Human Duchenne Muscular Dystrophy Patients Research Articles

Modeling Duchenne Muscular Dystrophy Cardiomyopathy with Patients' Induced Pluripotent Stem-Cell-Derived Cardiomyocytes.

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease caused by mutations in the dystrophin gene, resulting in death by the end of the third decade of life at the latest. A key aspect of the DMD clinical phenotype is dilated cardiomyopathy, affecting virtually all patients by the end of the second decade of life. Furthermore, despite respiratory complications still being the leading cause of death, with advancements in medical care in recent years, cardiac involvement has become an increasing cause of mortality. Over the years, extensive research has been conducted using different DMD animal models, including the mdx mouse. While these models present certain important similarities to human DMD patients, they also have some differences which pose a challenge to researchers. The development of somatic cell reprograming technology has enabled generation of human induced pluripotent stem cells (hiPSCs) which can be differentiated into different cell types. This technology provides a potentially endless pool of human cells for research. Furthermore, hiPSCs can be generated from patients, thus providing patient-specific cells and enabling research tailored to different mutations. DMD cardiac involvement has been shown in animal models to include changes in gene expression of different proteins, abnormal cellular Ca2+ handling, and other aberrations. To gain a better understanding of the disease mechanisms, it is imperative to validate these findings in human cells. Furthermore, with the recent advancements in gene-editing technology, hiPSCs provide a valuable platform for research and development of new therapies including the possibility of regenerative medicine. In this article, we review the DMD cardiac-related research performed so far using human hiPSCs-derived cardiomyocytes (hiPSC-CMs) carrying DMD mutations.

Biomarkers in Duchenne Muscular Dystrophy.

This review highlights the key studies investigating various types of biomarkers in Duchenne muscular dystrophy (DMD). Several proteomic and metabolomic studies have been undertaken in both human DMD patients and animal models of DMD that have identified potential biomarkers in DMD. Although there have been a number of proteomic and metabolomic studies that have identified various potential biomarkers in DMD, more definitive studies still need to be undertaken in DMD patients to firmly correlate these biomarkers with diagnosis, disease progression, and monitoring the effects of novel treatment strategies being developed.

CRISPR/Cas9 editing of directly reprogrammed myogenic progenitors restores dystrophin expression in a mouse model of muscular dystrophy.

SummaryGenetic mutations in dystrophin manifest in Duchenne muscular dystrophy (DMD), the most commonly inherited muscle disease. Here, we report on reprogramming of fibroblasts from two DMD mouse models into induced myogenic progenitor cells (iMPCs) by MyoD overexpression in concert with small molecule treatment. DMD iMPCs proliferate extensively, while expressing myogenic stem cell markers including Pax7 and Myf5. Additionally, DMD iMPCs readily give rise to multinucleated myofibers that express mature skeletal muscle markers; however, they lack DYSTROPHIN expression. Utilizing an exon skipping-based approach with CRISPR/Cas9, we report on genetic correction of the dystrophin mutation in DMD iMPCs and restoration of protein expression in vitro. Furthermore, engraftment of corrected DMD iMPCs into the muscles of dystrophic mice restored DYSTROPHIN expression and contributed to the muscle stem cell reservoir. Collectively, our findings report on a novel in vitro cellular model for DMD and utilize it in conjunction with gene editing to restore DYSTROPHIN expression in vivo.

The BDNF rs6265 Polymorphism is a Modifier of Cardiomyocyte Contractility and Dilated Cardiomyopathy.

