Mammalian animal models for Duchenne muscular dystrophy

Neuromuscular Disorders 19 (2009) 241–249

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Neuromuscular Disorders journal homepage: www.elsevier.com/locate/nmd

Review

Mammalian animal models for Duchenne muscular dystrophy Raffaella Willmann a, Stefanie Possekel b, Judith Dubach-Powell b, Thomas Meier b, Markus A. Ruegg a,* a b

Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Santhera Pharmaceuticals, Hammerstrasse 47, CH-4410 Liestal, Switzerland

a r t i c l e

i n f o

Article history: Received 9 October 2008 Received in revised form 24 November 2008 Accepted 27 November 2008

Keywords: Duchenne muscular dystrophy Animal models Mouse Dog Cat GRMD mdx

a b s t r a c t Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease that affects boys and leads to early death. In the quest for new treatments that improve the quality of life and in the search for a possible definitive cure, the use of animal models plays undoubtedly an important role. Therefore, a number of different mammalian models for DMD have been described. Much knowledge on the molecular mechanisms underlying the disease has arisen from studies in these animals. However, the use of different models does not often allow a direct comparison of results obtained in preclinical trials and therefore hinders a straightforward translational research. In the frame of ‘‘TREAT-NMD”, a European Network of Excellence addressing the fragmentation in the assessment and treatment of neuromuscular diseases, we compare here the currently used mammalian animal models for DMD with the aim of selecting and recommending the most appropriate ones for preclinical efficacy testing of new therapeutic strategies. Ó 2008 Elsevier B.V. All rights reserved.

1. Introduction Neuromuscular disorders cover a broad range of diseases associated with progressive muscle weakness and/or neurodegeneration, and are frequently affecting patients at young age. Almost all of these hereditary diseases have an incidence of less than 5/ 10,000 and are often referred to as orphan diseases. In Duchenne muscular dystrophy (DMD) the underlying genetic defects are now well understood. However, current treatment options remain limited. Challenges encountered in the attempt to develop efficacious therapeutic interventions are often based on the fact that the gene defect per se cannot be corrected because of considerable technical hurdles. Thus, alternative approaches including pharmacological treatment options or exon-skipping are currently under evaluation as potential therapeutic strategies. In contrast to most neuromuscular pathologies, several distinct mammalian models are available for DMD. One of the goals of ‘‘TREAT-NMD”, a European Network of Excellence, is to evaluate the multitude of animal models, readouts and measurement protocols used in preclinical research with the aim of reaching a consensus within the international community as to which models, readouts and protocols are most appropriate for efficacy testing of potential new treatments. This will open the possibility to design and conduct experiments that can be compared between

* Corresponding author. Tel.: +41 61 267 22 23; fax: +41 61 267 22 08. E-mail address: [email protected] (M.A. Ruegg). 0960-8966/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2008.11.015

different laboratories thus allowing proof-of-concept studies. We believe that these measures will increase the value of preclinical data collected for regulatory filings with health authorities, and will accelerate the preclinical stage of drug development. In a first step, animal models need to be thoroughly analyzed and compared with a special emphasis on their suitability in proof-of-concept studies. An appropriate animal model should allow a reliable prediction of the response to a given therapeutic intervention in humans. In the case of genetic diseases, the underlying genetic defect in the animal model should be comparable to the defect in patients. Moreover, a profound knowledge of all pathological characteristics of the animal model is crucial for its reliable use. The present review addresses these points by providing a comprehensive overview of the mammalian models currently in use and proposing a choice of animal models most appropriate for testing treatment options at the preclinical stage. For this reason, the review will not discuss animal models created to study basic mechanisms of the disease. Suggestions and guidelines to standardize readouts and assessment protocols in the selected animal models (see also [1]) represent the next goals for TREAT-NMD and will therefore be subject of future consensus papers. 2. Human pathology DMD is an X-linked recessive disease that occurs with an incidence of 1 in 3500 boys. Human dystrophin, which is mutated in DMD, was identified more than 20 years ago. Dystrophin is a protein of 427 kDa encoded by a giant gene of more than 2.5 Mb.

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Fig. 1. The dystrophin gene and its protein products (adapted form [87]). There are seven promoters of the dystrophin gene. The corresponding protein product, Dp427, is found in brain (B), muscle (M) and Purkinje neurons (P) and results in ‘‘full-length” dystrophin. The other promoters lead to shortened dystrophin. The C-terminal region, expressed in all isoforms, contains protein domains that form the binding sites for some of the components of the dystrophin-associated protein complex: the WW domain (WW), the cysteine-rich ZZ domain (ZZ) and the coiled coil domain (CC).

