Animals
Animals were used in accordance with standard ethical guidelines. Both sexes of C57BL/6 mice (Jackson Laboratories) were used in this study at an equal ratio. The number of days since birth was counted from postnatal day 0 (P0). Prestin KO mice were kindly provided by Dr. Zhi-yong Liu from CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and then were housed under a 12 h light/dark cycle at a room temperature of 22 ± 1 °C with food and water available ad libitum. The primers used for Prestin KO mice genotyping are F1: 5’-CCACCACGTTTAGTAGCATC-3’, R1: 5’-ACTGTGATGAACATGAGCCA-3’, F2: 5’-AGAGCACACCTGCGCTCTTC-3’, R2: 5’-AGTGTGGATGTCAGGCAGAGTA-3’. All experiments were approved by the Institutional Animal Care and Use Committee of ShanghaiTech University, China, and all efforts were made to minimize the number of mice used and their suffering.
Virus preparation
The C-terminal Flag-tagged NLS-mNeonGreen was cloned into the AAV plasmid containing the cytomegalovirus enhancer/chicken β-actin (CAG) promoter and the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) cassette, which was flanked by AAV2 inverted terminal repeats. All AAV serotype vectors were produced in HEK 293T cells co-transfected with rep-cap fused plasmid and a helper plasmid. AAVs were purified by iodixanol gradient ultracentrifugation. In brief, cultured medium was collected twice every 48 h after transfection. The cell lysate was treated with chloroform, and the supernatant was collected. The medium and supernatant were combined and concentrated by precipitation with 10% PEG 8000 and 1.0 M NaCl at 4 °C overnight. After centrifugation, the pellet was resuspended in 1 × PBS buffer with Benzonase. 15%, 25%, 40%, and 60% iodixanol solutions were carefully layered and then the generated viral suspension was overlaid, followed by centrifugation at 350,000 × g for 90 min at 10 °C. Following ultracentrifugation, the AAV-containing 40% fraction was collected. The buffer was exchanged to remove the iodixanol and concentrate the purified virus. The genome-containing titers of AAVs were determined by SYBR (Roche) analysis using primers targeting the WPRE region. The qPCR primers for WPRE are listed as follows: forward, 5′-GTCAGGCAACGTGGCGTGGTGTG-3′; reverse, 5′-GGCGATGAGTTCCGCCGTGGC-3′.
Structure analysis of AAV-ie capsid
For amino acid phosphorylation and ubiquitination site prediction, we followed a previously published method.43 In brief, phosphorylation sites in AAV-ie capsid were predicted with NetPhosK (http://www.cbs.dtu.dk/services/NetPhosK), Phosida (Phosphorylation Site Database; http://www.phosida.com), KinasePhos (http://kinasephos.mbc.nctu.edu.tw), and Scansite (http://scansite.mit.edu). Ubiquitination sites were predicted with UbiPred (http://iclab.life.nctu.edu.tw/ubipred/) prediction servers.
Site-directed mutagenesis
Serine (S)→alanine (A) and lysine (K)→arginine (R) mutations were introduced into AAV-ie rep/cap plasmid with Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Cat.P505) according to the manufacturer’s protocol. Briefly, a one-step PCR amplification of the target sites was performed for 28 cycles with the primers (Supplementary Table S1). The PCR products were digested by Dpn1 enzyme (NEW ENGLAND Bio Labs, cat. R0176S) for 2 h. Then ten microliters of the recombinant product were transformed into DH5α chemically competent cell (Shanghai Weidi Biotechnology CO., Ltd, cat. DL1001). Plasmids were isolated with TIANprep Mini Plasmid Kit (TIANGEN BIOTECH(BEIJING) CO., LTD, cat.DP118) and verified by DNA sequencing (Applied Biosystems 3730XL genetic analyzer; BioSune, Shanghai, China).
AAV injection by round window injection
We followed a previously published surgery procedure. Briefly, the neonatal mice were anesthetized by low temperature. P2–3 mice were placed in an ice bath for 2–3 min until loss of consciousness and then removed to an ice pad for subsequent surgical procedures. The surgery was limited to 5–10 min. Surgery was performed only on the right ear of each animal. Upon anesthesia, a post-auricular incision was made to expose the otic bulla and visualize the cochlea. Guided by the relative position between the temporal bone and the facial nerve, the round window was exposed. Special care was taken to avoid damage to the facial nerve during surgery. Injections were performed through the RWM with a glass micropipette (25 μm) controlled by micromanipulator UMP3 UltraMicroPump (World Precision Instruments). The volume of the injected materials was controlled at ~1.5 μL per cochlea within 1 min per AAV. After the injection, the skin incision was closed using veterinary tissue adhesive (Millpledge Ltd, UK). Pups were subsequently returned to the 37 °C warming pad for 10 min and then returned to their mother for continued nursing.
