Micro-utrophin Therapy for Duchenne Muscular Dystrophy
Authors: Dongsheng Duan
Journal: Molecular Therapy (2019)

Duchenne muscular dystrophy (DMD) is a deadly muscle disease caused by the loss of dystrophin, a critical subsarcolemmal protein that maintains muscle integrity during contraction. Dystrophin protects muscle cells by linking the cytoskeleton and the extracellular matrix. Interestingly, this function can also be accomplished by another cellular protein called utrophin. Transgenic overexpression of utrophin or mini-utrophin markedly prevented muscle disease in dystrophin-deficient mdx mice. Since utrophin is not a foreign protein to DMD patients (thus minimizing the risk of immune responses) and can functionally substitute for dystrophin, there has been a substantial interest in developing utrophin-based gene therapy. The therapeutic potential of the massively miniaturized utrophin gene (Figure 1) was demonstrated by the Chamberlain lab in the phenotypic dystrophin/utrophin double knockout mouse via intravenous injection of the adeno-associated virus (AAV) micro-utrophin vector.1Odom G.L. Gregorevic P. Allen J.M. Finn E. Chamberlain J.S. Microutrophin delivery through rAAV6 increases lifespan and improves muscle function in dystrophic dystrophin/utrophin-deficient mice.Mol. Ther. 2008; 16: 1539-1545Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar To translate AAV micro-utrophin therapy to DMD patients, an immediate next step is to scale up and validate the finding in the symptomatic canine model. A recent study by Song et al.,2Song Y. Morales L. Malik A.S. Mead A.F. Greer C.D. Mitchell M.A. Petrov M.T. Su L.T. Choi M.E. Rosenblum S.T. et al.Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.Nat Med. 2019; 25: 1505-1511Crossref PubMed Scopus (39) Google Scholar published in Nature Medicine, addresses this important question. A highly promising therapy for DMD is the delivery of a synthetic micro-dystrophin to all muscles in the body using an AAV vector. Several clinical trials are ongoing to evaluate the safety and efficacy of systemic AAV micro-dystrophin therapy in affected boys (Figure 1).3Duan D. Micro-dystrophin gene therapy goes systemic in Duchenne muscular dystrophy patients.Hum. Gene Ther. 2018; 29: 733-736Crossref PubMed Scopus (49) Google Scholar One concern of dystrophin replacement therapy is the induction of the cellular immune response by the newly generated dystrophin protein.4Mendell 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, 5Vila M.C. Novak J.S. Benny Klimek M. Li N. Morales M. Fritz A.G. Edwards K. Boehler J.F. Hogarth M.W. Kinder T.B. et al.Morpholino-induced exon skipping stimulates cell-mediated and humoral responses to dystrophin in mdx mice.J. Pathol. 2019; 248: 339-351PubMed Google Scholar While application of transient immune suppression at the time of gene therapy may minimize the concern of dystrophin immunity, avoiding dystrophin would be a more radical solution. Utrophin and dystrophin arise from the duplication of the same ancestral gene during early evolution of vertebrates. They share high sequence homology at both DNA and protein levels. In mature muscle, dystrophin is expressed at the sarcolemma, while utrophin is highly enriched at the neuromuscular junction and myotendinous junction. However, utrophin is expressed at the sarcolemma during early embryonic development and in regenerating myofibers. Structural similarity and overlapping distribution suggest that there may be functional redundancy between dystrophin and utrophin. Indeed, dystrophin and utrophin double knockout mice show a much more severe phenotype than mdx mice. The immune advantage of utrophin was first demonstrated in a side-by-side comparison study using an adenoviral vector that expressed either a mini-dystrophin gene or a mini-utrophin gene.6Ebihara 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 In immune-competent mdx mice, persistent expression was achieved with the mini-utrophin, but not mini-dystrophin, vector. Since adenoviral vectors are not suitable for systemic DMD gene therapy, the highly abbreviated micro-utrophin gene was engineered for AAV delivery (Figure 1). Song et al.2Song Y. Morales L. Malik A.S. Mead A.F. Greer C.D. Mitchell M.A. Petrov M.T. Su L.T. Choi M.E. Rosenblum S.T. et al.Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.Nat Med. 2019; 25: 1505-1511Crossref PubMed Scopus (39) Google Scholar built an AAV vector in which the optimized synthetic micro-utrophin gene was transcribed from the ubiquitous cytomegalovirus promoter (Figure 1). They then conducted blinded studies in neonatal mdx mice, newborn and infant golden retriever muscular dystrophy (GRMD) puppies, and adult German short-haired pointer muscular dystrophy (GSHPMD) dogs. GRMD dogs carry a splice site point mutation in intron 6 and exhibit rare dystrophin-positive revertant myofibers. The entire dystrophin gene is deleted in the genome of GSHPMD dogs and they therefore do not show revertant myofibers. Consistent with previous studies in the mouse model,1Odom G.L. Gregorevic P. Allen J.M. Finn E. Chamberlain J.S. Microutrophin delivery through rAAV6 increases lifespan and improves muscle function in dystrophic dystrophin/utrophin-deficient mice.Mol. Ther. 2008; 16: 1539-1545Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar intraperitoneal injection of the AAV micro-utrophin vector successfully prevented muscle disease in neonatal mdx mice.2Song Y. Morales L. Malik A.S. Mead A.F. Greer C.D. Mitchell M.A. Petrov M.T. Su L.T. Choi M.E. Rosenblum S.T. et al.Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.Nat Med. 2019; 25: 1505-1511Crossref PubMed Scopus (39) Google Scholar When systemic AAV micro-dystrophin therapy was tested at the dose of 1.5 × 1014 viral genome (vg) particles/kg in 4-day-old newborn GRMD puppies, treatment resulted in widespread micro-dystrophin expression in multiple muscles throughout the body without causing a T cell response. However, treated dogs (n = 2) showed weight loss, muscle atrophy, and contracture and had to be euthanized at the age of 120 days.7Kornegay J.N. Li J. Bogan J.R. Bogan D.J. Chen C. Zheng H. Wang B. Qiao C. Howard Jr., J.F. Xiao X. Widespread muscle expression of an AAV9 human mini-dystrophin vector after intravenous injection in neonatal dystrophin-deficient dogs.Mol. Ther. 2010; 18: 1501-1508Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar A similar response was observed in dystrophin/utrophin double knockout mice when the AAV micro-dystrophin vector was administered at the same dose. Interestingly, no adverse reactions were seen at a 10-fold lower dose.7Kornegay J.N. Li J. Bogan J.R. Bogan D.J. Chen C. Zheng H. Wang B. Qiao C. Howard Jr., J.F. Xiao X. Widespread muscle expression of an AAV9 human mini-dystrophin vector after intravenous injection in neonatal dystrophin-deficient dogs.Mol. Ther. 2010; 18: 1501-1508Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar The exact reason for this toxicity is not known, but the authors suspected that it might relate to an innate immune response triggered by supraphysiological-level human micro-dystrophin expression in dystrophic animals.7Kornegay J.N. Li J. Bogan J.R. Bogan D.J. Chen C. Zheng H. Wang B. Qiao C. Howard Jr., J.F. Xiao X. Widespread muscle expression of an AAV9 human mini-dystrophin vector after intravenous injection in neonatal dystrophin-deficient dogs.Mol. Ther. 2010; 18: 1501-1508Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar In the new micro-utrophin study, Song et al.2Song Y. Morales L. Malik A.S. Mead A.F. Greer C.D. Mitchell M.A. Petrov M.T. Su L.T. Choi M.E. Rosenblum S.T. et al.Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.Nat Med. 2019; 25: 1505-1511Crossref PubMed Scopus (39) Google Scholar performed intravenous injection in 6- to 10 day-old puppies at doses that were 15-fold (1.0 × 1013 vg/kg, n = 2) and ∼5-fold (3.16 × 1013 vg/kg, n = 2) lower than the dose used in the micro-dystrophin study.7Kornegay J.N. Li J. Bogan J.R. Bogan D.J. Chen C. Zheng H. Wang B. Qiao C. Howard Jr., J.F. Xiao X. Widespread muscle expression of an AAV9 human mini-dystrophin vector after intravenous injection in neonatal dystrophin-deficient dogs.Mol. Ther. 2010; 18: 1501-1508Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar The treated puppies were euthanized at the age of ∼130 days. In contrast to what was seen in the micro-dystrophin study, micro-utrophin treated puppies did not show weight loss. Consistent with the results of the micro-dystrophin study, no T cell response was detected in micro-utrophin-treated puppies. Micro-utrophin treatment normalized the myofiber size in the vastus lateralis muscle. However, there was no obvious improvement in muscle histology by H&E staining. No data were presented on whether systemic micro-utrophin therapy in newborn puppies reached all muscles in the body, for example, the diaphragm and the heart. Next, Song at al.2Song Y. Morales L. Malik A.S. Mead A.F. Greer C.D. Mitchell M.A. Petrov M.T. Su L.T. Choi M.E. Rosenblum S.T. et al.Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.Nat Med. 2019; 25: 1505-1511Crossref PubMed Scopus (39) Google Scholar performed systemic injection of the AAV micro-utrophin vector in two preweaning 7.5 week-old dogs. This age is equivalent to ∼2 years in humans. One dog received 5.0 × 1013 vg/kg vector and the other dog received a 2.5-fold higher dose (1.25 × 1014 vg/kg). Transient immune suppression (prednisolone 1 mg/kg) was applied starting 3 days before injection until 25 days after injection. Treated dogs were euthanized at 7 weeks after AAV administration. Robust micro-utrophin expression was detected in the biceps brachii, biceps femoris, cranial sartorius, diaphragm, temporalis, and the heart on western blot. Micro-utrophin expression restored the dystrophin-associated glycoprotein complex (sarcoglycans and dystroglycans) to the sarcolemma. Muscle histology was greatly improved on H&E staining. Calcification and embryonic myosin heavy chain-positive myofibers were completely eliminated. Masticatory muscles express MYH16, a unique powerful myosin isoform. MYH16 was reduced in untreated dogs but was restored following micro-utrophin therapy. Compared with that of pre-injection, the serum creatine kinase level dropped ∼5-fold by 1 week post-injection and stabilized thereafter. As has been reported in AAV micro-dystrophin therapy in the canine model,8Yue Y. Pan X. Hakim C.H. Kodippili K. Zhang K. Shin J.-H. Yang H.T. McDonald T. Duan D. 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, 9Shin J.-H. Pan X. Hakim C.H. Yang H.T. Yue Y. Zhang K. Terjung R.L. Duan D. Microdystrophin ameliorates muscular dystrophy in the canine model of duchenne muscular dystrophy.Mol. Ther. 2013; 21: 750-757Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 10Le 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 AAV micro-utrophin therapy did not induce a cellular immune response. To establish the immunological advantage of micro-utrophin therapy, Song et al.2Song Y. Morales L. Malik A.S. Mead A.F. Greer C.D. Mitchell M.A. Petrov M.T. Su L.T. Choi M.E. Rosenblum S.T. et al.Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.Nat Med. 2019; 25: 1505-1511Crossref PubMed Scopus (39) Google Scholar performed a side-by-side comparison in two 7 year-old GSHPMD dogs. One side of the tibialis muscle received the micro-utrophin vector (2.0 × 1012 vg/kg), and the contralateral muscle received an equal amount of the micro-dystrophin vector. Injected muscles were biopsied 4 weeks later. On H&E staining, the micro-dystrophin treated muscle showed severe dystrophic histology. In sharp contrast, dystrophic histology was greatly attenuated in the micro-utrophin-treated muscle. Immunostaining revealed robust micro-utrophin, but not micro-dystrophin, expression. Abundant CD3+ and CD8+ T cells were detected in the micro-dystrophin-treated, but not micro-utrophin-treated, muscle. In the interferon-γ ELISPOT assay, peripheral blood mononuclear cells responded to the micro-dystrophin peptide library at 2, 4, 6, and 8 weeks post-injection. However, no response was detected to the micro-utrophin peptide library. In summary, the results from Song et al.2Song Y. Morales L. Malik A.S. Mead A.F. Greer C.D. Mitchell M.A. Petrov M.T. Su L.T. Choi M.E. Rosenblum S.T. et al.Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.Nat Med. 2019; 25: 1505-1511Crossref PubMed Scopus (39) Google Scholar suggest that AAV-mediated micro-utrophin expression can prevent muscle disease in newborn mdx mice, avoid the untoward innate immune response in neonatal GRMD dogs, and mitigate pathological lesions without inducing the T cell response in adult dogs that lack the entire dystrophin gene. The Song et al.2Song Y. Morales L. Malik A.S. Mead A.F. Greer C.D. Mitchell M.A. Petrov M.T. Su L.T. Choi M.E. Rosenblum S.T. et al.Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.Nat Med. 2019; 25: 1505-1511Crossref PubMed Scopus (39) Google Scholar study has provided the much-needed large animal data for translating AAV micro-utrophin gene therapy. The dystrophin-independent nature of this therapy completely eliminates dystrophin-related immune reactions. Both dystrophin and utrophin are large complex proteins. While nearly 40 configurations of micro-dystrophin have been tested in animal models,11Duan D. Systemic AAV Micro-dystrophin Gene Therapy for Duchenne Muscular Dystrophy.Mol. Ther. 2018; 26: 2337-2356Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar only one form of micro-utrophin has been tested.1Odom G.L. Gregorevic P. Allen J.M. Finn E. Chamberlain J.S. Microutrophin delivery through rAAV6 increases lifespan and improves muscle function in dystrophic dystrophin/utrophin-deficient mice.Mol. Ther. 2008; 16: 1539-1545Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 2Song Y. Morales L. Malik A.S. Mead A.F. Greer C.D. Mitchell M.A. Petrov M.T. Su L.T. Choi M.E. Rosenblum S.T. et al.Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.Nat Med. 2019; 25: 1505-1511Crossref PubMed Scopus (39) Google Scholar It is possible that molecular engineering may yet yield a more potent micro-utrophin. All ongoing AAV micro-dystrophin gene therapy trials use a muscle-specific promoter to drive transgene expression,11Duan D. Systemic AAV Micro-dystrophin Gene Therapy for Duchenne Muscular Dystrophy.Mol. Ther. 2018; 26: 2337-2356Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar whereas Song et al.2Song Y. Morales L. Malik A.S. Mead A.F. Greer C.D. Mitchell M.A. Petrov M.T. Su L.T. Choi M.E. Rosenblum S.T. et al.Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.Nat Med. 2019; 25: 1505-1511Crossref PubMed Scopus (39) Google Scholar used the ubiquitous cytomegalovirus promoter. The authors tried to manufacture a vector that carries the muscle-specific Spc5-12 promoter, but vector yield was reduced by 10-fold. Future studies are needed to test a micro-utrophin vector that is expressed from an alternative muscle-specific promoter. More importantly, additional large sample-size studies are needed to measure muscle and heart function improvements in the canine model. It will also be important to conduct long-term studies to evaluate the durability of the therapy. Despite the structural similarity and functional redundancy, there are important differences between dystrophin and utrophin. Dystrophin can anchor neuronal nitric oxide synthase to the sarcolemma to prevent functional ischemia during muscle contraction, but utrophin cannot.12Li D. Bareja A. Judge L. Yue Y. Lai Y. Fairclough R. Davies K.E. Chamberlain J.S. Duan D. Sarcolemmal nNOS anchoring reveals a qualitative difference between dystrophin and utrophin.J. Cell Sci. 2010; 123: 2008-2013Crossref PubMed Scopus (71) Google Scholar, 13Lai 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 (283) Google Scholar Dystrophin can also organize the subsarcolemmal microtubule lattice, whereas utrophin cannot.14Prins K.W. Humston J.L. Mehta A. Tate V. Ralston E. Ervasti J.M. Dystrophin is a microtubule-associated protein.J. Cell Biol. 2009; 186: 363-369Crossref PubMed Scopus (141) Google Scholar, 15Belanto J.J. Mader T.L. Eckhoff M.D. Strandjord D.M. Banks G.B. Gardner M.K. Lowe D.A. Ervasti J.M. Microtubule binding distinguishes dystrophin from utrophin.Proc. Natl. Acad. Sci. USA. 2014; 111: 5723-5728Crossref PubMed Scopus (92) Google Scholar Microtubule lattice disorganization contributes to eccentric contraction-induced injury.15Belanto J.J. Mader T.L. Eckhoff M.D. Strandjord D.M. Banks G.B. Gardner M.K. Lowe D.A. Ervasti J.M. Microtubule binding distinguishes dystrophin from utrophin.Proc. Natl. Acad. Sci. USA. 2014; 111: 5723-5728Crossref PubMed Scopus (92) Google Scholar These differences may have important clinical consequences. Micro-dystrophin gene therapy is already being tested in DMD patients.3Duan D. Micro-dystrophin gene therapy goes systemic in Duchenne muscular dystrophy patients.Hum. Gene Ther. 2018; 29: 733-736Crossref PubMed Scopus (49) Google Scholar Song et al.2Song Y. Morales L. Malik A.S. Mead A.F. Greer C.D. Mitchell M.A. Petrov M.T. Su L.T. Choi M.E. Rosenblum S.T. et al.Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.Nat Med. 2019; 25: 1505-1511Crossref PubMed Scopus (39) Google Scholar have moved micro-utrophin gene therapy one step closer to a human trial. These gene therapies have brought hope to dramatically change the disease course and improve the quality of life of DMD patients. D.D. is a member of the scientific advisory board for Solid Biosciences and an equity holder of Solid Biosciences. D.D.'s lab has received research support from Solid Biosciences and Edgewise Therapeutics. The author thanks the support from the NIH (AR-70517 and NS-90634), the Jackson Freel DMD Research Fund, and Parent Project Muscular Dystrophy.