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

使用病毒进行基因治疗已经在临床前环境中进行了广泛的测试，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类型，它显示出极大的潜力，因为它可以在细胞培养系统中实现非常有效的基因传递，但在体内它表现出一些毒性，限制了它的应用，特别是当它通过血液传递时。最常用的腺病毒来源于5型血清，但目前还有更多的血清型正在评估中。使用来自其他血清型的腺病毒将细胞运送到对传统5型血清型介导的基因转移无效的组织中是有用的。在病毒表面(病毒衣壳)上的病毒蛋白中，纤维蛋白(从衣壳表面投射出来)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一特性来设计特定应用的基因传递系统，例如在癌症、心血管疾病或疫苗接种中。当通过血流腺病毒5型载体传递时，主要感染肝和脾，我们最近发现了一种新的途径，决定了这种感染性特征-本质上，病毒非常紧密地结合在血液中循环的不同凝血因子家族，这种相互作用将病毒靶向肝和脾中的特定受体。这种相互作用是由纤维蛋白介导的。我们现在发现，这是一种非常常见的途径，因为来自不同血清型腺病毒的许多其他纤维也结合了这些凝血因子。这对于在基因治疗应用中使用这些交替的腺病毒作为基因传递载体具有广泛的意义，而对此进行表征是我们当前拨款申请的中心主题。我们与总部位于荷兰的生物技术公司Crucell合作，评估了凝血途径对腺病毒感染性的影响，这些病毒正在开发用于癌症、心血管疾病和疫苗接种的病毒(腺病毒主要来自B和D亚组，但也来自A和F亚组)，初步数据显示：(A)凝血因子直接与每个腺病毒结合，(B)这会影响肝脏衍生细胞系的体外传染性，(C)体内试验数据表明，调节血液中的凝血因子水平对静脉注射时病毒的分布有很大影响。因此，在这项研究中，我们希望通过一系列的体外和体内实验，全面描述这一途径对这些交替的腺病毒的生物学、感染性和毒理学的影响。对此的描述将对致力于在基因治疗和疫苗接种程序中使用这些病毒的研究人员和临床医生产生广泛影响，并进一步加深我们对腺病毒-宿主相互作用的理解，这种相互作用强烈地决定了这些病毒在临床上的使用。最终，我们相信调节凝血因子：病毒的相互作用将改善体内的基因递送情况，并严重降低迄今为止在一些使用系统递送病毒的腺病毒临床试验中观察到的剂量限制毒性。反过来，这将使腺病毒载体成为临床应用更具吸引力的病毒。

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.