Evaluating the impact of coagulation factors on gene delivery using pseudotyped adenoviruses: receptor usage bioavailability and immunogenicity

利用病毒进行基因治疗已在临床前进行了广泛试验，并取得了一些临床进展。腺病毒是这一过程中非常常用的一种病毒，它显示出极好的潜力，因为它可以在细胞培养系统中非常有效地将基因传递给细胞，但在体内，它显示出一些毒性，限制了它的应用，特别是在通过血液传递时。最常用的腺病毒来自血清5型，但目前正在评估更多的血清型。使用来自其他血清型的腺病毒可将细胞递送到难以通过传统血清型5介导的基因转移的组织中。在病毒表面的病毒蛋白（病毒衣壳）中，纤维蛋白（从衣壳表面伸出）是病毒如何与靶细胞上的受体相互作用的主要决定因素。许多血清型结合不同的受体，因此人们可以利用这种特征来设计基因传递系统，用于特定的应用，例如癌症、心血管疾病或疫苗接种。当通过血液传递时，腺病毒血清5型载体主要感染肝脏和脾脏，我们最近发现了一种新的途径，它决定了这种传染性——本质上，病毒与血液中循环的一个独特的凝血因子家族紧密结合，这种相互作用将病毒靶向肝脏和脾脏的特定受体。这种相互作用是由纤维蛋白介导的。我们现在表明这是一个非常常见的途径，因为来自不同血清型腺病毒的许多其他纤维也会结合这些凝血因子。这对于在基因治疗应用中使用这些替代腺病毒作为基因传递载体具有广泛的意义，并且表征这是我们当前资助申请的中心主题。我们与荷兰的一家生物技术公司Crucell合作，评估了凝血途径对腺病毒传染性的影响，这些病毒正在开发用于癌症、心血管疾病和疫苗接种（主要来自B和D亚群的腺病毒，但也来自a和F亚群），并获得了初步数据显示(a)凝血因子直接与每种腺病毒结合；(B)这影响肝源性细胞系的体外感染性和(C)体内试验数据表明，血液中凝血因子水平的调节在静脉注射时强烈影响病毒的分布。因此，在这项研究中，我们希望通过一系列体外和体内实验，充分表征这一途径对这些交替腺病毒的生物学、感染性和毒理学的影响。这种描述将广泛影响研究人员和临床医生在基因治疗和疫苗接种程序中使用这些病毒，并进一步我们对腺病毒-宿主相互作用的理解，这些相互作用强烈地指示了这些病毒在临床环境中的使用。最终，我们相信调节凝血因子：病毒相互作用将改善基因在体内的传递谱，并严重降低剂量限制性毒性，迄今为止在一些使用全身传递病毒的腺病毒临床试验中观察到。反过来，这将使腺病毒载体成为临床应用中更有吸引力的病毒。

The use of viruses to delivery genetic therapy has been widely tested in the pre-clinical setting and some clinical progress has been made. Adenoviruses, a very commonly used type of virus for this procedure, shows excellent potential since it can achieve very efficient gene delivery to cells in cell culture systems but in the body it shows some toxicity that limits its application, particularly when delivered through the bloodstream. The most commonly used adenovirus is derived from serotype 5 but there are many more serotypes that are currently under evaluation. The use of adenoviruses derived from other serotypes is useful for delivery of cells to tissues that are refractory to gene transfer mediated by the traditional serotype 5 form. Of the virus proteins on the surface of the virus (the virus capsid) the fiber protein (that projects from the capsid surface) is the main determinant of how a virus interacts with receptors on target cells. Many serotypes bind different receptors therefore one can exploit this profile to engineer gene delivery systems for specific applications e.g.in cancer, cardiovascular disease, or for vaccination. When delivered via the bloodstream adenovirus serotype 5 vectors predominantly infect liver and spleen and we have recently identified a new pathway that dictates this infectivity profile - essentially the virus binds very tightly to a distinct family of coagulation factors that circulate in the blood and this interaction targets the virus to specific receptors in the liver and spleen. This interaction is mediated by the fiber protein. We now show that this is a very common pathway in that many other fibers from different serotypes of adenovirus also bind these coagulation factors. This has broad implications for the use of these alternate adenoviruses as gene delivery vectors in gene therapy applications and characterisation of this is the central theme of our current grant application. We have worked with Crucell, a biotechnology company based in the Netherlands, to assess the influence of the coagulation pathway on adenovirus infectivity on viruses being developed for cancer, cardiovasccular disease and vaccination (adenoviruses derived from subgroups B and D mainly but also from A and F) and have preliminary data showing that (A) coagulation factors bind directly to each adenovirus, that (B) this influences in vitro infectivity of liver-derived cells lines and (C) pilot data in vivo to show that modulating coagulation factor levels in the blood strongly influences the distribution of the viruses when delivered intravenously. In this study we therefore wish to characterise fully the influence of this pathway on the biology, infectivity and toxicology of these alternate adenoviruses using a range of in vitro and in vivo experiments. Description of this will broadly impact researchers and clinicians working on the use of these viruses in gene therapy and vaccination procedures and further our understanding of adenovirus-host interactions that strongly dictates the use of these viruses in the clinical setting. Ultimately, we believe that modulating the coagulation factor:virus interaction will improve gene delivery profiles in vivo and severely reduce the dose limiting toxicity observed to date in some adenovirus clinical trials using systemic delivery of virus. In turn, this will make the adenovirus vector a more attractive virus for clinical applications.