Micro-dystrophin Genes Bring Hope of an Effective Therapy for Duchenne Muscular Dystrophy

Systemic delivery of genes to muscle using vectors based on recombinant adenovirus-associated virus (rAAV) has been explored extensively in animal models of Duchene muscular dystrophy (DMD) to replace the missing dystrophin gene in both skeletal and cardiac muscles.1Chamberlain J.R. Chamberlain J.S. Progress toward Gene Therapy for Duchenne Muscular Dystrophy.Mol Ther. 2017; 25: 1125-1131Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 2Duan D. Systemic AAV Micro-dystrophin Gene Therapy for Duchenne Muscular Dystrophy.Mol Ther. 2018; 26: 2337-2356Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar A major challenge in bringing this approach to the clinic for DMD is the low gene capacity of rAAV (∼5 kb) and the large size of the dystrophin mRNA (14 kb). Although dystrophin mini-genes and micro-genes were reported almost three decades ago, many lacked important binding sites of the protein. In this issue of Molecular Therapy, Ramos et al.3Ramos J.N. Hollinger K. Bengtsson N.E. Allen J.M. Hauschka S.D. Chamberlain J.S. Development of Novel Micro-dystrophins with Enhanced Functionality.Mol Ther. 2019; 27 (this issue): 623-635Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar report the functional evaluation of eight different micro-dystrophin genes whose function is optimized by varying the rod domain structure of dystrophin. This work is significant because, although these dystrophin micro-genes are not fully functional, they are showing sufficient promise to be included in clinical trials in DMD patients. DMD (OMIM: 310200) is an X-linked recessive disorder affecting 1 in 5,000 newborn males and is caused by mutations that lead to the loss of the large cytoskeletal muscle protein, dystrophin.4Guiraud S. Aartsma-Rus A. Vieira N.M. Davies K.E. van Ommen G.J. Kunkel L.M. The Pathogenesis and Therapy of Muscular Dystrophies.Annu. Rev. Genomics Hum. Genet. 2015; 16: 281-308Crossref PubMed Scopus (202) Google Scholar Patients are generally wheelchair bound by the age of 12 years and die in their late 20s or early 30s due to respiratory-cardiac failure. There is currently no effective treatment, although promising genetic approaches that extend the ambulatory phase of the disease, such as exon-skipping and stop codon read through, are in the clinic. Nevertheless, these therapies are limited because they are only applicable to a subset of patients with particular mutations. Dystrophin provides an important structural link between the internal cytoskeleton via F-actin and the dystrophin-associated protein complex (DAPC), which lies across the sarcolemma and links to laminin.4Guiraud S. Aartsma-Rus A. Vieira N.M. Davies K.E. van Ommen G.J. Kunkel L.M. The Pathogenesis and Therapy of Muscular Dystrophies.Annu. Rev. Genomics Hum. Genet. 2015; 16: 281-308Crossref PubMed Scopus (202) Google Scholar Acting as a shock absorber, dystrophin protects the sarcolemma from damage and stress developed during muscle contraction. Dystrophin has four domains: (1) the N-terminal region (NTD) contains the binding sites for F-actin, (2) the central rod domain contains 24 spectrin-like repeats interspersed by four hinge regions, and (3) the cysteine-rich domain (CRD) binds the dystroglycoprotein complex and the C-terminal domain (CTD) (Figure 1). Loss of either the DAPC or F-actin binding results in DMD, but other in-frame deletions of the gene result in milder forms of the disease, Becker muscular dystrophy (BMD; OMIM: 300376), since the truncated dystrophin molecules are partially functional. The binding of neuronal nitric oxide synthase (nNOS) involves both the N-terminal and rod domains and has been correlated with the severity of disease, but loss of the nNOS localization is consistent with a mild phenotype in some BMD patients. The concept of a mini-gene being used for the therapy of DMD is based on a very mildly affected BMD patient that had 46% of the dystrophin protein missing with the deletion lying across the central rod domain.5England 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 The patient presented with muscle weakness in his 30s but was still able to walk with a stick in his 60s. A biopsy taken from an affected male in another branch of this family confirmed that this deletion results in the localization of the truncated dystrophin at the sarcolemma. Delivery of a reconstruction of this mini-gene to the mdx mouse model of DMD ameliorated the pathology,6Wells D.J. Wells K.E. Walsh F.S. Davies K.E. Goldspink G. Love D.R. Chan-Thomas P. Dunckley M.G. Piper T. Dickson G. Human dystrophin expression corrects the myopathic phenotype in transgenic mdx mice.Hum. Mol. Genet. 