Cabp2-Gene Therapy Restores Inner Hair Cell Calcium Currents and Improves Hearing in a DFNB93 Mouse Model

FIGURE 1. Experimental design and hearing improvement in calcium-binding protein 2 (Cabp2)–/– animals injected with AAV2/1-Cabp2. (A) Schematic representation of the plasmid construct: An hCMV enhanced hBA promotor with the WPRE enhancer drives the expression of Cabp2 and eGFP, which are separated by a self-cleaving P2A peptide. The construct was inserted in two viral vectors, AAV2/1 and synthetic AAV-PHP.eB. (B) Mice of either sex received virus injections at postnatal day P5-7 and were tested for hearing at 5–8 weeks of age. Afterward, cochleae were harvested for patch-clamp experiments or processed for immunohistochemistry. (C) Representative image of an apical coil of the organ of Corti from an animal injected with AAV2/1-Cabp2-eGFP as observed under a fluorescent microscope. Several images were taken and stitched together to reconstruct the whole apical turn. A row of fluorescent IHCs can be observed, indicating high transduction efficiency of the virus. Please note that the fluorescence line of the IHCs is broken three times due to the nylon grid holding the explant in place. (D) Representative images of the midcochlear region of two AAV2/1-Cabp2-eGFP injected cochleae immunolabeled…

FIGURE 2. High transduction efficiency of PHP.eB-calcium-binding protein 2 (Cabp2) in Cabp2–/– mice. (A) Examples of PHPeB-Cabp2 injected and contralateral non-injected cochleae immunolabeled for eGFP and parvalbumin alpha [note that the apical portion of the injected side (represented by white dotted line) was used for patch-clamp experiments and is thus missing in the reconstruction]. When using a PHP.eB vector, eGFP expression was also observed in the non-injected contralateral ears albeit at reduced levels. EGFP immunosignal is very strong, but not limited to IHCs (also labeled by parvalbumin alpha). (B) Based on eGFP immunofluorescence signal, the transduction efficiency in the IHCs was close to 100% over the entire length of the injected cochleae. In the contralateral ears, the transduction rates were more variable and decreased progressively from the apex toward the base. (C) Overall transduction rates were 98 ± 1% (N = 10) for injected and 56 ± 6% (N = 11) for non-injected cochleae. All values are given as mean ± SEM.

FIGURE 3. PHPeB-mediated calcium-binding protein 2 (Cabp2) gene transfer improves ABR thresholds and amplitude responses in Cabp2–/– animals. (A) Mean ABR thresholds of 5-week-old mice plotted as a function of stimulus frequency for WT (black), Cabp2–/– controls (blue), and Cabp2–/– mice injected with PHP.eB-Cabp2 (injected ears – green; non-injected contralateral ears – cyan). Partial improvement of hearing was observed in injected Cabp2–/– mice. See Table 1A for statistical analysis. (B) Seven out of nine injected Cabp2–/– animals showed hearing thresholds, which were significantly decreased as compared to non-injected Cabp2–/– animals (six animals) or on the border of significance (one animal). ABR click responses of these animals are displayed in panels (C–E) (see Supplementary Figure 3 for all animals). Overexpression of Cabp2 (or eGFP) in WT animals resulted in increased tone burst ABR thresholds (yellow asterisk). See Tables 1B,C for the statistical analysis. (C) Increased average ABR responses to an 80-dB click stimulus (presented at 20 Hz repetition rate) in Cabp2–/– animals injected with PHP.eB-Cabp2. (SP, summating potential; I-IV, ABR waves I-IV) (D,E) ABR wave I (D) and…

FIGURE 4. PHP.eB-calcium-binding protein 2 (Cabp2)-mediated restoration of IHC CaV 1.3 channel inactivation kinetics. (A) Representative brightfield and corresponding fluorescence image showing eGFP-expressing IHCs of a 5-week old Cabp2–/– animal injected with PHP.eB-Cabp2. Scale bar = 10 μm. (B) Current-voltage relationships show slightly decreased calcium current amplitudes in IHCs of injected Cabp2–/– animals. Recordings were performed at 33 ± 0.1°C with 1.3 mM Ca2+ in the bath solution. Cells were held at a slightly depolarized voltage of −65 mV. (C) Average absolute and (D) normalized calcium current responses elicited by 500-ms depolarization steps to the peak calcium current potential. Cabp2-deficient IHCs show significantly enhanced calcium current inactivation that recovers almost completely upon virally induced expression of Cabp2. (E) Residual calcium currents after 500 ms of ongoing depolarization (Student’s t-test or Wilcoxon rank test; p < 0.05; asterisks; see also main text). (F) Representative calcium current traces (top) and respective capacitance increments (ΔCm; bottom) of non-injected Cabp2+/+, Cabp2–/– IHCs and IHCs from PHP.eB-Cabp2-injected Cabp2–/– animals upon…