Antisense oligonucleotide therapy for spinocerebellar ataxia type 2

Journal name:
Nature
Volume:
544,
Pages:
362–366
Date published:
DOI:
doi:10.1038/nature22044
Received
Accepted
Published online

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.

At a glance

Figures

  1. Effect of ASO7 on motor phenotypes.
    Figure 1: Effect of ASO7 on motor phenotypes.

    a, Eight-week-old ATXN2-Q127 mice were treated with 210 μg ASO7 or saline ICV and tested on the rotarod at the indicated ages. n = 15 mice per group. b, Eight-week-old BAC-Q72 mice and wild-type (WT) littermates were treated with 175 μg ASO7 or saline ICV and tested on the rotarod at the indicated ages. n = 13 and 13, for wild-type mice treated with ASO7 and saline, respectively; n = 11 and 14, for BAC-Q72 mice treated with ASO7 and saline, respectively. Values are mean ± s.e.m. Probabilities of significance were determined using the method of generalized estimating equations. *P < 0.05; **P < 0.01; NS, not significant.

  2. Cerebellar gene expression for ASO7-treated SCA2 mice following rotatod tests.
    Figure 2: Cerebellar gene expression for ASO7-treated SCA2 mice following rotatod tests.

    ac, Expression in ATXN2-Q127 mice. a, ATXN2-Q127 mice were treated as in Fig. 1a and the cerebellar expression of the indicated genes was determined by qPCR relative to Actb at 22 weeks of age. The number of mice for saline and ASO7 treatments, respectively, were: ATXN2, 11 and 11; Atxn2, 11 and 11; Aif1, 7 and 8; Gfap, 11 and 11; Rgs8, 4 and 4; Pcp2, 11 and 11; Pcp4, 10 and 11; Cep76, 10 and 11; Homer3, 10 and 11; Fam107b, 10 and 10 mice. b, c, Cerebellar expression in ATXN2-Q127 mice for ATXN2, mouse Atxn2, Rgs8, Pcp2, Pcp4, Cep76, Homer3, Fam107b and β-actin (b), with abundances expressed relative to β-actin, determined by densitometry (c). Numbers indicate mouse identifiers. df, Expression in BAC-Q72 mice. d, BAC-Q72 mice were treated as in Fig. 1b and the cerebellar expression of the indicated genes was determined at 19 weeks of age. The number of mice per group in qPCR analyses was 14 saline-treated mice and 11 ASO7-treated mice for all genes except Rgs8 and Fam107b, where the n was 14 saline-treated and 10 ASO-treated mice. e, Cerebellar expression in BAC-Q72 mice of expanded ATXN2 detected with anti-1C2 antibody, and expression of mouse Atxn2, Rgs8, Pcp2, Pcp4, Cep76, Homer3, Fam107b and β-actin (e), with abundances expressed relative to β-actin, determined by densitometry (f). Values are mean ± s.d. from the number of mice indicated (a, d) or from three replicate blots (c, f). *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant; by Student’s t-test.

  3. Slow firing frequency of Purkinje cells from ATXN2-Q127 mice was restored by ASO7.
    Figure 3: Slow firing frequency of Purkinje cells from ATXN2-Q127 mice was restored by ASO7.

