Abstract
CDKL5 deficiency disorder (CDD) is a rare X-linked epileptic encephalopathy caused by de novo mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene located on the X chromosome (Xp22). Loss of function mutations in CDKL5 lead to CDD and is clinically defined by early onset of seizures, developmental delay, and intellectual disability. To date, there is still limited understanding of how quasi-haploinsufficiency of CDKL5 affects brain function. Females have a mosaic of neurons that randomly express either the maternal or paternal X chromosome for dosage compensation through X Chromosome Inactivation (XCI). Reactivation of the inactive X-chromosome containing the silenced, healthy CDKL5 in neurons has become an attractive therapeutic strategy. However, due to males carrying only one copy of CDKL5 gene, patients with a hemizygous CDKL5 variants would not be candidates for this approach. The first goal of this project was to model a CDKL5 variant in human induced pluripotent stem cells that would mimic the genetic cause of the disorder. As a proof-of-concept, we modeled CDD in an easy to transfect cell, HEK293T. CRISPR/Cas9-mediated Homology-Direct Repair (HDR) was used to insert the CDKL5 variant (Arg178Gln). This variant has been reported to be pathogenic and common in CDD patients. CDD-HEK293T cells and their isogenic clones were isolated through limiting dilutions. Kompetitive Allele-Specific PCR (KASP) was used to identify HEK293T clones that contained the CDD variant. KASP uses a common reverse primer and two allele-specific forward primers, one targeting Arg-178 (nucleotide 533-G) and the other targeting the variant Gln-178 (nucleotide 533-A). CDD variant lines were called following cluster analysis. Though we did not find the predicted HEK293T 178G clone, we have created pools of insertions and deletions in the catalytic domain of CDKL5 that resulted in decreased CDKL5 expression. The second goal of this project was to generate a CDKL5 isoform 1 lentivirus (CDKL_1LV) to restore CDKL5 via traditional gene therapy.CDD-HEK293T cells were used to assess the restoration of CDKL5 in vitro following transduction with CDKL5_1LV. With additional optimization, our CDD-models and gene therapy approach could potentially contribute to the future of CDD therapeutic treatments.