II. Action Plan for the Muscular Dystrophies. Additional strong evidence of the importance of canine DMD models for developmental therapeutics is provided through the Action Plan for the Muscular Dystrophies developed by the Muscular Dystrophy Coordinating Committee Scientific Working Group. Formed in the spring of 2005 by the Department of Health and Human Services, this committee included representatives from academia, federal agencies, and both advocacy and patient groups. The committee was charged with developing recommendations for specific Research Objectives for the muscular dystrophies. They focused on scientific opportunities in the areas of disease mechanisms, diagnosis and screening, therapy, living with muscular dystrophy, and research infrastructure. The resulting Action Plan, approved in December, 2005, emphasized the key role that animal models must play in DMD research. Relevant points are excerpted here.

• The absence of adequate access to appropriate animal models of muscular dystrophy is a potential obstacle to progress in understanding the mechanisms and pathogenesis of these diseases.
• There is no animal model that perfectly mirrors human muscular dystrophy. The mouse models serve an important role in early therapeutic development, but dog models better reflect both disease severity and the immunological problems that are associated with several therapeutic development strategies.
• While dystrophic dog colonies are important for pre-clinical translational projects in therapeutic development, only limited numbers of animals are available from existing sources.
• Available dog models include the Golden Retriever (CXMD; a naturally occurring mutation that has been bred), German Shorthair Pointer (a dystrophin knockout), Welsh Corgis (a naturally occurring mutation that has been bred), and beagle (CXMDJ; artificially inseminated with frozen-thawed spermatozoa derived from an affected golden retriever). Careful comparisons of these models have not yet been done. (Note, see updated information on these models below).
• Muscle functional testing core facilities, for both mouse and dog, are essential to mechanistic and therapeutic development studies in the muscular dystrophies

The Action Plan also included high-priority Research Objectives that could be used by the Muscular Dystrophy Coordinating Committee and the muscular dystrophy scientific community to coordinate research activities to achieve the goal of timely detection, diagnosis, treatment, and prevention of all of the muscular dystrophies. Several objectives spoke specifically to the need to expand access to canine models of DMD.

Therapy of Muscular Dystrophy Section, Research Objective 9: Conduct large animal model testing to determine optimal AAV serotypes for human gene therapy clinical trials.
Therapy of Muscular Dystrophy Section, Research Objective 10: Improve the efficiency of gene therapy delivery in the muscular dystrophies, while minimizing the immune response to both gene product and delivery vehicle.
Therapy of Muscular Dystrophy Section, Research Objective 19: Develop the animal models, assays, and tools necessary for preclinical translational research projects that focus upon rapidly moving the accumulated mechanistic knowledge into clinical practice.
Therapy of Muscular Dystrophy Section, Research Objective 21: Ensure the availability and use of large animal models for the later stages of preclinical development.
Research Infrastructure Needs Section, Research Objective 2: Establish standardized endpoints for preclinical trials in both mouse models, and the dog model, and ensure that facilities are available that enable testing of drugs and other therapeutic approaches.

Other objectives from the Action Plan referred to the critical role that animal models must play in addressing potential treatments.

Therapy of Muscular Dystrophy Section, Research Objective 5: Identify alternative mechanisms of myostatin inhibition and establish their potential as therapeutics through preclinical testing in animal models of various types of muscular dystrophy.
Therapy of Muscular Dystrophy, Research Objective 7: Define, through basic and preclinical translational studies, the therapeutic potential of alternative muscle progenitor cells.
Therapy of Muscular Dystrophy Research Objective 8: Define, through basic and preclinical translational studies, the therapeutic potential of embryonic stem cells.
Therapy of Muscular Dystrophy, Research Objective 12: Evaluate the safety and efficacy of stop codon read-through and exon skipping agents through additional translational studies and clinical trials.

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1. GRMD. 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 6.⁸ 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^9-11 and pathophysiologic features^12-14 of GRMD, together with those of Dr. Barry Cooper’s group at Cornell, done at times in collaboration,^15,16 established this condition as a valid model of DMD. An affected dog studied by our group until 40 months of age² 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 UNC-CH in 2007. See a more detailed discussion of the GRMD model below.
2. GSHPMD. German shorthaired pointers (GSHPMD) have a large DNA deletion, essentially amounting to a “dystrophin knock out”¹⁷ (Figure 1). Dogs with this condition were first identified in 1998 at the North Carolina State University College of Veterinary Medicine by Drs. Scott Schatzberg, Nick Sharp, and Natasha Olby. 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.
   
