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 evaluate the feasibility of a characterization of the cochlear susceptibility in the Alport X-linked mouse model, with the aim of defining its peculiarities. Indeed, an audiological characterization of this 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 evaluate if comparison of hearing threshold levels of 10 Alport mice and 11 wild-type (WT) mice to the audiological test of ABR (Auditory Brainstem Response) would be statistically significant. ABR examinations should be 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 would show if Alport mice exhibit greater cochlear fragility toward high intensity sounds with a higher transient hearing threshold shift (TTS). Conclusions. The mouse model does not show hearing impairment and may not develop deafness during their lifetime. Using noise exposure, we could show whether the Alport mouse is 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, therefore, could indicate if the Alport 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.