The essential product of the Duchenne muscular dystrophy (DMD) gene is dystrophin1, a rod-like protein2 that protects striated myocytes from contraction-induced injury3,4. Dystrophin-related protein (or utrophin) retains most of the structural and protein binding elements of dystrophin5. Importantly, normal thymic expression in DMD patients6 should protect utrophin by central immunologic tolerance. We designed a codon-optimized, synthetic transgene encoding a miniaturized utrophin (µUtro), deliverable by adeno-associated virus (AAV) vectors. Here, we show that µUtro is a highly functional, non-immunogenic substitute for dystrophin, preventing the most deleterious histological and physiological aspects of muscular dystrophy in small and large animal models. Following systemic administration of an AAV-µUtro to neonatal dystrophin-deficient mdx mice, histological and biochemical markers of myonecrosis and regeneration are completely suppressed throughout growth to adult weight. In the dystrophin-deficient golden retriever model, µUtro non-toxically prevented myonecrosis, even in the most powerful muscles. In a stringent test of immunogenicity, focal expression of µUtro in the deletional-null German shorthaired pointer model produced no evidence of cell-mediated immunity, in contrast to the robust T cell response against similarly constructed µDystrophin (µDystro). These findings support a model in which utrophin-derived therapies might be used to treat clinical dystrophin deficiency, with a favorable immunologic profile and preserved function in the face of extreme miniaturization.
This is a preview of subscription content
Subscribe to Nature+
Get immediate online access to the entire Nature family of 50+ journals
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
All data generated or analyzed during this study are included in this published article or in the Supplementary Information files.
Hoffman, E. P., Brown, R. H. Jr. & Kunkel, L. M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51, 919–928 (1987).
Koenig, M., Monaco, A. P. & Kunkel, L. M. The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 53, 219–226 (1988).
Petrof, B. J., Shrager, J. B., Stedman, H. H., Kelly, A. M. & Sweeney, H. L. Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc. Natl Acad. Sci. USA 90, 3710–3714 (1993).
Ibraghimov-Beskrovnaya, O. et al. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature 355, 696–702 (1992).
Tinsley, J. M. et al. Primary structure of dystrophin-related protein. Nature 360, 591–593 (1992).
Mesnard-Rouiller, L., Bismuth, J., Wakkach, A., Poea-Guyon, S. & Berrih-Aknin, S. Thymic myoid cells express high levels of muscle genes. J. Neuroimmunol. 148, 97–105 (2004).
Clemens, P. R. et al. In vivo muscle gene transfer of full-length dystrophin with an adenoviral vector that lacks all viral genes. Gene Ther. 3, 965–972 (1996).
Wang, 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 97, 13714–13719 (2000).
Harper, S. Q. et al. Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Nat. Med. 8, 253–261 (2002).
Gregorevic, P. et al. Systemic delivery of genes to striated muscles using adeno-associated viral vectors. Nat. Med. 10, 828–834 (2004).
Gregorevic, P. et al. rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice. Nat. Med. 12, 787–789 (2006).
Moore, M. J. & Flotte, T. R. Autoimmunity in a genetic disease—a cautionary tale. N. Engl. J. Med. 363, 1473–1475 (2010).
Mendell, J. R. et al. Dystrophin immunity in Duchenne’s muscular dystrophy. N. Engl. J. Med. 363, 1429–1437 (2010).
Kornegay, J. N. et al. Widespread muscle expression of an AAV9 human mini-dystrophin vector after intravenous injection in neonatal dystrophin-deficient dogs. Mol. Ther. 18, 1501–1508 (2010).
Duan, D. & Systemic, A. A. V. Micro-dystrophin gene therapy for Duchenne muscular dystrophy. Mol. Ther. 26, 2337–2356 (2018).
Muthu, M., Richardson, K. A. & Sutherland-Smith, A. J. The crystal structures of dystrophin and utrophin spectrin repeats: implications for domain boundaries. PLoS One 7, e40066 (2012).