   Figure 1 - GSHPMD.   A - Affected GSHP littermates. B - Western blot of normal canine and GSHPMD skeletal muscle.  The expected 427 kDa dystrophin band is seen in the normal muscle when labeled with rod (lane A) and C-terminal (lane B) antibodies, while GSHPMD dogs have no bands (lanes D and E).  Both normal and GSHPMD dogs display the 43 kDa b-dystroglycan band (Lanes C and F).  C -  FISH analysis of the dam (Left - A,B) and affected male (Right - C,D) in which Xp- specific plasmid clones mapping to Xp21 were labeled with digoxygenin (green) and two clones proximal and distal to Xp21 were labeled with biotin (red).  In A and B, the normal X (right) of the carrier dam shows signal due to hybridization of all four clones, whereas the deleted X (left) has hybridization from only the flanking clones.  This pattern of hybridization is also seen in the X chromosome of the affected dog (C,D).  From reference 17; color photos courtesy of Dr. Scott Schatzberg.
   
   GSHPMD carriers were subsequently provided to the University of Missouri-Columbia to establish a colony. We have worked extensively to identify and improve molecular methods for detection of carriers to enhance development of our colony. Four dinucleotide repeat markers that exhibit length polymorphism from within the canine DMD gene were identified (Johnson GS, unpublished observations). Flanking PCR primer pairs with fluorescent labels were designed such that they can all be amplified together in a multiplexed microsatellite assay. Although the primers span over 1.4 Mb, affected GSHP dogs have a negative allele at all four marker positions. With the distinct maternal and paternal haplotypes, carrier females can be distinguished from normal females because carrier females have only the paternal haplotype, while normal females have both the paternal and maternal haplotype. With the establishment of a solid foundation colony, we have begun producing sufficient numbers of GSHPMD dogs to begi n use in independent and parallel studies of the GRMD model. This colony has now been moved to UNC-CH and is the only source of affected dogs.

The GSHPMD “dystrophin knockout” model will be advantageous for two main reasons. First, scattered dystrophin-positive (revertant) fibers¹⁸ 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.¹⁹ 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.²⁰ In keeping with this species dichotomy and in contrast to findings in mdx mice, studies of intramuscular AAV-mediated dystrophin gene therapy in GRMD dogs have documented a marked immune response to either components of the transgene (to include dystrophin)²¹ and/or viral capsid proteins.²² 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.²³ 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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IV. Biomarkers for Assessment of the Natural History and Response to Treatment in DMD. The study of muscle diseases has evolved from a classical period in which a diagnosis was based on clinical and pathologic features, to a modern period when muscle biopsies were further characterized through histo- and cytochemical techniques, to the current era of molecular diagnosis.^24,25 With the advent of techniques such as multiplex polymerase chain reaction and Southern blotting, DMD can be diagnosed non-invasively without the need for muscle biopsy.²⁶ As a result, baseline and follow-up pathologic data are not typically available to assess disease progression or response to therapy. Other surrogate biomarkers must be utilized to ensure that results of treatment trials are interpreted appropriately. Most DMD natural history studies have included measurements of muscle strength, joint contractures, and timed function tests. Results from these tests are used to track disease progression and offer insight on clinical milestones, such as the loss of ambulation and the need for ventilatory support. Both muscle weakness and joint contractures contribute to postural instability and ultimate loss of ambulation.²⁷ Contracture and muscle strength scores generally correlate and deteriorate synchronously over time.²⁸

Magnetic resonance imaging (MRI) has been used increasingly to provide meaningful data on the natural history and response to therapy of a number of diseases, including DMD.^29-41 Principal MRI changes in DMD include an increase in T2 and decrease in T1 relaxation times due to accumulation of fat in affected muscles and an associated increase in whole body fat and decrease in muscle mass. Objective grading systems allow data to be compared over the disease course.^31,32 Results from these grading systems have correlated with those of clinical functional tests. One serial study suggested that MRI is more sensitive than functional tests in predicting disease progression.³² MRI has also been used to monitor DMD disease progression in treatment trials.^42,43

1. 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 contractures (Figure 2)⁴⁴ and demonstrate weakness of individual⁴⁵ and grouped⁴⁶ muscles. As with mdx mice, weakness is exacerbated by eccentric contractions.⁴⁷ Tibiotarsal joint angles and torque force measurements correlate.⁴⁸ Dogs with weak extension and strong flexion force values tend to have tibiotarsal joint flexor contractures. Values in heterozygous males and homozygous females do not differ statistically, so both genders can be assessed in parallel using these tests in treatment trials.⁴⁹ By comparing serial measurements, one can document improvement or delayed progression of disease.

