**2. Animal models to study the pathogenesis of DMD**

The most widely used and well described animal model for Duchenne muscular dystrophy (DMD) research is the *mdx* mouse. Spontaneous X chromosomelinked mutation arose in inbred C57BL/10 colony of mice and produced viable and fertile homozygous animals. Mutant mice exhibited specific features similar to human DMD such as elevated plasma pyruvate kinase and CK levels and histological lesions of skeletal muscles. Later, the nature of the mutation was established. Nonsense point mutation caused by a single base substitution of C for T within an exon 23 leads to a premature termination of the dystrophin translation [24]. In addition to the absence of dystrophin all proteins of the DAPC such as sarcoglycans, syntrophin, nNOS, dystrobrevin, α-dystroglycan are significantly reduced at the sarcolemma in *mdx* skeletal muscle [9]. The absence of dystrophin and destabilization of the DAPC complex are believed to make muscle cells susceptible to stretch-induced damage and increased intracellular calcium influx. These pathological processes lead to skeletal and cardiac muscle degeneration [9]. Despite the absence of full-length dystrophin, *mdx* mice have mild symptoms of muscular dystrophy compared to DMD patients or the golden retriever muscular dystrophy (GRMD) dog model [24]. The pathogenesis of muscular dystrophy, physiological, biochemical and histological characteristics have been well studied in *mdx* mice of various ages. Birth body weight and neonatal death rates do not differ from their wild type counterparts. Significant histopathological abnormalities begin to be observed in *mdx* muscles at 3–4 weeks. The occurrence of extensive necrosis followed by regeneration and involving skeletal muscles was documented in *mdx* mice as young as 16–17 days [25]. In humans DMD is characterized by muscle hypertrophy in the early ages and atrophy in the late stages of disease. Contrary, in *mdx* mice myofibers pass through progressive hypertrophy from week 24 till the end of life without atrophy signs. Myofiber branching increased with the age and contributed to the hypertrophy. Aged *mdx* myofibers are also hypernucleated. The "extra" nuclei are central nuclei which highlight that the muscle undergoes continuous cycles of degeneration-regeneration. The estimation of synapse number indicated significant myofiber loss in *mdx* mice with the age [26]. The damaged skeletal muscle fibers with impaired function lead to a 20–30% loss in maximum specific force depending on mice age. The weakness is more severe in muscles of old *mdx* than in younger mice and healthy control mice [27]. *Mdx* muscle also demonstrates high susceptibility to contraction-induced injury [28]. Except skeletal muscles the diaphragm is severely damaged in *mdx* mice showing progressive deterioration, as is also typical for affected humans [24]. Compared to the voluntarily moving limb muscles, diaphragm fibers in *mdx* mice are subjected to early contraction-induced membrane rupture due to continuous action in the absence of dystrophin [24]. Histopathological changes of *mdx* diaphragm start to be observed at 4 weeks and include myofiber degeneration, necrosis, mineralization and large areas of fibrosis. But in contrast to the limb skeletal muscles, which are constantly affected to cycles of degeneration and regeneration, diaphragm undergoes progressive degeneration. By 16 months of age the *mdx* diaphragm looks pale due to extensive myofiber necrosis and replacement fibrosis. Changes in the physiological properties of *mdx* diaphragm correlate to histopathological lesions. Another muscular organ that is affected in *mdx* mice as in DMD patients is the heart. Echocardiographic signs of cardiomyopathy arise after ~8 months of age, while histological evidence of interstitial cardiac fibrosis does not appear until about 17 months [29].

Similar to DMD patients, *mdx* mice have increased levels of CK, marker of muscle damage, wherein CK levels were shown to increase with age, exercise, and male gender [30].

