Biology subjects

Schorge, S.

Publications and source records attributed to Schorge, S..

15 recordsLinked to original sources

bioRxiv2026

MicroRNA-sensing plasmid system for dynamic control of functional ion channel expression

Gene therapy offers the potential for long-term treatment or cures for a range of chronic diseases. However, permanent gene therapy expression may not be desirable. Efforts have been made to create systems which can be switched on/off by stimuli including light, designer drugs, or cellular contexts such as increased electrical activity. Here, we designed a novel plasmid system in which ion channel expression, and therefore function, is regulated by microRNA (miR) - an endogenous class of short noncoding RNAs which negatively regulate gene expression via binding the 3 untranslated region of target transcripts. We modified an existing voltage-gated potassium channel gene therapy with a binding cassette for miR-193a-3p, and transfected this miR-193-OFF system in neuro2A cells. Co-transfection with an inhibitor or mimic of miR-193a-3p respectively enhanced or repressed expression of our transgene, assessed using a GFP marker. Using whole-cell voltage clamp, we observed enhanced voltage-gated potassium currents in cells co-transfected with a miR-193a-3p inhibitor, compared with a non-targeting control oligonucleotide. Together, this demonstrates the concept of a novel miR-mediated molecular switch which can bias therapeutic ion channel expression based on a specific miR signal. As miRs are a ubiquitous molecular mechanism, our approach could be applied to a wide range of cellular and disease contexts, potentially expanding gene therapy to new patient populations.

neuroscience↗

bioRxiv2025

AAV delivery of RNA editing machinery rescues SUDEP and seizure phenotype in a mouse model of Dravet Syndrome

Dravet syndrome (DS) is a severe childhood genetic epilepsy, caused by de novo heterozygous mutations in SCN1A, resulting in a loss-of-function of the voltage-gated sodium ion channel, Nav1.1. Nav1.1 is expressed in the brain, and at a lower level, in the heart. DS manifests in the first year of life. Patients exhibit tonic-clonic seizures, febrile seizures, cognitive decline, developmental delays, ataxia, and sudden unexpected death from epilepsy (SUDEP). We have developed a novel AAV-F mediated CRISPR-Cas-inspired RNA targeting system (CIRTS) preclinical treatment to increase endogenous Scn1a and ameliorate the disease phenotype in a clinically-relevant heterozygous loss-of-function mouse model of DS. We designed novel guide RNAs (gRNAs) to target the long non-coding RNA, (or natural antisense transcript) of Scn1a to increase the expression of Scn1a mRNA in DS mice. We show that intracerebroventricular and intravenous administration of AAV-F-CIRTS-gRNA9 to target the brain and the heart to neonatal Scn1a+/-mice resulted in a significant increase in survival, and a reduction in SUDEP, febrile seizures and seizure duration. These findings provide proof-of-concept evidence that an AAV-F-CIRTS mediated therapy hold promise as a potential treatment for DS.

neuroscience↗

bioRxiv2024

AAV9-mediated targeting of natural antisense transcript as a novel treatment for Dravet Syndrome

Dravet syndrome (DS) is a severe childhood onset developmental and epileptic encephalopathy which leads to life-long disability. Symptoms usually manifest in the first year of life and include prolonged severe seizures, developmental delay and severe intellectual disability. DS patients have an increased mortality rate, including sudden unexpected death in epilepsy (SUDEP). Approximately 90% of patients carry a heterozygous loss-of-function mutation in the SCN1A gene, which encodes a voltage-gated sodium ion channel, NaV1.1. The NaV1.1 channel is expressed in the brain and at a lower level, in the heart. Previous studies have identified a long non-coding RNA (lncRNA) which specifically downregulates SCN1A gene expression. This natural antisense transcript (NAT) can be modulated by AntagoNATs, small synthetic oligonucleotides developed to inhibit NAT function. In a DS mouse model, AntagoNATs were shown to modulate Scn1a expression by targeting the Scn1a NAT, improving seizure frequency after repeated administration. Here, we have developed novel AntagoNATs and incorporated these into a clinically relevant adeno-associated virus serotype 9 (AAV9) gene therapy vector, to test in a DS mouse model (Scn1a+/-) and provide a one-off treatment approach. Eighteen AntagoNATs were tested in vitro; from the best performing candidates, we selected two AntagoNAT sequences (K & H) for in vivo testing as they had the highest homology (90%) to human SCN1A NAT. We administered both vectors to newborn Scn1a+/- mice via intracerebroventricular (ICV) and intravenous (IV) injection to target the brain and heart. AAV9-AntagoNAT-H significantly increased survival, decreased febrile seizures and reduced spontaneous seizure frequency compared to the PBS control group. When administered at P14 by ICV and IV injection, AAV9-AntagoNAT-H increased survival. In this proof-of-concept study, we have demonstrated for the first time the delivery of AntagoNAT technology via an AAV9 vector and thus offering the possibility of a one-time treatment for DS patients.