1992; 1: 35-40Crossref PubMed Scopus (61) Google Scholar confirming that the mild phenotype is a result of the truncated dystrophin produced rather than genetic background. Several groups have modified the original BMD patent's dystrophin mini-gene to obtain a construct that would fit into an rAAV vector together with the appropriate regulatory sequences, but none show full functional recovery.7Wang 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, 8Harper 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 The efficacy of these mini-genes has improved with the addition of nNOS binding sites by including spectrin repeats 16/179Lai Y. Thomas G.D. Yue Y. Yang H.T. Li D. Long C. Judge L. Bostick B. Chamberlain J.S. Terjung R.L. Duan D. Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy.J. Clin. Invest. 2009; 119: 624-635Crossref PubMed Scopus (284) Google Scholar and codon optimization.10Foster H. Sharp P.S. Athanasopoulos T. Trollet C. Graham I.R. Foster K. Wells D.J. Dickson G. Codon and mRNA sequence optimization of microdystrophin transgenes improves expression and physiological outcome in dystrophic mdx mice following AAV2/8 gene transfer.Mol Ther. 2008; 16: 1825-1832Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar However, what determines the severity of clinical phenotype is not fully understood. For example, the way in which the spectrin-like repeats assemble relative to each other is more complex than first thought,11Delalande O. Molza A.E. Dos Santos Morais R. Chéron A. Pollet É. Raguenes-Nicol C. Tascon C. Giudice E. Guilbaud M. Nicolas A. et al.Dystrophin's central domain forms a complex filament that becomes disorganized by in-frame deletions.J. Biol. Chem. 2018; 293: 6637-6646Crossref PubMed Scopus (14) Google Scholar since in-frame deletions of the rod domain result in differing severities of BMD. Ramos et al.3Ramos J.N. Hollinger K. Bengtsson N.E. Allen J.M. Hauschka S.D. Chamberlain J.S. Development of Novel Micro-dystrophins with Enhanced Functionality.Mol Ther. 2019; 27 (this issue): 623-635Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar report the optimization of micro-dystrophin genes further by exploring configurations of the rod domain and hinge regions of dystrophin in the mdx4cv mouse model. Their previously reported most robust micro-dystrophin gene, μDysH3, was included for comparison.8Harper 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 Many of these truncated genes protect the sarcolemma from contraction-induced injury and increase force generation, as assayed initially by direct intramuscular injection (IM) to mouse muscle and then by systemic delivery. Both micro-dystrophin μDYs3 (which also contained hybrid spectrin repeats) and μDYs4 lacked the nNOS binding site in repeats 16 and 17, performed less well than μDysH3 when tested by IM, and were not tested further by systemic delivery. These initial studies were undertaken using the cytomegalovirus (CMV) promoter, but the systemic delivery was performed using a muscle-specific promoter smaller in size than CMV that resulted in a smaller overall gene size and better viral titers. The micro-dystrophin genes tested are summarized in Figure 1, alongside additional micro-dystrophins that have been tested by other groups. As expected, the micro-dystrophins lacking the spectrin repeats 16 and 17 were less effective since they could not localize nNOS. Micro-dystrophin constructs were also tested in mice for the longevity of expression. The configuration μDYs5 with five spectrin repeats was the most functional. Interestingly, no single micro-dystrophin construct performed optimally in all tests and therefore the choice of the best configuration was a compromise. Functional efficacy of the μDYs5 gene in the skeletal muscle of the dog model of the disease was recently reported by the same group, suggesting that μDYs5 works well in animals with large muscles and is therefore a good candidate for patient trials (Hakim et al., 2018, Mol Ther., conference). It is also important to consider the ability of these constructs to restore cardiac function. This was not tested by Ramos et al.,3Ramos J.N. Hollinger K. Bengtsson N.E. Allen J.M. Hauschka S.D. Chamberlain J.S. Development of Novel Micro-dystrophins with Enhanced Functionality.Mol Ther. 2019; 27 (this issue): 623-635Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar although expression in the heart was observed. Wasala et al.12Wasala N.B. Shin J.H. Lai Y. Yue Y. Montanaro F. Duan D. Cardiac-Specific Expression of ΔH2-R15 Mini-Dystrophin Normalized All Electrocardiogram Abnormalities and the End-Diastolic Volume in a 23-Month-Old Mouse Model of Duchenne Dilated Cardiomyopathy.Hum. Gene Ther. 2018; 29: 737-748Crossref PubMed Scopus (15) Google Scholar have reported the importance of spectrin repeats 16–19 as being protective for the heart. It should be noted that the original BMD patient with the large deletion that included exons 16–19 lived into his 70s with no obvious cardiac problems. In addition, increased function of the diaphragm has been shown to be beneficial to cardiac function in the mouse and may improve cardiac function in DMD patients.13Crisp A. Yin H. Goyenvalle A. Betts C. Moulton H.M. Seow Y. Babbs A. Merritt T. Saleh A.F. Gait M.J. et al.Diaphragm rescue alone prevents heart dysfunction in dystrophic mice.Hum. Mol. Genet. 