Antisense oligonucleotide precision therapy in KCNT1 - severe epilepsy

© 2019 Lisseth Estefania Burbano PortillaIn 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