    ad, ATXN2-Q127 mice were treated with 210 μg ASO7 or saline by ICV injection at 8 weeks, and the firing frequency of Purkinje cells was evaluated at 14 weeks of age. Sal, saline. a, Mean distribution of firing frequencies for Purkinje cells from saline- and ASO7-treated mice. b, c, Representative traces of 1 s duration from Purkinje cells (b) and interspike-interval histogram (c) of the same cell as in b, calculated for a 2 min duration, with coefficients of variation (CV) shown. d, The firing frequencies (mean ± s.e.m.) were 25 ± 2 Hz for the saline-treated mouse (n = 50 neurons, n = 1 mouse) and 46 ± 2 Hz for ASO7-treated mice (n = 88 neurons, n = 2 mice). eh, ATXN2-Q127 mice were treated with 210 μg ASO7 or saline by ICV injection at 8 weeks. The firing frequency of Purkinje cells was evaluated at 22 weeks of age. e, Mean firing frequencies of all Purkinje cells measured from saline- and ASO7-treated mice and age-matched wild-type mice. f, g, Representative traces of 1 s duration of Purkinje cells (f) and interspike-interval histogram (g) of the same cell as in f, calculated for a 2 min duration, with values for the coefficient of variation shown. h, The firing frequencies (mean ± s.e.m.) were 16 ± 1 Hz for saline-treated ATXN2-Q127 mice (n = 107 neurons, n = 2 mice), 42 ± 2 Hz for the ASO7-treated ATXN2-Q127 mice (n = 102 neurons, n = 2 mice), and 40 ± 1 Hz for an age-matched wild-type mouse (n = 50 neurons, n = 1 mouse). All recordings were measured at 34.5 ± 1 °C. ****P < 0.0001, Student’s t-test.

  4. Slow firing frequency of Purkinje cells from BAC-Q72 mice was restored by ASO7.
    Figure 4: Slow firing frequency of Purkinje cells from BAC-Q72 mice was restored by ASO7.

    ac, Firing frequencies of BAC-Q72 mice at the indicated ages. a, Distributions of the firing frequency for wild-type and BAC-Q72 Purkinje cells. The number of Purkinje cells were (the age of mice is indicated in brackets): n = 33 wild-type and 59 BAC-Q72 (4 months); 49 wild-type and 101 BAC-Q72 (6 months); 55 wild-type and 92 BAC-Q72 (12 months) cells. b, Example recordings from Purkinje cells for wild-type and BAC-Q72 mice. c, Interspike intervals of the same cell over 2 min with mean firing frequency and coefficients of variation indicated. df, The firing frequency of Purkinje cells was restored in ten-month-old BAC-Q72 mice treated with 210 μg ASO7 for 10 weeks. d, The firing frequency of Purkinje cells in BAC-Q72 mice, BAC-Q72 mice treated with ASO7 and wild-type littermates. e, f, Representative 1-s trace of Purkinje cells (e) and interspike intervals (f) of the same cells over 2 min in saline-treated BAC-Q72 mice, ASO-treated BAC-Q72 and wild-type littermates with Purkinje cell firing frequency and coefficients of variation indicated. g, h, Mean firing frequency of Purkinje cells in a and d. g, Age-dependent firing frequency of Purkinje cells from BAC-Q72 mice. At 4 months the mean firing frequencies in Hz were 51 ± 2 for wild-type (n = 33 Purkinje cells, n = 1 mouse) and 54 ± 2 for BAC-Q72 (n = 59 Purkinje cells, n = 1 mouse). At 6 months the values were 41 ± 2 Hz for wild-type (n = 49 Purkinje cells, n = 2 mice) and 32 ± 1 Hz for BAC-Q72 (n = 101 Purkinje cells, n = 2 mice). At 12 months, values were 49 ± 2 Hz for wild-type (n = 55 Purkinje cells, n = 1 mouse) and 34 ± 1 for BAC-Q72 (n = 92 Purkinje cells, n = 1 mouse). h, ASO7 treatment restored the firing frequency of Purkinje cells from BAC-Q72 mice. The mean firing frequency of Purkinje cells from ten-month-old BAC-Q72 mice treated with saline was 36 ± 1 Hz (n = 148 Purkinje cells, n = 3 mice), whereas the mean firing frequency of BAC-Q72 mice treated with ASO7 was 49 ± 1 Hz (n = 134 Purkinje cells, n = 4 mice), similar to that of wild-type mice (49 ± 1 Hz, n = 155 Purkinje cells, n = 3 mice). ****P < 0.0001, ANOVA followed by Tukey’s multiple-comparison post hoc testing.

  5. In vitro screen for ATXN2 ASOs by qPCR.
    Extended Data Fig. 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.

  6. Positive hit ASOs evaluated in vivo.
    Extended Data Fig. 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.

  7. Effects of ASO7 on ATXN2 expression in vivo by dose and time.
    Extended Data Fig. 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.

