Animal models: an overview

Safety considerations discussed above point to the importance of careful design of preclinical studies, to include animal model selection. In this regard, a 1985 National Research Council report emphasized that biological modeling could be by either analogy or homology [66]. Analogy implies a point-by-point relationship between one structure or process to another, while homology suggests a shared evolutionary history and matching DNA makeup. Before the discovery of the DMD gene in 1987, it was not possible to identify true genetically homologous animal models. Investigators had to rely on models that were neither analogous phenotypically nor genetically homologous [3]. Naturally occurring genetic DMD mammalian models have subsequently been defined in mice [67–69], dogs [70], cats [71, 72], and pigs [73]. As would be expected, given inherent biologic differences between quadruped animals and humans, none of these mammalian DMD models are fully analogous clinically. Most notably, mdx mice are mildly affected, with near-normal life expectancy. Dystrophic cats have a curious hypertrophic myopathy and are prone to a malignant-hyperthermia like syndrome [74]. Dogs have a severe phenotype that more closely mirrors that of DMD. But, as discussed below, dystrophic dogs may stabilize after ~6 months of age and often live well into adulthood [75]. Although knockout and spontaneous dystrophic pigs have not yet been fully characterized, one knock out model had an unexpectedly severe phenotype (Rogers C, personal communication, 2014). The lack of analogy between the phenotypes of different animal models and DMD inherently limits conclusions that can be reached from mechanistic and preclinical studies. A rat model, created by molecularly targeting DMD gene exon 23, could have advantages over the thus-far described mdx and larger spontaneous mammalian models [76].

Potential DMD treatments have generally been tested initially in the mdx mouse, often establishing proof of concept for the particular approach [77]. Major advantages of the mdx mouse include its consistent phenotype and relatively modest expense, which allows multiple variables to be tested through reasonably powered studies. On the other hand, the mouse’s small size limits assessment of scalable variables such as cell migration or drug diffusion [78]. Perhaps even more importantly, mdx mouse preclinical trials have generally not identified treatment complications. This has been particularly problematic with their failure to predict immunologic side effects of gene and cell therapies. These advantages and limitations are essentially reversed for dystrophic dogs. Expense of maintaining dogs limits the number of variables that can be tested and phenotypic variation further reduces the power that can be achieved. With that said, the dog’s larger size and outbred nature allows for better modeling of scalable variables such as cell diffusion and the immune response to biologics. A general paradigm has evolved, whereby initial testing is done in mdx mice and, pending positive results, follow-up studies in dystrophic dogs are considered.

Animal models: the two cultures of drug discovery

Considerable attention has recently been focused on the failure of preclinical treatment trials to translate to human patients. Various reasons have been offered to account for this disconnect. A key factor relates to what has been termed the “two cultures phenomenon” [79]. Put simply, preclinical studies are often as loose as clinical trials are rigorous. Too little attention is paid to basic tenets of experimental design, extending from power analysis of the biomarker used to assess efficacy to the need for blinding [80, 81]. This problem extends to the level of detail provided on experimental design in grant applications and also to the degree to which it is considered in the review process. Understandably, the review process places considerable emphasis on a proposal’s innovative approach and potential impact. Unfortunately, less attention is sometimes paid to experimental design. Canine and other large animal models are particularly vulnerable in this area. Legitimate animal availability and budgetary issues consistently preclude conduct of a perfect study that is sufficiently powered and allows all variables to be considered. Accordingly, large animal studies must be focused and resist the temptation to explore a tangential issue.

Animal models: a change in mindset

As detailed above, preclinical animal studies help to inform the drug discovery process for both efficacy and safety. Safety is well represented in classical toxicologic assays that demonstrate potential off-target effects of a drug, as with hepatotoxicy. However, safety has often taken a backseat to efficacy in preclinical treatment trials. Investigators seem overly motivated to show that a treatment “works” versus pointing out that it failed or had deleterious consequences. Results that identify limitations of a treatment are said to be “disappointing” and, potentially, not even deserving of publication [81]. One could argue that this is precisely the wrong attitude. The greatest shortcoming of an animal model is not that it failed to demonstrate efficacy but that it failed to identify a potential risk. That it did not identify a worst-case scenario that led to a set of serious side effects. Stated another way, an animal model has failed when it did not identify a risk that caused serious complications in the 131st patient in a phase 3 treatment trial after all had gone well with the first 130. To address the apparent disconnect between animal and human clinical studies, preclinical investigators must be more rigorous in developing their experimental designs and place more importance on identifying treatment complications.

