Authors :
Presenting Author: Morgan Robinson, PhD – Purdue University
Maria Olivero-acosta, PhD – Purdue University
Kyle Wettschurack, BS, MEng – Purdue University
Zhefu Que, PhD – Purdue University
Manasi Halurkar, BS – Purdue University
Jingliang Zhang, PhD – Purdue University
Xiaoling Chen, PhD – Purdue University
Ye-Eun yoo, PhD – Purdue University
Chongli Yuan, PhD – Purdue University
Yang Yang, PhD – Purdue University
Rationale: Variants in the SCN2A gene are a leading cause of epilepsy and autism. The SCN2A gene encodes the voltage-gated alpha subunit II (Nav1.2), which is critical for the initiation, propagation and back-propagation of action potentials. The recurrent SCN2A-L1342P variant is a confirmed cause of DEE and has been identified in 6 patients worldwide. This variant is associated with early-onset intractable seizures (beginning at 4 to 6 months of age), progressive brain atrophy, and early childhood mortality. There is an urgent need to develop new therapies to treat monogenic brain disorders, especially the most severe cases that are often treatment resistant and have high infant mortality. Prime Editing, a new generation of CRISPR-based gene editing systems developed in the past five years, offers a safer alternative to traditional CRISPR. Traditional CRISPR relies on generating double-stranded DNA breaks, whereas Prime Editing introduces only a single stranded nick in the DNA, which improves its safety profile for therapeutic use. Meanwhile, human pluripotent stem cells (iPSCs) enable researchers to test formulations and editing directly on cells carrying disease-causing gene variants.
Methods: Previously, our lab established disease phenotypes for the epilepsy-related SCN2A-L1342P model in two-dimensional iPSC-derived neurons. Here we extend our findings to three-dimensional cortical brain organoids applying, patch clamp current clamp electrophysiology and Transcriptomic analyses. We also applied prime editing to edit the SCN2A-L1342P mutation site using nucleofection of KOLF2 iPSCs, we report next generation sequencing analysis of several prime editing systems with variable editing efficiencies.
Results: Our results indicate that L1342P brain organoids exhibit pronounced hyperexcitability at the intrinsic and network level. Bulk RNA sequencing and transcriptomics results suggest that mechanisms related to delayed neurodevelopment, synaptic neurotransmission as well as senescence and apoptosis are involved in the underlying mechanisms of disease. Prime Editing of iPSCs using nucleofection displayed a range of editing efficiencies from 20-67% depending on the specific design, location of the guide RNAs, and the presence of mismatch repair inhibition.
Conclusions: This work establishes disease-related phenotypes in 3D brain organoids, and establishes that our Prime Editing designs are effective at editing the L1342P mutation. In future work, we aim to establish that gene correction will restore neuronal function, unlocking a path toward curative therapies for monogenic brain disorders.
Funding:
The authors gratefully acknowledge support from the FamilieSCN2A Foundation for Hodgkin-Huxley Research Grant, and PIDD and PIIN for additional funding support, as well as the Purdue Lillian Gilbreth Engineering Postdoctoral Fellowship to Morgan Robinson. The research reported in this study was supported by the NINDS of the NIH (R01NS117585 and R01NS123154 to Yang Yang).