Phenotypic features of genetically modified DMD-XKOXWT pigs

Introduction Duchenne muscular dystrophy (DMD) is a hereditary neuromuscular disorder caused by mutation in the dystrophin gene (DMD) on the X chromosome. Female DMD carriers occasionally exhibit symptoms such as muscle weakness and heart failure. Here, we investigated the characteristics and representativeness of female DMD carrier (DMD-XKOXWT) pigs as a suitable disease model. Methods In vitro fertilization using sperm from a DMD-XKOY↔XWTXWT chimeric boar yielded DMD-XKOXWT females, which were used to generate F2 and F3 progeny, including DMD-XKOXWT females. F1–F3 piglets were genotyped and subjected to biochemical analysis for blood creatine kinase (CK), aspartate aminotransferase, and lactate dehydrogenase. Skeletal muscle and myocardial tissue were analyzed for the expression of dystrophin and utrophin, as well as for lymphocyte and macrophage infiltration. Results DMD-XKOXWT pigs exhibited various characteristics common to human DMD carrier patients, namely, asymptomatic hyperCKemia, dystrophin expression patterns in the skeletal and cardiac muscles, histopathological features of skeletal muscle degeneration, myocardial lesions in adulthood, and sporadic death. Pathological abnormalities observed in the skeletal muscles in DMD-XKOXWT pigs point to a frequent incidence of pathological abnormalities in the musculoskeletal tissues of latent DMD carriers. Our findings suggest a higher risk of myocardial abnormalities in DMD carrier women than previously believed. Conclusions We demonstrated that DMD-XKOXWT pigs could serve as a suitable large animal model for understanding the pathogenic mechanism in DMD carriers and developing therapies for female DMD carriers.


Introduction
Duchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disorder, affecting up to 1/3600 male births worldwide [1].Major symptoms of DMD include progressive muscle weakness and wasting [2].Deficiency of dystrophin caused by mutation in the dystrophin gene (DMD) on the X-chromosome results in degeneration and necrosis of muscle fibers due to loss of integrity of the sarcolemma, thereby leading to respiratory insufficiency [3], heart failure [4], and premature death in the second to fourth decade of life [5].
Although females possessing heterozygous DMD mutations (DMD carriers) rarely exhibit clinical symptoms [6], they can sporadically develop muscle weakness and heart failure [6e8].The incidence of cardiomyopathy in DMD carriers increases with age [9,10].Adult DMD carriers therefore have been recommended to undergo echocardiography every 5 years [11,12].The unique characteristics of carriers, including onset and sites of pathology, and severity of the symptoms, have yet to be investigated.However, studies involving DMD carriers can be hindered due to biased selection of subjects and the intentional avoidance of invasive examinations such as myocardial biopsy.
Animal disease models manifesting symptoms extrapolatable to human patients are indispensable for understanding pathogenic mechanisms and developing therapies [13,14].Novel disease models in pigs have been intensively developed in recent studies due to pigs' advantages as an experimental animal with physiological and anatomical similarity to humans [15,16].We have demonstrated that DMD-knockout (KO) pigs that faithfully manifest DMD symptoms [17e20] can serve as a large animal model for translational studies, including gene therapy [21].Thus, DMD carrier pigs may also provide clinically relevant insights.
We have developed a stable procedure for reproducing DMD carrier pigs through the use of cryopreserved boar sperm carrying the DMD mutation on the X-chromosome [22].In the present study, we performed physiological and pathological analyses of female DMD carrier pigs to evaluate their characteristics and ability to model female patients.

Experimental animals
All animal experiments were approved by the Institutional Animal Care and Use Committee of Meiji University (approval numbers: MUIACUC2020-111 and MUIACUC2020-125).All recombinant DNA experiments performed in this study were approved by the Genetic Modification Safety Committee of Meiji University (2018-6).All experiments were performed in accordance with institutional guidelines and regulations.Pigs were kept in a temperature-controlled room, fed food appropriate for their growth stage, and given free access to water.