molecular biology↗

bioRxiv2024

Optimizing clinical interpretability of functional evidence in epilepsy-related ion channel variants

Variants in genes encoding the voltage-gated ion channels are among the most common monogenic causes of epilepsy and neurodevelopmental disorders. Functional effects of a variant are increasingly important for diagnosis and therapeutic decisions. To incorporate knowledge regarding functional consequences in formal clinical variant interpretation, we developed an approach for evaluating multiple functional measurements within the Bayesian framework of the modified ACMG/AMP guidelines. We analyzed 216 functional assessments of 191 variants in SCN1A (n=74), SCN2A (n=66), SCN3A (n=18), and SCN8A (n=33). Of 20 commonly measured biophysical parameters, the most frequent drivers of overall functional consequence were persistent current (f=0.54), voltage dependence of activation (f=0.51), and voltage dependence of fast inactivation (f=0.40) for gain-of-function and peak current (f=0.87) for loss-of-function. By comparing measurements of 23 benign variants, we determined thresholds by which published data on these four parameters confer Strong evidence of variant pathogenicity (likelihood ratio > 18.7) under the ACMG/AMP rubric. Similarly, we delineated evidence weights for the most common epilepsy-related potassium channel gene, KCNQ2, through reports of 80 pathogenic and 24 benign variants, accounting for heterozygous and homozygous experimental conditions. We collected the resulting categorization of functional data into FENICS, a biomedical ontology of 152 standardized terms for coherent annotation of electrophysiological results. Across 271 variants in SCN1A/2A/3A/8A and KCNQ2, 1,731 annotations are available in ClinVar, facilitating use of this evidence in variant classification. In summary, we introduce and apply an ACMG/AMP-calibrated framework for electrophysiological studies in epilepsy-related channelopathies to delineate the impact of functional evidence on clinical variant interpretation.

genetics↗

bioRxiv2024

Molecular dynamics simulations reveal molecular mechanisms for the gain and loss of function effects of four SCN2A variants

SCN2A gene disorders cover a wide range of medical conditions, from epileptic encephalopathies to neurodevelopmental disorders. The variants of these disorders, studied through electrophysiology, show complex behaviors that go beyond simple classification as either gain or loss of function. In our study, we simulated the biophysical effects of variants (R937C, V208E, S1336Y, and R853Q) to understand their impact. Our findings reveal that all these variants negatively affect the structural stability of the gene, with R937C being the most unstable. Specifically, R937C disrupts important charged interactions affecting sodium ion flow, while S1336Y introduces a new interaction that impacts the channels inactivation gate. Conversely, the variants V208E and R853Q, which are located in the voltage-sensing domains, have opposite effects: R853Q increases compactness and interaction, whereas V208E shows a decrease. Our computer-based method offers a scalable way to gain crucial insights into how genetic variants influence channel dysfunction and contribute to neurodevelopmental disorders. AUTHOR SUMMARYDespite numerous advancements in computational methods for predicting variant pathogenicity in the SCN2A gene, understanding the precise biophysical molecular mechanisms associated with each variant at the atomic level remains a challenge. Presently, variants are predominantly categorized as either gain or loss of function, often overlooking critical structural details associated with these variants. This study focuses on elucidating the molecular mechanisms linked to the four most common SCN2A variants using all-atom molecular dynamics simulations, employing three replicas for each system. Our findings offer insights into the potential mechanisms underlying these four variants, thereby providing explanations for the observed electrophysiological outcomes. This investigation significantly contributes to enhancing our comprehension of how SCN2A variants manifest in various diseases. It underscores the importance of unraveling the biophysical properties underlying potential disease mechanisms, which could potentially enhance diagnostic and therapeutic strategies for patients afflicted with SCN2A-related disorders.

