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.

A single-cell level comparison of human inner ear organoids with the human cochlea and vestibular organs

“…Apparently healthy inner ear tissue was collected after elective termination of pregnancy by vacuum aspiration as previously described. 130 Embryonic or fetal age was determined by obstetric ultrasonography prior to termination, i.e., gestational age minus two weeks, with a standard error of two days. No other characteristics were documented in line with legislation and medical ethical approval.…”

Engineering of the AAV-Compatible Hair Cell-Specific Small-Size Myo15 Promoter for Gene Therapy in the Inner Ear

“…Generally speaking, AAV1, AAV2/1, AAV5, AAV8, AAV9, AAV-PHP.eB, AAV2.7m8, AAV-ie, and AAV-ie-K558R can consistently transduce IHCs in mice, and some AAV variants have high transduction efficiency [ 34 , 35 , 55 – 57 ]; for example, AAV-PHP.eB, Anc80L65, AAV-ie, AAV2.7m8, and AAV-ie-K558R consistently transduce OHCs with a relatively high efficiency [ 34 , 35 , 55 – 57 ], while AAV.DJ, AAV-ie, AAV2.7m8, and AAV-ie-K558R transduce SCs with high efficiency [ 33 – 35 , 55 – 57 ] and AAV1 and AAV2/1 can transduce cells in spiral ganglion neurons [ 18 , 58 ]. In addition, several studies have demonstrated that AAV9-PHP.B and AAV-S efficiently transduce IHCs, OHCs, and SCs in nonhuman primates [ 59 , 60 ] and that AAV9-PHP.B can deliver GFP to human cochlear HCs and vestibular HCs [ 61 ]. While these findings show that some AAV variants transduce cochlear cells in an efficient manner, the cell-type specificity of these AAV variants need to be further improved.…”

Systemic administration of novel engineered AAV capsids facilitates enhanced transgene expression in the macaque CNS

“…For example, while PHP.B and PHP.eB are widely regarded to have a low potential for therapeutic use—at least in the context of systemic administration—they have proven useful in a variety of research applications. These two vectors have recently been used for purposes as diverse as gene therapy in human inner ear explants, 51 delivering shRNA to elucidate the role of SPC25 in microglial outgrowth, 52 and a novel method for retrograde tracing to study neural circuits. 53 The properties of our mouse-specific CNS-tropic vectors, which have the added benefit over PHP.B and PHP.eB of being active in multiple mouse strains, may prove useful in similarly varied applications.…”