Molecular Medicine

, Volume 18, Issue 3, pp 466–476 | Cite as

Effect of Nuclear Factor κB Inhibition on Serotype 9 Adeno-Associated Viral (AAV9) Minidystrophin Gene Transfer to the mdx Mouse

  • Daniel P Reay
  • Gabriela A Niizawa
  • Jon F Watchko
  • Molly Daood
  • Ja’Nean C Reay
  • Eugene Raggi
  • Paula R Clemens
Research Article


Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-κB signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that inhibition of pathological NF-κB activation in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. Systemic gene transfer using serotype 9 adeno-associated viral (AAV9) vectors is promising for treatment of preclinical models of DMD because of vector tropism to cardiac and skeletal muscle. In quadriceps of C57BL/10ScSn-Dmdmdx/J (mdx) mice, the addition of octalysine (8K)-NF-κB essential modulator (NEMO)-binding domain (8K-NBD) peptide treatment to AAV9 minidystrophin gene delivery resulted in increased levels of recombinant dystrophin expression suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of expression. Indices of necrosis and regeneration were diminished with AAV9 gene delivery alone and to a greater degree with the addition of 8K-NBD treatment. In diaphragm muscle, high-level transgene expression was achieved with AAV9 minidystoophin gene delivery alone; therefore, improvements in histological and physiological indices were comparable in the two treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer, which may be highly significant for clinical applications of muscle gene delivery.



This work was supported by a Department of Veterans Affairs (VA) Merit Review Grant (PR Clemens). We extend special thanks to Xiao Xiao, Bing Wang and Hiroyuki Nakai for the expression cassette plasmid and technical advice on AAV9 minidystrophin vector production. The authors take full responsibility for the contents of this report, which do not represent the views of the Department of Veterans Affairs or the U.S. Government.