biophysics↗

bioRxiv2023

Spinal cord pathology in a Dravet Syndrome mouse model

SummaryO_ST_ABSObjectivesC_ST_ABSDravet syndrome is a severe epileptic encephalopathy that begins in early childhood. More than 80% of patients with Dravet syndrome exhibit a haploinsufficiency in SCN1A, which encodes the voltage-gated sodium ion channel NaV1.1. The epilepsy is believed be caused by specific deficit of SCN1A in inhibitory interneurons of the hippocampus. However, the aetiology of other symptoms including gait disturbances, ataxia, cardiac issues and dysautonomia is less clear. MethodsIn an Scn1a knock-out (Scn1a-/-) mouse model which recapitulates clinical phenotypes, we assessed NaV1.1 and neuroinflammation throughout the central nervous system. ResultsConsistent with current understanding, wild-type expression of NaV1.1 transcript and protein were absent in knock-out mice in the prefrontal cortex, striatum, hippocampus, thalamus, and cerebellum. Increased GFAP was detected in the brain only in the hippocampus. Transcript and protein were detected in wild-type cervical, thoracic and lumbar spinal cord but not in knock-out mice. Unexpectedly, GFAP was increased in all three spinal regions. Therefore, we proceeded to perform transcriptomic analysis of cortex, hippocampus and spinal cord. Pathways associated with monooxygenase activity, fatty acid ligases and lactate transporters were highly dysregulated in the spinal cord. ConclusionThe existence and relevance of pathology of the spinal cord in Dravet syndrome has received scant attention. Our findings are consistent with some systemic symptoms of Dravet syndrome, with the benefits of treatments which may modulate the astrocyte-neuron lactate shuttle such as Stiripentol and ketogenic dietary regimes, and with the efficacy of intrathecal delivery of therapeutics. Key PointsO_LIDecrease of endogenous Scn1a and NaV1.1 expression in Scn1a-/- mice has a widespread impact on the gene expression profile in the spinal cord. C_LIO_LIIncreased GFAP expression observed in the spinal cord of Scn1a-/- mice. C_LIO_LIDifferentially expressed genes related to monooxygenase activity, fatty acid ligases and lactate transporters in cervical spinal cord of Scn1a-/- mice. C_LI

molecular biology↗

bioRxiv2023

Anti-seizure Gene Therapy for Focal Cortical Dysplasia

Focal Cortical Dysplasias (FCDs) are a common subtype of malformation of cortical development, which frequently present with a spectrum of cognitive and behavioural abnormalities as well as pharmacoresistant epilepsy. FCD type II is typically caused by somatic mutations resulting in mTOR hyperactivity, and is the commonest pathology found in children undergoing epilepsy surgery. However, surgical resection does not always result in seizure freedom, and is often precluded by proximity to eloquent brain regions. Gene therapy is a promising potential alternative treatment and may be appropriate in cases that represent an unacceptable surgical risk. Here, we evaluated a gene therapy based on overexpression of the Kv1.1 potassium channel in a mouse model of frontal lobe FCD. An engineered potassium channel (EKC) transgene was placed under control of a human promoter that biases expression towards principal neurons (CAMK2A) and packaged in an adeno-associated viral vector (AAV9). We used an established FCD model generated by in utero electroporation of frontal lobe neural progenitors with a constitutively active human RHEB plasmid, an activator of mTOR Complex 1. First, we further characterised this by quantifying electrocorticograms and behavioural abnormalities, both in mice developing spontaneous generalised seizures and in mice only exhibiting abnormal interictal discharges. Then, using continuous video-electrocorticogram recordings from epileptic mice before and after injection of AAV9-CAMK2A-EKC in the dysplastic region, we observed a robust decrease in the frequency of seizures and in interictal activity, compared to mice injected with a control viral vector. Despite the robust anti-epileptic effect of the treatment, there was neither an improvement nor a worsening of performance in behavioural tests sensitive to frontal lobe function. AAV9-CAMK2A-EKC had no effect on interictal activity or behaviour in non-epileptic mice. AAV9-CAMK2A-EKC gene therapy is a promising therapy with translational potential to treat the epileptic phenotype of mTOR-related malformations of cortical development. Cognitive and behavioural co-morbidities may, however, resist an intervention aimed at reducing circuit excitability.

