Antisense oligonucleotides increase Scn1a expression and reduce seizures and SUDEP incidence in a mouse model of Dravet syndrome

2020 ◽  
Vol 12 (558) ◽  
pp. eaaz6100 ◽  
Author(s):  
Zhou Han ◽  
Chunling Chen ◽  
Anne Christiansen ◽  
Sophina Ji ◽  
Qian Lin ◽  
...  
Keyword(s):  
Mouse Model ◽  
Antisense Oligonucleotides ◽  
De Novo ◽  
Nuclear Gene ◽  
Epileptic Encephalopathy ◽  
Dravet Syndrome ◽  
Unexpected Death ◽  
Α Subunit ◽  
Gene And Protein Expression ◽  
Electrographic Seizures

Dravet syndrome (DS) is an intractable developmental and epileptic encephalopathy caused largely by de novo variants in the SCN1A gene, resulting in haploinsufficiency of the voltage-gated sodium channel α subunit NaV1.1. Here, we used Targeted Augmentation of Nuclear Gene Output (TANGO) technology, which modulates naturally occurring, nonproductive splicing events to increase target gene and protein expression and ameliorate disease phenotype in a mouse model. We identified antisense oligonucleotides (ASOs) that specifically increase the expression of productive Scn1a transcript in human cell lines, as well as in mouse brain. We show that a single intracerebroventricular dose of a lead ASO at postnatal day 2 or 14 reduced the incidence of electrographic seizures and sudden unexpected death in epilepsy (SUDEP) in the F1:129S-Scn1a+/− × C57BL/6J mouse model of DS. Increased expression of productive Scn1a transcript and NaV1.1 protein was confirmed in brains of treated mice. Our results suggest that TANGO may provide a unique, gene-specific approach for the treatment of DS.

scholarly journals Scn1a gene reactivation after symptom onset rescues pathological phenotypes in a mouse model of Dravet syndrome

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Nicholas Valassina ◽  
Simone Brusco ◽  
Alessia Salamone ◽  
Linda Serra ◽  
Mirko Luoni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Model ◽  
Symptom Onset ◽  
Epileptic Encephalopathy ◽  
Dravet Syndrome ◽  
Neurological Deficits ◽  
Behavioral Alterations ◽  
Unexpected Death ◽  
Physiological Level ◽  
Behavioral Abnormalities

AbstractDravet syndrome is a severe epileptic encephalopathy caused primarily by haploinsufficiency of the SCN1A gene. Repetitive seizures can lead to endurable and untreatable neurological deficits. Whether this severe pathology is reversible after symptom onset remains unknown. To address this question, we generated a Scn1a conditional knock-in mouse model (Scn1a Stop/+) in which Scn1a expression can be re-activated on-demand during the mouse lifetime. Scn1a gene disruption leads to the development of seizures, often associated with sudden unexpected death in epilepsy (SUDEP) and behavioral alterations including hyperactivity, social interaction deficits and cognitive impairment starting from the second/third week of age. However, we showed that Scn1a gene re-activation when symptoms were already manifested (P30) led to a complete rescue of both spontaneous and thermic inducible seizures, marked amelioration of behavioral abnormalities and normalization of hippocampal fast-spiking interneuron firing. We also identified dramatic gene expression alterations, including those associated with astrogliosis in Dravet syndrome mice, that, accordingly, were rescued by Scn1a gene expression normalization at P30. Interestingly, regaining of Nav1.1 physiological level rescued seizures also in adult Dravet syndrome mice (P90) after months of repetitive attacks. Overall, these findings represent a solid proof-of-concept highlighting that disease phenotype reversibility can be achieved when Scn1a gene activity is efficiently reconstituted in brain cells.

scholarly journals Dravet syndrome associated mutations in GABRA1, GABRB2 and GABRG2 define the genetic landscape of defects of GABAA receptors

2021 ◽  
Author(s):  
Ciria C Hernandez ◽  
XiaoJuan Tian ◽  
Ningning Hu ◽  
Wangzhen Shen ◽  
Mackenzie A Catron ◽  
...  
Keyword(s):  
Gabaa Receptor ◽  
De Novo ◽  
Epileptic Encephalopathy ◽  
Dravet Syndrome ◽  
First Year ◽  
Genes Encoding ◽  
Genetic Landscape ◽  
Clonic Seizures ◽  
Trafficking Defects ◽  
First Year Of Life