Brain-derived neurotrophic factor (BDNF) is a neuronal growth and survival factor that harbors cardioprotective qualities that may attenuate dilated cardiomyopathy. In ~30% of the population, BDNF has a common, nonsynonymous single nucleotide polymorphism rs6265 (Val66Met), which might be correlated with increased risk of cardiovascular events. We previously showed that BDNF correlates with better cardiac function in Duchenne muscular dystrophy (DMD) patients. However, the effect of the Val66Met polymorphism on cardiac function has not been determined. The goal of the current study was to determine the effects of rs6265 on BDNF biomarker suitability and DMD cardiac functions more generally. We assessed cardiovascular and skeletal muscle function in human DMD patients segregated by polymorphic allele. We also compared echocardiographic, electrophysiologic, and cardiomyocyte contractility in C57/BL-6 wild-type mice with rs6265 polymorphism and in mdx/mTR (mDMD) mouse model of DMD. In human DMD patients, plasma BDNF levels had a positive correlation with left ventricular function, opposite to that seen in rs6265 carriers. There was also a substantial decrease in skeletal muscle function in carriers compared to the Val homozygotes. Surprisingly, the opposite was true when cardiac function of DMD carriers and non-carriers were compared. On the other hand, Val66Met wild-type mice had only subtle functional differences at baseline but significantly decreased cardiomyocyte contractility. Our results indicate that the Val66Met polymorphism alters myocyte contractility, conferring worse skeletal muscle function but better cardiac function in DMD patients. Moreover, these results suggest a mechanism for the relative preservation of cardiac tissues compared to skeletal muscle in DMD patients and underscores the complexity of BDNF signaling in response to mechanical workload.

The Role of Innate Immunity in Dystrophic Diaphragm Pathology

The pathophysiology of Duchenne muscular dystrophy (DMD) is complex and incompletely understood. However it is clear that the primary defect, lack of dystrophin, is necessary but not sufficient on its own to fully account for the onset of muscle fiber destruction, leading to eventual replacement by fibrotic tissue. Dystrophin restoration strategies are currently under clinical development, but the only treatment clearly shown to slow disease progression thus far in human DMD patients is the use of anti‐inflammatory corticosteroids. This presentation will focus on the role of the innate immune system in promoting the dysregulated inflammatory and aberrant muscle repair responses found in DMD. Using the mdx mouse diaphragm as a preclinical model due to its resemblance to the human DMD phenotype, the importance of monocyte‐derived macrophages in driving early disease progression will be outlined. This is exemplified by the therapeutic benefits (improved force generation and reduced fibrosis) noted when monocyte/macrophage recruitment to the dystrophic diaphragm is prevented through genetic or pharmacologic inhibition of the chemokine receptor CCR2. In addition, preventing activation of Toll‐like receptors (TLRs) is similarly beneficial and suggests significant involvement of damage‐associated molecular patterns (DAMPs) in DMD pathogenesis. Data will also be presented indicating that epigenetic alterations occur at the level of macrophage precursors in the bone marrow, which are likely stimulated by TLR4 recognition of DAMPs, and that subsequently play a role in dictating monocyte/macrophage phenotypic characteristics following their entry into the diseased muscles. Although the ideal treatment for DMD would be restoration of dystrophin to all muscles of the body including the diaphragm, significant hurdles remain for this ultimate therapy. In the meanwhile, there are serious drawbacks to the current practice of treating DMD children with corticosteroids, which have major side effects in DMD such as obesity, diabetes and osteoporosis. Future development of a more targeted therapeutic approach based on innate immune system modulation could represent an important advance in the field.Support or Funding InformationCanadian Institutes of Health Research

Blockade of IGF2R improves muscle regeneration and ameliorates Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a debilitating fatal X‐linked muscle disorder. Recent findings indicate that IGFs play a central role in skeletal muscle regeneration and development. Among IGFs, insulinlike growth factor 2 (IGF2) is a key regulator of cell growth, survival, migration and differentiation. The type 2 IGF receptor (IGF2R) modulates circulating and tissue levels of IGF2 by targeting it to lysosomes for degradation. We found that IGF2R and the store‐operated Ca2+ channel CD20 share a common hydrophobic binding motif that stabilizes their association. Silencing CD20 decreased myoblast differentiation, whereas blockade of IGF2R increased proliferation and differentiation in myoblasts via the calmodulin/calcineurin/NFAT pathway. Remarkably, anti‐IGF2R induced CD20 phosphorylation, leading to the activation of sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) and removal of intracellular Ca2+. Interestingly, we found that IGF2R expression was increased in dystrophic skeletal muscle of human DMD patients and mdx mice. Blockade of IGF2R by neutralizing antibodies stimulated muscle regeneration, induced force recovery and normalized capillary architecture in dystrophic mdx mice representing an encouraging starting point for the development of new biological therapies for DMD.

