Different tropism of adenoviruses and adeno-associated viruses to corneal cells: implications for corneal gene therapy
Different tropism of adenoviruses and adeno-associated viruses to corneal cells: implications for corneal gene therapy
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发表时间：
2008-11
期刊：
影响因子：
2.2
通讯作者：
J. Liu;M. Saghizadeh;S. Tuli;A. Kramerov;A. Lewin;D. Bloom;W. Hauswirth;M. Castro;G. Schultz;A. Ljubimov
中科院分区：
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作者：
J. Liu;M. Saghizadeh;S. Tuli;A. Kramerov;A. Lewin;D. Bloom;W. Hauswirth;M. Castro;G. Schultz;A. Ljubimov
目的病变的角膜是以病毒为基础的基因治疗的潜在靶点，以使特定蛋白的表达正常化(刺激或抑制)。病毒载体的选择是达到最佳效果的重要因素。本研究的目的是比较重组腺病毒(RAV)和重组腺相关病毒(RAAV)构建的重组腺病毒(RAV)和重组腺相关病毒(RAAV)对不同角膜细胞的趋向性。方法在早期巨细胞病毒(CMV)主要启动子的控制下，构建携带绿色荧光蛋白(GFP)基因的RAV。在鸡β-肌动蛋白启动子和巨细胞病毒增强子的作用下，1、2、5、7和8型病毒的重组腺病毒载体具有绿色荧光。器官培养采用16例健康人和糖尿病人死后角膜。将5~15个μL按107个/1μL空斑形成单位加入未损伤角膜培养液中，继续培养5~32d。每只角膜分别加入1.2~7.8×1010个载体基因组，连续3天，继续培养14~23天。用免疫组织化学方法检测角膜冰冻切片。活体兔角膜用于准分子激光消融角膜上皮后，保留基底层。将相同数量的rAAV颗粒(2x1011载体基因组)应用于角膜10min。在允许角膜愈合和基因表达的七天后，动物被安乐死，切除角膜，并通过免疫组织化学分析切片。结果活体培养的人角膜上皮细胞经RAV基因转导后，直接荧光显微镜下可见较强的GFP信号，且呈剂量依赖关系。在角膜切片上，绿色荧光蛋白在所有上皮层和部分内皮细胞均可见，但大多数角膜基质细胞呈阴性。在rAAV转导的器官培养的人角膜中，只有抗GFP抗体免疫组织化学才能检测到GFP信号。绿色荧光蛋白见于上皮细胞、角质形成细胞和内皮细胞，rAAV1的基底上皮细胞染色比其他血清型更明显。在正常角膜和糖尿病角膜之间，绿色荧光蛋白的表达模式或水平没有差异。兔角膜表现出与人角膜非常相似的GFP分布模式。在所有rAAV型载体中，非转导的角膜上皮细胞的绿色荧光蛋白染色明显高于背景染色(p=0.007)，且rAAV1和rAAV8的染色强度明显高于rAAV2、rAAV5(p=0.03；rAAV5对rAAV1)和rAAV7。RAAV血清型载体也不同程度地转导了兔角膜基质细胞和内皮细胞。结论rAAV比RAV能到达更多的角膜细胞，尤其是角质形成细胞，尽管GFP表达水平低于RAV。在需要高蛋白表达水平的基因治疗应用中，RAV可能比rAAV更有用，但在角质细胞靶向方面，rAAV可能更好。
Purpose Diseased corneas are potential targets for viral-based gene therapy to normalize (stimulate or inhibit) the expression of specific proteins. The choice of viral vectors is important to achieve optimal effect. The purpose of this study was to compare the tropism to different corneal cells of recombinant adenovirus (rAV) and recombinant adeno-associated virus (rAAV) constructs using live rabbit and organ-cultured human corneas. Methods rAV constructs harbored the green fluorescent protein (GFP) gene under the control of major immediate early cytomegalovirus (CMV) promoter. rAAV constructs from virus serotypes 1, 2 5, 7, and 8 had GFP under the chicken β-actin promoter and CMV enhancer. For organ culture, 16 healthy and diabetic postmortem human corneas were used. Five or fifteen μl rAV at 107 plaque forming units per 1 μl were added for 2 days to culture medium of uninjured corneas that were further cultured for 5–32 days. rAAV were added at 1.2–7.8×1010 vector genomes per cornea for 3 days to each cornea; the culture then continued for another 14–23 days. Corneal cryostat sections were examined by immunohistochemistry. Live rabbit corneas were used following excimer laser ablation of the corneal epithelium with preservation of the basal cell layer. Equal numbers of rAAV particles (2x1011 vector genomes) were applied to the cornea for 10 min. After seven days to allow for corneal healing and gene expression the animals were euthanized, the corneas were excised, and sections analyzed by immunohistochemistry. Results By direct fluorescence microscopy of live organ-cultured human corneas GFP signal after rAV transduction was strong in the epithelium with dose-dependent intensity. On corneal sections, GFP was seen in all epithelial layers and some endothelial cells but most keratocytes were negative. In rAAV-transduced organ-cultured human corneas GFP signal could only be detected with anti-GFP antibody immunohistochemistry. GFP was observed in the epithelium, keratocytes, and endothelium, with more pronounced basal epithelial cell staining with rAAV1 than with other serotypes. No difference in the GFP expression patterns or levels between normal and diabetic corneas was noted. The rabbit corneas showed very similar patterns of GFP distribution to human corneas. With all rAAV serotype vectors, GFP staining in the epithelium was significantly (p=0.007) higher than the background staining in non-transduced corneas, with a trend for rAAV1 and rAAV8 to produce higher staining intensities than for rAAV2, rAAV5 (p=0.03; rAAV5 versus rAAV1), and rAAV7. rAAV serotype vectors also transduced stromal and endothelial cells in rabbit corneas to a different extent. Conclusions rAAV appears to reach many more corneal cells than rAV, especially keratocytes, although GFP expression levels were lower compared to rAV. rAV may be more useful than rAAV for gene therapy applications requiring high protein expression levels, but rAAV may be superior for keratocyte targeting.