Interrogation of the adenovirus hexon: A new paradigm for virus biology and application to therapeutic gene delivery

基因治疗正在迅速发展成为治愈或阻止许多人类疾病进展的有效方法。第一批基因治疗产品最近在中国(用于癌症治疗)获得许可。因此，利用基因治疗人类疾病是一个现实的机会，可以提供有效和持久的治疗，特别是那些缺乏最佳药物治疗的疾病。基因治疗的一个绝对关键的方面是基因输送，即将治疗货物输送到体内正确的细胞和组织的方法。基因传递是通过具有将治疗性DNA转移到细胞中的能力的“载体”实现的--这些载体可以是非病毒的，也可以是基于病毒的。腺病毒载体通常被用作基于病毒的递送系统，目前在所有正在进行的基因治疗临床试验中约有25%被使用。这些病毒的优点是它们具有将基因转移到广泛细胞的能力，因此使它们适合于广泛的基因治疗应用。然而，尽管这种病毒在临床上得到了广泛的应用，但定义该病毒实际如何感染细胞的基本机制尚未解开。在细胞培养中，病毒结合到一个确定的受体(称为柯萨奇和腺病毒受体，CAR)，并通过与称为整合素的蛋白质相互作用进入细胞。然而，当我们最近将病毒注射到血液中时，我们发现病毒实际上是使用血液中的蛋白质来感染细胞，而不是病毒与肝脏中的靶细胞直接相互作用。病毒将凝血因子FX(FX)招募到病毒表面称为六邻体的蛋白质上，该病毒：FX复合体负责通过FX蛋白上的一个结构域(丝氨酸蛋白酶结构域)与细胞结合。这对于腺病毒生物学和将这种病毒应用于人类的基因治疗是一个关键的新概念。Fx与Hexon的相互作用是意想不到的，并为该病毒蛋白在介导病毒感染性方面赋予了重要的新功能。因此，我们的研究旨在通过使用高分辨率技术(结晶学)并通过对六邻体突变的分子分析来生成不具有FX结合能力的病毒来全面记录这种新的相互作用。我们还将审问与FX的互动。腺病毒载体的主要问题之一是，许多人在自然界中接触过这种病毒，因此对该病毒具有预先存在的免疫力。其中很大一部分是针对六邻体蛋白的，因此我们将评估FX和腺病毒六邻体突变体对中和抗体阻止感染的能力的影响。我们的目标是创造没有外汇结合和中和能力的载体。这将创造新一代腺病毒载体，广泛应用于人类基因治疗。

Gene therapy is rapidly developing into an effective method to cure or halt the progression of many human diseases. The first gene therapy products have been licensed recently in China (for cancer therapy). Therefore, the use of genes to treat human disease is a realistic opportunity to provide efficient and lasting therapies, especially those diseases that lack optimal drug-based therapies. An absolutely critical aspect of gene therapy is gene delivery, i.e. the method that is used to deliver the therapeutic cargo to the correct cells and tissues in the body. Gene delivery is achieved by a 'vector' that has the capability of transferring the therapeutic DNA into cells - these vectors can either be non-viral or viral-based. Adenovirus vectors are commonly used as a viral-based delivery system and are currently utilised in some 25% of all ongoing clinical trials in gene therapy. The advantages of these viruses is that they have the ability to transfer genes to a broad range of cells, hence this makes them suitable for a wide range of applications to gene therapy. However, despite the extensive use of this virus in the clinic, basic mechanims that define how the virus actually infects cells have not been unravelled. In cell culture the virus binds to a defined receptor (called the coxsackie and adenovirus receptor, CAR) and enters the cell by interaction with proteins called integrins. However, when injected into the bloodstream we recently showed that the virus actually uses proteins from the blood to infect cells rather than a direct interaction of the virus with the target cells in the liver. The viruses recruit coagulation factor FX (FX) to a protein on the virus surface called the hexon and this virus:FX complex is responsible for cell binding through a domain on the FX protein (the serine protease domain). This is a critical new concept for adenovirus biology and the application of this virus to gene therapy in humans. The interaction of FX with the hexon was not expected and has assigned an important new function to this virus protein in mediating infectivity of the virus. Our study is therefore designed to fully document this novel interaction by using high resolution techniques (crystallography) and through the molecular analysis of hexon mutants to generate viruses that are devoid of FX binding capacity. We will also interrogate the interaction with FX. One of the major issues with adenovirus vectors is that many humans have been exposed to the virus in nature and thus have pre-existing immunity to the virus. A substantial proportion of this is against the hexon protein and hence we will assess the impact of FX and the adenovirus hexon mutants on the ability of neutralising antibodies to block infection. Our aim is to create vectors devoid of FX binding and devoid of neutralising capacity. This will create a new generation of adenovirus vectors for wide ranging applications to human gene therapy.