Blocking Cryptic Splicing In Usher Syndrome Using Antisense Oligonucleotides

Abstract

Purpose: : Usher syndrome (Usher) is the leading genetic cause of combined blindness and deafness. 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 type 1 Usher that does exhibit both phenotypes was developed based on a mutation in the USH1C gene. This mutation, Ush1c.216G>A (c.216G>A), causes the creation of 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 pre-mRNA 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 caused by mutations that affect splicing such as c.216G>A.

Methods: : Antisense oligonucleotides (ASOs) were used to block the c.216G>A cryptic 5’ splice site. ASOs were screened for activity using an ASO-tiling approach and Ush1c.216G and A minigenes expressed in HEK-293T cells. ASOs that most effectively blocked cryptic splicing of the minigene transcripts were subsequently tested in a lymphoblast cell line from an Usher1C patient with the 216AA mutation. ASOs were also injected into c.216G>A mice and correction of splicing in liver, kidney and eye tissue was quantitated by RT-PCR.

Results: : ASOs effectively blocked cryptic splicing and increased the amount of normal splicing in an Ush1c.216G>A minigene system, in cells from a 216AA Usher 1C patient and in kidney and liver tissues from c.216G>A mice injected with the ASOs.

Conclusions: : Our results show that ASOs can effectively block cryptic splicing of the Ush1c.216G>A transcript in vivo. These results suggest the therapeutic potential of ASOs in Usher syndrome and other diseases caused by mutations that disrupt splicing.

Keywords: gene modifiers • mutations • transgenics/knock-outs