Previously we found that inhibitor of differentiation 3 (Id3) gene, a transcriptional repressor, efficiently inhibits corneal keratocyte differentiation to myofibroblasts in vitro. This study evaluated the potential of adeno-associated virus 5 (AAV5)-mediated Id3 gene therapy to treat corneal scarring using an established rabbit in vivo disease model. Corneal scarring/fibrosis in rabbit eyes was induced by alkali trauma, and 24 h thereafter corneas were administered with either balanced salt solution AAV5-naked vector, or AAV5-Id3 vector (n = 6/group) via an optimized reported method. Therapeutic effects of AAV5-Id3 gene therapy on corneal pathology and ocular health were evaluated with clinical, histological, and molecular techniques. Localized AAV5-Id3 gene therapy significantly inhibited corneal fibrosis/haze clinically from 2.7 to 0.7 on the Fantes scale in live animals (AAV5-naked versus AAV5-Id3; p < 0.001). Furthermore, AAV5-Id3 treatment significantly reduced profibrotic gene mRNA levels: α-smooth muscle actin (α-SMA) (2.8-fold; p < 0.001), fibronectin (3.2-fold; p < 0.001), collagen I (0.8-fold; p < 0.001), and collagen III (1.4-fold; p < 0.001), as well as protein levels of α-SMA (23.8%; p < 0.001) and collagens (1.8-fold; p < 0.001). The anti-fibrotic activity of AAV5-Id3 is attributed to reduced myofibroblast formation by disrupting the binding of E-box proteins to the promoter of α-SMA, a transforming growth factor-β signaling downstream target gene. In conclusion, these results indicate that localized AAV5-Id3 delivery in stroma caused no clinically relevant ocular symptoms or corneal cellular toxicity in the rabbit eyes.

此前我们发现分化抑制剂3（Id3 ）基因是一种转录抑制因子，在体外能有效抑制角膜角质细胞向肌成纤维细胞的分化。本研究使用已建立的兔体内疾病模型评估了腺相关病毒 5 (AAV5) 介导的Id3基因疗法治疗角膜疤痕的潜力。碱创伤诱导兔眼角膜疤痕/纤维化，24小时后通过优化的报道方法向角膜施用平衡盐溶液AAV5裸载体或AAV5-Id3载体（n = 6/组）。通过临床、组织学和分子技术评估 AAV5-Id3 基因治疗对角膜病理学和眼健康的治疗效果。在活体动物中，局部 AAV5-Id3 基因治疗在临床上显着抑制角膜纤维化/混浊，从 Fantes 量表的 2.7 降至 0.7（AAV5 裸露与 AAV5-Id3；p < 0.001）。此外，AAV5-Id3 治疗显着降低促纤维化基因 mRNA 水平：α-平滑肌肌动蛋白 (α-SMA)（2.8 倍；p < 0.001）、纤连蛋白（3.2 倍；p < 0.001）、胶原蛋白 I（0.8 倍） ；p < 0.001）和 III 型胶原蛋白（1.4 倍；p < 0.001），以及 α-SMA 的蛋白质水平（23.8%；p < 0.001）和胶原蛋白（1.8 倍；p < 0.001）。AAV5-Id3 的抗纤维化活性归因于通过破坏 E-box 蛋白与 α-SMA 启动子（转化生长因子-β 信号传导下游靶基因）的结合来减少肌成纤维细胞形成。总之，这些结果表明基质中的局部 AAV5-Id3 递送不会在兔眼中引起临床相关的眼部症状或角膜细胞毒性。