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Antisense oligonucleotide therapy for spinocerebellar ataxia type 2


There are no disease-modifying treatments for adult human neurodegenerative diseases. Here we test RNA-targeted therapies1 in two mouse models of spinocerebellar ataxia type 2 (SCA2), an autosomal dominant polyglutamine disease2. Both models recreate the progressive adult-onset dysfunction and degeneration of a neuronal network that are seen in patients, including decreased firing frequency of cerebellar Purkinje cells and a decline in motor function3,4. We developed a potential therapy directed at the ATXN2 gene by screening 152 antisense oligonucleotides (ASOs). The most promising oligonucleotide, ASO7, downregulated ATXN2 mRNA and protein, which resulted in delayed onset of the SCA2 phenotype. After delivery by intracerebroventricular injection to ATXN2-Q127 mice, ASO7 localized to Purkinje cells, reduced cerebellar ATXN2 expression below 75% for more than 10 weeks without microglial activation, and reduced the levels of cerebellar ATXN2. Treatment of symptomatic mice with ASO7 improved motor function compared to saline-treated mice. ASO7 had a similar effect in the BAC-Q72 SCA2 mouse model, and in both mouse models it normalized protein levels of several SCA2-related proteins expressed in Purkinje cells, including Rgs8, Pcp2, Pcp4, Homer3, Cep76 and Fam107b. Notably, the firing frequency of Purkinje cells returned to normal even when treatment was initiated more than 12 weeks after the onset of the motor phenotype in BAC-Q72 mice. These findings support ASOs as a promising approach for treating some human neurodegenerative diseases.

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Figure 1: Effect of ASO7 on motor phenotypes.
Figure 2: Cerebellar gene expression for ASO7-treated SCA2 mice following rotatod tests.
Figure 3: Slow firing frequency of Purkinje cells from ATXN2-Q127 mice was restored by ASO7.
Figure 4: Slow firing frequency of Purkinje cells from BAC-Q72 mice was restored by ASO7.

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We thank P. Jafar-Nejad for his contributions to interpreting results and for reading and editing the manuscript, L. Pflieger for contributing to the production of Supplementary Data. This work was supported by grants R01NS33123, R56NS33123 and R37NS033123 from the National Institutes of Neurological Disorders and Stroke (NINDS) to S.M.P., the Noorda foundation to S.M.P., NINDS grants RC4NS073009 and R21NS081182 to D.R.S. and S.M.P., NINDS grant NS090930 to T.S.O., and a gift from Ionis Pharmaceuticals. S.M.P. received grant support from the Target ALS Foundation.

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Authors and Affiliations



D.R.S. conceived and designed the study, performed experiments, conducted all ICV injections, analysed all data and wrote the manuscript. M.S. performed all motor-testing experiments and with D.R.S. contributed to blinding of all mouse trials including ASO treatments, motor testing and electrophysiological evaluations. M.D.S. also conducted all qPCR analyses of mouse tissues. P.M. designed and performed all electrophysiological experiments, analysed and interpreted the resulting data, and prepared figures. S.P. prepared all western blots. W.D. conducted the study of SCA2 patient-derived fibroblasts. K.P.F. was in charge of mouse breeding. G.H. led the ASO in silico design, ASO in vitro screening, advised the in vivo screening approach, and provided ASOs. F.R. and C.F.B. contributed to the in vivo screening approach, design of motor phenotype studies, and interpretation of results. T.S.O. designed and helped interpret the electrophysiological analyses. S.M.P. conceived and designed the study with D.R.S. and contributed SCA2 patient-derived fibroblasts. All authors contributed to the writing of the manuscript.

Corresponding authors

Correspondence to Daniel R. Scoles or Stefan M. Pulst.

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Competing interests

S.M.P. is a consultant for Progenitor Life Sciences and Ataxion Pharmaceuticals. T.S.O. is an employee of F. Hoffmann-La Roche, Ltd. G.H., F.R. and C.F.B are employed by Ionis Pharmaceuticals, which supplied the ASOs used in the study.

Additional information

Reviewer Information Nature thanks R. L. Juliano, J. Rothstein and T. Siddique for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 In vitro screen for ATXN2 ASOs by qPCR.

A total of 152 ASOs were delivered at 4.5 μM to HepG2 cells by electroporation in two 384-well plates. ATXN2 expression was evaluated by qPCR (n = 3 wells per ASO). Shown is the evaluation of the eight best positive hit ASOs for half maximal inhibitory concentration (IC50) determination. Values are mean ± s.d. of ATXN2 quantity relative to total RNA. Cont., scrambled control ASO.

