Robert C. Wykes

Optogenetic and Potassium Channel Gene Therapy in a Rodent Model of Focal Neocortical Epilepsy

Light-activated gene therapy acutely suppresses seizures, and gene therapy with a potassium channel prevents epileptogenesis and treats established epilepsy in a rodent model. Casting Light on the Shadow of Epilepsy Epilepsy affects 1% of the population and is often resistant to medication. Surgery to remove the region of the brain that generates seizures is only feasible in a minority of cases because of risks to movement, language, vision, and other essential functions. There is an urgent need for alternative treatments. In a new study, Wykes et al. used a virus to express a therapeutic gene in a small number of brain neurons in the seizure-generating zone in a rat model of drug-resistant epilepsy. They also developed new wireless technology to monitor and detect seizures using a miniaturized implanted transmitter and advanced algorithms. They took two approaches to reduce brain circuit excitability and hence epileptic seizures in the rat model. First, to suppress neuronal firing acutely, they expressed the light-sensitive chloride transporter halorhodopsin in the seizure-generating zone. When laser light was delivered via an optic fiber to this region and the halorhodopsin was activated, they observed a decrease in electrical seizure activity. The success of this “optogenetic” approach implies that a device could be developed to detect and stop seizures “on demand” akin to an implantable defibrillator for heart rhythm disturbances. For longer-term suppression of epilepsy, the investigators overexpressed a brain potassium ion channel that normally regulates both neuronal excitability and neurotransmitter release. This gene therapy treatment fully prevented epilepsy from developing in the rat model. When it was applied during established epilepsy, potassium channel gene therapy progressively diminished the frequency of seizures until they stopped after a few weeks. Neither of the gene therapy approaches tested interfered with normal behavior, most likely because only a small number of neurons were targeted. Although still in the earliest stages of study, this gene therapy approach may hold promise for treating drug-resistant epilepsy. Neocortical epilepsy is frequently drug-resistant. Surgery to remove the epileptogenic zone is only feasible in a minority of cases, leaving many patients without an effective treatment. We report the potential efficacy of gene therapy in focal neocortical epilepsy using a rodent model in which epilepsy is induced by tetanus toxin injection in the motor cortex. By applying several complementary methods that use continuous wireless electroencephalographic monitoring to quantify epileptic activity, we observed increases in high frequency activity and in the occurrence of epileptiform events. Pyramidal neurons in the epileptic focus showed enhanced intrinsic excitability consistent with seizure generation. Optogenetic inhibition of a subset of principal neurons transduced with halorhodopsin targeted to the epileptic focus by lentiviral delivery was sufficient to attenuate electroencephalographic seizures. Local lentiviral overexpression of the potassium channel Kv1.1 reduced the intrinsic excitability of transduced pyramidal neurons. Coinjection of this Kv1.1 lentivirus with tetanus toxin fully prevented the occurrence of electroencephalographic seizures. Finally, administration of the Kv1.1 lentivirus to an established epileptic focus progressively suppressed epileptic activity over several weeks without detectable behavioral side effects. Thus, gene therapy in a rodent model can be used to suppress seizures acutely, prevent their occurrence after an epileptogenic stimulus, and successfully treat established focal epilepsy.

Gene therapy in epilepsy—is it time for clinical trials?

Epilepsy represents a major burden to society, not least because approximately 25% of patients do not respond satisfactorily to antiepileptic medication, and only a minority with pharmacoresistant epilepsy are eligible for potentially curative surgery. Several studies have explored gene therapy as a treatment strategy. The translation of scientific breakthroughs into the clinic faces several challenges, including the validation of experimental models of human pharmacoresistant epilepsy, establishment of sensitive and specific measures of therapeutic efficacy, and evaluation of the long-term safety of gene therapy. On the basis of successful reports of gene therapy in experimental models of epilepsy, a roadmap toward clinical trials is proposed.

