“Inner Ear Drug Delivery Route Selection: A Cross-Species Translation Framework." Sébastien Diot, Molly Kalker, Marion Souchal, Carolanne Coyat, Aurore Marie, Elodie Flaszka, Véronique Baudoux, Camille Dejean, Sylvie Pucheu, Gaëlle Naert, Benny Muraj, Ellie Khadir, Donald Hodges, Rami Tzafriri, Misty J Williams-Fritze.

Background: Gene or cell therapy targeting inner ear represents a challenge due to the blood-labyrinth barrier, necessitating local delivery approaches, especially for expensive therapy with limited therapeutic range, or, susceptibility to systemic side effects. This is particularly relevant for viral gene transfer and cell-based therapies. Adeno-associated virus (AAV) vectors were selected for inner ear applications due to their favorable safety characteristics, broad cellular targeting capability, and sustained transgene expression. This study aimed to compare different intracochlear (IC) injection techniques in mice, guinea pigs, and mini-swine.

Methods: Anatomical differences limit the options for IC for each species, Mice were administered via posterior semicircular canal (PSCC) and round window (RW) injections, while guinea pigs received cochleostomy infusion, RW injection (RWI) or RW deposit (RWD) of the therapeutic compound. For swine, RWI are already being performed in laboratories and transtympanic injection (TT) is still a relevant approach to evaluate diffusion across the RW membrane. Auditory function effects of IC administration were evaluated in both species via auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) measurements. Following AAV delivery, GFP (Green Fluorescent Protein, biological marker to visualize gene expression or protein localization) expression from AAV-mediated cell transfection enabled the identification of transfected cochlear regions.

Results: All injection methods successfully targeted hair cells, although transfection patterns and intensity varied depending on the method and substance used. AAV injection provided efficient transduction via both the round window (RW) and posterior semicircular canal (PSCC) routes in mice. Finally, the impact on auditory function differed between injection methods, with PSCC, intracochlear (IC), and transtympanic (TT) approaches showing less effect on DPOAE amplitudes and ABR thresholds compared to the RW route.

Conclusions: This study highlights the importance of refined intracochlear delivery method in animals according to the delivered product and targeted inner ear disorders. While all methods effectively reached cochlear hair cells, variations in transfection patterns, product concentration at targeted cells, and effects on auditory functions, highlight the need for careful consideration of injection strategies. It is worth mentioning that PSCC and cochleostomy routes, though effective in animals, lack translational relevance to humans. Developing these methods in swine (a large- species) would expand research opportunities while avoiding the use of non-human primates, while fostering more ethical practices. Moving forward, further research into optimizing delivery methods will be essential for advancing therapeutic interventions in auditory disorders.