Lentiviral Vector-Mediated Expression of Exoenzyme C3 Transferase Lowers Intraocular Pressure in Monkeys

Abstract

Primary open-angle glaucoma (POAG) is considered a lifelong disease characterized by optic nerve deterioration and visual field damage. Although the disease progression can usually be controlled by lowering the intraocular pressure (IOP), therapeutic effects of current approaches do not last long. Gene therapy could be a promising method for persistent treatment of the disease. Our previous study demonstrated that gene transfer of exoenzyme C3 transferase (C3) to the trabecular meshwork (TM) to inhibit Rho GTPase (Rho), the upstream signal molecule of Rho-associated kinase (ROCK), resulted in lowered IOP in normal rodent eyes. In the present study, we show that the lentiviral vector (LV)-mediated C3 expression inactivates RhoA in human TM cells by ADP ribosylation, resulting in disruption of the actin cytoskeleton and altered cell morphology. In addition, intracameral delivery of the C3 vector to monkey eyes leads to persistently lowered IOP without obvious signs of inflammation. This is the first report of using a vector to transduce the TM of an alive non-human primate with a gene that alters cellular machinery and physiology. Our results in non-human primates support that LV-mediated C3 expression in the TM may have therapeutic potential for glaucoma, the leading cause of irreversible blindness in humans.

Keywords: C3 transferase; Rho GTPases; intraocular pressure; lentivirus; rhesus monkey; trabecular meshwork.

Figure 1

Effects of Lentiviral Vector-Mediated C3 Expression on HTM Cells (A) qRT-PCR analysis of C3 mRNA at 48 h post-vector transduction. (B and C) Western blot (B) and corresponding densitometric analysis (C) for total RhoA expression at 48 h post-transduction. ADP-rib. RhoA, ADP-ribosylated RhoA. Results were normalized to reference protein GAPDH and gene GAPDH, and these values were further standardized to that value of the mock group. (D) The changes of morphology and GFP expression in HTM cells at 3 or 24 h after LV-C3-GFP or LV-GFP transduction (MOI of 5). (E) The actin labeling of HTM cells at 48 h after LV-C3-GFP or LV-GFP transduction (MOI of 10). White arrowheads indicate the cells showing LV-C3-GFP-induced changes, and images show green fluorescence comparison before and after adjusting the parameters (brightness and contrast) by Photoshop CS5 software. The amount of cell in each 40× field (290 × 290 μm) was counted in terms of DAPI-stained cells. (F) Percentage of actin cytoskeleton-disruptive cells (AC-disruptive cells) in the three groups (n = 5). Scale bars, 200 μm (D) and 50 μm (E). Error bars show SEM, and the significance of difference between the three groups was calculated using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.

Figure 2

GFP Expression in the Anterior Segment of Monkeys (A) Photographs of fluorescence detection device in the monkey eye. (B and C) Schematic (B) showing the fluorescence detection in different areas (nasal, anterior, and temporal quadrants) and GFP expression (C) of these areas in the anterior chamber angle, at 14 days after injection with LV-C3-GFP (left eye) or LV-GFP (right eye). Arrows indicate fluorescence in the region of the TM. (D) GFP expression in the temporal quadrant of all monkey eyes at 3, 35, and 70 days after injection with LV-GFP or LV-C3-GFP. Arrowheads indicate the representative fluorescence of the outer puncture site. (E) GFP expression in the anterior segment of monkey eyes at 35 days post-injection. Fluorescence was visualized as a green ring in all TM. (F) The IOD calculating in Image-Pro Plus software for two regions, TM or other regions (iris and/or endothelia), and its enlarged views (labeled by white dashed line boxes). The areas within the green lines indicate the areas of interest (AOIs, excluding non-measuring area). The areas within the red lines indicate the IOD-calculated areas and are labeled by green numbers. The fluorescence in OPS and IPS were excluded from green lines. OPS, outer puncture site. IPS, inner puncture site. (G) IOD in the TM and other regions of the temporal anterior chamber angle in the contralateral LV-C3-GFP and LV-GFP groups. Error bars show SEM, and the significance of IOD difference in the TM between LV-GFP- and LV-C3-GFP-treated eyes was calculated using two-tailed paired t test. n = 4 from days 0 to 70, n = 3 from days 70 to 112; *p < 0.05, **p < 0.01. (H) Histologic assessment of GFP expression in the anterior segment at day 70 after LV transduction. Scale bar, 200 μm. C, cornea; P, pupil; I, iris; TM, trabecular meshwork; SC, Schlemm’s canal; L, lens.

Figure 3

Slit-Lamp Examination and Time Course of IOP Response after Vector Treatment (A and D) Representative images of the anterior segment of eyes at different time points after transduction with LV-C3-GFP or LV-GFP, including the general status at days 1, 63, and 94 (A) and the slit-lamp examination at days 3 and 7 (D). (B and C) Photographs of slit-lamp (B) and IOP-measuring (C) examinations. (E) Time course of IOP response following LV-C3-GFP or LV-GFP transduction. Horizontal dashed line indicates the baseline IOP in the LV-C3-GFP group. (F–H) The correlation between IOP response (ΔIOP) and fluorescence intensity (IOD-C3) after LV-C3-GFP transduction. ΔIOP1 indicates the IOP difference between the LV-C3-GFP and LV-GFP groups. ΔIOP2 indicates the difference between IOP before and after LV-C3-GFP transduction. Horizontal dashed line indicates no difference between IOP before and after C3 transduction or in IOP between LV-GFP-injected and LV-C3-GFP-injected eyes (F and H). (G) Time course of IOD in each monkey after LV-C3-GFP injection. (H) Time course of ΔIOP1 in each monkey after LV-C3-GFP injection. Vertical dashed lines indicate each time point of GFP examination. Error bars show SEM, and the significance of difference between before and after LV-C3-GFP injection or in IOP between LV-GFP-injected and LV-C3-GFP-injected eyes was calculated using two-tailed paired t test. n = 4 from days 0 to 70, n = 3 from days 70 to 112; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Pathological Analysis of Various Ocular Tissues on Monkey 333 and Ultrasound Biomicroscopy Examination at 150 Days on Monkeys 362, 367, and 264, following Vector Treatment (A) Top: representative UBM of the eyes of monkey 367 at 150 days post-injection. Bottom: the thickness quantification of the central cornea (CCT) or central iris (CIT) between the two groups is shown (n = 3, including monkeys 362, 367, and 264). The red arrows represent the sites of anterior chamber paracentesis in the temporal side of the cornea for vector delivery. (B–E) Representative images of H&E staining in the whole cornea (B), corneal endothelium (C), iris (D), and trabecular meshwork (E) on monkey 333. (F) Phalloidin labeling of actin in the anterior chamber angle. Right column: the enlarged views of actin labeling in the TM (labeled by white dashed line boxes) are shown. Schlemm’s canal (SC) was outlined in an irregular white dashed line. I, iris; CM, ciliary muscle; TM, trabecular meshwork. Scale bars, 1,000 μm (A and B) and 100 μm (C–F). Error bars show SEM, and the significance of difference between the two groups was calculated using two-tailed paired t test. NS, not significant.