Nonviral ocular gene transfer
Nonviral ocular gene transfer
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DOI：
10.1038/sj.gt.3302475
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发表时间：
2005-05-01
期刊：
影响因子：
5.1
通讯作者：
Campochiaro, PA
中科院分区：
文献类型：
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作者：
Kachi, S;Oshima, Y;Campochiaro, PA
在这项研究中，我们探讨了使用电穿孔或媒体，促进lipoplex形成的非病毒基因转移的眼睛。视网膜下、玻璃体内或眼周注射含有LacZ CMV启动子表达盒的质粒后，没有检测到LacZ染色，但当三个位点中的每个位点的质粒注射与电穿孔相结合时，存在有效的转导。视网膜下注射含有卵黄状黄斑营养不良2（VMD 2）启动子表达盒的质粒后，在视网膜色素上皮（RPE）细胞中观察到LacZ的特异性染色，表明这种方法可用于体内评估据称的组织特异性启动子。10 V/mm的电穿孔导致强烈的LacZ染色，但对光感受器有损伤;使用3.4 V/mm的电穿孔，未见视网膜损伤证据的实质性转导。在视网膜下或眼周注射含40% Lipofectamine 2000（Lf）的培养基中的质粒DNA后，也可见LacZ染色，但未在玻璃体内注射。将40%Lf注射到视网膜下间隙中引起感光细胞损伤，但视网膜下注射含有10%NeuroPorter的培养基中的质粒DNA导致RPE细胞的转导，如通过视网膜电图（ERG）评估的，对视网膜形态或功能没有不良影响。无论是电穿孔或脂质体转染后，LacZ染色可检测至少14天，并可以通过第二个程序重新诱导。这些数据表明，电穿孔或脂质体转染可用作眼部基因转移的实验工具，以评估组织特异性启动子片段或评估转基因在视网膜中表达的影响。此外，通过额外的优化，非病毒基因转移可能被证明是一种有价值的方法，用于治疗视网膜和脉络膜疾病。
In this study, we explored the use of electroporation or media that promote lipoplex formation for nonviral gene transfer in the eye. There was no detectable staining for LacZ after subretinal, intravitreous, or periocular injection of a plasmid containing a CMV promoter expression cassette for LacZ, but when plasmid injection in each of the three sites was combined with electroporation, there was efficient transduction. Specific staining for LacZ was seen in retinal pigmented epithelial (RPE) cells after subretinal injection of a plasmid containing a vitelliform macular dystrophy 2 (VMD2) promoter expression cassette, demonstrating that this approach can be used to evaluate purported tissue-specific promoters in vivo. Electroporation with 10 V/mm resulted in strong LacZ staining, but was damaging to photoreceptors; substantial transduction with no evidence of retinal damage was seen using 3.4 V/mm. Staining for LacZ was also seen after subretinal or periocular, but not intravitreous, injection of plasmid DNA in medium containing 40% Lipofectamine2000 (Lf). Injection of 40% Lf into the subretinal space caused damage to photoreceptors, but subretinal injection of plasmid DNA in medium containing 10% NeuroPorter resulted in transduction of RPE cells with no adverse effects on retinal morphology or function as assessed by electroretinograms (ERGs). After either electroporation or lipofection, LacZ staining was detectable for at least 14 days, and could be reinduced by a second procedure. These data suggest that electroporation or lipofection can be used as experimental tools for ocular gene transfer to evaluate tissue-specific promoter fragments or to evaluate the effects of transgene expression in the retina. Also, with additional optimization, nonviral gene transfer may prove to be a valuable approach for the treatment of retinal and choroidal diseases.