Treatment of Usher syndrome with antisense oligonucleotides

Abstract

Purpose: Usher syndrome (Usher) is the leading genetic cause of combined deafness and blindness. Type 1 Usher (Usher 1) is characterized by profound hearing impairment and vestibular dysfunction at birth, and the development of retinitis pigmentosa (RP) in early adolescence. The 216G>A (216A) mutation in USH1C creates a cryptic splice site that is used preferentially over the correct site and results in a truncated harmonin protein. We created a mouse model for Usher 1C by knocking in the 216A mutation. 216AA mice exhibit circling behavior indicative of severe vestibular dysfunction and deafness, have retinal dysfunction by 1 month of age and begin to lose photoreceptors after 6 months of age. This mouse model provides an opportunity to test the feasibility of correcting the disease-associated genetic defect using antisense oligonucleotides (ASOs).

Methods: Antisense oligonucleotides (ASOs) were used to target Ush1c mutations in vitro and in vivo by systemic or local injection. Correction of splicing was evaluated by RT-PCR and western blot. Immunofluorescence was used to analyze Harmonin expression and ASO localization. Hearing and vestibular function were evaluated by auditory-evoked brain stem response (ABR) and open-field chamber analysis, respectively. Visual function was evaluated by electroretinogram (ERG) analyses and optical coherence tomography (OCT) imaging was used to examine ocular structures.

Results: ASOs effectively corrected splicing of 216A RNA in an Usher patient cell line, and in the cochleae and retinas of 216AA mice when delivered systemically or locally. Cell-free and cellular assays also demonstrated that ASOs targeted to the Ush1c.238dupC mutation result in in-frame skipping of exon 3. Treatment with 216A-targeted ASOs to neonate mice corrected protein expression with an improvement in harmonin localization in the hair cells and photoreceptors; rescued vestibular and hearing function, and demonstrated a small improvement in visual function.

Conclusions: Our results show that ASOs can effectively target Ush1c mutations both in vitro and in vivo. These results suggest the therapeutic potential of ASOs in Usher syndrome and other diseases of the ear and eye.

Keywords: 702 retinitis • 494 degenerations/dystrophies