Preclinical development of antisense oligonucleotide therapy for RIPOR2-associated adultonset hearing loss
Royal National Institute for Deaf People
Radboud University Nijmegen Medical Centre
We recently identified a 12-nucleotide deletion in RIPOR2 (rs760676508) as a frequent cause of dominantly inherited adult-onset sensorineural hearing loss (DFNA21). Based on the allele frequencies in gnomAD, it is estimated that ~30,000 individuals in Northwest Europe are at risk to develop hearing loss due to this RIPOR2 variant. The mutated protein exhibits an aberrant localization in stereocilia. The proposed project builds on these findings and aims to put a genetic treatment on the horizon of DFNA21 patients. The age of onset and progressive nature of the hearing loss provide a window of opportunity for therapeutic intervention. This, together with the high prevalence, makes DFNA21 an important target for therapy development.  As the 12-nucleotide deletion in RIPOR2 is indicated to have a dominant negative or toxic gain of function effect, we hypothesize that suppression of mutant RIPOR2 synthesis will prevent or delay the onset and/or progression of the hearing loss. To test this therapeutic strategy, the aims of the project are:  1)_x0001_To design and test antisense oligonucleotides (AONs) for effective and specific targeting of the mutant RIPOR2 (pre-)mRNA for degradation in HEK293T and patient-derived cells.  2)_x0001_To provide insight into the feasibility of AON-mediated therapy for DFNA21 in Δ12-nt Ripor2 mutant mice .   Allele-specific quantitative PCR will be employed to demonstrate AON-effectivity and specificity in HEK293T cells transfected with plasmids encoding mutant and wildtype RIPOR2 and in patient-derived fibroblasts and otic progenitor cells. Audiometric testing and (immuno)histology will be the readouts for therapeutic efficacy in the DFNA21 mouse model.
5.2 Cellular and gene therapies