Dusty and Rusty

The GRMD model can be traced to a litter of golden retrievers, three males and one female, born July 21, 1981, outside Athens, Georgia, the home of the University of Georgia. Stiffness and simultaneous advancement of the pelvic limbs (bunny hopping) was noted in all three males at 9–11 weeks of age. Clinical signs of muscle disease were not seen in the dam, sire, or female littermate or in previous or subsequent litters of the dam. However, the same mating was purposely not repeated. A single male sibling from both the dam’s own litter and that of the grand dam had similar clinical signs. Cardinet and Holiday reported partial results from histopathologic studies of the male sibling from the dam’s litter in their 1979 monograph on canine muscle disease [82]. The sibling from the grand dam’s litter was euthanized after only a minimal evaluation and a necropsy was not done.

A veterinarian in private practice near Athens evaluated the dogs and submitted blood for routine analysis through the Veterinary Medical Diagnostic Laboratory at the University of Georgia on September 29, 1981. The most significant abnormality was dramatic elevation of serum CK (U/l) in two of the males, Dusty (16,770) and Rusty (24,442). Values for the other male (798) and female (529) were within normal limits. An inherited degenerative myopathy was suspected, and the owners were given a guarded prognosis. The male dog with the normal CK value died acutely when it was 10 weeks old, and a congenital diaphragmatic hernia compatible with an apparently unrelated syndrome seen in golden retrievers [83] was diagnosed at necropsy. Histopathologic lesions were not seen on assessment of selected skeletal muscles.

Rusty (Fig. 3) was moved to the College of Veterinary Medicine at Cornell University at 41 months of age (see below). Clinical and pathologic data collected until his death at 6 years have been published [86, 87].

Early reports of golden retrievers with apparent myopathy (Table 1)

Meier described a 10-day-old male golden retriever with an apparent myopathy in a monograph on canine muscle disease in 1958 [88]. The dog was born with an enlarged tongue that became bigger over time and “crowded the oral cavity and interfered with normal respiration and nursing.” Bradycardia and gross enlargement of the muscles of the neck and shoulders were identified on clinical examination. The most striking features at necropsy were body stunting and muscle hypertrophy. Variation in myofiber size, hyaline fibers with calcification, and myofiber basophilia consistent with regeneration were noted microscopically. Milder lesions were seen in the heart. There was a remarkable inflammatory cell infiltrate that included histiocytes and giant cells. Meier speculated that the findings were consistent with vitamin E deficiency at birth. Obviously, one cannot say definitively that this dog had a dystrophinopathy. While muscle hypertrophy, especially involving the tongue, is a feature of both DMD and GRMD, generalized muscle enlargement from birth is unprecedented in my experience. Otherwise, this dog bears some resemblance to the fulminant form of disease reported by Valentine et al. [86] and seen by others, in which respiration is compromised at birth. This condition may be analogous to pulmonary hypoplasia seen with congenital diaphragmatic hernia in humans [89]. Normal lung development is dependent on contractile activity of the diaphragm [90] so would likely be compromised in severely affected GRMD fetuses.

Alexander (Sandy) deLahunta provided clinical data on two male golden retrievers evaluated at Cornell University in his 1977 textbook, Veterinary Neuroanatomy and Clinical Neurology [91]. The syndrome was discussed together with a similar condition in Irish Terriers (see below) under a subheading of “Hereditary Myotonic Myopathy in Puppies.” Stiff gait was seen at 6 to 8 weeks of age, with progression to a stilted, shuffling gait by 3 months. Other clinical features included fatigue with exercise, occasional respiratory distress, neck stiffness, resistance to jaw opening, and enlargement of the base of the tongue. The dogs were still able to walk at 6 to 9 months when euthanasia was performed. Additional key findings included elevation of serum muscle enzymes, “myotonia” on EMG, and evidence of “severe degenerative muscle disease” on muscle biopsy or necropsy (necrosis of muscle cells, mononuclear phagocytosis, giant cells, and calcification). Reference was made to the more thoroughly studied condition in Irish Terriers, which had been shown by pedigree analysis to be X-linked [92] (see below).

