Allele-selective inhibition of huntingtin expression by switching to an miRNA-like RNAi mechanism

Abstract

Inhibiting expression of huntingtin (HTT) protein is a promising strategy for treating Huntington's disease (HD), but indiscriminant inhibition of both wild-type and mutant alleles may lead to toxicity. An ideal silencing agent would block expression of mutant HTT while leaving expression of wild-type HTT intact. We observe that fully complementary duplex RNAs targeting the expanded CAG repeat within HTT mRNA block expression of both alleles. Switching the RNAi mechanism toward that used by miRNAs by introducing one or more mismatched bases into these duplex RNAs leads to potent (<10 nM) and highly selective (>30-fold relative to wild-type HTT) inhibition of mutant HTT expression in patient-derived cells. Potent, allele selective inhibition of HTT by mismatched RNAs provides a new option for developing HD therapeutics.

Figure 1. Allele-selective inhibition by mismatch-containing duplex RNAs

Schemes showing recognition of a (a) perfectly complementary duplex leading to cleavage of mRNA and (b) mismatch-containing duplex leading to inhibition of translation. Effect on HTT expression from adding duplex RNAs (c) P9, (d) REP (e) PM3 to GM04281 cells or f) P9 to GM04717 cells. Data describing the selectivities and potencies of other duplexes can be found in Table 1. See also Figure S1.

Figure 2. Mechanism of allele-selective RNAs

Effect on mutant and wild-type HTT expression after treatment with duplex RNAs that contain a mismatched base in either the (a) passenger strand, P9a, or (b) guide strand, P9b. (c) qPCR analysis of effect on HTT mRNA levels after treatment of increasing amount of siRNAs siHdh1, REP, and P9. (d) Effect of varied siRNAs (12 nM) on HTT mRNA levels measured by qPCR. (e) Effect of allele-selective duplex RNAs (25 nM) on expression of other genes containing regions with CAG repeats. CM: a duplex RNA not complementary to HTT mRNA. siHdh1: an siRNA that is complementary to HTT mRNA outside of the CAG repeat region (Omi et al., 2005). See also Figure S2.