Engineered tRNAs efficiently suppress CDKL5 premature termination codons

Abstract

The CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental disorder characterized by early-onset epilepsy, intellectual disability, motor and visual dysfunctions. The causative gene is CDKL5, which codes for a kinase required for brain development. There is no cure for CDD patients; treatments are symptomatic and focus mainly on seizure control. Several pathogenic variants are loss-of-function, but recent studies suggest that the CDD phenotype is sensitive to the CDKL5 gene dosage. Therefore, mRNA-targeted correction strategies that respect the physiological regulation of CDKL5 could be a valid alternative to augmentative gene therapy. Nonsense mutations cause ~ 11% of CDD cases, and these patients might benefit from readthrough therapies. We proved that drug-mediated readthrough efficiently suppresses premature CDKL5 nonsense codons, but the recoded kinase remained highly hypomorphic, curtailing the translational value of this pharmacological approach. In this study we explored if the recently developed Anticodon-edited tRNAs (ACE-tRNAs) offer an alternative readthrough strategy for CDD. Transfecting cells expressing different CDKL5 nonsense variants, we demonstrated that ACE-tRNAs efficiently restore full-length kinase synthesis. The recoded CDKL5 is correctly localized and catalytically active, thereby bringing tRNA-based therapy back into the spotlight for future investigations to assess the efficacy of this approach in correcting the pathological phenotype of CDD.

Fig. 1

ACE-tRNA therapy restores full-length CDKL5 protein synthesis. (a) Western blot of GFP CTRL, GFP-CDKL5 WT, R59X, R134X and R550X constructs co-transfected in HEK293T cells with or without 1X or 4X ACE-tRNA^Arg_UGA. Fusion proteins were detected using the α-GFP antibody. Black arrowheads indicate full-length GFP-CDKL5. Total protein content was visualized by TGX Stain-Free technology (lower panel). (b,c) Western blot of endogenous VSNL1 (b) and MeCP2 (c) in untransfected HEK293T cells or in HEK293T cells transfected with GFP with or without 1X or 4X ACE-tRNA^Arg_UGA. In panel b, the asterisk indicates a non-specific band recognized by the antibody. Images of original blots can be retrieved in the Supplementary Information file.

Fig. 2

ACE-tRNAs stabilize the GFP-CDKL5 R134X transcripts. The graph shows GFP-CDKL5 mRNA levels in transfected HEK293T cells, expressed as a percentage with respect to GFP-CDKL5 WT – transfected cells. Data are shown as mean ± SEM. *p < 0.05, by two-way ANOVA, followed by Sidak’s multiple comparison test.

Fig. 3

CDKL5 PTC variants recover proper nuclear/cytosolic distribution following delivery of ACE-tRNA^Arg_UGA. (a) Representative immunofluorescence images of GFP-CDKL5 in HEK293T cells transfected with GFP-CDKL5 WT and its truncated derivatives R59X and R550X in the presence or absence of the 1X ACE-tRNA^Arg_UGA expressing vector. (b) Violin plots indicate the median (dashed line) and 25th and 75th percentiles (dotted lines) of the relative distribution in the nucleus and cytoplasm of CDKL5 WT, R59X and R550X variants left untreated (UT) or following treatment with 1X ACE-tRNA^Arg_UGA. Values are expressed as percentages compared to CDKL5 WT. *p < 0.05, ***p < 0.001 and ****p < 0.0001 by Kruskal-Wallis test followed by Dunn’s multiple comparison test. §§p < 0.0001 indicates a difference between R550X treated with 1X ACE-tRNA^Arg_UGA with respect to UT WT. (c) The subcellular distributions of GFP-CDKL5 WT and of its derivative R550X were compared in cells co-transfected or not with the 4X ACE-tRNA^Arg_UGA expressing plasmid. (d) Violin plots were obtained as in a. ****p < 0.0001 and ***p < 0.001 by Kruskal-Wallis test followed by Dunn’s multiple comparison test. §p < 0.05 indicates a difference between R550X treated with 4X ACE-tRNA^Arg_UGA with respect to UT WT.

Fig. 4

Phosphorylation of the exogenous GFP-CDKL5 TEY motif and endogenous EB2 are restored following ACE-tRNA^Arg_UGAdelivery. (a, c) Representative western blots for CDKL5 pTyr¹⁷¹ (a) and EB2 pSer²²² (c) in HEK293T co-transfected with GFP, WT GFP-CDKL5 or -R59X, -R134X, -R550X constructs with or without 1X or 4X ACE-tRNA^Arg_UGA. CDKL5 phosphorylation at Tyr¹⁷¹ was revealed together with full-length CDKL5 (through an anti-GFP antibody). White arrowheads show the premature truncated GFP-CDKL5 R550X derivative that was not subjected to readthrough. Original images of blots in a are shown in the Supplementary Information file. (b,d) Histograms depict the percentages of full-length GFP-CDKL5 phosphorylation at Tyr¹⁷¹(b) and of EB2 phosphorylation at Ser²²² (d) with respect to untreated (UT) GFP-CDKL5 WT. For analysis of phosphorylated EB2, for each sample data were normalized to the total levels of corresponding protein. Data are expressed as mean ± SEM. *p < 0.05 by Kruskal-Wallis test followed by Dunn’s multiple comparison test.