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. 2021 Dec 1;131(23):e152079.
doi: 10.1172/JCI152079.

Antisense oligonucleotide therapy reduces seizures and extends life span in an SCN2A gain-of-function epilepsy model

Melody Li  1 , Nikola Jancovski  1 , Paymaan Jafar-Nejad  2 , Lisseth E Burbano  1 , Ben Rollo  1 , Kay Richards  1 , Lisa Drew  1 , Alicia Sedo  1 , Jacqueline Heighway  1 , Svenja Pachernegg  1 , Armand Soriano  2 , Linghan Jia  1 , Todd Blackburn  1   3 , Blaine Roberts  1 , Alex Nemiroff  3   4 , Kelley Dalby  3   4 , Snezana Maljevic  1 , Christopher A Reid  1 , Frank Rigo  2 , Steven Petrou  1   3   4
Affiliations

Antisense oligonucleotide therapy reduces seizures and extends life span in an SCN2A gain-of-function epilepsy model

Melody Li et al. J Clin Invest. .

Abstract

De novo variation in SCN2A can give rise to severe childhood disorders. Biophysical gain of function in SCN2A is seen in some patients with early seizure onset developmental and epileptic encephalopathy (DEE). In these cases, targeted reduction in SCN2A expression could substantially improve clinical outcomes. We tested this theory by central administration of a gapmer antisense oligonucleotide (ASO) targeting Scn2a mRNA in a mouse model of Scn2a early seizure onset DEE (Q/+ mice). Untreated Q/+ mice presented with spontaneous seizures at P1 and did not survive beyond P30. Administration of the ASO to Q/+ mice reduced spontaneous seizures and significantly extended life span. Across a range of behavioral tests, Scn2a ASO-treated Q/+ mice were largely indistinguishable from WT mice, suggesting treatment is well tolerated. A human SCN2A gapmer ASO could likewise impact the lives of patients with SCN2A gain-of-function DEE.

Keywords: Epilepsy; Neuroscience.