Since the pathogenesis of DMD in the *mdx* mice is genetically, biochemically and histologically similar to DMD patients, they have been extensively used as a preclinical model for DMD over the last 20 years. These mice are used to study the mechanisms of disease occurrence and dystrophin function, to test pharmaceutical drugs and to establish proof-of-concept for gene and cell therapy focusing on restoration of dystrophin expression [24, 30, 31]. The efficacy of a large number of pharmacological agents such as prednisone, deflazacort and other immunosuppressive and anti-inflammatory drugs currently used in therapy of DMD patients was tested in *mdx* mice in preclinical trials [30]. Also *mdx* mice were used in preclinical trials of replacement gene therapy on adeno-associated viruses carrying the dystrophin microgene/minigene. This therapy is currently in clinical trials [17].

Although *mdx* mice are the most commonly used animal model for DMD, its main disadvantage is the mild phenotype compared to DMD patients. To enhance muscular dystrophy pathology a lot of animal models with a more severe phenotype were created. Several approaches were used to create new murine models with DMD symptoms: N-ethylnitrosourea (ENU) mutagenesis (*mdx*2Cv, *mdx*3Cv, *mdx*4Cv, *mdx*5Cv mice models), generation of humanized transgenic mice with yeast artificial chromosomes (YAC) (hDMD mice), CRISPR/Cas9 (Clustered Regularly Interspaced Palindromic Repeats/CRISPR associated protein 9) and homologous recombination in embryonic stem cells (different murine models with exons deletion/duplication), Cre-loxP (Cre is from gene name *cre* that means "causes recombination"; loxP is for Locus of Crossover in P1) recombination system (*Dmd*-null mice), breeding *mdx* mice with other backgrounds (DBA/2-*mdx* mice, albino-*mdx* mice, BALB/c-*mdx* mice, immune deficient *mdx* mice) or other knockout (KO) murine models (*hDMD*/*mdx* mice, *hDMD*/*Dmd* null mice, *mdx*/Cmah−/−, *hDMD*/*mdx*/*Utrn*−/−, *mdx*/*Utrn*−/−, *mdx*/a7−/−, *mdx*/*MyoD*−/−).

Four new mdx murine models (*mdx*2Cv, *mdx*3Cv, *mdx*4Cv, *mdx*5Cv) were generated with ENU chemical mutagenesis [32]. Nature of these mutations was characterized. It was established that *mdx*2Cv allele results from mutation affecting mRNA splicing, and is located in the splice acceptor of intron 42 [33]. The *mdx*3Cv allele arises from a mutant splice acceptor site in intron 65 [32]. Similar to the *mdx*2Cv allele, the *mdx*3Cv splice acceptor mutation generates a complex pattern of aberrant splicing that generates multiple transcripts. But, in contrast to the *mdx*3cv mutation, alternative transcripts generated from *mdx*2Cv allele do not preserve the normal open reading frame [33]. In the case of the *mdx*4Cv allele, mutation is a C to T transition in exon 53, creating a stop codon (CAA to TAA). In the *mdx*5Cv allele, the dystrophin mRNA contains a 53 base pairs deletion and a single A to T transversion in exon 10 which does not alter the encoded amino acid. But a new splice donor was created (GTGAG) that generates a frameshifting deletion in the processed mRNA [33]. Despite all four new mutants show elevated serum CK level and muscle pathology similar to original *mdx* mice [32], each strain of mutant mice has unique features. Although each strain of mutant mice has unique features. The *mdx*3Cv mice exhibit abnormal breeding behavior and cognitive defects in addition to dystrophic muscle pathology. The levels of DAPC proteins and full-length dystrophin were decreased. So *mdx*3Cv mice may act as a useful model for studying the effect of subtherapeutic level of dystrophin on DMD phenotype recovery. Surprisingly, skeletal muscle strength was only slightly reduced compared to wild type mice and muscles were partially protected from eccentric contraction-induced injury [34]. Histopathological analysis of skeletal muscles, heart and diaphragm of the *mdx*4Cv and *mdx*5Cv mutants indicates 10-fold fewer revertants than in the muscles of *mdx*

#### *Duchenne Muscular Dystrophy Animal Models DOI: http://dx.doi.org/10.5772/intechopen.96738*

mice [24]. Also *mdx*5Cv mice have a more severe skeletal muscle phenotype than *mdx* mice. These mice showed pronounced functional deficits and lower interindividual variability in motor activity tests compared with *mdx* mice which is a great advantage in studies with small numbers of animals [24, 30, 31]. Both of these murine models *mdx*4Cv and *mdx*5Cv are currently used in preclinical trials of gene therapy [35].