Extended Data Figure 2 Positive hit ASOs evaluated in vivo.

ac, 250 μg of the indicated ASOs in a total of 7 μl was delivered by ICV injection. After 7 days treatment, the expression of mouse Atxn2 or human ATXN2 and Aif1 was determined by qPCR relative to Actb. a, Wild-type FVB mice. Whereas ASO7 reduced mouse Atxn2 the most by 50%, significant reduction of mouse Atxn2 was not indicated by ANOVA testing for any of ASOs. ASO8 significantly elevated Aif1 expression (P < 0.05). b, BAC-Q72 mice. ATXN2 expression was significantly reduced by ASOs 1, 3 and 7 compared to saline (P < 0.001, 0.01 and 0.01, respectively). Elevations of Aif1 was not observed compared to saline-treated mice. c, ATXN2-Q127 mice. Compared to saline treated mice, ASOs 1, 3, 7 and 8 all significantly lowered ATXN2 expression by 40% or greater (P < 0.001), whereas ASOs 3 and 8 increased Aif1 expression (P < 0.001). Values are mean ± s.d. relative to those determined from normal saline-treated mice. Statistical tests were ANOVA followed by the Bonferroni correction. Technical replication was inserted by employing qPCR with triplicate determinations, and biological replication was made by evaluating multiple mice and/or mouse lines. The number of mice (left-to-right in each chart) was as follows: a, n = 2, 2, 2, 3, 2, 2, 2, 2, 1; b, n = 2, 1, 1, 2, 2, 2, 1, 2; c, n = 2, 3, 1, 1, 1. d, e, ASOs localized to the cerebellar Purkinje cell layer of treated mice. Mice were treated by ICV injection into the right lateral ventricle of the indicated lead ASOs for 7 days in BAC-Q72 mice (ASO3 and ASO7 used at 250 μg) or 10 weeks in ATXN2-Q127 mice (ASO1 used at 200 μg). ASOs were localized in paraffin embedded sections by immunohistochemical peroxidase staining using an anti-ASO antibody. Saline, ATXN2-Q127 mice treated by ICV injection of 7 μl saline for 10 weeks. d, 10× objective. e, 3× digital zoom of a region of the Purkinje cell layer in the corresponding 10× image, indicated by the box. Scale bars, 100 μm (d), 25 μm (e). fi, Distribution of ASO7 in the cerebellum. f, ASO7 was distributed in Purkinje cell layers throughout the cerebellum (2× objective). gi, Higher power images for regions indicated in f showed ASO7 localization in Purkinje cells across the cerebellum: g, 10× objective; h, i, 40× objective.

Extended Data Figure 3 Effects of ASO7 on ATXN2 expression in vivo by dose and time.

ac, Dose response for ASO7 on ATXN2 expression in BAC-Q72 mice treated with ASO7 by ICV injection for 14 days. a, Expression of cerebellar human ATXN2 and mouse Atxn2 determined by qPCR. The 210 μM dose reduced ATXN2 by 63.2% (P < 0.01) and mouse Atxn2 by 44.5% (P < 0.001) compared to the 0 μM dose. The statistical test used was ANOVA followed by the Bonferroni correction. b, Cerebellar Aif1 expression determined by qPCR demonstrated that Aif1 levels were not significantly altered by ASO7 treatment. c, Cerebellar Gfap expression determined by qPCR demonstrated that Gfap levels were not significantly altered by ASO7 treatment. ac, The replicate number of mice for the saline, 52 μg, 105 μg and 210 μg treatments was 3, 2, 3 and 3, respectively, and the values indicated are mean ± s.d. The experiment was performed once.

Source data

Extended Data Figure 4 Weights of mice before and after rotarod testing.

a, Rotarod test of ATXN2-Q127 mice treated with a single ICV dose of 210 μg ASO7. b, Rotarod test of BAC-Q72 mice treated with a single ICV dose of 175 μg ASO7. Weeks of ASO treatments are indicated on the x axes. Mouse weights were unaffected by ASO7 treatment. Significant differences between weights of BAC-Q72 mice compared to wild-type littermates were observed (P < 0.001 for any age group, Student’s t-test). The relevance of mouse weights on motor phenotype testing is discussed in the Supplementary Discussion.

Source data

Extended Data Figure 5 ASO7 lowered expression of wild-type and mutant ATXN2 in cultured SCA2 patient-derived fibroblasts.

a, Patient-derived SCA2(CAG35) fibroblasts were transfected with the indicated quantities of ASO7. After 72 h RNA was prepared and total ATXN2 expression was determined by qPCR. Values are mean ± s.d. of 3 technical replicates from single cultures. ATXN2 was reduced by 79% for the 2 μM dose compared to 0 μM (P < 0.001, Student’s t-test). b, To determine ASO7 effect on the expression of non-mutant (CAG22) and mutant (CAG35) ATXN2, RT–PCR reactions were evaluated by agarose gel electrophoresis, with loading controlled for by GAPDH. Both a and b were replicated once yielding nearly the same result.

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Scoles, D., Meera, P., Schneider, M. et al. Antisense oligonucleotide therapy for spinocerebellar ataxia type 2. Nature 544, 362–366 (2017).

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