Joe N. Kornegay, DVM, PhD, DACVIM (Neurology)
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder affecting approximately 1 of 3,500 newborn human males in which absence of the protein dystrophin causes progressive degeneration of skeletal and cardiac muscle.1 No treatment halts or reverses progression of DMD. Although cellular and gene therapies are promising, key questions must first be addressed in relevant animal models. Spontaneous forms of X-linked muscular dystrophy due to dystrophin deficiency have been identified in mice, multiple dog breeds, and cats. Unlike the dystrophin-deficient mdx mouse, which remains relatively normal clinically,2 affected dogs develop progressive, fatal disease strikingly similar to the human condition.3 Accordingly, studies in the canine dystrophin-deficient models are more likely than those in mdx mice to predict pathogenesis and outcome of treatment in DMD.
While numerous dog breeds with dystrophin-deficient muscular dystrophy have been characterized clinically,4,5 few have been studied at the molecular level. Over the past 20 years, we have conducted extensive studies in golden retrievers with muscular dystrophy (GRMD). An RNA processing error in GRMD dogs results from a single base change in the 3' consensus splice site of intron 6.6 Exon 7 is consequently skipped during RNA processing. The resulting transcript predicts that the dystrophin reading frame is terminated within its N-terminal domain in exon 8. A truncated, apparently unstable dystrophin molecule is produced. Our initial studies of the genomic and pathophysiologic features of GRMD, together with those of Dr. Barry Cooper's group at Cornell done at times in collaboration,7,8 established this condition as a valid model of DMD. An affected dog studied by our group until 40 months of age3 is the common sire of all dogs in GRMD colonies both in the US (UNC-CH and the Fred Hutchinson Cancer Center in Seattle) and around the world (Brazil, France, Japan, and the Netherlands). We initially established our colony at North Carolina State University in the late '80s. It was moved to the University of Missouri-Columbia in 1994 and to the University of North Carolina-Chapel Hill (UNC-CH) in 2007. We currently maintain colonies of golden retrievers and German shorthaired pointers9 (GSHPMD, see further discussion of this condition at the end of these proceedings) with muscular dystrophy. Most of our research using these dogs is focused on preclinical studies of potential treatments for DMD and is done in collaboration with scientists at UNC-CH and other universities.
Use of the GRMD Model in Treatment Development for DMD
Potential treatments for DMD may be broadly categorized as molecular, cellular, or pharmacologic.10 The two most common molecular approaches are gene therapy, whereby the dystrophin gene is introduced into muscle either locally or through the vasculature, generally through use of plasmids or viral vectors; and gene correction, which involves introduction of oligonucleotides (chimeric or antisense) to induce either inherent repair mechanisms or exon skipping to reestablish the correct nucleotide sequence (reading frame). With cell-based therapies, normal cells such as myoblasts or stem cells are transplanted into diseased muscle. Pharmacologic approaches do not deliver the defective gene and/or protein to the diseased muscle(s). Instead, specific pathogenetic mechanisms that contribute to the dystrophic phenotype are targeted. Examples include compounds that reduce inflammation (NF-kB inhibition), increase muscle mass (insulin-like growth factor, myostatin inhibition), read through stop codons in the defective dystrophin gene (aminoglycoside antibiotics), or increase production of utrophin (the autosomal form of dystrophin).
