scAAV2-Mediated Expression of Thioredoxin 2 and C3 Transferase Prevents Retinal Ganglion Cell Death and Lowers Intraocular Pressure in a Mouse Model of Glaucoma

**Background:** Glaucoma is a leading cause of irreversible blindness, affecting up to 80 million people worldwide. Elevated intraocular pressure (IOP) is the primary modifiable risk factor, but even with IOP-lowering treatments, retinal ganglion cell (RGC) degeneration can progress. Current therapies target IOP alone, leaving a need for treatments that also provide neuroprotection. The trabecular meshwork (TM) regulates aqueous humor outflow; its fibrosis and increased contractility contribute to IOP elevation. RhoA signaling and oxidative stress are key pathogenic mechanisms. Exoenzyme C3 transferase inactivates RhoA, reducing TM contractility and IOP, while thioredoxin 2 (Trx2) is a mitochondrial antioxidant that protects RGCs. This study aimed to develop a dual-action gene therapy using a self-complementary AAV2 vector expressing a Trx2-C3 fusion protein (scAAV2-Trx2-C3) and evaluate its effects in a dexamethasone (DEX)-induced mouse model of glaucoma.

**Methods:** A self-complementary AAV2 vector was constructed carrying a fusion gene of human Trx2 and C3 transferase linked by six glycine residues. In vitro studies used HeLa cells transduced with scAAV2-Trx2-C3 or scAAV2-GFP (control). RhoA activity was measured by GST-Rhotekin-RBD pull-down assay. Cofilin phosphorylation at Ser3 was assessed by western blot. Oxidative stress was evaluated using CellROX fluorescent dye. Fibronectin expression was examined by immunocytochemistry. For in vivo studies, 8-week-old male C57BL/6 mice received topical 0.1% dexamethasone sodium phosphate or PBS twice daily for 8 weeks to induce ocular hypertension. After IOP exceeded 20 mmHg, mice received a single intracameral injection of scAAV2-Trx2-C3 or scAAV2-GFP (1 μL, 5.0 × 10^7 vg). IOP was measured weekly using a rebound tonometer (TONOVET Plus). At 4 weeks post-injection, eyes were harvested for immunohistochemistry (fibronectin, alpha-SMA, NeuN) and TUNEL assay. Statistical analysis used unpaired two-tailed Student's t-test or ANOVA with Student–Newman–Keuls post hoc test; significance set at p < 0.05.

**Key Results:** In vitro, scAAV2-Trx2-C3 significantly reduced GTP-bound active RhoA and decreased phospho-cofilin Ser3 levels compared to scAAV2-GFP. DEX treatment increased fibronectin expression and oxidative stress in HeLa cells; scAAV2-Trx2-C3 significantly reduced both. In vivo, baseline IOP was 12.25–13.25 mmHg. After DEX treatment, IOP rose to 21.6 ± 1.9 mmHg (scAAV2-GFP) and 20.4 ± 1.7 mmHg (scAAV2-Trx2-C3) at 1–2 weeks. scAAV2-Trx2-C3-treated mice showed significantly lower IOP for up to 4 weeks post-injection compared to scAAV2-GFP/DEX controls. Immunohistochemistry revealed that scAAV2-Trx2-C3 reduced fibronectin and alpha-SMA expression in the TM. NeuN staining showed significantly more RGCs in scAAV2-Trx2-C3/DEX mice than in scAAV2-GFP/DEX mice. TUNEL assay demonstrated fewer apoptotic cells in the ganglion cell layer of scAAV2-Trx2-C3-treated eyes.

**Clinical Implications:** This study provides proof-of-concept for a gene therapy that simultaneously lowers IOP and protects RGCs by targeting RhoA signaling and oxidative stress. The scAAV2-Trx2-C3 vector offers a single-administration approach that could improve patient compliance and provide sustained therapeutic benefit. The reduction of fibronectin and alpha-SMA suggests anti-fibrotic effects on the TM, addressing a key pathological mechanism in steroid-induced and primary open-angle glaucoma. The neuroprotective effect on RGCs is particularly important because many patients continue to lose vision despite adequate IOP control. Limitations include the short observation period (4 weeks) and the use of a DEX-induced model, which may not fully replicate human POAG. Future studies should evaluate long-term efficacy, safety, and translation to larger animal models and humans.