The Role of Immunosuppression in Gene- and Cell-Based Treatments for Duchenne Muscular Dystrophy (2007)

Duchenne muscular dystrophy (DMD) is a progressive and lethal, X-linked muscle-wasting disease caused by mutations in the dystrophin gene, affecting 1 in 3,500 males born worldwide. Clinically, most boys with DMD exhibit muscle weakness by the age of 5 years and lose independent ambulation by 10–12 years. During the late teen years, respiratory failure occurs and pulmonary ventilatory support is often required. Although this strategy allows patients to survive into their third decade, the onset of cardiomyopathy and worsening of respiratory function prevent longer-term survival. Although there is currently no cure for the disease, 20 years of study of the molecular characteristics of dystrophin is leading to new hope for the possibility of a specific treatment for DMD. Two new studies in the golden retriever muscular dystrophy (GRMD) model, including one in this issue of Molecular Therapy, provide encouragement for the potential of cell- and/or gene-based therapy coupled with immunosuppression (IS) for the treatment for DMD.1Wang Z Kuhr CS Allen JM Blankinship M Gregorevic P Chamberlain JS et al.Sustained AAV-mediated dystrophin expression in a canine model of Duchenne muscular dystrophy with a brief course of immunosuppression.Mol Ther. 2007; 18: 1160-1166Abstract Full Text Full Text PDF Scopus (180) Google Scholar,2Sampaolesi M Blot S D'Antona G Granger N Tonlorenzi R Innocenzi A et al.Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs.Nature. 2006; 444: 574-579Crossref PubMed Scopus (596) Google ScholarMost cases of DMD result from either the absence of full-length forms of dystrophin or expression of nonfunctional protein due to mutations or large gene deletions of the dystrophin gene. In humans the gene is expressed in all muscle types, although dystrophin levels are much higher in skeletal and cardiac muscle than in smooth muscle. The protein is also found in nervous tissue in large quantity. Dystrophin is an essential component of a multiprotein complex—the dystrophin-glycoprotein complex (DGC)—that links the cytoskeleton of the muscle fiber to the extracellular matrix. Dystrophin is thought to be an elastic and flexible protein that probably protects the muscle cell from the stresses caused by mechanical forces. Therefore, dystrophic myofibers are susceptible to damage induced by muscle contraction and undergo progressive degeneration and necrosis.3Nowak KJ Davies KE Duchenne muscular dystrophy and dystrophin: pathogenesis and opportunities for treatment.EMBO Rep. 2004; 5: 872-876Crossref PubMed Scopus (220) Google ScholarAlthough the capacity for muscle regeneration is significant at early stages of the disease, this property diminishes following years of continuous muscle damage, and muscle tissue is eventually replaced by fibrosis and adipose cells. These findings form the basis for current strategies designed to reduce the severity of muscular dystrophy by (i) reducing muscle necrosis, (ii) enhancing muscle regeneration, and (iii) and combating fibrosis using a variety of drugs and nutritional interventions.4Radley HG De Luca A Lynch GS Grounds MD Duchenne muscular dystrophy: focus on pharmaceutical and nutritional interventions.Int J Biochem Cell Biol. 2007; 39: 469-477Crossref PubMed Scopus (74) Google ScholarDMD is characterized by aggressive inflammation that is thought to contribute to myofiber necrosis. These findings motivated strategies based on IS to ameliorate the disease, and the use of IS therapy has now become a common practice. The long-term use of corticosteroids has improved pulmonary and cardiac outcomes in DMD patients.5Markham LW Spicer RL Khoury PR Wong BL Mathews KD Cripe LH Steroid therapy and cardiac function in Duchenne muscular dystrophy.Pediatr Cardiol. 2005; 26: 768-771Crossref PubMed Scopus (106) Google Scholar Moreover, early clinical studies demonstrate that cyclosporine (CSP) improves parameters of muscle force in boys with DMD.6Miller RG Sharma KR Pavlath GK Gussoni E Mynhier M Yu P et al.Myoblast implantation in Duchenne muscular dystrophy: the San Francisco study.Muscle Nerve. 