2011; 20: 413-421Crossref PubMed Scopus (59) Google Scholar What does this mean for the development of rAAV therapy for DMD patients? The study by Ramos et al.3Ramos J.N. Hollinger K. Bengtsson N.E. Allen J.M. Hauschka S.D. Chamberlain J.S. Development of Novel Micro-dystrophins with Enhanced Functionality.Mol Ther. 2019; 27 (this issue): 623-635Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar together with studies from other groups suggest that there may not be an optimal micro-dystrophin gene and that each one is a compromise. The configurations currently in clinical trials are shown in Figure 1. The μDysH2 configuration is being used by Nationwide Children's Hospital and Sarepta Therapeutics with a rAAV-rh74 and by Genethon and Sarepta Therapeutics with a rAAV2/8 in current clinical trials in DMD patients and it remains to be seen whether ringbinden myofibers are a major issue. Ringbinden fibers are aberrant myofibers that wrap themselves around an existing fiber in a tight spiral, impairing function. A ΔR3-19/R20-21/ΔCTD mini-gene and μDYs5 both encapsidated within a rAAV9 are in clinical trials (Pfizer and Solid Biosciences). These constructs show excellent functional performance in the dog model of the disease (Hakim et al., 2018, Mol Ther., conference).14Le Guiner C. Servais L. Montus M. Larcher T. Fraysse B. Moullec S. Allais M. François V. Dutilleul M. Malerba A. et al.Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy.Nat. Commun. 2017; 8: 16105Crossref PubMed Scopus (144) Google Scholar Preliminary data presented at the World Muscle Society Conference (http://investorrelations.sarepta.com/news-releases/news-release-details/sarepta-therapeutics-announces-its-first-rd-day-jerry-mendell-md) shows expression of the micro-dystrophin in DMD patients after systemic delivery of a micro-dystrophin gene using rAAV. We are at an exciting point in the development of therapy for DMD and eagerly await the final outcome of these trials. However, there will be other challenges in addition to the configuration of the micro-dystrophin. There is a need not only to effectively target skeletal and cardiac muscle, but also to target muscle satellite cells, which are less efficiently transduced by certain rAAV serotypes.15Arnett A.L. Konieczny P. Ramos J.N. Hall J. Odom G. Yablonka-Reuveni Z. Chamberlain J.R. Chamberlain J.S. Adeno-associated viral (AAV) vectors do not efficiently target muscle satellite cells.Mol. Ther. Methods Clin. Dev. 2014; 1: 1Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Very high viral titers are required to see a beneficial clinical effect. Timing of delivery may also be crucial as, unlike in the mouse, muscle growth may lead to loss of the virus in young DMD patients, but administration later will be less efficient because of the significantly reduced muscle mass remaining in which to restore function. Re-administration of virus and an immune response to the dystrophin may also be issues, necessitating the exclusion of some patients with pre-existing immunity. These challenges could be addressed through the delivery of micro-genes modeled on the structure of the dystrophin-related protein utrophin (Figure 1), which would not evoke an immune response,16Ebihara S. Guibinga G.H. Gilbert R. Nalbantoglu J. Massie B. Karpati G. Petrof B.J. Differential effects of dystrophin and utrophin gene transfer in immunocompetent muscular dystrophy (mdx) mice.Physiol. Genomics. 2000; 3: 133-144Crossref PubMed Google Scholar and the development of non-viral delivery routes. At present, additional obstacles, such as producing sufficient high titer virus and the price of treatment, are preventing rAAV therapy from becoming a routine therapy for all DMD patients. Nevertheless, the field has come a long way on its journey from gene to therapy, even if there is still a long way to go. Patients and their families now have reason to be optimistic since a truly transformative therapy is on the horizon that will improve the lives of all DMD patients. The authors declare no competing interests. We acknowledge the Medical Research Council MR/NO10698 for funding. Development of Novel Micro-dystrophins with Enhanced FunctionalityRamos et al.Molecular TherapyFebruary 1, 2019In BriefGene therapy for DMD is complicated by the enormous dystrophin gene. AAV vectors can only deliver partially functional micro-dystrophin genes. In this report, improved micro-dystrophin cassettes are described that increase muscle strength, avoid muscle-tendon injury, and persist more than 2 years. These improved vectors have great promise for DMD therapy. Full-Text PDF Open Access