Whole Body Skeletal Muscle Transduction in Neonatal Dogs with AAV-9

  • Yongping Yue
  • Jin-Hong Shin
  • Dongsheng Duan
Part of the Methods in Molecular Biology book series (MIMB, volume 709)


Gene therapy of muscular dystrophy requires systemic gene delivery to all muscles in the body. Adeno-associated viral (AAV) vectors have been shown to lead to body-wide muscle transduction after a single intravascular injection. Proof-of-principle has been demonstrated in mouse models of Duchenne muscular dystrophy and limb girdle muscular dystrophy. Before initiating clinical trials, it is important to validate these promising results in large animal models. More than a dozen canine muscular dystrophy models have been developed. Here, we outline a protocol for performing systemic AAV gene transfer in neonatal dogs. Implementing this technique in dystrophic dogs will accelerate translational muscular dystrophy research.

Key words

AAV: adeno-associated virus Muscular dystrophy Alkaline phosphatase Gene therapy Dog Systemic gene transfer 



The protocols were developed with the grant support from the National Institutes of Health (HL-91883, AR-49419 and AR-57209 to DD), the Muscular Dystrophy Association (DD) and the Parent Project Muscular Dystrophy (DD). We thank Drs. Dietrich Volkmann, Bruce Smith and Joe Kornegay for helpful discussion. We thank Robert J. McDonald, Jr., M.D. for the generous support to Duchenne muscular dystrophy research in the Duan lab.


