Novel targeted adenovirus

摘要 实现有效基因治疗的中心任务是实现细胞特异性靶向治疗的能力。 交付.利用腺病毒载体（Ad）的体内递送功效，最近的研究 已经强调了有针对性的广告的能力，以实现细胞的选择性，在这个严格的 交付上下文。然而，系统地使用Ad进行基因递送目前受到以下限制： 载体颗粒在肝脏中的隔离。然而，最近几个实验室的工作， 确定了载体嗜肝性的生物学指示。基于这一认识， 已经有可能“非靶向”肝脏，从而促进旨在实现细胞增殖的策略。 在系统性载体施用的情况下的特异性基因递送。值得注意的是，我们最近 表明这种肝脏非靶向策略可以与所述载体靶向协同作用 例如基于限制递送的转基因表达至靶细胞的那些方法 用组织/肿瘤选择性启动子（“转录靶向”）。戏剧性的协同效应 结合这两种方法注意到的特异性增益逻辑上表明， 如果结合使用其他目标确定方法，可能会产生收益。在这方面，委员会注意到， 已经提出了基于重定向载体与靶细胞的结合来靶向Ad的策略 特异性细胞表面标志物。这种“转导”靶向方法将提供潜在的 与我们上面提到的定向方法协同作用。这种奋进仅限于此 目前的载体工程不能实现抗体的衣壳掺入 目标物种。在这里，我们试图解决这个关键限制。首先，我们开发了一种方法 用缺乏天然纤维结的替代嵌合体替换天然腺病毒纤维 结合域这种操作消除了天然向性，并允许掺入一种 更广泛的大/复杂的候选靶向配体。第二，我们已经证明， 来源于骆驼科动物的单域抗体种类（sdAb）具有独特的属性， 允许与腺病毒衣壳合成和组装的生物相容性。总的来说， 这两种技术现在允许抗体靶向物质的功能性掺入 用于实现细胞特异性靶向。这种额外的目标定位 提供了与定义的肝脏非靶向和转录载体的潜在协同作用 我们探索的方法。

ABSTRACT A central mandate to realize effective gene therapy is the ability to accomplish cell specific delivery. Capitalizing on the in vivo delivery efficacy of adenoviral vectors (Ad), recent studies have highlighted the capacity of targeted Ad to accomplish cell selectivity in this stringent delivery context. Systemic employment of Ad for gene delivery, however, is currently limited by vector particle sequestration in the liver. Recent work in several laboratories, however, has identified the biologic dictates of vector hepatotropism. Based on this understanding, it has now been possible to "un-target" the liver thereby facilitating strategies designed to achieve cell specific gene delivery in the context of systemic vector administration. Of note, we have recently shown that such liver un-targeting strategies can synergize with described vector targeting methods such as those based upon restricting delivered transgene expression to target cells with a tissue/tumor selective promoter ("transcriptional targeting"). The dramatic synergistic specificity gains noted with combination of these two approaches logically suggests that further gains may accrue additional targeting methods exploited in combination. In this regard, strategies have been proposed to target Ad based upon re-directing vector binding to target cell specific cell surface markers. Such "transductional" targeting methods would offer potential synergies with the targeting methods we note above. Such an endeavor has been limited to this point by the inability of current vector engineering to achieve capsid incorporation of antibody targeting species. Herein we seek to address this key limit. First, we have developed a method to replace the native adenovirus fiber with a substitute chimera devoid of the native fiber's knob binding domain. This maneuver eliminates native tropism and allows for the incorporation of a wider range of large/complex candidate targeting ligands. Second, we have demonstrated that the single domain antibody species derived from camelids (sdAb) possess the unique attributes allowing biologic compatibility with adenovirus capsid synthesis and assembly. In the aggregate, these two technologies now allow for the functional incorporation of antibody targeting species into the Ad capsid for the achievement of cell-specific targeting. This additional level of targeting provides for potential synergies with the defined liver un-targeting and transcriptional vector targeting methods we have explored.