TY - JOUR
T1 - A microRNA embedded AAV alpha-synuclein gene silencing vector for dopaminergic neurons
AU - Han, Ye
AU - Khodr, Christina E.
AU - Sapru, Mohan K.
AU - Pedapati, Jyothi
AU - Bohn, Martha C.
N1 - Funding Information:
The technical assistance of Jianping Xie, Xue Song Wang, David George and Brian Corstange is greatly appreciated. The authors acknowledge support from the Department of Defense ( W81XWH-06-1-0774 ; MCB), the National Institutes of Health ( NS054989 ; MCB), Children's Memorial Research Center (YH), NIH Training grant T32 NS041234 (CEK) and the Medical Research Institute Council of Children's Memorial Hospital (MCB).
PY - 2011/4/22
Y1 - 2011/4/22
N2 - Alpha-synuclein (SNCA), an abundantly expressed presynaptic protein, is implicated in Parkinson's disease (PD). Since over-expression of human SNCA (hSNCA) leads to death of dopaminergic (DA) neurons in human, rodent and fly brain, hSNCA gene silencing may reduce levels of toxic forms of SNCA and ameliorate degeneration of DA neurons in PD. To begin to develop a gene therapy for PD based on hSNCA gene silencing, two AAV gene silencing vectors were designed, and tested for efficiency and specificity of silencing, as well as toxicity in vitro. The same hSNCA silencing sequence (shRNA) was used in both vectors, but in one vector, the shRNA was embedded in a microRNA backbone and driven by a pol II promoter, and in the other the shRNA was not embedded in a microRNA and was driven by a pol III promoter. Both vectors silenced hSNCA to the same extent in 293T cells transfected with hSNCA. In DA PC12 cells, neither vector decreased expression of rat SNCA, tyrosine hydroxylase (TH), dopamine transporter (DAT) or the vesicular monoamine transporter (VMAT). However, the mir30 embedded vector was significantly less toxic to both PC12 and SH-SY5Y cells. Our in vitro data suggest that this miRNA-embedded silencing vector may be ideal for chronic in vivo SNCA gene silencing in DA neurons.
AB - Alpha-synuclein (SNCA), an abundantly expressed presynaptic protein, is implicated in Parkinson's disease (PD). Since over-expression of human SNCA (hSNCA) leads to death of dopaminergic (DA) neurons in human, rodent and fly brain, hSNCA gene silencing may reduce levels of toxic forms of SNCA and ameliorate degeneration of DA neurons in PD. To begin to develop a gene therapy for PD based on hSNCA gene silencing, two AAV gene silencing vectors were designed, and tested for efficiency and specificity of silencing, as well as toxicity in vitro. The same hSNCA silencing sequence (shRNA) was used in both vectors, but in one vector, the shRNA was embedded in a microRNA backbone and driven by a pol II promoter, and in the other the shRNA was not embedded in a microRNA and was driven by a pol III promoter. Both vectors silenced hSNCA to the same extent in 293T cells transfected with hSNCA. In DA PC12 cells, neither vector decreased expression of rat SNCA, tyrosine hydroxylase (TH), dopamine transporter (DAT) or the vesicular monoamine transporter (VMAT). However, the mir30 embedded vector was significantly less toxic to both PC12 and SH-SY5Y cells. Our in vitro data suggest that this miRNA-embedded silencing vector may be ideal for chronic in vivo SNCA gene silencing in DA neurons.
KW - Cell death
KW - Gene therapy
KW - Neurodegeneration
KW - Parkinson's disease
KW - Tyrosine hydroxylase
KW - Vesicular monoamine transporter
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UR - http://www.scopus.com/inward/citedby.url?scp=79953862206&partnerID=8YFLogxK
U2 - 10.1016/j.brainres.2011.02.041
DO - 10.1016/j.brainres.2011.02.041
M3 - Article
C2 - 21338582
AN - SCOPUS:79953862206
SN - 0006-8993
VL - 1386
SP - 15
EP - 24
JO - Brain Research
JF - Brain Research
ER -
