Muscular Dystrophy Human Disease and Animal Models

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive, progressive muscle-wasting disease affecting approximately 1:3,500 boys. A milder form of the disease, Becker muscular dystrophy (BMD), has a later onset and longer survival rates. Both disorders result from mutations in the dystrophin gene. Approximately 65% of DMD and BMD patients have gross deletions of the dystrophin gene resulting in the absence of full-length molecules of dystrophin. In humans the gene is expressed in all muscles, with the highest dystrophin levels in skeletal and cardiac muscles (Blake et al. 2002). Dystrophin is an essential component of a multiprotein complex, the dystrophin-glycoprotein complex (DGC), which links the cytoskele-ton of the muscle fiber to the extracellular matrix which protects the muscle cell from stress caused by mechanical force. Thus, dystrophic muscle is susceptible to damage induced by muscle contraction and undergoes progressive degeneration and necrosis. Current strategies designed to reduce the severity of muscular dystrophy are based on reducing muscle necrosis, enhancing muscle degeneration, and combating fibrosis using a variety of drugs and nutritional interventions (Nowak and Davies 2004). For most of the patients, curative treatments will involve cell therapy and/or gene replacement, and for a subgroup of patients, corrections of specific mutations are envisioned.

The main goal of gene replacement therapy for DMD is the production of endogenous dystrophin protein in a previously dystrophin-deficient muscle. Immunostaining of muscle sections is essentially negative in DMD patients; whereas in BMD muscle samples, sporadic cell fibers are positive (Hoffman et al. 1992). By western blot, DMD patients have little or no detectable dystrophin, and those with BMD have reduced levels of dystrophin with an abnormal size due to deletions of a specific coding region. Thus, it is possible that expression of the curative dystrophin will be perceived by the immune system as a neoantigen (foreign antigen), which might induce destruction of recently transduced cells and accelerate the loss of muscle fibers in patients who have already lost many of their muscles due to the dystrophic pathological process.

The replacement of the full-length dystrophin gene is challenging due to its large coding sequence (14-kb mRNA transcript). Structure-function analyses of dystrophin variants expressed in the milder form of the disease (BMD) demonstrate that large in-frame deletions in the central rod domain minimally affect the functional capacity of dystrophin (Blake et al. 2002). The development of truncated dystrophins (m-dystrophins) has enabled the design of expression cassettes to be highly effective at preventing muscle degeneration in models of DMD using gene therapy. However, it is possible that the formation of novel-junction sequences within the m-dystrophin protein could potentially be presented as neoantigens with increased risk of immune responses.

