Expression of human nerve growth factor beta gene in central nervous system mediated by recombinant adeno-associated viruses type-2 vector.
BACKGROUND
Neurone atrophy and loss are major causes of chronic neurodegenerative disorders such as Alzheimer's disease. Despite many pharmacotherapies for neurodegeneration, there are no accepted treatments. We investigated the feasibility of human nerve growth factor beta (hNGFbeta) gene expression mediated by recombinant adeno-associated viruses type-2 (rAAV-2) vector in the central nervous system (CNS) after blood brain barrier (BBB) disruption.


METHODS
rAAV-2 containing hNGFbeta gene was constructed. The ability of hNGFbeta gene mediated by rAAV-2 vector (rAAV-2/hNGFbeta) to transfect cells in vitro was confirmed by both ELISA and bioassay of hNGFbeta in the culture supernatant of BHK-21 cells infected by rAAV-2/hNGFbeta. rAAV-2/hNGFbeta and rAAV-2/green fluorescence protein (GFP) were administrated separately to rat brains through internal carotid intubation after BBB disruption with hypertonic mannitol. Brain hNGFbeta concentration was measured by ELISA and GFP in brain sections was examined by laser scan confocal microscope.


RESULTS
After 48 hours, hNGFbeta content in supernatant was up to (188.0 +/- 28.6) pg/ml when BHK-21 cells were infected by rAAV-2/hNGFbeta at multiplicity of infection (MOI) 1.0 x 10(6) vector genome. Neurone fibre outgrowths were obvious in dorsal root ganglion neurone assays by adding serum free culture medium harvested from BHK-21 cells exposed to rAAV-2/hNGFbeta. Whole brain hNGFbeta content in rAAV-2/hNGFbeta transferred group was up to (636.2 +/- 140.6) pg/ml. hNGFbeta content of BBB disruption in rAAV-2/hNGFbeta infused group increased significantly compared to the control group (P <0.05). GFP expression was clearly observed in brain sections of rAAV-2/GFP transferred group.


CONCLUSIONS
rAAV-2/hNGFbeta successfully expresses in the CNS after BBB disruption induced by hypertonic mannitol.
Gao
Kai
K
National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China.
Wu
Yong-jie
YJ
Wu
Xiao-bing
XB
Rao
Chun-ming
CM
Wang
Jun-zhi
JZ
D000818:Animals; D001812:Blood-Brain Barrier; D001921:Brain / Q000378:metabolism*; D006224:Cricetinae; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020932:Nerve Growth Factor / Q000235:genetics*; D011995:Recombination, Genetic
D020932:Nerve Growth Factor