Its transcription is regulated in a tissue-specific manner by several promoters (Fig. 1). In healthy muscle, dystrophin is located at the subsarcolemma and interacts with a number of membrane proteins forming the dystrophin glycoprotein complex (DGC) (for review see [2]), whose function is to stabilize muscle cell membrane during cycles of contraction and relaxation. Moreover, several lines of evidence indicate a role of the DGC in the regulation of mechanosensitive calcium channels, especially in the subsarcolemmal space, that influence calcium homeostasis and signaling (for review see [3]). About two thirds of all DMD patients have large deletions in the dystrophin gene, mainly involving two ‘‘hot spots” located around exons 45–53 and exons 2–30. As a consequence, the essential C-terminal region that binds to the DGC is often truncated. In cases where portions of the central part of the protein are missing but the C-terminal region is maintained, the protein is partially functional. The resulting disease is milder and defined as Becker muscular dystrophy [4]. In one third of DMD cases, the disease is caused by small deletions or point mutations that introduce a stop codon, resulting in very little or no dystrophin production [5], either because of transcript or protein instability. Absence of dystrophin, as encountered in DMD, leads to disintegration of the DGC, instability of the muscle cell membrane and uncontrolled influx of calcium. This initiates a set of downstream pathological processes that ultimately lead to disintegration of muscle cell proteins, cell damage and progressive muscle wasting. The subsequent regeneration causes the appearance of fibers with different sizes and with centralized nuclei. Later, muscles become replaced by fibroblasts and fat cells, which results in collagen deposition (fibrosis). DMD patients typically appear normal at birth but display elevated levels of creatine kinase in the blood. The disease usually manifests at the age of 2–5 years when the delay in reaching motor milestones (e.g. climbing stairs) becomes apparent. Later, pseudohypertrophy of the calf muscles and weakness of proximal limb muscles develop, inevitably leading to wheelchair dependence. Respiratory insufficiency and cardiomyopathy are predominant factors contributing to early morbidity and mortality.

3. Mouse models for DMD 3.1. mdx mouse A naturally occurring dystrophin-deficient mutant was first described in 1984 in a colony of C57BL/10 mice (C57BL/10ScSnJ) [6] and has since been referred to as the ‘‘mdx-mouse”. This mouse, now called C57BL/10ScSn-Dmdmdx/J, is readily available from commercial breeders and thus widely used in basic and translational research. It carries a point mutation in exon 23 of the mouse dystrophin gene introducing a premature stop codon, which leads to the absence of full-length dystrophin. This type of mutation accounts for approximately one third of the mutations found in DMD patients (see above). Mdx mice have a slightly shorter life span as compared to wildtype controls [7] (Table 1). The muscle degeneration in mdx mice differs from DMD patients in that it appears in waves and is not a continuum. At first, a marked degeneration and regeneration of muscle fibers is observed at a young age (2–4 weeks [8]), which results in an increase in the number of newly differentiating myofibers characterized by centralized nuclei and an increased heterogeneity in myofiber size. In parallel, necrosis is observed in muscles at this early stage [9,10] while it decreases again after 60 days [11]. Subsequently, loss of muscle tissue is slow and general muscle weakness in cage-reared animals is not evident until later in life [12]. Fibrosis in most mdx mouse muscles is less pronounced than in DMD patients with the exception of the diaphragm muscle [13–15]. Although absolute muscle force of limb muscles remains comparable to unaffected mice, the relative force normalized to body weight decreases by approximately 20% and 50% at the age of 8–10 weeks and 4 months, respectively [10,14,16]. However, others could not confirm this finding [17,18], which is probably based on variations in the mice strains (e.g. body weight, cooperation of the animals in force measurement assays) and on different experimental procedures. Similarly, a reduced force of contraction has also been reported in isolated muscle preparations although the lack of standardized conditions makes it again difficult to compare the results obtained by different groups [19–22]. These discrepancies clearly show that the availability of standardized experimental

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R. Willmann et al. / Neuromuscular Disorders 19 (2009) 241–249 Table 1 Qualitative and quantitative measurements in mdx mouse model Parameter General Life span

Clinical signs at onset First signs of pathology First signs of cardiomyopathy Breeding Kyphosis Body weight Muscle Physiology Fore-limb strength (g)

Fore-limb strength per body weight (g strength/g BW)

Twitch force (mN)

Normalized twitch force (mN/mm2 CSA)

Tetanic force (mN)

Normalized tetanic force (mN/mm2 CSA)

Muscle Physiology Force drop after eccentric contractions

Force drop after fatigue protocol Normalized peak force (mN/mm2 CSA) Biochemistry CK

Hydroxyproline (fibrosis marker)

Intracellular [Ca2+]