Abstract Dravet syndrome is a rare, catastrophic epileptic encephalopathy that begins in the first year of life, usually with febrile or afebrile hemiclonic or generalized tonic-clonic seizures followed by status epilepticus. De novo variants in genes that mediate synaptic transmission such as SCN1A and PCDH19 are often associated with Dravet syndrome. Recently, GABAA receptor subunit genes (GABRs) encoding α1 (GABRA1), β3 (GABRB3) and γ2 (GABRG2), but not β2 (GABRB2) or β1 (GABRB1), subunits are frequently associated with Dravet syndrome or Dravet syndrome-like phenotype. We performed next generation sequencing on 870 patients with Dravet syndrome and identified nine variants in three different GABRs. Interestingly, the variants were all in genes encoding the most common GABAA receptor, the α1β2γ2 receptor. Mutations in GABRA1 (c.644T>C, p.L215P; c.640C>T, p.R214C; c.859G>A; V287I; c.641G>A, p.R214H) and GABRG2 (c.269C>G, p.T90R; c.1025C>T, p.P342L) presented as de novo cases, while in GABRB2 two variants were de novo (c.992T>C, p.F331S; c.542A>T, p.Y181F) and one was autosomal dominant and inherited from the maternal side (c.990_992del, p.330_331del). We characterized the effects of these GABR variants on GABAA receptor biogenesis and channel function. We found that defects in receptor gating were the common deficiency of GABRA1 and GABRB2 Dravet syndrome variants, while mainly trafficking defects were found with the GABRG2 (c.269C>G, p.T90R) variant. It seems that variants in α1 and β2 subunits are less tolerated than in γ2 subunits, since variant α1 and β2 subunits express well but were functionally deficient. This suggests that all of these GABR variants are all targeting GABR genes that encode the assembled α1β2γ2 receptor, and regardless of which of the three subunits are mutated, variants in genes coding for α1, β2 and γ2 receptor subunits make them candidate causative genes in the pathogenesis of Dravet syndrome.

scholarly journals Gabra2 is a genetic modifier of Dravet syndrome in mice

2020 ◽  
Author(s):  
Nicole A. Hawkins ◽  
Toshihiro Nomura ◽  
Samantha Duarte ◽  
Robert W. Williams ◽  
Gregg E. Homanics ◽  
...  
Keyword(s):  
Protein Expression ◽  
De Novo ◽  
Genetic Modifier ◽  
Clinical Severity ◽  
Dravet Syndrome ◽  
Unexpected Death ◽  
Increased Risk ◽  
Hippocampal Synapses ◽  
Severe Epilepsy ◽  
Strain Dependent

AbstractPathogenic variants in epilepsy genes result in a spectrum of clinical presentation, ranging from benign phenotypes to intractable epilepsies with significant co-morbidities and increased risk of sudden unexpected death in epilepsy (SUDEP). One source of this phenotypic heterogeneity is modifier genes that affect penetrance, dominance or expressivity of a primary pathogenic variant. Mouse models of epilepsy also display varying degrees of clinical severity on different genetic backgrounds. Mice with heterozygous deletion of Scn1a (Scn1a+/−) model Dravet syndrome, a severe epilepsy most often caused by SCN1A haploinsufficiency. Scn1a+/− heterozygous mice recapitulate key features of Dravet syndrome, including febrile and afebrile spontaneous seizures, SUDEP, and cognitive and behavioral deficits. The Scn1a+/− mouse model also exhibits strain-dependent phenotype severity. Scn1a+/− mice maintained on the 129S6/SvEvTac (129) strain have normal lifespan and no overt seizures. In contrast, admixture with C57BL/6J (B6) results in severe epilepsy and premature lethality in [B6×129]F1.Scn1a+/− mice. In previous work, we identified Dravet Survival Modifier loci (Dsm1-Dsm5) responsible for strain-dependent differences in survival. Gabra2, encoding the GABAA α2 subunit, was nominated as the top candidate modifier at the Dsm1 locus on chromosome 5. Direct measurement of GABAA receptors found lower abundance of α2-containing receptors in hippocampal synapses of B6 mice relative to 129. We also identified a B6-specific single nucleotide intronic deletion within Gabra2 that lowers mRNA and protein by nearly 50%. Repair of this de novo deletion reestablished normal levels of Gabra2 transcript and protein expression. In the current study, we used B6 mice with the repaired Gabra2 allele to validate it as a modifier of phenotype severity in Scn1a+/− mice. Repair of Gabra2 restored transcript and protein expression, increased abundance of α2-containing GABAA receptors in hippocampal synapses, and improved seizure and survival phenotypes of Scn1a+/− mice. These findings validate Gabra2 as a genetic modifier of Dravet syndrome.

