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AAV-Mediated Gene Therapy to the Isolated Limb in Rhesus Macaques

  • Louise R. Rodino-Klapac
  • Chrystal L. Montgomery
  • Jerry R. Mendell
  • Louis G. ChicoineEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 709)

Abstract

The development of a nonhuman primate (NHP) model for vascular delivery of therapeutic transgenes with adeno-associated viral (AAV) vectors is crucial for successfully treating muscular dystrophies. Current animal models for Duchenne muscular dystrophy (DMD) gene therapy have species limitations related to assessing function, immune response, and distribution of the micro- and minidystrophin transgenes in a clinically relevant manner. In addition, there are many forms of muscular dystrophy for which there are no available disease models. NHPs provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for local or broadly targeted gene therapy studies. The vascular anatomy NHPs more clearly parallels humans providing an appropriate substrate for translational experiments. Here we outline the development of a rhesus macaque isolated focal limb perfusion (IFLP) protocol targeting the vascular bed of the gastrocnemius. This protocol serves as a model with broad implications for other muscle diseases along with the capability of targeting multiple muscle groups. To overcome the partial homogeneity between portions of the human microdystrophin transgene and those of the NHP dystrophin gene, we utilized a FLAG tag for tracking distribution of microdystrophin. We also provide methods for assessing transduction efficiency of microdystrophin.FLAG following the IFLP vascular delivery protocol.

Key words

Adeno-associated virus (AAV) Gene therapy Muscle disease Duchenne muscular ­dystrophy Microdystrophin mdx Mice Nonhuman primates Isolated focal limb perfusion 

Notes

Acknowledgments

We thank the Viral Vector Core at Nationwide Children’s for vector production. Research supported by the Children’s Hospital Foundation (J.R.M.); National Institutes of Health U54 (1U54NS055958-01A1), Muscular Dystrophy Association (J.R.M.); Jesse’s Journey Foundation for Gene and Cell Therapy (J.R.M.), Ruth L. Kirschstein NRSA postdoctoral fellowship (1F32AR055008 to L.R.K.).

References

  1. 1.
    Rodino-Klapac, L. R., Chicoine, L. G., Kaspar, B. K., and Mendell, J. R. (2007). Gene therapy for duchenne muscular dystrophy: expectations and challenges. Arch Neurol 64, 1236–1241.PubMedCrossRefGoogle Scholar
  2. 2.
    Wang, Z., Chamberlain, J. S., Tapscott, S. J., and Storb, R. (2009). Gene therapy in large animal models of muscular dystrophy. ILAR J 50, 187–198.PubMedGoogle Scholar
  3. 3.
    Harper, S. Q., et al. (2002). Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Nat Med 8, 253–261.PubMedCrossRefGoogle Scholar
  4. 4.
    Liu, M., Yue, Y., Harper, S. Q., Grange, R. W., Chamberlain, J. S., and Duan, D. (2005). Adeno-associated virus-mediated microdystrophin expression protects young mdx muscle from contraction-induced injury. Mol Ther 11, 245–256.PubMedCrossRefGoogle Scholar
  5. 5.
    Rodino-Klapac, L. R., et al. (2007). A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy. J Transl Med 5, 45.PubMedCrossRefGoogle Scholar
  6. 6.
    Gregorevic, P., et al. (2006). rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice. Nat Med 12: 787–789.PubMedCrossRefGoogle Scholar
  7. 7.
    Yue, Y., Ghosh, A., Long, C., Bostick, B., Smith, B. F., Kornegay, J. N., et al. (2008) A single intravenous injection of adeno-associated virus serotype-9 leads to whole body skeletal muscle transduction in dogs. Mol Ther 16, 1944–1952.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Louise R. Rodino-Klapac
    • 1
  • Chrystal L. Montgomery
    • 1
  • Jerry R. Mendell
    • 1
  • Louis G. Chicoine
    • 1
    Email author
  1. 1.Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital and Department of PediatricsThe Ohio State UniversityColumbusUSA

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