Five siRNAs targeting three SNPs may provide therapy for three-quarters of Huntington's disease patients

Abstract

Among dominant neurodegenerative disorders, Huntington's disease (HD) is perhaps the best candidate for treatment with small interfering RNAs (siRNAs) [1-9]. Invariably fatal, HD is caused by expansion of a CAG repeat in the Huntingtin gene, creating an extended polyglutamine tract that makes the Huntingtin protein toxic [10]. Silencing mutant Huntingtin messenger RNA (mRNA) should provide therapeutic benefit, but normal Huntingtin likely contributes to neuronal function [11-13]. No siRNA strategy can yet distinguish among the normal and disease Huntingtin alleles and other mRNAs containing CAG repeats [14]. siRNAs targeting the disease isoform of a heterozygous single-nucleotide polymorphism (SNP) in Huntingtin provide an alternative [15-19]. We sequenced 22 predicted SNP sites in 225 human samples corresponding to HD and control subjects. We find that 48% of our patient population is heterozygous at a single SNP site; one isoform of this SNP is associated with HD. Several other SNP sites are frequently heterozygous. Consequently, five allele-specific siRNAs, corresponding to just three SNP sites, could be used to treat three-quarters of the United States and European HD patient populations. We have designed and validated selective siRNAs for the three SNP sites, laying the foundation for allele-specific RNA interference (RNAi) therapy for HD.

Figure 1

Analysis of SNPs in the human Huntingtin mRNA. (A) We sequenced PCR amplicons from genomic DNA from 109 HD patients and 116 controls spanning 22 SNP sites within the Huntingtin mRNA. The SNP at nucleotide 9,633 (rs362307, shown in red) is associated with HD and sites for which we have designed siRNAs are in bold. (B) The maximum percentage of patients to have at least one heterozygous SNP using any combination of 1 to 7 SNPs was calculated using the experimentally determined frequency of heterozygosity for the SNP sites in our study. Three SNPs cover ~75% of the patient population analyzed here.

Figure 2

Representative data for the development of isoform-specific siRNAs targeting two additional SNP sites. (A) siRNAs mismatched at position 16 discriminated between luciferase reporter mRNAs bearing either the C or the A isoform of the rs363125 SNP site. (B) siRNAs bearing a mismatch to the SNP site at position 10 and an additional position 5 mismatch discriminated between the G and A isoforms of the rs362273 SNP site.