Doxycycline-controlled splicing modulation by regulated antisense U7 snRNA expression cassettes.

Area of Science:

• Molecular Biology
• Gene Regulation
• RNA Therapeutics

Background:

• Alternative splicing is crucial for proteome diversity and gene expression regulation.
• Dysregulation of splicing contributes to various human diseases.
• Targeted control of splicing events is essential for understanding and treating these pathologies.

Purpose of the Study:

• To develop a tightly regulated system for controlling specific pre-mRNA splicing events.
• To adapt modified U7 small nuclear RNAs (snRNAs) for inducible and repressible exon manipulation.
• To demonstrate the therapeutic potential of this system in disease models.

Main Methods:

• Development of a doxycycline-inducible system utilizing Krüppel-associated box (KRAB)/KAP1 transcriptional silencing.
• Co-transduction of target cells with lentiviral vectors encoding KRAB and the regulatable U7 cassette.
• Assessment of U7 snRNA expression and its correlation with splicing correction in disease models.

Main Results:

• Achieved tight, doxycycline-dependent regulation of modified U7 snRNA expression with no observed leakage in the repressed state.
• Demonstrated efficient induction of exon skipping or inclusion at low doxycycline concentrations (ng/mL).
• Showcased successful splicing correction in models of beta-globin thalassaemia and spinal muscular atrophy (SMN2 gene).

Conclusions:

• The developed regulatable U7 snRNA system provides precise control over alternative splicing.
• This technology enables efficient and reversible modulation of specific splicing events.
• The system holds significant promise for research into splicing-associated diseases and the development of novel RNA-based therapeutics.