RNAi reduces human htt expression in vitro. (A) RNA sequence of shHD2.1. The 21-nt antisense strand is cognate to nucleotides 416–436 of human htt mRNA (GenBank accession no. NM_002111). (B and C) Northern and Western blots demonstrate shHD2.1-mediated reduction of HD-N171-82Q mRNA and protein expression 48 h after transfection of target- and shRNA-expressing plasmids. GAPDH and actin serve as loading controls. (D) Western blots show that shHD2.1 inhibits expression of full-length human htt protein, 48 h after transfection. (E) shHD2.1 induces dose-dependent reduction of human htt mRNA. Cells were transfected with either shLacZ- or shHD2.1-expressing plasmids in the indicated amounts. Relative htt expression was determined by using quantitative PCR 24 h later.

AAV.shHD2.1 delivers widespread RNAi expression to mouse striatum. (A) AAV.shHD2.1 viral vector. ITR, inverted terminal repeat. (B) Northern blot showing shHD2.1 transcripts are expressed in vivo. Processed antisense (lower band) and unprocessed (upper band) shHD2.1 transcripts in three different AAV.shHD2.1-injected mice are shown. L, ladder; +, positive control oligonucleotide. The blot was probed with radiolabeled sense probe. (C) Typical AAV1 transduction pattern (hrGFP) in mouse brain. CC, corpus callosum; LV, lateral ventricle.

AAV.shHD2.1 eliminates the accumulation of htt-reactive neuronal inclusions and reduces HD-N171-82Q mRNA in vivo.(A) Representative photomicrographs show htt-reactive inclusions (arrows) in HD striatal cells transduced with AAV.shLacZ but not AAV.shHD2.1. (Scale bar, 20 μm.) (B) Higher-magnification photomicrograph from A (Lower Right) showing lack of htt-reactive inclusions in cells transduced by AAV.shHD2.1. * serves as a marker for orientation. (Scale bar, 20 μm.) (C) Representative Western blot demonstrates decreased HD-N171-82Q expression in mouse striata transduced with AAV.shHD2.1, compared with uninjected or AAV.shLacZ-injected striata. Prion protein was used as a loading control to normalize for tissues expressing the HD-N171-82Q transgene. (D) AAV.shHD2.1-treated HD mice showed a 55% average reduction in HD-N171-82Q mRNA, compared with AAV.shLacZ or uninjected HD mice. Data are means + SEM relative to uninjected HD samples. *, Difference from AAV.shHD2.1 samples, P < 0.05; ANOVA. (E) Mice were injected with either AAV.shHD2.1 or AAV.shLacZ directly into the cerebellum. Cerebellar sections confirm that AAV.shHD2.1, but not AAV.shLacZ, reduces htt immunoreactivity. GCL, granule cell layer; ML, molecular layer. (Scale bar, 100 μm.)

AAV.shHD2.1 improves behavioral deficits in HD-N171-82Q mice. (A) The box plot shows that the bilateral striatal delivery of AAV.shHD2.1 improved stride length in HD-N171-82Q mice. HD mice had significantly shorter stride lengths, compared with WT mice. AAV.shHD2.1 mediated significant gait improvement, compared with control-treated HD mice. *, P < 0.0001; ANOVA with Scheffé's post hoc test. (B) Bilateral striatal delivery of AAV.shHD2.1 significantly improved rotarod performance in HD-N171-82Q mice. Only AAV.shLacZ-injected and uninjected HD-N171-82Q mice declined significantly with time. Data are means ± SEM.