Correction of Cryptic Splicing in Usher Syndrome Using Antisense Oligonucleotides

Abstract

Purpose: : Usher syndrome (Usher) is the leading cause of combined blindness and deafness. All Usher patients develop retinitis pigmentosa, with the age of onset, and the severity of deafness and presence of vestibular defects differing among subtypes. Patients with Usher 1 suffer retinitis pigmentosa beginning in early adolescence with congenital deafness and vestibular dysfunction. An obstacle to developing treatment strategies for the disease has been the lack of animal models that develop both auditory and visual defects. Recently, a mouse model for Usher that does exhibit both phenotypes was developed based on the human mutation in the USH1C gene responsible for Usher type 1C. The Ush1c216AA knock-in mice exhibit retinal degeneration that begins after deafness and vestibular dysfunction. Abnormal electroretinograms (ERGs) are evident as early as 1 month of age, however, the loss of rod photoreceptors begins between 6 and 12 months of age. The Ush1c.216G>A (c.216G>A) mutation introduces a cryptic 5’ splice site that is used preferentially over the normal site, producing a truncated mRNA and protein product. This mouse model provides a valuable tool to investigate therapeutic strategies for Usher and other diseases associated with mutations in splice sites. Antisense oligonucleotides (ASOs) are powerful tool that can be used to correct aberrant splicing and may be a useful therapeutic approach to treat Usher.

Methods: : Antisense oligonucleotides (ASOs) were used to block the c.216G>A cryptic 5’ splice site in vitro and in vivo. ASOs that most effectively blocked cryptic splicing of Ush1c.216A minigene transcripts were subsequently tested in cell lines generated from Usher1C patients (216AA) and the c.216AA mice. ASOs were also injected into c.216AA neonatal mice and correction of splicing in the retina and cochleae were quantitated by RT-PCR and western blot. Hearing and visual function were evaluated by auditory-evoked brainstem response (ABR) and ERG analyses, respectively.

Results: : ASOs effectively blocked cryptic splicing and increased the amount of normal splicing in an Ush1c.216A minigene system, in cells from 216AA Usher 1C patients and the c.216AA mice. A single systemic treatment with ASOs to neonate mice corrected splicing and protein expression in the retina and cochlea. ASO-treated mice had no circling behavior characteristic of the 216AA mice. Mice are currently being evaluated for restoration of hearing and vision by ERG and ABR analysis.

Conclusions: : Our results demonstrate that ASOs can effectively block cryptic splicing of the c.216A transcript in vivo. These results suggest the therapeutic potential of ASOs in Usher syndrome and other diseases caused by mutations that disrupt splicing.

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