Gene Therapy for Neurological Disease: State of the Art and Opportunities for Next-generation Approaches

Gene therapy for rare monogenetic neurological disorders is reaching clinics and offering hope to families affected by these diseases. There is also potential for gene therapy to offer new and effective treatments for common, non-genetic disorders. Treatments for Parkinson's Disease are in clinical trials, and treatments for refractory epilepsies are due to enter first-in-human clinical trials in 2022. Gene therapies for these disorders are based on delivering genes that address the mechanism of the disease, not repairing a mutated gene. Similar 'mechanistic' gene therapies could offer treatments to a wide range of neurological and neuropsychiatric diseases where there is a known mechanism that could be restored using gene therapy. However, the permanent nature of most gene therapies is a serious drawback for translation of gene therapies to a wide-range of diseases because it could present risk of irreversible adverse effects. Several lines of research are aimed at developing gene therapy approaches that allow for the treatment to be turned on and off, including: using proteins activated by exogenous ligands, and promoters turned on by activators. We review these approaches and propose an overall de-risking strategy for gene therapy for common neurological and psychiatric diseases. This approach is based on using a temporary mRNA-based treatment to initially assess efficacy and safety of the planned manipulation, and only following with permanent, virally-delivered treatment if the approach appears safe and effective

CNS gene therapy: present developments and emerging trends accelerating industry-academia pathways

The recent success of first central nervous system gene therapies has reinvigorated the growing community of gene therapy researchers and strengthened the field's market position. We are witnessing an increase of clinical trials with long-term efficiency mainly for neurometabolic, neurodegenerative and neurodevelopmental diseases caused by loss-of-function mutations. The ever-expanding knowledge and accessibility to the most advanced tools allow enrichment of applications to more complex diseases. This gradually contributes towards sealing the gap between top diseases impacting current global health and those towards which gene therapy development is currently aimed. Here, we highlight innovative therapeutic approaches that have reached the clinics and outline the latest improvements of vector design and targeting. Finally, we address the pressing challenges faced by clinical trials and the direction they are heading

Dysfunction of the CaV2.1 calcium channel in cerebellar ataxias

Mutations in the CACNA1A gene are associated with episodic ataxia type 2 (EA2) and spinocerebellar ataxia type 6 (SCA6). CACNA1A encodes the α-subunit of the P/Q-type calcium channel or CaV2.1, which is highly enriched in the cerebellum. It is one of the main channels linked to synaptic transmission throughout the human central nervous system. Here, we compare recent advances in the understanding of the genetic changes that underlie EA2 and SCA6 and what these new findings suggest about the mechanism of the disease

BICS01 Mediates Reversible Anti-seizure Effects in Brain Slice Models of Epilepsy

Drug-resistant epilepsy remains a significant clinical and societal burden, with one third of people with epilepsy continuing to experience seizures despite the availability of around 30 anti-seizure drugs (ASDs). Further, ASDs often have substantial adverse effects, including impacts on learning and memory. Therefore, it is important to develop new ASDs, which may be more potent or better tolerated. Here, we report the preliminary preclinical evaluation of BICS01, a synthetic product based on a natural compound, as a potential ASD. To model seizure-like activity in vitro, we prepared hippocampal slices from adult male Sprague Dawley rats, and elicited epileptiform bursting using high extracellular potassium. BICS01 (200 μM) rapidly and reversibly reduced the frequency of epileptiform bursting but did not change broad measures of network excitability or affect short-term synaptic facilitation. BICS01 was well tolerated following systemic injection at up to 1,000 mg/kg. However, we did not observe any protective effect of systemic BICS01 injection against acute seizures evoked by pentylenetetrazol. These results indicate that BICS01 is able to acutely reduce epileptiform activity in hippocampal networks. Further preclinical development studies to enhance pharmacokinetics and accumulation in the brain, as well as studies to understand the mechanism of action, are now required

Gene variant effects across sodium channelopathies predict function and guide precision therapy

