Molecular Cloning

Full-length mouse otoferlin (fl-Otof) was subcloned from a previously generated cDNA clone (pcDNA3-mOtof-IRES-EGFP) into a pAAV vector. The cloning procedure was done by in-fusion cloning according to manufacturer instructions (TaKaRa/Clontech, In-Fusion HD Cloning kit). Owing to the size of fl-Otof we amplified three overlapping otoferlin fragments which complement fl-Otof by high fidelity PCR using the HiFi-PCR-premix provided by the kit and fused into the linearized pAAV target vector to generate pAAV_fl-Otof. The corresponding vector map of pAAV_fl-Otof with ubiquitous CMV-β-Actin (CMV/hbA) hybrid promoter is shown in Supplementary Figure 1. Due to the lack of suitable restriction sites within the otoferlin coding region and the substantial length of the fl-Otof cDNA we refrained from using traditional T4 ligase-based cloning techniques. Instead, we applied in fusion cloning strategy to generate a fl-Otof plasmid. To this end we linearized the target vector for virus expression (pAAV) by digestion with the restriction enzymes NheI and HindIII (Fermentas). We amplified three otoferlin fragments complementing fl-Otof [fragment A (primer pair A_F2/R1), fragment B (B_F1/R1), and fragment C (C_F1/R2)], which contained overlapping regions with each other and with the linearized target vector (see Appendix A1). This step required fragment and primer optimization [e.g., by determining optimal fragment sizes and ratios and by using “split overlaps” to reduce primer lengths (primers see Appendix A1.1 and A1.2)]. Thus, the linearized vector and three fragments were simultaneously fused together using the TaKaRa/Clontech In-Fusion HD Cloning kit, following manufacturer's instructions. This approach yielded the final fl-Otof viral vector (pAAV_fl-Otof) used for subsequent virus production (Supplementary Figure 1B). Target pAAV vector linearized by NheI/HindIII digest and PCR-amplified otoferlin fragments A, B, C using the primer pairs are shown in Supplementary Figure 1A and Appendix A1.2. Note that primer A_F2 contains a 15 bp overlap with the target vector at the 5'-end and encodes the START-codon for expression of otoferlin, while C_R2 encodes the STOP codon and a 15 bp overlap with the AAV vector at the 3'-end. Restriction enzyme digestions and Sanger sequencing (SeqLab, Germany) was employed to verify the correct fl-Otof insert. The length of the otoferlin coding region is 5,979 kb comprising the mouse transcript variant 1 which contains the alternative Exon5B (NCBI accession numbers {"type":"entrez-protein","attrs":{"text":"NP_001093865.1","term_id":"154240702","term_text":"NP_001093865.1"}}NP_001093865.1, Appendix A4). This sequence shows 94% identity to the human otoferlin sequence ({"type":"entrez-protein","attrs":{"text":"NP_001274418.1","term_id":"566559996","term_text":"NP_001274418.1"}}NP_001274418.1), as determined by using the BLASTP algorithm (NCBI). A protein alignment of mouse and human otoferlin is shown in the Appendix A4 [CLUSTAL O(1.2.4)]. The CDS size (5,979 kb) is beyond the packaging capacity of adeno-associated virus of 4.7 kb (according to (Grieger and Samulski, 2005). A truncated otoferlin, consisting only of the C2E and C2F including the transmembrane domain (TM)—named in this study as “miniOtoferlin” (Supplementary Figures 2, 3A)—was generated in a similar way. Briefly, two overlapping fragments covering the C-terminal part of Otof were amplified from the cDNA clone (pcDNA3-mOtof-IRES-EGFP) by high fidelity PCR using the HiFi-PCR-premix provided (TaKaRa/Clontech, In-Fusion HD Cloning kit) and overlapping primer pairs (G_F1/B_R1 and C_F1/R1 for fragments G and C2, respectively) (see Appendix A1). For linking to EGFP, both fragments were fused simultaneously into pEGFP-C2 linearized by SacI (Thermo Fisher Scientific) using the in-fusion reaction according to the manufacturer's instructions (TaKaRa/Clontech, In-Fusion HD Cloning kit). The resulting EGFP-miniOtof fusion construct was subcloned to the linearized pAAV vector. Insert and vector were restricted using NheI and HindIII (Thermo Fisher Scientific) and complementary ends were ligated using T4 ligase. This approach yielded the final EGFP-miniOtof viral vector (pAAV_EGFP-miniOtof) used for subsequent virus production. Restriction enzyme digestion and Sanger sequencing (SeqLab, Germany) was employed to verify the correct EGFP-miniOtof insert.