Conference Agenda

Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. We have previously shown that a catalytically dead Cas9 (dCas9) fused to a transcriptional activation domain and delivered in vivoby intracerebroventricular injection of a dual adeno-associated virus (AAV) tool can boost the endogenous Scn1a gene expression mouse and ameliorate febrile seizures in a mouse model of DS.

While it revealed useful to provide a proof of principle of the applicability, this tool needs to be optimized to proceed toward therapeutic translation. In particular, smaller activatory tools should be exploited to be better accommodated into AAVs and enhance brain transduction efficiency. To this aim we explored the possibility to use a smaller dCas9, deleted of nucleasic domain or, alternatively, Zinc Finger based transcription factors. The effect of these two novel activatory tools on the expression of Scn1a gene and on some distinctive features of DS mice will be presented.

In addition, exploiting a reversible mouse model of DS in which Cre mediated Scn1a reactivation can be efficiently achieved, we are mimicking an ideal gene therapy and gaining information on cell type timing of therapy delivery and preferential brain areas to target to achieve significant phenotypic amelioration.

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/intermingled neurons. We describe an activity‑dependent gene therapy strategy that downregulates the excitability of overactive neurons in closed loop, and test it 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 anti‑epileptic effect without interfering with normal behaviours. Activity‑dependent gene therapy is a promising on‑demand cell-autonomous treatment for brain circuit disorders.

Alexander disease (AxD) is an autosomal dominant leukodystrophy caused by missense mutations in the glial fibrillary acidic protein (GFAP) gene. Accumulation of GFAP aggregates in Rosenthal fibers leads to impairment of proteasomal activity and hyperactivation of the stress response, thus compromising astrocyte functions and altering the homeostasis of the central nervous system (CNS). Currently, this disease lacks a cure.

Here, we aim at developing novel, single-dose gene editing strategies to treat AxD. By in vitro screening of single guide RNAs (sgRNAs) targeting the Gfap gene, we selected a CRISPR/Cas9 system inducing a robust downregulation of the GFAP protein with no off-target activity at the predicted genomic loci. We identified an AAV serotype and optimized intracerebral injection protocols for maximal in vivo delivery of the editing machinery in CNS astrocytes. Neonatal intracerebroventricular injections of Cas9 nuclease and sgRNA reduced GFAP expression and mitigated the formation of Rosenthal fibers in white matter regions of AxD mice, providing in vivo proof-of-concept of the potential efficacy of a CRISPR/Cas9 editing approach in recovering disease-associated phenotypes. We are currently optimizing allele-specific editing approaches to target GFAP mutation hotspots, thus overcoming the potential side effects associated with permanent GFAP knock-out (e.g. altered synaptic transmission and reduced myelination). We identified Cas9 nucleases inducing allele-specific knock-out of Gfap sequences harboring the R76H and R236H mutations, homologs of the human mutation hotspots. Also, we identified adenine base editors to correct the R76H mutation, thus reducing the potential genotoxicity induced by Cas9 nucleases.

Overall, these results pave the way for advanced preclinical studies aimed at safe and effective delivery of editing systems targeting the mutated Gfap allele in the CNS using AAV vectors or nanoparticles. Novel editing platforms for in vivo targeting of CNS astrocytes could benefit AxD and other neurodegenerative disorders characterized by primary astrocyte degeneration or dysfunctional/maladaptive astrogliosis.