Dravet syndrome is caused by dominant loss-of-function mutations in SCN1A which cause reduced activity of Nav1.1 leading to lack of neuronal inhibition. On the other hand, gain-of-function mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating Na_V1.6 channels. These observations suggest that Nav1.1 and Nav1.6 represent two opposing sides of the neuronal balance between inhibition and activation. Here, we hypothesize that Dravet syndrome may be treated by either enhancing Nav1.1 or reducing Nav1.6 activity. To test this hypothesis we generated and characterized a novel DS zebrafish model and tested new compounds that selectively activate or inhibit the human Na_V1.1 or Na_V1.6 channel respectively. We used CRISPR/Cas9 to generate two separate Scn1Lab knockout lines as an alternative to previous zebrafish models generated by random mutagenesis or morpholino oligomers. Using an optimized locomotor assay, spontaneous burst movements were detected that were unique to Scn1Lab knockouts and disappear when introducing human SCN1A mRNA. Besides the behavioral phenotype, Scn1Lab knockouts show sudden, electrical discharges in the brain that indicate epileptic seizures in zebrafish. Scn1Lab knockouts showed increased sensitivity to the GABA antagonist pentylenetetrazole and a reduction in whole organism GABA levels. Drug screenings further validated a Dravet syndrome phenotype. We tested the Na_V1.1 activator AA43279 and two novel Na_V1.6 inhibitors MV1369 and MV1312 in the Scn1Lab knockouts. Both type of compounds significantly reduced the number of spontaneous burst movements and seizure activity. Our results show that selective inhibition of Na_V1.6 could be just as efficient as selective activation of Na_V1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish however, should always be further validated in other model systems for efficacy in mammals and to screen for potential side effects.

Novel genome editing and transient gene therapies have been developed the past ten years, resulting in the first in-human clinical trials for monogenic disorders. Syndromic autism spectrum disorders can be caused by mutations in a single gene. Given the monogenic aspect and severity of syndromic ASD, it is an ideal candidate for gene therapies. Here, we selected 11 monogenic ASD syndromes, validated by animal models, and reviewed current gene therapies for each syndrome. Given the wide variety and novelty of some forms of gene therapy, the best possible option must be decided based on the gene and mutation.

The heterozygous Phospholamban p.Arg14del mutation is found in patients with dilated or arrhythmogenic cardiomyopathy. This mutation triggers cardiac contractile dysfunction and arrhythmogenesis by affecting intracellular Ca²⁺ dynamics. Little is known about the physiological processes preceding induced cardiomyopathy, which is characterized by sub-epicardial accumulation of fibrofatty tissue, and a specific drug treatment is currently lacking. Here, we address these issues using a knock-in Phospholamban p.Arg14del zebrafish model. Hearts from adult zebrafish with this mutation display age-related remodeling with sub-epicardial inflammation and fibrosis. Echocardiography reveals contractile variations before overt structural changes occur, which correlates at the cellular level with action potential duration alternans. These functional alterations are preceded by diminished Ca²⁺ transient amplitudes in embryonic hearts as well as an increase in diastolic Ca²⁺ level, slower Ca²⁺ transient decay and longer Ca²⁺ transients in cells of adult hearts. We find that istaroxime treatment ameliorates the in vivo Ca²⁺ dysregulation, rescues the cellular action potential duration alternans, while it improves cardiac relaxation. Thus, we present insight into the pathophysiology of Phospholamban p.Arg14del cardiomyopathy.