Generation of DMD-X KO X WT female pigs
We previously generated a male DMD-X KO Y4X WT X WT chimeric pig by blastocyst complementation using DMD-X KO Y male cells and wild-type (WT) female embryos [22].This chimeric male pig produced fertile gametes: DMD-X KO sperm and normal Y sperm [22].To generate DMD-X KO X WT female pigs, cryopreserved epididymal sperm collected from the DMD-X KO Y4X WT X WT chimeric boar were subjected to in vitro fertilization (IVF) as previously described [23] with slight modifications.Briefly, sperm (5 Â 10 5 cells/mL) and in vitro matured oocytes were cocultured in porcine fertilization medium (PFM; Research Institute for Functional Peptides, Yamagata, Japan) at 38.5 C for 8 h under the following conditions: 5% CO 2 , 5% O 2 , 90% N 2 , and saturated humidity.Presumed zygotes were cultured using porcine zygote medium-5 (PZM-5, Research Institute for Functional Peptides) under the following conditions: 5% CO 2 , 5% O 2 , 90% N 2 , 38.5 C, and saturated humidity.After the morula stage, 10% fetal bovine serum was added to the culture medium.Blastocyst-stage embryos cultured in vitro for 5e6 d after fertilization were surgically transferred into the uterine horns of estrus-synchronized female recipient pigs, which generated the F1 generation of DMD-X KO X WT progeny.Natural mating or artificial insemination of the F1 DMD-X KO X WT sows with wild-type boars, produced the F2 generation.DMD-X KO X WT pigs from our previous study [22] were also used to produce the F2 generation.F2 DMD-X KO X WT pigs were crossed with wild-type boars to produce the F3 generation.

Genotyping
Genomic DNA was extracted from the tail tissue of the F1eF3 piglets using a DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany).PCR analysis was performed using standard methods.The primers 5 0 -GTCTTTCAGCCACTGATTGT-3 0 and 5 0 -TTTATGAG-TATTGAATTTCCATCCC-3 0 were used for the detection of DMD exon 52, which is deleted on the mutated X chromosome.The PCR product was subjected to agarose gel electrophoresis to confirm whether the test individuals were WT, DMD-X KO X WT , or DMD-X KO Y.

Blood biochemical analysis
Blood samples were collected from the ear vein and allowed to clot before the serum was separated by standard methods.Creatine kinase (CK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were measured using a dry-chemistry analyzer (FUJI DRI-CHEM 7000, FUJIFILM, Tokyo, Japan).

Tissue analysis
The biceps femoris and the left ventricle of DMD-X KO X WT female pigs were used in the analysis of skeletal muscle and myocardial tissue, respectively.Control tissues were obtained from WT and DMD-X KO Y littermates.The tissues were mounted on cork bases using tragacanth gum (FUJIFILM Wako Pure Chemical, Osaka, Japan) and frozen in isopentane cooled in liquid nitrogen.Frozen tissues were sliced at a thickness of 8 mm and stained.
We analyzed the expression of the following three proteins by immunofluorescence staining: (i) dystrophin, an essential cytoskeletal protein for the maintenance of skeletal muscle and myocardium [24]; (ii) utrophin (UTRN), a homolog of dystrophin whose expression is upregulated in DMD patients [25]; and (iii) laminin subunit alpha-2 (LAMA2), a component of the basal lamina of the extracellular matrix [26].
Immunohistochemical staining was performed to examine the presence of lymphocyte and macrophage infiltration in muscle tissue.As primary antibodies, anti-CD3 (IS503, Dako, Santa Clara, CA, USA) was used for the detection of T lymphocytes, and anti-Iba1 (019-19741, FUJIFILM Wako Pure Chemical) was used for the detection of macrophages.Primary antibodies were detected using horseradish peroxidase conjugated secondary antibodies (K4061, Dako).A liquid DAB þ substrate chromogen system (K3468, Dako) was used as the chromogenic substrate.Hematoxylineeosin (HE) staining was performed by standard procedures.

Quantification of dystrophin expression levels in muscle tissue and the fibrotic area in the myocardium
Dystrophin expression in the skeletal muscle and myocardium of DMD-X KO X WT female pigs was quantified from immunostaining images.Laminin, similar to dystrophin, is expressed in the sarcolemma.Normal muscle fibers of WT pigs are positive for both laminin and dystrophin staining.In DMD-X KO X WT pigs, muscle fibers lacking dystrophin expression are positive for laminin and negative for dystrophin staining.Thus, differences in dystrophin expression patterns between DMD-X KO X WT and WT pigs can be quantitatively compared by calculating the difference in the dystrophin/laminin fluorescence intensity ratio.We performed double staining of dystrophin and laminin, and 10 random fields were photographed at 20 Â magnification using a fluorescence microscope (BZ-X700; Keyence).The luminance of dystrophin and laminin was measured using ImageJ software v1.53 (National Institutes of Health, Bethesda, MD, USA), the luminance ratio of dystrophin/ laminin was determined, and the value relative to the ratio in WT samples was calculated.
Sirius Red staining was performed to detect myocardial fibrosis, and the fibrotic area was quantified using ImageJ software.

