NOVEL APPROACHES OF ¿PERSONALISED MEDICINE¿ AS PROOF-OF-PRINCIPLE FOR CDKL5-RELATED PATHOLOGIES

Alterations of CDKL5 give rise to several forms of neurological disorders generally characterised by epileptic encephalopathy, severe developmental delay, hypotonia and RTT-like features. To date no cure exists and only secondary symptoms can be treated. About 15% of CDKL5 patients carry a nonsense mutation and might benefit of a readthrough strategy as “personalised” medicine approach. The read-through process occurs when a near-cognate aminoacyl-tRNA binds a premature stop codon (PTC), allowing its suppression and the subsequent protein elongation. This mispairing event can rarely occur, but can be facilitated using a wide range of drugs. In order to test PTC suppression, we have chosen some human pathogenic CDKL5 nonsense mutations located in the two main domains of the protein: the catalytic N-terminus (R59X, R134X) or the C-terminal tail (Q347X, E364X, R550X, S855X). We then evaluated the read-through process using aminoglycoside and non-aminoglycoside drugs in cells transfected with the mutagenized constructs. In this study, we have demonstrated that tested CDKL5 PTCs can be suppressed by gentamicin and geneticin (G418) in a dose-dependent manner and that PTC position can be critical for read-through. In particular, G418 was found to be more effective than gentamicin. Considering the known aminoglycosides toxicity, we evaluated the activity of PTC124 and GJ072 but no PTC suppression was detectable in our experimental conditions. Finally, in order to understand whether the full-length derivatives may maintain the proper function of WT CDKL5, we analysed some features of read-through products compared to the WT protein. In particular, while premature truncated proteins showed an altered subcellular localisation, read-through products demonstrated a nucleo-cytoplasmic distribution more similar to the WT one. Moreover, by evaluating the auto-phosphorylation of the TEY motif, the read-through derivatives demonstrated to recover some catalytic activity, although remaining highly hypomorphic. Nevertheless, preliminary studies on Cdkl5-null neurons transfected with R134X construct suggested that G418 treatment can ameliorate impaired neuronal morphology. Collectively, our results indicate that: (i) aminoglycosides are able to induce read-through of different CDKL5 PTCs; (ii) the read-through derivatives recover some features characterizing the WT protein; (iii) PTC position can be crucial for read-though and for rescue of a proper function and (iv) neuronal morphological defects might be rescued by small amount of a possible hypomorphic CDKL5, therefore supporting the potential validity of a read-through therapy.