There are several models of mice obtained by crossing *mdx* mice with other genetic backgrounds such as albino mice [36], BALB/c mice, DBA2 mice [37], C57BL/6 mice, C3H mice [38], FVB mice and immune deficient mice [24]. In some cases background does not dramatically alter dystrophic phenotype of *mdx* mice (BALB/c-*mdx* mice, C57BL/6-*mdx* mice, FVB-*mdx* mice). But some murine models obtained during the crossing showed new phenotypic features and more severe phenotypes than *mdx* mice (albino *mdx* mice, DBA2/*mdx* mice). For example, albino-*mdx* mice combined signs of muscular dystrophy (histopathology of skeletal muscles, increased serum CK level, body and muscle weights) with signs of oculocutaneous albinism (skin, fur and eye depigmentation) [36]. In contrast to original black *mdx* mice, albino-*mdx* mice showed slow geotaxis, which can indicate a deterioration of neurological state of DMD [39], and increased circulating cytokines levels [40].

The most phenotypically relevant to the human DMD murine model was created on the DBA2 background. The DBA2 inbred mouse strain carries a naturally occuring in-frame deletion within the latent TGFβ-Binding Protein 4 (LTBP4) gene. This promotes enhanced inflammation and loss of ambulation in DMD patients [41]. The DBA2-*mdx* (D2-*mdx*) mice showed progressive development of muscular dystrophy. These mice had severe histopathological features, including the rapid progression of fibrosis in diaphragm and skeletal muscles. In addition, all muscles of these mice had zones of extensive calcification. In contrast to original *mdx* mice D2-*mdx* mice developed cardiomyopathy at an earlier age, moreover, more fibrous tissue was observed in the hearts of D2-*mdx* mice [37]. The more pronounced dystrophic phenotype and faster progression of the disease in D2-*mdx* mice compared to *mdx* mice on C57BL/10 background makes D2-*mdx* mouse strain more suitable for evaluation of treatment efficacy in preclinical trials [42]. Immune-deficient *mdx* mice are used to test cell therapies as one of the approaches to treating muscular dystrophy. These strains were created by crossing of *mdx* mice with different strains of mice with mutations in different genes (c-kit receptor gene, IL-2 receptor gene, DNA-dependent protein kinase catalytic subunit deficient and others) and deficiency of B cells, T cells and NK cells [43], cytokine signaling deficiency [43], hematopoietic cells deficiency [44] or with severe combined immunodeficiency [45]. Severity of phenotypical features in immune-deficient dystrophic mice are usually similar to *mdx* mice. But these murine strains are a good model for preclinical trials of cell transplantation therapies.

*Mdx* murine model lacking dystrophin expression demonstrates less pronounced degenerative changes in comparison with DMD in humans. This may be attributed to various species-specific compensatory mechanisms in mice, increased expression of other membrane proteins in murine muscles, or the characteristics of the skeletal and cardiac muscles themselves. To study the effect of compensatory mechanisms in mice, double-knockout (DKO) murine models and humanized murine models were created. Compensation for the lack of dystrophin with structurally related proteins possibly leads to a milder DMD phenotype in *mdx* mice than in DMD patients. In *mdx* mice, unlike humans, the expression of utrophin, in skeletal muscles, diaphragm, heart and non-muscular tissues persists throughout life [46]. The amino acid sequence of utrophin repeats largely dystrophin and can hypothetically substitute it on the sarcolemma and participate in muscle contraction [9].

