Identification of new features and evaluation of antimir-based seizure therapies in the Scn1a(+/-)tm1Kea mouse model of Dravet syndrome

Dravet syndrome (DS) is a rare and intractable form of paediatric epilepsy characterized by the early onset of seizures caused mainly by mutations in the SCN1A gene encoding the alpha subunit of Nav1.1 channels. Patients with DS suffer with several comorbidities and severe life-threatening seizures that are refractory to antiseizure medication. Several DS mouse models have been generated which have served as valuable tools for understanding underlying mechanisms and identifying new treatments. However, the discovery and preclinical development of new therapies requires full understanding of the epilepsy and behaviour phenotypes in DS mouse models. The Scn1a(+/-)tm1Kea mouse line has demonstrated translational value in identifying known and novel anti-seizure molecules. However, certain epilepsy-related features at the different stages of DS progression, long-term neuropsychiatric comorbidities, and the influence of sex on these parameters have not been fully explored in this DS mouse line. Among novel approaches being pursued for the treatment of drug-resistant epilepsy is the targeting of small noncoding RNAs called microRNA. One of these, microRNA-134 (miR-134), has been shown to target gene networks controlling neuronal microstructure and brain excitability. Levels of miR-134 have been found to be elevated in several rodent models and in human brain tissue resected from temporal lobe epilepsy (TLE) patients. Notably, the use of antisense oligonucleotides (locked nucleic acid (LNA), antimirs/antagomirs) targeting miR-134 (Ant-134) reduced evoked and spontaneous seizures and conferred a sustained neuroprotective effect in multiple rat and mouse models of epilepsy. Based on that, we hypothesized that antimirs might be protective in DS. In this thesis, we proposed three specific aims. First, to characterise the epilepsy phenotype over different developmental stages and associated comorbidities in F1.Scn1a(+/-)tm1Kea mice. Second, to functionally assess the role of miR-134 in the epilepsy phenotype of F1.Scn1a(+/-)tm1Kea mice. Finally, to characterise novel miRNAs that may be relevant for the treatment of DS. Experiments in the first chapter comprised a comprehensive characterisation of phenotypes in F1.Scn1a(+/-)tm1Kea mice. At P18, F1.Scn1a(+/-)tm1Kea mice experience sensitivity to hyperthermia-induced seizures. Between P21 and P28, EEG recordings in F1.Scn1a(+/-)tm1Kea mice combined with video monitoring revealed a high frequency of spontaneous recurrent seizures (SRS) and sudden unexplained death in epilepsy (SUDEP). Power spectral analyses of background EEG activity also revealed that low EEG power in multiple frequency bands was associated with SUDEP risk in F1.Scn1a(+/-)tm1Kea mice during the worsening stage of DS. Later, SRS and SUDEP rates stabilized and then declined in F1.Scn1a(+/-)tm1kea mice. The incidence of SRS ending with death in F1.Scn1a(+/-)tm1kea mice displayed variations with the time of day and sex, with female mice displaying higher numbers of severe seizures resulting in greater SUDEP risk. At ~6 month-old, F1.Scn1a(+/-)tm1kea mice displayed fewer behavioural impairments than expected including hyperactivity, impaired exploratory behaviour and poor nest building performance. These results reveal new features of this model that will optimize use and selection of phenotype assays for future studies on the mechanisms, diagnosis, and treatment of DS. Experiments in the second results chapter investigated the levels of miR-134 and whether Ant-134 could protect against hyperthermia-induced seizures, SRS and mortality (SUDEP) in F1.Scn1a(+/-)tm1kea mice. Hippocampal levels of miR-134 were similar between wildtype and F1.Scn1a(+/-)tm1kea mice. At P17, animals were intracerebroventricular (i.c.v) injected with 0.1 – 1 nmol of Ant-134 and subject to a hyperthermia challenge at P18. A second cohort of P21 F1.Scn1a(+/-)tm1kea mice received Ant-134 and were followed by video and EEG monitoring until P28 to track the incidence of spontaneous seizures and SUDEP. Ant-134 had no effect on hyperthermia-induced seizures, spontaneous seizures or SUDEP incidence. These findings suggest that targeting miR-134 does not have therapeutic applications in DS. In the final results chapter, we investigated a rational-targeting approach, selecting miRNAs that target the SCN1A transcript for inhibition. In addition, we performed small RNA sequencing of miRNAs in DS brain samples. Two miRNAs, miR-582-5p and miR-374a-5p were identified as potential conserved SCN1A/Scn1a targeting miRNAs and antimirs were designed against these. However, a combination of Anti-582-5p and Anti-374a-5p did not protect against hyperthermia-induced seizures in F1.Scn1a(+/-)tm1kea mice. Additionally, Scn1a transcript levels remained unchanged in the antimir-treated F1.Scn1a(+/-)tm1kea mice. Finally, sequencing of miRNAs bound to the RNA silencing component Ago revealed several differentially expressed miRNAs in F1.Scn1a(+/-)tm1kea mice, three of which are predicted to target the SCN1A transcript. Overall, the findings in this thesis enhance our understanding of the phenotypes of the Scn1a(+/-)tm1Kea DS mouse line and identify limitations as well as opportunities for a miRNA targeting approach that may be relevant in DS treatment.