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.


| INTRODUCTION
Gene transfection is a critical procedure in both genetic studies and gene therapy. Gene transfection methods can be divided into two categories: non-viral and viral. Viral methods of gene transfection are more efficient, despite recent rapid progress in non-viral gene transfection methods. [1][2][3][4][5][6][7][8][9][10] Among what have been tested, recombinant adeno-associated viruses (rAAV) exhibit clear advantages such as low immunogenicity, long-lasting transfected gene expression in various host cells, and non-exogenous DNA insertion into the genomes of transfected cells. 3 This viral vector has been used in the gene therapy studies of auditory system in animal models [11][12][13][14][15] and human trials. 11,16 The inner ear is highly isolated from surrounding organs and tissues. This unique feature makes it an ideal organ for genetic manipulation, with a low risk of side effects. However, this feature also makes it difficult to access. Generally, rAAV vectors must be injected into the inner ear, either via the round window membrane (RWM) or by cochleostomy. However, the injection disrupts the integrity of the inner ear, and might impair the hearing function.
The RWM has been explored as an approach to deliver drugs to the inner ears. 17,18 Unfortunately, the intact RWM is not permeable to rAAVs, 19 and therefore rAAV-mediated gene transfection via the RWM requires an injection. 16,[20][21][22] This barrier could be overcome by increasing the RWM permeability temporarily. In one of our previous studies, we reported that this could be realized by treating the RWM with digestive enzymes. 23,24 Consequently, temporary RWM damage allows the rAAV to diffuse across the RWM. Since the treatment itself does not cause hearing loss, this method has a potential in cochlear gene therapy for the purpose of protection. However, the effectiveness of this treatment varied among individual subjects, likely due to variations in the RWM thickness and local tissue reactions to the enzyme solution.
Ultrasound-mediated microbubble (USMB) cavitation can create small pores on the cell membrane (sonoporation). [25][26][27] This temporary injury significantly increases the permeability of the cell membrane to large molecules. 28,29 The wound created by the USMB cavitation is self-healable, 30 and therefore such treatments do not permanently impair the normal functions of the treated cells. In addition to medication delivery, 26,31 the use of USMB cavitation for gene transfection via plasmid DNA, siRNA, and miRNA has been investigated. 27,28,32 USMB-mediated AAV gene transfection in the rat retina has also been reported. 33 In that application, however, AAV was injected into the subretinal space before USMB was applied. Such an approach is not safe if applied in cochlear gene transfection.
Two studies have applied the USMB method for drug delivery via the RWM. 29,34 USMB effectively increased the permeability of the guinea pig RWM to large molecules such as biotin-FITC. 29 This method successfully facilitated the delivery of dexamethasone across the RWM and protected the cochleae against noise damage. 34 However, the US probes used in these studies had a diameter of 6 mm.
Such a large probe could not be inserted near the RWM even in human's ears. The long working distance needs a larger amount of working solution and a higher acoustic power, which may be potentially harmful.
In addition, no previous study has investigated the usefulness of this method for AAV-mediated gene transfection via the RWM. As viral vectors are highly expensive, MB-vector packaging or coadministration of virus with MB appears to be impractical. In addition, the packing of vectors into MBs may deteriorate the activity of the virus. Therefore, the viral vector must be administered after USMB was applied to the RWM. This required that the wound would not be sealed quickly. Up to date, there is no data whether the damage by USMB will last. In one study, the sonoporation created by a single shot of USMB was healed in seconds. In other study, RWM damage by USMB was observed with electron microscopy with information how long the wound will be recovered. 35 In this study, we developed a new ultrasound probe with a considerably smaller diameter (1.5 mm). By using this small probe, we were able to create intense, focalized damage to the RWM with a lower ultrasound power and a smaller amount of MB solution. The damage was limited to the outer epithelial layer of the RWM and lasted for more than a day. Effective eGFP gene transfection was observed when rAAV-eGFP was administered after USMB treatment.
Additionally, a new-generation rAAV vector (AAV2/Anc80L65) was used to get satisfactory transfection. 36,37 This approach should be useful for the future development of cochlear gene therapies and the translation to humans.

