CAR-dependent and CAR-independent pathways of adenovirus vector-mediated gene transfer and expression in human fibroblasts
CAR-dependent and CAR-independent pathways of adenovirus vector-mediated gene transfer and expression in human fibroblasts
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DOI：
10.1172/jci5309
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发表时间：
1999-02-01
影响因子：
15.9
通讯作者：
Crystal, RG
中科院分区：
文献类型：
--
作者：
Hidaka, C;Milano, E;Crystal, RG
原代成纤维细胞不能被 C 亚型腺病毒 (Ad) 载体有效转导，因为它们表达低水平的高亲和力柯萨奇病毒和腺病毒受体 (CAR)。在本研究中，我们使用原代人真皮成纤维细胞作为模型来探索设计Ad载体进入CAR缺陷细胞的策略。使用在高感染复数下表达人类 CAR cDNA (AdCAR) 的 Ad 载体，原代成纤维细胞从 CAR 缺陷转变为 CAR 充足。评估了基于标准 Ad5 的载体和具有五邻体和纤维改变的基于 Ad5 的载体的后续基因转移的效率。标准 Ad5 载体在 CAR 充足的成纤维细胞中实现了结合和转基因表达的显着增强。 Ad Delta RGD beta gal 是一种基于 Ad5 的载体，其五邻体碱基缺乏精氨酸-甘氨酸-天冬氨酸 (RGD) α(v) 整合素识别位点，在 CAR 充足但非 CAR 缺陷的细胞中实现了表达。纤维改变的基于 Ad5 的 Meters，包括 (a) AdF(pK7)beta gal（纤维末端带有 7 个赖氨酸）、(b) AdF(RGD)beta gal（纤维末端带有高亲和力 RGD 序列）和 (c) AdF9sK beta gal（带有短纤维和 Ad9 旋钮），在 CAR 缺陷的成纤维细胞中表现出增强的基因转移，但没有进一步增强CAR 充足的成纤维细胞。总之，这些观察结果表明，Ad 靶标上的 CAR 缺陷可以通过提供 CAR 或通过修饰 Ad 纤维以与其他细胞表面受体结合来规避。
Primary fibroblasts are not efficiently transduced by subgroup C adenovirus (Ad) vectors because they express low levels of the high-affinity Coxsackie virus and adenovirus receptor (CAR). In the present study, we have used primary human dermal fibroblasts as a model to explore strategies by which Ad vectors can be designed to enter cells deficient in CAR. Using an Ad vector expressing the human CAR cDNA (AdCAR) at high multiplicity of infection, primary fibroblasts were converted from being CAR deficient to CAR sufficient. Efficiency of subsequent gene transfer by standard Ad5-based vectors and Ad5-based vectors with alterations in penton and fiber was evaluated. Marked enhancement of binding and transgene expression by standard Ad5 vectors was achieved in CAR-sufficient fibroblasts. Expression by Ad Delta RGD beta gal, an Ad5-based vector lacking the arginine-glycine-aspartate (RGD) alpha(v) integrin recognition site from its penton base, was achieved in CAR-sufficient, but not CAR-deficient, cells. Fiber-altered Ad5-based Meters, including (a) AdF(pK7)beta gal (bearing seven lysines on the end of fiber) (b) AdF(RGD)beta gal (bearing a high-affinity RGD sequence on the end of fiber), and (c) AdF9sK beta gal(bearing a short fiber and Ad9 knob), demonstrated enhanced gene transfer in CAR-deficient fibroblasts, with no further enhancement in CAR-sufficient fibroblasts. Together, these observations demonstrate that CAR deficiency on Ad targets can be circumvented either by supplying CAR or by modifying the Ad fiber to bind to other cell-surface receptors.