Abstracts

Rationale: Drug-resistance remains a major hurdle for clinical management of epilepsy. Thirty percent of patients are refractory to current antiseizure medications underlining the urgent need for new, disease-modifying drugs to eliminate drug-resistant seizures and cure the disease. RNA therapeutics comprise an emerging drug modality for the treatment of neurological disorders. Through their ability to directly target RNA molecules such as messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs), RNA drugs can be utilised to modulate targets previously deemed undruggable by small molecules. Indeed, targeting of microRNAs (miRNA) enables modulation of pathological networks and provide treatments with potential disease-modifying properties for multifactorial diseases, such as drug-resistant focal-onset epilepsy. Antisense oligonucleotides (ASOs) can be designed to inhibit miRNA function and RNA splicing (so-called mixmers) or to deploy endogenous RNAse H activity to knock down mRNA targets (gapmers). We have developed chemically modified mixmer ASOs to inhibit specific miRNAs known to be important regulators of pathological pathways in drug-resistant focal-onset epilepsy. Here, we demonstrate the efficacy of these inhibitors in the intra-amygdala kainic acid (IAKA) mouse model. We also report pilot data on ASO-mediated knockdown of a protein-coding gene associated with promotion hyperexcitable neuronal networks through epigenetic changes.

Methods: All ASOs were designed by a proprietary AsoDesign program developed at Aalborg University, Copenhagen, Denmark. Lead candidates were identified through in vitro screening in human cell lines for target affinity (luciferase assay) and functional effects on downstream target mRNA (qPCR) and protein levels (Western blot). RNA-sequencing was used to confirm target selectivity. The lead compounds were then tested in dose-range studies in vivo and evaluated in the IAKA mouse model for anti-seizure and anti-epileptogenic properties.

Results: NEUmiRNA’s lead ASO potently suppressed levels of target miRNA in vivo and reduced seizure severity in the IAKA mouse model. Preclinical proof-of-concept studies indicated long-lasting and potentially disease-modifying properties.

Conclusions: The studies demonstrate the potential of the ASO drug platform in therapeutic targeting of miRNAs and mRNAs implicated in pathological disease pathways.

Funding: NEUmiRNA Therapeutics is supported by a grant from Innovation Fund, Denmark.