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Figures

Figure 1
Figure 1. Disease phenotype of the Q/+ mouse model.
(A) Schematic presentation of SCN2A channel depicting 4 domains (D1–D4), each comprised of 6 transmembrane regions, and the intracellular N and C-terminus of the channel. The p.R1882Q variant is predicted to affect the C-terminus of the channel. (B) Images of Q/+ mice undergoing spontaneous seizure at P1 (left) and P25 (right). (C) Survival curves of Q/+ and +/+ mice. (+/+ n = 19, Q/+ n = 22). ****P < 0.0001, log rank test. (D) Body weight measured on P10 and P21 (+/+ n = 7–20, Q/+ n = 7–8). (E) Representative voltage traces from a neuron injected with 100 pA current. Scale bar applies to all traces. (F) Input-output relationship generated for each injected current step (+/+ n = 3 mice, 20 cells, Q/+ n = 3 mice, 14 cells). *P < 0.05 (F (15, 512) = 6.301, 2-way ANOVA with Sidak’s multiple comparison. (G) Input resistance. **P < 0.005 (t = 3.22, df = 31). (H) Rheobase. **** P < 0.0001 (t = 5, df = 31), unpaired t test. Data are represented as mean ± SEM.
Figure 2
Figure 2. Scn2a mRNA and protein expression were reduced in mice i.c.v. injected with Scn2a ASO ED80 at P1.
(A) Percentage of voltage-gated sodium channel isoform mRNA remaining in the cortex. n = 3–5 +/+ mice for each treatment group. **** P < 0.0001 (F (2, 9 = 212.6)), **P < 0.01 (F (2, 9 = 10.30)), 1-way ANOVA with Tukey’s multiple comparison. (B) Level of Scn2a protein normalized to actin as determined by mass spectrometry, n = 5 to 6 mice for each treatment group. **** P < 0.0001 (F(2, 14 = 79.80)), 1-way ANOVA with Tukey’s multiple comparison. (C) Z-stack images of cortical slices labelled by AnkG (magenta) and Scn2a protein (green). One independent experiment, n = 2–3 +/+ mice for each treatment group. Scale bar: 20 μm. Data are represented as mean ± SEM.
Figure 3
Figure 3. Scn2a ASO ED50 extends survival of Q/+ mice.
(A) Survival curves of Q/+ mice i.c.v. injected with Scn2a ASO ED50 or the negative control ASO at P1. Control ASO (50 μg, n = 39), Scn2a ASO ED50 (2 μg, n = 49). (B) Survival curves of Q/+ mice i.c.v. injected with Scn2a ASO ED50 (2 μg) at P1, then redosed with control ASO (170 μg, n = 11) or Scn2a ASO ED50 (33 μg, n = 15). (C) Survival curves of Q/+ mice i.c.v. injected with Scn2a ASO ED50 or control ASO at P14–P16. Control ASO (85 μg, n = 8), Scn2a ASO ED50 (20 μg, n = 15). Syringes indicate time of i.c.v. injection. ****P < 0.0001, ***P < 0.005, log rank test.
Figure 4
Figure 4. Scn2a ASO ED50 suppresses seizure phenotype of Q/+ mice.
(A) Representative ECoG traces recorded at P40–P45. Scale bar applies to all traces. (B) Number of spikes during 24-hour ECoG recorded at P40–P45 (+/+ n = 6, Scn2a ASO ED50 Q/+ n = 4). (C) Representative ECoG traces recorded at P56–P78 in Q/+ mice treated with Scn2a ASO ED50 at P1. Top panel: arrows indicate inter-ictal spikes. Bottom panel: seizure followed by post-ictal depression recorded on ECoG. Trace in box showed time expanded view of spike and seizure. (D and E) Number of spikes (D) or seizures (E) determined at different time points in Q/+ mice i.c.v. injected with Scn2a ASO ED50 at P1 (P40–P45 n = 5, P56–P78 n = 3). ***P < 0.005 (t = 19.25, df = 6), *P < 0.05 (t = 2.739, df = 6), unpaired 2 tailed t test. (F) Time course of Scn2a mRNA reduction in mice i.c.v. injected with Scn2a ASO ED50 at P1, n = 5–6 per group per time point. Data are represented as mean ± SEM.
Figure 5
Figure 5. Behavioral comparison between Scn2a ASO ED50 treated Q/+ mice and untreated +/+ mice.
(A) Timeline for behavioral studies. (B) Body weight measured at P30. (C) Grip force. (D) Number of foot faults in grid walk test. (E) Ambulatory time measured in locomotor chamber. (F) Time spent in the different compartments of 3-chamber social interaction test. (G) Duration spent in different arms of the elevated plus maze. ***P < 0.001, *P < 0.05 (F (2, 78) = 11.94), 2-way ANOVA with Sidak’s multiple comparison, n = 10–21 per genotype. Data are represented as mean ± SEM.
Figure 6
Figure 6. Effect of administering Scn2a ASO ED80 in Q/+ mice at P1.
(A) Survival curves of Q/+ mice i.c.v. injected with Scn2a ASO ED80 or the negative control ASO at P1. Control ASO (50 μg, n = 39), Scn2a ASO ED80 (10 μg, n = 22). (B) Left: number of spikes during 24-hour ECoG recorded at P40–P45 (+/+ n = 6, Scn2a ASO ED80 Q/+ n = 4). Right: representative ECoG traces. Scale bar applies to all traces. (C) Body weight measured at P30. (D) Grip force. (E) Number of foot faults in grid walk test. (F) Ambulatory time measured in locomotor chamber. (G) Duration spent in different arms of the elevated plus maze. (H) Time spent in the different compartments of 3-chamber social interaction test. ##P < 0.005 (t = 3.557, df = 22), #P < 0.05 (t = 2.518, df = 24), unpaired 2 tailed t test. ****P < 0.0001, (F (2, 63) = 36.72), 2-way ANOVA with Sidak’s multiple comparison, n = 10 to 26 per genotype. Data are represented as mean ± SEM.
Figure 7
Figure 7. Effect of Scn2a ASO ED50 administration in P1 +/+ mice.
(A) Body weight measured at P21. (B) Grip force. (C) Number of foot faults in grid walk test. #P < 0.05 (t = 2.207, df = 17), unpaired 2-tailed t test. (D) Ambulatory time measured in locomotor chamber. (E) Time spent in the different compartments of 3-chamber social interaction test. (F) Percentage duration spent in different arms of the elevated plus maze. **P < 0.01 (F (5, 54) = 8.512), 2-way ANOVA with Sidak’s multiple comparison, n = 8 to 20 per genotype. Data are represented as mean ± SEM.
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