Epigenetic Reactivation of CDKL5 Rescues CDKL5 Deficiency Disorder

Abstract CDKL5 deficiency disorder (CDD) is a severe X-linked neurodevelopmental condition characterized by early-onset epilepsy, intellectual disability, and motor dysfunction. Here, we present a dual-effector CRISPR-based epigenome editing platform that enables targeted reactivation of the silenced CDKL5 allele. Using a split dCas9 system compatible with AAV9 delivery, we achieved simultaneous transcriptional activation and DNA demethylation of the CDKL5 promoter in both murine and human models of disease. In heterozygous Cdkl5 E6del mice, intracerebroventricular delivery of the editor restored Cdkl5 protein expression, re-engaged downstream signaling, and rescued motor and cognitive deficits. In patient-derived neural stem cells and cortical organoids, CDKL5 reactivation normalized gene expression and restored neuronal network activity. These findings establish a scalable, mutation-independent strategy for treating CDD and highlight the therapeutic potential of epigenetic reprogramming for X-linked disorders.