Controlled delivery of glial cell line-derived neurotrophic factor by a single tetracycline-inducible AAV vector.
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.
Chtarto
A
A
Laboratory of Experimental Neurosurgery, Multidisciplinary Research Institute (IRIBHM) Université Libre de Bruxelles, Hôpital Erasme, Belgium.
Yang
X
X
Bockstael
O
O
Melas
C
C
Blum
D
D
Lehtonen
E
E
Abeloos
L
L
Jaspar
J-M
JM
Levivier
M
M
Brotchi
J
J
Velu
T
T
Tenenbaum
L
L
D000818:Animals; D001921:Brain / Q000378:metabolism; D045744:Cell Line, Tumor; D003342:Corpus Striatum / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004318:Doxycycline / Q000008:administration & dosage / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000187:drug effects*; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D007267:Injections; D051381:Rats; D017207:Rats, Sprague-Dawley; D013754:Tetracyclines / Q000494:pharmacology*; D013997:Time Factors; D014161:Transduction, Genetic; D019076:Transgenes; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D018076:DNA, Complementary; D051100:Glial Cell Line-Derived Neurotrophic Factor; D013754:Tetracyclines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014446:Tyrosine 3-Monooxygenase; D004318:Doxycycline