Figure EV2. Dual‐AAV vector strategies for full‐length otoferlin gene transfer.

• A, B
  Schematic representation of the otoferlin dual‐AAV‐TS (A) and dual‐AAV‐Hyb (B) half‐vectors used for postnatal cochlear injections. Both otoferlin dual‐AAV half‐vector systems contain the first half of the otoferlin coding sequence (CDS) in the 5′‐AAV and the other half in the 3′‐AAV half‐vector. The correct reconstitution of the full‐length otoferlin mRNA in the dual‐AAV‐TS strategy is mediated by non‐homologous end joining of the inverted terminal repeats (ITRs). In the dual‐AAV‐Hyb strategy, the reassembly is mediated by non‐homologous end joining of the ITRs and/or homologous recombination of the highly recombinogenic AK sequence. Splice donor (SD) and splice acceptor (SA) sites facilitate the excision of the ITRs via trans‐splicing. The woodchuck hepatitis virus post‐transcriptional regulatory element (WPRE) stabilizes the mRNA. To ensure the production of two separate proteins, a P2A peptide inducing ribosomal skipping is introduced between the eGFP and the otoferlin CDS. hbA: human beta‐actin promoter, CMVe: cytomegalovirus enhancer, pA: polyadenylation signal.