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   Figure 2. GRMD dog at 3 months (top photo) and 6 months (bottom photo, same dog) of age. By 6 months, pelvic limbs of affected pups appear stiff, with flexor contractures of the tibiotarsal joints (hocks), hyperextension of the carpi, and abduction of the paws. Analogous findings in DMD boys have been described. From reference 44 We have focused principally on measurement of torque force generated by the tibiotarsal joint.46 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. 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 (Figure 3). 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).46 Importantly, by comparing serial measurements from treated and untreated groups, one can document improvement or delayed progression of disease. Treatment may be administered either locally (gene or cell therapy) or systemically (pharmacologic management). Force measurements are especially valuable in assessing efficacy of treatments localized to a single muscle or group of muscles. A wider array of phenotypic parameters, such as body weight and serum creatine kinase (CK) concentration, can be used to monitor systemic treatments.

   
   Figure 3.  Joint Force. Tibiotarsal force measurements from normal (open bars) and GRMD (closed bars) at 3, 4.5, 6, and 12 months of age.  GRMD force is less than normal at all ages.  Differential involvement is seen according to age, with flexion affected early and extension late. From Reference 46.

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   Functional outcome values have varied considerably, even among dogs within the same litter, suggesting that modifier genes significantly influence the phenotype.^44-46 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.⁵⁰ Extension forces in GRMD dogs treated daily with 1 and 2 mg/kg prednisone measured 2.349 ± 0.92 and 3.486 ± 0.67 N/kg, respectively, compared to 1.927 ± 0.63 N/kg in untreated GRMD controls (p < 0.05 for 2 mg/kg group); GRMD flexion forces measured 0.435 ± 0.13 and 0.303 ± 0.08 N/kg, respectively, compared to 0.527 ± 0.01 N/kg in untreated GRMD controls (p < 0.05 for both groups). 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.⁵¹ Treatment with prednisone could have attenuated this early necrosis and functional hypertrophy. This finding is compatible with the increase in tibiotarsal joint flexor force seen over time (Figure 3) and suggests that increased flexor force could actually predict a deleterious outcome.
   
   
2. Golden Retriever Muscular Dystrophy (GRMD) Model – Pathologic Studies: We have conducted histopathologic studies to determine both the natural history^5,51,52 and response to treatment of GRMD dogs.⁵⁰ One natural history study evaluated the degree of gross muscle atrophy and hypertrophy of pelvic limb muscles.⁵¹ 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, 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 play a role analogous to iliotibial band tightness in DMD.⁵³ These natural history data must be considered carefully in interpreting phenotypic effects of myostatin inhibition.
   
   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 (Figure 4).⁵⁰ 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.

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   Figure 4.  Prednisone Pathologic Effects.  Vastus lateralis muscle biopsies taken from 6-month-old dogs with GRMD showing changes in response to chronic 2 mg/kg daily oral prednisone treatment. Compared to untreated GRMD controls (A, C, and E), cross sections from prednisone-treated GRMD dogs show differences in myofiber calcification (B & F) and fetal myosin expression (D).  A & B, H&E stain; C & D, antibody to fetal myosin; E & F, alizarin red stain. Bar = 100 µm). From reference.50 C. 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.54 Regression of MRI lesions has been seen in both mdx mice54 and other murine models55,56 of muscular dystrophy after gene therapy. In one recent study, the thoracic limbs of 2-month-old GRMD and normal dogs were scanned at 4 T.57 Standard T2- and T-1-weighted images and fat-saturated T1-weighted images pre- and post-gadolinium chelate injection were acquired and kinetics of muscle enhancement were studied for 2 hours. GRMD dogs had abnormally high T2-weighted/T1-weighted signal ratio, T2-weighted image heterogeneity, and maximal signal enhancement post-gadolinium.

