711. Allele-Specific Silencing of Mutant Huntingtin for Huntington's Disease Therapy

Huntington's disease (HD) is a dominant genetic neurodegenerative disease. Studies in a tetracycline-responsive mouse model showed that continuous expression of the mutant allele is required for the development of behavioral and pathological symptoms; when mutant huntingtin expression was turned off, disease phenotypes resolved. More recently, gene therapy approaches using interference RNA (RNAi) in HD mouse models successfully prevented HD phenotypes. Because we do not know if reduction of both normal and mutant huntingtin alleles will be tolerated in humans, we investigated an allele-specific RNAi approach for HD. Here we show two different strategies for specific silencing of the mutant huntingtin allele. One is based on structural changes of the mutant huntingtin mRNA as a consequence of the massive CAG-expansion within exon 1 relative to the wild type allele. A second approach is based on a polymorphism in exon 58 that reportedly resides in |[sim]|40% of mutant huntingtin alleles in a sample South American population. To perform our analyses, we developed a novel assay design. Briefly, we generated two plasmids expressing full-length wild type or mutant huntingtin (CAG expansion in exon 1 plus exon 58 polymorphism), each possessing distinct epitope tags and either renilla (WT htt) or firefly (mutant htt) luciferase to normalize transfection efficiencies. This design allowed us to assess allele specificity in the same cell after co-transfection. Sequences of short interfering RNA (siRNA) targeting 5' and 3' of the CAG-repeat region, or the exon 58 polymorphism, were generated; some siRNA sequences were altered according to recent guide-strand rules for optimized RISC loading. Our initial studies with sequences targeting either the exon-58 polymorphism or the CAG repeat region showed specific silencing of the mutant allele. With some siRNAs, mutant protein levels were reduced to 60% with no reduction in wildtype protein levels. Other siRNAs preferentially silenced the mutant huntingtin allele up to 70% while reducing wild type protein levels to only 30% of control values. Together, our data indicate that targeting sequences flanking the CAG-repeat expansion region in exon-1, or the polymorphism in exon 58, may be viable strategies for allele specific silencing for HD therapy.