Janet R. Bogan

Widespread Muscle Expression of an AAV9 Human Mini-dystrophin Vector After Intravenous Injection in Neonatal Dystrophin-deficient Dogs

Duchenne (DMD) and golden retriever (GRMD) muscular dystrophy are caused by genetic mutations in the dystrophin gene and afflict striated muscles. We investigated systemic gene delivery in 4-day-old GRMD dogs given a single intravenous injection of an AAV9 vector (1.5 x 10(14) vector genomes/kg) carrying a human codon-optimized human mini-dystrophin gene under control of the cytomegalovirus (CMV) promoter. One of the three treated dogs was euthanized 9 days later due to pre-existing conditions. Scattered mini-dystrophin-positive myofibers were seen by immunofluorescent (IF) staining in numerous muscles. At the end of the 16-week study, the other two dogs showed generalized muscle expression of mini-dystrophin in ~15% to nearly 100% of myofibers. Western blot and vector DNA quantitative PCR results agreed with the IF data. Delayed growth and pelvic limb muscle atrophy and contractures were seen several weeks after vector delivery. T-2 weighted magnetic resonance imaging (MRI) at 8 weeks showed increased signal intensity compatible with inflammation in several pelvic limb muscles. This marked early inflammatory response raised concerns regarding methodology. Use of the ubiquitous CMV promoter, extra-high vector dose, and marked expression of a human protein in canine muscles may have contributed to the pathologic changes seen in the pelvic limbs.

Canine Models of Duchenne Muscular Dystrophy and Their Use in Therapeutic Strategies

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder in which the loss of dystrophin causes progressive degeneration of skeletal and cardiac muscle. Potential therapies that carry substantial risk, such as gene- and cell-based approaches, must first be tested in animal models, notably the mdx mouse and several dystrophin-deficient breeds of dogs, including golden retriever muscular dystrophy (GRMD). Affected dogs have a more severe phenotype, in keeping with that of DMD, so may better predict disease pathogenesis and treatment efficacy. Various phenotypic tests have been developed to characterize disease progression in the GRMD model. These biomarkers range from measures of strength and joint contractures to magnetic resonance imaging. Some of these tests are routinely used in clinical veterinary practice, while others require specialized equipment and expertise. By comparing serial measurements from treated and untreated groups, one can document improvement or delayed progression of disease. Potential treatments for DMD may be broadly categorized as molecular, cellular, or pharmacologic. The GRMD model has increasingly been used to assess efficacy of a range of these therapies. A number of these studies have provided largely general proof-of-concept for the treatment under study. Others have demonstrated efficacy using the biomarkers discussed. Importantly, just as symptoms in DMD vary among patients, GRMD dogs display remarkable phenotypic variation. Though confounding statistical analysis in preclinical trials, this variation offers insight regarding the role that modifier genes play in disease pathogenesis. By correlating functional and mRNA profiling results, gene targets for therapy development can be identified.

Hydrodynamic Limb Vein Injection of Adeno-Associated Virus Serotype 8 Vector Carrying Canine Myostatin Propeptide Gene into Normal Dogs Enhances Muscle Growth

Inhibition or blockade of myostatin, a negative growth factor of skeletal muscle, enhances muscle growth and therefore is considered a promising strategy for the treatment of muscle-wasting diseases such as the muscular dystrophies. Previously, we showed that myostatin blockade in both normal and dystrophin-deficient mdx mice by systemic delivery of the myostatin propeptide (MPRO) gene by an adeno-associated virus serotype 8 (AAV8) vector could enhance muscle growth and ameliorate dystrophic lesions. Here, we further investigate whether the muscle growth effect of myostatin blockade can be achieved in dogs by gene transfer. First, we cloned the canine MPRO gene, packaged it in the AAV8 vector, and showed robust muscle-enhancing effects after systemic delivery into neonatal mice. This vector was then further tested in two 3-month-old normal dogs (weighing 9.7 and 6.3 kg). The vector was delivered to one limb by hydrodynamic vein injection, and the contralateral limb served as a control. The delivery procedure was safe, without discernible adverse effects. AAV vector DNA and MPRO gene expression were detected by quantitative polymerase chain reaction, Western blotting, and immunofluorescence staining of muscle biopsies. Overexpression of MPRO resulted in enhanced muscle growth without a cytotoxic T lymphocytic immune response, as evidenced by larger myofibers in multiple muscles, increased muscle volume determined by magnetic resonance imaging, and the lack of CD4+ and CD8+ T cell infiltration in the vector-injected limbs. Our preliminary study thus supports further investigation of this therapeutic strategy in the dystrophin-deficient golden retriever muscular dystrophy dog model.

The cranial sartorius muscle undergoes true hypertrophy in dogs with golden retriever muscular dystrophy

The degree of atrophy or hypertrophy of selected pelvic limb muscles was determined in the canine homologue of Duchenne muscular dystrophy. While most muscles were atrophied, the caudal and cranial sartorius were hypertrophied. Cranial sartorius weights were corrected for body weight and endomysial space to determine true muscle weights (g/kg; mean+/-SD) in three golden retriever muscular dystrophy age groups, 4-10 (Group 1; n=15), 13-26 (Group 2; n=4), and 33-66 (Group 3; n=4) months and grouped normal dogs (6-20 months; n=12). Group 1 golden retriever muscular dystrophy weights (2.2063+/-0.6884) were greater than those of normal dogs (1.2699+/-0.1966), indicating that young golden retriever muscular dystrophy dogs have true cranial sartorius muscle hypertrophy. Values of Group 2 (1.3758+/-0.5078) and Group 3 (0.5720+/-0.2423) golden retriever muscular dystrophy dogs were less than those of Group 1, suggesting that the cranial sartorius muscle atrophies over time. Given that cranial sartorius muscle weight correlated with tarsal joint angle in affected dogs (r=-0.817), the hypertrophied muscle may play a role analogous to iliotibial band tightness in Duchenne muscular dystrophy.