Ortega, E. et al. The structure of the plakin domain of plectin reveals an extended rod-like shape. J. Biol. Chem. 291, 18643–18662 (2016).
Ishikawa-Sakurai, M., Yoshida, M., Imamura, M., Davies, K. E. & Ozawa, E. ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan. Hum. Mol. Genet. 13, 693–702 (2004).
Hnia, K. et al. ZZ domain of dystrophin and utrophin: topology and mapping of a beta-dystroglycan interaction site. Biochem. J. 401, 667–677 (2007).
Zinn, E. et al. In silico reconstruction of the viral evolutionary lineage yields a potent gene therapy vector. Cell Rep. 12, 1056–1068 (2015).
Foster, H. et al. Codon and mRNA sequence optimization of microdystrophin transgenes improves expression and physiological outcome in dystrophic mdx mice following AAV2/8 gene transfer. Mol. Ther. 16, 1825–1832 (2008).
Odom, 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. 16, 1539–1545 (2008).
Sonnemann, K. J. et al. Functional substitution by TAT-utrophin in dystrophin-deficient mice. PLoS Med. 6, e1000083 (2009).
Kennedy, T. L. et al. Micro-utrophin improves cardiac and skeletal muscle function of severely affected D2/mdx mice. Mol. Ther. Methods Clin. Dev. 11, 92–105 (2018).
Song, Y. et al. Suite of clinically relevant functional assays to address therapeutic efficacy and disease mechanism in the dystrophic mdx mouse. J. Appl. Physiol. 122, 593–602 (2017).
Kobayashi, Y. M. et al. Sarcolemma-localized nNOS is required to maintain activity after mild exercise. Nature 456, 511–515 (2008).
Nichols, T. et al. Translational data from adeno-associated virus-mediated gene therapy of hemophilia B in dogs. Hum. Gene Ther. Clin. Dev. 26, 5–14 (2015).
Calcedo, R. et al. Adeno-associated virus antibody profiles in newborns, children and adolescents. Clin. Vaccine Immunol. 18, 1586–1588 (2011).
Yiu, E. M. & Kornberg, A. J. Duchenne muscular dystrophy. J. Paediatr. Child Health 51, 759–764 (2015).
Liu, J. M. et al. Effects of prednisone in canine muscular dystrophy. Muscle Nerve 30, 767–773 (2004).
Stedman, H. H. et al. Myosin gene mutation correlates with anatomical changes in the human lineage. Nature 428, 415–418 (2004).
Toniolo, L. et al. Masticatory myosin unveiled: first determination of contractile parameters of muscle fibers from carnivore jaw muscles. Am. J. Physiol. Cell Physiol. 295, C1535–C1542 (2008).
Hinderer, C. et al. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an adeno-associated virus vector expressing human SMN. Hum. Gene Ther. 29, 285–298 (2018).
Hordeaux, J. et al. The neurotropic properties of AAV-PHP.B are limited to C57BL/6J mice. Mol. Ther. 26, 664–668 (2018).
Stedman, H. H. et al. The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature 352, 536–539 (1991).
Schatzberg, S. J. et al. Molecular analysis of a spontaneous dystrophin ‘knockout’ dog. Neuromuscul. Disord. 9, 289–295 (1999).
VanBelzen, D. J., Malik, A. S., Henthorn, P. S., Kornegay, J. N. & Stedman, H. H. Mechanism of deletion removing all dystrophin exons in a canine model for DMD implicates concerted evolution of X chromosome pseudogenes. Mol. Ther. Methods Clin. Dev. 4, 62–71 (2017).
Schatzberg, S. J. et al. Alternative dystrophin gene transcripts in golden retriever muscular dystrophy. Muscle Nerve 21, 991–998 (1998).
