Suppression of Kv3.3 channels by antisense oligonucleotides reverses biochemical effects and motor impairment in spinocerebellar ataxia type 13 mice

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder characterized by cerebellar and retinal degeneration, and is caused by a CAG-polyglutamine repeat expansion in the ATAXIN-7 gene. Patients with SCA7 develop progressive cone-rod dystrophy, typically resulting in blindness. Antisense oligonucleotides (ASOs) are single-stranded chemically modified nucleic acids designed to mediate the destruction, prevent the translation, or modify the processing of targeted RNAs. Here, we evaluated ASOs as treatments for SCA7 retinal degeneration in representative mouse models of the disease after injection into the vitreous humor of the eye. Using Ataxin-7 aggregation, visual function, retinal histopathology, gene expression, and epigenetic dysregulation as outcome measures, we found that ASO-mediated Ataxin-7 knockdown yielded improvements in treated SCA7 mice. In SCA7 mice with retinal disease, intravitreal injection of Ataxin-7 ASOs also improved visual function despite initiating treatment after symptom onset. Using color fundus photography and autofluorescence imaging, we also determined the nature of retinal degeneration in human SCA7 patients. We observed variable disease severity and cataloged rapidly progressive retinal degeneration. Given the accessibility of neural retina, availability of objective, quantitative readouts for monitoring therapeutic response, and the rapid disease progression in SCA7, ASOs targeting ATAXIN-7 might represent a viable treatment for SCA7 retinal degeneration.

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A chlorzoxazone-baclofen combination improves cerebellar impairment in spinocerebellar ataxia type 1

10.1101/2020.08.14.251330 ◽

2020 ◽

Author(s):

David D. Bushart ◽

Haoran Huang ◽

Luke J. Man ◽

Logan M. Morrison ◽

Vikram G. Shakkottai

Keyword(s):

Ion Channels ◽

Spinocerebellar Ataxia ◽

Brain Slices ◽

Motor Impairment ◽

Purkinje Neuron ◽

Muscle Relaxants ◽

Disease Stage ◽

Spinocerebellar Ataxia Type ◽

Target Engagement ◽

Cerebellar Dysfunction

AbstractBackgroundA combination of central muscle relaxants, chlorzoxazone and baclofen (chlorzoxazone-baclofen), has been proposed for treatment of cerebellar symptoms in human spinocerebellar ataxia (SCA). However, central muscle relaxants can worsen balance. The optimal dose for target engagement without toxicity remains unknown.ObjectivesUsing the genetically precise Atxn1154Q/2Q model of SCA1, we determine the role of cerebellar dysfunction in motor impairment. We also aim to identify appropriate concentrations of chlorzoxazone-baclofen needed for target engagement without toxicity to plan for human clinical trials.MethodsWe use patch clamp electrophysiology in acute cerebellar slices and immunostaining to identify the specific ion channels targeted by chlorzoxazone-baclofen. Behavioral assays for coordination and grip strength are used to determine specificity of chlorzoxazone-baclofen for improving cerebellar dysfunction without off-target effects in Atxn1154Q/2Q mice.ResultsWe identify irregular Purkinje neuron firing in association with reduced expression of the ion channels Kcnma1 and Cacna1g in Atxn1154Q/2Q mice. Using in vitro electrophysiology in brain slices, we identify concentrations of chlorzoxazone-baclofen that improve Purkinje neuron spike regularity without reducing firing frequency. At a disease stage in Atxn1154Q/2Q mice when motor impairment is due to cerebellar dysfunction, orally administered chlorzoxazone-baclofen improves motor performance without affecting muscle strength.ConclusionWe identify a tight relationship between baclofen-chlorzoxazone concentrations needed to engage target, and levels above which cerebellar function will be compromised. We propose to use this information for a novel clinical trial design, using sequential dose escalation within each subject, to identify dose levels that are likely to improve ataxia symptoms while minimizing toxicity.

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Removal of the Polyglutamine Repeat of Ataxin-3 by Redirecting pre-mRNA Processing

International Journal of Molecular Sciences ◽

10.3390/ijms20215434 ◽

2019 ◽

Vol 20 (21) ◽

pp. 5434 ◽

Cited By ~ 1

Author(s):

Craig S. McIntosh ◽

May Thandar Aung-Htut ◽

Sue Fletcher ◽

Steve D. Wilton

Keyword(s):

Spinocerebellar Ataxia ◽

Conformational Changes ◽

Antisense Oligonucleotides ◽

Therapeutic Benefit ◽

Spinocerebellar Ataxia Type ◽

Gene Transcript ◽

Spinocerebellar Ataxia Type 3 ◽

Polyglutamine Tract ◽

Type 3

Spinocerebellar ataxia type 3 (SCA3) is a devastating neurodegenerative disease for which there is currently no cure, nor effective treatment strategy. One of nine polyglutamine disorders known to date, SCA3 is clinically heterogeneous and the main feature is progressive ataxia, which in turn affects speech, balance and gait of the affected individual. SCA3 is caused by an expanded polyglutamine tract in the ataxin-3 protein, resulting in conformational changes that lead to toxic gain of function. The expanded glutamine tract is located at the 5′ end of the penultimate exon (exon 10) of ATXN3 gene transcript. Other studies reported removal of the expanded glutamine tract using splice switching antisense oligonucleotides. Here, we describe improved efficiency in the removal of the toxic polyglutamine tract of ataxin-3 in vitro using phosphorodiamidate morpholino oligomers, when compared to antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone. Significant downregulation of both the expanded and non-expanded protein was induced by the morpholino antisense oligomer, with a greater proportion of ataxin-3 protein missing the polyglutamine tract. With growing concerns over toxicity associated with long-term administration of phosphorothioate oligonucleotides, the use of a phosphorodiamidate morpholino oligomer may be preferable for clinical application. These results suggest that morpholino oligomers may provide greater therapeutic benefit for the treatment of spinocerebellar ataxia type 3, without toxic effects.

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