In vivo Genome Editing Therapeutic Approaches for Neurological Disorders: Where Are We in the Translational Pipeline?

In vivo genome editing tools, such as those based on CRISPR, have been increasingly utilized in both basic and translational neuroscience research. There are currently nine in vivo non-CNS genome editing therapies in clinical trials, and the pre-clinical pipeline of major biotechnology companies demonstrate that this number will continue to grow. Several biotechnology companies commercializing in vivo genome editing and modification technologies are developing therapies for CNS disorders with accompanying large partnering deals. In this review, the authors discuss the current genome editing and modification therapy pipeline and those in development to treat CNS disorders. The authors also discuss the technical and commercial limitations to translation of these same therapies and potential avenues to overcome these hurdles

Sustainability of a university designed and developed media annotation tool to prepare learners with skills needed for future employment

RMIT University‘s media annotation tool (MAT) is a computer software program dedicated to incorporating video footage to the student learning experience in a novel manner. In addition to the usual functions associated with videos, MAT allows users to enter written comments at strategic and key positions to emphasise the required learning points. Innovatively introduced to creatively support learning for work-ready skills, in 2011 MAT was integrated into courses across nine student cohorts, over six disciplines, in the Vocational and Higher Education (undergraduate and post graduate) sectors of the university. This paper will focus on analysis of insights of teacher experiences using MAT, highlighting sustainable ways forward with university designed innovations. It will introduce the context of implementing MAT and discuss the process of evaluating the requirements for promoting MAT to the wider university community and, more specifically, to embed and sustain MAT into the long-term

Arx acts as a regional key selector gene in the ventral telencephalon mainly through its transcriptional repression activity

AbstractThe homeobox-containing gene Arx is expressed during ventral telencephalon development and required for correct GABAergic interneuron tangential migration from the ganglionic eminences to the olfactory bulbs, cerebral cortex and striatum. Its human ortholog is associated with a variety of neurological clinical manifestations whose symptoms are compatible with the loss of cortical interneurons and altered basal ganglia-related activities. Herein, we report the identification of a number of genes whose expression is consistently altered in Arx mutant ganglionic eminences. Our analyses revealed a striking ectopic expression in the ganglionic eminences of several of these genes normally at most marginally expressed in the ventral telencephalon. Among them, Ebf3 was functionally analyzed. Thus, its ectopic expression in ventral telencephalon was found to prevent neuronal tangential migration. Further, we showed that Arx is sufficient to repress Ebf3 endogenous expression and that its silencing in Arx mutant tissues partially rescues tangential cell movement. Together, these data provide new insights into the molecular pathways regulated by Arx during telencephalon development

Scn1a gene reactivation after symptom onset rescues pathological phenotypes in a mouse model of Dravet syndrome

Dravet syndrome is a severe epileptic encephalopathy caused primarily by haploinsufficiency of the SCN1A gene. Repetitive seizures can lead to endurable and untreatable neurological deficits. Whether this severe pathology is reversible after symptom onset remains unknown. To address this question, we generated a Scn1a conditional knock-in mouse model (Scn1a Stop/+) in which Scn1a expression can be re-activated on-demand during the mouse lifetime. Scn1a gene disruption leads to the development of seizures, often associated with sudden unexpected death in epilepsy (SUDEP) and behavioral alterations including hyperactivity, social interaction deficits and cognitive impairment starting from the second/third week of age. However, we showed that Scn1a gene re-activation when symptoms were already manifested (P30) led to a complete rescue of both spontaneous and thermic inducible seizures, marked amelioration of behavioral abnormalities and normalization of hippocampal fast-spiking interneuron firing. We also identified dramatic gene expression alterations, including those associated with astrogliosis in Dravet syndrome mice, that, accordingly, were rescued by Scn1a gene expression normalization at P30. Interestingly, regaining of Nav1.1 physiological level rescued seizures also in adult Dravet syndrome mice (P90) after months of repetitive attacks. Overall, these findings represent a solid proof-of-concept highlighting that disease phenotype reversibility can be achieved when Scn1a gene activity is efficiently reconstituted in brain cells

In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy

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

Conspicuity and muscle-invasiveness assessment for bladder cancer using VI-RADS: a multi-reader, contrast-free MRI study to determine optimal b-values for diffusion-weighted imaging

To (1) compare bladder cancer (BC) muscle invasiveness among three b-values using a contrast-free approach based on Vesical Imaging-Reporting and Data System (VI-RADS), to (2) determine if muscle-invasiveness assessment is affected by the reader experience, and to (3) compare BC conspicuity among three b-values, qualitatively and quantitatively

dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice

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. Here we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Nav1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage

Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+

Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced egocytosis. Thus, the observed requirement of external Ca2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+ from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct

Temporal evolution of the microbiome, immune system and epigenome with disease progression in ALS mice

Amyotrophic lateral sclerosis (ALS) is a terminal neurodegenerative disease. Genetic predisposition, epigenetic changes, aging and accumulated life-long environmental exposures are known ALS risk factors. The complex and dynamic interplay between these pathological influences plays a role in disease onset and progression. Recently, the gut microbiome has also been implicated in ALS development. In addition, immune cell populations are differentially expanded and activated in ALS compared to healthy individuals. However, the temporal evolution of both the intestinal flora and the immune system relative to symptom onset in ALS is presently not fully understood. To better elucidate the timeline of the various potential pathological factors, we performed a longitudinal study to simultaneously assess the gut microbiome, immunophenotype and changes in ileum and brain epigenetic marks relative to motor behavior and muscle atrophy in the mutant superoxide dismutase 1 (SOD1G93A) familial ALS mouse model. We identified alterations in the gut microbial environment early in the life of SOD1G93A animals followed by motor dysfunction and muscle atrophy, and immune cell expansion and activation, particularly in the spinal cord. Global brain cytosine hydroxymethylation was also altered in SOD1G93A animals at disease end-stage compared to control mice. Correlation analysis confirmed interrelationships with the microbiome and immune system. This study serves as a starting point to more deeply comprehend the influence of gut microorganisms and the immune system on ALS onset and progression. Greater insight may help pinpoint novel biomarkers and therapeutic interventions to improve diagnosis and treatment for ALS patients. This article has an associated First Person interview with the joint first authors of the paper