Antisense-mediated down-regulation of the human huntingtin gene

The present study determines whether the expression of the huntingtin gene might be subject to antisense (AS)-mediated down-regulation. A series of AS oligodeoxynucleotides (ODNs) complementary to the huntingtin transcript [i .e., nucleotide (nt) -25 to 35] were designed and synthesized, and the AS e fficacy was investigated by using a combination of in vitro transcription a nd translation to mimic in vivo conditions. An oligomer directed to nt -1 t o 15 (ODN III) markedly reduced the incorporation of [H-3] leucine into the huntingtin gene product in a dose-dependent manner (ED50 of similar to 11. 5 mu M). ODNs that overlap with ODN III on both 5'- and 3'-flanking regions also produced translation arrest of the huntingtin protein; however, the A S-mediated effect of these ODNs represented similar to 50% of the effect of ODN III. In contrast, an ODN directed to nt 19 to 35 had no AS effect. The efficacy of ODN III also was investigated in an inducible, stably transfec ted PC-12 cell line expressing a truncated huntingtin exon 1 protein. In ac cordance with the cell free translation studies, ODN III (1-10 mu M) marked ly decreased the abundance of the huntingtin-green fluorescence fusion prot ein to 40 to 46% of the control levels. In summary, a series of putative AS candidates were screened for down-regulation of the huntingtin gene, and a n ODN molecule directed to the methionine initiation codon was identified w ith maximum AS effects.