1997; 20: 469-478Crossref PubMed Scopus (165) Google Scholar Continuing clinical studies of combined CSP and prednisone therapy will be informative as to the role of IS as a strategy for the treatment of DMD. However, specific treatment of DMD will require the replacement of the dystrophin gene, rescue of dystrophin mRNA, or upregulation of utrophin by cell- and/or gene-based strategies. A challenge for these treatments is to achieve widespread distribution of the therapeutic cell or gene product to all affected muscles.In this issue, Wang et al. demonstrate long-term expression of dystrophin by direct intramuscular (i.m.) injection of an adeno-associated virus 6 (AAV-6) vector encoding the canine micro-dystrophin gene in a GRMD model.1Wang Z Kuhr CS Allen JM Blankinship M Gregorevic P Chamberlain JS et al.Sustained AAV-mediated dystrophin expression in a canine model of Duchenne muscular dystrophy with a brief course of immunosuppression.Mol Ther. 2007; 18: 1160-1166Abstract Full Text Full Text PDF Scopus (180) Google Scholar The expression of dystrophin was sufficient for the restoration of the DGC assembly at the sarcolemma. Despite the small numbers of animals, these data provide useful information for further studies on the use of AAV vectors for correction of DMD. The choice of a large and immunocompetent animal model, combined with the use of a species-specific transgene, was a major asset of this study. The GRMD dog model exhibits a very severe form of dystrophy that affects limb, respiratory, heart, and pharyngeal muscles; by 8 months of age most dogs are unable to walk normally. The molecular basis of the dystrophin deficiency is a single base change in the 3′ consensus splice site of intron 6. The seventh exon is then skipped, which predicts a termination of the dystrophin reading frame within its N-terminal domain in exon 8. Thus, the GRMD model resembles the human disease at both the clinical and molecular levels.7Cooper BJ Winand NJ Stedman H Valentine BA Hoffman EP Kunkel LM et al.The homologue of the Duchenne locus is defective in X-linked muscular dystrophy of dogs.Nature. 1988; 334: 154-156Crossref PubMed Scopus (339) Google Scholar,8Sharp NJH Kornegay JN Van Camp SD Herbstreith MH Secore SL Kettle S et al.An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy.Genomics. 1992; 13: 115-121Crossref PubMed Scopus (283) Google Scholar The generation of the canine micro-dystrophin as the transgene was a critical factor for the sustained expression of the therapeutic protein. Previous gene- or cell-based therapies for GRMD have met with limited or no success.9Cerletti M Negri T Cozzi F Colpo R Andreetta F Croci D et al.Dystrophic phenotype of canine X-linked muscular dystrophy is mitigated by adenovirus-mediated utrophin gene transfer.Gene Ther. 2003; 10: 750-757Crossref PubMed Scopus (113) Google Scholar,10Dell'Agnola C Wang Z Storb R Tapscott SJ Kuhr CS Hauschka SD et al.Hematopoietic stem cell transplantation does not restore dystrophin expression in Duchenne muscular dystrophy dogs.Blood. 2004; 104: 4311-4318Crossref PubMed Scopus (67) Google Scholar,11Howell JM Lochmüller H O'Hara A Fletcher S Kakulas BA Massie B et al.High-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscle of dystrophic dogs: prolongation of expression with immunosuppression.Hum Gene Ther. 1998; 9: 629-634Crossref PubMed Scopus (76) Google ScholarEarlier work by this group demonstrated T-cell–mediated immune responses following i.m. injection of AAV-2 or AAV-6 in naive normal dogs, which occurred regardless of the identity of the transgene or vector promoter.12Wang Z Allen JM Riddell SR Gregorevic P Store R Tapscott SJ et al.Immunity to adeno-associated virus-mediated gene transfer in a random-bred canine model of Duchenne muscular dystrophy.Hum Gene Ther. 2007; 18: 18-26Crossref PubMed Scopus (121) Google Scholar Thus, in the current experiments using the GRMD model, the authors sought to prevent immune responses by short-term IS (up to 3 months after vector injection). The endpoints of the immune response were restricted to the determination of local transgene expression and the presence of CD8+ T-cell infiltrates during and after IS therapy. As expected, dogs injected with AAV-6 vector encoding non-species-specific transgenes exhibit local immune responses, as judged by T-cell infiltrates in muscle biopsy samples harvested after removal of IS. In another