**Abstract**

Duchenne muscular dystrophy is a complex and severe orphan disease. It develops when the organism lacks the expression of dystrophin - a large structural protein. Dystrophin is transcribed from the largest gene in the human genome. At the moment, there is no cure available. Dozens of groups all over the world search for cure. Animal models are an important component of both the fundamental research and therapy development. Many animal models reproducing the features of disease were created and actively used since the late 80's until present. The species diversity spans from invertebrates to primates and the genetic diversity of these models spans from single mutations to full gene deletions. The models are often non-interchangeable; while one model may be used for particular drug design it may be useless for another. Here we describe existing models, discuss their advantages and disadvantages and potential applications for research and therapy development.

**Keywords:** Duchenne muscular dystrophy, DMD, dystrophin, animal models, *mdx*, genome editing, exon-skipping, gene therapy

## **1. Introduction**

Duchenne muscular dystrophy (DMD) was primarily described in 1834–1836 by Neapolitan physicians Giovanni Semmola and Gaetano Conte. Dr. Guillaume Duchenne de Boulogne made a significant contribution to the description of the disease in 1860s [1]. DMD is considered a rare, or orphan, disease but it is definitely one of the most frequent among muscular dystrophies. About one male in 3500 is diagnosed with DMD. DMD is an X-linked recessive disease so women are affected with a frequency of 1 case per 50 million [2–4]. Many attempts of various groups and organizations are set towards the search for the cure. Different strategies such as genome editing, replacement therapy, anti-inflammatory and antioxidative drug treatment are developed [5]. These therapies target different components of an extremely complex scheme of DMD pathogenesis. So animal models are important for study of the disease, research and development of the therapies. Many animal models were created or, in some cases, adapted from natural sources.

It is important to understand the mechanism of DMD pathogenesis and progression in order to discuss origins, purposes and potential uses of animal disease models. DMD develops when the organism lacks dystrophin expression. Dystrophin is encoded by the largest gene in the genome (*DMD*) that consists of more than 2.3

megabases (Mb). The gene contains 7 promoters and two polyadenylation signal sequences which orchestrate expression of 17 known isoforms. Three large isoforms are produced by three distant promoters. These isoforms are brain isoform Dp427c, muscle isoform Dp427m and Purkinje isoform Dp427p [6]. Each of them consists of 79 exons which include the first unique exon and 78 common exons. Several smaller isoforms are operated by 4 internal promoters and some of them have alternatively spliced variants. These isoforms are retinal Dp260, Dp140 which is prevalent in central nervous system (CNS) and kidney, Dp116 which is expressed in Schwann cells and ubiquitously expressed Dp71 and Dp40 [6, 7]. Muscle isoform Dp427m is the most characterized and widely studied due to its crucial role in DMD manifestation. Most of the mutations that lead to DMD progression are large insertions, exon deletions or duplications which lead to the shift of the reading frame in the Dp427m [8]. Usually these mutations produce preliminary stop codon leading to the complete absence of the protein [8]. Point mutations (deletions, insertions or substitutions) are responsible for a small portion of all DMD cases [8]. Other isoforms are studied less. The deficiency of most of them is usually linked to CNS and behavioral disorders while Dp260 deficiency is linked to retinal impairment [6, 7].

Muscle dystrophin is a very complicated molecular machine. The function of muscle dystrophin is formation of dystrophin-associated protein complex (DAPC) and absorption of mechanical tensions which occur due to muscle constriction [9]. Muscle dystrophin is 427 kDa protein that consists of 3685 amino acids [10]. The protein is usually divided in four functional and structural superdomains. The N-terminal superdomain consists of two calpain-homology domains and provides binding of the protein to actin. The second superdomain is called rod domain. It is the largest domain that includes 24 spectrin-like repeats and 4 unstructured hinge domains. It acts as a spring that adsorbs mechanical tensions. The third superdomain (referred to as cysteine-rich domain, or CR) includes WW-motif, two EF motifs and ZZ-motif. This domain binds dystrophin to the sarcolemmal proteins being the central driver of DAPC formation. C-terminal domain binds to several proteins performing mostly signal functions [10].