neuroscience↗

bioRxiv2022

Conserved patterns across ion channels correlate with variant pathogenicity and clinical phenotypes

Clinically identified genetic variants in ion channels can be benign or cause disease by increasing or decreasing the protein function. Consequently, therapeutic decision-making is challenging without molecular testing of each variant. Our biophysical knowledge of ion channel structures and function is just emerging, and it is currently not well understood which amino acid residues cause disease when mutated. We sought to systematically identify biological properties associated with variant pathogenicity across all major voltage and ligand-gated ion channel families. We collected and curated 3,049 pathogenic variants from hundreds of neurodevelopmental and other disorders and 12,546 population variants for 30 ion channel or channel subunits for which a high-quality protein structure was available. Using a wide range of bioinformatics approaches, we computed 163 structural features and tested them for pathogenic variant enrichment. We developed a novel 3D spatial distance scoring approach that enables comparisons of pathogenic and population variant distribution across protein structures. We discovered and independently replicated that several pore residue properties and proximity to the pore axis were most significantly enriched for pathogenic variants compared to population variants. Using our novel 3D scoring approach, we showed that the strongest pathogenic variant enrichment was observed for pore-lining residues and alpha-helix residues within 5[A] distance from the pore axis center and not involved in gating. Within the subset of residues located at the pore, the hydrophobicity of the pore was the feature most strongly associated with variant pathogenicity. We also found an association between the identified properties and both clinical phenotypes and fucntional in vitro assays for voltage-gated sodium channels (SCN1A, SCN2A, SCN8A) and N-methyl-D-aspartate (NMDA) receptor (GRIN1, GRIN2A, GRIN2B) encoding genes. In an independent expert-curated dataset of 1,422 neurodevelopmental disorder pathogenic patient variants, and 679 electrophysiological experiments that pore axis distance is associated with seizure age of onset and cognitive performance as well as differential gain vs. loss-of-channel function. In summary, we identified biological properties associated with ion-channel malfunction and show that these are correlated with in vitro functional read-outs and clinical phenotypes in patients with neurodevelopmental disorders. Our results suggest that clinical decision support algorithms that predict variant pathogenicity and function are feasible in the future.

genetics↗

bioRxiv2020

Alternative splicing tunes sodium channels to support channel- and neuron- specific effects

Neuronal excitability is tightly regulated, requiring rapidly activating and inactivating voltage-gated sodium channels to allow accurate temporal encoding of information. Alternative splicing greatly broadens the repertoire of channels, but the adaptive significance of this phenomenon is incompletely understood. An alternative splicing event that is conserved across vertebrates affects part of the first domain of sodium channels and modulates their availability after inactivation. Here we use this conserved splicing event to ask whether this modulation has consistent effects in different neuronal backgrounds, or whether a conserved splicing event can be exploited to produce distinct effects in different cell types. We show that the consequences of alternate splicing of human Nav1.1 and Nav1.2 for neuronal activity depend on whether they are expressed in the cell types where they normally predominate (interneurons or excitatory neurons, respectively). Splicing in the adult isoform in both channels is sufficient to slow action potential rise times in all neurons. However, changes to both action potential half width and maximal firing rate are specific to cell type and channel, with each channel appearing tuned to mediate effects in its predominant neuronal background. Finally, we use dynamic clamp to demonstrate that alternative splicing in Nav1.1 changes how interneurons fire during epileptiform events. Our data show that, for sodium channels, despite conserved amino acid changes and similar effects on channel gating, alternative splicing has distinct impacts on neuronal properties, thus highlighting how closely sodium channels are tuned to distinct cellular backgrounds.