dog, i.m. injection of AAV encoding canine FIX (a transgene to which the animal is tolerant) under less intensive IS (CPS and mycophenolate mofetil, MMF), local expression of FIX was also accompanied by CD8+ T-cell infiltrates. The next experiment consisted of two modifications: the addition of anti-thymocyte globulin to the CPS/MMF regimen and the use of the canine micro-dystrophin gene. This strategy resulted in long-term sustained expression of micro-dystrophin and corrected the assembly of the DGC at the sarcolemma even after 4 months' discontinuation of the IS drugs. In this dog, T-cell infiltrates were not detected in muscle biopsy samples obtained during IS, but CD8+ T cells were present in samples collected 4 months after removal of IS. The authors argue that the T-cell infiltrates constitute evidence of an immune response to AAV-6 capsid or capsid-related proteins present at the injection site after IS was discontinued.There are several points to consider in interpreting these findings. First, the capsid proteins would have had to be present for periods longer than 7 months following i.m. injection if CD8+ T cells were indeed directed against AAV capsid. This is possible, although detailed studies of the duration of the persistence of capsid protein following vector injection are lacking. Second, novel junction sequences within the micro-dystrophin protein could potentially be presented as neoantigens. Third, the continuing muscle damage of DMD provides a pro-inflammatory environment in which T-cell infiltrates are commonly present, although the location and intensity of infiltrates vary.7Cooper BJ Winand NJ Stedman H Valentine BA Hoffman EP Kunkel LM et al.The homologue of the Duchenne locus is defective in X-linked muscular dystrophy of dogs.Nature. 1988; 334: 154-156Crossref PubMed Scopus (339) Google Scholar,13Spencer MJ Montecino-Rodriguez E Dorshkind K Tidball JG Helper (CD4(+)) and cytotoxic (CD8(+)) T cells promote the pathology of dystrophin-deficient muscle.Clin Immunol. 2001; 98: PubMed Scopus Google Scholar Thus, the T-cell infiltrates after removal of IS the muscle studies on the course and of the T-cell responses are to the of the transgene, and the muscle disease in CD8+ T-cell or cell-based strategies for the treatment of DMD require an to of skeletal muscle and AAV vectors to local gene and transgene expression and long-term expression following i.m. injection into human skeletal CS S et factor gene transfer to skeletal muscle in patients with severe 2003; PubMed Scopus Google Scholar However, a major of i.m. of AAV vectors is the small muscle of AAV leading to widespread of skeletal muscle of Stedman M et of to skeletal muscle long-term correction of B in a large animal 2005; PubMed Scopus Google Scholar as by K Wang Z A et gene transfer to muscle after of 2005; PubMed Scopus Google Scholar of skeletal and cardiac muscle of a dog following of vector a or for study on the GRMD model using stem cells also the of of and for DMD. This study combined (i) canine stem by a vector encoding human micro-dystrophin or (ii) canine dystrophin coupled with M Blot S D'Antona G Granger N Tonlorenzi R Innocenzi A et al.Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs.Nature. 2006; 444: 574-579Crossref PubMed Scopus (596) Google Scholar of the by dystrophin expression was with of both muscle and dogs a as judged by the in the contraction force and of is not these are by the use of species-specific and/or full-length dystrophin in the In is that IS for the use of was a role in the of the disease KE Grounds MD muscular dystrophy with stem 2006; Full Text Full Text PDF PubMed Scopus Google CSP in a clinical of transfer therapy long-term in boys with DMD.6Miller RG Sharma KR Pavlath GK Gussoni E Mynhier M Yu P et al.Myoblast implantation in Duchenne muscular dystrophy: the San Francisco study.Muscle Nerve. 