Serena Gea Giannelli

Objective: Dravet syndrome (DS) is a developmental and epileptic encephalopathy primarily caused by haploinsufficiency of the SCN1A gene, which encodes the alpha subunit of Na _V 1.1 voltage‐gated sodium channel. The disease manifests with febrile and spontaneous seizures, developmental delay, cognitive impairment, and increased risk of sudden unexpected death in epilepsy. Most preclinical therapeutic strategies aim to enhance endogenous Scn1a gene expression or deliver a functional copy via perinatal interventions. Although promising, these approaches face challenges in achieving efficient brain‐wide delivery, targeting all interested brain areas. Methods: In this study, we used a conditional DS reversible mouse model ( Scn1a ^Stop/+ ) to assess the therapeutic potential of postsymptomatic restoration (postnatal day 30) of Na _V 1.1 physiological levels in all neuronal populations but restricted to minimal brain regions. Results: We found that hippocampal correction alone reduces seizure susceptibility, whereas simultaneous restoration in the hippocampus, thalamus, and overlying somatosensory and visual cortex was associated with a trend toward further reduction in seizure burden and improved survival. Significance: These findings indicate that hippocampal restoration accounts for a substantial component of the therapeutic benefit, and broader network correction may confer additional advantages.

The overexpression of MECP2 leads to severe neurological deficits in MECP2 duplication syndrome, and its dosage is considered a risk factor in gene therapy for Rett syndrome. However, in MECP2 duplication syndrome patients, MECP2 dysregulation arises at the embryonic stage while in Rett syndrome gene therapy, MECP2 is delivered into the mature brain. Here, we show that MeCP2 overexpression induces transcriptional alterations in neural progenitor cells, but has minimal effects in neurons in both mouse and human contexts. Consequently, MeCP2 overexpression in neural progenitor cells, but not mature neurons, leads to functional changes. Mechanistically, we observe that both endogenous and overexpressed Mecp2 bind to the same CpG island repertoire. In neurons, where endogenous Mecp2 is highly expressed, ectopic protein expression leads to reduced CpG island binding and accelerated protein degradation. In contrast, in neural progenitor cells, lower endogenous Mecp2 levels facilitate stronger deposition of the ectopic protein onto CpG islands, driving the transcriptional activation of many developmental bivalent genes. We show that this activation is mediated by the interaction with the SWI/SNF chromatin remodeling complex. Our findings establish that increased gene dosage-dependent effects are highly influenced by cell type, levels of proteins and their mechanisms of action.

Neuroinflammation plays a key role in exacerbating dopaminergic neuron (DAN) loss in Parkinson’s disease (PD). However, it remains unresolved how to effectively normalize this immune response given the complex interplay between the innate and adaptive immune responses occurring within a scarcely accessible organ like the brain. In this study, we uncovered a consistent correlation between neuroinflammation, brain parenchymal lymphocytes, and DAN loss among several commonly used mouse models of PD generated by a variety of pathological triggers. We validated a viral therapeutic approach for the microglia-specific expression of interleukin 10 (IL-10) to selectively mitigate the excessive inflammatory response. We found that this approach induced a local nigral IL-10 release that alleviated DAN loss in mice overexpressing the human SNCA gene in the substantia nigra. Single-cell transcriptomics revealed that IL-10 induced the emergence of a molecularly distinct microglial cell state, enriched in markers of cell activation with enhanced expression of prophagocytic pathways. IL-10 promoted microglial phagocytotic and clearance activities in vitro and reduced αSYN aggregate burden in the nigral area in mice overexpressing SNCA . Furthermore, IL-10 stimulated the differentiation of CD4 ⁺ T lymphocytes into active T regulatory cells and promoted inhibitory characteristics in CD8 ⁺ T cells. In summary, our results show that local and microglia-specific IL-10 transduction elicited strong immunomodulation in the nigral tissue with enhanced suppression of lymphocyte toxicity that was associated with DAN survival. These results offer insights into the therapeutic benefits of IL-10 and showcase a promising gene delivery approach that could minimize undesired side effects.

Huntington’s disease (HD) is a neurodegenerative disorder caused by CAG-repeat expansions in the huntingtin (HTT) gene. The resulting mutant HTT (mHTT) protein induces toxicity and cell death via multiple mechanisms and no effective therapy is available. Here, we employ a genome-wide screening in pluripotent mouse embryonic stem cells (ESCs) to identify suppressors of mHTT toxicity. Among the identified suppressors, linked to HD-associated processes, we focus on Metal response element binding transcription factor 1 (Mtf1). Forced expression of Mtf1 counteracts cell death and oxidative stress caused by mHTT in mouse ESCs and in human neuronal precursor cells. In zebrafish, Mtf1 reduces malformations and apoptosis induced by mHTT. In R6/2 mice, Mtf1 ablates motor defects and reduces mHTT aggregates and oxidative stress. Our screening strategy enables a quick in vitro identification of promising suppressor genes and their validation in vivo, and it can be applied to other monogenic diseases.

Adeno-associated Viral vectors (AAVs) have been successful exploited in gene therapy applications for the treatment of several genetic disorders. AAV is considered an episomal vector but it has been shown to integrate within the host-cell genome following generation of double-strand DNA breaks or nicks. While AAV integration raises some safety concerns, it can also provide therapeutic benefit; the direct intrathymic injection of an AAV harboring a therapeutic transgene results in integration in T cell progenitors and long-term T cell immunity. To assess the mechanisms of AAV integration, we retrieved and analyzed hundreds of AAV integration sites from lymph node-derived mature T-cells as compared to liver and brain tissue from treated mice. Notably, we found that while AAV integrations in the liver and brain were distributed across the entire mouse genome, >90% of the integrations in T-cells were clustered within the T cell receptor α, β and γ genes. More precisely, the insertion mapped to DNA breaks created by the enzymatic activity of recombination activating genes (RAG) during V(D)J recombination. Our data indicate that RAG activity during T-cell receptor maturation induces a site-specific integration of AAV genomes and opens new therapeutic avenues for achieving long-term AAV-mediated gene transfer in dividing cells.

Rett syndrome is an incurable neurodevelopmental disorder caused by mutations in the gene encoding for methyl-CpG binding-protein 2 (MeCP2). Gene therapy for this disease presents inherent hurdles since MECP2 is expressed throughout the brain and its duplication leads to severe neurological conditions as well. Herein, we use the AAV-PHP.eB to deliver an instability-prone Mecp2 (iMecp2) transgene cassette which, increasing RNA destabilization and inefficient protein translation of the viral Mecp2 transgene, limits supraphysiological Mecp2 protein levels. Intravenous injections of the PHP.eB-iMecp2 virus in symptomatic Mecp2 mutant mice significantly improved locomotor activity, lifespan and gene expression normalization. Remarkably, PHP.eB-iMecp2 administration was well tolerated in female Mecp2 mutant or in wild-type animals. In contrast, we observed a strong immune response to the transgene in treated male Mecp2 mutant mice that was overcome by immunosuppression. Overall, PHP.eB-mediated delivery of iMecp2 provided widespread and efficient gene transfer maintaining physiological Mecp2 protein levels in the brain.

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.