| Animals and research design
Twenty-seven 2-month-old male guinea pigs (albino Hartley) were obtained for this experiment from Shanghai Songlian Lab Animal Field (Shanghai, China) with body weight between 250 and 350 g. All animals passed Preyer's reflex test, an otoscope inspection and a baseline hearing evaluation with an auditory brainstem response (ABR) test.
The guinea pigs were then randomly assigned into six different groups To evaluate the structural changes in the RWM by USMB, the middle ear was filled with the fixative immediately after ultrasound treatment to fixed the RWM. The cochlea was further fixed after the animal was sacrificed. To evaluate AAV transfection, the animals were subjected to a repeated ABR after a 2-week interval prior to sacrifice.
The cochleae were harvested and treated, to investigate either the structure of the RWM or the transfection of AAV across the neuroepithelium. All the experimental procedures were approved by the Institutional Animal Care and Use Committee of the Shanghai Sixth People's Hospital affiliated to Shanghai Jiaotong University (permit number DWLL2017-0295).

| ABR recording
The animals were anesthetized via an intraperitoneal injection of ketamine and xylazine (40 and 10 mg/kg, respectively) and placed on a thermostatic heating pad to maintain the body temperature at~38 C.
The ABR tests were performed in an acoustically and electrically

| Surgery for gene transfection or RWM treatment
The subjects were anesthetized with inhaled isoflurane (4% for induction, 2% for maintenance, 0.3 L/min O 2 flow rate). The animal's head was placed in the lateral position and fixed with a stereotaxic restraint.
The body temperature was maintained using a thermostatic heating pad at 38 C. The animal was laid laterally, and the head was held in position using a custom-made holder ( Figure 3a). The tympanic bony bulla was exposed using a post-auricular approach. After administering local analgesia with lidocaine, a 2 cm arc incision was made along the root of the earlobe, and the mastoid was exposed via blunt dissection.
A hole with a diameter of 3-4 mm was made on the bulla to expose the RW niche and the bony cochlear wall. Next, the animal was laid in the lateral supine position, and the head orientation was adjusted such that the RW surface faced up ( Figure 3b). The ultrasound probe was inserted into the correct position against the RW niche with assistance from a manipulator. The lower edge of the probe front was placed on the RW niche ( Figure 3c). The estimated distance between the front surface of the probe lens and the RW was 0.5-1 mm (Figure 3c).
For the USMB treatment of the RWM, the ultrasound contrast agent (Definity, USA, DIN:02243173) was prepared and injected into the RW niche to fill the space between the probe lens and RWM completely.
The US generator was turned on to yield 5 min of sonication. After the US exposure, the MB solution was suctioned, and the middle ear cavity was irrigated with sterile saline and the residual solution was cleaned.
To observe the damage created by the USMB treatment, a fixative solution (2.5% glutaraldehyde) was used to fill the middle ear cavity immediately after washing. The animal was then sacrificed with an overdose of injected pentobarbital (100 mg/kg, i.p.). The animal was then decapitated under deep anesthesia, and the cochlea was harvested.
F I G U R E 1 Ultrasound probe and acoustic measurements. (a) Photograph of the tip of the finished ultrasound probe, showing the aluminum lens and copper layer. (b) Impedance and phase response curves. The resonance peak at 1.55 MHz (indicated by the arrow, i.e., phase peak) was targeted for probe activation by the pulse pattern. (c) Raw acoustic pressure waveform at a fixed distance of 5.8 mm from the probe after electrical activation. This distance allowed the complete distinction of the acoustic response from the electrical stimulus artifact. The probe was stimulated with five cycles of ±30 V square waves at the indicated frequencies. (d) The data from (c) are presented after low-pass filtering with a cutoff of 3.5 MHz. The greatest filtered amplitude was observed at 1.70 MHz, and this frequency was selected as the best MB cavitation frequency for the probe. (e) The peak negative pressure over a volume was recorded in response to a 1.70-MHz stimulus. The maximum axial slice is shown. (f) The data from E are shown after low-pass filtering of the waveform at each location as in (d) relative to (c) For AAV transfection via the RWM, a piece of gelfoam was placed in the RW niche after the US treatment. Ten microliters of an AAV solution were injected into the gelfoam. For AAV transfection via cochleostomy, a small hole (diameter: 0.3 mm) was drilled via the bone shell of the basal turn. Ten microliters of viral vector were injected into the scala tympani (rate: 20 nL/s) through a 34-gauge glass tip (microfil) connected to a picrosyringe pump (Micro4; WPI, Kissimmee) by a polyethylene tube. The cochleostomy hole was then sealed with muscle tissue, and the hole of the bulla was closed by suturing the muscle and skin.
The adapted AAV2/Anc80L65 backbone was similar to the vector in a previous report. 36,37 The rAAV vector was constructed to carry an AAV2 ITR-flanked genome encoding CAG-driven eGFP, a Woodchuck Hepatitis Virus Regulatory Element (WPRE) and a bovine Growth Hormone poly-adenylation site (Taitool Bioscience, China).
The vector was presented at a titer of 1.16 × 10 13 .