  1. 1.
    Wang, Z., Zhu, T., Qiao, C., Zhou, L., Wang, B., Zhang, J., Chen, C., Li, J., and Xiao, X. (2005) Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart. Nat Biotechnol 23, 321–328.PubMedCrossRefGoogle Scholar
  2. 2.
    Gregorevic, P., Blankinship, M. J., Allen, J. M., Crawford, R. W., Meuse, L., Miller, D. G., Russell, D. W., and Chamberlain, J. S. (2004) Systemic delivery of genes to striated muscles using adeno-associated viral vectors. Nat Med 10, 828–834.PubMedCrossRefGoogle Scholar
  3. 3.
    Ghosh, A., Yue, Y., Long, C., Bostick, B., and Duan, D. (2007) Efficient whole-body transduction with trans-splicing adeno-associated viral vectors. Mol Ther 15, 750–755.PubMedCrossRefGoogle Scholar
  4. 4.
    Pacak, C. A., Mah, C. S., Thattaliyath, B. D., Conlon, T. J., Lewis, M. A., Cloutier, D. E., Zolotukhin, I., Tarantal, A. F., and Byrne, B. J. (2006) Recombinant adeno-associated virus serotype 9 leads to preferential cardiac transduction in vivo. Circ Res 99, e3–e9.PubMedCrossRefGoogle Scholar
  5. 5.
    Shelton, G. D., and Engvall, E. (2005) Canine and feline models of human inherited muscle diseases. Neuromuscul Disord 15, 127–138.PubMedCrossRefGoogle Scholar
  6. 6.
    Emery, A. E. H., and Muntoni, F. (2003) Duchenne muscular dystrophy. Oxford University Press, Oxford.Google Scholar
  7. 7.
    Flanigan, K. M., von Niederhausern, A., Dunn, D. M., Alder, J., Mendell, J. R., and Weiss, R. B. (2003) Rapid direct sequence analysis of the dystrophin gene. Am J Hum Genet 72, 931–939.PubMedCrossRefGoogle Scholar
  8. 8.
    Bradley, D. M., Parsons, E. P., and Clarke, A. J. (1993) Experience with screening newborns for Duchenne muscular dystrophy in Wales. BMJ 306, 357–360.PubMedCrossRefGoogle Scholar
  9. 9.
    Drousiotou, A., Ioannou, P., Georgiou, T., Mavrikiou, E., Christopoulos, G., Kyriakides, T., Voyasianos, M., Argyriou, A., and Middleton, L. (1998) Neonatal screening for Duchenne muscular dystrophy: a novel semiquantitative application of the bioluminescence test for creatine kinase in a pilot national program in Cyprus. Genet Test 2, 55–60.PubMedCrossRefGoogle Scholar
  10. 10.
    Parsons, E. P., Clarke, A. J., Hood, K., Lycett, E., and Bradley, D. M. (2002) Newborn screening for Duchenne muscular dystrophy: a psychosocial study. Arch Dis Child Fetal Neonatal Ed 86, F91–F95.PubMedCrossRefGoogle Scholar
  11. 11.
    Duan, D., Yue, Y., Yan, Z., Yang, J., and Engelhardt, J. F. (2000) Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus. J Clin Invest 105, 1573–1587.PubMedCrossRefGoogle Scholar
  12. 12.
    Bartlett, R. J., Winand, N. J., Secore, S. L., Singer, J. T., Fletcher, S., Wilton, S., Bogan, D. J., Metcalf-Bogan, J. R., Bartlett, W. T., Howell, J. M., Cooper, B. J., and Kornegay, J. N. (1996) Mutation segregation and rapid carrier detection of X-linked muscular dystrophy in dogs. Am J Vet Res 57, 650–654.PubMedGoogle Scholar
  13. 13.
    Van Belle, H. (1976) Alkaline phosphatase. I. Kinetics and inhibition by levamisole of purified isoenzymes from humans. Clin Chem 22, 972–976.PubMedGoogle Scholar
  14. 14.
    Miller, M. E., and Evans, H. E. (1993) Miller’s anatomy of the dog. Saunders, Philadelphia.Google Scholar
  15. 15.
    Goody, P. C. (1997) Dog anatomy : a pictorial approach to canine structure. J.A. Allen, London.Google Scholar
  16. 16.
    Budras, K.-D., McCarthy, P. H., Fricke, W., and Richter, R. (2002) Anatomy of the dog: an illustrated text. Schlèutersche, Hannover.Google Scholar
  17. 17.
    Kainer, R. A., and McCracken, T. (2003) Dog anatomy: a coloring atlas. Teton NewMedia, Jackson.Google Scholar
  18. 18.
    Evans, H. E., and DeLahunta, A. (2004) Guide to the dissection of the dog. Saunders, St. Louis.Google Scholar
  19. 19.
    Yue, Y., Ghosh, A., Long, C., Bostick, B., Smith, B. F., Kornegay, J. N., and Duan, D. (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
  20. 20.
    Bostick, B., Ghosh, A., Yue, Y., Long, C., and Duan, D. (2007) Systemic AAV-9 transduction in mice is influenced by animal age but not by the route of administration. Gene Ther 14, 1605–1609.PubMedCrossRefGoogle Scholar
  21. 21.
    Goldstein, D. J., Rogers, C., and Harris, H. (1982) Evolution of alkaline phosphatases in primates. Proc Natl Acad Sci U S A 79, 879–883.PubMedCrossRefGoogle Scholar
  22. 22.
    Moak, G., and Harris, H. (1979) Lack of homology between dog and human placental alkaline phosphatases. Proc Natl Acad Sci U S A 76, 1948–1951.PubMedCrossRefGoogle Scholar
  23. 23.
    Kornegay, J. N., Cundiff, D. D., Bogan, D. J., Bogan, J. R., and Okamura, C. S. (2003) The cranial sartorius muscle undergoes true hypertrophy in dogs with golden retriever muscular dystrophy. Neuromuscul Disord 13, 493–500.PubMedCrossRefGoogle Scholar
  24. 24.
    Valentine, B. A., and Cooper, B. J. (1991) Canine X-linked muscular dystrophy: selective involvement of muscles in neonatal dogs. Neuromuscul Disord 1, 31–38.PubMedCrossRefGoogle Scholar
  25. 25.
    Nguyen, F., Cherel, Y., Guigand, L., Goubault-Leroux, I., and Wyers, M. (2002) Muscle lesions associated with dystrophin deficiency in neonatal golden retriever puppies. J Comp Pathol 126, 100–108.PubMedCrossRefGoogle Scholar
  26. 26.
    Le Du, M. H., Stigbrand, T., Taussig, M. J., Menez, A., and Stura, E. A. (2001) Crystal structure of alkaline phosphatase from human placenta at 1.8 A resolution. Implication for a substrate specificity. J Biol Chem 276, 9158–9165.PubMedCrossRefGoogle Scholar
  27. 27.
    Bell, P., Limberis, M., Gao, G., Wu, D., Bove, M. S., Sanmiguel, J. C., and Wilson, J. M. (2005) An optimized protocol for detection of E. coli beta-galactosidase in lung tissue following gene transfer. Histochem Cell Biol 124, 77–85.PubMedCrossRefGoogle Scholar
  28. 28.
    Xu, Z., Yue, Y., Lai, Y., Ye, C., Qiu, J., Pintel, D. J., and Duan, D. (2004) Trans-splicing adeno-associated viral vector-mediated gene therapy is limited by the accumulation of spliced mRNA but not by dual vector coinfection efficiency. Hum Gene Ther 15, 896–905.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Yongping Yue
    • 1
  • Jin-Hong Shin
    • 1
  • Dongsheng Duan
    • 1
  1. 1.Department of Molecular Microbiology and Immunology, School of MedicineThe University of MissouriColumbiaUSA

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