Approaching PCDH19 clustering epilepsy in vitro: CRISPR/Cas9-mediated gene editing and characterization of patient-derived hiPSC-neurons

Loss of function mutations in PCDH19 gene cause an X-linked, infant-onset clustering epilepsy, associated with intellectual disability and autistic features. The unique pattern of inheritance includes random X-chromosome inactivation, which leads to pathological tissue mosaicism. Females carrying PCDH19 mutations are affected, while males have normal phenotype. No cure is presently available for this disease. We obtained heterozygous cells from the affected patient and performed transfection experiments creating two double-strand breaks and deleting the intervening DNA segment of PCDH19 gene. We generated a CRISPR/Cas9 mediated knockout of the PCDH19 gene in patient-derived cells. Targeting different parts of the PCDH19 gene, we obtained 72,9% of editing efficiency using the AAV-based vector with sgRNAs flanking the ends of the first exon, and with sgRNAs flanking the ends of the gene – 48,5%, employing a lentivirus-based vector. Fibroblasts from a female patient carrying frameshift mutation c.2133delG were reprogrammed into human induced pluripotent stem cells (hiPSC). We created a cell model of PCDH19-clustering epilepsy (PCDH19-CE) where both cell populations co-exist, by mixing wild-type and mutated human iPSC clones, and differentiated them into mature neurons with overexpression of the transcriptional factor Neurogenin 2. We set up the conditions for neuronal differentiation of hiPSC using an overexpression with transcriptional factor Neurogenin 2. With a preliminary electrophysiological experiment we observed that PCDH19 mosaic neurons tend to have an elevated excitability, representing the situation in PCDH19-CE brain. We confirmed and completed existing data showing that PCDH19 mutation c.2133delG affects proper metaphases and leads to an increased number of centrosomes in stem cells. In conclusion, we provide an efficient and reproducible strategy of CRISPR/Cas9 targeted PCDH19 knockout and demonstrate that this is a promising therapeutic tool for the treatment of PCDH19-CE. We suggest Ngn-2 hiPSC-derived PCDH19 neurons as an informative, efficient, and reproducible experimental tool for understanding the pathogenesis of PCDH19-CE and a suitable approach for use in targeted drug screening strategies