Therapeutic strategy to rescue mutation-induced exon skipping in rhodopsin by adaptation of U1 snRNA.
Retinitis pigmentosa (RP) is a degenerative retinopathy leading to visual impairment in more than 1.5 million patients worldwide. Splice site (SS) mutations cause various diseases including RP. Most exonic donor splice-site (DS) mutations are reported at the last nucleotide of an exon and over 95% of them are predicted to result in missplicing. A novel human mutation at the last nucleotide of exon 4 in rhodopsin (RHO, c.936G>A) is shown to generate two misspliced transcripts in COS 7 cells and retinal explants. One of these transcripts skips exon 4 whereas the other activates a cryptic DS. Both are predicted to result in truncated RHO, explaining the pathogenic mechanism underlying the patient's RP phenotype. U1 snRNA-mediated DS recognition is a key step in the splicing process. As a therapeutic strategy, U1 snRNAs were adapted to the novel RHO mutation and tested for its potential to reverse missplicing. The rescue efficiency for misspliced transcripts of RHO was examined by quantitative RT-PCR. Using mutation-adapted U1 snRNA, we observed significantly reduced exon skipping that reached wild-type levels. Nevertheless, activation of the cryptic splice site (CS) was still detected. To test the feasibility of the strategy for mutations that only cause exon skipping, we inactivated the CS. Indeed, adapted U1 snRNA was able to rescue almost 95% [corrected] of misspliced transcripts. This study shows that modified U1 snRNAs constitute a promising therapeutic strategy to treat DS mutations. Our findings have implications for various diseases caused by similar mutations.
Division of Medical Molecular Genetics and Gene Diagnostics, Institute of Medical Genetics, University of Zurich, Schwerzenbach, Switzerland.
Gaby
G
Tanner
Esther
E
Glaus
Daniel
D
Barthelmes
Marius
M
Ader
Johannes
J
Fleischhauer
Franco
F
Pagani
Wolfgang
W
Berger
John
J
Neidhardt
D000328:Adult; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D005091:Exons / Q000235:genetics*; D015316:Genetic Therapy; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics; D012342:RNA, Small Nuclear / Q000235:genetics*; D012243:Rhodopsin / Q000235:genetics*
D022821:RNA Splice Sites; D012342:RNA, Small Nuclear; C405483:U1 small nuclear RNA; D012243:Rhodopsin