Respiratory Physiology Respiratory rate Response to hypercapnia/hypoxia

Parameter

mdx

mdx reduced by 17% in females and 19% in males [7] 23m [65] CK levels increase, necrosis, regeneration 2-3w 6m normal 9m [22] normal, increases after 8w [14] drops by 25% after 6m [65] 12w: 35g [10,14,68] 4w: 95g; 8-12w: 156g [17] 10w: 30g [10] 4w: 103g; 8-12w: 138g [17] r 1.5 at 8w [14] 1.6 at 3w, 10w [69] 1.1 at 10w [10] 75 at 4m (whole body) [16] 5.34 at 4w, 6.5 at 8w [70] 4.59 at 4w, 5.07 at 8w [70]r Edl, 16w: 130 [72] Edl, 8w: 12 [20] Sol, 8w: 12 [20] Stm, 8w: 50 [46] Edl, 10w: 50 [19] Sol, 10w: 40 [19] Dia, 10w: 20 [19] Dia, 7w: 5.8 [22] Stm, 8w: 6.6 [46] Edl, 8w: 82 [20] Edl, 90d: 50.2 [16] Edl, 16w: 370 [72] Edl, 8w: 52 [20] Sol, 8w: 57 [20] Edl, 10w: 170 [20] Edl, 6w: 200 [21] Sol, 10w: 200 [19] Dia, 10w: 130 [19] Dia, 7w: 32 [22] Edl, 3-32w: decreased [74] Edl, 90d: 147 [16] Edl, 16w: 72% [21] Edl, 90d: 69% [16] Edl, 10w: -41% [19] Sol, 10w: 75% [19] Edl: 272 [19] 1200 U [42] 5000 U [16]r8000 U/l [15]r10000/ 12000 U/l [17,40] Dia, 24w: 19 lg/mg [75] Dia, 7w: 2.5 lg/mg [22] Int, 7w: 0.9 lg/mg [22] normal in Fdb, Sol at 4-9w (in [76]) normal in isolated Fdb fibers [31] 60 lM (higher) in Edl at 8-12w [18] 80 nM (2x higher) in Edl at 8-12 w [18] r

Ca2+ sparks after hypotonia Parvalbumin

Table 1 (continued)

Fdb: irreversible at 4w [77] fast muscles: mRNA increases [32] TA, 24w: 9 lg/mg [33] Gas, 24w: 7 lg/mg [33] Sol, 24w: 50 ng/mg [33] Edl, 32w normal levels [74] altered at 16m [23] reduced at 7m [78]

Histology % of centronucleated fibers

Histology Fiber size

Fibrosis

Necrosis

Fiber type composition

Cardiac Physiology Dilated cardiomyopathy (LVEDD/LVESD in mm by echocardiography)

Heart rate

Force output (mN/mm2) Heart fibrosis (% of area)

Hydroxyproline content ECG Left ventricular developed pressure (mm Hg) Rate of pressure development (mm Hg/s) Rate of relaxation (mmHg/s)

TA, 7w: 50% [79] Dia, 7w: 25% [79] Sol, 7w: 56% [79] hind-limb, 4-14w: average 35% [8] Edl, 6w: 58%; TA, 90d : 75% [16] Edl, 10-12w: 64% [80] TA, 10-12w: 74% [80] Dia, 10-12w: 60% [80] Fdb, 12w: 25% [81] Gas, 8-10w: 50% [40] r TA, 3w: 302 [79] t Dia, 3w: 254 [79] t TA, 7w: 403 [79] t Dia, 7w: 433 [79] t Sol, 7w: 267 [79] t Dia at 16m [13] Glut at 24w [14] TA, 8-13w: 6% [15] TA, 8-13w: 9% [15] r TA, 3w: 20% [9] Edl, Bic, 10w: 5% [10] TA, Sol, Edl: peaks at 3060d [84], decreases after 60d [11] Gas, 8-10w: 20% [17] r Edl: 50% I, 50% IIA [20] Edl: 80% IIB, 20% IIX [19] Sol: 65% IIA, 35% I [19] Dia: 90% IIX, 5% IIB [19] TA 90d: 57% IIB [16] 3.6/2 at 42w (increased) [24] 3.7/2.6 at 24m (no change) [29] 3.9/2.9 at 9-10m [27] 3.83 at 10m [30] decreased, 612 bpm at 42w [24] 460 bpm at 9-10m [27] 454 bpm at 10m [30] 25 at 8w [85] 7% at 7m [24] 8% at 12m [29] 12% at 24m [29] 1.68% at 11w [41] 6.23% at 11w [41] r 0.7-3.4% at 10m [30] 4.5 lg/mg at 15m [28] 4 lg/mg at 12-14m [26] abnormal at 6 and 12m [25] 80 at 15m [28] 2500 at 15m [28] 2000 at 15m [28]

