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

使用病毒来递送基因治疗已经在临床前环境中进行了广泛的测试，并且已经取得了一些临床进展。腺病毒是一种非常常用的病毒类型，显示出极好的潜力，因为它可以在细胞培养系统中实现非常有效的基因递送到细胞，但在体内它显示出一些限制其应用的毒性，特别是当通过血流递送时。最常用的腺病毒来源于血清型5，但目前正在评估更多的血清型。使用衍生自其它血清型的腺病毒可用于将细胞递送至对由传统血清型5形式介导的基因转移难治的组织。在病毒表面（病毒衣壳）的病毒蛋白中，纤维蛋白（从衣壳表面突出）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一特征来设计基因递送系统，用于特定的e.g.in、心血管疾病或疫苗接种。当通过血流递送时，腺病毒血清型5载体主要感染肝脏和脾脏，并且我们最近已经确定了一种决定这种感染性特征的新途径-基本上病毒与血液中循环的不同凝血因子家族非常紧密地结合，并且这种相互作用将病毒靶向肝脏和脾脏中的特定受体。这种相互作用由纤维蛋白介导。我们现在表明，这是一个非常常见的途径，因为许多其他纤维从不同血清型的腺病毒也结合这些凝血因子。这对于在基因治疗应用中使用这些替代腺病毒作为基因递送载体具有广泛的意义，并且这一点的表征是我们当前资助申请的中心主题。我们与荷兰生物技术公司Crucell合作，评估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（腺病毒主要来源于亚组B和D，但也来源于A和F），并且具有初步数据显示（A）凝血因子直接结合到每种腺病毒，（B）这影响肝来源的细胞系的体外感染性，和（C）体内试验数据表明调节血液中的凝血因子水平强烈影响静脉内递送时病毒的分布。因此，在本研究中，我们希望使用一系列体外和体内实验来详细研究该途径对这些替代腺病毒的生物学、感染性和毒理学的影响。这一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的工作，并进一步了解腺病毒-宿主相互作用，强烈要求在临床环境中使用这些病毒。最后，我们认为，调节凝血因子：病毒相互作用将改善体内基因递送概况，并严重降低迄今为止在使用病毒全身递送的一些腺病毒临床试验中观察到的剂量限制性毒性。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.