AAV DJ-CAG-GFP

The use of adeno-associated vectors (AAVs) to deliver genes into tissues in vivo has revolutionized the field of gene therapy. However, although neurological hearing loss is one of the most common sensory disorders worldwide, gene therapy for the human inner ear is still in its infancy. Recent advances in the development of recombinant AAVs have significantly improved their cell tropism and transduction efficiency in different inner ear cell types, making them valuable for conditionally manipulating gene expression in studies of mouse inner ear developmental biology. Here, researchers describe a protocol for intrauterine microinjection of AAV into the embryonic inner ear using AAV-PHP.eB and AAV-DJ serotypes, targeting sensory hair cells and supporting cells of the auditory sensory epithelium, respectively. These findings indicate that AAV-PHP.eB and AAV-DJ provide effective and reliable tools for conditional gene expression targeting cochlear sensory cells and supporting cells in the mouse inner ear starting from late embryonic stages.

At E13.5, six embryos from the same dam were injected with approximately 1013 genome copies/ml (GC/ml) of AAV-DJ::CAG-GFP. Only one cochlea out of the six contained a small number of GFP-positive cells that were identified as sensory cells based on their morphology and location within the apical and middle regions of the organ of Corti (Figure 1A). Thus, unilateral delivery of AAV-DJ::CAG-GFP to the inner ear at E13.5 did not result in significant transduction of supporting cells or any other cell type in the cochlea analyzed at P0. Given that AAV-DJ efficiently and specifically transduces supporting cells at the neonatal stage, this suggests that the receptor for this serotype may only be expressed at late embryonic stages of supporting cell differentiation. Injection of five embryos from the same litter with AAV-DJ::CAG-GFP at E14.5 also did not result in transduction of viable cochlear cells. However, injection of AAV-DJ::CAG-GFP at E15.5 and E16.6 resulted in consistent and comparable transduction profiles of supporting cells (Figure 1C) and inner sulcus cells in the basal zone of the left and right cochlear epithelium (Figure 1B). P0 cochleae injected at E15.5 or E16.5 had similar transduction rates, with peaks ranging from 10-50% for inner phalanx cells, pillar cells, and inner border cells. However, Deiters cells in these cochleae remained poorly transduced, with peak transduction rates not exceeding 5% (Figure 1C). These results reveal a pattern of transduction that extends from the base to the apex of the cochlea, reflecting the differentiation dynamics of the cochlear epithelium.

Figure 1. Inner ear transduction efficiency of AAV-DJ administered at embryonic stages. (Barbosa Spinola C M, et al., 2024)