| Statistics
All data are expressed as means ± SE of the mean (SEM). ANOVAs followed by post hoc testing (Holm-Sidak method) were performed using SigmaPlot (ver. 14; Systat Software Inc., San Jose, CA). In all analyses, p < 0.05 was taken to indicate statistical significance. In all analyses, a p value <0.05 was considered to indicate statistical significance. In the RWM observed immediately after the USMB treatment, a focused region could be identified as being damaged, while the other regions appeared to be normal. The damaged region took approximately 1/3 of the total RWM area and located anteriorly. A square region was circulated in Figure 4d, which was magnified in E and F to show the detail of the damaged epithelial layer. The damaged cells frequently contained round-shaped, scar-like structures, which were likely the residuals of large MBs. Figure 4g is also a magnified image of Figure Figure 5 also showed the image of a control sample observed immediately in which RWM was only exposed to ultrasound but no microbubbles (Figure 5g-i), the epithelial layer of RWM was nearly intact in those images.  (Figure 6d-f) however, the continuity of the outer epithelial was interrupted (as shown in the white cycle in Figure 6d). This interruption of intercellular continuity was not extended to the deeper layers.

| ABR threshold
ABRs were tested to examine the hearing threshold at the baseline (i.e., before surgery) and 2 weeks after the transfection surgery. The results in Figure 7 show that AAV delivery via the RWM after USMB treatment does not cause a shift in the ABR threshold (Figure 7a). In contrast, a small ABR threshold elevation was observed in subjects treated with cochleostomy, in which the post hoc pairwise test revealed a significant threshold shift at 16 kHz (Figure 7b) relative to the baseline (q = 3.336, p = 0.023). A significant between-group difference was revealed by a two-way ANOVA (F 1,48 = 6.391, p = 0.015).

| Short summary
In this study, we used a homemade ultrasound probe with a transducer diameter as small as 1.5 mm. When the probe was inserted against the RWM niche, we managed to maintain the structural integrity of middle ears necessary for good hearing.
USMB-mediated cavitation caused controllable, focused, and reversible RWM damage (Figures 4 and 5) that was limited to the outer epithelial layer (Figure 6). This treatment effectively increased the permeability of RWM to the rAAV, which could not normally pass across the RWM to transfect cochlear cells (Figure 8c,d), resulting in the satisfactory transfection of cochlear sensory cells (Figure 8a and 9) by using AAV2/Anc80L65. Although the RWM approach yielded a lower transfection rate than cochleostomy, the former approach did not affect the hearing thresholds of treated animals. The small probe size allowed us to insert the probe in touch with the RWM niche in the guinea pig ear. Therefore, the estimated distance between the probe lens and the RWM was within 0.5-1 mm (Figure 3c). At this distance, our tests indicated that the peak negative acoustic pressure delivered by our probe typically reached 0.3-0.5 MPa, and MI adjusted to 0.5. Placing the probe against the RW niche enabled a much better focus on the target and required a reduced device output, as much less energy was lost to attenuation.
The focalized damage to RWM was shown in both Figures 4d and 5a. In fact, in all the sample treated with USMB, the damage was limited to an oval shape region in the anterior 1/3 of RWM. This is well corresponding to the pointing direction of our probe as shown in

| USMB methods for cochlear drug delivery
Collectively, only two studies published by one group have addressed the use of USMB in drug delivery across the RWM. 29,34 In these reports, the ultrasound probe diameter was 6 mm. Moreover, the probe was placed outside the middle ear, which must then be filled fully with MB solution. The estimated distance between the front surface of the probe and the RWM was 5 mm. The acoustic intensity was 1-3 W/cm 2 , which corresponded to a MI of 0.147-0.283. In this setting, USMB considerably enhanced the transportation of biotin-FITC, which can permeate the intact RWM. The integrity of the RWM in response to this USMB treatment method was reported more recently in a separate paper by the same group. 35 4.4 | USMB in rAAV-mediated cochlear gene therapy US has long been recognized as a useful tool for targeting material delivery for therapeutic applications, including gene transfection (see reviews 26,31,32,[39][40][41]. MBs have been used as imaging enhancers since the 1990s. 41,42 Shortly thereafter, the application of MBs was extended to therapeutic areas. 43,44 Several potential mechanisms have been proposed to explain how USMB methods enhance cell permeability and drug uptake. Depending on the magnitude of the US driving pressure, the MB response may shift from linear spherical to nonlinear or nonspherical oscillations and eventually to inertial cavitation. 31 At a driving pressure greater than 300 kPa, the fluid inertia will overcome the pressure inside the MBs, resulting in bubble collapse and/or fragmentation. 45,46 The surrounding cells exposed to the shock waves and jet formation associated with cavitation can incur damage ranging from small and temporary pores (~1 μm in diameter), which heal quickly, 30,[47][48][49][50] to large damage (>10 μm) associated with cell death. 49

