Adenovirus-mediated silencing of huntingtin expression by shRNA.
Huntington's disease (HD) is an inherited autosomal dominant, neurodegenerative disease that is caused by a gain of function mutation characterized by the expansion of a CAG trinucleotide repeat in exon 1 of the huntingtin (htt) gene. Since hairpin small interference RNA (shRNA) technology allows inhibition of specific gene expression in vitro and in vivo, vector-mediated expression of an shRNA directed to htt mRNA could form the basis of a new treatment modality for HD. By initial plasmid transfection of 293 cells, we identified one exon 1-targeted shRNA, which efficiently inhibited expression of an htt exon 1-GFP fusion protein and the endogenous htt gene. A replication-deficient adenovirus (Ad) vector Adie-1-1 was constructed to express this shRNA from the U6 promoter. In A549 cells expressing exon 1 of htt with an expanded CAG allele, Adie- 1-1 efficiently prevented htt exon 1 expression and htt aggregate formation. In addition, in different neuronal and nonneuronal cell lines, Adie-1-1 efficiently inhibited the expression of endogenous htt. Together, this data indicates the delineation of an shRNA strategy that may become the basis for treatment of HD.
Huang
Bin
B
Division of Gene Therapy, University of Ulm, D-89081 Ulm, Germany.
Kochanek
Stefan
S
D000256:Adenoviridae / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D005091:Exons; D005470:Fluorometry; D020868:Gene Silencing; D018390:Gene Targeting; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D009447:Neuroblastoma / Q000473:pathology; D009474:Neurons / Q000378:metabolism; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012342:RNA, Small Nuclear / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014161:Transduction, Genetic; D014162:Transfection
C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D012342:RNA, Small Nuclear; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins