Florey Department of Neuroscience and Mental Health - Theses
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ItemA platform for analysis of in vitro neuronal networks for the development of precision therapeutics in SCN2A disease( 2019)Background and Purpose Developmental and epileptic encephalopathies are a group of devastating neurological disorders in which the patients have developmental impairment as well as refractory seizures. Comorbid states are common and include cognitive and movement disorders. SCN2A, which encodes the brain sodium channel Nav1.2, has emerged as one of the most prominent developmental and epileptic encephalopathy genes. Based on the onset of disease, patients with SCN2A epilepsy variants can be divided into two major groups. In the early onset group, seizures start within the first three months of life, whereas in the second group, the onset is after three months of age. Sodium channel blockers such as phenytoin are effective in some of the early onset patients. In contrast phenytoin is ineffective and may in fact worsen seizure outcomes in late onset disease. This suggests different molecular pathomechanisms. The lack of efficacious therapies underscores an urgent need for novel treatment strategies. Experimental Approach Two knock-in mouse lines were generated carrying Scn2a p.R1883Q and p.R854Q variants corresponding to human SCN2A p.R1882Q and p.R853Q variants. These are the most common recurrent variants found in the early and the late onset group of SCN2A developmental and epileptic encephalopathies, respectively. In vitro signatures of neuronal network behaviour were assessed using multi-electrode array analysis of the primary cortical cultures obtained from postnatal day 0-1 animals carrying the respective variants up to 28 days in vitro. Acute pharmacological effects were evaluated around 22 days in vitro. Key Results After 2-4 weeks network analysis in culture showed increased activity for neurons harbouring the heterozygous p.R1882Q variant associated with early onset disease. Conversely, a decreased firing rate was observed in cultures in which neurons carried the heterozygous p.R853Q variant associated with late-onset disease. The excitability in both cultures was reduced by phenytoin, which resulted in shifting the p.R1882Q in vitro phenotype towards the wild types and p.R853Q away from the wild types, consistent with clinical observations. Interestingly, one of the tested antiepileptic drugs changed the activity of both cultures was towards the wild type phenotype indicating potential benefits of this drug for both early and late onset SCN2A developmental and epileptic encephalopathies. Conclusion and Implications The assumption that early onset SCN2A variants are more likely to cause gain-of-function and the late onset SCN2A variants to a loss-of-function of the Nav1.2 channel was confirmed for the two studied variants using in vitro neuronal cultures. Moreover, the clinical observations regarding the effectiveness of the sodium channel blocker phenytoin in patients with early and late onset seizures was corroborated by our in vitro models. Lastly, an antiepileptic drug was identified as a potential treatment for both early and late onset SCN2A developmental and epileptic encephalopathies.
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ItemAntisense oligonucleotide precision therapy in KCNT1 - severe epilepsy( 2019)In recent years, the advancement of sequencing technology used in conjunction with thorough clinical evaluation has enabled clinicians to attribute genetic factors to the etiology of epilepsy. A significant number of mutations in genes encoding ion channels have been identified as the cause of the developmental and epileptic encephalopathies (DEE), a group of severe epilepsy syndromes of childhood and infancy characterized by the presence of abundant epileptiform activity, refractory seizures, intellectual disability, developmental regression, movement disorders, and increased mortality. The gene KCNT1 encodes the sodium activated potassium channel subunit KNa1.1. De novo mutations in this gene have been identified in patients with both severe and milder forms of epilepsy. The most commonly associated phenotypes are epilepsy of infancy with migrating focal seizures (EIMFS) and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). EIMFS is situated on the severe end of the spectrum and manifests during the first six months of life with frequent multifocal seizures in addition to developmental plateau or regression. Patients with KCNT1 associated epilepsy typically respond poorly to conventional anti-seizure medications, resulting in an unfavorable prognosis and compromising their quality of life. Biophysical studies in heterologous expression systems have shown that KCNT1 pathogenic variants found in epilepsy result in strongly increased potassium currents. This overall gain of function (GoF) is currently accepted as the primary mechanism of disease in KCNT1 associated epilepsy. To directly address this pathologic mechanism, antisense oligonucleotide (ASO) mediated knockdown was used to test the idea that reducing Kcnt1 expression would be therapeutic in a mouse model of KCNT1 associated epilepsy. This approach was supported by the tolerance for KCNT1 loss of function (LoF) observed in the general population and the mild phenotype found in Kcnt1 knockout mice. Using the CRISPR Cas9 system, an orthologous pathogenic variant found in KCNT1 EIMFS was inserted in Kcnt1 to develop a rodent model. Mice, homozygous for the mutation display frequent spontaneous seizures, abundant interictal activity in the electrocorticogram (ECoG), behavioral abnormalities and early death. Thus, recapitulating key aspects of KCNT1 severe epilepsy and offering a valuable tool for therapeutic screening. Then, an ASO was designed to specifically hybridize with Kcnt1. An additional nontargeting sequence was used as a control. After a single intracerebroventricular bolus injection, homozygous mice showed a marked knockdown of Kcnt1 mRNA and in comparison to control treated animals, displayed almost complete abolition of seizures, prolonged survival, and improved cognition. Exaggerated pharmacology was explored in wild type mice treated with a dose of ASO that produced more than 90 percent knockdown. Here, behavioral measures revealed some anxiety like traits in ASO treated mice, but knockdown was otherwise tolerated. Additional seizure susceptibility in LoF was tested in Kcnt1 knock out mice, which indicated that LoF was not related to a reduction in seizure threshold. To conclude, the preclinical evidence presented in this thesis supports ASO based gene silencing as a therapeutic approach in KCNT1 GoF epilepsies. This body of work provides proof of concept for such approach and encourages the translation of ASO based therapies for genetic epilepsies, in particular for those with an underlying GoF pathomechanism.