   
   

   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 (Figure 5; Table 2). With the recent relocation of our colony, we did not have access to normal dogs for comparative studies. For these preliminary 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 [rectus femoris and vastus heads evaluated separately], biceps femoris, adductor, gracilis, semimembranosus, and semitendinosus). 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 (Figure 5) and quantitatively (Table 2). 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 (Figure 5D). Fatty changes in the older GRMD dog were more prominent in the semimembranosus and semitendinosus muscles (Figure 5J). Volumetric changes did not vary dramatically between GRMD and carrier dogs. Additional studies completed through the NCDMD should define the role of MRI as a biomarker for assessing natural history and treatment efficacy.
   
   Figure 5.  The four panels from left to right are MRI images from a 2-month-old GRMD carrier (A,C,E,G) and affected littermate (B,D,F,H) and 5-year-old GRMD carrier (I,K,M) and affected dog (J,L,N).  A, B, I, and J are TSE-fat percentage and C, D, K, and L are TSE-fat saturation.  Transverse sections of muscle have been segmented in E, F, M, and N for region-of-interest measurements (Table 2) and are shown in three dimensions in G and H (2-month-old dogs only).  Note, particularly, the signal-intense lesions in several muscles in D and J, representing fluid accumulation, acutely, and fatty change, chronically, respectively.  Signal-intense lesions seen in J reverse with fat saturation in L.

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   Wolff and his colleagues have conducted MRI and magnetic resonance angiography (MRA) studies in monkeys after high-pressure intravascular naked DNA plasmid injection.⁵⁸ Intramuscular fluid signal intensity correlated with DNA expression of a luciferase reporter gene. Vascular effects were limited and reversible. Working in collaboration with Dr. Xiao Xiao, we have administered AAV-dystrophin minigene constructs by both intramuscular injection and regional limb perfusion using different constructs, perfusion rates, and volumes.⁵⁹ Strong, near-uniform expression of canine mini-dystrophin gene and other components of the glycoprotein complex has been seen in some GRMD dogs. Less pronounced expression was noted in the other dogs. Viral batch appears to be a key variable in myofiber transduction. Other variables, such as perfusion pressure and volume and the degree of immunologic reaction, must be further clarified. Building on the natural history studies discussed above, we have evaluated MRI in one 11-week-old GRMD dog following regional limb delivery of an AAV-dystrophin minigene construct (Figure 6). A tourniquet was placed in the inguinal area and the saphenous vein was catheterized. The AAV construct was suspended in saline and infused (20 ml/kg, 1 ml/sec, total dwell time of 10 minutes). There was reversible gross limb enlargement and tightening during the perfusion. Using a T2-weighted, TSE protocol, increased signal intensity was seen within both the intermuscular interstitium and muscle bellies. This signal did not reverse with fat saturation and was judged to be fluid. Muscles proximal and distal to the stifle (knee) were evaluated. Resolution was better in the proximal muscles (Figure 6; also see Figure 5 above). The biceps femoris muscle of both the perfused and non-perfused limbs was segmented for volumetric and statistical analysis. The volume and signal intensity were higher in the perfused (31,941.5 mm³ and 111.375, respectively) versus non-perfused limb (18,116 mm³ and 80.6553, respectively). Distal to the stifle, muscles of the cranial and caudal tibial compartments also had increased signal intensity, with the long digital extensor showing the most prominent changes. These results provide further evidence for the value of MRI in defining optimal parameters of regional limb perfusion.

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   Figure 6.  MRI 50 Minutes after Limb Perfusion of an 11-week-old GRMD Dog. T2-weighted TSE MRI images without (A) and with (B,C) fat saturation.  Composite images have been created to orient the left and right limbs in the same plane.  As a result, some proximal structures such as the colon are duplicated. Longitudinal (top) and transverse (bottom) sections proximal to the stifle (knee) are seen in A and B.  In each image, the perfused limb is on the left. Increased signal intensity is consistently seen in the perfused limb.  Intensity could be either fat or fluid in A but is due to fluid alone in the fat saturation images in B and C.  Intensity is greatest in the distal third of the femur and is particularly prominent in the biceps femoris muscle which is highlighted in C.  In the fat saturation images in C, the perfused (red) and non-perfused (green) biceps femoris muscles were segmented for quantitative measurements.  The volume and signal intensity of the biceps femoris were 31,941.5 mm³ and 111.375, respectively, in the perfused limb and 18,116 mm³ and 80.6553 in the non-perfused limb (also see discussion in text).
   

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