溶瘤病毒治疗是靶向肿瘤免疫治疗的重要分支，近期在临床研究中取得了较大进展，成为了研究热点之一。溶瘤病毒联用其它肿瘤治疗手段可以进一步提高肿瘤治疗效果，表现出巨大的临床应用价值。但由于调控机制比较单一，现有溶瘤病毒疗法不能通过精准调控肿瘤治疗的强度、位置和时间应对复杂的肿瘤微环境，存在安全风险和优化空间。本研究尝试利用合成生物学手段，综合考虑肿瘤微环境信息，构建精细的可编程基因线路开关，调控溶瘤腺病毒在肝癌细胞中的选择性复制和免疫效应因子的精准表达，为肝癌临床治疗提供新的思路。首先，我们构建了调控溶瘤腺病毒的可编程基因线路开关。通过总结之前的实验证据和数学模型分析，确定利用双向互抑制闭环基因线路作为开关骨架；通过基因表达差异分析和细胞功能验证，筛选、改造启动子和微小RNA标志物作为输入信号；通过细胞功能实验，验证利用不同互抑制蛋白对构建的基因线路对输入信号的响应效率。在此基础上，我们针对确定的基因线路设计了模块化的快速组装方法并构建了响应不同癌种的输入信号库，为快速开发靶向不同类型肿瘤的溶瘤腺病毒提供了高效的技术平台。第二，比较基因线路调控的合成溶瘤腺病毒和传统溶瘤腺病毒的安全性和治疗效果。在细胞和裸鼠水平的实验结果表明，合成溶瘤腺病毒在同等可比的治疗效果下具有更高的鲁棒性。第三，在细胞水平验证合成溶瘤腺病毒的生物功能。实验结果表明，合成溶瘤腺病毒可以有效杀伤多种肝癌细胞，而不影响正常肝细胞生长。同时，合成溶瘤腺病毒可以在癌细胞中表达具有生物功能的外源效应因子，并成功释放到细胞外。第四，在动物水平检测了合成溶瘤腺病毒的治疗效果。实验结果表明，溶瘤腺病毒可以有效杀伤小鼠模型中的肿瘤细胞，并且在免疫健全的C57小鼠模型中产生系统的抗肿瘤免疫反应。该过程中，病毒携带的免疫效应因子，腺病毒剂量以及腺病毒在靶向肿瘤细胞中的包装效率是影响溶瘤腺病毒杀伤肿瘤效果的重要因素。本论文利用合成生物学的理念和技术手段构建了靶向肝细胞癌的更加稳定，精准的合成溶瘤腺病毒。通过细胞水平和小鼠水平的实验验证了合成溶瘤腺病毒的疗效和安全性，为提高溶瘤腺病毒在临床水平的应用提供了新的研究思路和技术手段。
As an important branch of targeted tumor immunotherapy, oncolytic adenovirus has made great progresses in clinical research recently and become one of the research hotspots. Combination of oncolytic virus and other tumor treatment methods can improve the tumor therapeutic effect greatly and show great value in clinical applications. However, due to the oversimplified regulation mechanism, existing oncolytic virus therapy cannot cope with the complex tumor microenvironment by precisely regulating the intensity, location and time of tumor treatment, which leaves safety risks and optimization space. This study attempts to construct an oncolytic adenovirus programmed by synthetic gene circuit switch which integrates the information of the tumor microenvironment and selectively replicates in hepatocellular carcinoma. The synthetic oncolytic adenovirus will provide new strategy for hepatocellular carcinoma clinical treatment. Firstly, for the strict control of oncolytic adenovirus, we constructed a programmable gene circuit switch. By summarizing the previous experimental evidences and analyzing the mathematical model, we chose the mutual inhibition closed gene circuit as the switch skeleton. Gene expression differential analysis and cell function verification were used to screen and modify promoters and microRNA markers as input signals. Cell function experiments were conducted to verify the response efficiency of the constructed gene circuits to input signals by using different inter-inhibitory proteins. On this basis, we designed a modular rapid assembly method for the identified gene circuits and constructed an input signal library in response to different cancer species, providing an efficient technology platform for the rapid development of oncolytic adenovirus targeting different types of tumors.Secondly, the safety and therapeutic effect of synthetic oncolytic adenovirus and existing oncolytic adenovirus were compared. Experimental results at the cellular and nude mouse levels indicate that the synthetic oncolytic adenovirus is more robust with comparable therapeutic effects.Thirdly, validate the biological function of synthetic oncolytic adenovirus at the cellular level. The results showed that the synthetic oncolytic adenovirus could kill many kinds of hepatocellular carcinoma cells effectively without affecting the growth of normal hepatocytes. Meanwhile, synthetic oncolytic adenovirus can express exogenous effectors with biological functions in cancer cells and release them successfully to the outside of cells.Fourthly, the therapeutic effect of synthetic oncolytic adenovirus was tested at the animal level. The results showed that the oncolytic adenovirus could kill tumor cells effectively in the mouse model and produce a systematic anti-tumor immune response in the immune competent C57 mouse model. The immune effectors carried by the virus, adenovirus dose and the packaging efficiency of adenovirus in targeted tumor cells are the important factors affecting the killing effect of oncolytic adenovirus on tumor.This thesis constructs a stable and accurate synthetic oncolytic adenovirus targeting hepatocellular carcinoma using the ideas and techniques of synthetic biology. The efficacy and safety of synthetic oncolytic adenovirus were verified by cell level and mouse level experiments, which providing new interventions for HCC.