Yue, Y. et al. Safe and bodywide muscle transduction in young adult Duchenne muscular dystrophy dogs with adeno-associated virus. Hum. Mol. Genet. 24, 5880–5890 (2015).
Le Guiner, C. et al. Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy. Nat. Commun. 8, 16105 (2017).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).
Solovyev, V., Kosarev, P., Seledsov, I. & Vorobyev, D. Automatic annotation of eukaryotic genes, pseudogenes and promoters. Genome Biol. 7, S10.11–S10.12 (2006).
Shi, Y., Falahati, R., Zhang, J., Flebbe-Rehwaldt, L. & Gaensler, K. M. Role of antigen-specific regulatory CD4+ CD25+ T cells in tolerance induction after neonatal IP administration of AAV-hF.IX. Gene Ther. 20, 987–996 (2013).
Davey, M. G. et al. Induction of immune tolerance to foreign protein via adeno-associated viral vector gene transfer in mid-gestation fetal sheep. PLoS One 12, e0171132 (2017).
Vandenberghe, L. H. et al. Efficient serotype-dependent release of functional vector into the culture medium during adeno-associated virus manufacturing. Hum. Gene Ther. 21, 1251–1257 (2010).
Lock, M., Alvira, M. R. & Wilson, J. M. Analysis of particle content of recombinant adeno-associated virus serotype 8 vectors by ion-exchange chromatography. Hum. Gene Ther. Methods 23, 56–64 (2012).
Zincarelli, C., Soltys, S., Rengo, G. & Rabinowitz, J. E. Analysis of AAV serotypes 1–9 mediated gene expression and tropism in mice after systemic injection. Mol. Ther. 16, 1073–1080 (2008).
Song, Y., Forsgren, S., Yu, J., Lorentzon, R. & Stal, P. S. Effects on contralateral muscles after unilateral electrical muscle stimulation and exercise. PloS One 7, e52230 (2012).
Mishra, M. K., Loro, E., Sengupta, K., Wilton, S. D. & Khurana, T. S. Functional improvement of dystrophic muscle by repression of utrophin: let-7c interaction. PLoS One 12, e0182676 (2017).
Mingozzi, F. et al. AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells. Blood 114, 2077–2086 (2009).
Aarts, E., Verhage, M., Veenvliet, J. V., Dolan, C. V. & van der Sluis, S. A solution to dependency: using multilevel analysis to accommodate nested data. Nat. Neurosci. 17, 491–496 (2014).
Li, X., Eastman, E. M., Schwartz, R. J. & Draghia-Akli, R. Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences. Nat. Biotechnol. 17, 241–245 (1999).
This work was supported by grants from the NIH (H.S., R01NS042874, U01NS052476 and S10RR028027; L.M., T32AR053461; C.D.G., T32HL007954; J.K., U24NS059696; A.M., F.B. and M.P., T32AR053461; Y.S. and histology, P30AR050950), the Muscular Dystrophy Association (H.S. and J.K.) and the families of O. Soot and M. Haider, to whom this work is dedicated. Additional services were supported by P30DK047757 and the NHLBI Gene Therapy Resource Center as well as the University of Pennsylvania Vector and Immunology Cores. We thank L. Vandenberghe for providing helper plasmid (AAP2) for Anc80 production, A. Stout and J. Zhao as well as the UPenn CDB microscopy core for their technical assistance and resource access, and V. Arruda, J. Bennett, J. Johnston, R. Calcedo, F. Wright and J. Plotkin for support and expertise. Dedicated to the memory of Mohammed Haider and Beth Stedman.
H.H.S. is an inventor on several patents assigned to the University of Pennsylvania and subject to the institutional patent policies, which may include royalty distribution in the event of licensure. None of the patents are currently licensed, their potential valuation may go up or down with the publication of this manuscript, all are in the public domain, some have recently expired.
