Rescue of Mutation-Induced Exon Skipping in Rhodopsin by Adaptation of U1snRNA

Abstract

Purpose: : Splice site mutations constitute a major cause of disease-associated mis-splicing. We aim to develop a therapeutic intervention for diseases caused by donor splice site mutations.

Methods: : Direct sequencing of PCR products was used to screen for mutation in patients' DNAs. Splice assays were performed with minigene and U1snRNA expression constructs that were cotransfected into COS7 cells. RT-PCR and fragment analysis were applied for transcript analysis.

Results: : We identified a novel donor splice site mutation at the last base of exon 4 of RHO (c.936G>A). The mutation causes both, skipping of RHO exon 4 and activation of a cryptic splice site. These splice defects were found to be similar in cultured cells and retinal explants. Both mis-spliced RHO transcripts are predicted to result in truncated proteins explaining the retinitis pigmentosa phenotype diagnosed in the patient.With the minigene splice assay we tested the feasibility and efficiency of a therapeutic strategy to rescue splice defects: donor splice sites are recognized by complementary base pairing of the U1snRNA to the pre-mRNA, which assists in exon recognition and defines the exon-intron boundary. We tested whether adaptation of U1snRNA to the mutation rescues the splice defect and cotransfected the minigene constructs and the adapted U1snRNA. RT-PCR and fragment analysis showed a significant reduction in exon 4 skipping. Nevertheless, activation of the cryptic splice site was still detected. Inactivation of the cryptic splice site in the minigenes and co-transfection with adapted U1snRNA demonstrated a rescue of exon skipping without activation of a cryptic splice site.

Conclusions: : Our results demonstrate for the first time the feasibility and high efficiency of an U1snRNA-mediated therapeutic intervention to treat splice donor site mutations affecting the last base of an exon with the aim to rescue normal splicing in patients with retinal degeneration.

Keywords: mutations • gene transfer/gene therapy • retinal degenerations: hereditary