Significant inhibition of corneal scarring in vivo with tissue-selective, targeted AAV5 decorin gene therapy

Abstract

Purpose: This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the stroma with adeno-associated virus serotype 5 (AAV5) inhibits corneal fibrosis in vivo without significant side effects.

Methods: An in vivo rabbit model of corneal fibrosis was used. Targeted decorin gene therapy was delivered to the rabbit cornea by a single topical application of AAV5 (100 μL; 6.5 × 10(12) μg/mL) onto the bare stroma for 2 minutes. The levels of corneal fibrosis were determined with stereomicroscopy, slit lamp biomicroscopy, α-smooth muscle actin (αSMA), fibronectin, and F-actin immunocytochemistry, and/or immunoblotting. CD11b, F4/80 immunocytochemistry, and TUNEL assay were used to examine immunogenicity and cytotoxicity of AAV5 to the cornea. Transmission electron microscopy (TEM) was used to investigate ultrastructural features. Slot-blot-quantified the copy number of AAV5-delivered decorin genes.

Results: Selective decorin delivery into the stroma showed a significant (P < 0.01) decrease in corneal haze (1.3 ± 0.3) compared with the no-decorin-delivered control rabbit corneas (3 ± 0.4) quantified using slit lamp biomicroscopy. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA, F-actin, and fibronectin proteins (59%-73%; P < 0.001 or <0.01) in decorin-delivered rabbit corneas compared with the no-decorin-delivered controls. The visual clinical eye examination, slit lamp clinical studies, TUNEL, CD11b, and F4/80 assays revealed that AAV5-mediated decorin gene therapy is nonimmunogenic and nontoxic for the cornea. TEM studies suggested that decorin gene delivery does not jeopardize collagen fibrillogenesis as no significant differences in collagen fibril diameter and arrangement were observed in decorin-delivered and no-decorin-delivered control corneas.

Conclusions: Tissue-targeted AAV5-mediated decorin gene therapy is effective and safe for treating corneal fibrosis in vivo.

Figure 1.

Representative stereomicroscopy (A, C, E) and slit lamp (B, D) biomicroscopy images showing haze levels in no-decorin-delivered control (A, B) and decorin-delivered (C, D) rabbit corneas. Haze was produced with PRK surgery, AAV5 viral titer (6.5 × 10¹² μg/mL) was applied immediately to the cornea for 2 minutes after PRK, and corneal tissues were imaged 4 weeks after PRK (the time at which peak in haze is reported). (F) Quantification of corneal haze. Significantly less haze (P < 0.01) was observed in decorin-delivered corneas than in no-decorin-delivered corneas. No haze was observed in the contralateral naïve rabbit corneas (E). dcn, decorin.

Figure 2.

Effect of AAV5-mediated decorin gene delivery on markers of corneal fibrosis, as detected by immunostaining in rabbit corneal tissue. Immunohistochemistry showing levels of αSMA (A, B), F-actin (C, D), and fibronectin (E, F) in no-decorin-delivered control and decorin-delivered rabbit corneal tissue, collected at 4 weeks after PRK-induced corneal fibrosis. Blue: DAPI-stained nuclei; green: αSMA staining; red: F-actin and fibronectin staining. Decorin-treated corneas showed significant decreases in αSMA (P < 0.001) and notable decreases in F-actin and fibronectin levels in the stroma compared with that in control corneas. Scale bar, 100 μm.

Figure 3.

The effect of AAV5-mediated decorin gene therapy on fibrosis in rabbit corneal tissues quantified by counting αSMA-positive cells at 400× magnification. Decorin treatment significantly (P < 0.001) decreased αSMA-positive cells.

Figure 4.

Western blot analysis for αSMA, to detect the effect of AAV5-mediated decorin gene therapy on fibrosis in rabbit corneas. A significant (75%–86%) decrease in the expression of αSMA was detected in decorin-delivered corneas compared with control corneas, suggesting that AAV5-mediated decorin gene therapy is highly efficient in treating corneal fibrosis. β-Actin was used to confirm equal loading of protein in each well and to normalize the data.

Figure 5.

Effect of AAV5-decorin gene therapy on immune response in rabbit corneas using CD11b and F4/80 immunocytochemistry. The lack of a significant difference in the presence of CD11b- and F4/80-stained cells in the stroma of no-decorin-delivered and decorin-delivered rabbit corneas suggests that AAV5-mediated decorin gene therapy does not induce an immune response in the cornea. Blue: DAPI-stained nuclei; red: CD11b or F4/80 stained cells. Alkali-burned rabbit corneal tissue sections at 4 hours were used as the positive control. Scale bar, 100 μm.

Figure 6.

Effect of AAV5-decorin gene delivery on rabbit corneal toxicity tested with TUNEL assay. The low number of TUNEL⁺ cells (n = 2–5) in the stroma of no-decorin-delivered control (A) and decorin-delivered (B) rabbit corneal sections, the comparable number of TUNEL⁺ cells in the epithelium, and no change in overall keratocyte density in the stroma determined with DAPI⁺ nuclei suggests that decorin gene therapy is noncytotoxic and safe for the cornea. Red: TUNEL⁺ cells; blue: DAPI⁺ nuclei. Scale bar, 100 μm.

Figure 7.

Effect of decorin gene therapy on collagen fibril diameter determined by transmission electron microscopy of no-decorin-delivered control (A) and decorin-delivered (B) rabbit corneas. (C) Quantification of collagen fibril diameter in nanometers. No significant difference in the collagen fibril diameter was detected between control and decorin-delivered rabbit corneas. This result suggests that AAV5-delivered decorin did not alter the collagen fibril status in the rabbit cornea. dcn, decorin.

Figure 8.

Slot-blot analysis showing the decorin gene copy number delivered by the AAV5 vector to the rabbit cornea. Densitometric analysis detected 10⁹ copies of the decorin gene delivered by the vector to the rabbit corneas,. Left lane: the pTRUF11-dcn plasmid DNA blotted at different concentrations; right lane: corneal DNA samples isolated from two animals that received AAV5-dcn.