Suzanne de Bruijn

149 The development of a genetic therapy for DFNA21 INTRODUCTION DFNA21, caused by an in-frame deletion in the RIPOR2 gene, is a progressive form of dominantly inherited adult-onset hearing loss (HL). Recently, this deletion (c.1696_1707del, p.(Gln566_Lys569del), NM_014722.3) was identified as the most frequent cause of this type of HL within Europe. It is estimated that >30,000 individuals within Northwest Europe carry this variant, and therefore are at risk to develop DFNA21. 1 DFNA21 is highly variable; the age of onset ranges from congenital to the 7 th decade of life, and HL can progress from mild in early stages of the disease, to severe or profound later in life. 1-3 Currently, there is no cure available to treat DFNA21. RIPOR2 encodes the RHO Family Interacting Cell Polarisation Regulator 2 protein (RIPOR2). The protein is expressed in a wide variety of tissues and cell types, including the cochlea. 4 Inmouse cochlear hair cells, RIPOR2 is described to be localized at the basal taper region of stereocilia, where it is organized in a ring-like fashion. In the absence of RIPOR2, development, function and maintenance of murine hair cells is severely affected and morphological defects in both hair cells and stereociliary structures can be observed. Ripor2 knockout mice are already deaf at 4 weeks of age, and ripor2 knockdown in zebrafish leads to profound HL and loss of saccular hair cells. 4-6 Pathogenic variants in RIPOR2 have not only been associated with dominantly inherited, but with recessively inherited HL (DFNB104) as well. 6 A loss-of-function variant in exon 3 of the gene is associated with profound, prelingual HL that corresponds with the phenotypes observed in the mouse knockout and zebrafish knockdown models. 4,6 The c.1696_1707del variant that affects exon 14, on the other hand, can be considered a milder variant and is associated with a less severe, later onset, phenotype. 1 The mutated RIPOR2 was demonstrated to lack complete functionality due to aberrant localization in the stereocilia of wildtype mouse cochlear hair cells and the inability to rescue morphological defects of the hair bundle of RIPOR2-deficient hair cells. Although the pathogenic mechanismof the in-frame deletion is not yet completely understood, there are strong indications that the variant acts via a non-haploinsufficiency mechanism. The mislocalization of mutant RIPOR2 in the stereocilia suggests a toxic gain-of-function effect. In line with this, both humans and mice carrying heterozygous loss-of-function alleles do not display HL. 4,6 The non-haploinsufficiency disease mechanism implies that blocking the synthesis of mutant RIPOR2 proteins can alleviate their negative effects on auditory function. The lack of HL in heterozygous carriers of loss-of-function mutations, both in human and mice 4-6 , indicates that RIPOR2 synthesis from a single wildtype allele is sufficient for