(A) Schematic of the construct delivered to cells via AAV9 containing either non-targeted shRNA or shRNA against torsinA. Arrows are used to show promoters. (B) There was strong GFP expression in the basal ganglia (left image) of mice injected with the shRNA against torsinA. The right image is a sagittal section of a representative mouse injected with AAV-TorsinA shRNA-GFP. The dotted line delineates the separation between basal ganglia and cerebral cortex. CX = cerebral cortex, BG = basal ganglia, V = ventricle. Scale bar = 2.5 mm in left image, 500 µm in right image. (C) Example western blot (left) and quantification (right) from three animals in which AAV9 containing shRNA against torsinA was injected into the basal ganglia of adult mice. There is an average knockdown of 81.3% ± 6.2% (Mean ±S.E.M). (D) Observers blinded to the condition of the animals rated the basal ganglia injected animals on a previously published dystonia scale. On the graph each point represents an individual animal and the bars represent the average of all animals. All animals up to 13 weeks post-injection had an average score <1 (Mean ± S.E.M, NT shRNA, N = 8; Tor1A KD BG, N = 6; Tor1A KD BG2, N = 8). (E) There was strong GFP expression throughout the cerebellum (left image) of mice injected with the shRNA against torsinA. The right image is a sagittal section of a representative mouse injected with AAV-TorsinA shRNA-GFP. The dotted line delineates the cerebellum (CB). Scale bar = 2.5 mm in left image, 500 µm in right image. (F) Representative Western blot (left) and quantification (right) showing knockdown of torsinA from cerebellar lysates prepared from animals injected with a shRNA against torsinA when compared to WT animals or animals injected with a non-targeted (NT) shRNA. TorsinA KD 77.9% ± 4.7% (Mean ± S.E.M, Tor1A KD N = 6). (G) Adult mice with knockdown of torsinA in the cerebellum showed symptoms consistent with dystonia as rated by observers blinded to the condition of the animals. On the dystonia scale, a score of ≥2 is considered dystonic. Adult mice with knockdown of torsinA, using two shRNAs targeted to different regions of the protein, exhibited dystonic symptoms beginning around 7–9 weeks. (*=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001, Mean ± S.E.M, NT shRNA, N = 10; Tor1A KD CB, N = 20; Tor1A KD CB2, N = 15). By 13 weeks post-injection animals on average exhibited symptoms consistent with dystonia on this scale. (H) An example mouse 13 weeks after the injection of shRNA against torsinA into the cerebellum exhibiting an abnormal hind limb posture (arrow). (I) EMGs performed in three animals confirmed that abnormal postures are due to co-contraction of agonist and antagonist muscle pairs. In this example trace, the gastrocnemius (Ga, red) and anterior tibialis (AT, black) are shown during a dystonic co-contraction (arrow). Scale bar represents 5 s (x) by 100 μV (y). (J) The severity of motor symptoms, quantified by the dystonia scale 13 weeks after injection of torsinA in the cerebellum, is correlated with the level of shRNA mediated knockdown of torsinA. As the percent of torsinA knockdown increases, so does the severity of the motor phenotype (Torsin A KD CB (N = 22, purple) and torsin A KD CB2 (N = 15, pink). Black symbols show the average of the binned combined data from both shRNAs against torsinA (Spearman r = 0.63, 95% CI 0.3759 to 0.7960, p value < 0.0001). knockdown is presented as within-blot loading control-normalized values. The data are presented as mean ±S.E.M.