Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels.
Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels.
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基因治疗的成功取决于获得足够的转基因表达。为了确保转基因的高效表达，许多基因治疗载体都包含了高度活跃的病毒衍生转录元件。其他载体包括组织特异性真核转录元件，旨在将转基因表达限制在特定细胞类型，避免毒性，并防止免疫反应。不幸的是，组织特异性往往伴随着较低的转基因表达。在这里，我们使用真核(小鼠)转录元件和病毒衍生的转录后元件来构建盒式磁带，以在大血管内皮细胞(EC)中表达潜在的治疗性基因(IL-10)，其水平与CMV立即早期启动子获得的水平一样高，同时保持EC的特异性。将其导入辅助性腺病毒载体，体外转导牛主动脉内皮细胞，体内转导兔颈动脉内皮细胞。小鼠内皮素-1启动子具有EC特异性，但表达的IL-10mRNA仅为CMV的3%。在体内，恰好包含4个EC特异性增强子和转录后调控元件的拷贝将IL-10的表达增加到等于或高于CMV启动子的水平，同时保留-并可能增强-EC特异性，根据体外测量。本文报道的试剂盒可能有助于最大化转基因在大血管EC中的表达，同时将全身影响降至最低。
The success of gene therapy hinges on achievement of adequate transgene expression. To ensure high transgene expression, many gene-therapy vectors include highly active virus-derived transcriptional elements. Other vectors include tissue-specific eukaryotic transcriptional elements, intended to limit transgene expression to specific cell types, avoid toxicity, and prevent immune responses. Unfortunately, tissue specificity is often accompanied by lower transgene expression. Here we use eukaryotic (murine) transcriptional elements and a virus-derived posttranscriptional element to build cassettes designed to express a potentially therapeutic gene (interleukin-10) in large vessel endothelial cells (EC) at levels as high as obtained with the CMV immediate-early promoter, while retaining EC-specificity. The cassettes were tested by incorporation into helper-dependent adenoviral vectors, and transduction into bovine aortic EC in vitro and rabbit carotid EC in vivo. The murine endothelin-1 promoter showed EC-specificity, but expressed only 3% as much IL-10 mRNA as CMV. Inclusion of precisely 4 copies of an EC-specific enhancer and a posttranscriptional regulatory element increased IL-10 expression to a level at or above the CMV promoter in vivo, while retaining—and possibly enhancing—EC specificity, as measured in vitro. The cassette reported here will likely be useful for maximizing transgene expression in large vessel EC, while minimizing systemic effects.
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影响因子：
5.1
作者：
Modlich, U;Pugh, CW;Bicknell, R
通讯作者：
Bicknell, R
影响因子：
1.6
作者：
Baker, AH
通讯作者：
Baker, AH
影响因子：
4.2
作者：
De Palma, M;Venneri, MA;Naldini, L
通讯作者：
Naldini, L
影响因子：
8.3
作者：
Nicklin, SA;Reynolds, PN;Baker, AH
通讯作者：
Baker, AH
影响因子：
15.9
作者：
Newman, KD;Dunn, PF;Dichek, DA
通讯作者：
Dichek, DA