DAPC is located in sarcolemma and provides the linkage between dystrophin and external proteins such as laminin and collagen. The complex includes ɑ- and β-dystroglycans, ɑ-,β-, -,δ-,ε-sarcoglycans which interact with CR domain of dystrophin; and dystrobrevin, α1, β1, and β2-syntrophins, neuronal nitric oxide synthase (nNOS) and several other proteins which interact with C-terminal domain. The deficiency of these proteins also induces several pathologies such as limb-girdle muscular dystrophy, myotonia and some others [9].

The loss of dystrophin leads to several consequences. The initial one is the loss of membrane integrity and toughness. This causes membrane damage during muscle contractions and consequent membrane leakage. The homeostasis of extra- and intracellular components (calcium ions being the most important of all) is disrupted. This leads to calcium signaling imbalance, mitochondrial dysfunction (as mitochondria acts as calcium depo), proinflammatory and apoptotic signaling activation and other damaging consequences [11]. Finally, this results in muscle cell death and its replacement by new muscle cells originating from satellite predecessor cells that finally leads to depletion of the pool of satellite cells. Damaged and regenerating muscle tissue is characterized by central nuclei. The fraction of central nucleated myofibers is a quantitative marker of DMD progression and therapeutic treatment [12]. Normal muscular tissue is also replaced by connective tissue (fibrosis) and adipose tissue in addition to regeneration. Neutrophil and macrophage infiltration also accompanies the disease progression [13].

The first symptoms of DMD usually arise at the age of 16–18 months. The children may experience issues with walking, running or rising, toe walking or Gower's

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

sign. At the age of 2–3 years old the muscles of lower limbs begin to degrade. The children suffer from extensive weakness and obtain specific gait patterns. Scoliosis and flexion contractures of the limbs also develop in DMD patients. At the age of 10–12 years old children begin to use a wheelchair. Later, at 14 y.o., some patients develop dilated cardiomyopathy and arrhythmia. Patients usually die at 20 years due to heart failure or respiratory distress in absence of proper treatment. Female carriers do not suffer from severe symptoms; they usually have cardiomyopathy, mild respiratory issues, creatine kinase (CK) level enhancement and pseudo hypertrophy of the backside of the shin [14].

If any suspicious symptoms are observed CK level estimation is the first diagnostic procedure. This is a cheap and fast but not selective test as CK growth is a symptom of various muscle and nonmuscle (i.e. liver) diseases. So further diagnostics is required. If the CK is elevated the screening for exon deletion or duplication should be performed. About 30% of mutations may not be identified by these techniques (multiplex ligation-dependent probe amplification or comparative genomic hybridisation array) and full sequencing of the gene is required. The mutation location and character may help to predict the type and severity of the disease. If the mutation is still unidentified the muscle biopsy sample should be tested for dystrophin protein presence by immunohistochemistry or western blot [14].

In some cases, mutations in *DMD* gene do not lead to reading frame shift or do not cause severe instability or protein dysfunction. If the function of the protein is slightly affected the milder form of muscular dystrophy develops. This disease is referred to as Becker muscular dystrophy (BMD). BMD is characterized by a very wide spectrum of symptoms. In some cases disease may be almost as severe as DMD while in other cases it may develop comparatively mild phenotype [15]. In 1990 a patient with a large part (>50%) of the *DMD* gene deletion was discovered [16]. The patient was active at 61-year-old and demonstrated mild myodystrophy phenotype further described as BMD. The analysis of the mutant gene and its product revealed extremely valuable data on the mechanism of dystrophin molecular action. The deletion of the part of the gene did not lead to reading frame shift and functional protein was expressed. This protein lacked most of the rod domain while N-terminal, cysteine rich and C-terminal domains remained intact. Obviously the rod domain which is the largest part of the protein may be truncated without complete function loss. The second important outcome is the frameshift rule formulation. The restoration of the reading frame may lead to the synthesis of truncated but still partly functional protein and shift the DMD type to BMD type. These findings set the initial point for development of several antiDMD therapies [5].

