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AAV-shRNA mediated knockdown of CDKL5 to generate a novel human in vitro model of CDKL5 Deficiency Disorder

C Pickering(1) F McLeod(2) A Pantziarou(1) N D Mazarakis(1)

1:Imperial College London; 2:Newcastle University

CDKL5 Deficiency Disorder (CDD) is a rare developmental epileptic encephalopathy, arising due to de novo mutations in the gene encoding the serine-threonine kinase CDKL5. Patients with CDD generally experience early onset recurrent seizures coupled with severe developmental delay. At present there is no effective cure for CDD. Whilst recent research has highlighted key functions of CDKL5 in neuronal cells in the brain, many of these findings are based on mouse models which do not accurately recapitulate key aspects of the human phenotype. As such, there is a need for novel human models of CDD to interrogate CDKL5 function in multiple cell types in the brain – from excitatory and inhibitory neurons to astrocytes and other glial cells. Here, we present an adeno-associated virus (AAV) -mediated short hairpin RNA (shRNA) knockdown of CDKL5 driven by the constitutive U6 promoter, in adult human cortical organotypic brain slices obtained from anonymous patients undergoing surgical removal of deep brain tumours. In human cortical organotypic brain slices transduced with the shAAV-CDKL5 vector, CDKL5 knockdown lead to functional deficits as assessed by disrupted phosphorylation of known targets of CDKL5 and network changes in extracellular field potential recordings. Thus, this novel model of CDD provides an interesting platform to interrogate both cell-type specific and network consequences of CDKL5 deficiency, and could be used in the future to screen novel pharmacological and gene therapy treatments for CDD.

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