Sustained therapeutic reversal of Huntington's disease by transient repression of huntingtin synthesis

Abstract

The primary cause of Huntington's disease (HD) is expression of huntingtin with a polyglutamine expansion. Despite an absence of consensus on the mechanism(s) of toxicity, diminishing the synthesis of mutant huntingtin will abate toxicity if delivered to the key affected cells. With antisense oligonucleotides (ASOs) that catalyze RNase H-mediated degradation of huntingtin mRNA, we demonstrate that transient infusion into the cerebrospinal fluid of symptomatic HD mouse models not only delays disease progression but mediates a sustained reversal of disease phenotype that persists longer than the huntingtin knockdown. Reduction of wild-type huntingtin, along with mutant huntingtin, produces the same sustained disease reversal. Similar ASO infusion into nonhuman primates is shown to effectively lower huntingtin in many brain regions targeted by HD pathology. Rather than requiring continuous treatment, our findings establish a therapeutic strategy for sustained HD disease reversal produced by transient ASO-mediated diminution of huntingtin synthesis.

Figure 1. Sustained reduction in human and mouse huntingtin mRNA and protein by transient ASO infusion into the CNS

(A) Dose response for HuASO in the brain of BACHD mice immediately following continuous two-week infusion of a human huntingtin targeting ASO (HuASO) at 10, 25 or 50 μg/day. Human huntingtin mRNA levels determined by qPCR and are expressed as mean ± SEM percent (%) of human huntingtin mRNA relative to saline treated controls (n=3 per dose). (B–E) Animals treated with 50 μg/day HuASO euthanized at 0, 4, 8, 12 and 16 weeks post-treatment termination (n=4 per time point per dose). (B) Concentrations of accumulated HuASO in brain tissue measured by capillary gel electrophoresis. (C) Levels of human mRNAs as measured by qPCR. (D) Soluble mutant huntingtin levels quantified with time resolved forester resonance energy transfer (TR-FRET) assay. (E) Immunoblot of total huntingtin levels in brains of HuASO infused mice. The more slowly migrating band is human huntingtin protein (BACHD Tg); the more rapidly migrating band is endogenous mouse huntingtin (Mouse Htt). (F–H) Animals treated with 75 μg/day MoHuASO euthanized at 0, 2, 4, 6, 8, 11 and 16 weeks post-treatment termination (n=4 per time point per dose). Levels of (F) human and mouse (G) huntingtin mRNAs as measured by qPCR. (H) Immunoblot of total huntingtin levels in brains of infused mice. Asterisks (*p<0.05, **p<0.01, and ***p<0.001) denote statistically significant changes relative to saline infused animals (Two-way ANOVA and Bonferroni’s post hoc tests). See also Figure S1.

Figure 2. ASOs infused into the cerebral spinal fluid distribute and are active throughout the CNS

(A and B) An ASO targeting human huntingtin (HuASO) was infused continuously for two weeks into the right lateral ventricle of 2-month-old non-transgenic mice. Immunohistochemical staining of antisense oligonucleotides (red), neurons (NeuN, green), astrocytes (GFAP, green) and nuclei (DAPI, Blue). Antisense oligonucleotides are present in most neuronal nuclei (B, top inset), astrocytes (B, bottom inset) and other non-neuronal cells (examples denoted with *). Scale bar: (A) 100μm, (B) 50μm and inset 5μm. Representative example from two independent experiments, n=4 per treatment. (C–E) HuASO was infused continuously for two weeks into the right lateral ventricle of 2-month old BACHD mice. Immunoblot of human (upper band) and mouse (lower band) huntingtin, and quantification of human huntingtin mRNA levels (mean % ± SEM relative to saline controls) 8 weeks post-treatment termination in BACHD (C) cortex ipsilateral and contralateral to the injection site, (D) ipsilateral and contralateral striatum (E) thalamus, midbrain, brainstem and cerebellum. Asterisks (*p<0.05, **p<0.01, and ***p<0.001) mark statistically significant changes relative to saline infused animals (n=5 per treatment, two-tailed unpaired t-tests). See also Figure S2.

