] 4-3.Results Four of the eight pigs tested showed reduced dystrophin expression, and three of them also showed muscle degeneration. These three pigs were selected as carrier sows. One of the carrier sows was selected as a sow, and it was confirmed that the piglets born from this sow also showed the same pathology. These results suggest that muscle biopsies from newborn pigs can identify piglets with abnormalities in dystrophin at an early stage, establishing an efficient method for selecting sows that are carriers of BMD cardiomyopathy.

[0057] 5. Collection and cryopreservation of sperm from BMD cardiomyopathy model pigs and oocytes and embryos from BMD cardiomyopathy carrier sows Overview We collected sperm from a pig model of BMD cardiomyopathy, whose causative gene is a dystrophin mutant gene lacking exons 21-28, and attempted to cryopreserve eggs and fertilized eggs from pigs carrying the BMD cardiomyopathy.

[0058] 5-2. Method Male pigs, which were BMD cardiomyopathy model pigs, were raised in a conventional manner until they reached sexual maturity (7 months of age). Ejaculated sperm were collected manually, and epididymal sperm were collected from the cauda epididymis during pathological autopsy. They were then frozen and stored in 0.25-0.5 mL straws using standard methods. In addition, female pigs that were carriers of the BMD cardiomyopathy model were raised normally until they reached sexual maturity (7 months of age), and eggs or fertilized eggs were surgically collected and vitrified using a porcine embryo vitrification and preservation solution kit (Functional Peptide Research Institute).

[0059] 5-3.Results The sperm, eggs, and fertilized eggs were collected in good condition and were cryopreserved. [Industrial Applicability]

[0060] The method of the present invention makes it possible to select sows that give birth to BMD cardiomyopathy pig models, thereby enabling the acquisition of novel BMD cardiomyopathy model boars and / or BMD cardiomyopathy carrier sows that give birth to such model boars, which have been difficult to find in the past. This makes it possible to obtain a variety of cardiomyopathy models based on different causes of onset, enabling multilayered research and elucidation of the causes of BMD cardiomyopathy, and therefore can be used in many industries related to the treatment and prevention of BMD cardiomyopathy. In particular, the BMD cardiomyopathy pig model of the present invention, which is caused by dystrophin lacking the exon 21-28 region, closely resembles the symptoms of human BMD cardiomyopathy. Therefore, by using this model pig as a pathological model of the disease, it will be easier to clarify the pathophysiology of Becker muscular dystrophy cardiomyopathy, which has many unknowns, or to research therapeutic drugs, and the model has extremely high potential for use in the fields of medicine and drug discovery. Furthermore, recent developments in drugs for treating DMD have made it possible to alleviate the severe symptoms of DMD patients to relatively mild BMD-like symptoms. Therefore, the use of the BMD cardiomyopathy pig model of the present invention is expected to lead to the development of not only BMD drugs but also novel drugs for treating DMD.

Claims

1. A method for selecting a sow that will give birth to a pig model of Becker muscular dystrophy cardiomyopathy, the method comprising administering an anesthetic to male piglets given birth to the sow, using as an indicator the induction of cardiac dysfunction, and further confirming that the mRNA of the dystrophin gene expressed by the male piglets or the sow in which dysfunction has been induced is deficient in the exon 21-28 region.

2. The selection method according to claim 1, wherein the administration of the anesthetic agent is intramuscular administration of a triple-dose anesthesia (medetomidine hydrochloride, midazolam, and butorphanol tartrate), and the cardiac dysfunction is heart failure.

3. The selection method described in claim 1, characterized in that the administration of anesthetic agents involves inducing anesthesia by intramuscular administration of alphaxalone and diazepam, followed by maintaining the anesthetized state by inhalation administration of isoflurane, and the cardiac dysfunction is cyanosis and / or arrhythmia.

4. A method for selecting female pigs that are carriers of Becker muscular dystrophy cardiomyopathy, comprising: carrying out muscle dystrophin expression analysis and / or muscle histopathological examination on female piglets given birth to sows selected by the selection method described in any one of claims 1 to 3; and selecting female piglets that have reduced dystrophin expression and / or muscle degenerative lesions as carriers of Becker muscular dystrophy cardiomyopathy.

5. A method for selecting male pigs to become model pigs for Becker muscular dystrophy cardiomyopathy, comprising: conducting dystrophin expression analysis in the muscles and / or muscle histopathological examination on male piglets born from sows selected by the selection method described in any one of claims 1 to 3; and selecting male piglets with reduced dystrophin expression and / or muscle degenerative lesions as male pigs to become model pigs for Becker muscular dystrophy cardiomyopathy.

6. A method for screening or evaluating therapeutic agents for Becker muscular dystrophy cardiomyopathy, comprising administering a drug to a cardiomyopathy model pig that is the offspring of a sow that gives birth to a Becker muscular dystrophy cardiomyopathy model pig selected by the method of any one of claims 1 to 3, and evaluating the drug using as an indicator the induction of cardiac dysfunction by administering an anesthetic and / or cardiac damage markers in the serum.

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