Peer review information Brett Benedetti and Kate Gao were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Evolution of dystrophin and utrophin and the framework for therapeutic vector design.
a, Reconstructed timeline of the dystrophin/utrophin divergence illustrating the timing of two independent duplications and three subsequent lineage-specific deletions (&, @, $, %, #). The timing of the dystrophin/utrophin duplication correlates with the emergence of hinged jaws, oligodendrocytes and membrane in-folding at the neuromuscular junctional site where utrophin is most highly expressed. MYA, million years ago. b, Delineation of domain conservation relative to recombinant proteins compatible with the size limit for AAV transgenes, including dystrophin-based constructs in early-stage clinical investigation and others described in the text. ClinicalTrials.gov reference numbers: *NCT03362502, ~NCT02376816 and ^NCT03368742. Symbols (@, $, %, #) indicate the positions of naturally occurring lineage-specific deletions; localization of epitopes recognized by anti-utrophin antibodies Utro N and C are depicted at the extreme lower edge of the figure.
a, Schematic representation of μUtro based on CnN3D renderings of calponin homology domain (green), four spectrin-like repeats (yellow/blue) and combined WW-EF hand-ZZ domain (red). Disordered domains (‘hinges’) 1, 2 and 4 not depicted. a′, Western blot analysis and quantification of μUtro expression in HEK 293 cells using eight distinct plasmid vectors. a′, Western blot analysis and quantification of μUtro expression one week post intramuscular injections in the tibialis anterior (TA) followed by electroporation of six distinct μUtro containing vectors, as labeled (UTO, μ-Utro cDNA; opt, optimized; SP, synthetic promoter C5-1253; loading control, α-actin; CMV, cytomegalovirus promoter). b,c, Immunofluorescence staining against utrophin N terminus (Utro_N) and laminin in mdx mice injected with either Anc80-μUtro or PBS. Muscles collected include diaphragm, heart (b), quadriceps (quad), TA and gastrocnemius (GM) (c). d, Western blot analysis of μUtro expression in tissue samples, with vinculin serving as a loading control. e, Western blot analysis comparing μUtro expression in the TA and GM after systemic deliver using an AAV9 or Anc80 vector. Vinculin serving as a loading control. (See Source Data for full uncropped gel images.) All experiments were repeated independently at least two or more times with similar results. (See Source Data Extended Data Fig. 2).
Extended Data Fig. 3 Neonatal systemic administration of AAV-μUtro in mdx mice shows histopathological amelioration in limb and diaphragm muscle eight weeks post injection.
a, Double immunofluorescence staining against an epitope shared by native and recombinant utrophin (Utro_N) and an epitope unique to native utrophin (Utro_C) of the entire tibialis anterior muscle. Scale bar, 100 μm. b, Representative immunofluorescence staining of sectioned diaphragm35 at two distinct vector doses per mouse. Scale bar, 50 μm. All experiments were repeated independently at least two times with similar results.
Extended Data Fig. 4 Neonatal systemic administration of AAV-µUtro eliminates active proteolysis, apoptosis and centrally nucleated myofibers in eight-week-old mdx limb muscle.
a, Representative immunofluorescence staining with MuRF1 (top panel) and TUNEL (bottom panel). Scale bar, 50 μm. b, Table summarizing mean and s.d. of centrally nucleated myofibers (Fig. 1d), MuRF1, TUNEL and embryonic positive myofibers (for example, see Fig. 1a) in mdx + PBS, mdx + AAV-µUtro and wild type + PBS. Data are presented as mean ± s.d.
