Albert Snowball

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.

Epilepsy is a major health burden, calling for new mechanistic insights and therapies. 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 hypersynchronziation. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding K_v1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated K_v1.1 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.

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.

Refractory focal 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 recovery of the channels from inactivation. For improved safety, this engineered potassium channel (EKC) gene was packaged into a nonintegrating lentiviral vector under the control of a cell type-specificCAMK2Apromoter. In a blinded, randomized, placebo-controlled preclinical trial, the EKC lentivector robustly reduced seizure frequency in a male rat model of focal neocortical epilepsy characterized by discrete spontaneous seizures. When packaged into an adeno-associated viral vector (AAV2/9), the EKC gene was also effective at suppressing seizures in a male rat model of temporal lobe epilepsy. 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 being ready for clinical translation in the treatment of refractory focal epilepsy.SIGNIFICANCE STATEMENTPharmacoresistant epilepsy affects up to 0.3% of the population. Although epilepsy surgery can be effective, it is limited by risks to normal brain function. We have developed a gene therapy that builds on a mechanistic understanding of altered neuronal and circuit excitability in cortical epilepsy. The potassium channel geneKCNA1was mutated to bypass post-transcriptional editing and was packaged in a nonintegrating lentivector to reduce the risk of insertional mutagenesis. A randomized, blinded preclinical study demonstrated therapeutic effectiveness in a rodent model of focal neocortical epilepsy. Adeno-associated viral delivery of the channel to both hippocampi was also effective in a model of temporal lobe epilepsy. These results support clinical translation to address a major unmet need.

Focal neocortical epilepsy is a common form of epilepsy and there is a need to develop animal models that allow the evaluation of novel therapeutic strategies to treat this type of epilepsy. Tetanus toxin (TeNT) injection into rat visual cortex induces 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 3 weeks. The TeNT model of occipital cortical epilepsy is a model of acquired focal neocortical epilepsy well suited for preclinical evaluation of novel anti-epileptic strategies. We provide here a detailed analysis of the epilepsy phenotypes, 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.

Chronic pain and epilepsy together affect hundreds of millions of people worldwide. While traditional pharmacotherapy provides essential relief to the majority of patients, a large proportion remains resistant, and surgical intervention is only possible for a select few. As both disorders are characterised by neuronal hyperexcitability, manipulating the expression of the most direct modulators of excitability – ion channels – represents an attractive common treatment strategy. A number of viral gene therapy approaches have been explored to achieve this. These range from the up‐ or down‐regulation of channels that control excitability endogenously, to the delivery of exogenous channels that permit manipulation of excitability via optical or chemical means. In this review we highlight the key experimental successes of each approach and discuss the challenges facing their clinical translation.