Recombinant AAV Delivery to the Central Nervous System
- 5.6k Downloads
Recombinant AAV-mediated gene delivery to the CNS can be performed either by direct delivery at the target site or from the periphery, using intramuscular injections and retrograde transport along motor neuron projections or intravenous injections and blood–brain barrier crossing.
Methods for recombinant virus administration, including stereotactic surgery, intramuscular, and intravenous administration.
Methods to evaluate the number and biodistribution of brain and spinal cord cells expressing the transgene by immunohistochemisty as well as the amount of transgene product by ELISA in the target region.
Methods to characterize the cellular specificity of transgene expression by double immunofluorescence.
Methods to quantify the amounts of viral DNA as well as of transgene mRNA by quantitative PCR and RT-PCR, respectively.
Key wordsStereotaxy Intravenous injection Retrograde transport Immunohistochemistry Confocal microscopy GFP GDNF Quantitative PCR mRNA Hirt DNA
O.B. was the recipient of a predoctoral fellowship from the Belgian “FRIA” (Fonds pour la Recherche dans l’Industrie et l’Agriculture and FNRS-Télévie). L.T. was the recipient of a “Crédit aux chercheurs” from the Belgian National Research Foundation. This work was also supported by grants from “Fonds National de la Recherche Scientifique Médicale,” “Région Bruxelles-Capitale,” and “Association Française contre les Myopathies.”
The Kaspar Laboratory is funded in part from The National Institute of Health (NIH).
- 6.Kirik D, Rosenblad C, Bjorklund A, Mandel RJ. Long-term rAAV-mediated gene transfer of GDNF in the rat Parkinson’s model: intrastriatal but not intranigral transduction promotes functional regeneration in the lesioned nigrostriatal system. J Neurosci 2000 Jun 15;20(12):4686–4700.PubMedGoogle Scholar
- 7.Mandel RJ, Spratt SK, Snyder RO, Leff SE. (1997) Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson’s disease in rats. Proc Natl Acad Sci USA 94, 14083–8.PubMedCrossRefGoogle Scholar
- 8.Yang X, Mertens B, Lehtonen E, Vercammen L, Bockstael O, Chtarto A, et al. (2009) Reversible neurochemical changes mediated by delayed intrastriatal glial cell line-derived neurotrophic factor gene delivery in a partial Parkinson’s disease rat model. J Gene Med 11, 899–912.PubMedCrossRefGoogle Scholar
- 12.Shen Y, Muramatsu SI, Ikeguchi K, Fujimoto KI, Fan DS, Ogawa M, et al. (2000) Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase, aromatic-L-amino-acid decarboxylase, and GTP cyclohydrolase I for gene therapy of Parkinson’s disease. Hum Gene Ther 11, 1509–19.PubMedCrossRefGoogle Scholar
- 20.Paxinos, G. and Watson, 1997. “The rat brain in stereotaxic coordinates”, 3rd compact edition, Academic Press, Orlando, FLAGoogle Scholar
- 21.Paxinos, G. and Franklin, K.B.J., 1997. “The mouse brain in stereotaxic coordinates” 2nd edition, Academic PressGoogle Scholar
- 32.Hirt B. (1967) Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 26: 365–9.Google Scholar