Progress toward Gene Therapy for Duchenne Muscular Dystrophy
Authors: Joel R. Chamberlain, Jeffrey S. Chamberlain
Journal: Molecular Therapy (2017)

Duchenne muscular dystrophy (DMD) has been a major target for gene therapy development for nearly 30 years. DMD is among the most common genetic diseases, and isolation of the defective gene (DMD, or dystrophin) was a landmark discovery, as it was the first time a human disease gene had been cloned without knowledge of the protein product. Despite tremendous obstacles, including the enormous size of the gene and the large volume of muscle tissue in the human body, efforts to devise a treatment based on gene replacement have advanced steadily through the combined efforts of dozens of labs and patient advocacy groups. Progress in the development of DMD gene therapy has been well documented in Molecular Therapy over the past 20 years and will be reviewed here to highlight prospects for success in the imminent human clinical trials planned by several groups. Duchenne muscular dystrophy (DMD) has been a major target for gene therapy development for nearly 30 years. DMD is among the most common genetic diseases, and isolation of the defective gene (DMD, or dystrophin) was a landmark discovery, as it was the first time a human disease gene had been cloned without knowledge of the protein product. Despite tremendous obstacles, including the enormous size of the gene and the large volume of muscle tissue in the human body, efforts to devise a treatment based on gene replacement have advanced steadily through the combined efforts of dozens of labs and patient advocacy groups. Progress in the development of DMD gene therapy has been well documented in Molecular Therapy over the past 20 years and will be reviewed here to highlight prospects for success in the imminent human clinical trials planned by several groups. Duchenne muscular dystrophy (DMD) was identified as a genetic disorder by several groups in the mid-19th century.1Emery A.E.H. Duchenne Muscular Dystrophy. Oxford Medical Publications, 1993Google Scholar The disease is inherited in an X-linked recessive pattern, and in-line with Haldane's hypothesis, one-third of all cases arise from spontaneous, new mutations. Accordingly, genetic counseling or even curing all current cases will not greatly reduce the incidence. Individuals with DMD display a progressive loss of skeletal muscle mass, increasing weakness, and a later-onset cardiomyopathy. Approximately one-third of patients display varying degrees of cognitive dysfunction, and in some cases, smooth muscle manifestations lead to gastrointestinal issues.1Emery A.E.H. Duchenne Muscular Dystrophy. Oxford Medical Publications, 1993Google Scholar A milder and more slowly progressing variant of the disorder is termed Becker muscular dystrophy (BMD). While DMD typically arises from genetic null allele mutations, BMD generally results from mutations that allow production of lower levels of, or partially functional, dystrophin protein. Patients from families without a prior history of the disorder are typically diagnosed between the ages of 2 and 6 years, but a family history enables early diagnosis, with the possibility for carrier testing and prenatal diagnosis. Increasing use of respiratory and cardiac support has extended lifespans over the past 20 years from the late teens up to the mid-30s, but these interventions do not by themselves significantly improve muscle function. The one treatment to date that has slowed muscle loss and extended ambulation is the use of corticosteroids, such as prednisone and deflazacort.2Griggs R.C. Miller J.P. Greenberg C.R. Fehlings D.L. Pestronk A. Mendell J.R. Moxley 3rd, R.T. King W. Kissel J.T. Cwik V. et al.Efficacy and safety of deflazacort vs prednisone and placebo for Duchenne muscular dystrophy.Neurology. 