Efficient Viral Transduction in Fetal and Adult Human Inner Ear Explants with AAV9-PHP.B Vectors

Abstract

Numerous studies have shown the recovery of auditory function in mouse models of genetic hearing loss following AAV gene therapy, yet translation to the clinic has not yet been demonstrated. One limitation has been the lack of human inner ear cell lines or tissues for validating viral gene therapies. Cultured human inner ear tissue could help confirm viral tropism and efficacy for driving exogenous gene expression in targeted cell types, establish promoter efficacy and perhaps selectivity for targeted cells, confirm the expression of therapeutic constructs and the subcellular localization of therapeutic proteins, and address the potential cellular toxicity of vectors or exogenous constructs. To begin to address these questions, we developed an explant culture method using native human inner ear tissue excised at either fetal or adult stages. Inner ear sensory epithelia were cultured for four days and exposed to vectors encoding enhanced green fluorescent protein (eGFP). We focused on the synthetic AAV9-PHP.B capsid, which has been demonstrated to be efficient for driving eGFP expression in the sensory hair cells of mouse and non-human primate inner ears. We report that AAV9-PHP.B also drives eGFP expression in fetal cochlear hair cells and in fetal and adult vestibular hair cells in explants of human inner ear sensory epithelia, which suggests that both the experimental paradigm and the viral capsid may be valuable for translation to clinical application.

Keywords: AAV; AAV9-PHP.B; ampulla; cochlea; gene therapy; hair cell; hearing loss; inner ear; saccule; utricle; vestibular.

Figure 1

The adult bony labyrinth, right ear. Adult vestibular specimens were collected from the opened semicircular canals (black arrow, left panel) and after further drilling of the vestibulum (black arrow, right panel). EAC, external auditory canal. LSC, lateral semicircular canal. PSC, posterior semicircular canal. SCC, superior semicircular canal. Te, tegmen.

Figure 2

Human adult and fetal dissected inner ears. (A) Adult utricle. (B) Adult ampulla. (C) Fetal cochlea, W14. (D) The otic capsule was removed to expose the modiolus and organ of Corti. (E) Fetal saccule, W14. Scale bars: 1 mm.

Figure 3

Experimental setup. Tissues were dissected and transferred to a flat cap of a PCR tube. Viral vector was added to the medium in a concentration of 1:10. After 48 h of incubation, 4 mL of medium was added, followed by fixation on day 4. FMC: full media change.

Figure 4

Overview of transduced adult utricle, immunostained with anti-MYO7A antibodies. Transduction was effective, as indicated by eGFP expression throughout the specimen. Scale bar applies to all images: 200 µm.

Figure 5

Details of transduced adult utricle (Utr) and ampulla (Amp), immunostained with anti-MYO7A antibodies. Cell counts were carried out on data from 2 ampullas and 3 utricles. Scale bars apply to all images in each row: 50 µm.

Figure 6

Details of transduced fetal organ of Corti (OC) showing inner and outer hair cells, respectively (IHC, OHC), and a fetal saccule (Sacc) and utricle (Utr), immunostained with anti-MYO7A antibodies. Fetal age = 14 weeks. Scale bars apply to all images in each row: 50 µm.

Figure 7

Transduction efficiency. The percentage of eGFP-positive hair cells was quantified by three blinded investigators. Cell counts on adult specimens were carried out on data from two ampullas and three utricles. Cell counts on fetal specimens were carried out on data from two cochleas and three utricles. Each black dot represents the mean of three individual cell counts. Bars represent the mean for each end organ, as indicated on the x-axis. eGFP-positive hair cells (%): adult ampulla, 58%; adult utricle, 63%; fetal cochlea, 37%; fetal utricle, 59%.