Smith Theodore A G, Idamakanti Neeraja, Rollence Michele L, Marshall-Neff Jennifer, Kim Jin, Mulgrew Kathy, Nemerow Glen R, Kaleko Michael, Stevenson Susan C
Genetic Therapy, Inc., a Novartis Company, Gaithersburg, MD 20878, USA.
Hum Gene Ther. 2003 May 20;14(8):777-87. doi: 10.1089/104303403765255165.
Adenoviral vectors used in gene therapy are predominantly derived from adenovirus serotype 5 (Ad5), which infects a broad range of cells. Ad5 cell entry involves interactions with the coxsackie-adenovirus receptor (CAR) and integrins. To assess these receptors in vivo, we mutated amino acid residues in fiber and penton that are involved in receptor interaction and showed that CAR and integrins play a minor role in hepatic transduction but that integrins can influence gene delivery to other tissues. These data suggest that an alternative entry pathway exists for hepatocyte transduction in vivo that is more important than CAR or integrins. In vitro data suggest a role for heparan sulfate glycosaminoglycans (HSG) in adenovirus transduction. The role of the fiber shaft in liver uptake was examined by introducing specific amino acid changes into a putative HSG-binding motif contained within the shaft or by preparing fiber shaft chimeras between Ad5 and Ad35 fibers. Results were obtained that demonstrate that the Ad5 fiber shaft can influence gene transfer both in vitro and to the liver in vivo. These observations indicate that the currently accepted two-step entry pathway, which involves CAR and integrins, described for adenoviral infection in vitro, is not used for hepatic gene transfer in vivo. In contrast, alpha(v) integrins influence gene delivery to the lung, spleen, heart, and kidney. The detargeted vector constructs described here may provide a foundation for the development of targeted adenoviral vectors.
用于基因治疗的腺病毒载体主要来源于5型腺病毒（Ad5），它能感染多种细胞。Ad5进入细胞涉及与柯萨奇病毒-腺病毒受体（CAR）和整合素的相互作用。为了在体内评估这些受体，我们对纤维蛋白和五邻体中参与受体相互作用的氨基酸残基进行了突变，结果表明CAR和整合素在肝脏转导中起次要作用，但整合素可影响基因传递至其他组织。这些数据表明，体内肝细胞转导存在一条比CAR或整合素更重要的替代进入途径。体外数据表明硫酸乙酰肝素糖胺聚糖（HSG）在腺病毒转导中发挥作用。通过将特定氨基酸变化引入纤维杆内假定的HSG结合基序或制备Ad5和Ad35纤维之间的纤维杆嵌合体，研究了纤维杆在肝脏摄取中的作用。结果表明，Ad5纤维杆在体外和体内对肝脏的基因转移均有影响。这些观察结果表明，目前体外描述的腺病毒感染所涉及的、包含CAR和整合素的两步进入途径，在体内肝脏基因转移中并未被采用。相反，α(v)整合素影响基因传递至肺、脾、心和肾。本文所述的去靶向载体构建体可能为靶向腺病毒载体的开发提供基础。
