✍🏼 Colin D. Robertson, Patrick Davis, Ryan R. Richardson, Philip H. Iffland II, Daiana C. O. Vieira, Marilyn Steyert, Paige N. McKeon, Andrea J. Romanowski, Garrett Crutcher, Eldin Jašarević, Steffen B. E. Wolff, Brian N. Mathur, Peter B. Crino, Tracy L. Bale, Ivy E. Dick, Alexandros Poulopoulos

🏠 Department of Pharmacology and UM-MIND, University of Maryland School of Medicine, Baltimore, MD, USA

📑 bioRxiv (2024)

Abstract
Generating animal models that mirror a patient’s seizures within clinically-useful timeframes is an important step toward advancing precision medicine for genetic epilepsies. Here we report a somatic cell genome editing approach that rapidly incorporated a patient’s genomic variant into mice, which developed seizures recapitulating elements of the patient’s pathology. This approach offers a versatile in vivo platform for clinical, preclinical, and basic research applications, including tailoring pharmacotherapy, assessing variants of uncertain significance, and screening compounds to develop drugs for rare epilepsies. As proof-of-principle, we modeled an epilepsy patient with an ultra-rare variant of the NMDA receptor subunit GRIN2A using prime editing in utero directly in the developing brain of wild-type mice. This methodology achieved high-fidelity genome editing in vivo sufficient to induce frequent spontaneous seizures without necessitating germline modification or extensive breeding. Leveraging the speed and versatility of this approach, we propose a generalizable workflow to generate bedside-to-bench animal models of individual patients within weeks. This advance holds promise for providing a cost-effective, expedient in vivo testing platform that reduces barriers to access for precision medicine, and accelerates drug development for rare and neglected neurological conditions.

How the WOLF was used in this study
The WOLF  cell sorter was used to isolate genetically modified neurons following in utero electroporation for downstream sequencing analysis. After electroporated brain regions were dissected from mouse cortex and enzymatically dissociated into single-cell suspensions, cells were sorted at low flow rates on the WOLF using red fluorescence from the myr-tdTomato reporter to specifically gate electroporated neurons, with gating confirmed by overlap with green fluorescence from the co-expressed EGFP-2A-PE2 prime editor. Approximately 3,000 fluorescent neurons were collected per sample, ensuring enrichment of successfully edited cells while preserving cell integrity. This gentle microfluidic sorting step enabled the isolation of a pure population of targeted neurons for RNA extraction and next-generation sequencing, allowing accurate quantification of prime editing outcomes in vivo.