br Materials and Methods br Results br Discussion At present

Materials and Methods

Results

Discussion
At present time the majority of genetic disorders involving haploinsufficiency in a single gene, such as Dravet syndrome, have no adequate treatment. Only partially effective symptomatic therapy targeted at seizure reduction is now available for Dravet patients. Furthermore, although initially Dravet syndrome was considered an epileptic encephalopathy (Wolff et al., 2006), recent studies in Dravet patients have shown that some aspects of the syndrome may arise independently of seizures and thus cannot be addressed by anticonvulsants (Nabbout et al., 2013). In rat models, siRNA-mediated Scn1a knockdown in adult animals selective for basal forebrain induced cognitive impairment without seizures (Bender et al., 2013). Notably, upregulation of the remaining normal copy of the damaged gene has the potential to improve all disease manifestations and represents an appealing therapeutic strategy.
In our in vitro and in vivo experiments we were able to upregulate SCN1A expression by using AntagoNATs (oligonucleotide-like compounds) that block the activity of SCN1ANAT. While AntagoNAT treatment upregulated SCN1A, it did not affect >90% of all expressed genes, including other highly homologous sodium channel subunits and genes immediately adjacent to SCN1A on the chromosome (Fig. 5; Supplementary File 1).
Furthermore, we have demonstrated that SCN1A upregulation can be effective in a mouse model of Dravet if applied at some time point after birth, when genetic disorders are usually diagnosed. Our experiments in a mouse model harboring a known Dravet mutation demonstrate that Scn1a upregulation during PW 7–11 leads to improvements in several aspects of disease phenotype, including frequency and severity of seizures. We observed that a 25% increase in tsa inhibitor Supplier Scn1a levels was sufficient to elicit a 70% reduction in seizure frequency and a decrease in seizure severity (Fig. 8). Additionally, upregulation of brain Scn1a levels was associated with decreased sensitivity to heat-induced seizures, which represent a hallmark of Dravet syndrome. AntagoNAT treatment also normalized increased firing threshold and reduced firing frequency of inhibitory interneurons observed in Dravet models (Fig. 9, Oakley et al., 2011; Yamakawa, 2011; Tai et al., 2014; Ogiwara et al., 2013). Importantly, in rat models with RNAi-induced Scn1a knockdown, disruption in neuronal firing was correlated with performance in working memory task (Bender et al., 2016). This underscores the importance of normalizing the neuronal firing for the treatment of cognitive deficits associated with Dravet syndrome. Overall, significant improvements of seizure phenotype and neuronal electrophysiology observed in our experiments provide an integral indicator that Scn1a upregulation achieved using AntagoNATs can adequately address the excitation/inhibition imbalance occurring in Dravet brain.
Besides seizures, another known aspect of Dravet syndrome is increased mortality (Dravet, 2011). Similar to human disease, the death rate in the Scn1aE1099X/+ mice peaks in early development (with approximately 70% of mice dying before PW7) and then stabilizes at relatively low levels. Additionally, Dravet mice are generally smaller and have elevated sensitivity to surgical interventions compared to WT. For these reasons, in our experiments EEG electrode implantation and IT injection of AntagoNATs were conducted at PW7–11, past the critical period. As a result, the small numbers of deaths observed in our studies did not allow for a conclusive statistical analysis at this time. Consequently, the reported results are only relevant to the more mildly affected surviving mice or potentially Dravet patients older than 3years. Further experiments, in which AntagoNAT treatment will be started prior to the PW3–5 critical window are needed to verify whether AntagoNAT-mediated upregulation of SCN1A could reduce the early mortality rate. Due to technical difficulties, these experiments would likely require development of an alternative administration route.