Statistical analysis
Statistical analysis was performed using SPSS software v26 (IBM SPSS, Chicago, IL, USA).Data are presented as the mean ± standard deviation (SD) or mean ± standard error of the mean (SEM).Welch's t-test was used to evaluate differences in numerical data between DMD-X KO X WT and WT pigs.Differences were considered statistically significant at p < 0.05.

Generation of DMD-X KO X WT female pigs
We surgically transferred 22 blastocysts produced by in vitro fertilization using sperm from the DMD-X KO Y4X WT X WT chimeric boar to one female recipient, which resulted in five F1 offspring, including three DMD-X KO X WT females and two WT males.All three F1 DMD-X KO X WT females obtained reached sexual maturity.One of them was mated with a WT boar to produce F2 progeny.The other two were used for collecting samples of sexually mature individuals.
Furthermore, F1 DMD-X KO X WT females from our previous study [22] were used to produce F2 progeny.Five F1 DMD-X KO X WT females that had farrowed six times in total produced 60 F2 progeny, including 17 (28.3%)DMD-X KO X WT individuals.One of the F2 DMD-X KO X WT females was mated to a WT boar, resulting in the birth of seven more F3 progeny, of which two (28.6%) were DMD-X KO X WT females.DMD-X KO X WT pigs and WT littermates produced through the above process were used in this study.

Sudden onset in DMD-X KO X WT female pigs
Although most human DMD carriers are asymptomatic, clinical symptoms can sporadically present at a wide range of ages [6].Accordingly, the DMD-X KO X WT pigs were reared for up to a maximum of 32 months, during which we monitored growth rate, CK level fluctuations, and DMD symptom development.
The birth weights of DMD-X KO X WT pigs and WT littermates were 1.174 ± 0.08 kg (n ¼ 29) and 1.254 ± 0.07 kg (n ¼ 27), respectively, with no significant difference between the two groups.DMD-X KO X WT pigs exhibited no differences in weight gain (Fig. 1a) or appearance compared to WT pigs.
Five (17.2%) of the 29 DMD-X KO X WT pigs died during rearing (Fig. 1b).One died from prostration shortly after birth, and three died suddenly at 1, 2, and 4 months of age.Autopsies were not performed due to the time elapsed postmortem.The hind limbs of a 7-month-old DMD-X KO X WT pig collapsed during mating with a WT male, and the female subsequently died.Autopsy of this individual revealed skeletal muscle degeneration and myocardial fibrosis (Supplementary Fig. S1).On the other hand, there were no deaths among WT littermates.
Multiple blood tests were performed on the 27 DMD-X KO X WT pigs that survived at least 1 month after birth, and a wide range of serum CK levels (207e75,990 U/L) was observed among individuals of the same age (Supplementary Fig. S2a).The mean CK levels of young DMD-X KO X WT pigs were significantly higher than those of age-matched WT littermates (1 month old: 10,167 ± 19,145 vs. 821 ± 561 U/L; 3 months old: 9376 ± 12,843 vs. 1031 ± 780 U/L, p < 0.05; Fig. 1c).Those with high CK levels tended to also have high AST and LDH levels (Supplementary Fig. S2b).The CK levels of DMD-X KO X WT pigs tended to decrease with age (Supplementary Fig. S2a).

Histological analysis of the skeletal muscles of asymptomatic DMD-X KO X WT female pigs
Immunohistological and histopathological analyses of skeletal muscle tissue were performed in 12 asymptomatic DMD-X KO X WT pigs at 1.5e32 months of age.The skeletal muscle tissue of 1.5month-old DMD-X KO X WT pigs (n ¼ 3) exhibited a mosaic pattern of dystrophin staining.In contrast, the skeletal muscle tissue of 3month-old individuals (n ¼ 3) exhibited a uniform pattern of staining (Fig. 2a), which indicated that dystrophin was continuously distributed around the muscle fibers (sarcolemma).A similar pattern of dystrophin staining was observed in the skeletal muscle tissue of all older individuals (14e32 months old, n ¼ 6; Fig. 2a).As in the WT animals, only a weak utrophin expression was detected in the skeletal muscle tissue of DMD-X KO X WT pigs, regardless of age.At young ages (1.5e3 months old), the dystrophin expression levels in the skeletal muscle of DMD-X KO X WT pigs were lower than those of WT individuals (p < 0.05, Fig. 2c, Supplementary Fig. S3) and tended to increase with age (Fig. 2c).Degeneration of muscle fibers was observed in 3 of the 6 young individuals (Supplementary Fig. S4a).On the other hand, the skeletal muscle tissue of adult pigs exhibited no degeneration, and infiltration of immune cells was observed only in 1 of 6 individuals (Supplementary Fig. S4b).