Cardinet and Holliday described an additional 8-month-old golden retriever dog in 1979 [82]. Clinical features were consistent with those described by deLahunta. High-frequency discharges that waxed and waned were seen on EMG. Histopathologic features included scattered necrotic and/or calcified fibers. Cardinet and Holliday noted that fiber type grouping was consistent with a potential neuropathic component. Another dystrophic golden retriever with similar clinical signs was subsequently identified at Cornell and studied until 8 months of age [93]. Muscle ultrastructure was studied for the first time. Notably, focal wedge-shaped subsarcolemmal “delta” lesions and plasma membrane defects were illustrated (Fig. 4), in keeping with the membrane theory of DMD pathogenesis.

Thus, counting Rusty and Dusty, a total of seven golden retrievers with an apparent primary, inherited myopathy had been reported by the mid-1980s (Table 1). Additional dogs were cited via personal communication in our 1988 paper [85]. Strikingly, all affected golden retrievers were males, suggesting X-linked inheritance and potential genetic homology with DMD. Clinicopathologic evidence of serum CK elevation and small group muscle fiber necrosis and regeneration were also consistent with DMD. On the other hand, presence of CRDs on EMG, with associated stilted gait, stiffness, and marked muscle hypertrophy, were more consistent with myotonia.

Cornell and canine X-linked muscular dystrophy

As detailed above, deLahunta and colleagues at Cornell played a key role in identifying early dystrophic golden retrievers. Barry Cooper, in particular, was motivated to establish a breeding colony of affected dogs and was aware of ongoing studies at NCSU. Rusty was sent to Cornell at 41 months of age, with an understanding that he would be used to establish a colony and then returned for follow on studies at NCSU. However, Rusty developed congestive heart failure at 5½ years of age and was euthanized at 6 years. So, instead, two obligate carriers (Dys and Trophy) and an affected male (Lewis) produced by Rusty were provided to NCSU to establish a second colony.

The studies by Dr. Cooper and his veterinary colleague, Beth Valentine, were instrumental in defining the phenotype of dystrophic dogs [86, 87, 94]. Rusty was initially bred with two female beagles and a larger retriever cross to produce mixed-breed obligate carriers. These carriers were then bred with Rusty or his male descendants to produce affected dogs. In this sense, GRMD is not a disease of pure-bred golden retrievers. We use the GRMD acronym to refer to the DMD gene splice site mutation present in affected dogs. Other acronyms, such as GSHPMD for German shorthaired pointer muscular dystrophy, refer to additional canine DMD gene mutations. Based on pedigree analysis of the dogs descendant from Rusty at Cornell, a likely recessive X-linked pattern of inheritance was established [95]. Phenotypic features in dogs from the Cornell colony were consistent with those reported in the previously described seven cases and included elevation of serum CK, CRDs on EMG, and histopathologic evidence of grouped muscle fiber necrosis and regeneration. Because this work was done before the DMD gene and dystrophin protein were identified, there was still a question as to whether the disease was a Duchenne genetic homologue. Genetic homology was established with reasonable certainly when neither DMD messenger ribonucleic acid (mRNA) nor dystrophin could be identified in dystrophic dogs [94]. Consistent with the GRMD site splice mutation later identified by Sharp et al. [96] (see below), endonuclease probes did not demonstrate RFLPS that would be expected with a large DNA deletion. Cooper and colleagues termed the disease canine X-linked muscular dystrophy (CXMD).

Canine dystrophinopathies

Prior to the definition of the DMD gene and dystrophin protein, potentially analogous canine models were proposed based on an apparent pattern of recessive X-linked inheritance and consistent phenotypic features. In his 1977 textbook [91], deLahunta drew parallels between the condition in golden retrievers and an analogous disease in Irish terrier dogs reported by Wentink et al. [92]. The Irish terrier study included phenotypic data, extending from serum enzymes to necropsies, on a group of five male littermates. Pedigree data, suggesting a pattern of recessive X-linked inheritance, were reported from two prior litters from the same bitch. Serum enzymes, including aldolase and CK, were elevated, in keeping with the membrane theory of DMD pathogenesis. Wentink et al. specifically noted that the dramatic increase in CK was typical of DMD. Bizarre high-frequency discharges were seen on EMG, consistent with other canine dystrophinopathies that have since been characterized. Notably, dogs were variably affected, with two having a severe phenotype that necessitated euthanasia between 13 and 20 weeks of age, while another mildly affected dog lived until 16 months before euthanasia. Limb muscles were atrophied but the tongue was hypertrophied. One of the dogs had congenital peritoneopericardial diaphragmatic hernia at necropsy consistent with a syndrome later seen in GRMD dogs. Histopathological lesions were not seen in the hearts, central nervous system, or visceral organs of affected dogs. Based on extensive muscle histochemical and ultrastructural studies, Wentink et al. concluded that mitochondria were abnormal and speculated that uncoupled oxidative phosphorylation could be involved in disease pathogenesis.