Extended Data Fig. 5 Systemic delivery of AAV-µUtro in neonatal mdx mice provides sustained expression of µUtro and results in in vivo and ex vivo functional improvement.
a, Quantification of µUtro expression via western blot, at eight (n = 2 mice) and 16 weeks (n = 2 mice) of age, in GM, quad (quadriceps) and heart muscle. (See Source Data for full uncropped gel images.) b, Quantification of total distance covered on a running wheel over a 24 h time period by mdx + AAV-µUtro (n = 3) 3,614.11 ± 300.08 and mdx + PBS (n = 2) 1,848.96 ± 259.15. Distance was covered via downhill treadmill until cessation (# criteria for constituting cessation in the Methods). The dashed line represents distance covered during acclimatization prior to a ramped speed increase. Wild type (n = 4) 183 ± 0, mdx + AAV-µUtro (n = 4) 174.05 ± 17.9 and mdx + PBS (n = 5) 122.28 ± 24.12. Data are presented as mean ± s.d. Each symbol represents data from an individual animal and the wide horizontal line represents the group mean with associated error bars representing s.d. NS, not significant; **P < 0.01; statistical significance was assessed by one-way ANOVA with Tukey test multiple group comparison. c, Absolute force after seven cycles of eccentric contractions (ECCs) of extensor digitorum longus (EDL) muscle in mdx + AAV-µUtro mice (n = 10) and mdx + PBS mice (n = 9). Boxed values represent the fold difference of absolute force for mdx + AAV-µUtro (ECC1 210.37 ± 73.86, ECC2 128.36 ± 76.6, ECC3 115.54 ± 57.08, ECC4 101.83 ± 49.34, ECC5 89.28 ± 45.41, ECC6 78.21 ± 38.08) and mdx + PBS (ECC1 98.19 ± 48.78, ECC2 28.46 ± 16.81, ECC3 26.93 ± 14.91, ECC4 19.8 ± 14.74, ECC5 15 ± 12.8, ECC6 13.58, s.d. = 10.57). Data are presented as mean ± s.d. Each symbol represents data from an individual animal and the wide horizontal line represents the group mean with associated error bars representing s.d. **P < 0.01, ***P < 0.001; statistical significance was assessed by two-tailed unpaired t-test. d, Seven serial whole limb force measurements of wild type (n = 6), mdx + AAV-µUtro (n = 8) and mdx + PBS (n = 11). (See Source Data Extended Data Fig. 5).
Extended Data Fig. 6 Normal growth of GRMD dogs randomized to AAV-µUtro as evidence against immune-mediated myositis.
a, Individual weights of dogs randomized to the highest doses (3.16 × 1013 vg kg−1) of AAV-cU (AAV-μUtro) without immunosuppression (F[b] AAV-cU, F[c] AAV-cU), as well as relevant controls including littermates randomized to PBS and other littermate carrier females (F-carrier3, F-carrier4, F-carrier6) and non-littermate GRMD males (M-GRMD7, M-GRMD8) and females (F-GRMD9, F-GRMD10). Also included for comparison are relevant weights of previously reported GRMD females receiving human μDystro (F[e] AAV-hD, F[f] AAV-hD) showing rapid weight loss immediately prior to euthanasia and necropsy showing signs of systemic myositis14. b, Representative results of PCR assays for GRMD genotype. c, Graphical depiction of Animal ID#, creatine kinase (CK) levels at birth, GRMD status by PCR, and weight at two time points for littermates of AAV-μUtro injected dogs.
Extended Data Fig. 7 Homogeneously expressed recombinant µUtro normalizes β-sarcoglycan, reverses myopathology and normalizes fiber size in GRMD dogs after systemic gene delivery.
