Development and evaluation of helper dependent adenoviral vectors for inner ear gene delivery

Abstract

Viral vector gene therapy is an attractive strategy to treat hearing loss. Since hearing loss is due to a variety of pathogenic signaling cascades in distinct cells, viral vectors that can express large or multiple genes in a cell-type specific manner are needed. Helper-dependent adenoviral vectors (HdAd) are safe viral vectors with a large packaging capacity (-36 kb). Despite the potential of HdAd, its use in the inner ear is largely unexplored. Therefore, to evaluate the utility of HdAd for inner ear gene therapy, we created two HdAd vectors that use distinct cellular receptors for transduction: HdAd Serotype Type 5 (HdAd5), the Coxsackie-Adenovirus Receptor (CAR) and a chimeric HdAd 5/35, the human CD46+ receptor (hCD46). We delivered these vectors through the round window (RW) or scala media in CBA/J, C57Bl6/J and hCD46 transgenic mice. Immunostaining in conjunction with confocal microscopy of cochlear sections revealed that multiple cell types were transduced using HdAd5 and HdAd 5/35 in all mouse models. Delivery of HdAd5 via RW in the C57Bl/6 J or CBA/J cochlea resulted in transduced mesenchymal cells of the peri‑lymphatic lining and modiolar region while scala media delivery resulted in transduction of supporting cells and inner hair cells. Hd5/35 transduction was CD46 dependent and RW delivery of HdAd5/35 in the hCD46 mouse model resulted in a similar transduction pattern as HdAd5 in the peri‑lymphatic lining and modiolar region in the cochlea. Our data indicate that HdAd vectors are promising vectors for use in inner ear gene therapy to treat some causes of hearing loss.

Keywords: Gene therapy; Hearing loss; Helper dependent adenovirus; Inner ear.

Figure 1:. Overview of study methodology.

A: Brief overview of experimental timeline B: Three viral vector constructs carrying fluorescent reported mCLover3 were produced. HdAd5 vector constructs expressing mClover3 under the control of both CAG and CMV promoters were produced as well as an HdAd 5/35 construct expressing mClover3 under control of a CAG promoter. C: Three mouse models were used in our experiments. CBA/CaJ wild type adult mouse was injected with all three viral vector constructs. CD46 transgenic mice on a C57/Bl6 background were injected with HdAd5-CAG-mClover3 and HdAd 5/35-CAG-mClover3 constructs. Neonatal C57/Bl6 mice were injected with HdAd 5-CAG-mCLover3 viral construct. D: Illustration of approaches used to deliver viral vector. Created with Biorender.com

Figure 2:. HdAd5 predominantly transduces cells lining the scala tympani and in the modiolar region when injected by RW+CF approach in adult CBA/CaJ mice.

A/B: organ of Corti whole mounts of apical and basal segments from CBA/CaJ mouse injected with HdAd5 under control of CMV promoter (3.44 ×10⁹ viral particles/animal) or HdAd5/35-CAG-mclover3 (1.21 ×10⁹ viral particles/animal). C/D: organ of Corti whole mounts of apical and basal segments from mouse injected with HdAd5 under control of CAG promoter(3.83×10⁹ viral particles/animal). E: Apical segment of organ of Corti whole mount illustrating basement membrane region transduction F: Mid-modiolar section of basal segment illustrating basement membrane region transduction. G: Modiolar region from whole mount of basal segment illustrating non-neuronal spiral ganglion transduction. H/I: Comparison of transduction efficiency achieved by HdAd5 under control of CAG vs. CMV promoters in CBA/CaJ mice. BM: basilar membrane region. SM: scala media. ST: scala tympani OHC: outer hair cells. IHC: inner hair cells.

Figure 3:. HdAd5 transduces inner hair cells, supporting cells and non-neuronal cells in the spiral ganglion when injected by scala media approach in adult CBA/CaJ mice.

A/B: organ of Corti whole mounts of apical and basal segments from adult CBA mice injected with HdAd5 under control of CMV promoter by scala media approach. C: organ of Corti whole mount of apical segment illustrating inner hair cell and supporting cell transduction. D: Mid-modiolar section of basal segment illustrating supporting cell transduction. E: Modiolar region from whole mount of basal segment illustrating non-neuronal transduction in the spiral ganglion. BM: Basilar membrane region. SM: Scala Media. ST: Scala tympani OHC: outer hair cells. IHC: inner hair cells. PC: Pillar cells. DC: Deiter cells.

Figure 4:. HdAd5 predominantly transduces cells lining the scala tympani and in the modiolar region when injected by RW approach in neonatal C57/Bl6 mice.

A/B: organ of Corti whole mounts of apical and basal segments from neonatal mice injected with HdAd5 under control of CAG promoter C/D: Apical and basal segments of organ of Corti whole mount illustrating basement membrane region transduction. E/F: Apical and basal segments of organ of Corti whole mount illustrating modiolar region transduction. G/H: Comparison of transduction efficiency achieved by HdAd5 under control of CAG in adult vs. neonatal mice. BM: Basement membrane.

Figure 5:. Human CD46 is expressed on multiple cell types in the inner ear of CD46 tg mice.

No expression is seen in wild-type controls. A: organ of Corti whole mount illustrating human CD46 expression CD46 Tg mice. CD46 is expressed in green and is seen on the surgace of inner and outer hair cells as well as supporting cells. B: CD46 staingin of wild-type CBA/CaJ mouse organ of corti reveals no expression of CD46. IHC: inner hair cells. OHC: Outer hair cells.

Figure 6:. HdAd5/35 transduces cells lining the scala tympani and in the modiolar region when injected by RW+CF approach with higher efficiency in adult CD46

Tg mice. A-D: organ of Corti whole mounts of apical and basal segments from CD46 Tg mouse injected with HdAd5/35 under control of CAG promoter (1.21 ×10⁹ viral particles/animal). E/F: organ of Corti whole mounts of apical and basal segments from CBA/CaJ mouse injected with HdAd5/35 under control of CAG promoter (1.21 ×10⁹ viral particles/animal). E: Apical segment of organ of Corti whole mount illustrating basement membrane region transduction. G/H: Comparison of transduction efficiency achieved by HdAd5/35 under control of CAG in CD46 Tg vs. CBA/CaJ mice.