Additional apparent dystrophinopathies have since been reported in a number of canine breeds [70, 96–105] (Fig. 5). Phenotypic findings, typical of those seen in GRMD, have included increased serum CK, CRDs on EMG, and histopathologic evidence of grouped muscle fiber necrosis and regeneration. When multiple dogs have been observed, disease severity has varied, in keeping with phenotypic variation seen in GRMD. As with GRMD, paradoxical muscle hypertrophy has seemed to play a role in the phenotype of affected dogs, with stiffness at gait, decreased joint range of motion, and trismus being common features. For the most part, dystrophinopathies in other canine breeds have not been defined beyond immunohistochemical and Western blot studies demonstrating the loss of dystrophin. Relatively few have been studied at the molecular level, with mutations largely paralleling the deletions, insertions, and splice-site mutations seen in DMD [70, 96–105].

The classification of canine dystrophinopathies is complicated by the tendency to associate DMD and BMD with severe and mild clinical phenotypes, respectively. All canine DMD deletions described to date have been out-of-frame, which would predict a severe DMD phenotype. As discussed above and further below, despite having an out-of-frame mutation, GRMD dogs may stabilize and live well into adulthood [75]. The GRMD phenotype could be influenced by spontaneous alternative splicing of the DMD transcript [106], with resultant production of truncated partially functional dystrophin isoforms expressed in so-called revertant fibers. Alternative splicing [107, 108] and revertant fibers [109] have also been reported in DMD. Conditions in which truncated isoforms of dystrophin are expressed have been reported in Japanese Spitz and Labrador retriever canine breeds. A 5.4-Mb fragment of the X chromosome is inverted at the level of intron 19 of the DMD gene and the retinitis pigmentosa GTPase regulator (RTGP) gene of Japanese Spitz dogs [99]. Transcription was demonstrated at the 5′ end but not beyond. A truncated 70–80 kDa protein, consistent with aberrant expression of Dp71 dystrophin-related protein, was identified. Baroncelli et al. described a 3-year-old male Labrador retriever with what was termed a Becker type syndrome [110]. The dog had a mild phenotype, normal serum CK, and widespread immunohistochemical evidence of dystrophin expression. Immunoblotting revealed two bands consistent with rod-domain and carboxyl-terminus fragments. The molecular mutation was not identified.

Similarly, in another report, Labrador retrievers with no dystrophin had increased serum CK and dystrophic lesions on biopsy but no clinical evidence of myopathy [111]. A somewhat analogous situation exists in German Shorthaired pointers that have a relatively mild phenotype even through the entire DMD gene is deleted [101, 102, 112]. Genetic mechanisms other than frame shift must underlie milder (or more severe) phenotypes in dystrophic dogs. A mild phenotype in two so-called escaper GRMD dogs from one of the Brazilian colonies was associated with a G>T mutation in the promoter region of the Jagged1 gene [113]. Given that this substitution was apparently introduced by an outcross, the Jagged1 mutation would not necessarily be found in other GRMD colonies. We routinely see mildly affected GRMD dogs that live beyond 3 years of age. In testing done to date, none of these dogs have had the Jagged1 mutation. Importantly, the original founder dog for all GRMD dogs worldwide, Rusty, lived until 6 years when he developed congestive heart failure and was euthanized [86, 87]. Using Rusty as a benchmark, the GRMD phenotype actually appears to have worsened over time, potentially due to excessive inbreeding and concentration of the effects of genetic modifiers. The genetic basis of phenotypic variation is a subject of active research in my laboratory, with a recent genome wide association study (GWAS) identifying several potential candidate genes that could influence disease phenotype [114].

With the advent of molecular therapies, particular interest has been focused on certain so-called hot spot areas at exons 3-7 and 45-53 of the DMD gene (see above). In GRMD and the further outbred condition, canine X-linked muscular dystrophy in Japan (CXMD_J) [115], an mRNA processing error results from a single base change in the 3′ consensus splice site of intron 6. Exon 7 is consequently skipped during mRNA processing [96]. The resulting transcript predicts that the dystrophin reading frame will be terminated within its N-terminal domain in exon 8. The dystrophin reading frame can be reestablished using antisense oligonucleotides that target exons 6 and 8 [42, 116]. With regard to the even more clinically relevant exon 45-53 hot spot area, Cavalier King Charles spaniels have a G-T missense mutation in the 5′ consensus splice site of intron 50 that results in deletion of exon 50 [103]. Antisense oligonucleotide-mediated skipping of exon 51 restored the reading frame and protein expression in cultured myoblasts from an affected dog. As mentioned above, the fascinating DMD gene deletional mutation in the German Shorthaired pointer breed bears particular mention. The entire DMD gene is deleted in affected dogs [101, 102], thus eliminating the possibility of alternative splicing and the confounding effects of revertant fibers.

