Aminoglycoside drugs induce efficient read-through ofCDKL5nonsense mutations, slightly restoring its kinase activity

Abstract

The X-linked CDKL5 gene codes for a kinase whose mutations have been associated with a suite of neurodevelopmental disorders generally characterized by early-onset epileptic encephalopathy and severe intellectual disability. The impact of these mutations on CDKL5 functions and brain development remain mainly unknown, although the importance of maintaining the catalytic activity is generally recognized. Since no cure exists for CDKL5 disorders, the demand for innovative therapies is a real emergency. The recent discovery that CDKL5 is dosage sensitive poses concerns on conventional protein and gene augmentative therapies. Thus, RNA-based therapeutic approaches might be preferred. We studied the efficacy of read-through therapy on CDKL5 premature termination codons (PTCs) that correspond roughly to 15% of all mutations. Our results provide the first demonstration that all tested CDKL5 nonsense mutations are efficiently suppressed by aminoglycoside drugs. The functional characterization of the restored full-length CDKL5 reveals that read-through proteins fully recover their subcellular localization, but only partially rescue their catalytic activity. Since read-through can cause amino acid substitution, CDKL5 patients carrying the PTC outside the catalytic domain might benefit more from a nonsense suppression therapy. Eventually, we demonstrate that non-aminoglycoside drugs, such as Ataluren (PTC124) and GJ072, are unable to induce read-through activity on CDKL5 PTCs. Although these drugs might be more effective in vivo, these results question the validity of the Ataluren phase 2 clinical trial that is currently ongoing on CDKL5 patients.

Splicing Mutations Impairing CDKL5 Expression and Activity Can be Efficiently Rescued by U1snRNA-Based Therapy

“…As expected, in transient transfection experiments in HEK293T cells, this construct drove the synthesis of correctly spliced CDKL5 transcripts (Figure 5B, lane 2) that were paralleled by the synthesis of CDKL5 protein (Figure 5C, lane 4). Moreover, pTEY assays, conventionally used to assess the CDKL5 kinase activity [21], demonstrated that the recombinant CDKL5 protein possessed an appreciable catalytic activity. Conversely, the c.99+5G>A mutation led to complete exon 3 skipping (Figure 5B, lane 3) and to a shorter protein which is completely dysfunctional (Figure 5C, lane 5).…”

“…In this light, correction approaches acting at the post-transcriptional level would be of great interest for CDKL5 deficiency since they would preserve the physiological gene regulation. Among them, induction of ribosome read-through by aminoglycoside drugs is an attractive option, as recently demonstrated for mutations introducing premature termination codons (PTCs) and reported in ~15% of patients with CDKL5 deficiency [21]. However, the overall low efficiency of this strategy and the impaired CDKL5 catalytic activity of the rescued protein suggest caution for the translatability of the nonsense suppression therapy.…”

Nonsense Suppression Therapy: New Hypothesis for the Treatment of Inherited Bone Marrow Failure Syndromes

“…Furthermore, changes in convulsive or drop seizure frequency, cognitive, motor, and behavioral functions will be evaluated upon twelve weeks of treatment. However, a recent publication by Landsberger's group casts doubt on the final effectiveness of that clinical trial, because preclinical results showed that both ataluren and another non-aminoglycoside drug, termed GJ072, failed to induce PTC-readthrough on nonsense mutated CDKL5 gene in vitro [172].…”

Cyclin-Dependent Kinase-Like 5 (CDKL5): Possible Cellular Signalling Targets and Involvement in CDKL5 Deficiency Disorder

“…Conversely, nonsense mutations in CDKL5 result in the formation of proteins lacking the C-terminus. The C-terminal region contains a sequence vital to the regulation of the intracellular localisation of CDKL5, and the mutations result in abnormal CDKL5 localisation patterns, as observed in an in vitro transient expression study [26]. Furthermore, nonsense mutations completely inhibit the ability of CDKL5 to interact with proteins that typically interact with its C-terminus.…”