Therefore, upregulation of utrophin may be one of the treatment options for DMD. To test such drugs as well as to study DMD pathogenesis, mice deficient in both dystrophin and utrophin were created. This double-knockout (*mdx/utrn*<sup>−</sup>/<sup>−</sup>, u-dko) murine model was derived from breeding dystrophin deficient *mdx* mice with utrophin deficient mice [47]. In contrast to *mdx* mice u-dko mice were smaller and weaker and developed severe muscular dystrophy phenotype similar to phenotype in DMD patients. All clinical signs of the disease (pathohistology of skeletal and cardiac muscles, muscle functions) were more pronounced in u-dko mice than in *mdx* mice. These mice also started to show DMD symptoms at an earlier age [47]. This murine model is currently used in preclinical trials of gene therapy drugs based on adeno-associated or adenoviruses carrying shortened utrophin genes. Several studies have shown that utrophin-delivering therapy is equally effective as micro/minidystrophin-delivering therapies [48]. Another protein that can replace the absent dystrophin and perform complementary function in *mdx* muscles is the membrane protein integrin α7. Dystrophin and integrin α7 double knockout mice (*mdx*/α7<sup>−</sup>/<sup>−</sup>), as well as u-dko mice, showed a more apparent dystrophic phenotype compared to original *mdx* mice [49]. Dystrophin- and integrin α7-deficient mice had reduced body mass compared to *mdx* mice and demonstrated early lethality (4 weeks after birth). Skeletal and cardiac muscles of double-knockout *mdx*/α7<sup>−</sup>/<sup>−</sup> mice were more severely affected and exhibited loss of membrane integrity, more prominent histopathological and functional characteristics [49].

Another explanation for the less pronounced dystrophic phenotype in *mdx* mice may be the increased regeneration of muscle fibers after necrosis which presents in formation of fibers with centrally located nuclei and muscle pseudohypertrophy [23]. To test this hypothesis, several murine models with reduced muscle regeneration were created. Since activated satellite cells are involved in the regeneration of skeletal muscle fibers, murine models with knockout of genes involved in the activation of satellite cells, were created to limit regenerative capacity. The first approach is a knockout of myogenic basic-helix–loop–helix transcription factors MyoD which plays an important role in myogenesis [50]. Mice lacking both MyoD and dystrophin (*mdx*/MyoD/<sup>−</sup>/<sup>−</sup>) created by breeding of *mdx* mice with MyoD mutant mice developed a severe cardiomyopathy and muscle hypertrophy leading to premature death [51]. Phenotypically these mice are much closer to DMD patients. The second approach to enhance DMD phenotype in mice is a modeling of the telomerase RNA absence. It was established that telomere length in human dystrophic cardiomyocytes and skeletal muscles is shorter than in normal muscles [52]. To create such a murine model, *mdx* mice were crossed with mice lacking telomerase RNA (*mdx*/mTR KO) [52]. *Mdx*/mTR KO showed a severe dystrophic phenotype and significantly reduced lifespan compared to *mdx* or mTR KO controls. Also aged mice showed explicit skeletal deformity (kyphosis) [52]. Double knockout mice make a significant contribution to the study of DMD pathogenesis and the assessment of DMD drug therapy effectiveness, however, these murine models do not directly explain the differences in phenotype between mice and humans. Therefore, so-called humanized murine models were created.

Humanization makes phenotype of *mdx* mice closer to the phenotype of DMD patients. Mice have reduced inflammatory and immunologic reactivity compared to humans. For example, mice, unlike humans, evolutionally retained the cytidine monophosphate-sialic acid hydroxylase (*Cmah*) gene. Introduction of human-like inactivating deletion of Cmah gene into *mdx* mice prevented synthesis of the sialic acid N-glycolylneuraminic acid [53]. The *mdx*/Cmah/<sup>−</sup>/<sup>−</sup> mice had genotypic and phenotypic similarities to human DMD, enhanced DMD severity and shortened lifespan compared to *mdx* mice. Cardiac muscle of mutant mice shows large areas of fibrosis and mononuclear infiltration. These features make

#### *Duchenne Muscular Dystrophy Animal Models DOI: http://dx.doi.org/10.5772/intechopen.96738*

*mdx/Cmah/*<sup>−</sup>/<sup>−</sup> murine model suitable for evaluating effects of new DMD therapy on dystrophic cardiac muscle.

