Structural and biological insights into novel adenovirus-based platforms for therapeutic applications.

为了开发安全有效的病毒平台，有必要了解病毒介导细胞进入和通过细胞运输到细胞核的结构和分子机制，在细胞核中发生反式和病毒基因表达。此外，了解治疗载体转导对细胞的生物学影响对于确保开发的平台对患者翻译安全有效至关重要。目前研究最好的腺病毒平台是基于C种腺病毒人Ad5。虽然这种病毒已经被很好地理解，并且在实验上对过表达研究很有用，但这种平台在临床应用方面的局限性已经很好地确立了，包括脱靶摄取的高潜力——主要是由肝脏摄取，以及普通人群中对Ad5的预先免疫率很高。因此，非常需要基于社区中自然较低的免疫率和较低的脱靶摄取的替代血清型的新平台。因此，该项目将对源自物种D和B的新型腺病毒的结构和生物学产生新的见解，其中许多平台已经在内部开发并准备进行评估。该项目将大致分为以下几个一般领域：对病毒结构的结构洞察和纤维旋钮结构域介导的细胞结合。主要的细胞附着蛋白，纤维旋钮结构域，可以在细菌中重组产生并纯化。从已研究的腺病毒物种中产生重组纤维旋钮蛋白，纯化并播种结晶。一旦结晶，三聚体的结构将被解决并用于与已知腺病毒受体复杂的预测建模。此外，在可能的情况下，我们将尝试将重组旋钮蛋白与受体复合物共结晶，并使用表面等离子体共振等硅分析来测量结合亲和力。这些研究将通过使用冷冻电子显微镜对病毒的完整结构进行补充。细胞摄取的生物学见解。为了补充第一个区域，将对携带新旋钮蛋白的病毒的结合和进入进行生物学评估。重组knob蛋白将用于IC50实验，使用过表达已知腺病毒受体（CHO-CAR, CHO-BC1, CHO-DSG2）的细胞系。这些研究将通过使用免疫荧光评估重组纤维旋钮蛋白的摄取和核内体的运输进行补充（重组纤维旋钮蛋白含有一个可以通过IF检测到的HIS标签）。将产生基于Ad5的球形假型，并评估其转导携带已知腺病毒受体的细胞的能力。同样，在可能的情况下，将使用整个血清型载体进行检测。为了评估感染细胞中的病毒转录组，将正常成纤维细胞或转化细胞感染wt病毒血清型或由其发展的非复制病毒载体。转录组将与David Matthews博士（Bristol）合作使用Nanopore进行分析，并对病毒转录进行量化。这些研究将在正常细胞和转化细胞的体外细胞杀伤试验中得到补充。作为疫苗载体的体内评估。最后，我们将评估矢量化病毒平台作为疫苗的潜力。小鼠将被肌内或鼻内接种GFP表达载体，并评估t细胞和抗体对GFP的反应。总的来说，这些数据将为治疗应用的新型和可专利的病毒平台提供重要的体外和体内见解。

To develop safe and effective viral platforms, it is necessary to understand the structural and molecular mechanisms by which viruses mediate cell entry and trafficking through the cell to the nucleus, where both trans- and viral gene expression occur. Furthermore, understanding the biological impact on the cell of being transduced by a therapeutic vector will be critical in ensuring that developed platforms are safe and effective for patient translation. The best studied adenovirus platform is based on human Ad5, a species C adenovirus. Whilst this virus is well understood and useful experimentally for overexpression studies, the limitations of this platform for clinical applications are well established and include the high potential for off target uptake - primarily by the liver, and high rates of pre-existing immunity against Ad5 in the general population. Generation of new platforms based on alterative serotypes with naturally lower rates of immunity in the community and lower off target uptake is therefore highly desirable. This project will therefore generate new insights into structure and biology of novel adenoviruses derived from species D and B, where numerous platforms have been developed in house and are ready for assessment. The project will be broadly split into the following general areas: Structural insights into viral structure and cellular binding mediated by the fiber knob domain. The major cellular attachment protein, the fiber knob domain, can be generated recombinantly in bacteria and purified. Recombinant fiber knob proteins from understudied adenoviral species will be generated, purified and seeded for crystallization. Once crystallized, the structure of the trimer will be solved and used for predictive modelling in complex with known adenoviral receptors. Furthermore, where possible we will attempt to co-crystallize recombinant knob protein in complex with the receptors, and binding affinities will be gauged using in silico assays such as surface plasmon resonance. These studies will be complemented by full structural insights of the virus using cryo electron microscopy. Biological insights into cellular uptake. To complement the first area, binding and entry of viruses harboring novel knob proteins will be assessed biologically. Recombinant knob protein will be used for IC50 experiments using cell lines overexpressing known adenoviral receptors (CHO-CAR, CHO-BC1, CHO-DSG2). These studies will be complemented by studies evaluating the uptake of recombinant fiber knob protein and the trafficking in endosomes using immunofluorescence (the recombinant knob protein contains a HIS tag which can be detected by IF). Ad5 based knob pseudotypes will be generated, and their ability to transduce cells bearing known adenovirus receptors will be assessed. Similarly, where available, assays will be performed using whole serotype vectors. To assess the viral transcriptome in infected cells, either normal fibroblasts or transformed cells will be infected with wt viral serotypes or the non-replicating viral vectors developed from them. The transcriptome will be analyzed using Nanopore, in collaboration with Dr David Matthews (Bristol) and viral transcripts quantified. These studies will be complemented by in vitro cell killing assays in normal and transformed cells. In vivo assessment as vaccine vectors. Finally, we will assess the potential of vectorized viral platforms as vaccines. Mice will be inoculated either intramuscularly or intranasally with GFP expressing vectors, and T-cell and antibody responses against GFP will be assessed. Collectively these data will provide critical in vitro and in vivo insights into novel and patentable viral platforms for therapeutic applications.