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

730. Permanent Correction of Diverse Muscular Dystrophy Mutations in Human Cardiomyocytes by Myoediting

Dilated cardiomyopathy (DCM) is one of the most common lethal features of Duchenne muscular dystrophy (DMD), caused by diverse mutations in the X-linked dystrophin gene (DMD). Without dystrophin, a large cytoskeletal protein, muscles degenerate, causing myopathy. Although several gene therapies have been tested, there is no curative treatment so far. In 2014, we first used CRISPR/Cas9-mediated genome editing (termed Myoediting) to correct the mutation in the germ line of mdx mice, the mouse model of DMD [Long et al. Science 345:1184-8]. Recently, we applied Myoediting to postnatal muscle tissues in mice by delivering gene editing components via a harmless adeno-associated virus. Cardiac and skeletal muscle showed progressive rescue of dystrophin protein [Long et al. Science doi: 10.1126/science.aad5725]. These studies paved the way for novel genome editing-based therapeutics in DMD. We have now advanced Myoediting to cells from human DMD patients by engineering the permanent skipping of mutant or out-of-frame exons in the genome of DMD patient-derived induced pluripotent stem cells (iPSCs). We have optimized Myoediting of DMD mutations using pools of sgRNAs to target the top 12 hot spot mutant exons. We targeted the conserved RNA splicing acceptor/donor sites of each exon. NHEJ-mediated indels efficiently abolished the splicing sites and skipped the mutated or out-of-frame exons. Based on the known DMD mutations, we established a publicly available online resource (Duchenne Skipper Database) for selecting the optimal target DMD sequences for Myoediting, which will rescue dystrophin function in 60-80% of DMD patients. We performed Myoediting on representative iPSC-derived cardiomyocytes from multiple patients with point, deletion or duplication mutations and efficiently restored dystrophin protein expression in cardiomyocytes. Rescued DMD cardiomyocyte shows enhanced function. Opportunities and obstacles in the path toward efficient and permanent elimination of the genetic cause of DMD and other DCM will be discussed.

617. Histologic Assessment of Geriatric Esophagus Revels Therapeutic Targets in Wild-Type and Dystrophic Mouse Models

Gastroesophageal disorders increase as a process of aging and are exacerbated in degenerative neuromuscular disease such as Duchenne muscular dystrophy (DMD). DMD affects ≈1/5000 male births and is a catastrophic and ultimately fatal muscle wasting disease resulting from a mutation in the gene encoding the integral muscle protein dystrophin. Advancements in the treatment for DMD patients has increased patient lifespan and underscores a need to assess the impact of the disease in a range of organ systems, including the esophagus, likely to present new therapeutic challenges over time. Severity of esophageal pathology is varied to include reflux disease, achalasia, and megaesophagus. Defined as a pathologic enlargement of the esophagus, megaesophagus can result in a failure to complete peristalsis, impaction, severe weight loss and increased morbidity. Although poorly defined in human DMD patients, megaesophagus has been documented in canine and mouse models of DMD, however, a detailed cellular and mechanistic understanding remains unclear. We have performed a comprehensive morphologic and histologic examination of wild-type (C57/BL6) and dystrophic (mdx4cv) mouse esophagus to determine onset, pathology and changes in cellular composition accompanying megaesophagus onset. Age-associated presentation of megaesophagus was highly penetrant in both mouse models, with a four-fold increase in lumen dilation and coincident rate of impaction seen in aged dystrophic mice. In comparison, skeletal muscle area increased in both models with age, however, wild-type mice presented significantly greater hypertrophy than that observed in dystrophic mice. Interestingly, strain specific differences were not identified in esophagus mucosa strata with both strains presenting significant and similar increases in mucosa area and aging-dependent changes in mucosa composition. Further, a pronounced, aging-specific degradation of smooth musculature was observed in both mouse strains independent of dystrophin expression within wild-type mice. We demonstrate that esophageal smooth muscle is targetable via AAV-mediated gene therapy with future efforts aimed at correcting degeneration present within geriatric wild-type and dystrophic mouse models.

