G5X Alport Syndrome mouse model hearing characterization for a future preclinic application of CRISPR/Cas9

Background. Alport syndrome (AS) is a rare genetic disorder due to mutations in the COL4A3/A4/A5 genes that code for collagen type IV heterotrimer, a key structural component of the basement membrane of the kidney (GBM), eye and inner ear. Auditory involvement in Alport syndrome shows a progressive sensorineural hearing loss with varying degrees of severity. To date, no effective therapies are available to prevent or treat the hearing loss related to AS. The purpose of our study is to provide a characterization of the cochlear susceptibility in the Alport X-linked mouse model - G5X, currently available at the IRET Foundation under the supervision of Prof. Laura Calzà - with the aim of defining its peculiarities. Indeed, an audiological characterization of the G5X mouse model of Alport syndrome has not been performed before to our knowledge. The final aim would be to determine whether such a model may present cochlear auditory and structural features that admit the possibility of administering a CRISPR/Cas9-based gene therapy using AAVs (adeno-associated viruses) via a localized intratympanic injection, to evaluate its safety and efficacy in preventing the onset of sensorineural hearing loss related to the X-linked form of Alport syndrome, through the activation of the molecular correction mechanism on affected cochlear structures. Materials and Methods. To carry out this study, we compared the results of hearing threshold levels of 10 Alport G5X mice and 11 wild-type (WT) mice to the audiological test of ABR (Auditory Brainstem Response). All mice were 4 months old. ABR examinations were performed under basal conditions and after a noise-exposure to a high-intensity sound for a predetermined time of 30 minutes to evaluate the cochlear impairment related to noise exposure using the noise-induced hearing loss (NIHL) related to a temporary threshold shift phenomenon. Results. The results obtained from the evaluations by ABR pre- and post-exposure to noise showed that Alport G5X mice exhibit greater cochlear fragility toward high intensity sounds with a higher transient hearing threshold shift (TTS). At the baseline, the Alport group and the WT-type group showed similar results of hearing thresholds with ABR hearing threshold at 39.05.7 dB for the Alport group and 39.13.2 dB for the control group (p=0.9634). After noise-exposure, both groups showed a worsening of the hearing threshold but in the Alport group, the ABR threshold were significantly higher compared to the control group (Alport: 71.511.6; WT: 476.7, p<0.0001). The transience of this phenomenon was observed by repeating the ABR at least 30 days after noise exposure. Conclusions. G5X mouse model does not show hearing impairment at the baseline and may not develop deafness during their lifetime. Using noise exposure, we showed that the Alport mouse was more prone to cochlear damage induced by loud sounds: this behavior may be related to a more fragile cochlear structure, caused by the genetic defect in the structure of the type IV collagen. The results obtained, therefore, indicate that the Alport G5X mouse model may represent a reference for the study of potential gene therapies for the treatment of hearing loss associated with Alport syndrome. A subsequent study evaluating auditory evoked potentials before and after auditory stimulation with high-intensity sounds in subjects who received intratympanic administration of the CRISPR/Cas9-based therapy could confirm its efficacy by showing a reduction in cochlear susceptibility and reduction in the difference in TTS compared with wild-type mice.