Objective
Epilepsy treatment falls short in ~30% of cases. A better understanding of epilepsy pathophysiology can guide rational drug development in this difficult to treat condition. We tested a low‐cost, drug‐repositioning strategy to identify candidate epilepsy drugs that are already
FDA
‐approved and might be immediately tested in epilepsy patients who require new therapies.
Methods
Biopsies of spiking and nonspiking hippocampal brain tissue from six patients with unilateral mesial temporal lobe epilepsy were analyzed by
RNA
‐Seq. These profiles were correlated with transcriptomes from cell lines treated with
FDA
‐approved drugs, identifying compounds which were tested for therapeutic efficacy in a zebrafish seizure assay.
Results
In spiking versus nonspiking biopsies,
RNA
‐Seq identified 689 differentially expressed genes, 148 of which were previously cited in articles mentioning seizures or epilepsy. Differentially expressed genes were highly enriched for protein–protein interactions and formed three clusters with associated
GO
‐terms including myelination, protein ubiquitination, and neuronal migration. Among the 184 compounds, a zebrafish seizure model tested the therapeutic efficacy of doxycycline, metformin, nifedipine, and pyrantel tartrate, with metformin, nifedipine, and pyrantel tartrate all showing efficacy.
Interpretation
This proof‐of‐principle analysis suggests our powerful, rapid, cost‐effective approach can likely be applied to other hard‐to‐treat diseases.
Objective
A significant number of epileptic patients fail to respond to available anticonvulsive medications. To find new anticonvulsive medications, we evaluated FDA‐approved drugs not known to be anticonvulsants. Using zebrafish larvae as an initial model system, we found that the opioid antagonist naltrexone exhibited an anticonvulsant effect. We validated this effect in three other epilepsy models and present naltrexone as a promising anticonvulsive candidate.
Methods
Candidate anticonvulsant drugs, determined by our prior transcriptomics analysis of hippocampal tissue, were evaluated in a larval zebrafish model of human Dravet syndrome (scn1Lab mutants), in wild‐type zebrafish larvae treated with the pro‐convulsant drug pentylenetetrazole (PTZ), in wild‐type C57bl/6J acute brain slices exposed to PTZ, and in wild‐type mice treated with PTZ in vivo. Abnormal locomotion was determined behaviorally in zebrafish and mice and by field potential in neocortex layer IV/V and CA1 stratum pyramidale in the hippocampus.
Results
The opioid antagonist naltrexone decreased abnormal locomotion in the larval zebrafish model of human Dravet syndrome (scn1Lab mutants) and wild‐type larvae treated with the pro‐convulsant drug PTZ. Naltrexone also decreased seizure‐like events in acute brain slices of wild‐type mice, and the duration and number of seizures in adult mice injected with PTZ.
Significance
Our data reveal that naltrexone has anticonvulsive properties and is a candidate drug for seizure treatment.
CRISPR systems enable targeted genome editing in a wide variety of organisms by introducing single- or double-strand DNA breaks, which are repaired using endogenous molecular pathways. Characterization of on- and off-target editing events from CRISPR proteins can be evaluated using targeted genome resequencing. We characterized DNA repair fingerprints that result from non-homologous end joining (NHEJ) after double-stranded breaks (DSBs) were introduced by Cas9 or Cas12a for >500 paired treatment/control experiments. We found that building biological understanding of the repair into a novel analysis tool (CRISPAltRations) improved the quality of the results. We validated our software using simulated, targeted amplicon sequencing data (11 guide RNAs [gRNAs] and 603 on- and off-target locations) and demonstrated that CRISPAltRations outperforms other publicly available software tools in accurately annotating CRISPR-associated indels and homology-directed repair (HDR) events. We enable non-bioinformaticians to use CRISPAltRations by developing a web-accessible, cloud-hosted deployment, which allows rapid batch processing of samples in a graphical user interface (GUI) and complies with HIPAA security standards. By ensuring that our software is thoroughly tested, version controlled, and supported with a user interface (UI), we enable resequencing analysis of CRISPR genome editing experiments to researchers no matter their skill in bioinformatics.
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