Dravet syndrome (DS) is a monogenic epileptic encephalopathy caused by loss-of-function mutations in the voltage-gated sodium channel (VGSC) gene SCN1A . DS has an age of onset within the first year of life and severe disease prognosis. In the past years, it has been shown that upregulation of endogenous SCN1A can be beneficial in animal models for DS, but a complete rescue was not observed. We hypothesized that upregulation during early development that precedes onset of first symptoms might improve disease outcome. To test this hypothesis, we first evaluated the CRISPR activating method for early upregulation of voltage gated sodium channels during early development. We injected CRISPRa components, which target the proximal or distal promoter region of the VGSC gene scn1Laa in the yolk of one-cell stage zebrafish embryos. The effect of both dCas9–VPR and dCas9–VP64 was evaluated. Both CRISPRa fusions showed toxicity in the majority of embryos, with or without guide RNAs. The few embryos that survived developed normally, and dCas9-VPR induces an upregulation of scn1Laa mRNA until 24 hours after fertilization. At 5 days post fertilization, CRISPRa-injected embryos showed an epileptic phenotype, including locomotor burst movements, hyperactivity, and epileptiform activity originating from the brain. In addition to previously published scn1Laa and scn1Lab loss-of-function models, we conclude that gain of scn1Laa function can have an equally severe phenotype. Upregulation of scn1Laa in the current zebrafish model for DS, scn1Lab -KO, aggravated the disease phenotype, highlighting that early-stage upregulation using CRISPRa can lead to both toxicity and a worsening of the disease phenotype.

Background: Pathogenic variants in SCN1A cause variable epilepsy disorders with different disease severities. We here investigate whether common variation in the promoter region of the unaffected SCN1A allele could reduce normal expression, leading to a decreased residual function of Nav1.1, and therefore to more severe clinical outcomes in patients affected by pathogenic SCN1A variants.Methods: Five different SCN1A promoter‐haplotypes were functionally assessed in SH‐SY5Y cells using Firefly and Renilla luciferase assays. The SCN1A promoter region was analyzed in a cohort of 143 participants with SCN1A pathogenic variants. Differences in clinical features and outcomes between participants with and without common variants in the SCN1A promoter‐region of their unaffected allele were investigated.Results: All non‐wildtype haplotypes showed a significant reduction in luciferase expression, compared to the wildtype promoter‐region (65%–80%, p = 0.039–0.0023). No statistically significant differences in clinical outcomes were observed between patients with and without common promoter variants. However, patients with a wildtype promoter‐haplotype on their unaffected SCN1A allele showed a nonsignificant trend for milder phenotypes.Conclusion: The nonsignificant observed trends in our study warrant replication studies in larger cohorts to explore the potential modifying role of these common SCN1A promoter‐haplotypes.

Background: Prime editing (PE) is the most recent gene editing technology able to introduce targeted alterations to the genome, including single base pair changes, small insertions, and deletions. Several improvements to the PE machinery have been made in the past few years, and these have been tested in a range of model systems including immortalized cell lines, stem cells, and animal models. While double nicking RNA (dncRNA) PE systems PE3 and PE5 currently show the highest editing rates, they come with reduced accuracy as undesired indels or SNVs arise at edited loci. Here, we aimed to improve single ncRNA (sncRNA) systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids driven by an EF-1α promoter, which is especially suitable for human-induced pluripotent stem cell (hiPSC) models.Results: pAIO-EF1α-PE2 and pAIO-EF1α-PE4max were used to edit the voltage gated potassium channel gene KCNQ2 and voltage gated sodium channel gene SCN1A. Two clinically relevant mutations were corrected using pAIO-EF1α-PE2 including the homozygous truncating SCN1A R612* variant in HEK293T cells and the heterozygous gain-of-function KCNQ2 R201C variant in patient-derived hiPSC. We show that sncRNA PE yielded detectable editing rates in hiPSC ranging between 6.4% and 9.8%, which was further increased to 41% after a GFP-based fluorescence-activated cell sorting (FACS) cell sorting step. Furthermore, we show that selecting the high GFP expressing population improved editing efficiencies up to 3.2-fold compared to the low GFP expressing population, demonstrating that not only delivery but also the number of copies of the PE enzyme and/or pegRNA per cell are important for efficient editing. Edit rates were not improved when an additional silent protospacer-adjacent motif (PAM)-removing alteration was introduced in hiPSC at the target locus. Finally, there were no genome-wide off-target effects using pAIO-EF1α-PE2 and no off-target editing activity near the edit locus highlighting the accuracy of snc prime editors.Conclusion: Taken together, our study shows an improved efficacy of EF-1α driven sncRNA pAIO-PE plasmids in hiPSC reaching high editing rates, especially after FACS sorting. Optimizing these sncRNA PE systems is of high value when considering future therapeutic in vivo use, where accuracy will be extremely important.