Controlled delivery of glial cell line-derived neurotrophic factor by a single tetracycline-inducible AAV vector.

Abstract

An autoregulated tetracycline-inducible recombinant adeno-associated viral vector (rAAV-pTet(bidi)ON) utilizing the rtTAM2 reverse tetracycline transactivator (rAAV-rtTAM2) was used to conditionally express the human GDNF cDNA. Doxycycline, a tetracycline analog, induced a time- and dose-dependent release of GDNF in vitro in human glioma cells infected with rAAV-rtTAM2 serotype 2 virus. Introducing the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) downstream to the rtTAM2 coding sequence, resulted in a more rapid induction and a higher basal expression level. In vivo, 8 weeks after a single injection of the rAAV-rtTAM2-GDNF vector encapsidated into AAV serotype 1 capsids in the rat striatum, the GDNF protein level was 60 pg/mg tissue in doxycycline-treated animals whereas in untreated animals, it was undistinguishable from the endogenous level ( approximately 4 pg/mg tissue). However, a residual GDNF expression in the uninduced animals was evidenced by a sensitive immunohistochemical staining. As compared to rAAV1-rtTAM2-GDNF, the rAAV1-rtTAM2-WPRE-GDNF vector expressed a similar concentration of GDNF in the induced state (with doxycycline) but a basal level (without doxycycline) approximately 2.5-fold higher than the endogenous striatal level. As a proof for biological activity, for both vectors, downregulation of tyrosine hydroxylase was evidenced in dopaminergic terminals of doxycycline-treated but not untreated animals. In conclusion, the rAAV1-rtTAM2 vector which expressed biologically relevant doses of GDNF in the striatum in response to doxycycline with a basal level undistinguishable from the endogenous striatal level, as measured by quantitative ELISA assay, constitutes an interesting tool for local conditional transgenesis.

• Authors: A Chtarto, X Yang, O Bockstael, C Melas, D Blum, E Lehtonen, L Abeloos, J-M Jaspar, M Levivier, J Brotchi, T Velu, L Tenenbaum
• Journal: Experimental neurology (Exp Neurol) Vol. 204 Issue 1 Pg. 387-99 (Mar 2007) ISSN: 0014-4886 [Print] United States
• PMID: 17223106 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• DNA, Complementary
• Glial Cell Line-Derived Neurotrophic Factor
• Tetracyclines
• enhanced green fluorescent protein
• Green Fluorescent Proteins
• Tyrosine 3-Monooxygenase
• Doxycycline

Topics

• Animals
• Brain (metabolism)
• Cell Line, Tumor
• Corpus Striatum (metabolism)
• DNA, Complementary (metabolism)
• Dependovirus (genetics, metabolism)
• Dose-Response Relationship, Drug
• Down-Regulation
• Doxycycline (administration & dosage, pharmacology)
• Enzyme-Linked Immunosorbent Assay
• Female
• Gene Expression
• Gene Transfer Techniques
• Genetic Vectors (drug effects)
• Glial Cell Line-Derived Neurotrophic Factor (genetics)
• Green Fluorescent Proteins (genetics)
• Humans
• Immunohistochemistry
• Injections
• Rats
• Rats, Sprague-Dawley
• Tetracyclines (pharmacology)
• Time Factors
• Transduction, Genetic
• Transgenes
• Tyrosine 3-Monooxygenase (metabolism)