Alterations in Notch signalling in skeletal muscles from mdx and dko dystrophic mice and patients with Duchenne muscular dystrophy

New Findings What is the central question of this study? The Notch signalling pathway plays an important role in muscle regeneration, and activation of the pathway has been shown to enhance muscle regeneration in aged mice. It is unknown whether Notch activation will have a similarly beneficial effect on muscle regeneration in the context of Duchenne muscular dystrophy (DMD). What is the main finding and its importance? Although expression of Notch signalling components is altered in both mouse models of DMD and in human DMD patients, activation of the Notch signalling pathway does not confer any functional benefit on muscles from dystrophic mice, suggesting that other signalling pathways may be more fruitful targets for manipulation in treating DMD. Abstract In Duchenne muscular dystrophy (DMD), muscle damage and impaired regeneration lead to progressive muscle wasting, weakness and premature death. The Notch signalling pathway represents a central regulator of gene expression and is critical for cellular proliferation, differentiation and apoptotic signalling during all stages of embryonic muscle development. Notch activation improves muscle regeneration in aged mice, but its potential to restore regeneration and function in muscular dystrophy is unknown. We performed a comprehensive examination of several genes involved in Notch signalling in muscles from dystrophin-deficient mdx and dko (utrophin- and dystrophin-null) mice and DMD patients. A reduction of Notch1 and Hes1 mRNA in tibialis anterior muscles of dko mice and quadriceps muscles of DMD patients and a reduction of Hes1 mRNA in the diaphragm of the mdx mice were observed, with other targets being inconsistent across species. Activation and inhibition of Notch signalling, followed by measures of muscle regeneration and function, were performed in the mouse models of DMD. Notch activation had no effect on functional regeneration in C57BL/10, mdx or dko mice. Notch inhibition significantly depressed the frequency-force relationship in regenerating muscles of C57BL/10 and mdx mice after injury, indicating reduced force at each stimulation frequency, but enhanced the frequency-force relationship in muscles from dko mice. We conclude that while Notch inhibition produces slight functional defects in dystrophic muscle, Notch activation does not significantly improve muscle regeneration in murine models of muscular dystrophy. Furthermore, the inconsistent expression of Notch targets between murine models and DMD patients suggests caution when making interspecies comparisons.

IGF signaling in muscle degenerative diseases

IGF signaling in muscle degenerative diseases

Dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle

Duchenne muscular dystrophy (DMD) is caused by mutations in the X-linked dystrophin (DMD) gene. The absence of dystrophin protein leads to progressive muscle weakness and wasting, disability and death. To establish a tailored large animal model of DMD, we deleted DMD exon 52 in male pig cells by gene targeting and generated offspring by nuclear transfer. DMD pigs exhibit absence of dystrophin in skeletal muscles, increased serum creatine kinase levels, progressive dystrophic changes of skeletal muscles, impaired mobility, muscle weakness and a maximum life span of 3 months due to respiratory impairment. Unlike human DMD patients, some DMD pigs die shortly after birth. To address the accelerated development of muscular dystrophy in DMD pigs when compared with human patients, we performed a genome-wide transcriptome study of biceps femoris muscle specimens from 2-day-old and 3-month-old DMD and age-matched wild-type pigs. The transcriptome changes in 3-month-old DMD pigs were in good concordance with gene expression profiles in human DMD, reflecting the processes of degeneration, regeneration, inflammation, fibrosis and impaired metabolic activity. In contrast, the transcriptome profile of 2-day-old DMD pigs showed similarities with transcriptome changes induced by acute exercise muscle injury. Our studies provide new insights into early changes associated with dystrophin deficiency in a clinically severe animal model of DMD.

