P.19.2 Targeting the Huntingtin gene with splice-switching AOs: An unusual result

Abstract

Huntington's disease is a late-onset neuromuscular disorder caused by the abnormal expansion of a trinucleotide repeat (CAG) that codes for glutamine in exon 1 of the Huntingtin gene (HTT). Both the mutated protein and RNA transcript induce a toxic effect, altering downstream interactions and causing neural degeneration and a decline in cognitive and muscle function. Antisense oligonucleotides have been used to knockdown the expression of mutant HTT, either by inducing RNase H or blocking translation. However, most of these approaches target the repeat itself, which could lead to non-specific effects. This study used 2′-O-methyl AOs, which do not induce RNase H activity and are often used to alter exon selection to target sequences surrounding the repeat region. The hypothesis was that the presence of the expanded repeat is likely to alter the way splicing factors interact, and targeting an AO to exon 1 could induce the removal of the exon in a mutation-specific manner, leaving the wild type allele intact while knocking down the expanded mutant allele. Fourteen oligomers targeted to exon 1 were tested initially, and three of these altered pre-mRNA processing, as RT-PCR amplification demonstrated the presence of smaller products that did not follow exon boundaries. This phenomenon, whereby nucleotides from the middle of an exon are removed, has not been reported elsewhere. In silico analysis of potential splice sites indicates there are no potential splice sites near the excluded region. Further studies are currently being conducted to determine possible mechanisms of exon excision, and the implications for AO targeting of GC rich sequences.