Histological assessment of the myocardium of asymptomatic DMD-X KO X WT female pigs
Immunohistological and histopathological analyses of myocardial tissue were performed in 12 asymptomatic DMD-X KO X WT pigs at 1.5e32 months of age.Dystrophin immunostaining in DMD-X KO X WT pigs revealed a mosaic pattern, exhibiting a mixture of dystrophin-positive and dystrophin-negative cells (Fig. 2b).The ratio of dystrophin/laminin fluorescent intensities indicated that the dystrophin levels in the myocardium of DMD-X KO X WT pigs at all ages were significantly lower than those of WT pigs (p < 0.05, Fig. 2c, Supplementary Fig. S3).
Histopathological analysis of the myocardial tissue of young DMD-X KO X WT pigs (1.5e3 months old) revealed no notable pathological changes other than slight fibrosis of muscle fibers.However, the myocardial tissue of older individuals (14e32 months old) exhibited phagocytosis in dystrophin-negative areas (Fig. 3a) and degeneration, including fibrosis (Fig. 3b).The fibrotic area in the myocardial tissue of adult DMD-X KO X WT pigs was expanded compared with that of young individuals (p < 0.05, Fig. 3c).

Discussion
Although DMD carriers have long been believed to be asymptomatic [6], recent studies have revealed a sporadic incidence of symptom manifestation, including sudden death, in latent patients with the DMD-X KO X WT genotype.Manifestation of the DMD phenotype in females possessing heterozygous DMD mutations has been correlated with a skewed X chromosome inactivation (XCI) pattern [29e31].This theory suggests that the X chromosomes containing the normal DMD are inactivated at a higher rate than those that carry the mutated gene [30].In contrast, some reports denied a correlation between symptomatic carriers and skewed XCI [32e34].Thus, a follow-up study on DMD carrier females could provide new information, but such a study would present difficulties from a practical and ethical perspective.
DMD model animals have been developed earlier in mice and dogs [35,36].Although the DMD-X KO X WT females in animal models have been reported to exhibit muscle degeneration [37e40], dissimilar pathological features to humans hinder the utility of these animals as faithful models for DMD carriers [41].Recent development of disease model pigs, including DMD, demonstrated extrapolability of their phenotypes to the symptoms of human patients [19,42,43].We therefore generated a cohort of DMD-X KO X WT female pigs in the present study and investigated their eligibility as a DMD carrier model.
DMD-X KO X WT female pigs were produced in a previous study by using the somatic cell cloning technique [18].However, most of the founder DMD-X KO X WT cloned pigs obtained were prematurely mortal, which hindered the production of a cohort of DMD carrier progeny for the study [18].The mortality of DMD-X KO X WT cloned pigs is ascribed to abnormal XCI, which is prone to occur in cloned animals [44e46].Therefore, we generated DMD carrier females as the next-generation offspring of a DMD-X KO Y4X WT X WT chimeric boar [22].The use of cryopreserved DMD-X KO sperm assures continuous, highly scalable, and economical production of DMD-X KO X WT female pigs.
Approximately 70% of DMD carrier women exhibit high serum CK levels regardless of clinical manifestation [47].DMD-X KO X WT pigs also exhibited frequent hyperCKemia as one of their phenotypic features.In DMD carriers, the reported onset frequency of musculoskeletal abnormalities that range from light muscular pain to severe astasia [7,48e50] varies widely from 2.5 to 19% [6].This inconsistency may be ascribed to a lack of unity in defining symptoms as a result of the avoidance of invasive muscle biopsy in  clinical settings.In the present study, we revealed pathological abnormalities in the skeletal muscles in one-quarter of the DMD-X KO X WT pigs examined.These data suggest a frequent incidence of pathological abnormalities in the musculoskeletal tissues of latent DMD carrier women.
Clerk et al. [51] reported mosaic expression of dystrophin in the skeletal muscle of asymptomatic DMD carrier infants.In contrast, adult DMD carriers exhibited uniform dystrophin staining in skeletal muscle tissues [51].These phenomena were explained by the regeneration of myofibers by the activation of dystrophin-positive satellite cells and compensatory dystrophin production by nearby dystrophin-positive myonuclei [52].A similar transition pattern of dystrophin staining was observed in our DMD-X KO X WT pigs.Sequential symptomatic analysis in DMD-X KO X WT female pigs has yet to be performed.
Sporadic manifestation of cardiomyopathy, including dilated cardiomyopathy (DCM), is one of the major features of DMD carriers, and its occurrence increases with age [9,10,53].Severe DCM cases include mortal consequences of heart failure in middle age [9,10].Dystrophin is known to be expressed in a mosaic fashion among the myocardial myofibers of DMD carrier females [54].Because of the lack of regenerative capacity in myocardial fibers [55], the mosaic expression pattern of dystrophin in myocardial tissue is considered to be constant throughout the life of DMD carriers.Necrosis and fibrosis of dystrophin-negative cardiomyocytes are likely to be involved in cardiomyopathy in DMD carriers, similar to male DMD patients [56].
Stirm et al. [18] reported that 15% (3/20) of the DMD-X KO X WT pigs of which were descended from DMD carrier sows (F1 and F2) exhibited myocardial lesions at 6 months old.In addition, the authors observed severe myocardial abnormalities in the DMD-X KO X WT sow, which served 7 years.We also noted histological abnormalities in the myocardial tissue in all of the asymptomatic DMD-X KO X WT pigs (6/6) that had grown up without apparent health problems for 14e32 months after birth.Together, these data may suggest a higher risk of myocardial abnormalities in DMD carrier women than previously thought.The increased workload on the heart of DMD carriers necessary to maintain skeletal muscle may induce cardiomyopathy [57].The frequent occurrence of histological alterations in the myocardial tissue of DMD-X KO X WT pigs may underlie the risk of cardiac incompetency in DMD carriers.