  1. 1.
    Emery AE. (1991) Population frequencies of inherited neuromuscular diseases: a world survey. Neuromuscul. Disord. 1:19–29.CrossRefGoogle Scholar
  2. 2.
    Emery AEH, Muntoni F. (2003) Duchenne Muscular Dystrophy. 3rd edition. Oxford; New York: Oxford University Press. 270 pp.Google Scholar
  3. 3.
    Ervasti JM, Campbell KP. (1993) Dystrophin-associated glycoproteins: their possible roles in the pathogenesis of Duchenne muscular dystrophy. Mol. Cell. Biol. Hum. Dis. Ser. 3:139–66.PubMedGoogle Scholar
  4. 4.
    Hoffman E, Brown R, Kunkel L. (1987) Dystophin: the protein product of the Duchenne Muscular Dystrophy locus. Cell. 51:919–28.CrossRefGoogle Scholar
  5. 5.
    Zubrzycka-Gaarn EE, et al. (1988) The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature. 333:466–9.CrossRefGoogle Scholar
  6. 6.
    Bulfield G, Siller WG, Wight PA, Moore KJ. (1984) X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc. Natl. Acad. Sci. U. S. A. 81:1189–92.CrossRefGoogle Scholar
  7. 7.
    Wang B, Li J, Xiao X. (2000) Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model. Proc. Natl. Acad. Sci. U. S. A. 97:13714–9.CrossRefGoogle Scholar
  8. 8.
    Watchko J, et al. (2002) Adeno-associated virus vector-mediated minidystrophin gene therapy improves dystrophic muscle contractile function in mdx mice. Hum. Gene Ther. 13:1451–60.CrossRefGoogle Scholar
  9. 9.
    Inagaki K, et al. (2006) Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol. Ther. 14:45–53.CrossRefGoogle Scholar
  10. 10.
    Zincarelli C, Soltys S, Rengo G, Rabinowitz JE. (2008) Analysis of AAV serotypes 1–9 mediated gene expression and tropism in mice after systemic injection. Mol. Ther. 16:1073–80.CrossRefGoogle Scholar
  11. 11.
    Eghtesad S, Zheng H, Nakai H, Epperly MW, Clemens PR. (2010) Effects of irradiating adult mdx mice before full-length dystrophin cDNA transfer on host anti-dystrophin immunity. Gene Ther. 17:1181–90.CrossRefGoogle Scholar
  12. 12.
    Zaiss AK, Muruve DA. (2008) Immunity to adeno-associated virus vectors in animals and humans: a continued challenge. Gene Ther. 15:808–16.CrossRefGoogle Scholar
  13. 13.
    Acharyya S, et al. (2007) Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy. J. Clin. Invest. 117:889–901.CrossRefGoogle Scholar
  14. 14.
    Kumar A, Boriek AM. (2003) Mechanical stress activates the nuclear factor-kappaB pathway in skeletal muscle fibers: a possible role in Duchenne muscular dystrophy. FASEB J. 17:386–96.CrossRefGoogle Scholar
  15. 15.
    Messina S, et al. (2006) Lipid peroxidation inhibition blunts nuclear factor-kappaB activation, reduces skeletal muscle degeneration, and enhances muscle function in mdx mice. Am. J. Pathol. 168:918–26.CrossRefGoogle Scholar
  16. 16.
    Messina S, et al. (2006) Nuclear factor kappa-B blockade reduces skeletal muscle degeneration 476 and enhances muscle function in Mdx mice. Exp. Neurol. 198:234–41.CrossRefGoogle Scholar
  17. 17.
    Monici MC, Aguennouz M, Mazzeo A, Messina C, Vita G. (2003) Activation of nuclear factor-kappaB in inflammatory myopathies and Duchenne muscular dystrophy. Neurology. 60:993–7.CrossRefGoogle Scholar
  18. 18.
    Reay DP, et al. (2011) Systemic delivery of NEMO binding domain/IKKgamma inhibitory peptide to young mdx mice improves dystrophic skeletal muscle histopathology. Neurobiol. Dis. 43:598–608.CrossRefGoogle Scholar
  19. 19.
    Tang Y, et al. (2010) Inhibition of the IKK/NF-kappaB pathway by AAV gene transfer improves muscle regeneration in older mdx mice. Gene Ther. 17:1476–83.CrossRefGoogle Scholar
  20. 20.
    Rothwarf DM, Karin M. (1999) The NF-kappa B activation pathway: a paradigm in information transfer from membrane to nucleus. Sci. STKE. 1999:RE1.PubMedGoogle Scholar
  21. 21.
    Peterson JM, et al. (2011) Peptide-based inhibition of NF-kappaB rescues diaphragm muscle contractile dysfunction in a murine model of Duchenne muscular dystrophy. Mol. Med. 17:508–15.CrossRefGoogle Scholar
  22. 22.
    Tilstra J, et al. (2007) Protein transduction: identification, characterization and optimization. Biochem. Soc. Trans. 35:811–5.CrossRefGoogle Scholar
  23. 23.
    Kornegay JN, et al. (2010) Widespread muscle expression of an AAV9 human mini-dystrophin vector after intravenous injection in neonatal dystrophin-deficient dogs. Mol. Ther. 18:1501–8.CrossRefGoogle Scholar
  24. 24.
    Watchko JF, Johnson BD, Gosselin LE, Prakash YS, Sieck GC. (1994) Age-related differences in diaphragm muscle injury after lengthening activations. J. Appl. Physiol. 77:2125–33.CrossRefGoogle Scholar
  25. 25.
    Pan D, et al. (2002) Biodistribution and toxicity studies of VSVG-pseudotyped lentiviral vector after intravenous administration in mice with the observation of in vivo transduction of bone marrow. Mol. Ther. 6:19–29.CrossRefGoogle Scholar
  26. 26.
    Martinez-Amat A, et al. (2005) Release of alpha-actin into serum after skeletal muscle damage. Br. J. Sports Med. 39:830–4.CrossRefGoogle Scholar
  27. 27.
    Stedman HH, et al. (1991) The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature. 352:536–9.CrossRefGoogle Scholar
  28. 28.
    Bostick B, et al. (2008) Adeno-associated virus serotype-9 microdystrophin gene therapy ameliorates electrocardiographic abnormalities in mdx mice. Hum. Gene Ther. 19:851–6.CrossRefGoogle Scholar
  29. 29.
    Shin JH, et al. (2011) Improvement of cardiac fibrosis in dystrophic mice by rAAV9-mediated microdystrophin transduction. Gene Ther. 18:910–9.CrossRefGoogle Scholar
  30. 30.
    Yue Y, et al. (2003) Microdystrophin gene therapy of cardiomyopathy restores dystrophin-glycoprotein complex and improves sarcolemma integrity in the mdx mouse heart. Circulation. 108:1626–32.CrossRefGoogle Scholar
  31. 31.
    Janssen PM, Hiranandani N, Mays TA, Rafael-Fortney JA. (2005) Utrophin deficiency worsens cardiac contractile dysfunction present in dystrophin-deficient mdx mice. Am. J. Physiol. Heart Circ. Physiol. 289:H2373–8.CrossRefGoogle Scholar
  32. 32.
    Xu Y, Delfin DA, Rafael-Fortney JA, Janssen PM. (2011) Lengthening-contractions in isolated myocardium impact force development and worsen cardiac contractile function in the mdx mouse model of muscular dystrophy. J. Appl. Physiol. 110:512–9.CrossRefGoogle Scholar
  33. 33.
    Sapp JL, Bobet J, Howlett SE. (1996) Contractile properties of myocardium are altered in dystrophin-deficient mdx mice. J. Neurol. Sci. 142:17–24.CrossRefGoogle Scholar
  34. 34.
    Townsend D, et al. (2007) Systemic administration of micro-dystrophin restores cardiac geometry and prevents dobutamine-induced cardiac pump failure. Mol. Ther. 15:1086–92.CrossRefGoogle Scholar
  35. 35.
    Qin L, et al. (1997) Promoter attenuation in gene therapy: interferon-gamma and tumor necrosis factor-alpha inhibit transgene expression. Hum. Gene Ther. 8:2019–29.CrossRefGoogle Scholar
  36. 36.
    Machado RV, et al. (2011) Eicosapentaenoic acid decreases TNF-alpha and protects dystrophic muscles of mdx mice from degeneration. J. Neuroimmunol. 232:145–50.CrossRefGoogle Scholar

Copyright information

© The Author(s) 2012

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it.

The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this license, visit (

Authors and Affiliations

  • Daniel P Reay
    • 1
    • 2
  • Gabriela A Niizawa
    • 1
    • 2
  • Jon F Watchko
    • 3
  • Molly Daood
    • 3
  • Ja’Nean C Reay
    • 2
    • 4
  • Eugene Raggi
    • 2
  • Paula R Clemens
    • 1
    • 2
  1. 1.Neurology ServiceDepartment of Veterans Affairs Medical CenterPittsburghUSA
  2. 2.Department of NeurologyUniversity of PittsburghPittsburghUSA
  3. 3.Department of Pediatrics, Magee-Women’s Research InstituteUniversity of PittsburghPittsburghUSA
  4. 4.Lake Erie College of Osteopathic Medicine (LECOM) at Seton Hill UniversityGreensburgUSA

Personalised recommendations