Figure 3. Motor coordination reverts to normal levels after suppression of mutant huntingtin with ASO therapy

(A) Schematic of experimental design (B–C). At 3 months of age YAC128 mice were infused into the right lateral ventricle with an ASO that targets human huntingtin (HuASO) or saline for two weeks at 50 μg/day. (B) Motor performance on a rotarod of mouse cohorts before (3 months old) and 1 and 2 months after ASO infusion (4 and 5 months of age) (n=7–16). (C) Human huntingtin protein levels in brains of YAC128 mice 6 weeks after treatment termination. Data are expressed as mean percentage ± SEM relative to saline treated controls (All animals in B were analyzed, two-tailed unpaired t-test). (D) Schematic of experimental design (E–F). Six-month-old YAC128 mice were intraventricularly infused for two weeks with 50 μg/day of HuASO or a vehicle control (saline). (E) Human huntingtin protein levels in brains of YAC128 mice sacrifice at 9 months of age, 3 months after treatment. Data are expressed as mean percentage ± SEM relative to saline treated controls (All animals in F were analyzed, two-tailed unpaired t-test). (F) Motor performance on a rotarod of mouse cohorts at ages between 6 and 9 months (n=8–12). Asterisks denote (*p<0.05, **p<0.01, and ***p<0.001) statistically significant differences of YAC128 HuASO treated mice (using one-way ANOVA and Tukey’s post hoc test for all behavioral tests). See also Figure S3.

Figure 4. Sustained phenotypic reversal from transient ASO infusion into the CNS of BACHD mice for 8 months after treatment termination

(A) Schematic of treatment paradigms (B-D). At 6 months of age, BACHD mice were infused for two weeks with 50μg/day of an ASO that targets human huntingtin (HuASO), ASOs that have no target in the mouse genome (CntASO) or vehicle (Saline). Non-transgenic littermates were infused with vehicle (Saline). (B) Levels of human huntingtin mRNAs 2 months post-treatment, as measured by qPCR in BACHD mice treated at 6 months of age (n=5 per treatment, data are expressed as mean % ± SEM, two-tailed unpaired t-test, p<0.0001). (C) Motor performance on a rotarod between ages 6 and 12 months, and measured every 4 weeks. Asterisks denote statistically significant changes compared to BACHD HuASO treated animals (n=8–13). (D) Open-field performance 2 (8 months old, p=0.111), 4 (10 months old, p=0.027) and 6 (12 months old, p=0.034) months post-treatment in BACHD mice (non-transgenic, n=3–6; BACHD, n=12–16). (E) Schematic of treatment paradigm (F–K). At 6 months of age, BACHD mice were infused for two weeks with 50μg/day of HuASO (red) or saline (black). Non-transgenic mice are included as behavioral controls (white). (F) Performance of mice 6 and 9 months after ASO infusion determined by time mobile in an open-field test, p=0.002 and p=0.016, respectively (n=6–8). (G) Rotarod performance after two consecutive days of training at 9 months post-treatment (15 months of age), p<0.0001 (n=6–8). (H) Light/dark choice at 15 months of age, p=0.016 (n=6–8). In all instances (F–H), saline treated BACHD mice performed significantly worse than non-transgenic and HuASO treated BACHD mice (One-way ANOVAs and Tukey’s post hoc tests). (I) Immunoblot of huntingtin protein 2 and 9 months post-treatment, GAPDH is used as a loading control. (J) 9 months post-treatment qPCR was used to quantify mouse and human huntingin mRNA levels. qPCR are expressed as mean ± SEM percent (%) of huntingtin mRNA relative to saline treated controls (n=6 per treatment). (K) Immunohistochemical (IHC) staining for mutant huntingtin aggregates with anti-polyglutamine antibody (3B5H10) (brown) and nuclear counterstain hematoxylin (blue), 9 months post-treatment. Scale bar: 50μm. Representative images, n=6 BACHD per treatment. See also Figure S4.

Figure 5. Simultaneous suppression of mutant and normal huntingtin does not attenuate sustained phenotypic reversal

