Ad3.0: Studying infection biology of the natural diversity of adenoviruses and implications for gene-based medicine

腺病毒被广泛用于治疗和疫苗研究，或仅仅作为一种有效的DNA转移工具。近年来，我们一直致力于分析腺病毒基因组在感染哺乳动物细胞后的命运和稳定性。然而，除了腺病毒DNA分子的命运之外，衣壳在腺病毒的细胞转导过程中也起着关键作用。衣壳的主要成分是纤维蛋白、五角蛋白和六角蛋白。直到最近，人们还认为腺病毒纤维和五色子蛋白主要参与了病毒的趋向性。当发现Hexon的高变区(HVR)除了是主要的免疫调节器外，也在趋向性中起着关键作用，这一范式被改变了。然而，到目前为止，由于缺乏足够的遗传操纵系统，仅产生了在HVR内修饰的有限数量的腺病毒。总体而言，由于缺乏有效的腺病毒基因组克隆和修饰性遗传系统，对已鉴定的53个具有不同特性的人腺病毒血清型的鉴定受到阻碍。因此，这项提议首次旨在利用一种系统的方法对腺病毒进行基因改造。基于我们已经建立的细菌人工染色体(BAC)克隆技术，我们已经开始产生一套重组腺病毒。在这项建议中，我们计划(1)从人腺病毒血清型的全谱中克隆六邻体嵌合序列到重组腺病毒载体中。为了确保Hexon的完整性和进行直接比较，我们还计划首次亚克隆野生型腺病毒基因组的全谱作为BAC。重组腺病毒将进行(2)体外评估，包括人血清的中和能力；(3)体内趋向性和先天免疫反应的分析。通过这种方法，我们将获得关于影响腺病毒趋向性的因素的关键信息。与来自不同血清型的全型腺病毒基因组的亚克隆相结合，这种方法将为设计新型治疗性腺病毒铺平道路，包括我们已建立的无肠腺病毒技术。

Adenoviruses are widely used in therapeutic and vaccination studies or simply as a tool for efficient DNA transfer. Over the recent years we have focused on analyzing the fate and stability of adenoviral genomes after infection of mammalian cells. However, besides the fate of the adenoviral DNA molecule the capsid also plays a key role during cellular transduction with adenovirus. The major capsid components are represented by the fiber, penton and hexon proteins. Until recently it was believed that the adenovirus fiber and penton proteins are predominantly involved in virus tropism. This paradigm was changed when it was found that also the hypervariable regions (HVRs) of the hexon, besides being a dominant immune-modulator, also play a key role in tropism. However, until now only a limited number of adenoviruses modified within the HVRs were generated due to the lack of a sufficient system for genetic manipulation. In general the characterization of the 53 identified human adenovirus serotypes with their distinct properties is hindered due to the absence of an efficient genetic system for cloning and modification of adenovirus genomes. Therefore, for the first time this proposal aims at utilizing a systematic approach to genetically modify adenovirus. Based on our established bacterial artificial chromosome (BAC)-cloning technology, we have begun to generate a set of recombinant adenoviruses. In this proposal we plan to (1) clone hexon-chimeric sequences from the entire spectrum of human adenovirus serotypes into a recombinant adenoviral vector. To ensure integrity of the hexon and for direct comparison we also plan for the first time to subclone the complete spectrum of wild-type adenovirus genome as BACs. Reconstituted adenoviruses will be (2) evaluated in vitro including the neutralizing capacity of human sera and (3) the tropism and innate immune responses will be analyzed in vivo. With this approach we will gain pivotal information about factors influencing tropism of adenoviruses. In concert with subcloning of complete adenovirus genomes derived from various serotypes, this approach will pave the way towards design of novel therapeutic adenoviruses including our established gutless adenovirus technology.