Long-term Restoration of Cardiac Dystrophin Expression in Golden Retriever Muscular Dystrophy Following rAAV6-mediated Exon Skipping

Although restoration of dystrophin expression via exon skipping in both cardiac and skeletal muscle has been successfully demonstrated in the mdx mouse, restoration of cardiac dystrophin expression in large animal models of Duchenne muscular dystrophy (DMD) has proven to be a challenge. In large animals, investigators have focused on using intravenous injection of antisense oligonucleotides (AO) to mediate exon skipping. In this study, we sought to optimize restoration of cardiac dystrophin expression in the golden retriever muscular dystrophy (GRMD) model using percutaneous transendocardial delivery of recombinant AAV6 (rAAV6) to deliver a modified U7 small nuclear RNA (snRNA) carrying antisense sequence to target the exon splicing enhancers of exons 6 and 8 and correct the disrupted reading frame. We demonstrate restoration of cardiac dystrophin expression at 13 months confirmed by reverse transcription-PCR (RT-PCR) and immunoblot as well as membrane localization by immunohistochemistry. This was accompanied by improved cardiac function as assessed by cardiac magnetic resonance imaging (MRI). Percutaneous transendocardial delivery of rAAV6 expressing a modified U7 exon skipping construct is a safe, effective method for restoration of dystrophin expression and improvement of cardiac function in the GRMD canine and may be easily translatable to human DMD patients.

Contraction force generated by tarsal joint flexion and extension in dogs with golden retriever muscular dystrophy

Force generated due to torque caused by tarsal joint flexion and extension was measured noninvasively at 3, 4.5, 6, and 12 months of age in dogs with the Duchenne homologue, golden retriever muscular dystrophy (GRMD). Absolute and body-weight-corrected GRMD twitch and tetanic force values were lower than normal at all ages (P<0.01 for most). 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 will 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 suggest that measurement of tarsal tetanic force should be most useful to document therapeutic benefit in GRMD dogs.

Effects of prednisone in canine muscular dystrophy.

Glucocorticoid use may provide short‐term functional improvement in boys with Duchenne muscular dystrophy (DMD). We report functional and histopathologic changes following a 4‐month course of daily oral prednisone in a canine model of DMD, termed golden retriever muscular dystrophy (GRMD). Muscle 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 2mg/kg group); GRMD muscle 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). Although cranial sartorius hypertrophy and tibiotarsal joint angles also tended to improve, myofiber calcification increased and fetal myosin expression decreased following prednisone. Thus, functional data indicate benefit but histopathologic changes following prednisone treatment in GRMD suggest possible deleterious consequences. Muscle Nerve, 2004

The Paradox of Muscle Hypertrophy in Muscular Dystrophy

Mutations in the dystrophin gene cause Duchenne and Becker muscular dystrophy in humans and syndromes in mice, dogs, and cats. Affected humans and dogs have progressive disease that leads primarily to muscle atrophy. Mdx mice progress through an initial phase of muscle hypertrophy followed by atrophy. Cats have persistent muscle hypertrophy. Hypertrophy in humans has been attributed to deposition of fat and connective tissue (pseudohypertrophy). Increased muscle mass (true hypertrophy) has been documented in animal models. Muscle hypertrophy can exaggerate postural instability and joint contractures. Deleterious consequences of muscle hypertrophy should be considered when developing treatments for muscular dystrophy.

A canine minidystrophin is functional and therapeutic in mdx mice

Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disorder lacking a curative treatment. We wish to use the dystrophin-deficient golden retriever muscular dystrophy (GRMD) dog, a canine model of DMD, to investigate adeno-associated virus (AAV) vector-mediated minidystrophin gene therapy. The dog model is useful in evaluating vector dose requirement and immunological consequences owing to its large size and outbred nature. In this study, we have cloned and constructed a canine minidystrophin gene vector. Owing to limited availability of the GRMD dogs, here we first examined the functions and therapeutic effects of the canine minidystrophin in the mdx mouse model. We observed efficient minigene expression without cellular immune responses in mdx mice after AAV1-cMinidys vector intramuscular injection. We also observed restoration of the missing dystrophin-associated protein complex (DPC) onto the sarcolemma, including sarcoglycans and dystrobrevin, and a partial restoration of α-syntrophin and neural nitric oxide synthase (nNOS). In addition, minidystrophin treatment ameliorated dystrophic pathology, such as fibrosis and myofiber central nucleation (CN). CN remained minimal (<2%) after AAV injection in the neonatal mdx mice and was reduced from more than 75% to about 25% after AAV injection in adult mdx mice. Finally, in vivo cell membrane leakage test with Evans blue dye showed that the canine minidystrophin could effectively protect the myofiber plasma membrane integrity. Our results, thus, demonstrated the functionality and therapeutic potential of the canine minidystrophin and paved its way for further testing in the GRMD dog model.

Golden retriever muscular dystrophy (GRMD): Developing and maintaining a colony and physiological functional measurements.

Studies of canine models of Duchenne muscular dystrophy (DMD) provide insight regarding disease pathogenesis and treatment efficacy. To take maximal advantage, colonies of affected dogs must be maintained and outcome parameters developed. In this chapter, we review our 25 years of experience with the golden retriever muscular dystrophy (GRMD) model. Key challenges in colony development (breeding, neonatal death, and the risk of inbreeding) and representative functional measurements (tibiotarsal joint angle and torque force; and eccentric contraction decrement) are discussed.