scholarly journals Sudden unexpected death in a mouse model of Dravet syndrome

2013 ◽  
Vol 123 (4) ◽  
pp. 1798-1808 ◽  
Author(s):  
Franck Kalume ◽  
Ruth E. Westenbroek ◽  
Christine S. Cheah ◽  
Frank H. Yu ◽  
John C. Oakley ◽  
...  
Keyword(s):  
Mouse Model ◽  
Sudden Unexpected Death ◽  
Dravet Syndrome ◽  
Unexpected Death

Dravet syndrome is a severe developmental and epileptic encephalopathy. Fenfluramine and gene therapy are promising

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Nuclear Gene ◽  
Epileptic Encephalopathy ◽  
Dravet Syndrome ◽  
Phase Iii ◽  
Preclinical Research ◽  
Cardiovascular Side Effects ◽  
Phase Iii Trials ◽  
Negative Effect ◽  
Disease Specific ◽  
Specific Impact

Dravet syndrome is a severe developmental and epileptic encephalopathy related to SCN1A. Resistant epilepsy despite polypharmacy will soon have new therapeutic options.In 2019, the treatment range was broadened to include cannabidiol after 2 phase III trials showed dramatically decreased seizure frequency. However, the results are slightly less convincing than for stiripentol or fenfluramine, and thus far no disease-specific impact appears (yet). Fenfluramine, formerly used to treat obesity, was authorized in 2020 by the Food and Drug Administration (FDA) to treat seizures in Dravet's syndrome. Fenfluramine was extremely successful in 2 randomized, placebo-controlled Phase III trials (one without stiripentol, one with stiripentol). It was generally well-tolerated-with a small drop in appetite being the most prevalent negative effect. No cardiovascular side-effects have been detected. While the anticonvulsant mechanism is not well known, it seems largely serotonergic. Preclinical research showed indications of disease-specific and potential disease-modifying impact on Dravet syndrome. The latter would support fenfluramine usage beyond its anticonvulsant properties, and needs additional research. Studies with additional serotonergic compounds (clemizole and lorcaserine) began. Unfortunately, existing medications do not target Dravet's syndrome's underlying genetic etiology and hence have no substantial influence on patient cognition or other comorbidities. Therapies focusing on amplifying Nav 1.1 channels based on TANGO technology (anti-sense oligonucleotides; "targeted increase in nuclear gene output") will begin shortly (phase I and II studies). This technology seems promising and marks the beginning of an interesting therapeutic phase for Dravet syndrome patients.

scholarly journals Electrophysiological Alterations of Pyramidal Cells and Interneurons of the CA1 Region of the Hippocampus in a Novel Mouse Model of Dravet Syndrome

Genetics ◽  
2020 ◽  
Vol 215 (4) ◽  
pp. 1055-1066
Author(s):  
David A. Dyment ◽  
Sarah C. Schock ◽  
Kristen Deloughery ◽  
Minh Hieu Tran ◽  
Kerstin Ure ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mouse Model ◽  
Hippocampal Slices ◽  
Pyramidal Cells ◽  
Epileptic Encephalopathy ◽  
Dravet Syndrome ◽  
Resting Membrane Potential ◽  
Sodium Currents ◽  
Spike Amplitude ◽  
Induced Neurons

Dravet syndrome is a developmental epileptic encephalopathy caused by pathogenic variation in SCN1A. To characterize the pathogenic substitution (p.H939R) of a local individual with Dravet syndrome, fibroblast cells from the individual were reprogrammed to pluripotent stem cells and differentiated into neurons. Sodium currents of these neurons were compared with healthy control induced neurons. A novel Scn1aH939R/+ mouse model was generated with the p.H939R substitution. Immunohistochemistry and electrophysiological experiments were performed on hippocampal slices of Scn1aH939R/+ mice. We found that the sodium currents recorded in the proband-induced neurons were significantly smaller and slower compared to wild type (WT). The resting membrane potential and spike amplitude were significantly depolarized in the proband-induced neurons. Similar differences in resting membrane potential and spike amplitude were observed in the interneurons of the hippocampus of Scn1aH939R/+ mice. The Scn1aH939R/+ mice showed the characteristic features of a Dravet-like phenotype: increased mortality and both spontaneous and heat-induced seizures. Immunohistochemistry showed a reduction in amount of parvalbumin and vesicular acetylcholine transporter in the hippocampus of Scn1aH939R/+ compared to WT mice. Overall, these results underline hyper-excitability of the hippocampal CA1 circuit of this novel mouse model of Dravet syndrome which, under certain conditions, such as temperature, can trigger seizure activity. This hyper-excitability is due to the altered electrophysiological properties of pyramidal neurons and interneurons which are caused by the dysfunction of the sodium channel bearing the p.H939R substitution. This novel Dravet syndrome model also highlights the reduction in acetylcholine and the contribution of pyramidal cells, in addition to interneurons, to network hyper-excitability.

