**3. MDs and pharmacological treatments**

**1. Introduction**

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treatment of muscular dystrophies.

**dystrophies**

Muscular dystrophies (MDs) are genetic diseases caused by the continuous degeneration/ regeneration cycles of skeletal muscle tissue. Mutations in genes encoding for proteins, either at the plasma membrane or within internal membrane, are responsible for MDs. During contractions, the affected muscle fibres degenerate and the molecular mechanisms are not yet fully understood. Fibre loss is compensated by the regeneration of new fibres, mainly sustained by satellite cells. These are localized underneath the basal lamina of muscle fibres [1]. Damaged dystrophic muscles engage in a remodelling process to generate novel fibres and to produce abundant extracellular matrix (ECM). ECM is necessary for adequate tissue repair. During periods of degeneration/regeneration, myofibroblasts accumulate in dystrophic muscles and are responsible for large amounts of extracellular matrix proteins, generating fibrosis. Addi‐ tionally, at the final stage, satellite cells become exhausted and are not able to generate new fibres. Cardiac muscle is less efficient in regeneration, compared to skeletal muscle and scar tissues in replacing damaged cardiomyocytes after injuries [2]. However, several research groups have demonstrated the presence of stem/progenitor cells that are able to differentiate into cardiac tissues [3-6], as well as skeletal muscle lineages [7-12]. This paper deals with novel therapeutic approaches for skeletal muscle dystrophies and explores pharmacological treatments. It also provides more recent gene and cell therapeutic protocols. Different sources of myogenic stem cells are discussed, highlighting their advantages and disadvantages, as well as underlining controversies in literature. Finally, we discuss autologous and heterologous cell therapy, considering the viral and non-viral technologies for *ex vivo* cell therapy in the

**2. Epidemiology, diagnosis and clinical management of muscular**

Muscular dystrophy was described for the first time in 1860s by the neurologist Guillaume-Benjamin-Amand Duchenne (de Boulogne). This followed a study of 13 boys who were affected by the most common type of muscular dystrophy, now carrying his name. Worldwide, Duchenne muscular dystrophy (DMD) affects 1/3,500 born males. Other isoforms of muscular dystrophies include Becker muscular dystrophy (less severe than DMD, with an incidence 3-6: 100,000 male births), limb-girdle muscular dystrophy (mainly affecting hip and shoulder muscles, occurring between 10 and 30 years of age, with an estimated range of incidence between 0.5-4:100,000), congenital muscular dystrophy (present at birth and not affecting the life span, incidence 1:21,500), facioscapulohumeral muscular dystrophy (inherited form of muscular dystrophy, initially affecting skeletal muscles of the face, scapula and upper arms, starting from teenage years. Incidence 4-12:100,000), myotonic dystrophy (an inherited form of muscular dystrophy, normally occurring in patients of any age. European incidence: 3-15:100,000) and finally, oculopharyngeal muscular dystrophy (a type of muscular dystrophy occurring in the middle age and, at the beginning, causes drooping of eyelids, dysphagia and

Pharmacological treatments for MDs aim to stabilize the structural integrity of the muscle fibre membrane by counteracting chronic inflammation. Indeed, lack or genetic mutations of dystrophin causes a chronic influx of calcium into the myofibres. This is largely responsible for cell death and inflammatory response (Figure 1). Accumulation of fibrotic tissues in the replacement of damaged myofibres is another pathophysiological feature of MDs that is responsible for decreasing the contraction force and increasing fatigue. These events charac‐ terize dystrophinopathies because dystrophin plays a pivotal role in the anchoring of the dystrophin-associated protein complex, which, in normal conditions, can stabilize the struc‐ tural integrity of membrane.

**Figure 1.** Histological features of muscular dystrophy. Upper panels show the histological architecture of the *tibialis anterior* muscle in a healthy control mouse (left panel) and dystrophic mouse model of human Duchenne muscular dystrophy (mdx, right panel). Haematoxylin and Eosin stain identifies the presence of a huge amount of infiltrating mononuclear cells within the *interstitum* of mdx muscle. Necrotic fibres, as well as centrally located nuclei fibres, are other hallmarks of dystrophic muscle (right panel) compared to healthy control (left panel). Lower panels show the immunofluorescence stain for dystrophin (red). Compared to the healthy control (lower left panel), muscle from mdx shows a strong reduction of dystrophin content. Only a small percentage of revertant fibres are positive for dystrophin expression (lower right panel).

