Fig. 1. siRNA-HDExon1 is effective and specific in vivo and in vitro. (A) cDNA sequence showing the target position for siRNA-HDExon1 (red underline) and CAG repeats (green underline) in the first exon of the human huntingtin gene. (B) Sequences and predicted structure for the siRNA-HDExon1. (C) Representative images showing the effects of siRNA-HDExon1 against the httQ72-d1EGFP fusion protein in Cos-7 cells (upper panels) and newborn mouse brain neurons (lower panels), respectively. Scale bar, 200 μm. (D) Quantitative RT-PCR results showing the knock-down of targeted httQ72-d1EGFP mRNA by siRNA-HDExon1 in vitro using cultured Cos-7 cells (left panel), and in vivo by transfection of newborn mouse brain (right panel). (E) Western blots showing down-regulation of the targeted httQ72-d1EGFP fusion protein in Cos-7 cell lysates and brain lysates. β-actin was used as a protein loading control.

Fig. 2. Effects of siRNA-HDExon1 on the symptomatic phenotypes of the HD mouse model. (A) Effect of siRNA-HDExon1 on body weight in R6/2 mice. Body weight loss was delayed in the siRNA-HDExon1-treated group compared with the mock-treated or untreated groups (statistically significant at 13, 14 and 15 weeks; p < 0.01 by ANOVA). Vertical bars indicate S.D. (B) Survival curves (Kaplan–Meier method) showing that siRNA-HDExon1 treatment extended the longevity of R6/2 mice significantly (p < 0.0001 by log-rank test; siRNA-treated, n = 39; mock-treated, n = 9; untreated, n = 65). Wild-type mice that had the same treatment had a normal lifespan. (C) Representative images showing the effect of siRNA-HDExon1 on the test for the feet-clasping phenotype during tail suspension. A 13-week-old untreated R6/2 mouse displayed the feet-clasping posture at 5 s (center), whereas a 14-week-old R6/2 mouse treated with siRNA-HDExon1 did not display it until 42 s (right). (D) The treatment of siRNA-HDExon1 significantly delayed the age of feet-clasping (siRNA-treated, n = 41; mock-treated, n = 12; untreated, n = 48). The y-axis of the graph shows the percentage of mice that displayed the feet-clasping posture during the 15 s test period.

Fig. 3. Motor function assessment using a rotarod and open-field equipment. (A) R6/2 mice treated with siRNA-HDExon1 showed significant improvement on the rotarod at 6–8 weeks of age (t-test, ^*p < 0.05; siRNA-treated, n = 17; mock-treated, n = 10; untreated, n = 11). (B) Open-field test. Trace comparison for age-matched groups of wild-type, siRNA-HDExon1-treated, mock-treated, or untreated R6/2 mice. siRNA-HDExon1-treated mice were more active than untreated or mock-treated R6/2 mice, especially at ages over 12 weeks.

Fig. 4. siRNA-HDExon1 significantly reduces huntingtin transgenic mRNA and protein levels in brain tissues of R6/2 mice. (A) RNA from whole brain extracts was isolated, and huntingtin transgenic mRNA was determined by real-time PCR. siRNA-HDExon1-treated mice (black bars) suppressed transgene mRNA compared with the control group (combined mock-treated and untreated mice, white bars). Transgene expression was maximally repressed at 4 days post-transfection in vivo, and its effects lasted for at least 7 days (n = 3 for each group, ^*p < 0.01). (B) Western blots of huntingtin aggregates in striatal tissue (50 μg per lane, nuclear extracts) from each group at 8 weeks of age. W, wild-type; U, untreated; M, mock-treated; T1, siRNA-treated mouse #1; T2, siRNA-treated mouse #2. Aggregated proteins remained in the gradient gel, as indicated (^*, >200 kDa).

Fig. 5. Brain atrophy was inhibited and huntingtin-immunoreactive intranuclear inclusions were significantly reduced in siRNA-HDExon1-treated R6/2 mice. (A) Effect of siRNA-HDExon1 on ventricular enlargement and striatal atrophy. Upper panel, wild-type; middle panel, siRNA-treated; lower panel, untreated. Scale bar: 100 μm. (B) Brains from 8-week-old mice were immunostained with an antibody against mutant huntingtin (MAB5374), which recognizes the N-terminal portion of this protein. Neuronal intranuclear inclusions of mutant huntingtin were abundant in the striatum of mock-treated and untreated R6/2 mice. Compared with those mice, siRNA-HDExon1-treated R6/2 mice showed a decrease in number of cells with intranuclear inclusions in the striatum. Scale bars: 200 μm for upper panels (magnification ×40) and 30 μm for lower panels (magnification ×1000).