Suzanne de Bruijn

151 The development of a genetic therapy for DFNA21 MATERIALS AND METHODS Study approval The study of human subjects was approved by the medical ethics committee of the Radboudumc and performed in accordance with the principles of the World Medical Association Declaration of Helsinki. Written informed consent was obtained from all participants or their legal representatives. Design of antisense oligonucleotides AONs were designed following previously described criteria. 11,17-19 Exon 14, harboring the 12-nucleotide (nt) target deletion, was examined for open configuration (mfold Web Server 20 ). Thermodynamic properties of potential 20-mer AON molecules (40-60% GC content) were assessed using the RNAstructure software as previously described. 19 Uniqueness of AON target sequences was validated using BLAST (NCBI), allowing a maximum of two mismatches. AONs were purchased from Eurogentec and dissolved in PBS before use. Sequences and AON chemistry are provided in Table S1 . Cell culture conditions and AON delivery Patient-derived primary fibroblast cells were cultured in standard fibroblast medium consisting of DMEM (Gibco) supplemented with 20% fetal calf serum, 1% sodium pyruvate and 1% penicillin-streptomycin. Prior to AON treatment, cells were seeded in 12 wells plates and cultured to a confluency of ~80%. Cells were transfected with AON molecules (final concentrations 50-250 nM in the culture medium) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, following a 1:2 ratio (1 mg AON:2 ml Lipofectamine reagent). After 24 hours, cells were harvested for RNA isolation and subsequent transcript analysis. For the assessment of AON efficacy in HEK293T cells, plasmids containing cDNA sequences encoding an N-terminally FLAG-tagged wildtype or mutant RIPOR2 (NM_015864.3) were generated with Gateway Technology (Life Technologies). RNA isolated from patient-derived EBV-transformed lymphoblastoid cells was used as input for Gateway-adapted RT-PCR. The sequence of both mutant and wildtype entry clones was verified with Sanger sequencing. HEK293T cells were co-transfected with AONs and the generated DNA constructs (500 ng per well) using 45 µl polyethyleneimine (PEI). 21 Treated cells were collected 24 hours after AON delivery for transcript and protein analyses.