Optogenetic approaches to treat epilepsy

BACKGROUNDnNovel treatments for drug-resistant epilepsy are required.nnnNEW METHODnOptogenetics is a combination of optical and genetic methods used to control the activity of specific populations of excitable cells using light with high temporal and spatial resolution. Derived from microbial organisms, opsin genes encode light-activated ion channels and pumps. Opsins can be genetically targeted to well-defined neuronal populations in mammalian brains using viral vectors. When exposed to light of an appropriate wavelength, the excitability of neurons can be increased or decreased optically on a millisecond timescale.nnnCOMPARISON WITH EXISTING METHOD(S)nAlternative treatments for drug-resistant epilepsy such as vagal, cortical or subcortical stimulation, focal cooling, callosotomy, or ketogenic diet have met with limited success, whereas optogenetic approaches have shown considerable pre-clinical promise.nnnCONCLUSIONSnSeveral groups have reported that optogenetic approaches successfully attenuated epileptiform activity in different rodent models of epilepsy, providing proof of the principle that this approach may translate to an effective treatment for epilepsy patients. However, further studies are required to determine the optimal opsin, in which types (or subtypes) of neurons it should be expressed, and what are the most efficient temporal profiles of photostimulation. Although invasive due to the need to inject a viral vector into the brain and implant a device to deliver light to opsin-transduced neurons, this approach has the potential to be effective in suppressing spontaneous seizures while avoiding the side-effects of anti-epileptic drugs (AEDs) or the need to permanently excise regions of the brain. Optogenetic approaches may treat drug-refractory epilepsies.

Gene therapy in status epilepticus

Gene therapy in human disease has expanded rapidly in recent years with the development of safer and more effective viral vectors, and presents a novel approach to the treatment of epilepsy. Studies in animals models have demonstrated that overexpression of inhibitory peptides can modify seizure threshold, prevent the development of epilepsy, and modify established epilepsy. More recently there has been a flurry of studies using optogenetics in which light‐activated channels expressed in neurons can transiently change neuronal excitability on exposure to light, thereby enabling the development of closed loop systems to detect and stop seizure activity. The treatment of status epilepticus presents its own challenges. Because of both the delay in gene expression following transfection and also the necessity of using focal transfection, there are a limited number of situations in which gene therapy can be used in status epilepticus. One such condition is epilepsia partialis continua (EPC). We have used gene therapy in a model of EPC and have shown that we can “cure” the condition. Recent evidence suggesting that gene therapy targeting subcortical regions can modify generalized or more diffuse epilepsies, indicates that the range of situations in status epilepticus in which gene therapy could be used will expand.

Seizures and disturbed brain potassium dynamics in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts

Loss of function of the astrocyte‐specific protein MLC1 leads to the childhood‐onset leukodystrophy “megalencephalic leukoencephalopathy with subcortical cysts” (MLC). Studies on isolated cells show a role for MLC1 in astrocyte volume regulation and suggest that disturbed brain ion and water homeostasis is central to the disease. Excitability of neuronal networks is particularly sensitive to ion and water homeostasis. In line with this, reports of seizures and epilepsy in MLC patients exist. However, systematic assessment and mechanistic understanding of seizures in MLC are lacking.

Focal cortical seizures start as standing waves and propagate respecting homotopic connectivity

Focal epilepsy involves excessive cortical activity that propagates both locally and distally. Does this propagation follow the same routes as normal cortical activity? We pharmacologically induced focal seizures in primary visual cortex (V1) of awake mice, and compared their propagation to the retinotopic organization of V1 and higher visual areas. We used simultaneous local field potential recordings and widefield imaging of a genetically encoded calcium indicator to measure prolonged seizures (ictal events) and brief interictal events. Both types of event are orders of magnitude larger than normal visual responses, and both start as standing waves: synchronous elevated activity in the V1 focus and in homotopic locations in higher areas, i.e. locations with matching retinotopic preference. Following this common beginning, however, seizures persist and propagate both locally and into homotopic distal regions, and eventually invade all of visual cortex and beyond. We conclude that seizure initiation resembles the initiation of interictal events, and seizure propagation respects the connectivity underlying normal visual processing.Focal cortical seizures result from local and widespread propagation of excitatory activity. Here the authors employ widefield calcium imaging in mouse visual areas to demonstrate that these seizures start as local synchronous activation and then propagate along the connectivity that underlies normal sensory processing.