Abbreviations used are: Bic: biceps, Dia: diaphragm, Edl: extensor digitorum longus, Fdb: flexor digitorum brevis, Gas: gastrocnemius, Glut: gluteus, Int: intercostals, Pl: plantaris, Sol: soleus, Stm: sternomastoid, TA: tibialis anterior. d: days; w: weeks; m: months; y: years; BW: body weight; CSA: cross sectional area; CK: creatine kinase; LVEDD; Left ventricular end diastolic diameter; LVESD: left venticular end systolic diameter; bpm: beats per minute. r exercised mdx. s after 5th elongation [19]. t measured as the minimal Feret’s diameter, units in variance coefficient (vc) [79].

protocols would be of advantage both to elucidate basic aspects of the pathology and to test emerging therapeutic strategies. Respiratory pathology is evident in 16-month-old mice but not in 4month-old mice, showing a worsening of respiratory capacity with age, in line with the increased histological damage of respiratory

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muscles [23]. Abnormal cardiac function is also seen in the mdx mouse sharing several characteristics of DMD-associated cardiomyopathy [24]. For example, changes in the electrocardiogram (ECG) and echocardiogram have been reported in mice that are older than 6 months [25–27]. This change in cardiac function is paralleled by an increase in myocardial fibrosis and the occurrence of foci of myocardial necrosis and inflammation [24,28–30]. Mdx mice have served as a model for several studies on calcium homeostasis and alterations thereof. Basal cytosolic calcium levels were reported to be identical in mdx and wild-type mice [31] showing a very robust calcium homeostasis, possibly due to elevated expression of the calcium-buffer parvalbumin [32,33] (see Table 1). Nevertheless, [Ca2+] in the subsarcolemmal compartment [34] and in the sarcoplasmic reticulum [35] was increased and levels of inositol 1,4,5-triphosphate (IP3) and of the IP3 receptor were higher in mouse and human dystrophic cell lines [36]. Muscle pathology in the mdx mouse can be aggravated by forced exercise, a method introduced to allow a better evaluation of the efficacy of experimental therapies. Several exercise paradigms have been described [37–39]. Muscle damage of exercised mdx animals is increased as reflected by an increased number of muscle fibers with centralized nuclei and increased fibrosis [17,40] (Table 1). Plasma creatine kinase levels are further elevated, calcium homeostasis is more severely impaired and fore-limb strength is generally reduced [15,17,18,40] while body weight remains unchanged. Cardiomyopathy has been described for the exercised mdx mouse [41], however, a systematic analysis of the impact of exercise on cardiac pathology is still lacking.

3.4. mdx double mutants

3.2. mdx2Cv–mdx5Cv

3.4.2. mdx; MyoD/ MyoD is a myogenic regulatory factor that plays an important role during muscle differentiation. Therefore, MyoD/ mice were crossed with mdx mice in an attempt to obtain mice with a reduced capacity to regenerate [47]. Mutant mdx; MyoD/- mice uniformly display marked muscle dystrophy. Specifically, by 3–5 months of age, mdx; MyoD/ mice develop a kyphosis/scoliosis of the spine similar to DMD patients and an abnormal waddling gait (Table 2). The animals become progressively less active with concomitant weight loss and they die prematurely at 12 months of age [47]. Moreover, they develop severe cardiomyopathy at the age of 10 months with foci of necrosis that are not observed in MyoD/ mice [48] and therefore this mutant has been proposed as a model for DMD-associated cardiomyopathy.

In 1989, a series of mdx variants (termed mdx2Cv, mdx3Cv, mdx4Cv, and mdx5Cv) were recovered from male mice (C3H.X25  C57BL/ 6Ros) treated with the chemical mutagen N-ethyl-nitrosourea that were crossed with C57BL/10Sn.mdx female animals [42]. The mice display muscle pathology and elevated serum creatine kinase levels that do not differ from the original mdx mutant (Table 2). Muscle cross-sections showed degeneration/regeneration, centrally nucleated fibers, necrosis, cellular infiltration and fibrosis in the diaphragm [42]. The variation in fiber size was found to be larger than in mdx mice [43]. Pseudomyotonia, detected by electromyography, and some fibrosis were detected in the hearts of mdx2Cv and mdx3Cv at the age of 4 weeks [42]. The mdx3Cv strain also displays an abnormal breeding phenotype with reduced neonatal survival [43]. The pathology in this series of mice (mdx2Cv–mdx5Cv) at older age (>6 months) is not reported. 3.3. mdx52 To create a mouse model with large deletions in the dystrophin gene like those found in two thirds of human patients, exon 52 was disrupted on a C57BL/6J background [44]. The mice do not show skeletal muscle weakness or behavioral changes until 18 months of age. At 4 months of age, tibialis anterior and extensor digitorum longus muscles of the mutant mice are paler and about 1.5 times larger than corresponding wild-type muscles (Table 2). Fibrosis is evident in diaphragm, soleus and extensor digitorum longus. A marked variation of fiber size with some necrotic spots is also observed at 4 months and centronucleated fibers make up 90% of the whole fibers at this age. Although heart and brain are partially deficient for dystrophin, no pathological changes are observed in these organs. Overall, the dystrophic phenotype of 4-month-old mdx52 mice is similar to age-matched mdx mice; no data are, however, available on disease progression with age [44].