1997; 20: 469-478Crossref PubMed Scopus (165) Google Scholar The continuing clinical of CSP and for boys with DMD will provide IS a in the of and for use of gene- or cell-based therapies coupled with IS will be for the combined therapy. in the of novel to widespread of and of immune of the disease, are all to to these strategies to The of IS and novel gene- and cell-based therapies in the GRMD model the of the replacement of or the of the gene encoding dystrophin for DMD and muscle Duchenne muscular dystrophy (DMD) is a progressive and lethal, X-linked muscle-wasting disease caused by mutations in the dystrophin gene, affecting 1 in 3,500 males born worldwide. Clinically, most boys with DMD exhibit muscle weakness by the age of 5 years and lose independent ambulation by 10–12 years. During the late teen years, respiratory failure occurs and pulmonary ventilatory support is often required. Although this strategy allows patients to survive into their third decade, the onset of cardiomyopathy and worsening of respiratory function prevent longer-term survival. Although there is currently no cure for the disease, 20 years of study of the molecular characteristics of dystrophin is leading to new hope for the possibility of a specific treatment for DMD. Two new studies in the golden retriever muscular dystrophy (GRMD) model, including one in this issue of Molecular Therapy, provide encouragement for the potential of cell- and/or gene-based therapy coupled with immunosuppression (IS) for the treatment for DMD.1Wang Z Kuhr CS Allen JM Blankinship M Gregorevic P Chamberlain JS et al.Sustained AAV-mediated dystrophin expression in a canine model of Duchenne muscular dystrophy with a brief course of immunosuppression.Mol Ther. 2007; 18: 1160-1166Abstract Full Text Full Text PDF Scopus (180) Google Scholar,2Sampaolesi M Blot S D'Antona G Granger N Tonlorenzi R Innocenzi A et al.Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs.Nature. 2006; 444: 574-579Crossref PubMed Scopus (596) Google Scholar cases of DMD result from either the absence of full-length forms of dystrophin or expression of nonfunctional protein due to mutations or large gene deletions of the dystrophin gene. In humans the gene is expressed in all muscle types, although dystrophin levels are much higher in skeletal and cardiac muscle than in smooth muscle. The protein is also found in nervous tissue in large quantity. Dystrophin is an essential component of a multiprotein complex—the dystrophin-glycoprotein complex (DGC)—that links the cytoskeleton of the muscle fiber to the extracellular matrix. Dystrophin is thought to be an elastic and flexible protein that probably protects the muscle cell from the stresses caused by mechanical forces. Therefore, dystrophic myofibers are susceptible to damage induced by muscle contraction and undergo progressive degeneration and necrosis.3Nowak KJ Davies KE Duchenne muscular dystrophy and dystrophin: pathogenesis and opportunities for treatment.EMBO Rep. 2004; 5: 872-876Crossref PubMed Scopus (220) Google Scholar Although the capacity for muscle regeneration is significant at early stages of the disease, this property diminishes following years of continuous muscle damage, and muscle tissue is eventually replaced by fibrosis and adipose cells. These findings form the basis for current strategies designed to reduce the severity of muscular dystrophy by (i) reducing muscle necrosis, (ii) enhancing muscle regeneration, and (iii) and combating fibrosis using a variety of drugs and nutritional interventions.4Radley HG De Luca A Lynch GS Grounds MD Duchenne muscular dystrophy: focus on pharmaceutical and nutritional interventions.Int J Biochem Cell Biol. 2007; 39: 469-477Crossref PubMed Scopus (74) Google Scholar DMD is characterized by aggressive inflammation that is thought to contribute to myofiber necrosis. These findings motivated strategies based on IS to ameliorate the disease, and the use of IS therapy has now become a common practice. The long-term use of corticosteroids has improved pulmonary and cardiac outcomes in DMD patients.5Markham LW Spicer RL Khoury PR Wong BL Mathews KD Cripe LH Steroid therapy and cardiac function in Duchenne muscular dystrophy.Pediatr Cardiol. 2005; 26: 768-771Crossref PubMed Scopus (106) Google Scholar Moreover, early clinical studies demonstrate that cyclosporine (CSP) improves parameters of muscle force in boys with DMD.6Miller RG Sharma KR Pavlath GK Gussoni E Mynhier M Yu P et al.Myoblast implantation in Duchenne muscular dystrophy: the San Francisco study.Muscle Nerve. 