TY - JOUR
T1 - Sleeping Beauty-mediated down-regulation of huntingtin expression by RNA interference
AU - Chen, Zongyu J.
AU - Kren, Betsy T.
AU - Wong, Phillip Y P
AU - Low, Walter C.
AU - Steer, Clifford J.
PY - 2005/4/8
Y1 - 2005/4/8
N2 - Huntington disease (HD) is a devastating neurologic disorder that is characterized by abnormal expansion of a CAG nt repeat in the first exon of the huntingtin (htt) gene, producing a mutant protein with an elongated polyglutamine stretch. The presence of this mutant protein is correlated with the characteristic loss of striatal neurons and the clinical manifestation of HD. Currently there is no effective treatment for the associated cell death. The aim of this study was to evaluate an innovative strategy combining RNA interference (RNAi) and gene transfer via the nonviral Sleeping Beauty (SB) transposon system to down-regulate Htt expression. siRNA expression vectors were designed to target exons 1, 4, 6, and 62 of the human htt gene. Real-time RT-PCR and Western blot analysis were used to quantify Htt mRNA and protein levels, respectively, in human cell lines. The results indicated that selected siRNA constructs significantly decreased Htt mRNA and protein levels relative to controls. In addition, SB transposition of the siRNA constructs into the genome reduced long-term protein expression of Htt by ∼90%. The combination of siRNA, the SB transposon, and an accurate transgenic mouse model may permit evaluation of this approach in preventing the pathogenesis associated with expression of mutant Htt.
AB - Huntington disease (HD) is a devastating neurologic disorder that is characterized by abnormal expansion of a CAG nt repeat in the first exon of the huntingtin (htt) gene, producing a mutant protein with an elongated polyglutamine stretch. The presence of this mutant protein is correlated with the characteristic loss of striatal neurons and the clinical manifestation of HD. Currently there is no effective treatment for the associated cell death. The aim of this study was to evaluate an innovative strategy combining RNA interference (RNAi) and gene transfer via the nonviral Sleeping Beauty (SB) transposon system to down-regulate Htt expression. siRNA expression vectors were designed to target exons 1, 4, 6, and 62 of the human htt gene. Real-time RT-PCR and Western blot analysis were used to quantify Htt mRNA and protein levels, respectively, in human cell lines. The results indicated that selected siRNA constructs significantly decreased Htt mRNA and protein levels relative to controls. In addition, SB transposition of the siRNA constructs into the genome reduced long-term protein expression of Htt by ∼90%. The combination of siRNA, the SB transposon, and an accurate transgenic mouse model may permit evaluation of this approach in preventing the pathogenesis associated with expression of mutant Htt.
KW - Genomic transposition
KW - Huntington disease
KW - Neuronal cell culture
KW - Nonviral gene transfer
KW - Polyglutamine
KW - Post-transcriptional gene silencing
KW - RNA interference
KW - Sleeping Beauty transposon
KW - Small interfering RNA
UR - http://www.scopus.com/inward/record.url?scp=14644445202&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=14644445202&partnerID=8YFLogxK
U2 - 10.1016/j.bbrc.2005.02.024
DO - 10.1016/j.bbrc.2005.02.024
M3 - Article
C2 - 15737634
AN - SCOPUS:14644445202
SN - 0006-291X
VL - 329
SP - 646
EP - 652
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 2
ER -