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 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.

Despite the introduction of more than one dozen new antiepileptic drugs in the past 20 years, approximately one-third of people who develop epilepsy continue to have seizures on mono- or polytherapy. Viral-vector-mediated gene transfer offers the opportunity to design a rational treatment that builds on mechanistic understanding of seizure generation and that can be targeted to specific neuronal populations in epileptogenic foci. Several such strategies have shown encouraging results in different animal models, although clinical translation is limited by possible effects on circuits underlying cognitive, mnemonic, sensory or motor function. Here, we describe an autoregulatory antiepileptic gene therapy, which relies on neuronal inhibition in response to elevations in extracellular glutamate. It is effective in a rodent model of focal epilepsy and is well tolerated, thus lowering the barrier to clinical translation.

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 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 tonicclonic 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.

In recent years, there has been a significant increase in preclinical studies to test genetic therapies for epilepsy. Some of these therapies have advanced to clinical trials and are being tested in patients with monogenetic or focal refractory epilepsy. This article provides an overview of the current state of preclinical studies that show potential for clinical translation. Specifically, we focus on genetic therapies that have demonstrated a clear effect on seizures in animal models and have the potential to be translated to clinical settings. Both therapies targeting the cause of the disease and those that treat symptoms are discussed. We believe that the next few years will be crucial in determining the potential of genetic therapies for treating patients with epilepsy.

Developmental and epileptic encephalopathies (DEEs) are a group of rare childhood disorders characterized by severe epilepsy and related cognitive deficits. Numerous DEE genes have been discovered thanks to advances in genomic diagnosis, yet putative molecular links between these disorders are not known. CDKL5 deficiency disorder (CDD, DEE2) is one of the most common forms of genetic epilepsy; it is caused by loss-of-function mutations in the brain-enriched kinase CDKL5. To elucidate CDKL5 function, we looked for CDKL5 substrates using a SILAC based phosphoproteomic screen. We identified the voltage-gated Ca2+ channel Cav2.3 (encoded by CACNA1E) as a novel physiological target of CDKL5 in mice and humans. Recombinant channel electrophysiology and interdisciplinary characterization of Cav2.3 phosphomutant mice revealed that the loss of Cav2.3 phosphorylation leads to channel gain-of-function via slower channel inactivation and enhanced acetylcholine-induced stimulation, resulting in increased neuronal excitability. These changes in Cav2.3 closely resemble those described for gain-of-function point-mutations in CACNA1E that cause DEE69, a disorder sharing clinical features with CDD. Our results show that these two single-gene disorders are mechanistically related. We suggest that CDD is partly a channelopathy with Cav2.3 gain-of-function, thus Cav2.3 inhibition could be therapeutic in these DEEs.

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In this paper we study the production-scheduling problem for a two-stage flow shop with outsourcing options. The maintenance of in-house machine and job-related discount for outsourcing cost are considered in model, in which total cost and makespan are regarded as bi-objective for the scheduling problem. Based on the analysis of model, we introduce a concept-packaging to reduce the complexity of problem and then develop heuristic algorithm to solve the problem. Computational experiments using different groups of data are conducted to test the algorithm.