The physiological response of protease inhibition in dystrophic muscle

Duchenne muscular dystrophy (DMD) is caused by the production of a non-functional dystrophin gene product and a failure to accumulate functional dystrophin protein in muscle cells. This leads to membrane instability, loss of Ca(2+) homoeostasis and widespread cellular injury. Associated with these changes are increased protease activities in a variety of proteolytic systems. As such, there have been numerous investigations directed towards determining the therapeutic potential of protease inhibition. In this review, evidence from genetic and/or pharmacological inhibition of proteases as a treatment strategy for DMD is systematically evaluated. Specifically, we review the potential roles of calpain, proteasome, caspase, matrix metalloproteinase and serine protease inhibition as therapeutic approaches for DMD. We conclude that despite early results to the contrary, inhibition of calpain proteases is unlikely to be successful. Conversely, evidence suggests that inhibition of proteasome, matrix metalloproteinases and serine proteases does appear to decrease disease severity. An important caveat to these conclusions, however, is that the fundamental cause of DMD, dystrophin deficiency, is not corrected by this strategy. Hence, this should not be viewed as a cure, but rather, protease inhibitors should be considered for inclusion in a therapeutic cocktail. Physiological Relevance. Selective modulation of protease activity has the potential to profoundly change intracellular physiology resulting in a possible treatment for DMD. However, alteration of protease activities could also lead to worsening of disease progression by promoting the accumulation of substrates in the cell. The balance of benefit and potential damage caused by protease inhibition in human DMD patients is largely unexplored.

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.

Bowman-Birk inhibitor attenuates dystrophic pathology in mdx mice

Bowman-Birk inhibitor concentrate (BBIC), a serine protease inhibitor, has been shown to diminish disuse atrophy of skeletal muscle. Duchenne muscular dystrophy (DMD) results from a loss of dystrophin protein and involves an ongoing inflammatory response, with matrix remodeling and activation of transforming growth factor (TGF)-β(1) leading to tissue fibrosis. Inflammatory-mediated increases in extracellular protease activity may drive much of this pathological tissue remodeling. Hence, we evaluated the ability of BBIC, an extracellular serine protease inhibitor, to impact pathology in the mouse model of DMD (mdx mouse). Mdx mice fed 1% BBIC in their diet had increased skeletal muscle mass and tetanic force and improved muscle integrity (less Evans blue dye uptake). Importantly, mdx mice treated with BBIC were less susceptible to contraction-induced injury. Changes consistent with decreased degeneration/regeneration, as well as reduced TGF-β(1) and fibrosis, were observed in the BBIC-treated mdx mice. While Akt signaling was unchanged, myostatin activitation and Smad signaling were reduced. Given that BBIC treatment increases mass and strength, while decreasing fibrosis in skeletal muscles of the mdx mouse, it should be evaluated as a possible therapeutic to slow the progression of disease in human DMD patients.

Transduction of Full-length Dystrophin to Multiple Skeletal Muscles Improves Motor Performance and Life Span in Utrophin/Dystrophin Double Knockout Mice

Duchenne muscular dystrophy (DMD) is a fatal, progressive, muscle-wasting disease caused by defects in the dystrophin. No viral vector except the helper-dependent adenovirus vector (HDAdv) can package 14-kilobase (kb) full-length dystrophin complementary DNA (cDNA), and HDAdv is considerably safer than old-generation adenovirus vectors because of the large-size deletion in its genome. We have generated HDAdv that carries myc-tagged murine full-length dystrophin cDNA (HDAdv-myc-mFLdys). We injected it into multiple proximal muscles of 7-day-old utrophin/dystrophin double knockout mice (dko mice) (which typically show symptoms quite similar to human DMD) because the proximal muscles are affected in DMD patients. Eight weeks after the injections, the transduced dystrophin was widely expressed, and we found a significant reduction in centrally nucleated myofibers and the restoration of the dystrophin-associated proteins, beta-dystroglycan (beta-DG) and alpha-sarcoglycan (alpha-SG), as well as neuronal nitric oxide synthase (nNOS). The injected dko mice also showed an increase in body weight, an improvement in motor performance, and a prolongation of life span. Using HDAdv, we could treat DMD model mice even by transferring the therapeutic gene into multiple skeletal muscles. Our results suggest that multiple intramuscular administrations of HDAdv carrying full-length dystrophin cDNA may reduce symptoms and compensate for lost functions in DMD patients.