3.4.1. mdx; utr/ In an attempt to aggravate the pathology of the mdx model and to study utrophin-based gene therapy strategies, utrophin mutant mice (on C57B5/129J background) were crossed with C57BL/10 mdx mice to obtain double knock-out mice [45,46]. Mice lacking both dystrophin and utrophin share many phenotypical hallmarks with DMD. The mice have a markedly reduced life span of 4–20 weeks, severe muscle weakness with joint contractures, kyphosis and a pronounced growth retardation that starts around weaning (Table 2). Necrotic fibers and connective tissue are visible in the diaphragm already 6 days after birth and centrally nucleated fibers are evident at 2 weeks. Interestingly, the diaphragm is not more affected than that of the mdx mice, suggesting that utrophin does not compensate for the lack of dystrophin in this muscle [45,46]. The skeletal muscles appear normal at birth but show first signs of degeneration/regeneration already after 2 weeks. Although the dystrophic pathology is similar to mdx mice at 4–5 weeks of age, it then remains severe in contrast to the improvements seen in mdx mice. By 10 weeks of age, fibrosis is evident in tibialis anterior muscle. Occasionally, larger necrotic areas are observed in the heart [46]. The neuromuscular and the myotendinous junctions in mdx; utr/ mice show a marked reduction in membrane folds, presumably as a result of the lack of utrophin [45,46]. Therefore, both the disease progression and the phenotype of these double mutant mice resemble the progression of muscle dystrophy in DMD patients and the interstitial fibrosis of skeletal muscle lends itself to assess the efficacy of anti-fibrotic treatments.

3.4.3. mdx; adbn/ Mdx mice that lack the DGC protein a-dystrobrevin were created to investigate the role of a-dystrobrevin in signaling and in conferring muscle stability [49]. The double mutant mice have a reduced life expectancy of about 8–10 months and show moderate dystrophic changes including necrotic fibers, mononuclear infiltrates, fiber size variability and fibrosis (Table 2). The percentage of centronucleated fibers in the tibialis anterior muscle is higher than 90% and some cardiomyopathy was also observed. In general, these mice show a more severe pathology than mdx mice but not as severe as that of mdx; utr/ mice. 3.4.4. mdx; a7 integrin/ Several lines of evidence indicate that a7b1 integrin, the main integrin expressed in skeletal muscle, acts in synergy with the DGC to connect the extracellular matrix to the cytoskeleton [50]. To test this, a7 integrin and dystrophin double mutant mice were generated [51]. The double knockout mice show no phenotypic difference from wild-type and mdx at birth, but their pathology worsens rapidly and they die prematurely at 24–28 days of age (Table 2). Mice start to lose weight at 15 days of age and show a marked

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R. Willmann et al. / Neuromuscular Disorders 19 (2009) 241–249 Table 2 Qualitative and quantitative measurements in mouse models other than mdx Parameter

mdx2Cv-5Cv

mdx52

mdx; utr-/-

mdx; myoD-/-

mdx; adbn-/-

mdx; a7 integrin-/-

mdx; PV-/-

General Life span Clinical signs at onset

nd nd

nd nd

24-28d [51] nd

1y [16] nd

18m [44] none [44] nd

12m [47] waddling gait, less active [47] nd nd nd

8-10m [49] nd

nd nd abnormal in mdx3Cv [43] nd nd

6-20w [45,46] small size, joint contractures [14] at birth 8 w [46] none

nd nd nd

nd nd nd

nd nd nd

nd nd

present [66] 14 g at 4w, 18 g at 8w [46,66]

3-5m [47] nd

nd nd

18d [51] reduced after 15d compared to mdx [51]

nd nd

nd nd

nd nd

250 g at 8w [14] 1.8 at 8w [14]

nd nd

nd nd

nd nd

nd nd

nd nd

nd nd

nd nd

nd nd

nd

nd

nd

nd

nd

Edl, 90d: 113.6 [16]

nd Force drop after eccentric contraction s Force drop after fatigue protocol nd nd Normalized peak force (mN/ mm2 CSA)

nd

nd

nd

nd

Edl, 90d: 54.3% [16]

nd nd

Stm, 8w: 30 [46] Edl, 9w: 5 [73] Edl, 10w: 40 [73] Sol, 10w: 25 [73] Dia, 10w:15 [19] Stm, 8w: 4.9 [46] Edl, 10w: 130 [19] Sol, 10w: 100 [19] Dia, 1w: 90 [19] Edl, 9w: 65% [19] Edl, 10w: -76% [19] Sol, 10w: 50% [19] Edl, 217 [19]

nd 5.1 at 4m (whole body) [16] nd Edl, 90d: 39.8 [16]

nd nd

nd nd

nd nd

nd nd

First signs of pathology First signs of cardiomyopathy Breeding Kyphosis Body weight

