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.

Homeostatic Plasticity in Epilepsy

“…Gene therapy for epilepsy is a promising approach to treat the chronic phase of the pathology (Kullmann et al, 2014). Recent gene therapies target the symptoms (seizures) rather than the cause of epilepsy, for example, decreasing the excitability of excitatory neurons or potentiating inhibitory tone (Richichi et al, 2004;Noè et al, 2008;Wykes et al, 2012;Krook-Magnuson et al, 2013;Kätzel et al, 2014;Lieb et al, 2018;Agostinho et al, 2019;Wickham et al, 2019;Colasante et al, 2020). These therapies have been efficient in decreasing intrinsic neuronal excitability, synaptic transmission, and the number of seizures, in rescuing cognitive defects and also FIGURE 1 | Graphical representation of the potential role of homeostatic plasticity in the epileptic process.…”

“…In these cases, no homeostatic compensations have been observed to counteract the decreased excitability induced by the therapeutic approach. Furthermore, a net positive effect at transcriptomic level induced by an increase of endogenous Kv1.1 using CRISPRa, suggests a compensatory mechanism in line with a response to an increased network activity (Colasante et al, 2020). This effect was surprising because of the uncertainty on the effect of gene therapy: does it only increase seizure threshold or does it also rescue the epileptogenic process pushing back the brain state within its physiological boundaries?…”

“…However is also possible that the Kv1.1 enhancement just increased the threshold for seizure generation (unlikely because not observed in acute seizure induction), that in turns allow a rearrangement of network activity with a consequent resetting of the transcriptional profile and physiological brain state (Colasante et al, 2020). Another possible explanation is that decreasing neuronal excitability to a certain extent, the system can be pushed back to a more stable interictal state, less likely to fluctuate go back into the ictal state.…”

Kv1.1 Channelopathies: Pathophysiological Mechanisms and Therapeutic Approaches

“…In view of the well-recognized role of Kv1.1 in modulating neuronal excitability, recent proof-of-concept studies in rodent models of focal refractory epilepsy supported the notion that up-regulation of Kv1.1 channels, by means of genetic manipulation, could hold promise to reduce seizure frequency, regardless of the presence of a primary Kv1.1 mutation [121,122]. Gene therapy, relying on non-integrating lentiviral vector mediating Kv1.1 expression, proved effective in reducing seizures in a rat model of focal neocortical epilepsy [121].…”

“…In addition, Colasante et al provided the first proof-of-principle that CRISPRa technology could be exploited to selectively increase endogenous kcna1 expression in mouse hippocampal excitatory neurons to dampen excitability. CRISPRa-mediated up-regulation of Kv1.1 channels successfully led to decreased spontaneous generalized tonic-clonic seizures and rescue of cognitive impairment in a mouse model of intractable chronic temporal lobe epilepsy [122]. Of course, further studies on other models of epilepsy will be required to assess the preclinical efficacy and safety of these gene-based therapeutic strategies before translation in the clinical setting.…”