GRMD in preclinical trials: overview

Rather than repeat recent reviews of GRMD preclinical trials and biomarkers [77, 97], this discussion will focus on three areas: (a) the overall preclinical approach to DMD treatment development; (b) nuances of GRMD disease progression that must be considered in experimental design; and (c) cautionary lessons learned from prior GRMD treatment studies.

The DMD research community is fortunate to have two well-defined genetically homologous animal models that can be used in tandem to advance therapy development. Mdx mice should logically be employed for initial in vivo preclinical experiments. Studies that fail to demonstrate potential efficacy bode poorly for translation of the therapeutic strategy to humans and could provide a logical “stopping point” for continued development. Alternatively, if mdx findings are encouraging, consideration should be given to extending the work to GRMD or another canine model. However, in reality, studies in dogs are limited because of their availability and expense. When dystrophic dogs have been studied, results from mdx mice have not been consistently reproduced. In such cases, inherent species differences preclude saying whether the mouse or dog would better predict the ultimate outcome in humans. But, at the very least, a failure to reproduce favorable data in both species should raise concerns and prompt reassessment of the therapeutic approach.

GRMD in preclinical trials: experimental design

For the sake of GRMD trials, we have long focused on the 3- to 6-month age period during which clinical signs progress particularly rapidly. Based on an epidemiologic study by Patronek et al. [117], the first year of a golden retriever’s life roughly equates to 20 years of a human. Therefore, the GRMD age quartiles of 0–3, 3–6, 6–9, and 9–12 months would logically correspond to 0–5, 5–10, 10–15, and 15–20 years of DMD. In comparing the relative severity of disease over these age periods, GRMD and DMD generally progress analogously up to 6 months (GRMD) [75] and 10 years (DMD) [118, 119] (Fig. 6). While GRMD pups may be weak at birth and require dietary supplementation [120], they tend to stabilize around 2 weeks and are mildly affected up to 3 months, when more progressive clinical involvement is seen. Similarly, DMD boys have relatively stable disease up to 6 years of age and then progressively decline for the next 5 years [118].

In our experience, the GRMD and DMD phenotypes diverge after 6 months and 5 years, respectively, with dystrophic dogs tending to stabilize and boys inexorably progressing [75, 118–130]. Even though GRMD dogs in our colony progress markedly up to 6 months, they rarely lose the ability to walk, which would be analogous to becoming confined to a wheelchair in DMD, and often live well into adulthood. With this said, other laboratories have reported a more severe GRMD phenotype. For instance, ~25% of GRMD dogs produced in the colony at Alfort in France lose ambulation by 6 months [131]. In a similar vein, GRMD dogs in the Brazilian colony directed by Mayana Zatz generally die within the first 2 years of life [113]. Notably, both the French and Brazilian colonies were derived from the same founder dog, Rusty (see above), identified by our group in 1981. This distinction in phenotypic severity likely occurs due to the relative role of genetic modifiers in each colony, potentially being concentrated by inbreeding or introduced by outbreeding. As an example, inbreeding tends to worsen the phenotype of GRMD pups [120].

Planning and interpretation of (pre)clinical trials in both DMD and GRMD is confounded by variable disease progression among individuals. As with the differing phenotypes seen in GRMD colonies, DMD phenotypic variation likely occurs due to genetic modifiers. Polymorphisms in two genes, secreted phosphoprotein 1 (SPP1) (osteopontin) and latent transforming growth factor β binding protein 4 (LTBP4), have been shown to influence the DMD phenotype [132]. Per the discussion above, Vieira et al. identified the Jagged1 gene as a modifier of the GRMD phenotype [113]. We recently published results of a GWAS that defined a number of genes associated with phenotype [114].