  8. Weights of mice before and after rotarod testing.
    Extended Data Fig. 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.

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

References

  1. Rigo, F., Seth, P. P. & Bennett, C. F. Antisense oligonucleotide-based therapies for diseases caused by pre-mRNA processing defects. Adv. Exp. Med. Biol. 825, 303352 (2014)
  2. Pulst, S. M., Nechiporuk, A. & Starkman, S. Anticipation in spinocerebellar ataxia type 2. Nat. Genet. 5, 810 (1993)
  3. Dansithong, W. et al. Ataxin-2 regulates RGS8 translation in a new BAC-SCA2 transgenic mouse model. PLoS Genet. 11, e1005182 (2015)
  4. Hansen, S. T., Meera, P., Otis, T. S. & Pulst, S. M. Changes in Purkinje cell firing and gene expression precede behavioral pathology in a mouse model of SCA2. Hum. Mol. Genet. 22, 271283 (2013)
  5. Pulst, S. M. Degenerative ataxias, from genes to therapies: The 2015 Cotzias Lecture. Neurology 86, 22842290 (2016)
  6. Houlden, H. & Singleton, A. B. The genetics and neuropathology of Parkinson’s disease. Acta Neuropathol. 124, 325338 (2012)
  7. Scoles, D. R. et al. Repeat associated non-AUG translation (RAN translation) dependent on sequence downstream of the ATXN2 CAG repeat. PLoS One 10, e0128769 (2015)
  8. Huynh, D. P., Figueroa, K., Hoang, N. & Pulst, S. M. Nuclear localization or inclusion body formation of ataxin-2 are not necessary for SCA2 pathogenesis in mouse or human. Nat. Genet. 26, 4450 (2000)
  9. Huynh, D. P., Maalouf, M., Silva, A. J., Schweizer, F. E. & Pulst, S. M. Dissociated fear and spatial learning in mice with deficiency of ataxin-2. PLoS One 4, e6235 (2009)
  10. Saugstad, J. A., Marino, M. J., Folk, J. A., Hepler, J. R. & Conn, P. J. RGS4 inhibits signaling by group I metabotropic glutamate receptors. J. Neurosci. 18, 905913 (1998)
  11. Abdul-Ghani, M. A., Valiante, T. A., Carlen, P. L. & Pennefather, P. S. Metabotropic glutamate receptors coupled to IP3 production mediate inhibition of IAHP in rat dentate granule neurons. J. Neurophysiol. 76, 26912700 (1996)
  12. Meera, P., Pulst, S. M. & Otis, T. S. Cellular and circuit mechanisms underlying spinocerebellar ataxias. J. Physiol. (Lond.) 594, 46534660 (2016)
  13. Mizutani, A., Kuroda, Y., Futatsugi, A., Furuichi, T. & Mikoshiba, K. Phosphorylation of Homer3 by calcium/calmodulin-dependent kinase II regulates a coupling state of its target molecules in Purkinje cells. J. Neurosci. 28, 53695382 (2008)
  14. Iscru, E. et al. Sensorimotor enhancement in mouse mutants lacking the Purkinje cell-specific Gi/o modulator, Pcp2(L7). Mol. Cell. Neurosci. 40, 6275 (2009)
  15. Wei, P., Blundon, J. A., Rong, Y., Zakharenko, S. S. & Morgan, J. I. Impaired locomotor learning and altered cerebellar synaptic plasticity in pep-19/PCP4-null mice. Mol. Cell. Biol. 31, 28382844 (2011)
  16. Tsang, W. Y. et al. Cep76, a centrosomal protein that specifically restrains centriole reduplication. Dev. Cell 16, 649660 (2009)
  17. Ingram, M. et al. Cerebellar transcriptome profiles of ATXN1 transgenic mice reveal SCA1 disease progression and protection pathways. Neuron 89, 11941207 (2016)
  18. Fittschen, M. et al. Genetic ablation of ataxin-2 increases several global translation factors in their transcript abundance but decreases translation rate. Neurogenetics 16, 181192 (2015)
  19. Lee, K. H. et al. Circuit mechanisms underlying motor memory formation in the cerebellum. Neuron 86, 529540 (2015)
  20. Lang, E. J. et al. The roles of the olivocerebellar pathway in motor learning and motor control. A consensus paper. Cerebellum 16, 230252 (2017)
  21. Liu, J. et al. Deranged calcium signaling and neurodegeneration in spinocerebellar ataxia type 2. J. Neurosci. 29, 91489162 (2009)
  22. Miller, T. M. et al. An antisense oligonucleotide against SOD1 delivered intrathecally for patients with SOD1 familial amyotrophic lateral sclerosis: a phase 1, randomised, first-in-man study. Lancet Neurol. 12, 435442 (2013)
  23. Skotte, N. H. et al. Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients. PLoS One 9, e107434 (2014)
  24. Carroll, J. B. et al. Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene/allele-specific silencing of mutant huntingtin. Mol. Ther. 19, 21782185 (2011)
  25. Xia, H. et al. RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat. Med. 10, 816820 (2004)
  26. Keiser, M. S., Boudreau, R. L. & Davidson, B. L. Broad therapeutic benefit after RNAi Expression vector delivery to deep cerebellar nuclei: implications for spinocerebellar ataxia type 1 therapy. Mol. Ther. 22, 588595 (2014)
  27. Rodriguez-Lebron, E., Liu, G., Keiser, M., Behlke, M. A. & Davidson, B. L. Altered Purkinje cell miRNA expression and SCA1 pathogenesis. Neurobiol. Dis. 54, 456463 (2013)
  28. Becker, L. A. et al. Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice. Nature http://dx.doi.org/10.1038/nature22038 (2017)
  29. Swayze, E. E. et al. Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals. Nucleic Acids Res. 35, 687700 (2007)
  30. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)
  31. Meera, P., Wallner, M. & Otis, T. S. Molecular basis for the high THIP/gaboxadol sensitivity of extrasynaptic GABAA receptors. J. Neurophysiol. 106, 20572064 (2011)