Most of the gene therapy trials for genetic diseases are aimed at sustained expression of therapeutic genes by introducing the vector in the target tissue with minimal or no tissue damage. Activation of T cells is dependent on the "danger" or inflammatory signal. Thus, the context of antigen presentation for the treatment of DMD has a significant role in T cell activation, since the health status of target tissue, the nature of the vector, and tissue injury associated with the vector delivery all impose additional risks of unwanted T cell activation. Early studies on the prevalence of the expression of MHC class I in skeletal muscle of DMD revealed a remarkable variation. More recently, muscle biopsies of humans with muscular dystrophy or idiopathic inflammatory myopathies revealed upregulation of MHC class I in the sarcolemma in 11% and >60% of patients, respectively (van der Pas et al. 2004). Interestingly, prolonged immunosuppression was associated with significant reduction in MHC class I detection. Studies demonstrated that MHC class I was upregulated in Golden Retriever muscular dystrophy (GRMD) dogs following IM injection of a viral vector (Yuasa et al. 2007). Therefore, it is possible that several underlying host- and vector-dependent factors may influence the MHC class I status on the skeletal muscle fibers of DMD, and potentially the modulation of cellular immune responses. Further preclinical studies are imperative to address the safety profile of such IS regimens and a careful evaluation of the data has to take into consideration the evolutionary level of the immune system of the model (rodent vs dogs vs nonhuman primates vs humans), the disease-specific model availability (normal animals vs. animals with underlying-specific disease relevant to humans), and lastly, the possibility of testing drugs developed specifically for humans that recognize therapeutic targets in the disease model. Preclinical studies for DMD are favored by the homologues of the disease identified in several animals, including mice and dogs (Banks and Chamberlain 2008; Wang et al. 2009; Wells et al. 2002). The mdx mouse is the most widely used model of DMD, and displays some features of moderate muscle degeneration. Apart from the diaphragm, mechanical function and cardiomyopathy are less affected than in humans, and this model displays only a 20% reduction in lifespan. There are five mdx models characterized by distinct underlying mutations which have resulted in premature stop codons; RNA splicing defect, mutation at splice acceptor site, nonsense mutation, and the creation of a new splice donor site that generates a premature stop codon in RNA transcript (Banks and Chamberlain 2008). Thus, this model is easily accessible and is versatile for the testing of several alternative therapies for DMD. However, the intrinsically inbred nature and the small size of the mouse model preclude the translation of studies on the immune responses and the determination of efficacy of delivery techniques to achieve widespread transduction of the disease target. The X-linked canine muscular dystrophy model resulted in a spontaneous mutation in the dystrophin gene identified in several breeds of dogs; the best characterized is the GRMD. The disease results from a mutation in the 3' consensus splice site of exon 6 of the dystrophin gene, which leads to skipping of exon 7, and consequent disruption in the open reading frame and premature termination of translation with no detectable protein (Cooper et al. 1988). The pathogenesis of GRMD is similar to humans with DMD already evident at the time of birth, such as extensive necrosis of the muscles of the limbs, and severe fibrosis and joint contractures developed by six months of life. Similar to humans, young GRMD models frequently die from cardiac or respiratory failure, although some survive to reach several years of age (Collins and Morgan 2003; Cooper et al. 1988). Since these dogs are immunocompetent animals, and at adult age they have a body mass that is comparable to DMD patients, they represent an ideal model to determine the immu-nogenicity and the ability of scaling up to novel therapeutic platforms for transla-tional studies for humans. However, these dogs are fragile and require frequent and close surveillance that precludes studies on large numbers of animals (Collins and Morgan 2003). Nevertheless, studies on immunosuppression coupled with cell-and/or gene-based strategies in the GRMD dogs are of fundamental importance for translational studies (Liu et al. 2004; Wang et al. 2009).

11.1.3 Preventive Strategies Tissue-Specific Promoters

Systemic delivery of vectors for DMD gene therapy has the risk of spreading to non-targeted tissues and overall toxicity and/or the initiation of a host immune response against tissues expressing the transgene or gene vector (Wells et al. 2002). Transgene expression restricted to the target tissue by using tissue-specific promoters has been extensively utilized to avoid immune responses to the transgene. Thus, the use of muscle-specific promoters is highly desirable in the development of gene therapy vectors for DMD to minimize the harmful effects of ectopic transgene expression (Cordier et al. 2001) and to prevent transgene expression within antigen-presenting cells (APCs), such as dendritic cells (DC) or macrophages. However, the uptake of exogenous protein by APC and presentation in the context of MHC class I, as well as class II, does not require direct transduction of DC by the recombinant vectors. Plasmid DNA appears to generate cytotoxic CD8+ lymphocytes using a cross-priming mechanism (Wells et al. 2002). Therefore the use of muscle-specific promoters would not prevent immune responses if cross-priming is involved, even if the parental vectors do not transduce APCs. Moreover, muscle-specific promoters are typically less active than viral promoters; subsequently, high vector doses will be required. Hence, it seems likely that the balance between vector dose and tissue specificity will define the optimal strategy. Hauschka and colleagues (Salva et al. 2007) developed a series of muscle-specific promoters by modification and optimization of a muscle creatine kinase (MCK)-based construct to fulfill the desirable tissue-specificity and high efficacy. The size of these promoter sequences may overcome the capacity of some promising vectors with restricted packing capacity. Further modifications resulted in new cassettes that drive high-level tissue-specific transgene expression in both cardiac and skeletal muscle with a packaging size constraint for accommodating the m-dystrophin cDNA in AAV vectors (Salva et al. 2007; Wang et al. 2008). Thus, tissue-specific promoters may provide an alternative to avoid immune responses but its effect is rather limited if high vector doses are required. Alternative Therapeutic Transgenes

