Sleeping Beauty-mediated down-regulation of huntingtin expression by RNA interference

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 Hit expression. siRNA expression vectors were designed to target exons 1, 4, 6, and 62 of the human fat 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 Hit mRNA and protein levels relative to controls. In addition, SB transposition of the siRNA constructs into the genome reduced long-term protein expression of Hit by similar to 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 Hyt. (c) 2005 Elsevier Inc. All rights reserved.