2026-06-10 | Astroglial Dysfunction in Models of CDKL5 Deficiency Disorder
Abstract CDKL5 Deficiency Disorder (CDD) is a rare developmental epileptic encephalopathy typically caused by loss of function variants in the gene encoding the X-linked serine-threonine kinase CDKL5. CDKL5 is highly expressed in the brain during development, and key neuronal functions of the kinase include cytoskeletal organisation and synaptic stability. However, at present, little is known about the function of astroglia in CDD. Given the importance of these cells in synaptic development and homeostasis, as well as dysfunction in other epileptic diseases, it was hypothesised that astrocytes may contribute to CDD pathology. Induced pluripotent stem cells harbouring a CDKL5 loss-of-function mutation (and isogenic controls) were derived from CDD patient fibroblasts and differentiated into astrocytes (iAstros). Analysis of iAstros revealed transcriptomic, proteomic and functional dysregulation in CDKL5-mutant iAstros relating to water transport and immunological function, including a diminished response to TNFα stimulation. Moreover, iAstros showed increased branching and reduced phosphorylation of the known CDKL5 target end-binding protein 2 (EB2) - indicative of disrupted cytoskeletal regulation in a manner similar to CDKL5-null neurons. Finally, we report the generation of novel in vitro models of CDD. CDKL5 was knocked down in adult and foetal human organotypic brain slices through transduction with an AAV encoding a novel CDKL5 shRNA. Slices transduced with the CDKL5 shRNA displayed increased spontaneous network activity, demonstrating the functionality of this model. Importantly, interrogation of these models revealed dysregulation of key astrocytic proteins congruous with the human glial stem cell model. Consequently, this study describes the generation of novel human models of CDD and their associated astrocytic dysfunction – paving the way for novel discovery and therapeutic intervention.
Open article ↗
2026-05-26 | From Sea to Therapy: Development and Analytical Control of Recombinant Human CDKL5 Production in the Marine Bacterium Pseudoalteromonas haloplanktis TAC125.
Marine bacteria are increasingly explored as alternative microbial platforms for the production of high-value biopharmaceuticals. In this study, we investigate the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125), an unconventional host capable of yielding soluble and biologically active human cyclin-dependent kinase-like 5 (hCDKL5). This serine/threonine kinase plays a crucial role in neuronal development, and its deficiency causes CDKL5 Deficiency Disorder, a severe and currently untreatable neurodevelopmental disease. Recombinant production of hCDKL5 is a prerequisite for the development of enzyme replacement therapy; however, current manufacturing processes remain insufficient for industrial translation, particularly in terms of product quality and functional consistency. To address these limitations, we developed dedicated analytical strategies: protein accumulation was quantified using a customised sandwich Enzyme-Linked Immunosorbent Assay (ELISA) designed to selectively detect full-length hCDKL5, while protein functionality was assessed by mass spectrometry-based quantification of autophosphorylation, a critical determinant of kinase activation. These complementary tools were applied to characterise hCDKL5 production under different growth conditions. Overall, this work establishes an integrated analytical framework aligned with a Quality by Design approach, enabling the simultaneous assessment of yield, structural integrity, and functional activation, and providing a robust basis for rational process optimisation towards scalable hCDKL5 manufacturing.
Open article ↗
2026-04-11 | Base editing restores CDKL5 expression and rescues neuronal deficits in a patient-derived model of CDKL5 deficiency disorder.
Cyclin-dependent kinase like 5 (CDKL5) deficiency disorder (CDD) is a rare monogenic neurodevelopmental disorder caused by pathogenic mutations in the CDKL5 gene, with approximately 50% of reported variants being point mutations. Base editing presents a promising therapeutic strategy to correct such mutations, restore endogenous CDKL5 expression, and pave the way for novel treatments for CDD. To assess the therapeutic potential of base editing for CDD, we applied adenine base editing (ABE) to correct a CDKL5-R550* (c.1648 C > T) mutation in induced pluripotent stem cells (iPSCs) derived from a CDD patient. Isogenic control, CDKL5-R550* mutant, and ABE-corrected iPSCs were differentiated into neurons and the restoration of CDKL5-related and functional recovery were assessed. In this study, we demonstrated that ABE successfully restored CDKL5 protein levels and CDKL5-dependent signalling pathways in edited iPSC-differentiated neurons to levels comparable to the isogenic control. Morphological deficits, and genes expression were normalized in the ABE-corrected neurons. This study provides evidence that ABE can precisely correct pathogenic mutation and functionally rescue some CDD-associated neuronal phenotypes in patient-derived cells, supporting its potential as a valuable gene therapy for CDD. Moreover, these findings underscore the broader applicability of base editing for treating other monogenic neurodevelopmental disorders caused by point mutations.
Open article ↗
