Significant Inhibition of Corneal Scarring In Vivo with Tissue-Selective, Targeted AAV5 Decorin Gene Therapy
Significant Inhibition of Corneal Scarring In Vivo with Tissue-Selective, Targeted AAV5 Decorin Gene Therapy
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DOI：
10.1167/iovs.11-7357
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发表时间：
2011-06-01
影响因子：
4.4
通讯作者：
Tovey, Jonathan C. K.
中科院分区：
文献类型：
--
作者：
Mohan, Rajiv R.;Tandon, Ashish;Tovey, Jonathan C. K.
目的。本研究测试了一个假设，即用腺相关病毒血清型 5 (AAV5) 向基质进行组织选择性靶向核心蛋白聚糖基因治疗可抑制体内角膜纤维化，且无明显副作用。使用角膜纤维化的体内兔模型。通过在裸基质上单次局部应用 AAV5（100 μL；6.5 x 10(12) mu g/mL）2 分钟，将靶向核心蛋白聚糖基因治疗递送至兔角膜。通过立体显微镜、裂隙灯生物显微镜、α-平滑肌肌动蛋白（α SMA）、纤连蛋白和 F-肌动蛋白免疫细胞化学和/或免疫印迹测定角膜纤维化水平。 CD11b、F4/80免疫细胞化学和TUNEL测定用于检查AAV5对角膜的免疫原性和细胞毒性。透射电子显微镜（TEM）用于研究超微结构特征。狭缝印迹定量 AAV5 传递的核心蛋白聚糖基因的拷贝数。结果。与使用裂隙灯生物显微镜定量的未递送核心蛋白聚糖的对照兔角膜（3+/-0.4）相比，选择性核心蛋白聚糖递送至基质显示角膜混浊度显着（P<0.01）降低（1.3+/-0.3）。免疫染色和免疫印迹分析检测到 α SMA、F-肌动蛋白和纤连蛋白水平显着降低（59%-73%；P < 0.001 或
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 mu L; 6.5 x 10(12) mu g/mL) onto the bare stroma for 2 minutes. The levels of corneal fibrosis were determined with stereomicroscopy, slit lamp biomicroscopy, alpha-smooth muscle actin (alpha SMA), 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 alpha SMA, F-actin, and fibronectin proteins (59%-73%; P < 0.001 or