Ultrasound‐microbubble cavitation facilitates adeno‐associated virus mediated cochlear gene transfection across the round‐window membrane

Abstract

The round window of the cochlea provides an ideal route for delivering medicines and gene therapy reagents that can cross the round window membrane (RWM) into the inner ear. Recombinant adeno-associated viruses (rAAVs) have several advantages and are recommended as viral vectors for gene transfection. However, rAAVs cannot cross an intact RWM. Consequently, ultrasound-mediated microbubble (USMB) cavitation is potentially useful, because it can sonoporate the cell membranes, and increase their permeability to large molecules. The use of USMB cavitation for drug delivery across the RWM has been tested in a few animal studies but has not been used in the context of AAV-mediated gene transfection. The currently available large size of the ultrasound probe appears to be a limiting factor in the application of this method to the RWM. In this study, we used home-made ultrasound probe with a decreased diameter to 1.5 mm, which enabled the easy positioning of the probe close to the RWM. In guinea pigs, we used this probe to determine that (1) USMB cavitation caused limited damage to the outer surface layer or the RWM, (2) an eGFP-gene carrying rAAV could effectively pass the USMB-treated RWM and reliably transfect cochlear cells, and (3) the hearing function of the cochlea remained unchanged. Our results suggest that USMB cavitation of the RWM is a good method for rAAV-mediated cochlear gene transfection with clear potential for clinical translation. We additionally discuss several advantages of the small probe size. K E Y W O R D S cochlea, cochlear gene therapy, gene delivery, Guinea pigs, recombinant adeno-associated virus, round window membrane, ultrasonic microbubbles cavitation Zhen Zhang and Zhengnong Chen contributed equally to this study.

Ultrasound Microbubble–Facilitated Inner Ear Delivery of Gold Nanoparticles Involves Transient Disruption of the Tight Junction Barrier in the Round Window Membrane

“…This is the first study to reveal that the middle and upper third areas of the RWM exhibit the strongest response to cavitation. Furthermore, the size of the affected area accounts for approximately one-quarter of the whole RWM, in agreement with a recent study conducted in guinea pigs that indicated that the damaged region was focused in the one-third area located anteriorly on the RWM (Zhang et al, 2021).…”

“…be 0.207 MI, and applied it in the mouse model. The resultant ultrastructural changes in the outer epithelium were evidently less severe compared with previous US damage observed in a guinea pig model (Lin et al, 2019;Zhang et al, 2021). The physical explanation for this discrepancy lies in the fact that, at the same high frequency of 1 MHz, increases in the sound pressure at certain ranges achieve a higher cavitation activity or strength-the so-called cavitation intensity (Wu et al, 2020).…”

“…*p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA, with post hoc Bonferroni tests). disruption of the outer epithelial cells; this damage occurred both following five courses of USMB sonication at an MI of 0.254 (Lin et al, 2019) and following continuous 5-min USMB sonication at 0.5 MI (Zhang et al, 2021). However, this damage may be limited to only the outer epithelium of the RWM.…”

“…The mechanisms underlying the enhanced permeability of the RWM mediated by USMBs are not yet fully understood, and only a few studies have investigated this phenomenon (Lin et al, 2019;Zhang et al, 2021). USMB-induced cavitation is generally acknowledged to damage the RWM through extensive FIGURE 8 | Series of confocal laser scanning microscope images of the RWM after FITC-CS-AuNP treatment with or without prior USMB exposure.…”

“…The mechanisms underlying the enhanced permeability of the RWM mediated by USMBs are not yet fully understood, and only a few studies have investigated this phenomenon ( Lin et al, 2019 ; Zhang et al, 2021 ). USMB-induced cavitation is generally acknowledged to damage the RWM through extensive disruption of the outer epithelial cells; this damage occurred both following five courses of USMB sonication at an MI of 0.254 ( Lin et al, 2019 ) and following continuous 5-min USMB sonication at 0.5 MI ( Zhang et al, 2021 ). However, this damage may be limited to only the outer epithelium of the RWM.…”