组织选择性靶向 AAV5 饰胶蛋白基因治疗显著抑制体内角膜瘢痕形成。
Significant inhibition of corneal scarring in vivo with tissue-selective, targeted AAV5 decorin gene therapy.
机构信息
Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, USA.
出版信息
Invest Ophthalmol Vis Sci. 2011 Jul 1;52(7):4833-41. doi: 10.1167/iovs.11-7357.
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.
目的
本研究旨在验证一个假设，即通过腺相关病毒血清型 5（AAV5）将组织选择性靶向的核心蛋白聚糖基因治疗递送至基质，可在体内抑制角膜纤维化，而不会产生明显的副作用。
方法
采用体内兔角膜纤维化模型。通过将 AAV5（100μL；6.5×10(12)μg/mL）单次局部滴注于裸基质上 2 分钟，将靶向核心蛋白聚糖基因治疗递送至兔角膜。通过体视显微镜、裂隙灯生物显微镜、α-平滑肌肌动蛋白（αSMA）、纤维连接蛋白和 F-肌动蛋白免疫细胞化学和/或免疫印迹检测角膜纤维化程度。用 CD11b、F4/80 免疫细胞化学和 TUNEL 检测 AAV5 对角膜的免疫原性和细胞毒性。采用透射电子显微镜（TEM）观察超微结构特征。Slot-blot 定量分析 AAV5 递送的核心蛋白聚糖基因的拷贝数。
结果
选择性将核心蛋白聚糖递送至基质可显著降低（P<0.01）角膜混浊度（1.3±0.3），与未递送核心蛋白聚糖的对照组兔角膜（3±0.4）相比，用裂隙灯生物显微镜定量分析。免疫染色和免疫印迹分析显示，与未递送核心蛋白聚糖的对照组相比，核心蛋白聚糖递送的兔角膜中αSMA、F-肌动蛋白和纤维连接蛋白蛋白水平显著降低（59%-73%；P<0.001 或<0.01）。视觉临床眼部检查、裂隙灯临床研究、TUNEL、CD11b 和 F4/80 检测均表明，AAV5 介导的核心蛋白聚糖基因治疗对角膜无免疫原性和毒性。TEM 研究表明，核心蛋白聚糖基因递送不会危及胶原原纤维的形成，因为在核心蛋白聚糖递送和未递送核心蛋白聚糖的对照组角膜中，胶原原纤维直径和排列没有显著差异。
结论
组织靶向的 AAV5 介导的核心蛋白聚糖基因治疗对体内角膜纤维化的治疗是有效且安全的。
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