Progress toward Gene Therapy for Duchenne Muscular Dystrophy
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. Genet. 1995; 4: 1251-1258Crossref PubMed Scopus (265) Google Scholar, 9Wells D.J. Wells K.E. Asante E.A. Turner G. Sunada Y. Campbell K.P. Walsh F.S. Dickson G. Expression of human full-length and minidystrophin in transgenic mdx mice: implications for gene therapy of Duchenne muscular dystrophy.Hum. Mol. Genet. 1995; 4: 1245-1250Crossref PubMed Scopus (149) Google Scholar Such studies enabled the design of smaller but highly functional mini- and micro-dystrophin cDNAs as short as 3.6 kb (Figure 1).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, 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. 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