Anti-seizure gene therapy for focal cortical dysplasia.

Focal cortical dysplasias are a common subtype of malformation of cortical development, which frequently presents with a spectrum of cognitive and behavioural abnormalities as well as pharmacoresistant epilepsy. Focal cortical dysplasia type II is typically caused by somatic mutations resulting in mammalian target of rapamycin (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 focal cortical dysplasia. 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 focal cortical dysplasia model generated by in utero electroporation of frontal lobe neural progenitors with a constitutively active human Ras homolog enriched in brain (RHEB) plasmid, an activator of mTOR complex 1. We characterized the model by quantifying electrocorticographic and behavioural abnormalities, both in mice developing spontaneous generalized seizures and in mice only exhibiting interictal discharges. Injection of AAV9-CAMK2A-EKC in the dysplastic region resulted in a robust decrease (∼64%) in the frequency of seizures. 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 discharges or behaviour in mice without generalized seizures. 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.

Progressive myoclonus epilepsy KCNC1 variant causes a developmental dendritopathy.

OBJECTIVE: Mutations in KCNC1 can cause severe neurological dysfunction, including intellectual disability, epilepsy, and ataxia. The Arg320His variant, which occurs in the voltage-sensing domain of the channel, causes a highly penetrant and specific form of progressive myoclonus epilepsy with severe ataxia, designated myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK). KCNC1 encodes the voltage-gated potassium channel KV 3.1, a channel that is important for enabling high-frequency firing in interneurons, raising the possibility that MEAK is associated with reduced interneuronal function. METHODS: To determine how this variant triggers MEAK, we expressed KV 3.1bR320H in cortical interneurons in vitro and investigated the effects on neuronal function and morphology. We also performed electrophysiological recordings of oocytes expressing KV 3.1b to determine whether the mutation introduces gating pore currents. RESULTS: Expression of the KV 3.1bR320H variant profoundly reduced excitability of mature cortical interneurons, and cells expressing these channels were unable to support high-frequency firing. The mutant channel also had an unexpected effect on morphology, severely impairing neurite development and interneuron viability, an effect that could not be rescued by blocking KV 3 channels. Oocyte recordings confirmed that in the adult KV 3.1b isoform, R320H confers a dominant negative loss-of-function effect by slowing channel activation, but does not introduce potentially toxic gating pore currents. SIGNIFICANCE: Overall, our data suggest that, in addition to the regulation of high-frequency firing, KV 3.1 channels play a hitherto unrecognized role in neuronal development. MEAK may be described as a developmental dendritopathy.

dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice.

Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Nav1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage.

On-demand cell-autonomous gene therapy for brain circuit disorders.

Several neurodevelopmental and neuropsychiatric disorders are characterized by intermittent episodes of pathological activity. Although genetic therapies offer the ability to modulate neuronal excitability, a limiting factor is that they do not discriminate between neurons involved in circuit pathologies and "healthy" surrounding or intermingled neurons. We describe a gene therapy strategy that down-regulates the excitability of overactive neurons in closed loop, which we tested in models of epilepsy. We used an immediate early gene promoter to drive the expression of Kv1.1 potassium channels specifically in hyperactive neurons, and only for as long as they exhibit abnormal activity. Neuronal excitability was reduced by seizure-related activity, leading to a persistent antiepileptic effect without interfering with normal behaviors. Activity-dependent gene therapy is a promising on-demand cell-autonomous treatment for brain circuit disorders.

Gene Editing and Modulation: the Holy Grail for the Genetic Epilepsies?

Epilepsy is a complex neurological disorder for which there are a large number of monogenic subtypes. Monogenic epilepsies are often severe and disabling, featuring drug-resistant seizures and significant developmental comorbidities. These disorders are potentially amenable to a precision medicine approach, of which genome editing using CRISPR/Cas represents the holy grail. Here we consider mutations in some of the most 'common' rare epilepsy genes and discuss the different CRISPR/Cas approaches that could be taken to cure these disorders. We consider scenarios where CRISPR-mediated gene modulation could serve as an effective therapeutic strategy and discuss whether a single gene corrective approach could hold therapeutic potential in the context of homeostatic compensation in the developing, highly dynamic brain. Despite an incomplete understanding of the mechanisms of the genetic epilepsies and current limitations of gene editing tools, CRISPR-mediated approaches have game-changing potential in the treatment of genetic epilepsy over the next decade.

Olanzapine: A 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 not only are a powerful tool to test causality in basic neuroscience but also are potentially amenable to clinical translation. A major obstacle, however, is 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 the European Medicines Agency, limiting its translational potential. Here, we report that the atypical antipsychotic drug olanzapine, widely available in various formulations, is a potent agonist of the human M4 muscarinic receptor-based DREADD, facilitating clinical translation of chemogenetics to treat central nervous system diseases.