Novel targeted adenovirus

摘要 实现有效的基因治疗的核心任务是能够完成细胞特异性 送货。利用腺病毒载体(Ad)的体内递送效率，最近的研究 我强调了定向广告在这一严格的条件下实现细胞选择性的能力 交付上下文。然而，系统地使用Ad进行基因传递目前受到以下因素的限制 媒介粒子在肝脏中的封存。然而，最近几个实验室的工作已经 确定了媒介嗜肝性的生物学规定。基于这种认识，它现在已经 有可能去靶向肝脏，从而促进旨在实现细胞的策略 全身性载体管理背景下的特定基因传递。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描述的载体靶向协同作用 基于将转基因表达限制在靶细胞上的方法 使用组织/肿瘤选择性启动子(“转录靶向”)。戏剧性的协同效应 从逻辑上讲，这两种方法的结合带来的特异性收益表明， 联合使用的其他目标定位方法可能会增加收益。在这方面， 已经提出了基于重定向载体绑定到靶细胞的靶向广告的策略 特定的细胞表面标记。这种“转导”的目标定位方法将提供潜在的 与我们上面提到的目标方法的协同作用。这样的努力仅限于此 目前的载体工程不能实现抗体的衣壳掺入 瞄准物种。在此，我们试图解决这一关键限制。首先，我们开发了一种方法 用不含原生腺病毒纤维旋钮的嵌合体替换原生腺病毒纤维 结合结构域。这种策略消除了本机取向，并允许合并一个 更广泛的大/复杂候选靶向配体。第二，我们已经证明了 骆驼源单域抗体(SdAb)具有独特的特性 允许与腺病毒衣壳合成和组装的生物相容性。总而言之， 这两种技术现在允许抗体靶向物种的功能性结合。 进入Ad衣壳以实现细胞特异性靶向。这一额外的目标水平 提供与已定义的肝脏非靶向和转录载体的潜在协同作用 我们已经探索了目标定位的方法。

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.