CRISPR/Cas9-based targeted genome editing for the treatment of CDKL5 deficiency disorder

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause CDKL5 deficiency disorder (CDD), which is a rare neurodevelopmental disease characterized by severe epilepsy and global developmental delay. Most children affected suffer from seizures beginning in the first months of life and severe impairment of cognitive and motor skills, with great impact on their quality of life. Most cannot walk, talk, or feed themselves, and many are confined to using a wheelchair. Although rare, CDKL5 deficiency disorder is one of the most common forms of genetic epilepsy. Currently, there is no cure or effective treatment for CDD, hence the great urge to develop novel and effective therapeutic strategies. Here, we present a methodology for the correction of a pathogenic variant in CDKL5 (c.1090G>T (p.Glu364*)), using CRISPR/Cas9 genome editing technology in patient-derived cell models, in order to expedite the discovery of new therapies for CDD. CRISPR/Cas9 is a precise and versatile method of genetic manipulation, and it only requires three components to target and correct genetic mutations: guide RNAs, Cas9 endonuclease, and homology-directed repair (HDR) templates. We first tested plasmid-based delivery of CRISPR/Cas9 for correction in primary fibroblasts. This system proved to be up to 66% efficient but it was associated with extremely variable and unpredictable editing efficiency (33±31%) in three separate experiments. Hence, we decided to test additional guides and to replace the plasmid-based system with a protein-based ribonucleoprotein (RNP) delivery system for more rapid action and greater stability. We tested the system in induced pluripotent stem cells (iPSCs) obtained by reprogramming the patient’s fibroblasts. We reported the generation of genetically corrected iPSCs, where the mutated CDKL5 c.1090G>T (p.Glu364*) was corrected to the wild-type, using RNP-mediated delivery of CRISPR/Cas9. Based on PCR cloning results of gene-corrected clones, we can state that our system is able to selectively target the p.Glu364* variant while preserving the wild-type CDKL5 allele in vitro. We then differentiated in parallel mutant and isogenic sets of cells into neural cells to assess the functional consequences of the edit in the affected cell type. We demonstrated that CRISPR/Cas9 gene editing restores the expression of CDKL5 protein in iPSC-derived neurons by Western blot. We also showed by RT-qPCR that mutant neurons carrying the c.1090G>T (p.Glu364*) in CDKL5 pre-sent reduced expression of CDKL5 mRNA compared to isogenic control. Our findings demonstrate that we can achieve targeted and allele-specific correction of CDKL5 (c.1090G>T (p.Glu364*)) variant using CRISPR/Cas9-RNP system in a patient-specific cell model. Moreover, we proved that correction of the mutation at the DNA level rescues CDKL5 protein expression and increases CDKL5 mRNA expression in isogenic neurons. The results of this study might be decisive in proving CRISPR/Cas9 potential to carry out genome editing in human cells, and ultimately for developing advanced therapies for CDD.