neuroscience↗

bioRxiv2019

A systems approach delivers a functional microRNA catalog and expanded targets for seizure suppression in temporal lobe epilepsy

MicroRNAs (miRNAs) are short noncoding RNAs that shape the gene expression landscape, including during the pathogenesis of temporal lobe epilepsy (TLE). In order to provide a full catalog of the miRNA changes that happen during experimental TLE, we sequenced Argonaute 2-loaded miRNAs in the hippocampus of three different animal models at regular intervals between the time of the initial precipitating insult to the establishment of spontaneous recurrent seizures. The commonly upregulated miRNAs were selected for a functional in vivo screen using oligonucleotide inhibitors. This revealed anti-seizure phenotypes upon inhibition of miR-10a-5p, miR-21a-5p and miR-142a-5p as well as neuroprotection-only effects for inhibition of miR-27a-3p and miR-431-5p. Proteomic data and pathway analysis on predicted and validated targets of these miRNAs indicated a role for TGF{beta} signaling in a shared seizure-modifying mechanism. Together, these results identify functional miRNAs in the hippocampus and a pipeline of new targets for seizure control in epilepsy.

neuroscience↗

bioRxiv2019

Altered Hippocampal-Prefrontal Neural Dynamics in Mouse Models of Down Syndrome

Altered neural dynamics in medial prefrontal cortex (mPFC) and hippocampus may contribute to cognitive impairments in the complex chromosomal disorder, Down Syndrome (DS). Here, we demonstrate non-overlapping behavioural differences associated with distinct abnormalities in hippocampal and mPFC electrophysiology during a canonical spatial memory task in three partially trisomic mouse models of DS (Dp1Tyb, Dp10Yey, Dp17Yey) that together cover all regions of homology with human chromosome 21 (Hsa21). Dp1Tyb mice showed slower decision-making (unrelated to the gene dose of DYRK1A, which has been implicated in DS cognitive dysfunction) and altered theta dynamics (reduced frequency, increased hippocampal-mPFC coherence, increased modulation of hippocampal high gamma); Dp10Yey mice showed impaired alternation performance and reduced theta modulation of hippocampal low gamma; while Dp17Yey mice were no different from wildtype mice. These results link specific hippocampal and mPFC circuit dysfunctions to cognitive deficits in DS models and, importantly, map them to discrete regions of Hsa21.

neuroscience↗

bioRxiv2018

Olanzapine: a full and potent agonist at the hM4D(Gi) DREADD amenable to clinical translation of chemogenetics

Designer receptors exclusively activated by designer drugs (DREADDs) derived from muscarinic receptors are a powerful tool to test causality in basic neuroscience, but are also potentially amenable to clinical translation. A major obstacle is however that the widely-used agonist clozapine-N-oxide undergoes conversion to clozapine, which penetrates the blood-brain barrier but has an unfavorable side effect profile. Perlapine has been reported to activate DREADDs at nanomolar concentrations, but is not approved for use in humans by the Food and Drug Administration or European Medicines Agency, limiting its translational potential. Here we report that the atypical antipsychotic drug olanzapine, widely available in various formulations, is a full and potent agonist of the human muscarinic-receptor M4-based DREADD, facilitating clinical translation of chemogenetics to treat CNS diseases.

neuroscience↗

bioRxiv2018

CRISPRa-mediated Kcna1 upregulation decreases neuronal excitability and suppresses seizures in a rodent model of temporal lobe epilepsy

