Peripheral androgen receptor gene suppression rescues disease in mouse models of spinal and bulbar muscular atrophy

Abstract

Spinal and bulbar muscular atrophy (SBMA) is caused by the polyglutamine androgen receptor (polyQ-AR), a protein expressed by both lower motor neurons and skeletal muscle. Although viewed as a motor neuronopathy, data from patients and mouse models suggest that muscle contributes to disease pathogenesis. Here, we tested this hypothesis using AR113Q knockin and human bacterial artificial chromosome/clone (BAC) transgenic mice that express the full-length polyQ-AR and display androgen-dependent weakness, muscle atrophy, and early death. We developed antisense oligonucleotides that suppressed AR gene expression in the periphery but not the CNS after subcutaneous administration. Suppression of polyQ-AR in the periphery rescued deficits in muscle weight, fiber size, and grip strength, reversed changes in muscle gene expression, and extended the lifespan of mutant males. We conclude that polyQ-AR expression in the periphery is an important contributor to pathology in SBMA mice and that peripheral administration of therapeutics should be explored for SBMA patients.

Figure 1. Dose-dependent suppression of AR expression by targeted ASO

(a) Human umbilical vein endothelial cells (top) and mouse b. END cells (bottom) were electroporated in the presence of AR targeted or control ASO at the indicated concentrations. Relative AR mRNA levels were determined after 16 hr and are reported as mean +/− SD. UTC, untransfected control. (b, c) Wild type male mice received subcutaneous injections of ASO1, control ASO or saline from six to 14 weeks (n=3/group). Animals were treated with ASO1 or control ASO at 50 mg/kg/week, or with two lower doses of ASO1 (25 mg/kg/day × 3 days, then 12.5 mg/kg/week; or 17.5 mg/kg/day × 3 days, then 10 mg/kg/week). Quadriceps muscle was harvested at 14 weeks for analysis of AR expression. (b) Relative AR mRNA levels (mean +/− SEM). ^*p<0.05, ^***p<0.001 (c) AR protein as detected by immunoprecipitation and western blot. (d) BAC fxAR121 males (n=4/group) received subcutaneous injections of control ASO (25 mg/kg/week), ASO2 (25, 12.5 or 6.25 mg/kg/week) or saline from seven to 15 weeks. Quadriceps muscle was harvested 48 hrs after the final dose and analyzed for human and mouse AR mRNA levels. Data are mean +/− SD. ^***p<0.001 compared to saline treatment.

Figure 2. Subcutaneous ASO administration suppresses AR expression in muscle but not spinal cord

(a) 11 week old BAC fxAR121 males (n=4/group) received subcutaneous injections of ASO (100 mg/kg, twice weekly) or saline for four weeks. Quadriceps muscle, brain and spinal cord were harvested 48 hrs after the final dose and analyzed for human (left panel) and mouse (right panel) AR mRNA levels. Data are mean +/− SD. ND, not detected. ^***p<0.001 compared to saline treatment. (b) Wild type (left panel) and BAC fxAR121 males (middle and right panels) received subcutaneous injections of ASO1 (50 mg/kg weekly, starting at 6 weeks) or saline for four weeks. The external urethral sphincter muscle was harvested and stained for AR by immunohistochemistry. Scale bar, 15 μm. (c, d) AR113Q (n=10/group) and wild type (n=3/group) males received subcutaneous injections of ASO1 (50 mg/kg, twice weekly for four weeks, and then once weekly) or saline, from eight until 26 weeks. Quadriceps muscle and spinal cord were harvested at the indicated ages and analyzed for (c) AR mRNA (mean +/− SEM) and (d) protein by immunoprecipitation and western blot. ^***p<0.001; n.s., not significant.

Figure 3. Dose-dependent rescue of grip strength and body mass in BAC fxAR121 males by ASO2

(a, b) BAC fxAR121 or wild type males (n=10/group) were treated with increasing doses of ASO2 (6.25 - 25 mg/kg/week), control ASO or saline from seven until 15 weeks. Grip strength (a) and body mass (b) are reported as mean +/− SD. Grip strength and body mass of tg mice treated with ASO2 at 12.5 and 25 mg/kg/week are different from saline treated in weeks 15–21, p<0.05. (Tg mice treated with saline show decreased grip strength compared to wt mice in weeks 11–21, p<0.05 by ANOVA, and decreased body mass in weeks 12–21, p<0.05 by ANOVA.) (c, d) BAC fxAR121 or wild type males (n=7 – 10/group) received subcutaneous injections of ASO2 (50 mg/kg/week, starting at 6 weeks) or saline for four weeks. Age-dependent changes in (c) grip strength and (d) body mass are reported as mean +/− SD. In panel c, ^**p<0.01, ^***p<0.001 compared to saline treated BAC fxAR121 starting at week 13 by ANOVA. In panel d, p<0.05 for ASO2 vs saline treated tg mice from weeks 14–16 by ANOVA. (e) BAC fxAR121 or wild type males (n=9–10/group) received subcutaneous injections of ASO2 (50 mg/kg/week, starting at 6 weeks) or saline for four weeks. MRI was performed at 16 weeks and used to determine lean body mass. Data are mean +/− SD. ^***p<0.001 compared to saline treated BAC fxAR121 by ANOVA.