(A) Schematic of experimental design. At 2 months of age BACHD mice and non-transgenic littermates were infused for two weeks with 50μg/day HuASO (red), 75μg/day of an ASO that targets mouse and human huntingtin (MoHuASO) (Blue) or saline (black) (B–D, non-transgenic, n=11–15; BACHD, n=15–20). (B) Motor coordination (rotarod) immediately before treatment (2 months old) and monthly for 11 months after treatment. (B, Top) HuASO treated BACHD animals, saline treated BACHD animals and saline treated non-transgenic littermates, asterisks denote significance relative to HuASO treated BACHD animals. (B, Middle) MoHu treated BACHD animals and HuASO treated BACHD animals, no significant differences were found. (B, Bottom) Non-transgenic animals treated with saline, HuASO or MoHuASO, no significant differences were found. (C–D) Time mobile in the open-field 3 months post-treatment in 5-month old (C) BACHD (p=0.025) and (D) non-transgenic littermates (p=0.515). (E) Immunoblot of huntingtin from (top) BACHD mice and (bottom) non-transgenic littermates, 1 month or 11 months post-treatment. (F) 11 months post-treatment qPCR was used to quantify mouse and human huntingin mRNA levels. qPCR are expressed as mean ± SEM percent (%) of huntingtin mRNA relative to saline controls (BACHD, ASO n=4 and saline n=3; Non-transgenic, ASO n=3 and saline n=2). Asterisks (*p<0.5, **p<0.01, ***p<0.001) denote statistically significant differences (using one-way ANOVA and Tukey’s post hoc test for all behavioral assays). See also Figure S5.

Figure 6. Delayed disease progression and prolonged survival in R6/2 mice after CNS infusion of an ASO to mutant human huntingtin

(A) Schematic of treatment paradigm. R6/2 mice and non-transgenic littermates (NonTg) received intraventricular infusion of 50μg/day of human huntingtin ASO targeting the R6/2 transgene (HuASO^Ex1), a control ASO (Cnt1ASO) or saline. Treatment began at 8 weeks of age and animals were euthanized at 12 weeks of age for RNA analysis, pathology, or aged for survival. (B) Human huntingtin mRNA levels in brain of R6/2 mice treated with ASO targeting human huntingtin compared to saline treated mice (n=4). qPCR data expressed as mean ± SEM; percent (%) of RNA is relative to saline treated controls (two-tailed unpaired t test, **p<0.01). (C) Total brain weight (mg) immediately following the 4-week ASO infusion. Untreated 8-week-old R6/2 mice are included for comparison of the disease state at the time of treatment initiation (HuASO^Ex1 treated R6/2, n=10; saline treated R6/2, n=11; control ASO treated R6/2, n=8; saline treated Non-tg, n=10; HuASO^Ex1 treated Non-tg, n=5; and untreated 8-week-old R6/2, n=4, p<0.0001). Asterisks (**p<0.01) denote statistically significant differences using one-way ANOVA, Tukey’s post hoc test. (D) Representative image of HuASO^Ex1 treated R6/2 brain (left) or saline treated R6/2 brain (right) stained with cresyl violet. The striatum is outlined (Red: HuASO^Ex1 treated, Black: saline treated). (E–F) Quantification of (E) striatal and (F) cortical volume in R6/2 both ipsilateral and contralateral to injection site using the cavaleri method (saline, n=9; HuASO^Ex1, n=10; CntASO, n=7). (G) Plots of ages of R6/2 mice at time of death (saline, n=7; HuASO^Ex1, n=9). Data are expressed as Kaplan-Meyer survival curves (p=0.049). See also Figure S6.

Figure 7. Broad ASO distribution and suppression of huntingtin mRNA in a non-human primate brain after ASO infusion into the cerebral spinal fluid

(A) Distribution of ASOs in the Rhesus monkey brain visualized with immunostaining (anti-pan ASO) and counterstained for nuclei with hematoxylin following a 21 day intrathecal infusion of 4 mg/day ASO targeting monkey huntingtin (MkHuASO), including (A, bottom right) pyramidal neurons of the cortex and (A, bottom left) medium spiny neurons in the caudate. (B) ASO distribution in the hippocampus and (B, bottom right) neurons in the dentate gyrus. (C) ASO accumulation in a coronal section including the pons and cerebral aqueduct, CA; ASO is present in pontine cell bodies despite low levels of ASO accumulation in the surrounding tissue (lower panel). (D) ASO accumulation in the cerebellum and surrounding brain regions. (E) ASO accumulation in motor neurons in the spinal cord. (F) mRNA levels of monkey huntingtin from various brain regions immediately following infusion, as determined by qPCR (n=3 per treatment, two-tailed unpaired t-tests). (G) Monkey huntingtin mRNA levels in the anterior (frontal) cortex (red), posterior (occipital) cortex (orange), and cervical spinal cord (yellow), 0, 2, 4 and 8 weeks post-treatment termination (n=3 per treatment, two-way ANOVA, Bonferroni post hoc test). Data expressed as mean ± SEM, percent (%) of RNA relative to saline treated controls (blue). Asterisk (*p<0.5, **p<0.01, ***p<0.001) denotes statistically significant differences. See also Figure S7.