Muscle physiology Fore-limb strength (g) Fore-limb strength per body weight (g strength/g BW) Twitch force (mN) Normalized twitch force (mN/ mm2 CSA)

Normalized tetanic force (mN/ mm2 CSA)

Biochemistry CK

nd

elevated

nd

nd

nd

8000U [16]

Intracellular [Ca2+]

1200-6000 U [42] nd

nd

nd

nd

nd

nd

Fdb fibers at 90d: 69.8 nM [16] Gas at 90d: 9 lmol/g dry weight [16]

Respiratory physiology Respiratory rate

nd

nd

nd

nd

nd

higher [51]

nd

Histology % of centronucleated fibers

yes [42]

present [47]

TA, 1m: 90% [49]

Pl 10 and 20d: 2.5% and 10% [51]

TA, 90d: 87% [16]

Fiber size

nd

Fibrosis

some [42]

hind-limb, 15d : variations [51] hind-limb from day 15 [51]

Edl, 90 and 180d : hypertrophy [16] nd

Necrosis

nd

Necrotic/regenerative areas

yes [42]

Fiber type composition

nd

Edl, TA, Dia, 4.5m: Dia, 8w : 40% [45] 90% [44] TA, 35d: 75% [82] TA, 10w: 90% [46] Edl, 4w: 30% [66] Edl, 8w: 70% [66] Edl, 9w: 90% [73] Sol, 4w: 60% [66] Sol, 8w: 70% [66] Dia, *: 65% [83] Edl, *: 85% [83] TA, *: 80% [83] * : age unknown TA & Edl, 4m: nd Hypertrophy [44] Dia & TA 4.5m: Sol, 8w: extensive minimal [44] [66] Sol, Bic, Glu, 24w: extensive [14] TA, 10w: present [46] Dia, TA present at begins at 2w [46] 4.5m [44] present in Dia at 6d [45] nd Edl, 4w: 12% [66] Edl, 8w: 8% [66] Sol, 4w: 25% [66] Sol, 8w: 20% [66] nd Edl: 65% IIB, 35% IIX [19] Sol: 55% IIA; 45% I [19] Dia: 80% IIX, 5% IIB [19]

attenuated nd hypertrophy [47] nd nd

nd

TA, 1m: small nd areas [49]

nd

nd

nd

nd

nd

nd

nd

nd

TA, 90d: 46% IIB [16]

(continued on next page)

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Table 2 (continued) mdx2Cv-5Cv

mdx52

mdx; utr-/-

mdx; myoD-/-

mdx; adbn-/-

mdx; a7 integrin-/-

mdx; PV-/-

Pseudo-myotonia [42]

nd

nd

5m [48]

nd

nd

nd

nd

nd

nd

Force output (mN/mm )

nd

nd

nd

nd

nd

nd

Heart fibrosis (% of area)

nd

nd

prominent 8w [46] 15 at 8w [85] nd

none at 20-24d [51] evident at 20-24d [51]

evident at 10m in 50% [48]

nd

nd

nd

Parameter Cardiac physiology Dilated cardiomyopathy (LVEDD/ LVESD in mm by echocardiography) Necrosis (histological) 2

nd

Abbreviations used are: Bic: biceps, Dia: diaphragm, Edl: extensor digitorum longus, Fdb: flexor digitorum brevis, Gas: gastrocnemius, Pl: plantaris, Sol: soleus, Stm: sternomastoid, TA: tibialis anterior. d: days; w: weeks; m: months; y: years; BW: body weight; CSA: cross sectional area; CK: creatine kinase; LVEDD: Left ventricular end diastolic diameter; LVESD: left ventricular end systolic diameter; nd: not determined. s after 5th elongation [19].

kyphosis at 18 days of age. Fiber size variation and fibrosis is observed in hind-limb muscles from day 15 onwards and a pervasive dystrophy develops in the following days. Histological analysis shows that flexor muscles are generally more affected than extensor muscles. Interestingly, in these muscles a loss of fiber number and a general size reduction is observed together with a low number of centronucleated fibers, suggesting that impaired regeneration may be responsible for the loss of muscle mass. Mice eventually die of respiratory failure and cardiomyopathy. The aggravated pathology of mdx; a7 integrin/ mice suggests that the fine tuning of a7 integrin levels may be responsible for the different clinical severity of dystrophin deficiency in human and mouse [51]. In conjunction with the finding that overexpression of a7 integrin in mdx mice ameliorates the disease [52], these data indicate a compensatory function of a7 integrin in dystrophindeficient muscle.

