Abstracts

Rationale: Pathogenic genetic variants in the KCNT1 gene, which encodes a sodium-activated potassium ion channel, drive a severe childhood developmental epileptic encephalopathy. KCNT1 gain-of-function manifestations include EIMFS (epilepsy of infancy with migrating focal seizures) and SHE (sleep related hypermotor epilepsy) and present with frequent tonic seizures and delayed or missed developmental milestones. No current therapy provides more than sporadic or incremental improvement. Here we report suppression of seizures in a genetic mouse model of KCNT1 epilepsy with divalent small interfering siRNA (di-siRNA), a novel branch of oligonucleotide technology that enables months-long knockdown of KCNT1 transcripts widely throughout the brain following a single dose into the cerebrospinal fluid.

Methods: siRNAs targeting KCNT1 were synthesized and tested in vitro and in mice for knockdown of KCNT1 transcript and protein by RT-PCR, Western blot, and patch-clamp electrophysiology, leading to the di-siRNA compound ATL-201. The ATL-201 molecule is two identical fully synthetic double-stranded siRNAs that are covalently linked. The siRNA antisense sequence is fully matched to both human KCNT1 and to mouse Kcnt1 sequence. ATL-201 was tested for effects on spontaneous seizures in mice homozygous for Kcnt1-Y777H, the mouse ortholog to the human pathogenic KCNT1-Y796H missense variant. Seizures were measured in freely behaving mice with EEG from implanted leads plus behavioral observations. The number and durations of seizures were measured in mice dosed once with ATL-201, in a durability study and in a two-month dose-efficacy study. Normal mouse behavior was assessed with an assay for nest-building.

Results: ATL-201 reduced KCNT1 transcript and completely suppressed K+ currents from KCNT1 expressed in 293 cells and eliminated sodium-activated potassium currents recorded from cultured mouse cortical neurons. ICV dosing of ATL-201 in Kcnt1-Y777H mice suppressed seizures in a dose-dependent fashion, with near-complete suppression at four months following a single well-tolerated dose. A dose-efficacy study showed that approximately 50% protein knockdown reduced seizures by approximately 70%. ATL-201 restored nest-building behavior in Kcnt1-Y777H mice to levels equivalent to wildtype mice.

Conclusions: Patients with KCNT1-driven epilepsy experience up to hundreds of seizures a day and have severe impairment in cognitive, motor, and language development. ATL-201 reduced KCNT1 channel function, suppressed seizures, and restored an aspect of mouse behavior. ATL-201 targets a sequence that does not encompass known pathogenic variants in KCNT1, suggesting it should be effective against any genotype of KCNT1 epilepsy. The long durability of action gives promise for a quarterly or biannually dosed disease-modifying treatment of KCNT1 epilepsy, a severe childhood disease that does not respond to existing treatments.


Funding: All work was sponsored and executed by Atalanta Therapeutics, Inc.