Antisense oligonucleotide therapy for KCNT1 encephalopathy
Lisseth Estefania Burbano, Melody Li, Nikola Jancovski, Paymaan Jafar-Nejad, Kay Richards, Alicia Sedo, Armand Soriano, Ben Rollo, Linghan Jia, Elena V. Gazina, Sandra Piltz, Fatwa Adikusuma, Paul Q. Thomas, Helen Kopsidas, Frank Rigo, Christopher A. Reid, Snezana Maljevic, Steven Petrou
Lisseth Estefania Burbano, Melody Li, Nikola Jancovski, Paymaan Jafar-Nejad, Kay Richards, Alicia Sedo, Armand Soriano, Ben Rollo, Linghan Jia, Elena V. Gazina, Sandra Piltz, Fatwa Adikusuma, Paul Q. Thomas, Helen Kopsidas, Frank Rigo, Christopher A. Reid, Snezana Maljevic, Steven Petrou
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Research Article Genetics Neuroscience

Antisense oligonucleotide therapy for KCNT1 encephalopathy

Abstract

Developmental and epileptic encephalopathies (DEEs) are characterized by pharmaco-resistant seizures with concomitant intellectual disability. Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe of these syndromes. De novo variants in ion channels, including gain-of-function variants in KCNT1, which encodes for sodium activated potassium channel protein KNa1.1, have been found to play a major role in the etiology of EIMFS. Here, we test a potential precision therapeutic approach in KCNT1-associated DEE using a gene-silencing antisense oligonucleotide (ASO) approach. We generated a mouse model carrying the KCNT1 p.P924L pathogenic variant; only the homozygous animals presented with the frequent, debilitating seizures and developmental compromise that are seen in patients. After a single intracerebroventricular bolus injection of a Kcnt1 gapmer ASO in symptomatic mice at postnatal day 40, seizure frequency was significantly reduced, behavioral abnormalities improved, and overall survival was extended compared with mice treated with a control ASO (nonhybridizing sequence). ASO administration at neonatal age was also well tolerated and effective in controlling seizures and extending the life span of treated animals. The data presented here provide proof of concept for ASO-based gene silencing as a promising therapeutic approach in KCNT1-associated epilepsies.

Authors

Lisseth Estefania Burbano, Melody Li, Nikola Jancovski, Paymaan Jafar-Nejad, Kay Richards, Alicia Sedo, Armand Soriano, Ben Rollo, Linghan Jia, Elena V. Gazina, Sandra Piltz, Fatwa Adikusuma, Paul Q. Thomas, Helen Kopsidas, Frank Rigo, Christopher A. Reid, Snezana Maljevic, Steven Petrou

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Figure 3

Kcnt1 ASO produces a dose-dependent knockdown of Kcnt1 mRNA in the mouse CNS.

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Kcnt1 ASO produces a dose-dependent knockdown of Kcnt1 mRNA in the mous...
Dose-response curves for Kcnt1 ASO in the brain cortex (A) and thoracic spinal cord (B) for +/+ mice injected at P40. The tissue was collected 2 weeks after injection and processed for mRNA quantification (n = 3 for each dose and PBS control, curves were fitted with the Motulsky regression). (C) Mouse cortex was collected 2 weeks after i.c.v. injection for mRNA quantification. The i.c.v. administration of Kcnt1 ASO reduced the levels of Kcnt1 mRNA (Kruskal-Wallis test P = 0.0027), without affecting the paralog gene Kcnt2 (Kruskal-Wallis test P = 0.4794); data are presented as bar plots with mean and SEM, untreated n = 4, Kcnt1 ASO n = 5, control ASO n = 3. (D) Western blot showing a reduction of Kcnt1 protein in WT mice left hemisphere 2 weeks after i.c.v. injection. (E) Average band signal for Kcnt1 protein. Data are presented as bar plots with mean and SD (n = 3 mice for each treatment condition, NS for all comparisons except PBS versus 500 μg, 1-way ANOVA with Dunnett’s multiple comparisons test). (F) Distribution of the Kcnt1 ASO in the mouse brain. Coronal brain sections of +/+ mice treated with Kcnt1 ASO 75 μg. Tissue was collected 2 weeks after i.c.v. injection and stained with an ASO antibody (red) and neuronal marker (NeuN; green) and counterstained with nuclear stain DAPI (blue). Kcnt1 ASO was found throughout the meninges, hippocampus, and cortical layers (n = 3 experiments). Scale bars represent 500 μm.