a, Immunofluorescence staining detecting the utrophin N terminus (Utro N, red) and β-sarcoglycan (green) of vastus lateralis muscle from wild type (WT), treated (AAV-μUtro) or untreated (PBS). The experiment was repeated independently at least two times with similar results. b, H&E staining of vastus lateralis muscle biopsies. Scale bar, 100 μm c, Distribution of minimum Feret diameter of myofibers in vastus lateralis muscle biopsies pooled from age-matched wild type (n = 920) 19 ± 4.01, GRMD littermates randomized to AAV-μUtro (n = 758) 17.63 ± 4.9, and PBS (n = 1,014) 14.49 ± 7.24. Data are presented as mean ± s.d. Coefficients of variation for all groups are reported in the box. d, Cumulative distribution (percentage) plot of minimum Feret diameter from c. e, Distribution of minimum Feret diameter of myofibers in vastus lateralis muscle biopsies obtained at necropsy, wild type (n = 569) 28.26 ± 8.39, AAV-μUtro (n = 542) 28.4 ± 7.54 and PBS (n = 317) 32.26 ± 12.01. Data are presented as mean ± s.d. Coefficients of variation for all groups are reported in the box. f, Cumulative distribution (percentage) plot of minimum Feret diameter from e. Coefficients of variation for all groups are reported in the box. (See Source Data Extended Data Fig. 7).
Extended Data Fig. 8 µUtro expression provides histological improvement without any signs of toxicity in injected GRMD dogs.
a, Representative images of temporalis muscle from age-matched AVV-μUtro (treated) and PBS (untreated) GRMD dogs stained against an N-terminal epitope shared by native and recombinant utrophin (Utro N) and a C-terminal epitope unique to native utrophin (Utro C) (top) and MYH16, BA-F8 (slow twitch myofiber) and Utro N (bottom). Scale bar, 20 μm. Experiment was repeated independently at least two or more times with similar results. b, H&E stain. Top: A whole view of vastus lateralis muscle obtained from a muscle biopsy four weeks post-injection. Botom: A higher magnification of the corresponding boxed region. The experiment was repeated independently at least two times with similar results. c, γ-Interferon production was quantified by counting the spot forming units (SFUs) per million peripheral blood mononuclear cells (PBMCs). A response above the dotted line is considered positive and above the background signal. Peptide libraries used represent the full sequence of AAV9 capsid (A–C), μUtro (E–J), Adenovirus5 (1–4) and LacZ (1–4). d, Measurement of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), hemoglobin, platelets and γ-interferon before and after AAV-μUtro injection in two juvenile GRMD dogs (Hann/Beetle). Non-human primate (16C176 and RA2152) data are incorporated to illustrate the absence of disseminated intravascular coagulation (DIC)33,34. (See Source Data Extended Data Fig. 7).
Extended Data Fig. 9 μDystro, but not μUtro, elicits a local cell mediated immune response after an intramuscular injection in the GSHPMD dogs.
Muscle biopsies were obtained four weeks post-intramuscular injection of μDystro and μUtro. a, Representative H&E stain of muscle biopsies. Right panel for each dog shows higher magnification of the corresponding boxed region. Scale bar, 50 μm. b, Immunofluorescence staining of muscle biopsies against Utro_N and Utro_C. Scale bar, 50 μm. c, Immunofluorescence staining of muscle biopsies against CD3 and CD8; the bottom merged panel offers a validation of both antibodies. All experiments were repeated independently at least two times with similar results.
Unprocessed western blots/gels.
Statistical source data.
Statistical source data.
Unprocessed western blots/gels.
Statistical source data.
Unprocessed western blots/gels.
Unprocessed western blots/gels.
Statistical source data.
Statistical source data.
Statistical source data.
About this article
Cite this article
Song, Y., Morales, L., Malik, A.S. et al. Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models. Nat Med 25, 1505–1511 (2019). https://doi.org/10.1038/s41591-019-0594-0
Therapy development for spinal muscular atrophy: perspectives for muscular dystrophies and neurodegenerative disorders
Neurological Research and Practice (2022)
Therapeutic potential of highly functional codon-optimized microutrophin for muscle-specific expression
Scientific Reports (2022)
Evaluating the potential of novel genetic approaches for the treatment of Duchenne muscular dystrophy
European Journal of Human Genetics (2021)
Duchenne muscular dystrophy cell culture models created by CRISPR/Cas9 gene editing and their application in drug screening
Scientific Reports (2021)
Nature Reviews Drug Discovery (2020)