HEMATOPOIETIC STEM CELL–MEDIATED DELIVERY OF A SECRETABLE CDKL5 PROTEIN RESCUES NEUROLOGICAL DEFICITS IN CDKL5 DEFICIENCY DISORDER

CDKL5 Deficiency Disorder (CDD) is a severe X-linked neurodevelopmental disorder caused by loss of CDKL5 kinase activity and characterized by early-onset epilepsy, cognitive impairment, and autistic-like behaviors. Current treatments are purely symptomatic, while conventional gene therapy approaches are limited by inefficient and heterogeneous protein distribution within the central nervous system (CNS). In addition, the lack of reliable biomarkers hampers objective evaluation of therapeutic efficacy. Here, we developed an innovative hematopoietic stem cell gene therapy (HSC-GT) strategy using a lentiviral vector (LV) encoding a secretable, cell-penetrating CDKL5 protein (Igκ-TATk-CDKL5), designed to promote cross-correction of neighboring brain cells. In vitro analyses demonstrated that Igκ-TATk-CDKL5 retains CDKL5 kinase activity and undergoes efficient secretion and cellular uptake. In vivo, transplantation of HSCs transduced with LV–Igκ-TATk-CDKL5 was well tolerated in Cdkl5 knockout (KO) mice and resulted in stable engraftment of donor-derived microglia-like cells expressing the therapeutic protein in the brain. Behavioral analyses showed significant improvements in disease-relevant phenotypes, including reduced repetitive behavior, decreased hindlimb clasping, and rescue of associative memory deficits. Golgi staining of the cortex of HSC-GT–treated Cdkl5 KO mice revealed normalization of dendritic spine density and maturation comparable to wild-type levels. Exploratory analyses of extracellular vesicle–associated miRNAs revealed genotype-dependent differences, suggesting potential biomarkers for therapy monitoring. Overall, these findings demonstrate that HSC-mediated delivery of a secretable CDKL5 protein enables widespread CNS correction, restores synaptic organization, and rescues cognitive and behavioral deficits in a mouse model of CDD, supporting the translational potential of HSC-based gene therapy for neurodevelopmental disorders.