Hearing Preservation Following Repeated Adenovector Delivery.

Factors influencing the damage to the inner ear can include the surgical approach used for vector delivery, the volume of vector used, the buffer that the vector is suspended in as well as the host response to the vector capsid and vector genes that are transferred. We evaluated the effect of Ad5 capsid adenovectors on hearing function after delivery to the perilymph of adult C57Bl/6 mice. Hearing was evaluated before surgery and 3 days post-surgery by auditory brain stem response (ABR) and distortion product otoacoustic emissions (DPOAE). A second group of mice underwent repeated delivery of adenovector two times to determine if a preliminary exposure to an Ad vector could induce an inflammatory response leading to further loss. The first adenovector (Ad.11D.LacZ) was delivered to the posterior semicircular canal or via round window. In the second surgery, a second adenovector (Ad.11D.gfp) was delivered to the horizontal semicircular canal. The functional outcome was tested prior, 7 days post first vector delivery, and 3 days post second vector delivery via ABR and DPOAE. Dual delivery via the semicircular canals resulted in preservation of hearing suggesting that pre-exposure to Ad5 capsid does not predispose to hearing loss. Delivery to the round window resulted in hearing loss that was worsened after the second vector delivery, suggesting that delivery route and prior injury to the inner ear rather than the repetition of delivery predisposes to further hearing loss. Anat Rec, 303:600-607, 2020. © 2019 American Association for Anatomy.

Canalostomy as a surgical approach for cochlear gene therapy in the rat.

This article presents a unique approach for the delivery of gene therapy vectors into the cochlea of the laboratory rat. Mice and guinea pigs are established in vivo models for cochlear gene therapy each of which has distinct advantages and disadvantages. The rat has some of the molecular advantages of a mouse model combined with size advantages for surgical approaches. Vector delivery via cochleostomy or injection through the round window causes concomitant sensorineural hearing loss and is therefore not suitable for studies where the change in hearing is being followed. Compared to the mouse, the rat does not demonstrate easily recognizable landmarks that allow for use of the semicircular canal as an approach to the inner ear. We analyzed sagittal and coronal temporal bone sections of Long Evans rats and identified the bony entrance of the facial nerve as a crucial landmark for canalostomy. Auditory brainstem response and distortion product otoacustic emission measurements revealed minimal differences in the hearing threshold after adenovirus vector application when large volumes of vector were infused to the inner ear. Canalostomy and infusion of adenoviral vectors also resulted in temporary balance disturbance in the rat. Immunohistochemical assessment after delivery of a green fluorescent protein expressing vector showed significant GFP expression in the cochlea.

Bcl-2 gene therapy prevents aminoglycoside-induced degeneration of auditory and vestibular hair cells.

To evaluate the protective effects of bcl-2, we have developed an in vivo model of gentamicin ototoxicity in C57BL/6 mice using intratympanic delivery of gentamicin. Hair cell survival was evaluated using myosin VIIa immunohistochemistry, cytocochleogram and auditory brainstem response (ABR) testing. At 10 days after gentamicin application, a consistent loss of outer hair cells was seen. Mice were pretreated with an adenovector expressing human bcl-2 (Ad.11D.bcl-2) or a control vector (Ad.11D). Seventy-two hours after vector delivery mice were treated with intratympanic gentamicin and evaluated at 10 days after ototoxin delivery. Pretreatment with Ad.11D.bcl-2 resulted in morphologic protection of hair cells and preservation of hearing thresholds measured by ABR.

Adenoviral vectors for improved gene delivery to the inner ear.

An important requirement for gene therapy in the inner ear is to achieve efficient gene delivery without damaging residual inner ear function. This can be achieved by delivering a high concentration of vector in a minimal volume. Adenovectors are well suited to meet these requirements since high quality concentrated vector with a high capacity for a gene payload can be produced. To reduce the number of vector particles and volume of delivery to the inner ear, we tested vectors with enhancements in cell binding and cell entry properties. We compared delivery of a marker gene to the inner ear using two different advanced generation serotype 5 adenovector designs. The first adenovector tested, AdRGD, has a restricted tropism of entry into cells. AdRGD is an Ad5 capsid vector with an arg-gly-asp (RGD) motif built into the adenovector fiber that has also been modified to abolish the fiber-CAR and penton-integrin interactions that provide the normal well characterized two-step entry pathway for adenovirus. The AdRGD vector has enhanced binding to alphanu integrins. The second vector, AdF2K, contains 7 lysine residues within the fiber knob and has been shown to have expanded tropism for cells in vitro and in vivo. AdF2K maintains its normal CAR and integrin receptors interactions and has an additional mechanism of entry via its ability to interact with heparan sulfate. Both vectors demonstrated effective delivery to the inner ear and more uniform labeling of the inner ear sensory epithelia than native capsid vector, when tested in vivo. Analysis of expression efficiency using quantitative PCR was tested in vitro on cultured macular organs and demonstrated that vector delivery with the AdF2K vector design yielded optimal delivery. The present study demonstrates that retargeting strategies can improve delivery to the inner ear.