Gene Delivery by Subconjunctival Injection of Adenovirus in Rats: A Study of Local Distribution, Transgene Duration and Safety

Abstract

Subconjunctival injection is a minimally invasive route for gene delivery to ocular tissues, but has traditionally been limited to use in the cornea. The accurate ocular distribution of virus has not, however, been previously investigated. Adenovirus is an attractive gene vector as it can deliver large genes and allow for short-term gene expression, but how safe it is when delivered via subconjunctival injection remains to be established. We have characterized the bio-distribution and safety of subconjunctivally administered adenovirus in Brown Norway rats. The bio-distribution and transgene duration of adenovirus carrying luciferase gene (Ad-Luci) at various time intervals were evaluated via bioluminescence imaging after subconjunctival injection. Adenovirus carrying a reporter gene, β-galactosidase (Ad-LacZ) or hrGFP (Ad-hrGFP) was administered subconjunctivally and the viral distribution in various ocular tissues was assessed by histological analysis and quantitative PCR (qPCR). Hepatic damage was assessed by biochemical and immunohistological analysis with TUNEL stain. Systemic immunogenicity was assessed by measuring serum level of TNF-α via ELISA, 2 hours and 14 days after administration of adenovirus. Retinal function was examined by electroretinography. Subconjunctival injection of Ad-Luci induced luciferase expression in the injected eyes within 24 hours, for at least 64 days. Histological analysis showed adenovirus distributed across anterior and posterior ocular tissues. qPCR demonstrated different amounts of adenovirus in different ocular tissues, with the highest amounts closest to the injection site Unlike the intravenous route, subconjunctivally delivered adenovirus did not elicit any detectable hepatic injury or systemic immunogenicity. Retinal function was unaffected by adenovirus irrespective of administration route. In conclusion, an adenoviral vector administered subconjunctivally can infiltrate into different ocular tissues and lead to short-term ocular transgene expression, without causing hepatic injury and immune activation. Therefore, subconjunctivally administered adenovirus may be a promising gene delivery approach for managing anterior and posterior segment eye diseases requiring short-term therapy.

Fig 1. Viral dosage and duration of adenovirus-mediated gene delivery after a subconjunctival injection in the rats.

(A) Bioluminescence images of rat eyes at day 1, 2, 7, 14, 28, 35 and 64 after a subconjunctival injection of Ad-Luci (1x10⁹ and 1x10¹⁰ GC/eye). (B) Luciferase activity was measured in unit of photons by bioluminescence analysis. Data are presented as the means ± SEM (n = 5).

Fig 2. Bio-distribution of adenovirus after a subconjunctival or intravenous injection in the rats.

(A) Bioluminescence images of rat at day 1, 2, 7, 14, 28, 35 and 64 after a subconjunctival or intravenous injection of Ad-Luci (1x10¹⁰ GC/eye). (B) Luciferase activity was measured in unit of photons by bioluminescence analysis. Data are presented as the means ± SEM (n = 5).

Fig 3. Local ocular distribution of adenovirus after a subconjunctival injection in the rats.

Histological sections of the rat eyes injected with Ad-LacZ (1x10¹⁰ GC/eye) in the subconjunctiva. The blue staining (arrows) in the image indicate the expression of β-galactosidase. V: vitreous; S: sclera. Scale bar = 200 μm (left) and 50 μm (right).

Fig 4. Effect of liver function in the rats received a single injection of adenoviral vector via different administration routes.

(A) Serum level of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were measured at 14 days after an intravenous or subconjunctival injection of Ad-Luci (1x10¹⁰ GC/eye). Data are presented as the means ± SEM (***: p<0.0001, n = 6). One-way ANOVA followed by a Tukey’s test. (B) Sections of liver tissue were stained for TUNEL (green) and DAPI (Blue) 14 days after injection of adenovirus through different administration route. Scale bar = 200 μm.

Fig 5. The serum level of IL-1β and CINC-1 in the rats received a single injection of adenoviral vector via different administration routes.

Serum level of interleukin-1β (IL-1β; A) and cytokine-induced neutrophil chemoattractant factor-1 (CINC-1; B) were measured at 2 hours and 7 days after an intravenous or subconjunctival injection of Ad-Luci (1x10¹⁰ GC/eye). Data are presented as the means ± SEM (*: p<0.05, ***: p<0.0001, n = 6). One-way ANOVA followed by a Tukey’s test.