were crossed with PV knockout (PV/) mice [16]. Such mdx; PV/ mice are very similar to mdx mice (Table 2). The extensor digitorum longus muscle shows a marked hypertrophy at 90 and 180 days that exceeds that observed in mdx. Moreover, the decrease in the number of type IIb fibers and the increase in the number of centronucleated fibers are more pronounced in mdx; PV/ mice than in mdx mice. In addition, muscles from 90-day-old double mutants are weaker than those from mdx when tested in vivo and in vitro. In summary, although the high levels of PV in mdx mice may contribute to the rather mild pathology, it is not sufficient to explain the difference to DMD patients.

3.4.5. mdx; PV/ Compared to DMD patients and dog models thereof, mdx mice show a relatively mild pathology. To test whether this difference might be due to the high concentrations of the potent calcium-buffer parvalbumin (PV) in the skeletal muscles of rodents, mdx mice

In the golden retriever, the best characterized dog model, the disease results from a single base pair change in the 30 consensus splice site of intron 6, leading to skipping of exon 7 and alteration of the reading frame in exon 8, which creates a premature stop [53]. Unlike the dystrophin-deficient mdx mouse, GRMD dogs suf-

4. Canine models for DMD 4.1. Golden retriever (GRMD)

Table 3 Qualitative and quantitative measurements in the Golden Retriever Muscular Dystrophy (GRMD) dog model Parameter

Severe/less severe

General Life span Clinical signs at onset First signs of pathology First signs of cardiomyopathy Breeding Kyphosis Body weight

10d [55]/1-3y, in rare cases up to 6y [55] weak, dehydrated, hypothermic [55]/inability to open claw, less active, abnormal gait [55] at birth/6-9w [55] nd/1 y or more [55] none/normal [55] nd/6m [55] reduced at all ages [55]/ reduced at all ages [55,67]

Muscle physiology Fore-limb strength per body weight (g strength/kg BW) Twitch force (N) Normalized twitch force (N/kg BW)

nd/hind-limb, 3m: 5.8 [71] nd/hind-limb, 6m: 0.8 (flexion), 1.57 (extension) [67] nd/hind-limb, 6m: 0.064 (flexion), 0.112 (extension) [67]

Muscle physiology Tetanic force (N) Normalized tetanic force (N/kg BW)

nd/hind-limb, 6m: 5.89 (flexion), 12.82 (extension) [67] nd/hind-limb, 6m: 0.47 (flexion), 0.965 (extension) [67]

Biochemistry CK

100-500 x normal level at 1-2d [55]/increased from 3w; 6-8w: 100 x normal level [55]

Histology Fiber size Fibrosis Necrosis

nd/trunk, face muscles, 9-12w: atrophy [55] nd/in prox. muscles at 14 m [55] in cranial sartorius at 5 m [56] severe, many muscles [55]/nd

Cardiac physiology Dilated cardiomyopathy (LVEDD/LVESD in mm by echocardiography) Heart fibrosis (% of area)

nd/34/20 at 25w [58] nd/6.3% collagen at 25w [58] prominent at 1-1.5y [86]

Abbreviations used are: d: days; w: weeks; m: months; y: years; BW: body weight; CK: creatine kinase; LVEDD: Left ventricular end diastolic diameter; LVESD: left ventricular end systolic diameter; nd: not determined.

