Lentiviral transduction of green fluorescent protein in retinal epithelium: evidence of rejection
Lentiviral transduction of green fluorescent protein in retinal epithelium: evidence of rejection
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DOI：
10.1016/s0042-6989(01)00237-1
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发表时间：
2002-02-01
期刊：
影响因子：
1.8
通讯作者：
Gouras, P
中科院分区：
文献类型：
--
作者：
Doi, K;Hargitai, J;Gouras, P
本论文证明了用慢病毒将维多利亚水母（Aequoria victoria）的绿色荧光蛋白（GFP）基因的人源化形式在体外转导到人胎儿视网膜色素上皮（RPE）中，并在体内转导到兔RPE中。在体外，培养的人胎儿RPE在12-16 h后的2 - 3天内开始表达GFP，病毒滴度为10（8）-10（9）感染单位（IU）/ml，使用具有巨细胞病毒（CMV）启动子的慢病毒载体转导静止细胞和分裂细胞。表达保持稳定至少三到四个月，没有毒性的证据，并通过细胞分裂继续。在体内表达在兔眼中使用扫描激光检眼镜（SLO），它可以检测单个荧光视网膜细胞的非侵入性。在将病毒溶液引入视网膜下腔后的几天内开始体内表达。10（9）IU/ml的溶液在三至四天内产生荧光。浓度较低的溶液导致表达较慢且较少。在10（6）IU/ml浓度下未检测到表达。在引入病毒溶液后的一至两周内，存在通过SLO观察到的排斥的证据，表现为GFP荧光的损失和RPE的破坏。组织学显示在接受病毒溶液的区域内的脉络膜和视网膜下腔中RPE层的损伤和单核细胞浸润。强烈的GFP表达导致两周内的排斥反应。随着表达的减少，排斥反应被延迟，在某些情况下至少六个月无法检测到。如果GFP基因不包含在病毒载体中，或者如果病毒浓度不足以产生可检测的GFP表达，则看不到排斥反应。使用视紫红质启动子或将病毒注射到视网膜内而不是视网膜下产生弱表达且没有排斥反应。慢病毒可以诱导RPE中外源基因的表达。病毒诱导的转导和GFP表达对体外RPE的活力没有影响。细胞分裂后GFP的持续表达意味着基因的染色体整合。GFP在RPE中的体内表达遭遇排斥。低GFP表达可能不会发生排斥反应。后者在低病毒滴度、视紫红质启动子或视网膜内注射病毒溶液时发生。这一结果为基因转导导致外源蛋白表达的视网膜基因治疗提供了理论依据。(C)2002爱思唯尔科技有限公司。保留所有权利。
This paper demonstrates lentiviral transduction of the humanized form of the Aequoria victoria gene for green fluorescent protein (GFP) into human fetal retinal pigment epithelium (RPE) in vitro and rabbit RPE in vivo.In vitro GFP expression of cultured human fetal RPE begins within two to three days after 12-16 h of maintained exposure to the virus at titers of 10(8)-10(9) infectious units (IU)/ml, Both stationary and dividing cells are transduced using a lenti viral vector with a cytomegalovirus (CMV) promoter. Expression remains stable for at least three to four months without evidence of toxicity and continues through cell division.In vivo expression is followed non-invasively in rabbit eye using a scanning laser ophthalmoscope (SLO), which can detect single fluorescing retinal cells. In vivo expression begins within a few days after a viral solution is introduced into the subretinal space. A solution of 10(9) IU/ml produces fluorescence within three to four days. Less concentrated solutions lead to slower and less expression. No expression is detectable at concentrations of 10(6) IU/ml. Within one to two weeks after introduction of the viral solution, there is evidence of rejection seen by SLO as a loss of GFP fluorescence and disruption of the RPE. Histology shows damage to the RPE layer and monocytic cell infiltrates in the choroid and subretinal space within the area receiving the viral solution. Strong GFP expression leads to rejection within two weeks. With less expression, rejection is delayed and in some cases undetectable for at least six months. If the GFP gene is not included in the viral vector or if the viral concentration is insufficient to produce detectable GFP expression, rejection is not seen, Using a rhodopsin promoter or injecting the virus intra rather than subretinally produces weak expression and no rejection.Lentivirus can induce expression of a foreign gene in the RPE. Viral induced transduction and GFP expression have no effect on the viability of the RPE in vitro. Continued expression of GFP after cell division implies chromosomal integration of the gene. In vivo expression of GFP in RPE encounters rejection. Rejection may not occur with low GFP expression. The latter occurs with low viral titers, a rhodopsin promoter or intra-retinal injection of viral solution. The results are relevant to gene therapy in retina when gene transduction leads to the expression of foreign proteins. (C) 2002 Elsevier Science Ltd. All rights reserved.