Improved neuronal transgene expression from an AAV-2 vector with a hybrid CMV enhancer/PDGF-beta promoter.
BACKGROUND
Adeno-associated virus type 2 (AAV-2) vectors are highly promising tools for gene therapy of neurological disorders. After accommodating a cellular promoter, AAV-2 vectors are able to drive sustained expression of transgene in the brain. This study aimed to develop AAV-2 vectors that also facilitate a high level of neuronal expression by enhancing the strength of a neuron-specific promoter, the human platelet-derived growth factor beta-chain (PDGF) promoter.


RESULTS
A hybrid promoter approach was adopted to fuse the enhancer of human cytomegalovirus immediately early (CMV) promoter to the PDGF promoter. In cultured cortex neurons, AAV-2 vectors containing the hybrid promoter augmented transgene expression up to 20-fold over that mediated by titer-matched AAV-2 vectors with the PDGF promoter alone and 4-fold over the CMV enhancer/promoter. Injection of AAV-2 vectors with the hybrid promoter into the rat striatum resulted in neuron-specific transgene expression, the level of which was about 10-fold higher than those provided by the two control AAV-2 expression cassettes at 4 weeks post-injection and maintained for at least 12 weeks. Gene expression in the substantia nigra through possible retrograde transport of the AAV-2 vectors injected into the striatum was not obvious. After direct injection of AAV-2 vectors into the substantia nigra, transgene expression driven by the hybrid promoter was observed specifically in dopaminergic neurons and its level was about 3 and 17 times higher than that provided by the PDGF promoter alone and the CMV enhancer/promoter, respectively.


CONCLUSIONS
Enhanced transgene capacity plus neuron-specificity of the AAV-2 vectors developed in this study might prove valuable for gene therapy of Parkinson's disease.
Wang
C Y
CY
Institute of Bioengineering and Nanotechnology, National University of Singapore, Singapore.
Guo
H Y
HY
Lim
T M
TM
Ng
Y K
YK
Neo
H P
HP
Hwang
P Y K
PY
Yee
W-C
WC
Wang
S
S
D000818:Animals; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D009474:Neurons / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D013378:Substantia Nigra / Q000378:metabolism; D014162:Transfection; D019076:Transgenes
D010982:Platelet-Derived Growth Factor