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Experimental therapeutics—preclinical
P03 Reducing toxic N-terminal huntingtin fragments in HD using exon skipping
  1. MM Evers1,
  2. H-D Tran1,
  3. L Zalachoras2,
  4. JT den Dunnen1,3,
  5. G-JB van Ommen1,
  6. OC Meijer2,
  7. A Aartsma-Rus1,
  8. WMC van Roon-Mom1
  1. 1Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands
  2. 2Department of Endocrinology and Metabolic Disease, Leiden University Medical Center, Leiden, The Netherlands
  3. 3Leiden Genome Technology Center, Leiden University Medical Center, Leiden, The Netherlands


Background Several studies have implicated the importance of proteolytic cleavage of mutant huntingtin in HD pathogenesis (Thornberry et al 1997). Huntingtin fragments within the striatum of human HD brains clearly differ from those of control brains (Mende-Mueller et al 2001), suggesting cleavage is disease specific. HD cell models with caspase-3 and caspase-6 resistant neuronal and non-neuronal cells showed reduced toxicity and were found to be less prone to aggregate formation (Wellington et al 2000). In YAC128 HD mice, expressing human genomic mutant huntingtin containing 128 glutamines, the HD phenotype was prevented by blocking caspase 6-specific cleavage by mutating the caspase-6 cleavage motif (Graham et al 2006; Pouladi et al 2009). This suggests that caspase-6 cleavage at position 586 is a key player in neuronal dysfunction and neurodegeneration.

Aims For our study we make use of 2’O-methyl modified antisense oligonucleotides (AONs) with a phosphorothioate (PS) backbone to induce in-frame exon skipping in huntingtin of the exons in which the proteolytic cleavage motifs are located.

Methods/techniques Patient derived fibroblast cells were transfected with AONs, RNA was isolated 1-day after transfection and skipping efficiency was determined. To assess the formation of a shorter, skipped protein, total protein lysates were obtained 3 days after transfection and shown by Western blot. A caspase-6 assay was used to determine the caspase-resistance of the truncated huntingtin protein.

Results/outcome Patient derived fibroblast cells transfected with an AON binding to the 3’ part of exon 12, resulted in a partial skip of 135 base pairs. This partial exclusion of the 3’ part of huntingtin exon 12 can be explained by the existence of an cryptic 5’ splice site AG|GTCAG (Zhang et al 1998). The in-frame skip results in a slightly shorter huntingtin protein resistant to proteolytic cleavage at the caspase-6 cleavage site. In-frame multi-exon skipping of exon 12 and 13 from huntingtin pre-mRNA resulted in a shorter huntingtin protein resistant to proteolytic cleavage at the caspase-3 and caspase-6 cleavage sites. Currently we are testing whether skipping of this caspase-6 motif results in a reduction of N-terminal huntingtin fragments and thus reduced toxicity.

Conclusions Above described preliminary results suggest a novel therapeutic approach to reduce toxic N-terminal huntingtin fragments using exon skipping while maintaining huntingtin protein levels. Currently we are testing in vivo whether a single bilateral injection of AONs in the mice striatum would result in skipping of the caspase motifs.

  • Gene therapy
  • exon skipping
  • antisense oligonucleotide
  • caspase cleavage
  • N-terminal fragments

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