Evaluation of a Model of Long-Term Middle Ear Catheterization for Repeat Infusion Administration and Cochlear Hair Cell Injury in Guinea Pigs.

Middle ear administration has numerous applications, including antibiotherapy and gene therapy, and is increasingly used to target the auditory and vestibular systems. In animal studies, investigating repeated exposure that mimics clinical dosing regimens has remained a challenge due to the lack of suitable models. Intratympanic injections are not suitable for long-term studies due to the increased risk related to tympanic membrane rupture or scarring and repeat anesthesia events. Surgical models of middle ear catheterization previously used have not been reliable for longer than 4 weeks, resulted in elevated stress levels, and have been associated with significant changes related to the surgery and/or the presence of the catheter such as local trauma and inflammatory and degenerative processes. These complications cause decreased hearing/deafness and greatly diminish the value and accuracy of ototoxicity studies. We describe here a procedure that permits repeat dosing into the middle ear of guinea pigs and can be used to produce a model of aminoglycoside-induced hair cell injury. The innocuity of the procedures and the efficacy of the ototoxicity model were confirmed using auditory brain stem response assessment, histopathological evaluation, and cytocochleograms. Procedure-related changes were limited to minimal inflammation in the middle ear.

Preclinical testing of AAV9-PHP.B for transgene expression in the non-human primate cochlea.

In a number of mouse models of hereditary deafness, therapeutic transgene delivery to the cochlea and vestibular organs using adeno-associated viral vectors (AAVs) has shown striking rescue of hearing and balance. However, only a subset of AAV capsids have shown efficacy in transducing both inner hair cells and outer hair cells, and it is also not clear which of these can be translated to treatment of human inner ear. We recently reported efficient transgene expression of a GFP reporter in a non-human primate cochlea, in both inner and outer hair cells, following injection of the AAV9 capsid variant PHP.B via the round window membrane (RWM). However efficiency was poor at a lower dose. To further define the transduction potential of AAV9-PHP.B, we have performed a dosing study in the cynomolgus monkey and assessed vector-encoded GFP expression. Three animals were injected in both ears and four doses were tested. We describe a transmastoid surgical approach needed to access the RWM of this common primate model. We found that AAV9-PHP.B transduced nearly 100% of both IHCs and OHCs, from base to apex, at the higher doses (3.5 × 1011 and 7 × 1011 vector genomes). However, at lower doses there was a steep reduction in viral transduction. Thus, AAV9-PHP.B efficiently transduces the IHCs and OHCs of nonhuman primates, and should be considered as an AAV capsid for inner ear gene therapy in humans.

Gene Transfer with AAV9-PHP.B Rescues Hearing in a Mouse Model of Usher Syndrome 3A and Transduces Hair Cells in a Non-human Primate.

Hereditary hearing loss often results from mutation of genes expressed by cochlear hair cells. Gene addition using AAV vectors has shown some efficacy in mouse models, but clinical application requires two additional advances. First, new AAV capsids must mediate efficient transgene expression in both inner and outer hair cells of the cochlea. Second, to have the best chance of clinical translation, these new vectors must also transduce hair cells in non-human primates. Here, we show that an AAV9 capsid variant, PHP.B, produces efficient transgene expression of a GFP reporter in both inner and outer hair cells of neonatal mice. We show also that AAV9-PHP.B mediates almost complete transduction of inner and outer HCs in a non-human primate. In a mouse model of Usher syndrome type 3A deafness (gene CLRN1), we use AAV9-PHP.B encoding Clrn1 to partially rescue hearing. Thus, we have identified a vector with promise for clinical treatment of hereditary hearing disorders, and we demonstrate, for the first time, viral transduction of the inner ear of a primate with an AAV vector.