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Preventing Formation of Toxic N-Terminal Huntingtin Fragments Through Antisense Oligonucleotide-Mediated Protein Modification

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posted on 2014-06-16, 08:14 authored by Melvin m Evers, Hoang-Dai Tran, Ioannis Zalachoras, Onno C Meijer, Johan T den Dunnen, Gert-Jan B van Ommen, Annemieke Aartsma-Rus, Willeke Van roon-momWilleke Van roon-mom

Huntington’s disease (HD) is a progressive autosomal dominant disorder, caused by a CAG repeat expansion in the HTT gene, which results in expansion of a polyglutamine stretch at the N-terminal end of the huntingtin protein. Several studies have implicated the importance of proteolytic cleavage of mutant huntingtin in the HD pathogenesis and it is generally accepted that N-terminal huntingtin protein fragments are more toxic than full-length huntingtin protein. Important cleavage sites are encoded by exon 12 of HTT. Recent publications have shown the feasibility of reducing huntingtin levels using antisense oligonucleotides, but concerns were raised towards possible unwanted side effects from lowering huntingtin protein levels too much. Our approach reduces mutant huntingtin toxicity by modifying the huntingtin protein without changing overall protein levels. We use 2’O-methyl modified antisense oligonucleotides with a phosphorothioate (PS) backbone to induce skipping of exon 12 in huntingtin pre-mRNA, thereby preventing the formation of toxic N-terminal huntingtin protein fragments. In vitro studies showed successful exon skipping and appearance of a shorter huntingtin protein. Cleavage assays showed reduced formation of the 586 amino acid N-terminal huntingtin fragment in the treated samples. In vivo studies revealed exon skipping after single injection of antisense oligonucleotides in the mouse striatum.

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