Figure 4. Peripheral polyQ AR suppression rescues survival of SBMA mice

(a) Survival of BAC fxAR121 or wild type males (n=10/group) treated with increasing doses of ASO2 (6.25 - 25 mg/kg/week), control ASO or saline from seven until 15 weeks. The dark blue/black line depicts overlapping survival curves of wild type (saline and ASO2 treated) and transgenic mice treated with ASO2 @ 25 mg/kg/week. (b) Survival of AR113Q (n=10/group) and wild type (n=3/group) males receiving subcutaneous injections of ASO1 or saline from eight until 26 weeks (as described in figure 1c). The red line depicts overlapping survival of wild type males treated with saline or ASO1. ASO1 treatment significantly extended lifespan of AR113Q males (p=0.016).

Figure 5. Phenotype rescue is dependent upon treatment duration and is observed when treatment begins at symptom onset

BAC fxAR121 or wild type males received subcutaneous injections of ASO2 or saline starting at 11 weeks. Mice were treated (n=6–10/group) for eight weeks with 50 mg/kg/week, or for two, four or eight weeks with 25 mg/kg/week. (a) Survival of BAC fxAR121 (Tg) is significantly extended by ASO treatment. p< 0.01. The light blue line depicts overlapping curves for wild type, transgenic mice treated with ASO2 @ 50 mg/kg/week for 8 weeks, and transgenic mice treated with ASO2 @ 25mg/kg/week for 8 weeks. (b) Grip strength at 22 weeks, four weeks after final treatment (n=3–5/group, except saline treated BAC fxAR121, where only one mouse survived at this time point). Data are mean +/− SD. ^**p<0.05 compared to saline treated wt males.

Figure 6. Peripheral ASO administration rescues muscle atrophy and gene expression changes

(a) Tibialis anterior (TA) muscle mass at 26 weeks from AR113Q or wild type males treated with ASO1 or saline (treated as described in Figure 1c), or post-treatment at age 36 or 46 weeks. Data are mean +/− SEM. ^*p<0.05; n.s., not significant. (b) Quadriceps muscle fiber size distribution (left panel) and mean +/− SEM (right panel) from AR113Q or wild type males at 26 weeks, treated as described in Figure 1c. ^***p<0.001 (c) AR immunofluorescence (red) of quadriceps and levator ani/bulbocavernosus (LA/BC) muscles of AR113Q and wild type males at 26 weeks, treated as described in Figure 1c. DAPI stains nuclei. Scale bar, 10 μm (d) External urethral sphincter (upper panel) and quadriceps muscle (lower panel) fiber size were determined from BAC fxAR121 males (n=4/group) that received subcutaneous ASO2 (50 mg/kg/week, starting at 6 weeks) for four weeks. Muscle was harvested from transgenic (Tg) and wild type males at 10 and 16 weeks. Data are mean +/− SD. ^*p<0.05 (e) BAC fxAR121 males (n=3–4/group) received subcutaneous injections of ASO2 (50 mg/kg/week, starting at 6 weeks) or saline for four weeks. Quadriceps muscle was harvested at 10 and 16 weeks, and AChR-α and myogenin mRNA expression determined. Data are mean +/− SD. ^**p<0.01, ^***p<0.001 (f) AChR-α and myogenin mRNA expression in quadriceps muscle of AR113Q or wild type males at 26 weeks (treated as described in Figure 1c), or following termination of treatment at 36 weeks. Data are mean +/− SEM. ^**p<0.01, ^*p<0.05

Figure 7. Intraventricular ASO administration does not provide added benefit to BAC fxAR121 mice receiving peripheral ASO

(a) BAC fxAR121 mice were dosed as indicated with saline (sal), intraventricular (ICV) ASO3 or control (cnl) ASO (100 μg at 8 weeks), and subcutaneous (SQ) ASO2 (12.5 mg/kg/week, starting at 8 weeks). Spinal cord and quadriceps muscle were harvested 4 or 8 weeks following ICV administration and analyzed for human AR mRNA levels. Data (mean +/− SEM) are reported relative to mice receiving ICV and SQ saline (n=4/group). ^***p<0.001. (b, c) Wild type or BAC fxAR121 males (n=8–10/group) were treated as indicated with saline (sal), intraventricular (ICV) ASO3 or control (cnl) ASO (100 μg at 8 weeks), and subcutaneous (SQ) ASO2 (12.5 mg/kg/week, starting at 8 weeks). Age dependent changes in grip strength (b) and survival (c) are shown. Data are mean +/− SEM.