Currently no ultimate cure for DMD exists. Several treatment strategies are currently applied and many approaches are waiting for approval or being developed [5]. Most of the approved treatments target the farther consequences of dystrophin loss [5, 11]. Glucocorticosteroids suppress fibrosis and inflammation and mechanical ventilation helps patients with respiratory deficits. Anti-inflammatory and antioxidant drugs are also used or being tested [11]. But these approaches do not target the primary issue and are capable of lengthening the lifespan for about a decade. Several more complex approaches are now being developed. One of the most promising candidate therapies is the gene replacement therapy [17]. The idea is the delivery of a shortened but still functional gene copy to the muscles lacking its natural variant. The delivery may be provided via various types of vectors such as viral vectors, nanoparticles or even plasmids [18]. Several difficulties complicate the path to success. These are extremely high research and production costs, immune response and comparatively large size of the protein and corresponding genetic construct. Another class of therapies being developed is restoration of the reading frame [19]. This may be achieved by introduction of antisense

oligonucleotide, genome editing or some other techniques. The next class of therapies is utrophin modulation. Utrophin is an autosomal paralog of dystrophin which shares almost similar domain organization and high sequence correlation with dystrophin. In embryonic muscles utrophin localizes similarly to dystrophin and performs the same functions. In muscles utrophin is replaced by dystrophin in early childhood and in adults it is present in such non-muscle tissues as renal epithelia. In the adult organism utrophin expression is extremely low. In the case of dystrophin deficiency the expression of utrophin starts to increase but its level is still insufficient for dystrophin replacement in humans. Several approaches such as transcription modulators may potentially increase utrophin expression and slow down the disease progression [20]. Interestingly, several species such as mice are able to increase utrophin expression to sufficient level without any modulators [21]. This may provide fundamental data about dystrophy compensation mechanisms. However, it questions the adequacy of the DMD model based on these species. Other strategies include cell-based therapies which are being developed for a long time and interesting exosome-based approach which originated from cell-based one [22].

As can be seen from the above, the existing and potent strategies for DMD therapy include genome editing, pre-mRNA splicing and cell modification, gene or cell delivery, and others [5]. All of them require animal models to be tested. In most cases these models are not interchangeable. For example, if one develops an exon-skipping strategy for a rare mutation, they will need an animal model with a corresponding mutation. So ideally a unique model is essential for every single mutation (at least for most common of them). The type and location of mutation is also important as, despite almost all mutations lead to absence of three major isoforms, the presence or absence of short isoforms depends on mutation location and type. So different mutations on similar backgrounds may have different phenotypes and may be valuable both for research and drug development. Many animal (mostly mouse) models with different specific mutations were developed both for fundamental studies of the gene and protein function and role of short isoforms and for proof-of-concept and preclinical studies of potential therapies.

Despite mouse models of DMD being the most common due to their relative cheapness they possess a significant disadvantage. All dystrophin-deficient animals have dystrophic symptoms but the severity of them does not often correlate with the disease severity in DMD patients. For example the lifespan of classic mouse model *mdx* is about 80% of normal [23, 24] while the lifetime of a human with DMD is not more than one third of healthy. To circumvent these demerits, several other species were used to reproduce the phenotype of DMD in animals. These are large animal models (dogs, pigs, primates) or mouse models with mutations in additional genes, or crossbreed models. In some cases the genetic structure of these models does not correspond to any known DMD mutation in humans but similar phenotype makes them useful for studies of the disease and several symptomatic therapies.

Here we describe animal models starting from classic *mdx* identified in 80's to the newest ones introduced in 2020. The list of model species includes species from such invertebrates as *D. melanogaster* and *C. elegans* to monkeys. The origin, genotype, phenotype and purpose of these models are very diverse. We basically divide the models into two large groups. Chapter 1 will focus mostly on phenotypic properties of the most common models, the comparison of their advantages and disadvantages and their use in research and drug development. Chapter 2 will focus on the models created for development of unique and precision therapies. These are mostly murine models with various spectrum of mutations suitable for targeted drug design such as exon skipping.
