UMass Chan Medical School Faculty Publications

UMMS Affiliation

RNA Therapeutics Institute; Department of Neurology; Department of Radiology; Program in Innate Immunity, Division of Infectious Diseases and Immunology, Department of Medicine; Department of Biochemistry and Molecular Pharmacology; Graduate School of Biomedical Sciences

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Article Preprint


Biochemistry | Medicinal Chemistry and Pharmaceutics | Molecular and Cellular Neuroscience | Nervous System | Nervous System Diseases | Neurology | Nucleic Acids, Nucleotides, and Nucleosides | Pharmacology | Toxicology


Antisense oligonucleotides (ASOs) are emerging as a promising class of therapeutics for neurological diseases. When injected directly into the cerebrospinal fluid, ASOs distribute broadly across brain regions and exert long-lasting therapeutic effects. However, many phosphorothioate (PS)-modified gapmer ASOs show transient motor phenotypes when injected into the cerebrospinal fluid, ranging from reduced motor activity to ataxia or acute seizure-like phenotypes. The effect of sugar and phosphate modifications on these phenotypes has not previously been systematically studied. Using a behavioral scoring assay customized to reflect the timing and nature of these effects, we show that both sugar and phosphate modifications influence acute motor phenotypes. Among sugar analogues, PS-DNA induces the strongest motor phenotype while 2’-substituted RNA modifications improve the tolerability of PS-ASOs. This helps explain why gapmer ASOs have been more challenging to develop clinically relative to steric blocker ASOs, which have a reduced tendency to induce these effects. Reducing the PS content of gapmer ASOs, which contain a stretch of PS-DNA, improves their toxicity profile, but in some cases also reduces their efficacy or duration of effect. Reducing PS content improved the acute tolerability of ASOs in both mice and sheep. We show that this acute toxicity is not mediated by the major nucleic acid sensing innate immune pathways. Formulating ASOs with calcium ions before injecting into the CNS further improved their tolerability, but through a mechanism at least partially distinct from the reduction of PS content. Overall, our work identifies and quantifies an understudied aspect of oligonucleotide toxicology in the CNS, explores its mechanism, and presents platform-level medicinal chemistry approaches that improve tolerability of this class of compounds.


pharmacology, toxicology, motor phenotypes, antisense Ooigonucleotides, medicinal chemistry

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bioRxiv 2021.02.14.431096; doi: Link to preprint on bioRxiv.


This article is a preprint. Preprints are preliminary reports of work that have not been certified by peer review.

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Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.