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fer from a rapidly progressing, fatal disease similar to DMD. There is, however, a large variation in disease severity as some pups survive only for a few days, while others are ambulant for months or even years [54]. To account for this variation, we distinguish between the severe and the less severe pathology in Table 3. Incomplete muscle repair in GRMD results in progressive weakness and gait abnormalities at the age of 6–9 weeks leading to muscle atrophy, fibrosis and contractures by 6 months (Table 3). The growth of affected pups is delayed and their walking ability is impaired. The dogs become less active than non-affected littermates at about 9 months of age. At this time, their gait is still uncoordinated and their limbs are abducted [55]. Severely affected dogs have difficulties to rise and can walk only a few steps. At the age of 12 months, pharyngeal and esophageal dysfunctions start to develop and respiratory capacity is decreased. Kyphosis develops by the 6th month of age. Moreover, as seen in DMD patients, GRMD dogs display selective muscle involvement although the most affected muscles in dogs (i.e. tongue, masticatory and trunk muscles) are different to those of humans [56]. Myocardial involvement is much more evident in the golden retriever than in other animal models, matching very closely the cardiac complications encountered in DMD patients. Atrophy already appears during the neonatal period [55], although histological changes are generally not present up to 3 months of age [57]. Systolic dysfunction and left ventricular dilation becomes apparent after several months or years [57], although Tissue Doppler Imaging has allowed the detection of severely impaired systolic and diastolic indices in young dogs (25 ± 11 weeks) [58]. Recently, NMR techniques have been applied to assess differences between dystrophic and healthy muscles in the dog, a method that may be useful to monitor a potential therapeutic response [59]. 4.2. Beagle (CXMDJ) Beagle dogs were mated with an affected golden retriever to obtain a breed of a smaller size, which is an advantage for some therapeutical approaches. One of these strains has been established in Japan (CXMDJ [60]). GRMD and CXMDJ present very similar phenotypes. The survival rate of beagle dogs carrying the dystrophin mutation is increased compared to golden retriever and pup mortality is low, although it tends to increase after a few generations. In general, large breed dogs develop more severe clinical signs than small breed dogs [55]. The diaphragm muscle is affected shortly after birth while in limb muscles abnormalities become evident only after 2 months of age [61]. Cardiac involvement in the CXMDJ dogs is milder and has a slower progression than that described for GRMD dogs. Although the CXMDJ model has not been widely used and thus is not yet characterized in detail, it would seem to be an attractive alternative to the golden retriever, particularly because of its improved survival rate and because the beagle dog is the species of choice for non-rodent toxicology studies in drug development. 4.3. Feline models for DMD The only other natural dystrophin deletion described in a mammalian model occurs in the dystrophic cat, which lacks approximately 200 kb of the dystrophin gene [62]. The ‘‘hypertrophic feline muscular dystrophy” (hfmd) animal model is characterized by increased levels of creatine kinase in the blood at the age of 4–5 weeks although muscle involvement becomes apparent only at the age of 10–14 weeks. The name of the disorder reflects the development of extensive muscle hypertrophy. The cats eventually die due to compression of the esophagus by the hypertrophied diaphragm or because of impaired water intake caused by glossal hypertrophy [63], which then leads to renal failure. Histological

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analysis has revealed the presence of local foci of necrosis and regeneration but no signs of fibrosis were seen [63]. Although cardiac failure is rare, the hearts are hypertrophic in 9-month-old cats [64]. Thus, the pathology of hfmd cats does not resemble DMD as closely as the GRMD dog because it lacks the hallmarks of generalized muscle wasting.

5. Conclusions Several aspects must be considered when evaluating the animal models for use in the preclinical testing of potential new treatment options. The following criteria play a significant role:  The genetic basis of the disease should be the same both for humans and the animal model.  The model should reiterate key hallmarks of the human disease. For DMD, these include: muscle weakness, respiratory insufficiency, cardiomyopathy, histological changes in skeletal muscle (e.g. centralized nuclei, fiber size variations).  The animals should be readily (i.e. commercially) available, easy to handle and maintainable in standard laboratory housing conditions.  Disease progression should be well characterized and reference values should be available in the scientific literature to allow a precise outcome analysis.  The phenotype should be robust and reproducible over generations to allow the use of standard readout parameters and ensure comparability of outcomes. Taking all these aspects into consideration, double mutant mice like mdx mice deficient for MyoD, utrophin, parvalbumin or a7 integrin do not resemble the genetic background of DMD patients and are therefore less appropriate to predict therapeutic effects. Mdx2Cv-mdx5Cv, mdx52, the beagle and the cat models, while bearing the genetic defect found in patients, are not yet characterized in detail and the available literature is limited; moreover, the pathology observed in mdx52 and in cats is milder and differs considerably from the human disease. Therefore, the mdx and GRMD mammalian model should be considered currently as the best suited animal models for preclinical therapy testing. The mdx mouse model has considerable advantages, particularly because of the large number of papers published on this animal model resulting in a good understanding of muscle, cardiac and respiratory pathology. Moreover, the reproducibility of the phenotype, the commercial availability and the relatively low costs of maintenance are further important advantages. Finally, this animal model has been used to demonstrate efficacy of several potential treatment strategies including small molecule pharmaceuticals as well as gene correction and cell replacement approaches. One important drawback of GRMD is the high degree of variability in disease severity between breeds and among littermates although they all carry the same genetic mutation. Therefore, special care must be taken in choosing the control animals and control experiments. Moreover, a rather high number of animals is required to substantiate any conclusions about the treatment efficacy. Additional disadvantages are the high expense of breeding, the necessity for a veterinary facility and the large amount of compound required to treat large-sized animals. However, dog models closely resemble the human disease, are beginning to be appropriately characterized and have already been used in a few tests on treatment efficacy. Therefore, we recommend the mdx mouse as the model of choice for preclinical tests and proof-of-concept studies, whereas we recommend the use of GRMD in well controlled experimental settings, when using complementary tests, such as those used in

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