Although there are no proficient cures for dystrophinopathies, several drugs have been used to delay their detrimental effects on muscle tissues, mainly through the attenuation of inflammation. In the treatment of MDs, several drugs are used for their ability to reduce circulating levels of TGF-β, known to play a crucial role in the fibrotic tissue deposition in dystrophic muscles. Among the drugs used to counteract the systemic burden of TGF-β are non-steroidal anti-inflammatory drugs (NSAIDs) such as nabumetone, ibuprofen and isosor‐ bide dinitrate. These drugs have beneficial effects, especially in the treatment of Duchenne, Becker and Limb-Girdle muscular dystrophies [13]. Promising results were recently obtained from phase I studies in healthy volunteers. These studies revealed an optimal tolerability and safety profiles for a combined administration of isosorbide dinitrate, a nitric oxide donor and ibuprofen (NSAIDs) for the treatment of muscular dystrophies [14]. Other established pharmacological approaches aim to hamper the elevated muscle inflammation and necrosis events linked with mitochondrial dysfunction and altered metabolism. In particular, αmethylprednisolone (a synthetic glucocorticoid) stimulates the reduction of cytosolic calcium in dystrophic muscle and prevents apoptosis and/or necrosis events that normally occur during muscle degeneration in dystrophinopathies [15] (Figure 2). Another challenge in the pharmacological treatment of MDs is counteracting the high susceptibility to muscle damage that characterizes dystrophinopathies. Indeed, among the corticosteroids drugs, prednisone and deflazacort show positive effects in reducing muscle damage and weakness, as well as in counteracting the loss of muscle contraction. Moreover, since the integrity of muscular membrane is a critical determinant of muscle degeneration during the illness progression, many efforts have been made to promote the sarcolemall stability of muscle fibres through the increase of native utrophin as a compensatory mechanism of dystrophin loss. In particular, nabumetone is a small molecule with anti-inflammatory properties, derived from its ability to inhibit cyclooxygenase. In addition, nabumetone can activate the transcription of utrophin. Aminoglycosides antibiotics, such as gentamicin and other small molecules under analysis (e.g*.*, RTC13, RTC14 and ataluren, PTC124) aim to restore full-length dystrophin in patients with stop codon mutations. This promising therapeutic approach arises from the ability of such agents to stimulate the ribosomal read-through. This leads to the suppression of nonsense mutations in Duchenne/Becker muscular dystrophy [13].

**Figure 1.** Histological features of muscular dystrophy. Upper panels show the histological architecture of the *tibialis anterior* muscle in a healthy control mouse (left panel) and dystrophic mouse model of human Duchenne muscular dystrophy (mdx, right panel). Haematoxylin and Eosin stain identifies the presence of a huge amount of infiltrating mononuclear cells within the *interstitum* of mdx muscle. Necrotic fibres, as well as centrally located nuclei fibres, are other hallmarks of dystrophic muscle (right panel) compared to healthy control (left panel). Lower panels show the immunofluorescence stain for dystrophin (red). Compared to the healthy control (lower left panel), muscle from mdx shows a strong reduction of dystrophin content. Only a small percentage of revertant fibres are positive for dystrophin

Although there are no proficient cures for dystrophinopathies, several drugs have been used to delay their detrimental effects on muscle tissues, mainly through the attenuation of inflammation. In the treatment of MDs, several drugs are used for their ability to reduce circulating levels of TGF-β, known to play a crucial role in the fibrotic tissue deposition in dystrophic muscles. Among the drugs used to counteract the systemic burden of TGF-β are non-steroidal anti-inflammatory drugs (NSAIDs) such as nabumetone, ibuprofen and isosor‐ bide dinitrate. These drugs have beneficial effects, especially in the treatment of Duchenne, Becker and Limb-Girdle muscular dystrophies [13]. Promising results were recently obtained from phase I studies in healthy volunteers. These studies revealed an optimal tolerability and safety profiles for a combined administration of isosorbide dinitrate, a nitric oxide donor and ibuprofen (NSAIDs) for the treatment of muscular dystrophies [14]. Other established pharmacological approaches aim to hamper the elevated muscle inflammation and necrosis events linked with mitochondrial dysfunction and altered metabolism. In particular, αmethylprednisolone (a synthetic glucocorticoid) stimulates the reduction of cytosolic calcium in dystrophic muscle and prevents apoptosis and/or necrosis events that normally occur during muscle degeneration in dystrophinopathies [15] (Figure 2). Another challenge in the pharmacological treatment of MDs is counteracting the high susceptibility to muscle damage

expression (lower right panel).

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**Figure 2.** Schematic representation of the strategies adopted in the study/treatment of muscular dystrophy. Pharmaco‐ therapeutics approaches include drugs that can increase the expression of utrophyn, anti-inflammatory and No/Ca2++ homeostasis regulator drugs and small molecules that can promote the read-through of dystrophin. NSAIDs= nonsteroidal anti-inflammatory drugs; STEDs= steroid drugs; ABX= antibiotics; COX 1/2= cyclooxygenase type 1 and 2. Gene therapy is based on both exon skipping and Adeno- associated virus (AAV) gene delivery strategies aiming to the increase of dystrophin content. Cell therapy investigates the myogenic potential observed in adult stem cells (such as satellite cells, FAPs, SP, MABs and iPS) and in ES cells, through the increase of both dystrophin content and the pool of resident satellite cells.
