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

Mutations in the gene lead to an incurable rare neurological condition characterized by the onset of seizures in the first weeks of life and severe intellectual disability. Replacement gene or protein therapies could represent intriguing options, however, their application may be inhibited by the recent demonstration that is dosage sensitive. Conversely, correction approaches acting on pre-mRNA splicing would preserve physiological regulation. Since ~15% of pathogenic mutations are candidates to affect splicing, we evaluated the capability of variants of the spliceosomal U1 small nuclear RNA (U1snRNA) to correct mutations affecting +1 and +5 nucleotides at the 5' donor splice site and predicted to cause exon skipping. Our results show that minigene variants expressed in mammalian cells are a valid approach to assess splicing pattern. The expression of engineered U1snRNA effectively rescued mutations at +5 but not at the +1 nucleotides. Importantly, we proved that U1snRNA-mediated splicing correction fully restores CDKL5 protein synthesis, subcellular distribution and kinase activity. Eventually, by correcting aberrant splicing of an exogenously expressed splicing-competent transgene, we provided insights on the morphological rescue of null neurons, reporting the first proof-of-concept of the therapeutic value of U1snRNA-mediated splicing correction.