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. 2020 Mar;87(3):339-346.
doi: 10.1002/ana.25676. Epub 2020 Feb 6.

Scn8a Antisense Oligonucleotide Is Protective in Mouse Models of SCN8A Encephalopathy and Dravet Syndrome

Guy M Lenk  1 , Paymaan Jafar-Nejad  2 , Sophie F Hill  1   3 , Lucas D Huffman  3   4 , Corrine E Smolen  1 , Jacy L Wagnon  1 , Hayley Petit  1 , Wenxi Yu  1 , Julie Ziobro  5 , Kritika Bhatia  5 , Jack Parent  5 , Roman J Giger  3   4   5 , Frank Rigo  2 , Miriam H Meisler  1   3   5
Affiliations

Scn8a Antisense Oligonucleotide Is Protective in Mouse Models of SCN8A Encephalopathy and Dravet Syndrome

Guy M Lenk et al. Ann Neurol. 2020 Mar.

Abstract

Objective: SCN8A encephalopathy is a developmental and epileptic encephalopathy (DEE) caused by de novo gain-of-function mutations of sodium channel Nav 1.6 that result in neuronal hyperactivity. Affected individuals exhibit early onset drug-resistant seizures, developmental delay, and cognitive impairment. This study was carried out to determine whether reducing the abundance of the Scn8a transcript with an antisense oligonucleotide (ASO) would delay seizure onset and prolong survival in a mouse model of SCN8A encephalopathy.

Methods: ASO treatment was tested in a conditional mouse model with Cre-dependent expression of the pathogenic patient SCN8A mutation p.Arg1872Trp (R1872W). This model exhibits early onset of seizures, rapid progression, and 100% penetrance. An Scn1a +/- haploinsufficient mouse model of Dravet syndrome was also treated. ASO was administered by intracerebroventricular injection at postnatal day 2, followed in some cases by stereotactic injection at postnatal day 30.

Results: We observed a dose-dependent increase in length of survival from 15 to 65 days in the Scn8a-R1872W/+ mice treated with ASO. Electroencephalographic recordings were normal prior to seizure onset. Weight gain and activity in an open field were unaffected, but treated mice were less active in a wheel running assay. A single treatment with Scn8a ASO extended survival of Dravet syndrome mice from 3 weeks to >5 months.

Interpretation: Reduction of Scn8a transcript by 25 to 50% delayed seizure onset and lethality in mouse models of SCN8A encephalopathy and Dravet syndrome. Reduction of SCN8A transcript is a promising approach to treatment of intractable childhood epilepsies. Ann Neurol 2020;87:339-346.

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Conflict of interest statement

P.J.‐N. and F.R. are employed by Ionis Pharmaceuticals, a for‐profit company that develops ASO therapies.

