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

腺病毒广泛用于治疗和疫苗接种研究，或仅作为有效DNA转移的工具。近年来，我们一直专注于分析腺病毒基因组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期间也起关键作用。 主要的衣壳组分由纤维、五邻体和六邻体蛋白表示。直到最近，人们还认为腺病毒纤维和五邻体蛋白主要参与病毒的嗜性。当发现六邻体的高变区（HVR）除了是显性免疫调节剂之外，还在向性中发挥关键作用时，这种范式发生了变化。然而，由于缺乏足够的遗传操作系统，到目前为止，只产生了有限数量的在HVR内修饰的腺病毒。一般来说，由于缺乏用于克隆和修饰腺病毒基因组的有效遗传系统，53种鉴定的人腺病毒血清型及其独特性质的表征受到阻碍。 因此，该提案首次旨在利用系统的方法来遗传修饰腺病毒。基于我们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。在这个提议中，我们计划（1）从人腺病毒血清型的整个谱中克隆六邻体嵌合序列到重组腺病毒载体中。为了确保六邻体的完整性和直接比较，我们还计划首次亚克隆野生型腺病毒基因组的完整谱作为BAC。将（2）在体外评价重组腺病毒，包括人血清的中和能力，和（3）在体内分析嗜性和先天免疫应答。通过这种方法，我们将获得关键的信息，腺病毒嗜性的影响因素。与来自各种血清型的完整腺病毒基因组的亚克隆相一致，这种方法将为设计新型治疗性腺病毒铺平道路，包括我们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.