Mutations in the CDKL5 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 CDKL5 is dosage sensitive. Conversely, correction approaches acting on pre-mRNA splicing would preserve CDKL5 physiological regulation. Since ~15% of CDKL5 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 CDKL5 minigene variants expressed in mammalian cells are a valid approach to assess CDKL5 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 CDKL5 transgene, we provided insights on the morphological rescue of CDKL5 null neurons, reporting the first proof-of-concept of the therapeutic value of U1snRNA-mediated CDKL5 splicing correction.

Splicing mutations impairing CDKL5 expression and activity can be efficiently rescued by U1snRNA-based therapy / D. Balestra, D. Giorgio, M. Bizzotto, M. Fazzari, B.B. Zeev, M. Pinotti, N. Landsberger, A. Frasca. - In: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. - ISSN 1661-6596. - 20:17(2019 Aug), pp. 4130.1-4130.16. [10.3390/ijms20174130]

Splicing mutations impairing CDKL5 expression and activity can be efficiently rescued by U1snRNA-based therapy

D. Balestra;M. Fazzari;M. Pinotti;N. Landsberger
;
A. Frasca
2019

Abstract

Mutations in the CDKL5 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 CDKL5 is dosage sensitive. Conversely, correction approaches acting on pre-mRNA splicing would preserve CDKL5 physiological regulation. Since ~15% of CDKL5 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 CDKL5 minigene variants expressed in mammalian cells are a valid approach to assess CDKL5 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 CDKL5 transgene, we provided insights on the morphological rescue of CDKL5 null neurons, reporting the first proof-of-concept of the therapeutic value of U1snRNA-mediated CDKL5 splicing correction.
CDKL5; Mutations; Splicing; U1snRNA; Alleles; Alternative Splicing; Cell Line; Epileptic Syndromes; Exons; Gene Expression; Genetic Loci; Genetic Therapy; Genotype; Humans; Neurons; Nonsense Mediated mRNA Decay; Protein-Serine-Threonine Kinases; RNA, Small Nuclear; Spasms, Infantile; Mutation; RNA Splicing; Targeted Gene Repair
Settore BIO/11 - Biologia Molecolare
Dipartimenti di Eccellenza 2018-2022 - Dipartimento di FILOSOFIA
Article (author)
File in questo prodotto:
File Dimensione Formato  
Balestra et al.pdf

accesso aperto

Tipologia: Publisher's version/PDF
Dimensione 1.73 MB
Formato Adobe PDF
1.73 MB Adobe PDF Visualizza/Apri
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/738478
Citazioni
  • ???jsp.display-item.citation.pmc??? 11
  • Scopus 15
  • ???jsp.display-item.citation.isi??? 13
social impact