Efficacious and safe tissue-selective controlled gene therapy approaches for the cornea.
Efficacious and safe tissue-selective controlled gene therapy approaches for the cornea.
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DOI：
10.1371/journal.pone.0018771
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发表时间：
2011-04-12
期刊：
影响因子：
3.7
通讯作者：
Sharma A
中科院分区：
文献类型：
--
作者：
Mohan RR;Sinha S;Tandon A;Gupta R;Tovey JC;Sharma A
非靶向和不受控制的基因传递是基因治疗失败的主要原因。本研究旨在确定有效和安全的组织选择性靶向基因治疗方法，将基因传递到正常或患病兔角膜模型的角膜角质细胞中。实验采用新西兰大白兔、腺相关病毒血清型5 （AAV5）和基于微创吹风机的载体传递技术。将50微升表达绿色荧光蛋白基因（GFP）的AAV5滴度（6.5×1012 vg/ml）用定制的载体传递技术局部涂于正常或病变（纤维化或新生血管化）兔角膜上2分钟。准分子激光光屈光性角膜切除术和微袋法测定VEGF （630 ng）分别诱导兔角膜纤维化和新生血管形成。采用裂隙灯生物显微镜和免疫细胞化学方法证实兔角膜纤维化和新生血管形成。用免疫细胞化学和/或western blotting检测gfp基因在兔基质中的表达水平、位置和持续时间。槽点印迹法测定传递gfp基因拷贝数。在整个角膜和厚角膜切片上进行共聚焦显微镜，确定了三维排列中递送的绿色荧光蛋白的几何和空间定位。采用临床眼科检查、体视显微镜、裂隙灯生物显微镜、H&E染色等方法评价AAV5的毒性和安全性。免疫细胞化学和共聚焦显微镜显示，通过定制的递送技术，单次2分钟的AAV5局部应用有效地和选择性地转导了正常和患病兔角膜前基质中的角化细胞。第3天首次检测到转基因表达，第7天达到峰值，并维持至16周（最长测试时间点）。活体家兔的临床和裂隙灯眼检查以及H&E染色均未发现aav5处理和未处理的对照角膜有明显变化。这些发现表明，明确的基因治疗方法对于将基因传递到体内角质细胞是安全的，并且具有治疗人类角膜疾病的潜力。
Untargeted and uncontrolled gene delivery is a major cause of gene therapy failure. This study aimed to define efficient and safe tissue-selective targeted gene therapy approaches for delivering genes into keratocytes of the cornea in vivo using a normal or diseased rabbit model. New Zealand White rabbits, adeno-associated virus serotype 5 (AAV5), and a minimally invasive hair-dryer based vector-delivery technique were used. Fifty microliters of AAV5 titer (6.5×1012 vg/ml) expressing green fluorescent protein gene (GFP) was topically applied onto normal or diseased (fibrotic or neovascularized) rabbit corneas for 2-minutes with a custom vector-delivery technique. Corneal fibrosis and neovascularization in rabbit eyes were induced with photorefractive keratectomy using excimer laser and VEGF (630 ng) using micropocket assay, respectively. Slit-lamp biomicroscopy and immunocytochemistry were used to confirm fibrosis and neovascularization in rabbit corneas. The levels, location and duration of delivered-GFP gene expression in the rabbit stroma were measured with immunocytochemistry and/or western blotting. Slot-blot measured delivered-GFP gene copy number. Confocal microscopy performed in whole-mounts of cornea and thick corneal sections determined geometric and spatial localization of delivered-GFP in three-dimensional arrangement. AAV5 toxicity and safety were evaluated with clinical eye exam, stereomicroscopy, slit-lamp biomicroscopy, and H&E staining. A single 2-minute AAV5 topical application via custom delivery-technique efficiently and selectively transduced keratocytes in the anterior stroma of normal and diseased rabbit corneas as evident from immunocytochemistry and confocal microscopy. Transgene expression was first detected at day 3, peaked at day 7, and was maintained up to 16 weeks (longest tested time point). Clinical and slit-lamp eye examination in live rabbits and H&E staining did not reveal any significant changes between AAV5-treated and untreated control corneas. These findings suggest that defined gene therapy approaches are safe for delivering genes into keratocytes in vivo and has potential for treating corneal disorders in human patients.
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影响因子：
3.5
作者：
Borrás, T;Gabelt, BT;Kaufman, PL
通讯作者：
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