1997; 20: 469-478Crossref PubMed Scopus (165) Google Scholar Continuing clinical studies of combined CSP and prednisone therapy will be informative as to the role of IS as a strategy for the treatment of DMD. However, specific treatment of DMD will require the replacement of the dystrophin gene, rescue of dystrophin mRNA, or upregulation of utrophin by cell- and/or gene-based strategies. A challenge for these treatments is to achieve widespread distribution of the therapeutic cell or gene product to all affected In this issue, Wang et al. demonstrate long-term expression of dystrophin by direct intramuscular (i.m.) injection of an adeno-associated virus 6 (AAV-6) vector encoding the canine micro-dystrophin gene in a GRMD model.1Wang Z Kuhr CS Allen JM Blankinship M Gregorevic P Chamberlain JS et al.Sustained AAV-mediated dystrophin expression in a canine model of Duchenne muscular dystrophy with a brief course of immunosuppression.Mol Ther. 2007; 18: 1160-1166Abstract Full Text Full Text PDF Scopus (180) Google Scholar The expression of dystrophin was sufficient for the restoration of the DGC assembly at the sarcolemma. Despite the small numbers of animals, these data provide useful information for further studies on the use of AAV vectors for correction of DMD. The choice of a large and immunocompetent animal model, combined with the use of a species-specific transgene, was a major asset of this study. The GRMD dog model exhibits a very severe form of dystrophy that affects limb, respiratory, heart, and pharyngeal muscles; by 8 months of age most dogs are unable to walk normally. The molecular basis of the dystrophin deficiency is a single base change in the 3′ consensus splice site of intron 6. The seventh exon is then skipped, which predicts a termination of the dystrophin reading frame within its N-terminal domain in exon 8. Thus, the GRMD model resembles the human disease at both the clinical and molecular levels.7Cooper BJ Winand NJ Stedman H Valentine BA Hoffman EP Kunkel LM et al.The homologue of the Duchenne locus is defective in X-linked muscular dystrophy of dogs.Nature. 1988; 334: 154-156Crossref PubMed Scopus (339) Google Scholar,8Sharp NJH Kornegay JN Van Camp SD Herbstreith MH Secore SL Kettle S et al.An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy.Genomics. 1992; 13: 115-121Crossref PubMed Scopus (283) Google Scholar The generation of the canine micro-dystrophin as the transgene was a critical factor for the sustained expression of the therapeutic protein. Previous gene- or cell-based therapies for GRMD have met with limited or no success.9Cerletti M Negri T Cozzi F Colpo R Andreetta F Croci D et al.Dystrophic phenotype of canine X-linked muscular dystrophy is mitigated by adenovirus-mediated utrophin gene transfer.Gene Ther. 2003; 10: 750-757Crossref PubMed Scopus (113) Google Scholar,10Dell'Agnola C Wang Z Storb R Tapscott SJ Kuhr CS Hauschka SD et al.Hematopoietic stem cell transplantation does not restore dystrophin expression in Duchenne muscular dystrophy dogs.Blood. 2004; 104: 4311-4318Crossref PubMed Scopus (67) Google Scholar,11Howell JM Lochmüller H O'Hara A Fletcher S Kakulas BA Massie B et al.High-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscle of dystrophic dogs: prolongation of expression with immunosuppression.Hum Gene Ther. 1998; 9: 629-634Crossref PubMed Scopus (76) Google Scholar work by this group demonstrated T-cell–mediated immune responses following i.m. injection of AAV-2 or AAV-6 in naive normal dogs, which occurred regardless of the identity of the transgene or vector promoter.12Wang Z Allen JM Riddell SR Gregorevic P Store R Tapscott SJ et al.Immunity to adeno-associated virus-mediated gene transfer in a random-bred canine model of Duchenne muscular dystrophy.Hum Gene Ther. 2007; 18: 18-26Crossref PubMed Scopus (121) Google Scholar Thus, in the current experiments using the GRMD model, the authors sought to prevent immune responses by short-term IS (up to 3 months after vector injection). The endpoints of the immune response were restricted to the determination of local transgene expression and the presence of CD8+ T-cell infiltrates during and after IS therapy. As expected, dogs injected with AAV-6 vector encoding non-species-specific transgenes exhibit local immune responses, as judged by T-cell infiltrates in muscle biopsy samples harvested after removal of IS. In another dog, i.m. injection of AAV encoding canine FIX (a transgene