Epilepsy is a major health burden, calling for new mechanistic and therapeutic insights. CRISPR-mediated gene editing shows promise to cure genetic pathologies, although hitherto it has mostly been applied ex-vivo. Its translational potential for treating non-genetic pathologies is still unexplored. Furthermore, neurological diseases represent an important challenge for the application of CRISPR, because of the need in many cases to manipulate gene function of neurons in situ. A variant of CRISPR, CRISPRa, offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters. We asked if this strategy can effectively treat acquired focal epilepsy, focusing on ion channels because their manipulation is known be effective in changing network hyperactivity and hypersynchronisation. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding Kv1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated Kv1.1 upregulation led to a substantial decrease in neuronal excitability. Continuous video-EEG telemetry showed that AAV9-mediated delivery of CRISPRa, upon doxycycline administration, decreased spontaneous generalized tonic-clonic seizures in a model of temporal lobe epilepsy, and rescued cognitive impairment and transcriptomic alterations associated with chronic epilepsy. The focal treatment minimizes concerns about off-target effects in other organs and brain areas. This study provides the proof of principle for a translational CRISPR-based approach to treat neurological diseases characterized by abnormal circuit excitability.

neuroscience↗

bioRxiv2018

Epilepsy gene therapy using non-integrating lentiviral delivery of an engineered potassium channel gene

Refractory focal neocortical epilepsy is a devastating disease for which there is frequently no effective treatment. Gene therapy represents a promising alternative, but treating epilepsy in this way involves irreversible changes to brain tissue, so vector design must be carefully optimized to guarantee safety without compromising efficacy. We set out to develop an epilepsy gene therapy vector optimized for clinical translation. The gene encoding the voltage-gated potassium channel Kv1.1, KCNA1, was codon-optimized for human expression and mutated to accelerate the channels recovery from inactivation. For improved safety, this engineered potassium channel (EKC) gene was packaged into a non-integrating lentiviral vector under the control of a cell type-specific CAMK2A promoter. In a blinded, randomized, placebo-controlled pre-clinical trial, the EKC lentivector robustly reduced seizure frequency in a rat model of focal neocortical epilepsy characterized by discrete spontaneous seizures. This demonstration of efficacy in a clinically relevant setting, combined with the improved safety conferred by cell type-specific expression and integration-deficient delivery, identify EKC gene therapy as ready for clinical translation in the treatment of refractory focal epilepsy.

neuroscience↗

bioRxiv2018

Semiology, clustering, periodicity and natural history of seizures in an experimental visual cortical epilepsy model

ObjectiveTo characterize a rat model of focal neocortical epilepsy for use in developing novel therapeutic strategies in a type of epilepsy that represents a significant unmet need.\n\nMethodsIntracortical tetanus toxin (TeNT) injection was used to induce epilepsy in rats. Seizures and their behavioural manifestations were evaluated with continuous video-electrocorticography telemetry.\n\nResultsTeNT injection into rat primary visual cortex induced focal neocortical epilepsy without preceding status epilepticus. The latency to first seizure ranged from 3 to 7 days. Seizure duration was bimodal, with both short (approximately 30s) and long-lasting (>100s) seizures occurring in the same animals. Seizures were accompanied by non-motor features such as behavioural arrest, or motor seizures with or without evolution to generalized tonic-clonic seizures. Seizures were commoner during the sleep phase of a light-dark cycle. Seizure occurrence was not random, and tended to cluster with significantly higher probability of recurrence within 24 hours of a previous seizure. Across animals, the number of seizures in the first week could be used to predict the number of seizures in the following 22 days.\n\nSignificanceThe TeNT model of visual cortical epilepsy is a robust model of acquired focal neocortical epilepsy, and is well suited for preclinical evaluation of novel anti-epileptic strategies. We provide here a detailed analysis of the epilepsy phenotype, seizure activity, electrographic features, and the semiology. In addition we provide a predictive framework that can be used to reduce variation and consequently animal use in pre-clinical studies of potential treatments.\n\nKey PointsO_LITetanus toxin injection into rat visual cortex induces focal cortical epilepsy.\nC_LIO_LIElectrographic seizures were associated with non-motor and motor features with or without evolution to generalized tonic-clonic seizures.\nC_LIO_LISeizures could not be provoked by intermittent photic stimulation.\nC_LIO_LISeizures were clustered in time and exhibited a circadian variation in frequency.\nC_LIO_LIThe number of seizures in first week after seizure onset could be used to predict the total number of seizures in the following 3 weeks.\nC_LI

neuroscience↗