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Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models


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.

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Fig. 1: AAV-µUtro treatment restores the DGC, prevents myofiber degeneration, normalizes serum CK level and improves muscle function in mdx mice.
Fig. 2: Systemic delivery of AAV-µUtro in GRMD dogs at seven weeks of age prevents myonecrosis and results in rapid reduction of serum CK levels.
Fig. 3: Widespread expression of μUtro rescues the DGC proteins in treated GRMD dogs after systemic delivery at the age of seven weeks.
Fig. 4: Focal expression of μDystro, but not μUtro, elicits a detectable peripheral and local immune response in the GSHP dystrophin deletional-null dog model.

Data availability

All data generated or analyzed during this study are included in this published article or in the Supplementary Information files.


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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.

Author information




All authors contributed to data acquisition and analysis. Y.S., L.M., A.F.M. and H.H.S. designed the experiments. Dystrophin/utrophin phylogeny was determined by A.F.M. and H.H.S., vector optimization was carried out by A.S.M, M.A.M. and F.J.B., animal experiments by M.T.P., L.M., Y.S., P.P.N, M.E.C., S.T.R., J.N.K. and H.H.S., tissue preparation by Y.S., M.T.P., L.M., M.E.C. and R.F., immunohistochemistry and microscopy by Y.S. and L.M., western blot by X.L., Y.S., C.D.G. and L.M., muscle functional assays by Y.S., M.T.P., L.M., S.T.R. and E.L., Elispot by L.M. and A.F.M., data analysis by Y.S., C.D.G., L.M., M.E.C., M.T.P., R.F., S.T.R. and T.S.K. and the manuscript was written by L.M., Y.S., A.F.M., M.T.P., C.D.G., S.T.R. and H.H.S. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Hansell H. Stedman.

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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.

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Extended data

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. 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.

Extended Data Fig. 2 Optimization of canine μUtro transgene expression.

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). Source data

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). Source data Source data

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). Source data

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). Source data

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.

Supplementary information

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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).

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