Quantifying and Mitigating Motor Phenotypes Induced by Antisense Oligonucleotides in the Central Nervous System

Abstract

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.

Change of intracellular calcium level causes acute neurotoxicity by antisense oligonucleotides via CSF route

“…Further, combinatorial treatment with NBQX potentiated decreases in intracellular free calcium levels induced by ASO2 ( Figure 6 A) and modified ASO5 ( Figure S10 ) previously reported to be neurotoxic ( Table S1 ). 22 Then, we confirmed the maximum tolerated dose, i.e., 13.1 nmol, at which NBQX itself did not induce neurotoxicity according to scoring with the ATSS ( Figure S11 A) and open-field tests ( Figure S12 ). Acute tolerability scores in mice injected with ASO1 after pretreatment with the tolerated dose of NBQX were higher than those of mice injected with ASO1 after pretreatment with PBS as a negative control (the total acute tolerability score of the NBQX and PBS group at 1 h were 6.8 and 4.8, respectively; Figure 6 B; Video S2 ).…”

G-rich motifs within phosphorothioate-based antisense oligonucleotides (ASOs) drive activation of FXN expression through indirect effects

“…Interestingly, at this second site, we observed that the steric blocker ASOs S30-6, S30-4, S30-2 and S30 induced a similar level of FXN activation to their gapmer ASOs, demonstrating that RNA cleavage was not required for FXN activation (Figure 2F ). Our previous work showed that gapmer ASOs might trigger more severe neurotoxicity in mouse brain than steric blocker ASOs targeting the same sequence ( 26 ). Thus, in moving forward, we focused our work on the steric blocker S30 at this region.…”

“…Unilateral intracerebroventricular (ICV) injections were carried out under UMass Chan Medical School IACUC protocol A-2551 as previously described ( 26 ). In brief, YG8R mice at ∼12 weeks old were anesthetized by intraperitoneal injection of a sterile saline solution containing fentanyl/midazolam/dexmedetomidine (0.1, 5 and 0.25 mg/kg, respectively).…”

Suppression of mutant C9orf72 expression by a potent mixed backbone antisense oligonucleotide

“…As a pilot behavioral study, we performed an intrathecal injection of ASO5-2 at 2 mg kg −1 in four sheep. For intrathecal injection in sheep, it was necessary to thread a microcatheter up though the intrathecal space and deliver the ASO directly into the cisterna magna (Methods) 34 . Intracisternal contrast injection and cone beam computed tomography confirmed the correct catheter position before ASO injection.…”

“…Both of these analogues showed reduced motor phenotypes and a higher MTD than ASO5. Elsewhere, we explore the mechanism of the motor phenotypes observed here and follow-up on the improvement observed by mixed backbone modification patterns 34 .…”