Dystrophin function, as well as pathogenesis and treatment strategies for DMD have been well studied in different murine models (*mdx*, *mdx/Utrn*<sup>−</sup>/<sup>−</sup> dko and many others). All these murine strains lack full-length dystrophin expression and show specific dystrophic features. However, the expression of small isoforms of dystrophin may remain in some models. To study the contribution of small isoforms to the DMD pathogenesis, a model with a completely deleted dystrophin gene was created. Using the Cre-loxP recombination system Dmd gene was completely removed in mice. The resulting mutants (*Dmd*-null mice) were viable, but the males were sterile. The mice showed an evident dystrophic phenotype and behavioral abnormalities [54].

Murine models are the most convenient and widely used for studying protein function, pathogenesis and treatment options for the disease. Many preclinical trials of drugs that are currently used or tested in clinical trials have been performed on DMD murine models. However, many laboratories use not only mice for their studies, but also other species of animals, including non-mammalian models, other rodents or large mammals. Non-mammalian DMD models were generated in zebrafish *Danio rerio*, *Drosophila melanogaster* and *Caenorhabditis elegans* [24, 30, 31, 55]. Non-mammalian DMD models have some advantages over mammals. Fishes, worms and insects are eukaryotic models and have some valuable features: small size, high reproduction rate, fast growth and development, a large number of offsprings and fully sequenced genomes. Dystrophin amino acid sequence and subcellular localization are highly conserved between humans and zebrafish. The zebrafish *dmdta222a* mutants (*sapje*) with dystrophin deficiency showed muscle degeneration which was more severe than in *mdx* mice and died at an early larval stage [56]. Zebrafish DMD model is a good model to test exon-skipping therapeutic strategy. For example, FDA approved drug Ataluren (Translarna) was tested on zebrafish and led to restoration of muscle contractile functions [57]. One more non-mammalian DMD model is dystrophin deficient *Drosophila melanogaster*. Muscle-specific RNAi-mediated knockdown of all dystrophin isoforms in flies led to severe muscle degeneration, cardiomyopathy phenotype and climbing deficits [58, 59]. Nematode worm *Caenorhabditis elegans* is also used for DMD model creation. These worms have dystrophin homolog gene dys-1. Loss-of-function in dys-1 resulted in worm hyperactivity and hypercontraction [55].

In addition to mice, larger animal models are now available. All DMD canine and feline models have been identified in natural populations. Porcine, rat, monkey and rabbit models were created with CRISPR/Cas9 technology [24, 31]. The most popular DMD models in large animals are canine models. Spontaneous mutations in the dystrophin gene causing the development of dystrophic phenotype have been identified in 14 dog breeds [60]. Some of them are currently bred in nurseries as a DMD canine model, others were discovered in natural populations as individual cases and described in the literature. The first group includes the well known golden retriever muscular dystrophy dog model (GRMD), Cavalier King Charles spaniel model [61], Welsh corgi model Australian Labradoodle model, German short-haired pointer and new labrador retriever model with inversion in dystrophin gene [60]. The most widely used and well described canine model of DMD is the GRMD model. The GRMD mutation was first reported in four animals in the early 1980s [62]. It was established that GRMD dogs had a splice site mutation (transition A > G) in intron 6 causing abnormal mRNA splicing and loss of exon 7 of dystrophin gene. GRMD dogs had severe dystrophic phenotype including elevated CK level, skeletal muscle atrophy with contractures, dyspnoea, dysphagia, dilated cardiomyopathy, large fibrosis and fat tissue areas. The GRMD dog population also showed heterogeneity

