Efficient in Utero Gene Transfer to the Mammalian Inner Ears by the Synthetic Adeno-Associated Viral Vector Anc80L65

Abstract

Sensorineural hearing loss is one of the most common sensory disorders worldwide. Recent advances in vector design have paved the way for investigations into the use of adeno-associated vectors (AAVs) for hearing disorder gene therapy. Numerous AAV serotypes have been discovered to be applicable to inner ears, constituting a key advance for gene therapy for sensorineural hearing loss, where transduction efficiency of AAV in inner ear cells is critical for success. One such viral vector, AAV2/Anc80L65, has been shown to yield high expression in the inner ears of mice treated as neonates or adults. Here, to evaluate the feasibility of prenatal gene therapy for deafness, we assessed the transduction efficiency of AAV2/Anc80L65-eGFP (enhanced green fluorescent protein) after microinjection into otocysts in utero. This embryonic delivery method achieved high transduction efficiency in both inner and outer hair cells of the cochlea. Additionally, the transduction efficiency was high in the hair cells of the vestibules and semicircular canals and in spiral ganglion neurons. Our results support the potential of Anc80L65 as a gene therapy vehicle for prenatal inner ear disorders.

Keywords: AAV2/Anc80L65; adeno-associated virus; gene therapy; hereditary deafness; in utero microinjection; inner ears.

Graphical abstract

Figure 1

Schematic Diagram of the Method for Intrauterine Delivery into Embryonic Otocysts (A) View of an E12 embryo after microinjection through the uterine wall. The teardrop-shaped otocyst injected with a combination of Anc80L65 and dye is indicated by the dotted line. (B) Gross view of the distribution of the delivered eGFP in the inner ear at P28. The asterisk indicates the cochlea, the triangle indicates the transduced semicircular canals, and the arrows indicate the vestibule (saccule and utricle).

Figure 2

High Transduction Efficiency of Anc80L65 in Cochlear HCs (A) The organ of Corti was mounted and divided into three parts: the apex, middle, and base. One row of IHCs and three rows of OHCs are clearly visible in the images. We compared myosin VIIA and eGFP expression between the Anc80L65-injected ears and the sham ears (injection of PBS solution only). Red indicates myosin VIIA, green indicates eGFP, and blue indicates DAPI. The scale bar represents 50 μm (n = 5). (B) The transduction efficiency was quantified in cochlear HCs with eGFP expression. No significant difference between the IHC and OHC transduction efficiency was found in any turn. (C) Supporting cell layer in a whole-mount preparation of the middle cochlear turn (same as in A). Sham ears were utilized as a positive control for Sox2 and a negative control for eGFP. Gray indicates Sox2-positive supporting cells, and green indicates eGFP. The scale bar represents 50 μm.

Figure 3

In Utero Microinjection Has Minimal Adverse Effects on Hearing ABRs of noninjected (wild-type, n = 6), PBS-injected (sham, n = 5), and Anc80L65-injected mice (n = 5). (A) Click-evoked ABRs of wild-type (upper), sham (middle), and Anc80L65-injected (lower) mice at P28. The amplitude of the response is expressed in microvolts (μV). The time is expressed in milliseconds (ms). (B) The ABR was assessed with clicks and with tone bursts at 8 kHz, 16 kHz, and 32 kHz. No significant differences were observed among any of the tested frequencies.

Figure 4

Anc80L65 Robustly Transduces the Vestibular and Semicircular Canals Sensory Epithelia and SGNs (A and B) The expression of myosin VIIA (red) indicates the positions of HCs in vestibules (represented by saccules; A) and semicircular canals (represented by posterior semicircular canals; B). The proportion of eGFP-positive cells among all HCs was quantified. The scale bar represents 100 μm in the images of vestibules and semicircular canals. The scale bars in the magnified views (40×) from the areas in the white rectangles in the vestibules and semicircular canals represent 50 μm. (C) The transduction efficiency was 89.6% ± 7.8% in vestibules (n = 5) and 71.4% ± 9.3% in semicircular canals (n = 5). (D) Cross section of the cochlea at P28. (E) Magnified views of from the area in the white rectangle in (C). Beta III tubulin staining (red) indicates the distribution of SGNs, and eGFP staining indicates cells transduced by Anc80L65. The scale bar in (D) represents 200 μm, while that in (E) represents 25 μm.