Download references

Author information

  1. Present address: Roche Pharma Research and Early Development, Neuroscience, Ophthalmology & Rare Diseases, Roche Innovation Center Basel, Grenzacherstrasse 124, CH-4070 Basel, Switzerland.

    • Thomas S. Otis

Affiliations

  1. Department of Neurology, University of Utah, 175 North Medical Drive East, 5th Floor, Salt Lake City, Utah 84132, USA

    • Daniel R. Scoles,
    • Matthew D. Schneider,
    • Sharan Paul,
    • Warunee Dansithong,
    • Karla P. Figueroa &
    • Stefan M. Pulst
  2. Department of Neurobiology, University of California Los Angeles, Los Angeles, California 90095, USA

    • Pratap Meera &
    • Thomas S. Otis
  3. Ionis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, California 92010, USA

    • Gene Hung,
    • Frank Rigo &
    • C. Frank Bennett

Contributions

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.

Competing financial 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.

Corresponding authors

Correspondence to:

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.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: In vitro screen for ATXN2 ASOs by qPCR. (166 KB)

    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.

  2. Extended Data Figure 2: Positive hit ASOs evaluated in vivo. (1,475 KB)

    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.

  3. Extended Data Figure 3: Effects of ASO7 on ATXN2 expression in vivo by dose and time. (87 KB)

    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.

  4. Extended Data Figure 4: Weights of mice before and after rotarod testing. (78 KB)

    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.

  5. Extended Data Figure 5: ASO7 lowered expression of wild-type and mutant ATXN2 in cultured SCA2 patient-derived fibroblasts. (68 KB)

    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.

Supplementary information

PDF files

  1. Supplementary Information (5.5 MB)

    This file contains the Supplementary Discussion and Supplementary Figure 1, the uncropped blots.

Excel files

  1. Supplementary Tables (45 KB)

    This file contains Supplementary Tables 1-2.

Additional data