For RWM injection in adult mice, 4-week-old C57/B6 mice were used. Before surgery, the mice were anesthetized with ketamine (100 mg/kg) and xylazine (25 mg/kg). The otic bulla was opened to expose the round window niche. Injections were performed through the RWM with a glass micropipette (25 μm) controlled by a micromanipulator UMP3 UltraMicroPump (World Precision Instruments). The volume of the injected materials was controlled at ~2.0 μL per cochlea within 1 min per AAV. After the injection, the skin incision was closed using veterinary tissue adhesive (Millpledge Ltd, UK).
Hearing tests via ABR measurements
ABR is a method to assess hearing by measuring the hearing threshold. Based on the Tucker-Davis Technology system III (Tucker-Davies Technologies, TDT, Gainesville, FL, USA), this test closed-field ABR was recorded from mice anesthetized with ketamine (100 mg/kg) and xylazine (25 mg/kg) in a sound-attenuated room, and changes in the electrical activity of the brain in response to sound were recorded via electrodes. The ABR responses were elicited in tone bursts at five frequencies (4, 8, 12, 16, 24, and 32 kHz). The acquired response signal of ABR was amplified (10,000 times), filtered (0.1–3 kHz), averaged, and presented in the computer-based data-acquisition system, BioSigRZ (TDT, Gainesville, FL, USA). The sound level was raised in 5 to 10 dB steps from 0 to 90 dB sound pressure level (decibels SPL). At each level, 1024 responses were averaged (with alternating stimulus polarity) after artifact rejection, using the lowest sound pressure level that elicited a visually detectable response as the rejection threshold.
Immunofluorescence staining
Injected cochleae were harvest on day 10 after injection. The temporal bone was fixed in 4% paraformaldehyde (36314ES76, Yeasen) for 2 h at room temperature and then decalcified in 0.5 M EDTA (ST066, Beyotime) for 1-2 h at room temperature. Specimens were then cut into apical, middle, basal regions.
For cross-section, cochleae were excised and fixed in 4% PFA for 2 h and then decalcified in 0.5 M EDTA for 2 h at room temperature. Specimens were cryo-protected in 30% sucrose in 1 × PBS (SH30256.FS, Hyclone) overnight at 4 °C and then embedded in tissue freezing medium (14020108926, Leica), frozen, and sectioned (14 μm).
Samples were blocked with 10% donkey serum in 0.3% Triton X-100 dissolved in 1 × PBS at room temperature for 1 h. Then, the samples were stained with antibodies against myosin 7 A (Myo7a, #25-6790 Proteus Biosciences, 1:1000), Sox2 (Sox2, #sc-17320, Santa Cruz Biotechnology, 1:1000), Flag (Flag, #F3165, Sigma-Aldrich, 1:1000), together with corresponding secondary antibodies. Samples were mounted with VECTASHIELD antifade mounting medium (H-1000-NB, Novios) and confocal microscopy was used for observation.
Scanning electron microscopy
Mice were perfused with 1 × PBS and temporal bones were fixed in 2.5% glutaraldehyde at 4 °C overnight. After rinsing three times with 1 × PBS, the temporal bones were decalcified with 0.5 M EDTA at room temperature for 1-2 h, and the organ of Corti was dissected into apical, middle, and basal regions. Specimens were then rinsed in 1 × PB three times and post-fixed with 1% osmium tetroxide at room temperature for 1 h, after complete rinse with 1 × PB three times, the specimens were treated with 2% tannic acid (V900190-100G, Sigma). Specimens were rinsed with 1XPB and dehydrated through a graded ethanol series for 10 min at each gradient: 30%, 50%, 70%, 90%, and 100% at 4 °C. After dehydration, samples were critical point dried (Leica EM CPD300) and coated with 8 nm gold (Leica EM ACE200 Vacuum Coater) and observed with Aquilos Cryo-FIB (Thermo Scientific).
Statistical analysis
All data are shown as the mean or mean ± SEM and all experiments were repeated at least three times. Statistical analyses were carried out using the Excel (Microsoft) and GraphPad Prism 8.0 software. ABR results were analyzed with a two-tailed, unpaired Student’s t tests. A value of p < 0.05 was considered statistically significant. Error bars and n values are defined in the respective figures and legends.