Immunity to Adeno-Associated Virus-Mediated Gene Transfer in a Random-Bred Canine Model of Duchenne Muscular Dystrophy

Recombinant adeno-associated virus (rAAV)-mediated gene transfer has shown promise for treating diseases in various animal models including the mdx mouse model of Duchenne muscular dystrophy (DMD). In many cases, however, preclinical studies in inbred mice have not successfully predicted human clinical responses. To assess the potential clinical utility of treating human DMD patients by AAV-mediated gene delivery, we performed a series of direct intramuscular injections in random-bred wild-type dogs. AAV serotypes 2 and 6 carrying different promoter-transgene cassettes were produced as previously described for murine studies and administered intramuscularly. The injection sites were biopsied at various time points and analyzed for transgene expression and immunohistochemical analysis. In contrast to the generally nonimmunogenic nature of these vectors in murine studies, both AAV2 and AAV6 vectors elicited robust cellular immune responses regardless of the transgene expressed, the cellular specificity of the promoter, and the muscle type injected. Viral purification by various methods did not diminish T cell-mediated infiltration. Our data indicate that AAV2 and AAV6 capsid proteins can elicit primary cellular immune responses when injected into the skeletal muscle of random-bred dogs, and suggest the possibility of cellular immunity to AAV vectors in humans.

792. Canine Mini-Dystrophin Gene Transfer by AAV1 in mdx Mice Ameliorates Dystrophic Pathology and Protects Membrane Integrity

Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disorders, affecting one in 3,500 males. No efficacious treatment is currently available for DMD. While the mdx mouse is the most widely used small animal model for DMD, the large animal model, golden retriever muscular dystrophy (GRMD) dog, displays more similarities to human DMD patients in clinical and pathological characteristics. Therefore, the GRMD dog is considered a clinically more relevant and therapeutically more challenging DMD model.

791. Immunity to AAV-Mediated Gene Therapy in a Random-Bred Canine Model of Duchenne Muscular Dystrophy

Top of pageAbstract Introduction: Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. Studies in the mdx mouse model of DMD have shown that muscle membrane integrity and function can be improved by AAV-mediated delivery of a functional dystrophin protein. To assess the potential clinical utility of treating human DMD patients with AAV-mediated gene delivery, we performed a series of direct intra-muscular injections in random-bred normal dogs and in dogs with muscular dystrophy caused by a dystrophin mutation (xmd dogs). Methods: AAV serotypes 2 and 6 carrying different promoter-transgene cassettes were produced as previously described for the murine studies and purified either on a heparin column or with a combination of heparin column and cesium chloride gradient. Direct intramuscular injections of virus (1012, 1010, or 108 viral genomes per site) in a total volume of 250 ul were performed. The injection sites were biopsied under anesthesia between 2 and 12 weeks after injection. Results: Injection of either AAV6 or AAV2 expressing CMV-b-galactosidase (b-gal) induced a strong inflammatory response containing both CD8+ and CD4+ T-lymphocytes with peak tissue destruction 4 weeks following viral injection. A similar robust immune response was seen with injection of AAV6-RSV-alkaline-phosphatase, AAV6-CMV-canine-factor-IX (cFIX) and with empty AAV6 capsid alone. Additional purification of the AAV6-CMV-cFIX by centrifugation through a cesium chloride gradient did not diminish the immune response. Continuous immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) largely prevented the immune response to AAV6-CMV-b-gal in a normal dog for up to four weeks and permitted robust transgene expression. The same immunosuppressive regimen did not prevent an immune response to AAV6-CMV-cFIX or AAV6-CMV-human-micro-dystrophin in an xmd dog, suggesting that a more aggressive immunosuppressive regimen might be necessary in the xmd dog. Conclusions: Taken together, our results suggest that AAV capsid proteins, or proteins associated with the capsid, elicit significant immune responses when directly injected into skeletal muscle of normal random-bred dogs. The combination of CSP and MMF effectively prevents the immune responses in a normal dog, but not in an xmd dog, possibly due to the pre-existing inflammatory nature of the DMD muscle disease or due to genetic variation in a random-bred animal population. The robust immune responses to AAV2 and AAV6 in random-bred dogs contrast with the lack of an immune response in studies by others in inbred mice and dogs. Further studies will be necessary to determine the nature of the immune responses and to develop appropriate immunosuppressive regimen for AAV gene delivery to xmd muscle.