Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disorder characterized by muscle wasting which is caused by mutations in the
Duchenne muscular dystrophy (DMD) is a lethal X-linked muscle disease characterized by progressive skeletal muscle atrophy and weakness [
Muscular dystrophies are inherited, and progressive muscle disorders are characterized by muscle fiber degeneration and necrosis. Duchenne muscular dystrophy (DMD) is the most severe and common X-linked disorder (1 in 3,500 male births) [
DMD is caused by a mutation in the
The
For the treatment of DMD, various experimental approaches such as gene therapy using viral vectors, exon-skipping therapy, stem cell transplantation, read-through therapy, and pharmacological agents have been extensively developed, but none of these have met with success in the clinic. Among these therapeutic strategies, exon skipping using antisense oligonucleotides (AOs) has been considered to be one of the most promising therapies for the restoration of dystrophin expression at the sarcolemma in dystrophin-deficient muscle. Exon skipping as treatment for DMD is developed based on the frame-shift theory. AOs are chemically-synthesized single-strand nucleic acids about 25 bases in length which are designed to recognize a specific sequence of the mRNA splicing pattern or of the binding protein. These agents can artificially change the translation of the nucleic acid. Among the various types of AOs, the 2′-
Gene therapy using viral vectors has been extensively investigated. Adeno-associated virus (AAV) vectors are the most appropriate tools for viral vector gene therapy because they are nonpathogenic due to a replication defect and have low immunity with an effective ability to infect nondividing cells. This strategy, however, is limited with respect to the size of the inserted exogenous gene. The upper limit of 4.9 kb prevents the full-length
Animal models are needed for elucidation of the pathogenesis and assessment of efficacy and toxicity during the development of therapies. In DMD, various animal models have been identified and utilized. In this review, we introduce and discuss not only the pathological characteristics of mammalian (murine, feline, or canine) models of DMD but also recent advances in therapeutic applications using these models.
Various mouse models with mutations in the mouse
X-linked muscular dystrophy mouse (
Recent advances in therapeutic applications include exon skipping, gene therapy, and cell therapy. In the development of exon skipping therapy, the
In the development of gene therapy, we and other groups have designed a shorter but functional microdystrophin to incorporate into the recombinant AAV2 (rAAV2). The microdystrophin, which has a large deletion in the central rod domain, was constructed on the basis of the sequence of the mutated
Although certain issues such as body size, genetic background, and pathological features should be addressed, the
Katsuki and colleagues successfully generated a new DMD mouse model known as
(a) A male
The targeting of exon 51 for exon skipping is theoretically applicable to the highest percentage (13%) of DMD patients with an out-of frame deletion mutation [
Several different canine models of DMD have been reported thus far [
GRMD is characterized by progressive skeletal muscle weakness and atrophy as well as cardiac involvement. These characteristics are similar to those of DMD. GRMD is caused by a point mutation at the intron 6 splice acceptor site of the canine
The skeletal and cardiac characteristics of GRMD are more similar to those of DMD than of
Preliminary gene therapy experiments on GRMD performed using adenovectors [
In exon-skipping therapy, prolonged maintenance of functional dystrophin in GRMD muscle has additionally been achieved through chimeric RNA/DNA oligonucleotide therapy [
As described in the previous section, GRMD is well characterized and attractive for research on DMD. However, Golden Retrievers are too large to be treated or raised easily and animal trials employing these dogs have substantial animal welfare implications and high costs associated with both maintenance and treatment. To address these issues, we developed a strain of medium-sized dystrophic Beagles by artificial insemination of two females with thawed spermatozoa obtained from a Golden Retriever with GRMD followed by interbreed crossing of the carrier female dogs with Beagle sires [
(a) A male canine with X-linked muscular dystrophy (CXMDJ) at 6 months of age. The atrophy of muscles throughout the body (including temporal muscle) is observed. Kyphosis, abnormal sitting posture, and contracture of hindlimb joints are seen. (b) The pathology (H and E) of tibialis cranialis muscle indicates muscle necrosis with infiltration of inflammatory cells, centrally nucleated fibers, and increased interstitial connective tissues.
In the cardiac manifestation, GRMD as well as DMD have particular characteristics such as distinct deep electrocardiogram Q-waves and fibrosis of the left ventricular wall [
Recently, in conjunction with our collaborators at the Children’s National Medical Center in the USA, we developed a multiexon-skipping technique for targeting exons 6 and 8 to convert an out-of-frame mutation into an in-frame mutation using PMOs. Systemic multiexon-skipping treatment in CXMDJ was found to restore dystrophin in the whole-body skeletal muscle (with the exception of heart). Furthermore, skeletal muscle function was notably improved without any adverse effects [
To examine therapeutic effects and safety in a larger animal model, we have examined the efficiency of rAAV2 infection of canine myotubes and expression of the
Thus, the clinical phenotype of CXMDJ has been well characterized as an appropriate animal model and the similarity of pathology of DMD is regarded as the most appropriate model for DMD in clinical trials. Other therapeutic approaches will be evaluated in the dog model with a view to establishing feasible protocols.
Very recently, it was reported that another dystrophic dog, the Cavalier King Charles Spaniel with dystrophin-deficient muscular dystrophy (CKCS-MD) has a severe phenotype. This canine model has a missense mutation in the 5′ donor splice site of exon 50 resulting in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein [
The therapeutic strategy of exon skipping in the GRMD or CXMDJ models provides a significant advantage in clinical trials, but also has the unfavorable characteristic that the disease-causing mutation does not include the region of the
Feline muscular dystrophy with dystrophin deficiency has been identified [
Murine models will continue to provide important findings for the basic study of pathogenesis and development of therapies, but the clinical phenotype is mild and this is a weak point. On the other hand, canine models have severe skeletal and cardiac defects resembling DMD and the body size and genetic background of canines are more similar to human beings than the murine models. We are, therefore, convinced that canine models will be more useful for future contributions to preclinical study of newly developed therapies.
These studies were supported by Health Sciences Research Grants for Research on Psychiatric and Neurological Diseases and Mental Health (nos. H12-kokoro-025, H15-kokoro-021, and H18-kokoro-019), the Human Genome and Gene Therapy (nos. H13-genome-001, and H16-genome-003), and the Health and Labor Sciences Research Grants for Translation Research (no. H19-translational research-003) from the Ministry of Health, Labor and Welfare of Japan, and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (no. 21300157 to A. Nakamura).