For local (intramuscular) or regional (single limb) therapeutic approaches, the opposite untreated limb can serve as a control. With systemic treatments, it is difficult to distinguish a treatment effect from inherently mild clinical disease. Speaking generally, the number of subjects required to establish benefit tracks directly with the standard deviation of the outcome parameter being used and inversely with the relative benefit anticipated. In a somewhat sobering review, Brooke et al. concluded that for a DMD clinical trial intended to “test a drug which might slow the disease to 25% of its original rate of progression, two groups (placebo and treatment) of 40 patients each would have to be followed for a year” [118]. Such a study typically determines the longitudinal effect of a treatment by comparing baseline and end-of-study values. This helps to remove the confounding effect of phenotypic variation because each individual essentially becomes his own control. In principle, even more cases would be required if only data from a final outcome parameter were compared between control and treatment groups.

Power analysis has been used on a limited basis to assess GRMD preclinical biomarkers, with only tibiotarsal joint (TTJ) tetanic torque and the 6-minute walk test (6MWT) being evaluated. The power analysis in our TTJ study anticipated that a single measurement would be done at the end of treatment and compared with a value from a control group [130]. Data for TTJ tetanic flexion torque showed that groups of 15 and 5 GRMD dogs would be necessary to demonstrate differences of 0.2 and 0.4 at 6 months, with associated powers of 0.824 and 0.856. Based largely on these natural history data, we have typically included 5 or 6 dogs in our GRMD preclinical trials. However, the natural relative recovery of TTJ flexion torque between 3 and 6 months complicates its use in GRMD preclinical trials [130]. Extension values decline over this same period but tend to vary more markedly among dogs, necessitating larger group sizes to establish significance. With more careful monitoring of inbreeding coefficient for sire-dam pairs in our colony, the overall GRMD phenotype is now less severe and not as variable as it was at the time the TTJ torque data were published [130]. A subsequent trial of prednisone in GRMD dogs demonstrated therapeutic benefit of ~60% (p < 0.05) for extension torque using groups of 6 treated and 10 untreated GRMD dogs [133].

For sake of the 6MWT, GRMD dogs walked significantly shorter distances than normal littermates at 6 and 12 months, generally supporting its use in preclinical trials [134]. We also determined that percent increase in CK after the 6MWT was higher in GRMD versus normal dogs, providing an additional potential biomarker. With this said, neither the 6MWT nor CK percent increase was particularly sensitive. Assuming a group size of 6 each for treated and control GRMD dogs, power analysis revealed that an 80% increase in mean height-adjusted distance walked or 55% decrease in mean post-exercise CK would be necessary to achieve a desired power of 80%.

As with longitudinal DMD trials, a GRMD study comparing values at baseline (3 months) and end of treatment (6 months) would lessen the effects of phenotypic variation, reducing the number of dogs required. Ideally, treatment groups would also be balanced between mildly and severely affected individuals, based on an early biomarker that tracks with disease. In a GRMD study focused on identification of early disease biomarkers, increased numbers of a subset of circulating T cells and decreased stride frequency on accelerometry at 2 months strongly associated with more severe clinical disease at 6 months [131]. While independent control groups for each preclinical trial should ideally be assessed, budgetary and animal number limitations may necessitate the use of natural history controls. In one such study, TTJ extensor torque and other indices were improved in six GRMD dogs treated with a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor compared to an independent control group [135].

Just as the overall disease course for DMD patients and GRMD dogs varies, individual muscles also have different patterns of progression. Proximal muscles are classically affected in DMD, with further selective involvement of extensors versus flexors. Muscles, such as the quadriceps, that undergo eccentric contraction are particularly vulnerable [136]. Differential muscle involvement was well illustrated in our natural history TTJ torque study, with flexors being preferentially affected at 3 months and extensors at 6 months [130]. Pointing to the challenges one faces in analyzing functional data, the prednisone-treated GRMD dogs discussed above had a paradoxical decrease in flexor torque. This presumably occurred because of a reduction in early necrosis that otherwise would have led to functional flexor hypertrophy [133]. A similar decrease in flexor torque was seen in another study of GRMD dogs treated with prednisone and cyclosporine [137].

The pattern of differential involvement among skeletal muscles extends to the heart, which has a much later onset of clinical disease. Reasons for preferential disease of skeletal muscles or sparing of the heart are unclear but likely also involve different patterns of gene expression. Through a collaboration with investigators at Vanderbilt, we showed that brain-derived neurotropic factor (BDNF) is preferentially increased in young GRMD dogs, perhaps in keeping with a palliative cardiac effect. Moreover, BDNF levels tracked with increased ejection fraction in DMD patients [138]. The relatively late onset of cardiac dysfunction complicates preclinical GRMD studies done over the 3- to 6-month age period. Definition of cardiac biomarkers that would distinguish early subclinical disease is an active area of investigation for my laboratory.

