Therapeutic Silencing of Mutant Huntingtin by Targeting Single Nucleotide Polymorphisms: A Dissertation

Abstract

Huntington’s disease (HD) is an autosomal dominant, progressive neurodegenerative disorder. Invariably fatal, HD is caused by expansion of the CAG repeat region in exon 1 of the Huntingtin gene which creates a toxic protein with an extended polyglutamine tract. Silencing mutant Huntingtin messenger RNA (mRNA) is a promising therapeutic approach. The ideal silencing strategy would reduce mutant Huntingtin while leaving the wild-type mRNA intact. Unfortunately, targeting the disease causing CAG repeat expansion is difficult and risks targeting other CAG repeat containing genes. We examined an alternative strategy, targeting single nucleotide polymorphisms (SNPs) in the Huntingtin mRNA. The feasibility of this approach hinges on the presence of a few common highly heterozygous SNPs which are amenable to SNP-specific targeting. In a population of HD patients from Europe and the United states, forty-eight percent were heterozygous at a single SNP site; one isoform of this SNP is associated with HD. Seventy-five percent of patients are heterozygous at least one of three frequently heterozygous SNPs. Consequently, only five allele-specific siRNAs are required to treat three-quarters of the patients in the European and U.S. patient populations. We have designed and validated siRNAs targeting these SNPs. We also developed artificial microRNAs (miRNAs) targeting Huntingtin SNPs for delivery using recombinant adeno-associated viruses (rAAVs). Both U6