scholarly journals Astrocyte Ca2+ Signaling is Facilitated in an Scn1a+/− Mouse Model of Dravet Syndrome

2021 ◽  
Author(s):  
Kouya Uchino ◽  
Wakana Ikezawa ◽  
Yasuyoshi Tanaka ◽  
Masanobu Deshimaru ◽  
Kaori Kubota ◽  
...  
Keyword(s):  
Mouse Model ◽  
Sodium Channel ◽  
Epileptic Encephalopathy ◽  
Dravet Syndrome ◽  
Heterozygous Mutation ◽  
Loss Of Function ◽  
Cell Type ◽  
Voltage Gated ◽  
A Subunit ◽  
The Brain

Dravet syndrome (DS) is an infantile-onset epileptic encephalopathy. More than 80% of DS patients have a heterozygous mutation in SCN1A, which encodes a subunit of the voltage-gated sodium channel, Nav1.1, in neurons. The roles played by astrocytes, the most abundant glial cell type in the brain, have been investigated in the pathogenesis of epilepsy; however, the specific involvement of astrocytes in DS has not been clarified. In this study, we evaluated Ca2+ signaling in astrocytes using genetically modified mice that have a loss-of-function mutation in Scn1a. We found that the slope of spontaneous Ca2+ spiking was increased without a change in amplitude in Scn1a+/− astrocytes. In addition, ATP-induced transient Ca2+ influx and the slope of Ca2+ spiking were also increased in Scn1a+/− astrocytes. These data indicate that perturbed Ca2+ dynamics in astrocytes may be involved in the pathogenesis of DS.

Targeted Augmentation of Nuclear Gene Output (TANGO) of SCN1A Rescues Parvalbumin Interneuron Excitability and Reduces Seizures in a Mouse Model of Dravet Syndrome

2021 ◽  
pp. 147743
Author(s):  
Eric R. Wengert ◽  
Pravin K. Wagley ◽  
Samantha M. Strohm ◽  
Nuha Reza ◽  
Ian C. Wenker ◽  
...  
Keyword(s):  
Mouse Model ◽  
Nuclear Gene ◽  
Dravet Syndrome ◽  
Parvalbumin Interneuron

Changing Landscape of Dravet Syndrome Management: An Overview

2020 ◽  
Vol 51 (02) ◽  
pp. 135-145 ◽  
Author(s):  
Debopam Samanta
Keyword(s):  
Motor Impairment ◽  
Current Treatment ◽  
Epileptic Encephalopathy ◽  
Dravet Syndrome ◽  
Loss Of Function ◽  
Unexpected Death ◽  
Autistic Behavior ◽  
Behavioral Abnormalities ◽  
Treatment Paradigm ◽  
Antisense Oligonucleotide Therapy

AbstractDravet syndrome (DS), previously known as severe myoclonic epilepsy of infancy, is a severe developmental and epileptic encephalopathy caused by loss-of-function mutations in one copy of SCN1A (haploinsufficiency), located on chromosome 2q24, with decreased function of Nav1.1 sodium channels in GABAergic inhibitory interneurons. Pharmacoresistant seizures in DS start in the infancy in the form of hemiclonic febrile status epilepticus. Later, other intractable seizure types develop including myoclonic seizures. Early normal development in infancy evolves into moderate to severe intellectual impairment, motor impairment, behavioral abnormalities, and later a characteristic crouching gait. Clobazam, valproate, levetiracetam, topiramate, zonisamide, ketogenic diet, and vagus nerve stimulation had been shown to be effective, but even with polytherapy, only 10% of patients get adequate seizure control. The author provides a narrative review of the current treatment paradigm as well as recent advances in the management of DS based on a comprehensive literature review (MEDLINE using PubMed and OvidSP vendors with appropriate keywords to incorporate recent evidence), personal practice, and experience. In recent years, the treatment paradigm of DS is changing with the approval of pharmaceutical-grade cannabidiol oil and stiripentol. Another novel antiepileptic drug (AED), fenfluramine, had also shown excellent efficacy in phase 3 studies of DS. However, these AEDs primarily control seizures without addressing the underlying pathogenesis and other important common comorbidities such as cognitive impairment, autistic behavior, neuropsychiatric abnormalities, and motor impairment including crouching gait. Several agents targeted for DS are in the developmental stage: TAK935, lorcaserin, clemizole, huperzine analog, ataluren, selective sodium channel modulators and activators, antisense oligonucleotide therapy, and adenoviral vector therapy. As DS is associated with a high risk of sudden unexpected death in epilepsy, seizure detection devices can be used in this population for testing and clinical validation of these devices.

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