Figures

Figure 1
Figure 1
Scn8a antisense oligonucleotide (ASO) treatment reduces the abundance of Scn8a transcript in wild‐type neurons and brain. (A) Cartoon of the 3′ end of the Scn8a gene. The last 2 exons are represented by boxes separated by a line representing the last intron. Coding sequences within the exons are shaded. The position of the stop codon that terminates translation is marked as “stop.” The sequence of the 20 base pair (bp) Scn8a ASO is shown. The ASO sequence is identical to the genomic sequence of the 3′ untranslated region (UTR) at a position approximately 500 bp downstream from the translation stop codon within the last exon of the gene. (B) Primary cortical neurons from E14 embryos of strain C57BL/6J were cultured for 3 days with the indicated concentration of Scn8a ASO. (C) The indicated dose of ASO was administered to C57BL/6J mice on postnatal day 2, and the abundance of Scn8a transcript in brain RNA was measured at postnatal day 21. Five or 6 animals were treated with each dose; each symbol represents data from 1 animal.
Figure 2
Figure 2
Scn8a antisense oligonucleotide (ASO) delays seizure onset and prolongs survival of mutant mice expressing the pathogenic mutation SCN8A‐R1872W. (A) Scn8a cond/+, EIIA‐CRE mice were treated on postnatal day 2 by intracerebroventricular injection with the indicated mount of control or Scn8a ASO. A second dose of 100μg of Scn8a ASO at postnatal day 35 further extended survival (p < 0.001) (dotted line). (B) Dose dependence of mean survival; values are mean ± standard error of the mean. (C) Scn8a cond/+, EIIA‐CRE mice treated with 45μg exhibit normal posture without hind limb clasping at P21. (D) Duration of the effect of Scn8a ASO on transcript level. Brain RNA was prepared from Scn8a cond/+, EIIA‐CRE mice treated with 45μg of ASO and untreated age‐matched wild‐type mice and analyzed by quantitative reverse transcription polymerase chain reaction. (E) Reduction of Nav1.6 protein in brain at 3 weeks of age in mice treated with 45μg of ASO as in (D).
Figure 3
Figure 3
Antisense oligonucleotide (ASO) treatment protects against electrographic seizures. Mutant mice of genotype Scn8a cond/+ , EIIA Cre were treated with 45μg Scn8a ASO by intracerebroventricular injection at postnatal (P) day 2 (P2) and monitored from the age of P31 to P39 by 24‐hour video electroencephalographic (EEG) recording. Traces recorded from right and left cortical electrodes of 6 mice are shown. Five mice were completely protected from electrographic and behavioral seizures. Mouse 2 exhibited a single, fatal, generalized tonic–clonic seizure at P37 that closely resembled the EEG profile in untreated mutants at P15.11 (P37 corresponds to the age of the earliest seizure in the cohort of 10 mice treated with 45μg of ASO in Fig 2.)
Figure 4
Figure 4
Motor activity of antisense oligonucleotide (ASO) treated mice. Scn8a cond/+ , EIIA Cre mice were treated with 45μg Scn8a ASO at postnatal (P) day 2 (P2). (A) Activity in an open field was monitored for 30 minutes at 45 days of age. Treated mutant mice did not differ significantly from wild‐type mice in average running speed (Student t test, p = 0.12) or percentage of time spent in the center of the open field (2‐way analysis of variance, p = 0.83). Rearing behavior of mutant and wild‐type mice was also comparable. (B) Wheel running activity was monitored 24 h/day during the 4‐day interval between P31 and P39, after habituation to the running cage for 10 days. The distance travelled by the treated mutant mice during the 96 hours of monitoring was significantly smaller than for wild‐type mice. There was no significant difference in the time spent running, but the average speed was lower for the mutant mice. (C–F) Cerebellar function was assessed at 5 weeks of age by analysis of ledge walking, hind limb clasping, gait, and kyphosis as described.30 The only deficit observed was impaired ledge walking in 3 of the 5 treated mutant mice. Each symbol represents 1 animal, and values are the mean of triplicate assays. The p values are for the comparisons between wild‐type and mutant mice (Student t test). WT = wild‐type C57BL/6J mice untreated with ASO; Mutant = Scn8a cond/+ , EIIA Cre mice treated with 45μg ASO.
Figure 5
Figure 5
Scn8a antisense oligonucleotide (ASO) rescues survival of Dravet syndrome mice. Scn1a +/− mice were treated on postnatal (P) day 2 (P2) with 45μg Scn8a ASO. (A) For untreated Scn1a +/− mice, median survival was 26 days with 50% penetrance of the lethal phenotype, consistent with the original description of this Dravet model.18 In contrast, all of the ASO treated mice have survived beyond 5 months of age (p = 0.004, Cox–Mantel log‐rank test). Tick marks represent living mice. (B) The Scn8a ASO reduces Scn8a transcript level with no effect on Scn1a transcript level. Brain and spinal cord RNA was prepared from untreated wild‐type P21 (solid symbols) or mice treated with 45μg Scn8a ASO (open symbols). (C) The Scn8a ASO protects against electrographic seizures in Scn1a +/− mice. Four ASO treated mice at 5 months of age were monitored for 5 successive days with 24‐hour electroencephalographic (EEG) recording. No electrographic seizures or other EEG abnormalities were observed (2 traces for each animal, from left and right cortex).
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