An alternative approach for gene transfer for DMD is the upregulation of therapeutic transgenes that are endogenously expressed (nonforeign proteins) and can substitute dystrophin function or increase muscle growth. These approaches have the potential to circumvent the risk of immune responses to dystrophin in most DMD patients with null mutations in the dystrophin gene. Pharmacological strategies also are able to further enhance the expression of these proteins as an alternative or coadjuvant therapy for DMD. Two alternative transgenes are discussed below.


Utrophin is the autosomal paralogue of dystrophin that might be able to serve as a surrogate for the dystrophin in DMD muscle fibers (Miura and Jasmin 2006). These proteins present a high degree of sequence identity and functional redundancy. Utrophin is also expressed in the muscle of DMD patients and animal models of the disease, albeit at very low levels (Karpati et al. 1993). Studies in mdx mice demonstrate that pre- or post-natal upregulation of utrophin in dystrophic muscle fibers can restore sarcolemmal expression of the DGC and alleviate the dystrophic pathology (Banks and Chamberlain 2008). An increase in expression levels of utrophin correlates with a delay in wheelchair use in DMD patients, validating this type of therapy in humans (Kleopa et al. 2006). In the GRMD model, expression of a synthetic truncated form of human utrophin was accompanied by local upregulation of the DGC with reduced fibrosis (Cerletti et al. 2003), which further supports this alternative for the treatment of DMD. In this model, the use of cyclosporine was required to sustain transgene expression with no cellular or humoral immune responses to the non-species specific transgene. Unfortunately, utrophin delivery to GRMD dogs using other vector systems is currently lacking.

Inhibitor of Myostatin

Myostatin is an endogenous negative regulator of muscle growth that belongs to the transforming growth factor beta superfamily. Myostatin normally exists in a latent complex in blood circulation, and the myostatin propeptide (MPRO) is one of the major negative regulators of the protein (Patel and Amthor 2005). The function of myostatin is conserved in several species, including mice, dogs and humans. In the absence of myostatin, muscle regeneration has been shown to occur earlier and more robust after tissue damage. Therefore, endogenous enhancement of expression of the MPRO gene offers the opportunity to treat the skeletal muscle disease of DMD without the hurdles imposed by immunological responses against a neotransgene in subjects with a null mutation in the dystrophin gene. The mdx mouse lacking myostatin exhibited increased muscle mass and strength, and decreased fibrosis. Notably, Whippet dogs lacking myostatin are muscular, healthy and have a typically faster racing speed. Therefore, the down-regulation of myosta-tin by MPRO has several potential advantages as a treatment for DMD: (1) the muscle wasting phenotype can be delayed or prevented; (2) muscle fibrosis can be reduced, and (3) as myostatin is a secreted protein, local expression of MPRO to the affected muscle is not required. Finally, ongoing clinical studies of the use of MY0-029, a neutralizing antibody to myostatin, have initially demonstrated safety in limited studies of adults with muscular dystrophy (Wagner et al. 2008). Xiao and colleagues (Qiao et al. 2008a) showed that a myostatin blockade in both normal and dystrophin-deficient mdx mice by systemic delivery of MPRO gene with an AAV serotype 8 (AAV-8) vector, could enhance muscle growth and ameliorate dystrophic lesions. Further studies in normal dogs showed that expression of MPRO resulted in enhanced muscle growth without immune responses as evidenced by the lack of CD4+ and CD8+ T-cell infiltration in the vector-injected limbs. MPRO-based strategies are not effective in the treatment of the underlying cardiac disease.

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