Conclusions
In conclusion, DMD-X KO X WT pigs exhibit various characteristics similar to DMD carrier patients.Those characteristics included asymptomatic hyperCKemia, dystrophin expression patterns in the skeletal and cardiac muscles, histopathological features of skeletal muscle degeneration, myocardial lesions in adulthood, and sporadic death.Thus, DMD-X KO X WT pigs could facilitate long-term follow-up studies with invasive interventions, thereby increasing our understanding of the pathogenic mechanisms in asymptomatic carriers and eventually developing biomarkers for stratification of DMD carriers regarding their risk to become symptomatic.

Declaration of competing interest
H.N. is a founder and shareholder of PorMedTec Co., Ltd.These associations do not alter the authors' adherence to the journal's policies on sharing data and materials.The other authors declare that they have no conflicts of interest.

Fig. 1 .
Fig. 1.Growth and serum CK levels of DMD-X KO X WT female pigs.a, b: Growth (a) and survival curve (b) of DMD-X KO X WT pigs (red line, n ¼ 29) compared to WT littermates (blue line, n ¼ 27).c: Serum CK levels of young DMD-X KO X WT female pigs.*p < 0.05 vs. WT individuals.Samples were collected from DMD-X KO X WT pigs at 1 month (n ¼ 27) and 3 months (n ¼ 21) of age (red plots).WT pigs at 1 month (n ¼ 24) and 3 months (n ¼ 15) of age were used as the respective controls (blue plots).CK: creatine kinase.WT: wild type.

Fig. 2 .
Fig. 2. Histological analysis of the skeletal muscle and myocardium of DMD-X KO X WT pigs.a, b: Immunostaining of dystrophin and utrophin in skeletal muscle (a) and myocardium (b) of DMD-X KO X WT pigs.The upper and middle images of both panels exhibit fluorescent immunohistochemical staining of dystrophin and utrophin, respectively, and the lower images indicate HE staining.Scale bars ¼ 100 mm.b: Partial upregulation of utrophin expression on the sarcolemma, a known myopathological feature of DMD.c: Dystrophin expression levels in DMD-X KO X WT pigs (red bars).Dystrophin/laminin fluorescence intensity ratios were normalized to WT muscle tissues (blue bars).The graph represents the mean ± SEM. *p < 0.05 vs. WT pigs.Samples were collected from DMD-X KO X WT pigs at 1.5, 3, and 14e32 months of age (n ¼ 3, 3, and 6, respectively).WT pigs at 1.5, 3, and 6e8 months of age (n ¼ 2, 3, 3, respectively) were used as the respective controls.HE: hematoxylineeosin.WT: wild type.