Novel targeted adenovirus

摘要 实现有效基因治疗的核心任务是能够实现细胞特异性 交货。最近的研究利用腺病毒载体（Ad）的体内递送功效 强调了靶向广告在这种严格的条件下实现细胞选择性的能力 交付上下文。然而，Ad 在基因传递中的系统应用目前受到以下因素的限制： 矢量粒子隔离在肝脏中。然而，最近几个实验室的工作已经 确定了载体向肝性的生物学决定。基于这样的认识，现在 有可能“非靶向”肝脏，从而促进旨在实现细胞目标的策略 系统载体管理背景下的特定基因递送。值得注意的是，我们最近 研究表明，这种肝脏非靶向策略可以与所述的载体靶向协同作用 方法，例如基于限制递送转基因表达至靶细胞的方法 具有组织/肿瘤选择性启动子（“转录靶向”）。戏剧性的协同作用 结合这两种方法所注意到的特异性增益从逻辑上表明，进一步 组合使用的目标方法可能会带来收益。对此， 已提出基于重定向载体与靶细胞结合来靶向 Ad 的策略 特定的细胞表面标记。这种“转导”靶向方法将提供潜在的 与我们上面提到的定位方法的协同作用。这样的努力仅限于此 当前的载体工程无法实现抗体衣壳的掺入 目标物种。在这里，我们寻求解决这个关键限制。首先，我们开发了一种方法 用没有天然纤维旋钮的替代嵌合体取代天然腺病毒纤维 结合域。这种策略消除了固有的向性，并允许结合 更广泛的大型/复杂候选靶向配体。其次，我们已经证明了 源自骆驼科动物的单域抗体 (sdAb) 具有独特的属性 允许与腺病毒衣壳合成和组装具有生物相容性。总的来说， 这两种技术现在允许抗体靶向物种的功能整合 进入Ad衣壳以实现细胞特异性靶向。这种额外的定位水平 与指定的肝脏非靶向和转录载体提供潜在的协同作用 我们探索过的靶向方法。

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.