| MB selection and RWM damage
MBs typically have diameters of 1-10 μm and comprise a gas core and lipid shell. For clinical applications, several features of MBs, such as high biodegradability, low immunogenicity, sufficient flexibility and stability, are of concern. Regarding cochlear gene transfection, the ability of MB to create RWM damage that would be sufficiently persistent but healable is the major concern. MB properties such as the shell material, size, and concentration are important because each may affect the induction of inertial cavitation under ultrasonic exposure. 57,58 Lipids, proteins, polymers, or a combination of these materials have all been used in the shells of MBs. MBs coated with lipids are among the most interesting and frequently used formulations in studies associated with drug delivery. [59][60][61][62][63][64] The MB cavitation effect appears to be related to the size and total volume of MBs in the solution. One study reported that both the inertial cavitation dose and the BBB opening volume were positively correlated with the diameters of the MBs. 65 However, another study reported that the microbubble gas volume dose, not the size, determined the effect. 61

| Safety concerns
In this study, the intense damage caused by the application of USMB was limited to the RWM epithelial cells facing the tympanic cavity but was not extended to the deeper layers. In one of our previous reports, the RWM could be damaged using digestive enzymes. 24 In this study, the damage was also limited to the outer epithelial layer. Functionally, we observed no hearing losses in subjects treated with either RWM digestion in the previous study or with USMB in the present study.
These results suggest that the application of USMB to the RWM is a safe method for cochlear gene transfection. Moreover, the USMB method is more controllable than our previously reported digestion method.
Other than RWM approach, cochleostomy and canalostomy have been evaluated for cochlear gene transduction by AAV. In the best scenario, cochleostomy can achieve a safe cochlear gene transfection mediated AAV in large animal model like guinea pig with less than 10 dB threshold shift. However, such a good hearing reservation is difficult to be reached in adult mice in cochleostomy. 15 AAV injection via canalostomy can effectively infect cells of cochleae and vestibular organs in neonatal mice without significant hearing loss. 11,14 However, the great recovery ability of neonatal mouse cochlea after intense surgical injury is not likely duplicable in adult mice. More importantly, both canalostomy and cochleostomy are less likely to be translated in human cochlear gene therapy, especially for the protection purpose in which hearing reservation is critical. Unlike rodents, human inner ears are deeply embedded in the temporal bone. Both cochleostomy and canalostomy require intense surgery, and likely risky in causing hearing loss. In humans, RWM approach is the only one that has been utilized for inner ear drug delivery (e.g., in the treatment of sudden sensorineural hearing loss and Meniere's disease 67,68 ).

| Limitations and future improvements
In this study, we compared the transfection efficiencies between the USMB-RWM approach and the cochlear injection of virus via cochleostomy. A slight hearing loss was observed in the subjects after cochleostomy but not after USMB. However, the transfection rate was significantly lower in the USMB group than in the cochleostomy group ( Figure 9). While our focus is on cochlear gene transfection in this study, RWM approach is likely useful for gene transfection in the vestibular system, considering the fact that RWM is closer to vestibule than cochlea. We intend to evaluate this potential in our further study especially after the delivery system is optimized. Several possibilities for further improvements are under consideration. The first possibility involves the use of a smaller probe. Although the 1.5 mm probe allowed us to place the probe on the ring of the round window niche, the surgery required to open the area for access remains quite invasive. The reduction of the probe size to 1 mm would enable the surgery to be performed more easily, and the probe could be inserted into the niche along with a smaller amount of MB solution. However, the difficulty of manufacturing these devices increases as the diameter decreases. The second improvement involves the packaging of the rAAVs into MBs or the coadministration of rAAVs with the MB solution. A reduced probe size would make this approach possible. The third improvement involves the use of recently reported novel AAVs that have a higher transfection rate. [11][12][13][14]16,69,70 We believe that with these improvements, USMB-mediated cochlear gene transfection via the RWM would become a useful tool that could be translated into human clinical applications.