Pathogenic variants in the voltage-gated sodium channel gene family lead to early onset epilepsies, neurodevelopmental disorders, skeletal muscle channelopathies, peripheral neuropathies and cardiac arrhythmias. Disease-associated variants have diverse functional effects ranging from complete loss-of-function to marked gain-of-function. Therapeutic strategy is likely to depend on functional effect. Experimental studies offer important insights into channel function but are resource intensive and only performed in a minority of cases. Given the evolutionarily conserved nature of the sodium channel genes, we investigated whether similarities in biophysical properties between different voltage-gated sodium channels can predict function and inform precision treatment across sodium channelopathies. We performed a systematic literature search identifying functionally assessed variants in any of the nine voltage-gated sodium channel genes until 28 April 2021. We included missense variants that had been electrophysiologically characterized in mammalian cells in whole-cell patch-clamp recordings. We performed an alignment of linear protein sequences of all sodium channel genes and correlated variants by their overall functional effect on biophysical properties. Of 951 identified records, 437 sodium channel-variants met our inclusion criteria and were reviewed for functional properties. Of these, 141 variants were epilepsy-associated (SCN1/2/3/8A), 79 had a neuromuscular phenotype (SCN4/9/10/11A), 149 were associated with a cardiac phenotype (SCN5/10A) and 68 (16%) were considered benign. We detected 38 missense variant pairs with an identical disease-associated variant in a different sodium channel gene. Thirty-five out of 38 of those pairs resulted in similar functional consequences, indicating up to 92% biophysical agreement between corresponding sodium channel variants (odds ratio = 11.3; 95% confidence interval = 2.8 to 66.9; P < 0.001). Pathogenic missense variants were clustered in specific functional domains, whereas population variants were significantly more frequent across non-conserved domains (odds ratio = 18.6; 95% confidence interval = 10.9-34.4; P < 0.001). Pore-loop regions were frequently associated with loss-of-function variants, whereas inactivation sites were associated with gain-of-function (odds ratio = 42.1, 95% confidence interval = 14.5-122.4; P < 0.001), whilst variants occurring in voltage-sensing regions comprised a range of gain- and loss-of-function effects. Our findings suggest that biophysical characterisation of variants in one SCN-gene can predict channel function across different SCN-genes where experimental data are not available. The collected data represent the first gain- versus loss-of-function topological map of SCN proteins indicating shared patterns of biophysical effects aiding variant analysis and guiding precision therapy. We integrated our findings into a free online webtool to facilitate functional sodium channel gene variant interpretation (http://SCN-viewer.broadinstitute.org).Peer reviewe

On-demand cell-autonomous gene therapy for brain circuit disorders

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 or intermingled neurons. We describe a gene therapy strategy that down-regulates the excitability of overactive neurons in closed loop, which we tested 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 antiepileptic effect without interfering with normal behaviors. Activity-dependent gene therapy is a promising on-demand cell-autonomous treatment for brain circuit disorders

Altered hippocampal-prefrontal neural dynamics in mouse models of Down syndrome

Altered neural dynamics in the medial prefrontal cortex (mPFC) and hippocampus may contribute to cognitive impairments in the complex chromosomal disorder Down syndrome (DS). Here, we demonstrate non-overlapping behavioral differences associated with distinct abnormalities in hippocampal and mPFC electrophysiology during a canonical spatial working memory task in three partially trisomic mouse models of DS (Dp1Tyb, Dp10Yey, and Dp17Yey) that together cover all regions of homology with human chromosome 21 (Hsa21). Dp1Tyb mice show slower decision-making (unrelated to the gene dose of DYRK1A, which has been implicated in DS cognitive dysfunction) and altered theta dynamics (reduced frequency, increased hippocampal-mPFC coherence, and increased modulation of hippocampal high gamma); Dp10Yey mice show impaired alternation performance and reduced theta modulation of hippocampal low gamma; and Dp17Yey mice are not significantly different from the wild type. These results link specific hippocampal and mPFC circuit dysfunctions to cognitive deficits in DS models and, importantly, map them to discrete regions of Hsa21

A systems approach delivers a functional microRNA catalog and expanded targets for seizure suppression in temporal lobe epilepsy

Temporal lobe epilepsy is the most common drug-resistant form of epilepsy in adults. The reorganization of neural networks and the gene expression landscape underlying pathophysiologic network behavior in brain structures such as the hippocampus has been suggested to be controlled, in part, by microRNAs. To systematically assess their significance, we sequenced Argonaute-loaded microRNAs to define functionally engaged microRNAs in the hippocampus of three different animal models in two species and at six time points between the initial precipitating insult through to the establishment of chronic epilepsy. We then selected commonly up-regulated microRNAs for a functional in vivo therapeutic screen using oligonucleotide inhibitors. Argonaute sequencing generated 1.44 billion small RNA reads of which up to 82% were microRNAs, with over 400 unique microRNAs detected per model. Approximately half of the detected microRNAs were dysregulated in each epilepsy model. We prioritized commonly up-regulated microRNAs that were fully conserved in humans and designed custom antisense oligonucleotides for these candidate targets. Antiseizure phenotypes were observed upon knockdown of miR-10a-5p, miR-21a-5p, and miR-142a-5p and electrophysiological analyses indicated broad safety of this approach. Combined inhibition of these three microRNAs reduced spontaneous seizures in epileptic mice. Proteomic data, RNA sequencing, and pathway analysis on predicted and validated targets of these microRNAs implicated derepressed TGF-\u3b2 signaling as a shared seizure-modifying mechanism. Correspondingly, inhibition of TGF-\u3b2 signaling occluded the antiseizure effects of the antagomirs. Together, these results identify shared, dysregulated, and functionally active microRNAs during the pathogenesis of epilepsy which represent therapeutic antiseizure targets