Abstract
Adeno-associated virus (AAV) is the preferred vector for gene therapy of hereditary deafness, and different viral serotypes, promoters and transduction pathways can influence the targeting of AAV to different types of cells and the expression levels of numerous exogenous genes. To determine the transduction and expression patterns of AAV with different serotypes or promoters in hair cells and supporting cells in the neonatal mouse cochlea, we examined the expression of enhanced green fluorescent protein (eGFP) for five different types of AAV vectors [serotypes 2, 9, and Anc80L65 with promoter cytomegalovirus (CMV)-beta-Globin and serotypes 2 and 9 with promoter chicken beta-actin (CBA)] in in vitro cochlear explant cultures and we tested the transduction of AAV2/2-CBA, AAV2/9-CBA, and AAV2/Anc80L65-CMV by in vivo microinjection into the scala media of the cochlea. We found that each AAV vector had its own transduction and expression characteristics in hair cells and supporting cells in different regions of the cochlea. There was a tonotopic gradient for the in vitro transduction of AAV2/2-CBA, AAV2/9-CBA, AAV2/2-CMV, and AAV2/9-CMV in outer hair cells (OHCs), with more OHCs expressing eGFP at the base of the cochlea than at the apex. AAV2/2-CBA in vitro and AAV2/Anc80L65-CMV in vivo induced more supporting cells expressing eGFP at the apex than in the base. We found that AAV vectors with different promoters had different expression efficacies in hair cells and supporting cells of the auditory epithelium. The CMV-beta-Globin promoter could drive the expression of the delivered construct more efficiently in hair cells, while the CBA promoter was more efficient in supporting cells. The in vitro and in vivo experiments both demonstrated that AAV2/Anc80L65-CMV was a very promising vector for gene therapy of deafness because of its high transduction rates in hair cells. These results might be useful for selecting the appropriate vectors for gene delivery into different types of inner ear cells and thus improving the effectiveness of gene therapy.
Highlights
Noise, ototoxic drugs, infections, autoimmune diseases, and hereditary factors can cause loss of hair cells (HCs) and/or functional deficiency of HCs and supporting cells (SCs)
Three of the associated virus (AAV) vectors had the CMV-beta-Globin promoter and the Woodchuck hepatitis virus post-transcriptional regulatory element cassette, namely AAV2/2-CMV-beta-Globin-enhanced green fluorescent protein (eGFP) [1 × 1013 viral genomes (VG) per ml], AAV2/9-CMV-beta-Globin-eGFP (1 × 1013 VG/ml) and AAV2/Anc80L65-CMV-beta-Globin-eGFP (2.08 × 1012 VG/ml), and these were purchased from the Biolink Company (Shanghai, China)
When the working titer was increased from 1 × 1011 VG/ml to 1 × 1012 VG/ml, the transduction efficiency of AAV2/2-chicken beta-actin (CBA) in inner hair cells (IHCs) and outer hair cells (OHCs) did not increase statistically significantly in the apical, middle, or basal turn of the cochlea
Summary
Ototoxic drugs, infections, autoimmune diseases, and hereditary factors can cause loss of hair cells (HCs) and/or functional deficiency of HCs and supporting cells (SCs). Yu et al (2014) inoculated modified AAV vectors into the scala media of early postnatal conditional Gjb knockout mice to drive exogenous connexin expression They found extensive virally expressed connexin in cells lining the scala media, and the intercellular gap junction network was re-established in the organ of Corti of the mutant mouse cochlea, auditory brainstem responses (ABRs) did not show significant hearing improvement. Akil et al (2012) reported the successful restoration of hearing in the Vglut knockout mouse using AAV-mediated gene delivery They found that cochlear delivery of Vglut using AAV1 led to transgene expression only in inner hair cells (IHCs), and within 2 weeks of AAV1-Vglut delivery the click ABR thresholds had almost normalized. These findings indicate the successful restoration of hearing by gene replacement in mice, which is an important step toward gene therapy for human deafness
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