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Updated: May 30, 2025
Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System
Published on: October 29, 2019
Characterizing Antisense Oligonucleotide-Induced Histopathology Findings in Spinal Cord of Mauritius Cynomolgus
Valérie Dubost1, Kuno Wuersch1, Kelley Penraat2
1Novartis Institute for Biomedical Research, Basel, Switzerland.
Abstract:
The safety of a 2'-O-methoxyethyl antisense oligonucleotide (ASO) was investigated in Mauritius cynomolgus monkeys in a 41-week Good Laboratory Practice (GLP) toxicity study after multiple intrathecal (IT) administrations. Histopathological examination revealed ectopic formation of lymphoid follicles in the spinal cord (SC) at the injection site at all doses and the presence of granular material in neurons of the SC in high-dose animals. The granular material was seen in all the segments of the SC, but mainly in the lumbar segment and persisted at the end of the 26-week recovery period, while the lymphoid follicles showed a reversibility trend. Findings associated with repeated IT administration of ASOs have been described in nonhuman primate (NHP) toxicity studies, specifically in the brain, but findings in the SC are rarely reported. In the present study, we report a high incidence of findings in the SC compared to brain, especially in the lumbar segment in proximity to IT injection sites. An extensive panel of immunohistochemistry markers showed that the ectopic lymphoid follicle formation (LFF) had a cellular composition and organization consistent with tertiary lymphoid structure (TLS) without associated axonal damage in the adjacent nervous tissue. In situ hybridization with an miRNA probe complementary to the ASO revealed that the granular material represented a dose-dependent ASO accumulation in the cytoplasm of neurons without inducing cell death or apoptosis. Glial and ependymal cells in the SC also showed dose-dependent accumulation of the ASO preceding detection of granular material by hematoxylin and eosin (H&E). Based on these molecular localization data, the presence of LFF in SC suggests a chronic local immune activation. Considering the absence of neuronal dysfunction or injury and transient clinical signs previously reported with other 2'-MOE ASOs, the presence of TLS and ASO was considered non-adverse.
Insights
Spinal cord findings, including lymphoid follicles and granular material in neurons, were observed in nonhuman primates after intrathecal antisense oligonucleotide administration. These changes were dose-dependent and considered non-adverse, with lymphoid follicles showing reversibility.
Area of Science:
- Neuroscience
- Toxicology
- Immunology
Background:
- Antisense oligonucleotides (ASOs) are therapeutic agents requiring safety evaluation.
- Intrathecal (IT) administration of ASOs can lead to central nervous system effects.
- Spinal cord (SC) toxicity findings from IT ASO administration are less commonly reported than brain findings.
Purpose of the Study:
- To investigate the safety of a 2'-O-methoxyethyl antisense oligonucleotide (ASO) after multiple IT administrations in Mauritius cynomolgus monkeys.
- To characterize histopathological and molecular changes in the spinal cord following IT ASO administration.
- To assess the reversibility and potential adverse nature of observed SC findings.
Main Methods:
- A 41-week Good Laboratory Practice (GLP) toxicity study was conducted in nonhuman primates.
- Multiple intrathecal administrations of a 2'-O-methoxyethyl ASO were performed.
- Histopathology, immunohistochemistry, and in situ hybridization were used to evaluate spinal cord tissues.
Main Results:
- Ectopic lymphoid follicle formation (LFF) and granular material in SC neurons were observed at all doses.
- Granular material represented dose-dependent ASO accumulation within neurons, persisting after a recovery period.
- LFF showed characteristics of tertiary lymphoid structures (TLS) without axonal damage, and demonstrated a trend towards reversibility.
Conclusions:
- IT administration of this 2'-O-methoxyethyl ASO in nonhuman primates induced spinal cord changes, including TLS and intraneuronal ASO accumulation.
- These findings suggest a chronic local immune response in the SC.
- Given the absence of neuronal injury and transient clinical signs, the observed spinal cord changes were considered non-adverse.
