C3 Transferase-Expressing scAAV2 Transduces Ocular Anterior Segment Tissues and Lowers Intraocular Pressure in Mouse and Monkey

Abstract

Glaucoma is a lifelong disease with elevated intraocular pressure (IOP) as the main risk factor, and reduction of IOP remains the major treatment for this disease. However, current IOP-lowering therapies are far from being satisfactory. We have demonstrated that the lentivirus-mediated exoenzyme C3 transferase (C3) expression in rat and monkey eyes induced relatively long-term IOP reduction. We now show that intracameral injection of self-complementary AAV2 containing a C3 gene into mouse and monkey eyes resulted in morphological changes in trabecular meshwork and IOP reduction. The vector-transduced corneal endothelium and the C3 transgene expression, not vector itself, induced corneal edema as a result of actin-associated endothelial barrier disruption. There was a positive (quadratic) correlation between measured IOP and grade of corneal edema. This is the first report of using an AAV to transduce the trabecular meshwork of monkeys with a gene capable of altering cellular structure and physiology, indicating a potential gene therapy for glaucoma.

Keywords: C3 transferase; intraocular pressure; monkey; mouse; scAAV2.

Figure 1

Effects of scAAV2-Mediated C3 Expression on HTM Cells (A) Detailed structure of the scAAV2 vectors. ITR, inverted terminal repeat; ΔITR, truncated ITR; C3, C3 gene; EGFP, enhanced GFP gene; CA promoter, a promoter that combined the CMV enhancer with the chicken beta-actin promoter; BGH polyA, a poly(A) signal from the bovine growth hormone gene; Amp, ampicillin gene. (B) Western blot and densitometric analysis for total RhoA expression at 48 h post-vector transduction at different MOIs (1.5 × 10⁴ and 7.5 × 10³). ADP-rib. RhoA, ADP-ribosylated RhoA. Results were normalized to the reference protein GAPDH, and these values were further standardized to that value in the mock group. (C) The morphological changes, EGFP expression, and actin labeling in HTM cells at 48 h after scAAV2 vector transduction (MOI = 1.25 × 10⁴). Scale bars, 100 μm. (D) Integrated optical density (IOD; top) and percentage of actin cytoskeleton-disruptive cells (bottom) in the three groups. The amount of cells in each field (263 μm × 263 μm) was counted in terms of DAPI-stained cells. All error bars indicate SEM, and the significance of difference was calculated using one-way analysis of variance (ANOVA). For (B), n = 3 per group; **p < 0.01; ***p < 0.001. For (D), n = 3; *p < 0.05 **p < 0.01 versus scAAV2-EGFP and mock groups.

Figure 2

EGFP Expression in the Anterior Segment of Animals (A, C, and D) In vivo light and fluorescence images were captured by Micron IV. (A) EGFP expression of whole anterior segment at 14 days after scAAV2 vector injection. Arrows indicate fluorescence in the anterior chamber angle (ACA). (B) Fluorescence images of postmortem mouse flat-mount cornea obtained at 21 days after scAAV2 vector transduction (taken with an Olympus CKX53 microscope). Scale bar, 500 μm. (C) EGFP expression in temporal quadrant of monkeys 352 and 368 at 3, 35, and 70 days after injection with scAAV2 vectors. Arrows indicate fluorescence in the region of the trabecular meshwork (TM). (D) Top: schematic diagram showing the way to distinguish whether the fluorescence was located in the corneal endothelium or on the anterior surface of the iris. Middle and bottom: EGFP expression of these areas in the temporal quadrant at 77 days after injection with scAAV2-EGFP. OS, left eye; OD, right eye; C, cornea; P, pupil; L, lens.

Figure 3

Slit-Lamp Examination (A) Representative images of anterior segments of mouse eyes at day 7. (B–D) Representative images of anterior segments of monkey eyes at different time points, including the general status at days 1 and 105 (B), and the slit-lamp examination at days 3 and 7 (C) and day 63 (D). Red arrows indicate corneal opacity presented in scAAV2-C3-injected monkey eyes (right eye). (D) The images of anterior segment are presented in descending order by severity of corneal edema at day 63 post-scAAV2-C3 injection (from left to right).

Figure 4

The IOP Changes from Baseline after Vector Delivery and the Correlation of IOP Ratio and Grade of Corneal Edema or CCT Ratio (A) Mean IOP percent changes in mouse eyes before (day 0) and after (day 7) vector transduction (n = 4). (B) Mean IOP percent changes in monkey eyes before (day 0) and after (days 3, 7, 14, 21, and 28) vector transduction (n = 5). Error bars represent SEM, and the statistical significance in mean IOP percent changes was calculated using the two-tailed paired t test. *p < 0.05 and **p < 0.01 versus baseline; ^†p < 0.05, ^††p < 0.01, and ^†††p < 0.001. (C) Time course of IOP changes in each monkey after scAAV2-EGFP or scAAV2-C3 injection. The horizontal dashed line indicates no difference in IOP before and after scAAV2 vector injection (baseline; IOP ratio = 1.0). n = 5 from day 0 to day 77. n = 4 from day 77 to now. (D) Representative UBM images and corresponding CCT analysis at day 175 post-injection. The red arrows represent the site of anterior chamber paracentesis in the temporal side of cornea for vector delivery. CC, central cornea; ACA, anterior chamber angle. Scale bar, 1,000 μm. Error bars represent SEM (n = 5), and the significance of difference among the four monkeys was calculated using one-way ANOVA. (E and F) Regression analysis between the IOP ratio and the grade of corneal edema (E) and between the IOP ratio and CCT ratio (F). ***p < 0.001.

Figure 5

Histological Analysis of Anterior Segment Tissues in Mice and Monkey 285, and TEM Examination of Corneal Endothelium in Monkey 285, following Vector Treatment (A–E) H&E staining of sections at the anterior segment from mice and monkey 285. (A) Representative images of anterior segment in mice at 21 days. C, cornea; SC, Schlemm’s canal; TM, trabecular meshwork; CB, ciliary body; I, iris. (B–E) Morphological changes of anterior segment tissues, including whole cornea (B), corneal endothelium (C), iris (D), and TM (E), in monkey 285 at 77 days. Open arrowheads indicate the vacuolation on the posterior face of iris. Red arrowheads and arrows indicate the corneal epithelium and endothelium, respectively. (F and G) Actin labeling on the corneal endothelium of mice (F) and the TM of monkey 285 (G). Asterisk denotes the widened spaces within juxtacanalicular tissues. White arrowheads indicate the representatively contracted cells. (H) Ultrastructural changes of the corneal endothelium on monkey 285. The C3-transduced corneal endothelium shows the loosening or separation of the cell-cell (yellow arrowheads) and cell-basement (yellow arrows) junctions. Scale bars: 100 μm in (A)–(G) and 5 μm in (H).

Figure 6

Schematic Diagram Showing the Capability of Two Vectors to Transduce Different Cells in the Monkey Anterior Segment after Intracameral Injection (A and B) scAAV2 vectors (A) had a high tropism for the cells of the TM, corneal endothelium (CE), posterior epithelial layer (PEL), and ciliary muscle (CM), while LVs (B) had a high tropism almost exclusively on the TM.^, SC, Schlemm’s canal; ABL, anterior border layer; CB, ciliary body; AH, aqueous humor.