GRMD dogs have been used in a variety of preclinical studies. Howell et al showed that GRMD myofibers can be transduced for at least 14 days using plasmids containing full length dystrophin and minidystrophin cDNA.11 Adenovirus-mediated dystrophin minigene12 and utrophin13 transfer have also been achieved in immunosuppressed GRMD dogs. In other GRMD studies, dystrophin minigene transfer with an adeno-associated virus (AAV) vector was limited by a marked immunologic response to viral capsid antigens14 that could be suppressed with brief immunosuppression.15 Others have suggested that the immune response is to components of the construct, including dystrophin itself (see discussion of GSHPMD at the end of these proceedings).16 We have found that this immune response is less pronounced when the canine dystrophin minigene is administered either locally or via retrograde regional limb perfusion. Additional forms of cell and gene therapy have also yielded mixed results in affected dogs. GRMD dogs treated with mesoangioblasts had immunohistochemical and Western blot evidence of dystrophin expression.17 Chimeric oligonucleotides were used to induce normal host cell mismatch repair mechanisms to correct the splice site mutation in an affected dog.18 However, we were unable to achieve either myoblast or mesenchymal stem cell implantation in GRMD dogs during the early '90s despite the fact that others had demonstrated success in the mdx mouse. Similarly, hematopoietic stem cell transplantation did not restore dystrophin expression in affected dogs despite promising results in mdx mice.19 Recently, in preparation for high-dose and early-treatment regimens that might be impractical in human patients, we have shown that prednisone increases extensor muscle force in GRMD dogs (see further discussion below).20 Additional unpublished studies have shown promise, further validating the GRMD model and setting the stage for DMD clinical trials in some cases.
Biomarkers for Assessment of the natural history and Response to Treatment in GRMD
Clinical signs in GRMD occur soon after birth, as pups are often ineffectual sucklers and must be supplemented. As a result, they are typically somewhat stunted. By 6 weeks of age, the pelvic limbs may be advanced simultaneously and trismus is noted. Dogs subsequently develop a progressively more stilted gait, atrophy of particularly the truncal and temporalis muscles, a plantigrade stance due to hyperextension of the carpal joints and flexor contractures in the tarsal joints, excessive drooling suggesting pharyngeal muscle involvement, and initial lumbar kyphosis that progresses to lordosis. As with DMD, some muscles paradoxically hypertrophy. Aspiration pneumonia may occur due to pharyngeal or esophageal muscle involvement. Cardiac failure due to cardiomyopathy can also occur. Dogs with GRMD develop pathologic changes in muscle similar to those described for DMD (see below). Because of the striking similarities between clinical and pathologic features of DMD and GRMD, studies in affected dogs may be more likely than those in mdx mice to predict pathogenesis and outcome of treatment in DMD. However, biomarkers must be developed to objectively document benefit in these preclinical studies.
Golden Retriever Muscular Dystrophy (GRMD) Model--Functional Studies
To better utilize the GRMD model in therapeutic trials, we have developed various phenotypic tests to objectively characterize disease progression. Affected dogs have marked joint contractures21 and demonstrate weakness of individual22 and grouped23 muscles. As with mdx mice, weakness is exacerbated by eccentric contractions.24 We have focused principally on measurement of torque force generated by the tibiotarsal joint.23 The peroneal and tibial nerves are stimulated percutaneously so that the paw pulls (peroneal nerve, flexion) or pushes against (tibial nerve, extension) a lever interfaced with a force transducer. In our initial study, force values were measured at 3, 4.5, 6, and 12 months of age.23 Absolute and body-weight-corrected GRMD twitch and tetanic force values were lower than normal at all ages (P<0.01 for most). However, tarsal flexion and extension were differentially affected. Flexion values were especially low at 3 months, whereas extension was affected more at later ages. Several other GRMD findings differed from normal. The twitch/tetany ratio was generally lower; post-tetanic potentiation for flexion values was less marked; and extension relaxation and contraction times were longer. The consistency of GRMD values was studied to determine which measurements would be most useful in evaluating treatment outcome. Standard deviation was proportionally greater for GRMD versus normal recordings. More consistent values were seen for tetany versus twitch and for flexion versus extension. Left and right limb tetanic flexion values did not differ in GRMD; extension values were more variable. These results suggested that measurement of tarsal tetanic flexion force should be most useful to document therapeutic benefit in GRMD dogs. Groups of 15 and five would be necessary to demonstrate differences of 0.2 and 0.4 in the means of treated and untreated GRMD dogs at 6 months of age, with associated powers of 0.824 and 0.856, respectively (Sigma Stat, Jandel Scientific, 2591 Kerner Blvd., San Rafael, CA, USA).23
Results from functional tests tend to correlate with one another and with other clinicopathologic features. By comparing serial measurements from treated and untreated groups, one can document improvement or delayed progression of disease. Functional outcome values have varied considerably, even among dogs within the same litter, suggesting that modifier genes significantly influence the phenotype.21-23 Importantly, phenotypic variation confounds data analysis, requiring larger group sizes to demonstrate significance. The effects of phenotypic variation on statistical analysis can be offset by establishing baseline outcome values prior to treatment so that each dog serves as its own control. With localized treatments, the effect of phenotypic variation is less of a concern because the untreated opposite limb can serve as the control. We have utilized tetanic tibiotarsal joint force measurements to evaluate effects of prednisone given to GRMD dogs for a 4 month period beginning at 2 months of age.20 Extension forces in GRMD dogs treated daily with 2 mg/kg prednisone increased, while flexion values paradoxically decreased. The paradoxical decline in flexor force measurements was attributed to the fact that some GRMD flexor muscles undergo necrosis early in life with subsequent functional hypertrophy.25 Treatment with prednisone could have attenuated this early necrosis and functional hypertrophy.