of dystrophic features between different individuals, what also makes this model similar to DMD in humans [60]. A clinical course of GRMD dogs is more similar to DMD patients in contrast to *mdx* mice. Large body size, severe muscular dystrophic phenotype, humoral and cellular immune response to viral vector and transgene, as well as transplanted cells similar to human, make GRMD dogs a more suitable model for preclinical trials to test pharmaceutical drugs, gene replacement therapies and cell therapies [31]. The GRMD dogs model was used in different preclinical trials of gene and cell therapies. The advantage of using GRMD dogs in these studies is the experiment design similar to clinical trials. For example, in clinical trials, the inclusion criterion is the intake of immunosuppressive drugs. In dogs, in contrast to mice, the immune reactivity is similar to that of humans, which makes it possible to reproduce this design as well as to study the obvious adverse reactions associated with the activation of host immunity [63]. The mutation of Cavalier King Charles spaniel (CKCS) model is a splice site mutation (transition G > T) in intron 50 causing the deletion of exon 50 [61]. CKCS dogs show elevated levels of serum CK and typical areas of necrosis and regeneration in skeletal muscles and heart. Dogs of this breed seem to be suitable for testing due to their small body mass and amiable temperament. CKCS canine model can be used to test exon 51 skipping, the therapy that may be suitable for many patients, as DMD mutation hotspot is located between exons 45 and 55 [61]. The mutation of Welsh corgi model, Australian Labradoodle model and German short-haired pointer model (GSHPMD) are LINE-1 insertion in intron 13, point mutation in exon 21 and whole *DMD* gene deletion respectively [60]. These dogs show severe dystrophic phenotype including muscle degeneration, mineralization and inflammatory infiltration. It is important to note that GSHPMD dog model with completely absent dystrophin is the most suitable preclinical model for the prediction of immune responses to gene therapy due to the lack of immunological tolerance to dystrophin [64]. One more interesting canine DMD model is the recently identified labrador retriever (LRMD) model with an inversion in dystrophin gene. 2.2-Mb spontaneous inversion disrupting the *DMD* gene within intron 20 was found in two young labrador retriever dogs. The clinical signs of disease included elevated CK level in serum, specific histopathological lesions of skeletal and cardiac muscles, myopathic electrodiagnostic profile, high neonatal lethality. The LRMD dogs had detected expression of Dp71 isoforms of dystrophin. But unlike the GRMD dogs with absent Dp71 isoform, the LRMD dogs have more severe dystrophic phenotype. This may indicate that the presence of the Dp71 isoform in muscles does not provide a functional advantage [60].

In addition to dystrophic dog colonies maintained in nurseries several cases of spontaneous mutations in dogs of different breeds have also been described. The interesting case is 7 base pair deletion in exon 42 in Cavalier King Charles spaniel, the second CKCS model with mutation in the *DMD* gene hotspot area. These dogs had generalized skeletal muscle atrophy of the temporal region, limbs and thoracolumbar spine [65]. One more case of spontaneous mutation in dystrophin gene was revealed in Miniature Poodle dog. Dogs had whole *DMD* gene deletion and showed all dystrophic clinical signs including muscle degeneration, lumbar kyphosis, stiff gait and abnormal posture. Neurological examination also revealed reluctance to exercise in these dogs [66]. One case of disease development was also recently detected in the Jack Russell Terrier population. The dog had deletion of exons 3–21 causing severe dystrophic phenotype and death at the young age [67]. Progressive muscle weakness was also detected in a male border collie dog. Its mutation was a single nucleotide deletion in canine DMD exon 20, minor DMD mutation hotspot, resulting in generalized muscle atrophy, muscle fatigue and dysphagia [68].

Unequivocally, canine models have a significant advantage over murine models due to their more pronounced dystrophic phenotype and possible immune response

#### *Duchenne Muscular Dystrophy Animal Models DOI: http://dx.doi.org/10.5772/intechopen.96738*

to treatment. However, as well as *mdx* mice, GRMD and other dogs have some disadvantages associated with the high cost of keeping dog colonies and training of personnel caring for sick animals. In addition, due to a greater body weight than in mice, large amounts of drugs are required for dogs, which is essential for gene therapy based on viral delivery. Nevertheless, studies in dogs are considered more informative than studies in mice. The results of the dog trials provide a better indication of future clinical trials. In this regard, it is important to use not only widespread mice but also dogs in the design of preclinical trials.