2016; 87: 2123-2131Crossref PubMed Scopus (104) Google Scholar DMD is among the most common single-gene disorders in humans, affecting ∼1 in 5,000 newborn males.3Mendell J.R. Shilling C. Leslie N.D. Flanigan K.M. al-Dahhak R. Gastier-Foster J. Kneile K. Dunn D.M. Duval B. Aoyagi A. et al.Evidence-based path to newborn screening for Duchenne muscular dystrophy.Ann. Neurol. 2012; 71: 304-313Crossref PubMed Scopus (547) Google Scholar Despite this relatively low incidence in the general population, it is one of the most well-known genetic disorders and has attracted enormous interest in the scientific and patient advocate communities. Much of this interest grew from early work by the Muscular Dystrophy Association (USA) (MDA) and a high-profile telethon (the first of its kind) that raised money under the leadership of the entertainer Jerry Lewis. The interest in DMD led the MDA to direct significant funding in the 1980s to finding the gene responsible for DMD. Funding efforts were an enormous success and, in a seminal series of publications by the laboratory of Louis Kunkel, resulted in the identification of the gene in 1986.4Monaco A.P. Neve R.L. Colletti-Feener C. Bertelson C.J. Kurnit D.M. Kunkel L.M. Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene.Nature. 1986; 323: 646-650Crossref PubMed Scopus (826) Google Scholar That work enabled highly accurate prenatal diagnosis and carrier detection, an understanding of the tissue-specific effect of mutations, and delineated the differences between DMD and BMD. Cloning of the DMD gene arguably represents the beginning of the human genome project, as the gene was isolated based on genetic studies that identified its chromosomal location on Xp21. The availability of the gene and the cDNA for the muscle isoform made DMD an early candidate for gene therapy.5Chamberlain J.S. Caskey C.T. Duchenne muscular dystrophy.in: Current Neurology. Volume 10. Yearbook Medical Publishers, 1990: 65-103Google Scholar Despite early enthusiasm for the development of genetic therapies, many features of DMD presented important obstacles to development of a therapy. The gene is 2.2 Mb in size, and numerous isoforms are expressed in muscle and non-muscle tissues from seven different promoters and via alternative splicing. The enormous size of the locus is likely a major reason that the gene displays the highest known spontaneous mutation frequency of any human gene. Fortunately, a number of discoveries suggested approaches to gene therapy that were simpler than initially envisioned. One was the identification of rare patients with large deletions within the gene, in one case encompassing almost half the gene, that were associated with extremely mild cases of BMD.6England S.B. Nicholson L.V. Johnson M.A. Forrest S.M. Love D.R. Zubrzycka-Gaarn E.E. Bulman D.E. Harris J.B. Davies K.E. Very mild muscular dystrophy associated with the deletion of 46% of dystrophin.Nature. 1990; 343: 180-182Crossref PubMed Scopus (491) Google Scholar A second came from isolation of the muscle cDNA, which was 14 kb but had an 11.2-kb open reading frame. These initial observations led to a series of studies establishing transgenic mouse lines on the mdx background, a model for DMD. In one transgenic line it was found that expression of the full-length dystrophin cDNA in a muscle-specific manner eliminated virtually all known muscle aspects of the disorder.7Cox G.A. Cole N.M. Matsumura K. Phelps S.F. Hauschka S.D. Campbell K.P. Faulkner J.A. Chamberlain J.S. Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity.Nature. 1993; 364: 725-729Crossref PubMed Scopus (264) Google Scholar From these studies, it became clear that an effective therapy could be developed if a synthetic gene derived from the muscle cDNA could be delivered to striated muscle, thus avoiding the need to deliver the entire gene, multiple isoforms, or to target tissues that expressed many of the smaller dystrophin isoforms. Truncated versions of the cDNA (∼6 kb in size) based on genetic deletions in mildly affected BMD patients were subsequently shown to almost completely prevent disease in the mdx mouse models, which was accompanied by intensive efforts to understand the overall structure and function of the dystrophin protein.8Phelps S.F. Hauser M.A. Cole N.M. Rafael J.A. Hinkle R.T. Faulkner J.A. Chamberlain J.S. Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice.Hum. Mol. 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Assembly of the dystrophin-associated protein complex does not require the dystrophin COOH-terminal domain.J. Cell Biol. 2000; 150: 1399-1410Crossref PubMed Scopus (187) Google Scholar, 12Rafael J.A. Cox G.A. Corrado K. Jung D. Campbell K.P. Chamberlain J.S. Forced expression of dystrophin deletion constructs reveals structure-function correlations.J. Cell Biol. 1996; 134: 93-102Crossref PubMed Scopus (158) Google Scholar, 13Wang B. Li J. Xiao X. Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model.Proc. Natl. Acad. Sci. USA. 2000; 97: 13714-13719Crossref PubMed Scopus (405) Google Scholar, 14Yuasa K. Miyagoe Y. Yamamoto K. Nabeshima Y. Dickson G. Takeda S. Effective restoration of dystrophin-associated proteins in vivo by adenovirus-mediated transfer of truncated dystrophin cDNAs.FEBS Lett. 1998; 425: 329-336Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar The next and enormously challenging task was finding a means to deliver the synthetic gene to the striated muscles that make up nearly 40% of human body mass. While transgenic animal studies led to important insights into dystrophin protein structure and function that informed the design of dystrophin expression cassettes needed for therapy, such technology is not directly applicable to human use without a way to administer the cassettes to patients. How could dystrophin mini-genes be delivered bodywide such that all muscles are rescued? The advent of mini-genes with a size of less than 7 kb allowed early studies of direct intramuscular gene transfer using retroviral and adenoviral vectors.14Yuasa K. Miyagoe Y. Yamamoto K. Nabeshima Y. Dickson G. Takeda S. 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Mini-dystrophin gene transfer in mdx4cv diaphragm muscle fibers increases sarcolemmal stability.Gene Ther. 1997; 4: 401-408Crossref PubMed Scopus (39) Google Scholar However, this route of administration resulted in localized, not systemic, gene delivery and did not appear amenable to whole-body therapy. Development of improved, helper-dependent adenoviral vectors overcame many immune-system-related barriers to vector delivery and allowed for delivery of cassettes expressing the full-length dystrophin protein, but these studies were also largely limited to administration via intramuscular injection.18Yang Y. Haecker S.E. Su Q. Wilson J.M. Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle.Hum. Mol. Genet. 1996; 5: 1703-1712Crossref PubMed Scopus (142) Google Scholar, 19Kochanek S. Clemens P.R. Mitani K. Chen H.H. Chan S. Caskey C.T. 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Gene Ther. 2001; 12: 1741-1755Crossref PubMed Scopus (53) Google Scholar A major advance came in 1997, when the lab of Hansell Stedman showed that the musculature of an entire limb could be transduced by infusing large quantities of adenoviral vectors into hindlimb blood vessels under elevated pressure.22Greelish J.P. Su L.T. Lankford E.B. Burkman J.M. Chen H. Konig S.K. Mercier I.M. Desjardins P.R. Mitchell M.A. Zheng X.G. et al.Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector.Nat. Med. 1999; 5: 439-443Crossref PubMed Scopus (197) Google Scholar While this approach did not target muscles outside of the limb, such as the heart and respiratory muscles, it suggested that vasculature delivery of vectors might be adapted for bodywide gene delivery. Further testing of adenoviral vectors revealed numerous disadvantages for muscle gene transfer, including residual induction of an immune response, slow loss of gene expression, and difficulties targeting widespread muscles due to the large vector size and its high tropism for liver.18Yang Y. Haecker S.E. Su Q. Wilson J.M. Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle.Hum. Mol. Genet. 