Improved retinal transduction in vivo and photoreceptor-specific transgene expression using adenovirus vectors with modified penton base
Improved retinal transduction in vivo and photoreceptor-specific transgene expression using adenovirus vectors with modified penton base
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DOI：
10.1038/sj.mt.6300203
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发表时间：
2007-09-01
影响因子：
12.4
通讯作者：
Kumar-Singh, Rajendra
中科院分区：
文献类型：
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作者：
Cashman, Siobhan M.;McCullough, Laura;Kumar-Singh, Rajendra
腺病毒（Ad）载体可以注射到人眼组织中而不会产生不良事件，因此是一种有前途的基因转移到视网膜的手段。然而，当视网膜下施用时，Ad载体主要覆盖视网膜色素上皮（RPE），而引起光感受器（PR）变性的大多数突变基因产物仅在PR细胞中表达。虽然先前已经显示Ad的假型化可以部分克服Ad 5的有限PR转导，但是我们发现Ad的假型化对于PR细胞的转导不是必需的。我们确定，在Ad的情况下，巨细胞病毒（CMV）启动子在PR中不显着活跃。我们比较了CMV和鸡β肌动蛋白（CBA）启动子在神经视网膜中的表达水平，发现CBA的效力比CMV高173倍。我们还研究了小鼠视网膜中Ad-RPE相互作用的性质，并确定了Ad五邻体中的RGD结构域在RPE向性中起关键作用。缺失的RGD结构域加上使用CBA启动子允许转基因表达在神经视网膜约667倍，更有效地比Ad 5载体。这些载体与4.7 kb视紫红质启动子的组合使用使得转基因仅在体内PR细胞中表达。
Adenovirus ( Ad) vectors can be injected into human ocular tissues without producing adverse events and are therefore a promising means of gene transfer to the retina. However, when administered subretinally, Ad vectors primarily transduce the retinal pigment epithelium ( RPE), whereas the majority of mutant gene products that cause photoreceptor ( PR) degeneration are expressed exclusively in the PR cells. While it has been shown previously that pseudotyping of Ad can partially overcome the limited PR transduction by Ad5, we found that pseudotyping of Ad is not necessary for transduction of PR cells. We determined that, in the context of Ad, the cytomegalovirus ( CMV) promoter is not significantly active in PRs. We compared expression levels from CMV and chicken beta actin ( CBA) promoters in neural retina and found that CBA has a 173-fold greater potency than CMV. We also investigated the nature of the Ad-RPE interaction in murine retina and determined that the RGD domain in Ad penton plays a key role in RPE tropism. Deletion of the RGD domain coupled with use of the CBA promoter permitted transgene expression in neural retina approximately 667 times more efficiently than with Ad5 vectors. The use of these vectors in combination with a 4.7 kilobase ( kb) rhodopsin promoter enabled transgene expression exclusively in PR cells in vivo.