Results of DMD [118] and GRMD [120, 139] functional tests track and correlate with each other, providing some assurance that they are valid markers of disease and can be used to document benefit. For sake of GRMD, mildly affected dogs have proportionally larger TTJ angles and tetanic extensor torque. In addition, cranial sartorius circumference measured at the time of surgical biopsy is proportionally smaller. The opposite pattern is seen in severely affected dogs. On the other hand, 6MWT results from a much smaller group of GRMD dogs did not correlate with other tests, causing concern about this test’s reliability [134].

GRMD in preclinical trials: cautionary lessons

Pharmacologic approaches

In developing drug therapies for DMD, approaches have been based on somewhat intuitive assumptions that targeting secondary effects of dystrophin loss, like inflammation, myofiber necrosis, and muscle atrophy, would have benefit. While such treatments are logical, they may inherently compromise or augment ongoing homeostatic mechanisms and potentially do more harm than good. Studies in the GRMD dog have sometimes brought side effects into clearer focus, potentially allowing the particular treatment to be modified.

Anti-inflammatories

Glucocorticoids, such as prednisone, have become the standard of care for DMD [140, 141], but also cause a range of side effects [140, 142]. We were motivated to study the effects of prednisone in GRMD dogs to establish a baseline for comparing other treatments and to explore alternative dosage regimens that might not be practical in DMD. The study employed a variation of what has become our standard approach to preclinical studies, with dogs being dosed daily from 2 to 6 months of age at either 1 or 2 mg/kg [133]. While this dose is higher than the typical regimen of 0.75 mg/kg/day used in DMD [140, 141], care must be taken in extrapolating drug dosages among species. Body surface area takes into account variables such as drug metabolism and, therefore, offers a better guide than body weight in considering appropriate dosing [143]. Several functional and histopathologic biomarkers were compared between treated and control GRMD dogs at 6 months. Although a dose-related increase in TTJ extensor force was seen, there was a paradoxical decrease in flexor values. The reduced flexor force was attributed to a reduction in early necrosis and a less pronounced regenerative response that would otherwise have led to functional hypertrophy. Dogs in the 2 mg/kg group also had dramatic myofiber mineralization, potentially due to decreased clearing by macrophages. A subsequent GRMD study by another group of combined prednisone, again at 2 mg/kg, and cyclosporine produced similar results [137]. The fact that high dose prednisone has deleterious effects is not surprising and consistent with broader side effects of the drug. The differential effect on flexor and extensor function is more concerning because of the potential that this could translate to DMD patients. As discussed further below in section “Myostatin inhibition”, treatments that cause differential extensor and flexor muscle improvement or decline could aggravate preexisting contractures and postural instability.

Prednisone acts, in part, by inhibiting the NF-κB signaling pathway [144], which is activated in DMD [145]. Accordingly, this pathway has been targeted in preclinical DMD studies. We conducted a GRMD trial that built on prior mdx mouse experiments in which the Nemo Binding Domain (NBD) peptide was used to inhibit NF-κB signaling [135]. As with the prednisone study, treatment began at 2 months of age and extended for 4 months, with dogs being dosed intravenously three times weekly and studied at 6 months using several biomarkers. Results paralleled those seen with prednisone, in that TTJ extensor force was increased in treated versus control dogs but flexor values were decreased. Of greater concern, NBD administration over time led to infusion reactions in both treated GRMD and wild-type dogs, pointing to the potential for recombinant proteins to induce an immune response.

Inhibition of protein degradation

Given that DMD is predominantly a muscle-wasting disorder, degradative enzymes of the ubiquitin-proteasome (UPS) and calpain systems have been targeted therapeutically. Promising results have generally been achieved in the mdx mouse with inhibition of the UPS [146] but not the calpain system [147]. In a GRMD study, only about half of the muscles studied had increased calpain activity and most actually had decreased proteasome activity [148]. Of greater concern, numerous UPS components were decreased in the heart, suggesting that pharmacologic inhibition of these systems in DMD could have unintended deleterious consequences. Not surprisingly, GRMD dogs treated with a novel calpain inhibitor did not improve [149].