Cortical seizure propagation respects functional connectivity underlying sensory processing

Focal epilepsy involves excessive cortical activity that propagates both locally and distally. Does this propagation follow the same routes as normal cortical activity? We pharmacologically induced focal seizures in primary visual cortex (V1) of awake mice, and compared their propagation to the retinotopic organization of V1 and higher visual areas. We used simultaneous local field potential recordings and widefield imaging of a genetically encoded calcium indicator to measure prolonged seizures (ictal events) and brief interictal events. Both types of event are orders of magnitude larger than normal visual responses, and both start as standing waves: synchronous elevated activity in the V1 focus and in homotopic locations in higher areas, i.e. locations with matching retinotopic preference. Following this common beginning, however, seizures persist and propagate both locally and into homotopic distal regions, and eventually invade all of visual cortex and beyond. We conclude that seizure initiation resembles the initiation of interictal events, and seizure propagation respects the connectivity underlying normal visual processing.Focal epilepsy involves excessive and synchronous cortical activity that propagates both locally and distally. Does this propagation follow the same routes as normal cortical activity? We induced focal seizures in primary visual cortex (V1) of awake mice, and compared their propagation to the retinotopic organization of V1 and higher visual areas. We measured activity through simultaneous local field potential recordings and widefield imaging of a genetically encoded calcium indicator, and observed both prolonged seizures (ictal events) and brief interictal events. Both types of event were orders of magnitude larger than normal visual responses, and both started as standing waves: synchronous elevated activity in the focal V1 region and in corresponding retinotopic locations in higher areas. Following this common beginning, however, seizures, persisted and propagated both locally and into distal regions. These regions matched each other in retinotopy. We conclude that seizure propagation respects the functional connectivity underlying normal visual processing.

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.

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

Objective To 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. Methods Intracortical tetanus toxin (TeNT) injection was used to induce epilepsy in rats. Seizures and their behavioural manifestations were evaluated with continuous video-electrocorticography telemetry. Results TeNT 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. Significance The 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. Key Points Tetanus toxin injection into rat visual cortex induces focal cortical epilepsy. Electrographic seizures were associated with non-motor and motor features with or without evolution to generalized tonic-clonic seizures. Seizures could not be provoked by intermittent photic stimulation. Seizures were clustered in time and exhibited a circadian variation in frequency. The number of seizures in first week after seizure onset could be used to predict the total number of seizures in the following 3 weeks.

The Enlightened Brain: Novel Imaging Methods Focus on Epileptic Networks at Multiple Scales

Epilepsy research is rapidly adopting novel fluorescence optical imaging methods to tackle unresolved questions on the cellular and circuit mechanisms of seizure generation and evolution. State of the art two-photon microscopy and wide-field fluorescence imaging can record the activity in epileptic networks at multiple scales, from neuronal microcircuits to brain-wide networks. These approaches exploit transgenic and viral technologies to target genetically encoded calcium and voltage sensitive indicators to subclasses of neurons, and achieve genetic specificity, spatial resolution and scalability that can complement electrophysiological recordings from awake animal models of epilepsy. Two-photon microscopy is well suited to study single neuron dynamics during interictal and ictal events, and highlight the differences between the activity of excitatory and inhibitory neuronal classes in the focus and propagation zone. In contrast, wide-field fluorescence imaging provides mesoscopic recordings from the entire cortical surface, necessary to investigate seizure propagation pathways, and how the unfolding of epileptic events depends on the topology of brain-wide functional connectivity. Answering these questions will inform pre-clinical studies attempting to suppress seizures with gene therapy, optogenetic or chemogenetic strategies. Dissecting which network nodes outside the seizure onset zone are important for seizure generation, propagation and termination can be used to optimize current and future evaluation methods to identify an optimal surgical strategy.