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Gene Delivery to Neurons of the Dorsal Root Ganglia Using Adeno-Associated Viral Vectors

  • Nitish D. Fagoe
  • Ruben Eggers
  • Joost Verhaagen
  • Matthew R. J. MasonEmail author
Protocol
Part of the Neuromethods book series (NM, volume 98)

Abstract

Viral vector-mediated gene transfer, especially using adeno-associated viral (AAV) vectors, is a powerful strategy to manipulate gene expression in vivo in injured neurons. In this chapter we provide two methods to efficiently transduce dorsal root ganglion (DRG) neurons in vivo using AAV. We describe detailed procedures to perform direct injections into specific DRG and delivery via the intrathecal space to transduce the lumbar DRG. Finally, we discuss the specific advantages and disadvantages of these two methods of delivery. The main advantages of direct injection are that high transduction rates can be achieved in specific ganglia (L4/L5) with low amounts (μl) of a viral vector stock; however, the procedure is complex, invasive, and time-consuming. Intrathecal injection has the advantage of being a fast and simple method to transduce multiple DRG bilaterally, and involves no surgical manipulation of the DRG. However, intrathecal delivery does require much larger amounts of viral stock (10–20 μl) and has the disadvantage that viral particles will leak from the cerebrospinal fluid to the spinal cord and/or peripheral tissues.

Key words

Dorsal root ganglia Peripheral nerve injury Gene delivery Adeno-associated viral vector 

Notes

Acknowledgements

The authors are grateful for research funding provided by the International Spinal Research Trust and the Dutch Organization for Scientific Research (NWO).

References

  1. 1.
    Gaudet D, Methot J, Dery S et al (2013) Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an open-label trial. Gene Ther 20:361–369PubMedCrossRefGoogle Scholar
  2. 2.
    Kaplitt MG, Feigin A, Tang C et al (2007) Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson’s disease: an open label, phase I trial. Lancet 369:2097–2105PubMedCrossRefGoogle Scholar
  3. 3.
    Hermens WT, Ter BO, Dijkhuizen PA et al (1999) Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system. Hum Gene Ther 10:1885–1891PubMedCrossRefGoogle Scholar
  4. 4.
    Kaplitt MG, Leone P, Samulski RJ et al (1994) Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain. Nat Genet 8:148–154PubMedCrossRefGoogle Scholar
  5. 5.
    McCown TJ, Xiao X, Li J et al (1996) Differential and persistent expression patterns of CNS gene transfer by an adeno-associated virus (AAV) vector. Brain Res 713:99–107PubMedCrossRefGoogle Scholar
  6. 6.
    Peel AL, Zolotukhin S, Schrimsher GW et al (1997) Efficient transduction of green fluorescent protein in spinal cord neurons using adeno-associated virus vectors containing cell type-specific promoters. Gene Ther 4:16–24PubMedCrossRefGoogle Scholar
  7. 7.
    Hoke A (2006) Mechanisms of Disease: what factors limit the success of peripheral nerve regeneration in humans? Nat Clin Pract Neurol 2:448–454PubMedCrossRefGoogle Scholar
  8. 8.
    Macgillavry HD, Stam FJ, Sassen MM et al (2009) NFIL3 and cAMP response element-binding protein form a transcriptional feedforward loop that controls neuronal regeneration-associated gene expression. J Neurosci 29:15542–15550PubMedCrossRefGoogle Scholar
  9. 9.
    Stam FJ, Macgillavry HD, Armstrong NJ et al (2007) Identification of candidate transcriptional modulators involved in successful regeneration after nerve injury. Eur J Neurosci 25:3629–3637PubMedCrossRefGoogle Scholar
  10. 10.
    Geeven G, Macgillavry HD, Eggers R et al (2011) LLM3D: a log-linear modeling-based method to predict functional gene regulatory interactions from genome-wide expression data. Nucleic Acids Res 39:5313–5327PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Arthur-Farraj PJ, Latouche M, Wilton DK et al (2012) c-Jun reprograms Schwann cells of injured nerves to generate a repair cell essential for regeneration. Neuron 75:633–647PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Li S, Liu Q, Wang Y et al (2013) Differential gene expression profiling and biological process analysis in proximal nerve segments after sciatic nerve transection. PLoS One 8:e57000PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Verhaagen J, Van Kesteren RE, Bossers KA et al (2012) Molecular target discovery for neural repair in the functional genomics era. Handb Clin Neurol 109:595–616PubMedCrossRefGoogle Scholar
  14. 14.
    Beutler AS, Reinhardt M (2009) AAV for pain: steps towards clinical translation. Gene Ther 16:461–469PubMedCrossRefGoogle Scholar
  15. 15.
    Mingozzi F, High KA (2011) Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet 12:341–355PubMedCrossRefGoogle Scholar
  16. 16.
    Ginn SL, Alexander IE, Edelstein ML et al (2013) Gene therapy clinical trials worldwide to 2012 - an update. J Gene Med 15:65–77PubMedCrossRefGoogle Scholar
  17. 17.
    Mason MR, Ehlert EM, Eggers R et al (2010) Comparison of AAV serotypes for gene delivery to dorsal root ganglion neurons. Mol Ther 18:715–724PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Naldini L, Blomer U, Gallay P et al (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263–267PubMedCrossRefGoogle Scholar
  19. 19.
    Yu H, Fischer G, Jia G et al (2011) Lentiviral gene transfer into the dorsal root ganglion of adult rats. Mol Pain 7:63PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Storek B, Harder NM, Banck MS et al (2006) Intrathecal long-term gene expression by self-complementary adeno-associated virus type 1 suitable for chronic pain studies in rats. Mol Pain 2:4PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Storek B, Reinhardt M, Wang C et al (2008) Sensory neuron targeting by self-complementary AAV8 via lumbar puncture for chronic pain. Proc Natl Acad Sci U S A 105:1055–1060PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Towne C, Pertin M, Beggah AT et al (2009) Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery. Mol Pain 5:52PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Xu Q, Chou B, Fitzsimmons B et al (2012) In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery. PLoS One 7:e32581PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Malkmus SA, Yaksh TL (2004) Intrathecal catheterization and drug delivery in the rat. Methods Mol Med 99:109–121PubMedGoogle Scholar
  25. 25.
    Yaksh TL, Rudy TA (1976) Chronic catheterization of the spinal subarachnoid space. Physiol Behav 17:1031–1036PubMedCrossRefGoogle Scholar
  26. 26.
    Vulchanova L, Schuster DJ, Belur LR et al (2010) Differential adeno-associated virus mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture. Mol Pain 6:31PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Parikh P, Hao Y, Hosseinkhani M et al (2011) Regeneration of axons in injured spinal cord by activation of bone morphogenetic protein/Smad1 signaling pathway in adult neurons. Proc Natl Acad Sci U S A 108:E99–E107PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Asato F, Butler M, Blomberg H et al (2001) Distribution of intrathecal catheter positions in rat. Acta Anaesthesiol Scand 45:608–611PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Nitish D. Fagoe
    • 1
  • Ruben Eggers
    • 1
  • Joost Verhaagen
    • 1
    • 2
  • Matthew R. J. Mason
    • 1
    Email author
  1. 1.Laboratory for NeuroregenerationNetherlands Institute for Neuroscience, an Institute of the Royal Academy of Arts and SciencesAmsterdamThe Netherlands
  2. 2.Center for Neurogenomics and Cognition ResearchVrije Universiteit AmsterdamAmsterdamThe Netherlands

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