The first case of hypertrophic feline muscular dystrophy (HFMD) in domestic cats was described in 1989 [69]. Spontaneous mutation causing dystrophic phenotype was established as a deletion of the dystrophin promoter and first exons corresponding to dystrophin from muscle and Purkinje cells. Dystrophic cats showed pronounced appendicular and axial muscle hypertrophy, involving of tongue and diaphragm, histopathological lesions in skeletal muscles, diaphragm and heart, including different fiber diameter and acute necrosis and cardiomyopathy [70]. The HFMD model is rarely used in DMD preclinical research because tongue hypertrophy and diaphragm defects lead to difficulties in feeding, animal welfare and early death.

The CRISPR/Cas9 technology has made it possible to create several more models of DMD in such animals as pigs, rats, rabbits and monkeys. Rats are the most convenient animals for biomedical research, therefore several rat models have been created. The first rat model was created using CRISPR/Cas9 gene editing [71] and had exon 3–6 deleted in dystrophin gene. Dystrophin deficient rats showed reduced muscle strength and specific dystrophic phenotype of skeletal muscles, diaphragm and heart. Also these rats showed age-dependent decline of cardiac functions similar to DMD patients [72]. Later, based on this model, another rat model with an in-frame mutation in the dystrophin gene was generated [73]. New mutant rats had reduced expression of truncated dystrophin and mild phenotype similar to BMD patients. These rats can be useful to study BMD pathogenesis and efficiency of dystrophin recovery. The third rat model was created using TALEN (Transcription activator-like effector nucleases) technology. Its mutation was a frame shifting 11 base pairs deletion in exon 23 generating premature stop codon [74]. Animals exhibited reduced muscle strength, cardiomyopathy, large muscle necrosis and fibrosis. This model can be used for preclinical research as a small DMD animal model.

Several mice models were created that may be suitable mostly for scientific use. One of them is the Dmdmdx−bgeo model [75]. It contains the beta-Geo marker inserted after exon 63. The protein product translated from the resulting allele lacks cysteine-rich and C-terminal domains and is not functional. The Dmdmdx−bgeo model mostly resembles the *mdx*3cv model as both of them lack all dystrophin isoforms including Dp71 and Dp40. Hemizygous Dmdmdx−bgeo animals demonstrate phenotypic properties similar to other *mdx* models. LacZ (β-galactosidasemediated) staining helps to visualize the expression of dystrophin in various tissues on different stages of development including embryonic. Nevertheless, the dysfunctionality of dystrophin-lacZ chimeric protein should always be taken into account.

DmdEGFP reporter mouse [76] lacks the disadvantage of Dmdmdx−bgeo model. The eGFP (enhanced green fluorescent protein) coding sequence was introduced behind the exon 79 and the chimeric protein remains functional. The transgenic mice did not show any signs of pathology. This approach allows us to observe almost all major dystrophin isoforms except for those having alternative C-terminal domain. The studies with this model may provide valuable data on dystrophin expression and localization in muscle and non-muscle tissues and shed the light on its functions.

In 1999 the Dp71-null mouse model was described [77]. The first and unique exon of Dp71 is located between exons 62 and 63 of the *Dmd* gene. It is replaced by promotorless b-geo gene in Dp71-null mice leaving all other dystrophin isoforms intact (except for Dp40). The resulting construction provided the expression of β-Galactosidase regulated by Dp71 promoter while the native product, Dp71, was absent. This model acts as a valuable tool for examination of the role and functions of Dp71 isoform both by Dp71 promoter activity estimation by LacZ staining and Dp71-null phenotype examination. The further experiments demonstrated that Dp71 deficiency causes retinal vascular inflammation, increases retinal vascular permeability. AAV-mediated delivery of Dp71 restored retinal homeostasis and prevented retinal oedema [78] and restored defective electroretinographic responses [79]. Dp71 expression in neurons plays a regulatory role in synapse organization, formation and function and inactivation of Dp71 may lead to increased severity of mental retardation and intellectual disability [80].