to which the animal is tolerant) under less intensive IS (CPS and mycophenolate mofetil, MMF), local expression of FIX was also accompanied by CD8+ T-cell infiltrates. The next experiment consisted of two modifications: the addition of anti-thymocyte globulin to the CPS/MMF regimen and the use of the canine micro-dystrophin gene. This strategy resulted in long-term sustained expression of micro-dystrophin and corrected the assembly of the DGC at the sarcolemma even after 4 months' discontinuation of the IS drugs. In this dog, T-cell infiltrates were not detected in muscle biopsy samples obtained during IS, but CD8+ T cells were present in samples collected 4 months after removal of IS. The authors argue that the T-cell infiltrates constitute evidence of an immune response to AAV-6 capsid or capsid-related proteins present at the injection site after IS was are several points to consider in interpreting these findings. First, the capsid proteins would have had to be present for periods longer than 7 months following i.m. injection if CD8+ T cells were indeed directed against AAV capsid. This is possible, although detailed studies of the duration of the persistence of capsid protein following vector injection are lacking. Second, novel junction sequences within the micro-dystrophin protein could potentially be presented as neoantigens. Third, the continuing muscle damage of DMD provides a pro-inflammatory environment in which T-cell infiltrates are commonly present, although the location and intensity of infiltrates vary.7Cooper BJ Winand NJ Stedman H Valentine BA Hoffman EP Kunkel LM et al.The homologue of the Duchenne locus is defective in X-linked muscular dystrophy of dogs.Nature. 1988; 334: 154-156Crossref PubMed Scopus (339) Google Scholar,13Spencer MJ Montecino-Rodriguez E Dorshkind K Tidball JG Helper (CD4(+)) and cytotoxic (CD8(+)) T cells promote the pathology of dystrophin-deficient muscle.Clin Immunol. 2001; 98: PubMed Scopus Google Scholar Thus, the T-cell infiltrates after removal of IS the muscle studies on the course and of the T-cell responses are to the of the transgene, and the muscle disease in CD8+ T-cell or cell-based strategies for the treatment of DMD require an to of skeletal muscle and AAV vectors to local gene and transgene expression and long-term expression following i.m. injection into human skeletal CS S et factor gene transfer to skeletal muscle in patients with severe 2003; PubMed Scopus Google Scholar However, a major of i.m. of AAV vectors is the small muscle of AAV leading to widespread of skeletal muscle of Stedman M et of to skeletal muscle long-term correction of B in a large animal 2005; PubMed Scopus Google Scholar as by K Wang Z A et gene transfer to muscle after of 2005; PubMed Scopus Google Scholar of skeletal and cardiac muscle of a dog following of vector a or for DMD. The study on the GRMD model using stem cells also the of of and for DMD. This study combined (i) canine stem by a vector encoding human micro-dystrophin or (ii) canine dystrophin coupled with M Blot S D'Antona G Granger N Tonlorenzi R Innocenzi A et al.Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs.Nature. 2006; 444: 574-579Crossref PubMed Scopus (596) Google Scholar of the by dystrophin expression was with of both muscle and dogs a as judged by the in the contraction force and of is not these are by the use of species-specific and/or full-length dystrophin in the In is that IS for the use of was a role in the of the disease KE Grounds MD muscular dystrophy with stem 2006; Full Text Full Text PDF PubMed Scopus Google Scholar In CSP in a clinical of transfer therapy long-term in boys with DMD.6Miller RG Sharma KR Pavlath GK Gussoni E Mynhier M Yu P et al.Myoblast implantation in Duchenne muscular dystrophy: the San Francisco study.Muscle Nerve. 1997; 20: 469-478Crossref PubMed Scopus (165) Google Scholar The continuing clinical of CSP and for boys with DMD will provide IS a in the of and for use of gene- or cell-based therapies coupled with IS will be for the combined therapy. in the of novel to widespread of and of immune of the disease, are all to to these strategies to The of IS and novel gene- and cell-based therapies in the GRMD model the of the replacement of or the of the gene encoding dystrophin for DMD and muscle