Myostatin inhibition

Inhibition of the myostatin gene (growth and differentiation factor 8), a key negative regulator of muscle growth [150, 151], offers another approach to reverse muscle atrophy. Mdx mice in which myostatin was knocked out [152] or postnatally inhibited [153] had a less severe phenotype. Data from normal [154] and GRMD [155] dogs seemed to substantiate potential value of myostatin inhibition. However, likely reflecting feedback mechanisms, myostatin gene levels are already markedly lower in DMD patients [156], mdx mice [157], and GRMD dogs [139], raising questions about whether further inhibition is desirable. Such concerns were substantiated by a study in our colony in which GRMD dogs were bred with whippets carrying a myostatin gene mutation. We expected that myostatin heterozygous GRMD dogs (GRippets) would have an improved phenotype, consistent with the prior mdx work. Surprisingly, the GRippets had more severe postural changes than their dystrophic myostatin-wild-type littermates, apparently due to differential effects on agonist and antagonist muscles. For almost all muscles, the degree of atrophy or hypertrophy in GRMD dogs was more pronounced in the GRippets (Fig. 7) [158]. In keeping with studies showing that myostatin is already downregulated in GRMD, mRNA and protein levels did not differ between the two groups. For sake of interpreting these data, it is critical to recognize that findings from a study in which myostatin was downregulated in utero will not necessarily extrapolate to postnatal treatments. But, these findings emphasize that the dystrophic body is already motivated to downregulate myostatin and that the effects of inhibition on muscle mass will not necessarily be uniform.

Genetic therapies

Genetic therapies have included AAV-mediated insertion of a truncated mini/micro-dystrophin to fit the vector’s limited ~4.5 kb carrying capacity, antisense oligonucleotides to induce exon skipping and reestablish the dystrophin reading frame, agents to read-through stop codon mutations, and replacement of dystrophin at the sarcolemma with surrogates such as utrophin. Studies in the mdx mouse have generally demonstrated efficacy and safety of these strategies. Some therapies have subsequently been extended to the GRMD model, providing further proof of concept [77, 97] (see discussion under canine dystrophinopathies above). Data from dystrophic dogs supported the antisense approach, and side effects were not seen [116, 183–186]. But, even with supportive mdx and GRMD preclinical data, human trials have thus far been inconclusive [59, 60].

Therapies employing AAV-mini/micro-dystrophin constructs should, in principle, offer similar therapeutic benefits and potential side effects as those employing exon-skipping strategies. With that said, while supportive data have again been demonstrated, greater safety concerns have been identified. Initial alarm was caused when localized (intramuscular) treatment elicited an immune response to either AAV capsid antigen [187] or dystrophin serving as a neoantigen [188] that could be blocked with immunosuppression [189]. Providing encouragement, dystrophin expression was demonstrated after intramuscular injection of an AAV-micro-dystrophin construct without immunosuppression in a single dystrophic dog [190]. And, in another study, immunosuppressed dystrophic dogs treated by intramuscular injection with AAV-micro-dystrophin had improved force and lower eccentric contraction decrement (ECD) despite a nonspecific lymphocytic infiltrate [191].

Interestingly, the immune response has generally been less pronounced with subsequent regional limb and systemic therapies, potentially because of a dilutional effect on the causative antigen [97]. However, one GRMD dog treated with AAV-mini/micro-dystrophin by regional limb delivery by my group in collaboration with Xiao Xiao at the University of North Carolina-Chapel Hill had increased signal intensity on MRI compatible with edema 3 months after treatment, and the level of dystrophin expression was actually higher in the contralateral limb (Kornegay JN and Xiao X, unpublished). This likely reflected the combined effects of dampened dystrophin expression due to the immune response and leakage at the tourniquet to allow systemic delivery. Given the generally promising results of regional limb delivery in GRMD dogs, phase 1-safety studies using saline versus active construct have been completed in both the lower [192] and upper [193] extremities of adult muscular dystrophy patients, with minimal side effects.

Results of systemic AAV-mini/micro-dystrophin therapy in dystrophic dogs have been mixed. On the one hand, our group and others have demonstrated long-term dystrophin expression, with [194] and without [195] immunosuppression. But, one group of dogs that we studied had delayed growth and pelvic limb muscle atrophy and contractures, seemingly due to an innate immune response (Fig. 8) [195]. Moreover, clear evidence of functional improvement akin to that seen in GRMD dogs with the exon-skipping strategies [186] has not been demonstrated after systemic therapy with AAV-mini/micro-dystrophins, giving rise to persistent questions about the ability of mini/micro-dystrophins to sustain myofiber integrity when stressed by larger mammals.