Using Chemically Modified Oligonucleotides to Modulate Gene Expression, Treat Genetic Diseases, and Probe Novel Mechanisms of RNA Interference

A number of inherited neurological disorders remain incurable despite having well-defined monogenic etiologies. One example is Huntington's disease (HD), which is caused by CAG trinucleotide expansion in the gene HUNTINGTIN (HTT) and production of toxic glutamine-expanded protein. Targeting HTT with siRNAs could be a powerful approach, but allele-selectivity is a major challenge: nearly all HD patients are heterozygous at the HTT locus, and expression of wild-type HTT may need to be preserved. One way to achieve allele-selectivity is by exploiting the fact that the mutant HTT allele contains more CAG repeats. Previous work with double-stranded siRNAs (dsRNA) and chemically modified antisense oligonucleotides (ASO) that target the poly-CAG sequence both showed promise but each had significant limitations. To combine the simplicity of ASO and high selectivity of dsRNA, we tested chemically modified, single-stranded small-interfering RNA (ss-siRNA) of sequences targeting CAG repeats in collaboration with ISIS Pharmaceutical, and showed them to have high potency (IC50 ~2 nM) and allele-selectivity (>30-fold) against mutant HTT in HD-patient-derived cell-lines. Mechanistically, CAG-targeting ss-siRNA functions through endogenous RNAi by recruiting Ago2 and GW182 to HTT mRNA in the absence of a passenger strand and reducing mutant HTT protein level without affecting its mRNA level. Selectivity is achieved through preferential cooperative binding of multiple RISC units to the longer poly-CAG tract on the mutant HTT mRNA versus that of the wild-type. Structural-activity relationship (SAR) studies showed that several ss-siRNAs tolerated significant structural modifications and still retained high potency and selectivity. Furthermore, intraventricular infusion of a candidate ss-siRNA in a HD knock-in mouse model yielded selective inhibition of mutant HTT in a wide range of brain regions. Finally, we showed that a subset of ss-siRNAs were also potent, allele-selective inhibitors of ATAXIN-3, the mutated gene in spinocerebellar ataxia type 3 (SCA3). Taken together, we have identified and characterized a novel class of mechanistically interesting and therapeutically promising nucleic-acid-based compounds that could open new doors to finding a cure for genetic diseases such as HD.