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

Area of Science:

• Genetics
• Molecular Biology
• Neuroscience

Background:

• Mutations in the CDKL5 gene cause a severe, incurable neurological disorder with early-onset seizures and intellectual disability.
• CDKL5 is dosage-sensitive, limiting gene or protein replacement therapies.
• Splicing correction offers a therapeutic avenue by preserving physiological gene regulation.

Purpose of the Study:

• To evaluate the potential of spliceosomal U1 small nuclear RNA (U1snRNA) variants to correct CDKL5 mutations affecting splicing.
• To assess the functional restoration of CDKL5 protein following splicing correction.
• To provide a proof-of-concept for U1snRNA-mediated splicing correction as a therapeutic strategy.

Main Methods:

• Utilized CDKL5 minigene variants in mammalian cells to analyze splicing patterns.
• Engineered U1snRNA variants were expressed to target mutations at the 5' donor splice site (+1 and +5 nucleotides).
• Assessed CDKL5 protein synthesis, subcellular localization, kinase activity, and neuronal morphology post-correction.

Main Results:

• CDKL5 minigene variants effectively modeled splicing defects.
• Engineered U1snRNA rescued splicing mutations at the +5 but not +1 nucleotide positions.
• U1snRNA-mediated splicing correction fully restored CDKL5 protein function and rescued neuronal morphology.

Conclusions:

• U1snRNA-mediated splicing correction is a viable therapeutic strategy for CDKL5 mutations affecting splicing.
• This approach demonstrates potential for treating CDKL5-related neurological disorders by restoring protein function and neuronal morphology.