1996; 5: 1703-1712Crossref PubMed Scopus (142) Google Scholar, 23Hartigan-O'Connor D. Kirk C.J. Crawford R. Mulé J.J. Chamberlain J.S. Immune evasion by muscle-specific gene expression in dystrophic muscle.Mol. Ther. 2001; 4: 525-533Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar Many labs thus embarked on studies to identify more effective vectors, and several studies showed that recombinant vectors derived from adeno-associated virus (AAV) could lead to long-term expression following intramuscular injection.13Wang B. Li J. Xiao X. Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model.Proc. Natl. Acad. Sci. USA. 2000; 97: 13714-13719Crossref PubMed Scopus (405) Google Scholar, 24Kessler P.D. Podsakoff G.M. Chen X. McQuiston S.A. Colosi P.C. Matelis L.A. Kurtzman G.J. Byrne B.J. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein.Proc. Natl. Acad. Sci. USA. 1996; 93: 14082-14087Crossref PubMed Scopus (534) Google Scholar, 25Xiao X. Li J. Samulski R.J. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector.J. Virol. 1996; 70: 8098-8108PubMed Google Scholar Adapting AAV vector technology for DMD still required significant work. AAV vectors have a carrying capacity of ∼5 kb, and the early studies all used vectors derived from serotype 2, which poorly transduced striated muscles and could only be administered by intramuscular injection. However, studies in transgenic mice had revealed that highly functional "micro-dystrophin" cassettes could be generated with a size less than 4 kb.10Harper S.Q. Hauser M.A. DelloRusso C. Duan D. Crawford R.W. Phelps S.F. Harper H.A. Robinson A.S. Engelhardt J.F. Brooks S.V. Chamberlain J.S. Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy.Nat. Med. 2002; 8: 253-261Crossref PubMed Scopus (459) Google Scholar, 11Crawford G.E. Faulkner J.A. Crosbie R.H. Campbell K.P. Froehner S.C. Chamberlain J.S. Assembly of the dystrophin-associated protein complex does not require the dystrophin COOH-terminal domain.J. Cell Biol. 2000; 150: 1399-1410Crossref PubMed Scopus (187) Google Scholar, 12Rafael J.A. Cox G.A. Corrado K. Jung D. Campbell K.P. Chamberlain J.S. Forced expression of dystrophin deletion constructs reveals structure-function correlations.J. Cell Biol. 1996; 134: 93-102Crossref PubMed Scopus (158) Google Scholar, 26Sakamoto M. Yuasa K. Yoshimura M. Yokota T. Ikemoto T. Suzuki M. Dickson G. Miyagoe-Suzuki Y. Takeda S. Micro-dystrophin cDNA ameliorates dystrophic phenotypes when introduced into mdx mice as a transgene.Biochem. Biophys. Res. Commun. 2002; 293: 1265-1272Crossref PubMed Scopus (101) Google Scholar A major breakthrough occurred when it was discovered that improved vectors could be generated from newly discovered AAV serotypes (such as AAV6, 8 and 9) which, when injected into the vasculature at high dose (in the range of 1014 vector genomes [vg] per kilogram) could transduce all the striated muscles in adult mice.27Gregorevic P. Blankinship M.J. Allen J.M. Crawford R.W. Meuse L. Miller D.G. Russell D.W. Chamberlain J.S. Systemic delivery of genes to striated muscles using adeno-associated viral vectors.Nat. Med. 2004; 10: 828-834Crossref PubMed Scopus (544) Google Scholar This led to the demonstration that dystrophy could be almost entirely halted and largely reversed in an adult mammal via systemic deliver of AAV/micro-dystrophin vectors (Figure 2).27Gregorevic P. Blankinship M.J. Allen J.M. Crawford R.W. Meuse L. Miller D.G. Russell D.W. Chamberlain J.S. Systemic delivery of genes to striated muscles using adeno-associated viral vectors.Nat. Med. 2004; 10: 828-834Crossref PubMed Scopus (544) Google Scholar, 28Gregorevic P. Allen J.M. Minami E. Blankinship M.J. Haraguchi M. Meuse L. Finn E. Adams M.E. Froehner S.C. Murry C.E. Chamberlain J.S. rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice.Nat. Med. 2006; 12: 787-789Crossref PubMed Scopus (250) Google Scholar Refinement of the gene delivery cassette through miniaturization of muscle-restricted gene regulatory cassettes provided greater vector functionality.29Li X. Eastman E.M. Schwartz R.J. Draghia-Akli R. Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences.Nat. Biotechnol. 1999; 17: 241-245Crossref PubMed Scopus (191) Google Scholar, 30Hauser M.A. Robinson A. Hartigan-O'Connor D. Williams-Gregory D.A. Buskin J.N. Apone S. Kirk C.J. Hardy S. Hauschka S.D. Chamberlain J.S. Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors.Mol. Ther. 2000; 2: 16-25Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Since the murine studies showed significant promise, AAV/micro-dystrophin studies moved to testing in the larger canine model for DMD (CXMD). The dystrophic dog models not only allowed for evaluating scalability in a larger animal, but also enabled more sensitive assessment of potential immune responses against the vector. Several early studies using AAV6 suggested that T cell immune responses against the vector were limiting in this model, but other studies with AAV8 and 9 revealed minimal immune reactivity.31Arruda V.R. Stedman H.H. Nichols T.C. Haskins M.E. Nicholson M. Herzog R.W. Couto L.B. High K.A. Regional intravascular delivery of AAV-2-F.IX to skeletal muscle achieves long-term correction of hemophilia B in a large animal model.Blood. 2005; 105: 3458-3464Crossref PubMed Scopus (137) Google Scholar, 32Wang Z. Kuhr C.S. Allen J.M. Blankinship M. Gregorevic P. Chamberlain J.S. Tapscott S.J. Storb R. Sustained AAV-mediated dystrophin expression in a canine model of Duchenne muscular dystrophy with a brief course of immunosuppression.Mol. 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Safe and bodywide muscle transduction in young adult Duchenne muscular dystrophy dogs with adeno-associated virus.Hum. Mol. Genet. 2015; 24: 5880-5890Crossref PubMed Scopus (85) Google Scholar, 40Yue Y. Ghosh A. Long C. Bostick B. Smith B.F. Kornegay J.N. Duan D. A single intravenous injection of adeno-associated virus serotype-9 leads to whole body skeletal muscle transduction in dogs.Mol. Ther. 2008; 16: 1944-1952Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 41Qiao C. Li J. Zheng H. Bogan J. Yuan Z. Zhang C. Bogan D. Kornegay J. Xiao X. Hydrodynamic limb vein injection of adeno-associated virus serotype 8 vector carrying canine myostatin propeptide gene into normal dogs enhances muscle growth.Hum. Gene Ther. 2009; 20: 1-10Crossref PubMed Scopus (74) Google Scholar These encouraging results have led to considerable interest in developing clinical gene therapy protocols involving administration of AAV/micro-dystrophin vectors for DMD. To date only one clinical trial has been completed, involving intramuscular injection of an AAV2.5 vector. This study, performed collaboratively between the laboratories of Jerry Mendell and R. Jude Samulski, involved six patients, and while the vector injection proved safe, none of the patients expressed significant levels of micro-dystrophin (only two had any detectable exogenous dystrophin).42Mendell J.R. Campbell K. Rodino-Klapac L. Sahenk Z. Shilling C. Lewis S. Bowles D. Gray S. Li C. Galloway G. et al.Dystrophin immunity in Duchenne's muscular dystrophy.N. Engl. J. Med. 2010; 363: 1429-1437Crossref PubMed Scopus (453) Google Scholar Two of the six patients displayed a low-level T cell immune response (assayed by ELISpot) against dystrophin, and one other patient displayed a clear T cell response against the AAV vector (defined as greater than 3× background on the ELISpot assay).42Mendell J.R. Campbell K. Rodino-Klapac L. Sahenk Z. Shilling C. Lewis S. Bowles D. Gray S. Li C. Galloway G. et al.Dystrophin immunity in Duchenne's muscular dystrophy.N. Engl. J. Med. 2010; 363: 1429-1437Crossref PubMed Scopus (453) Google Scholar, 43Bowles D.E. McPhee S.W. Li C. Gray S.J. Samulski J.J. Camp A.S. Li J. Wang B. Monahan P.E. Rabinowitz J.E. et al.Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector.Mol. Ther. 2012; 20: 443-455Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar These results indicated that immune responses against dystrophin and/or the AAV capsid were responsible for the poor transduction, but given the limited data available and the relative insensitivity of ELISpot assays, it is difficult to make firm conclusions. All the patients developed high-titer neutralizing antibodies against the vector, and two had pre-existing neutralizing antibodies.43Bowles D.E. McPhee S.W. Li C. Gray S.J. Samulski J.J. Camp A.S. Li J. Wang B. Monahan P.E. Rabinowitz J.E. et al.Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector.Mol. Ther. 2012; 20: 443-455Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar This, combined with a potential suboptimal vector serotype could have led to poor vector uptake by the injected muscle. In this study dystrophin expression was regulated by the ubiquitously active cytomegalovirus (CMV) immediate early enhancer plus promoter, which could have facilitated an immune response against dystrophin (through expression in immune effector cells) and/or resulted in loss of expression due to promoter shutdown. Finally, intramuscular injection tends to induce more inflammation and is better able to elicit a T cell immune response than is a vascular delivery method. These results suggested that better vectors (especially alternate capsid serotypes) coupled with a vascular delivery system and a gene regulatory cassette that is inactive in immune cells might lead to improved expression. Consequently, most current efforts to develop human clinical trials revolve around the use of AAV8 or 9 and a muscle-specific promoter/enhancer. With considerable advances in the types of AAV vectors available, muscle-specific gene regulatory cassettes, and production/purification protocols, several groups are now planning human clinical trials involving vascular delivery of AAV/micro-dystrophin to patients. These plans are supported by extensive new data involving large-scale vector delivery to CXMD dogs and, for safety studies, to wild-type non-human primates.44Toromanoff A. Adjali O. Larcher T. Hill M. Guigand L. Chenuaud P. Deschamps J.Y. Gauthier O. Blancho G. Vanhove B. et al.Lack of immunotoxicity after regional intravenous (RI) delivery of rAAV to nonhuman primate skeletal muscle.Mol. Ther. 2010; 18: 151-160Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 45Rodino-Klapac L.R. Montgomery C.L. Bremer W.G. Shontz K.M. Malik V. Davis N. Sprinkle S. Campbell K.J. Sahenk Z. Clark K.R. et al.Persistent expression of FLAG-tagged micro dystrophin in nonhuman primates following intramuscular and vascular delivery.Mol. Ther. 2010; 18: 109-117Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 46Duan D. Duchenne muscular dystrophy gene therapy in the canine model.Hum. Gene Ther. Clin. Dev. 2015; 26: 57-69Crossref PubMed Scopus (53) Google Scholar While most of these new data remain unpublished or proprietary, several trends are emerging that are being used to support upcoming clinical trial applications to the US Food and Drug Administration (FDA) or the European Medicines Agency (EMA). Planned trials have many similarities but differ in details including vector serotype, micro-dystrophin design, gene regulatory cassette usage, and the age of the patients. Depending on regulatory agency approval, some of these trials could begin within the next year. These impending phase 1 clinical trials will answer several critical questions about the long-term feasibility of AAV/micro-dystrophin gene therapy for DMD. While safety will be the primary focus, the systemic gene delivery approaches will also enable important data to be gathered on efficacy. Outcome measurements should provide initial data on the functional capacity of the different micro-dystrophin designs. For example, will such miniaturized proteins improve muscle physiology similarly to what has been observed in mice and dogs? If improvement is obtained, how soon can this be observed and will the effect be sustained? Will DMD patients tolerate high-dose vector delivery as has been observed in canine and non-human primate studies? Will age and disease progres