Cochlear Function and Transgene Expression in the Guinea Pig Cochlea, Using Adenovirus- and Adeno-Associated Virus-Directed Gene Transfer

Abstract

Development of a viral vector that can infect hair cells of the cochlea without producing viral-associated ototoxic effects is crucial for utilizing gene replacement therapy as a treatment for certain forms of hereditary deafness. In the present study, cochlear function was monitored using distortion-product otoacoustic emissions (DPOAEs) in guinea pigs that received infusions of either (E1(-), E3(-)) adenovirus, or adeno-associated virus (AAV), directly into the scala tympani. Replication-deficient (E1(-), E3(-)) adenovirus-directed gene transfer, using the cytomegalovirus (CMV) promoter, drove transgene expression to inner hair cells and pillar cells of the cochlea. AAV transduction was tested with several promoters, such as platelet-derived growth factor (PDGF), neuron-specific enolase (NSE), and elongation factor 1alpha (EF-1alpha) promoters; which drove transgene expression to cochlear blood vessels, nerve fibers, and certain spiral limbus cells, respectively. AAV transgene expression was visualized by green fluorescent protein immunostaining. Immunocytochemistry to heparan sulfate confirmed the absence of proteoglycans in guinea pig hair cells, indicating that the receptor for AAV was not present on these cells. However, the heparan sulfate proteoglycan expression pattern mimicked the AAV transduction pattern. An overall finding was that cochlear function was not altered throughout the infection period using AAV titers as high as 5 x 10(8) IP/infused cochlea. In contrast, cochlear function was severely compromised by 8 days postinfection with adenoviral titers of 5 x 10(8) PFU/infused cochlea, and outer hair cells were eliminated. Thus, cochlear hair cells are amenable to in vivo gene transfer using a replication-deficient (E1(-), E3(-)) adenovirus. However, replication-defective or gutted adenovirus vectors must be employed to overcome the ototoxic effects of (E1(-), E3(-)) adenovirus vectors.

Coxsackie adenovirus receptor and ανβ3/ανβ5 integrins in adenovirus gene transfer of rat cochlea

“…Indeed, gene expression in the sensory hair cells and strial cells has previously been reported using the same syringe inoculation protocol. 10 Intriguing, however, are the results of Luebke et al, 8,9 who reported that 7 days of delivery of adenovirus into the basal turn perilymph via an osmotic minipump led to preservation of hearing and b-gal expression specifically in the sensory hair cells, but not into the adjacent structures such as the supporting cells. In Accordance with Ishimoto et al, 6 who was unable to replicate this result, we suggest that the method used to detect b-gal expression may account for this unexpected result.…”

“…6,7 However, other studies have demonstrated that adenovirus vectors can indeed transduce sensory hair cells in the organ of Corti. [8][9][10] So far, no explanation has been provided to explain this discrepancy.…”

A modified adenovirus can transfect cochlear hair cells in vivo without compromising cochlear function

“…[3][4][5][6] HSV, vaccina virus, lentivirus, and AAV vectors have also failed to infect cochlear hair cells when tested in vivo. [7][8][9] Interestingly, replication-deficient adenoviral vectors (ie E1 − , E3 − ), using the Rous sarcoma virus (RSV) promoter to drive LacZ expression, have failed to transduce cochlear hair cells in vivo when approximately 10 7 plaque-forming units of virus were injected into scala tympani. 10 In addition, the studies using adenoviral constructs to infect cochlea cells in vivo, either did not assess cochlea function, or used a functional test (ie auditory brainstem response or ABR) that evaluated principally the ascending auditory system at the auditory nerve and brainstem level, thus, only grossly examined hair cell function.…”

Treatment of peripheral sensorineural hearing loss: gene therapy

“…38 AAV vectors can accommodate DNA of 3.5-4.0 kilobases (kb), 18 sufficient to accommodate the DNA (o3 kb) encoding cytokines and neurotrophins. 37 AAV vectors are less toxic than Ad vectors, 39 although damage to cochlear architecture has been observed in a 24-day study. 31 Like Ad vectors, there is variation in expression profiles among experiments.…”