Golden Retriever Muscular Dystrophy (GRMD) Model--Pathologic Studies
We have conducted histopathologic studies to determine both the natural history3,25,26 and response to treatment of GRMD dogs.20 One natural history study evaluated the degree of gross muscle atrophy and hypertrophy of pelvic limb muscles.25 While most muscles were atrophied, the caudal and cranial sartorius muscles were hypertrophied. Cranial sartorius muscle weights were corrected for body weight and endomysial space to determine true muscle weights (g/kg; mean ± SD) in three GRMD age groups (4-10 mos [Group 1; n=15], 13-26 mos [Group 2; n=4], and 33-66 mos [Group 3; n=4]) and grouped normal dogs (6-20 mos; n=12). Group 1 GRMD weights (2.2063 ± 0.6884) were greater than those of normal dogs (1.2699 ± 0.1966), indicating that young GRMD dogs have true cranial sartorius muscle hypertrophy. Values of Group 2 (1.3758 ± 0.5078) and Group 3 (0.5720 ± 0.2423) GRMD dogs were less than those of Group 1, suggesting that the cranial sartorius muscle atrophies over time. Taken together, these data showed that young dogs have true cranial sartorius muscle hypertrophy. Values from older dogs indicated that the cranial sartorius subsequently atrophies over time, with an associated increase in the endomysial space due to deposition of fat and connective tissue. Given that the cranial sartorius muscle weight correlated with tarsal joint angle in affected dogs (r = 0.817), the hypertrophied muscle might contribute to contractures and play a role analogous to iliotibial band tightness in DMD.27
In the prednisone treatment study discussed above, we quantified numbers of fetal-myosin and alizarin-red-positive fibers to assess the degree of myofiber regeneration and mineralization, respectively.20 In the cranial sartorius muscle of GRMD dogs given 1 and 2 mg/kg daily prednisone, 13.6 ± 5% and 10.6 ± 13% of myofibers stained positive for fetal myosin, respectively, compared to 20.8 ± 6% in untreated GRMD controls (p < 0.01 for both groups). In the cranial sartorius muscle of dogs given 1 and 2 mg/kg of prednisone, 2.4 ± 2% and 9.2 ± 5% of myofibers stained positive for alizarin red, respectively, compared to 1.9 ± 1% in untreated GRMD controls (p < 0.01 for the 2 mg/kg group). There were similar changes in the vastus lateralis muscle. Evidence of calcified fibers suggests that prednisone may have deleterious effects, in keeping with the well-documented Cushing's myopathy seen in dogs. The reduction in fetal myosin positive-fibers is also concerning, although this might simply reflect a reduced demand for regeneration.
