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

对基于腺病毒的新型治疗应用平台的结构和生物学见解。

• 批准号: 2604453
• 金额: --
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2604453
• 关键词: Structural; biological; insights; into; novel

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.

为了开发安全和有效的病毒平台，有必要了解病毒通过细胞介导细胞进入和运输到细胞核的结构和分子机制，在那里发生跨基因和病毒基因的表达。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是安全和有效的至关重要。研究最多的腺病毒平台是以人Ad5为基础的，人Ad5是一种C型腺病毒。虽然这种病毒在实验上对过度表达研究很有帮助，但这个平台在临床应用上的局限性也是众所周知的，包括很高的非靶标摄取潜力--主要是通过肝脏，以及普通人群中对Ad5的高免疫率。因此，基于可变血清型的新平台的产生是非常可取的，因为该平台在社区中具有自然较低的免疫率和较低的目标摄取率。因此，该项目将对来自物种D和B的新腺病毒的结构和生物学产生新的见解，这两个物种的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一般领域：对病毒结构的结构洞察和纤维节域介导的细胞结合。主要的细胞附着蛋白，纤维结节结构域，可以在细菌中重组产生并纯化。来自未被研究的腺病毒物种的重组纤维结节蛋白将被产生、纯化并播种用于结晶。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的复合体的预测建模。此外，在可能的情况下，我们将尝试在与受体的复合体中共结晶重组纽结蛋白，结合亲和力将用于电子分析，如表面等离子体共振。这些研究将得到使用低温电子显微镜对病毒的全面结构洞察的补充。对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。重组的纽结蛋白将用于IC50实验，使用过表达已知腺病毒受体的细胞系(CHO-CAR，CHO-BC1，CHO-DSG2)。这些研究将得到利用免疫荧光评价重组纤维结节蛋白的摄取和内体运输的研究的补充(重组纤维结节蛋白含有组氨酸标签，可通过IF检测)。将产生基于Ad5的纽结假型，并将评估它们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，无论是正常的成纤维细胞还是转化的细胞，都会被wt病毒血清型或从它们发展出来的非复制型病毒载体感染。转录组将与David Matthews博士(布里斯托尔)合作，使用Nanopore进行分析，并对病毒转录本进行量化。这些研究将得到正常细胞和转化细胞的体外细胞杀伤试验的补充。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。小鼠将通过肌肉或鼻腔接种GFP表达载体，并将评估针对GFP的T细胞和抗体反应。总的来说，这些数据将在体外和体内为治疗应用提供新的和可申请专利的病毒平台的关键见解。