| In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy
Abstract
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.
Introduction
Epilepsy affects up to 1% of the population, and 30% of patients continue to experience seizures despite the use of current medication (Kwan et al., 2011; Tang et al., 2017). Although the majority of drug-resistant epilepsies are focal, targeting drugs to a restricted brain region presents major challenges, and potentially curative surgery is limited to a minority of cases where the seizure focus is remote from eloquent cortex (Kullmann et al., 2014). Gene therapy holds promise as a rational replacement for surgery for intractable pharmaco-resistant epilepsy, and could in principle improve the prospect for seizure freedom in many people (Kullmann et al., 2014; Lieb et al., 2018). Several approaches have been proposed to interfere with epileptogenesis or to decrease seizure frequency in chronic epilepsy (Simonato, 2014). Current experimental gene therapies mainly rely on viral vector-mediated expression of genes encoding normal CNS proteins or exogenous non-mammalian proteins (Richichi et al., 2004; Noe et al., 2008; Wykes et al., 2012, 2016; Krook-Magnuson et al., 2013; Kätzel et al., 2014; Lieb et al., 2018; Agostinho et al., 2019). This approach has several potential limitations, including a finite packaging capacity of viral vectors, difficulty in ensuring normal splicing and post-transcriptional processing, and, in the case of non-mammalian membrane proteins, concerns about immunogenicity. Modulating the expression of endogenous genes, in contrast, would represent an important step toward safe and rational treatment of intractable epilepsy and other neurological diseases.
The DNA editor/regulator CRISPR/Cas9 (Konermann et al., 2015; Dominguez et al., 2016; Adli, 2018) represents a powerful tool to modify endogenous genes, not only in somatic cells but also in mammalian neurons (Heidenreich and Zhang, 2016; Suzuki et al., 2016). In addition to permanently altering endogenous gene sequences, CRISPR/Cas9 can regulate the activity of genes through promoter modulation, an approach known as CRISPR activation (CRISPRa) (Dominguez et al., 2016; Liao et al., 2017; ,Matharu et al., 2019 ). CRISPRa is therefore a promising tuneable tool to increase the expression of genes encoding, for instance, ion channels, in chronic epilepsy in order to restore physiological levels of network activity (Wykes et al., 2012; Wykes and Lignani, 2018; Colasante et al., 2020). The CRISPRa system consists of a nuclease-defective Cas9 (dCas9) fused to a transcription activator and a small guide RNA (sgRNA) that targets dCas9 to the promoter of the gene of interest (Dominguez et al., 2016). There are multiple advantages of this system. First, it is versatile because the targeted gene can be switched simply by changing the sgRNA. Second, CRISPRa preserves the full range of native splice variants and protein biogenesis mechanisms (Liao et al., 2017). Third, CRISPRa is, in principle, safe because it only alters the promoter activity of genes that are already transcribed in targeted neurons. Finally, CRISPRa can be targeted to specific neurons in the epileptic focus using established viral vectors (La Russa and Qi, 2015).
Here, we report the use of CRISPRa to treat a mouse model of temporal lobe epilepsy, from in vitro validation to demonstration of efficacy in reducing seizure frequency and rescuing cognitive impairment in vivo.
Materials and methods
Study design
This study aimed to test the hypothesis that upregulating endogenous genes (e.g. Kcna1) with CRISPRa can treat chemoconvulsant-induced temporal lobe epilepsy. The experiments were designed to achieve a power >0.8 with an α = 0.05. For in vivo experiments the 3Rs guidelines for animal welfare were followed. Outliers were not excluded and at least three independent repetitions were performed. Exclusion criteria were applied for all the recordings (see below). All the experiments were randomized and researchers were blinded during recordings and analysis.
https://academic.oup.com/brain/advance-art...awaa045/5780426
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