Golden Retriever Muscular Dystrophy (GRMD) Model--MRI Studies
MRI has been used infrequently in animal models of DMD. Signal-intense lesions corresponding to necrotic areas on histopathologic examination have been seen in T2-weighted MR images of 8- to 14-week-old mdx mice.28 Regression of MRI lesions has been seen in both mdx mice and other murine models of muscular dystrophy after gene therapy. In one recent study, when compared to normal dogs, GRMD dogs had an increased T2:T1 signal and greater T2 signal heterogeneity and contrast enhancement.29 We have completed preliminary MRI studies on a 3T scanner available through the UNC-CH Animal Imaging Center The animal imaging protocol was designed to be close to one used previously in DMD patients at UNC-CH (Fan J, Howard J, and Lin W, unpublished observations). Our animal protocol provides excellent resolution, thus allowing region-of-interest measurements of MRI parameters. With the recent relocation of our colony, we did not have access to normal dogs for comparative studies. For these initial studies, MRI findings from GRMD dogs were compared to those in carriers. Quantitative studies have been completed in two GRMD dogs (2 months and 8 years old) and two carriers (2 months and 5 years old). We have focused on seven muscles of the proximal pelvic limb (cranial sartorius, quadriceps femoris (vastus heads and rectus femoris separately), biceps femoris, adductor, semimembranosus, semitendinosus, and gracilis). Signal-intense lesions presumably corresponding to fluid accumulation in necrotic lesions were seen on T2-weighted images in the 2-month-old GRMD dog, while the 8-year-old dog had increased fat deposition. The severity of these changes varied among muscles both visually and quantitatively. Signal-intense lesions in the 2-month-old dog were particularly pronounced in the rectus femoris, adductor, biceps femoris, and vastus lateralis and medialis muscles. Fatty changes in the older GRMD dog were more prominent in the semimembranosus and semitendinosus muscles. Volumetric changes did not vary dramatically between GRMD and carrier dogs.
German Shorthaired Pointer Muscular Dystrophy (GSHPMD)--Characterization and Relevance
As discussed above, dystrophin-deficient forms of muscular dystrophy have been characterized in several other canine breeds.4,5 However, only a few of these conditions have been studied at the molecular level. German shorthaired pointers (GSHPMD) have a large DNA deletion, essentially amounting to a "dystrophin knock out."9 Dogs with this condition were first identified in 1998 at the North Carolina State University College of Veterinary Medicine by Drs. Scott Schatzberg and Nick Sharp. Two affected littermates were characterized with generalized muscle atrophy, dilated cardiomyopathy, elevated CK levels, and no dystrophin immunoreactivity. Molecular cytogenetic analysis revealed a major deletion in the p21 region of the X chromosome that encompasses the entire dystrophin gene. The GSHPMD "dystrophin knockout" model will be advantageous for two main reasons. First, scattered dystrophin-positive (revertant) fibers30 that occur due to aberrant splicing in DMD patients, mdx mice, and dystrophic dogs and otherwise confound results of gene therapy studies should be absent. Second, and more importantly, the lack of these revertant fibers provides a "cleaner" background on which to conduct studies of the immunologic aspects of gene therapy. Adeno-associated virus (AAV)-mediated mini- and micro-dystrophin gene therapy has shown promise in the mdx mouse.31 However, use of AAV-mediated gene therapy in murine models of other diseases such as hemophilia has not consistently predicted the degree of immunologic response.32 In keeping with this species dichotomy and in contrast to findings in mdx mice, studies of localized (intramuscular) AAV-mediated dystrophin mini (or micro) gene therapy in GRMD dogs have documented a marked immune response to either components of the transgene (to include dystrophin)16 and/or viral capsid proteins.14 Before AAV-mediated gene therapy can move forward in DMD, the relative roles that the transgene and viral capsid proteins play in this immune response must be defined. The nature of the immunological response to dystrophin is further complicated by the presence of revertant fibers (above). These revertant fibers may render patients and animals receiving dystrophin gene therapy at least somewhat tolerant to the otherwise new (neo) dystrophin antigen.33 In this way, revertant fibers introduce another variable that must be considered in studies to define mechanisms contributing to immunorejection of dystrophin gene products. Such immunological questions can better be addressed in a truly dystrophin-null random-bred animal such as the GSHPMD dog.
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