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A Reporter Based Cellular Assay for Monitoring Splicing Efficiency
Published on: September 15, 2021
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Exploring non-coding variants and evaluation of antisense oligonucleotides for splicing redirection in Usher syndrome
Belén García-Bohórquez1,2,3, Pilar Barberán-Martínez1,3, Elena Aller1,2,3,4
1Molecular, Cellular and Genomics Biomedicine, Health Research Institute La Fe, 46026 Valencia, Spain.
Molecular Therapy. Nucleic Acids
|December 4, 2024
Summary
Deep-intronic variants in the USH2A gene significantly contribute to inherited retinal dystrophies. Antisense oligonucleotides (ASOs) show therapeutic potential by correcting aberrant splicing caused by these variants.
Area of Science:
- Genetics
- Ophthalmology
- Molecular Biology
Background:
- Non-coding regions are crucial for diagnosing inherited retinal dystrophies (IRDs).
- Deep-intronic variants in the USH2A gene can cause aberrant splicing, leading to Usher syndrome (USH) or retinitis pigmentosa.
- Antisense oligonucleotides (ASOs) are being developed to modulate splicing defects.
Purpose of the Study:
- To screen for known USH2A deep-intronic variants in patients with USH or isolated retinitis pigmentosa.
- To identify novel deep-intronic variants affecting splicing in USH2A and other USH genes.
- To assess the therapeutic potential of ASOs for correcting splicing defects.
Main Methods:
- Screening of five known USH2A deep-intronic variants.
- Sequencing of USH2A and other USH genes in unresolved cases.
- Minigene assays to confirm splicing impact of variants.
- Design and in vitro testing of ASOs for splicing correction.
Main Results:
- Diagnosed 30.95% of screened patients.
- Identified 16 new variants predicted to affect splicing, with four confirmed by minigene assays.
- Two novel deep-intronic variants predicted to cause pseudoexon inclusion.
- ASOs successfully corrected splicing for three USH2A deep-intronic variants in vitro.
Conclusions:
- Analyzing non-coding regions improves genetic characterization of IRDs.
- Deep-intronic USH2A variants are significant contributors to pathogenicity.
- ASO-mediated splicing modulation demonstrates therapeutic promise for IRDs caused by deep-intronic variants.
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