肺癌的发生发展涉及多基因、多因素和多环节。联合基因治疗是肺癌基因治疗的新方向，其最大的挑战是目的基因及载体的筛选。我们前期研究证明CD40L-M可减少因产生可溶性CD40L而引起的副作用，并具有更好的抗肺癌效果。更重要的是我们预实验提示CD40L-M可能与klotho这一抗衰老、潜在抗癌基因发挥协同抗肺癌作用。腺相关病毒（AAV）是最具潜力的病毒载体。我们前期研究显示，自身互补AAV血清型5及6（scAAV5及scAAV6）转导肺癌细胞具有明显优势。新近研究发现AAV外壳酪氨酸残基磷酸化影响其转导效率。本项目拟在此基础上将AAV外壳蛋白酪氨酸残基点突变为苯丙氨酸，应用新一代scAAV5及scAAV6，分别介导CD40L-M和klotho，高效转导肺癌细胞，进行联合基因治疗，探讨其抗肿瘤免疫及抑制肺癌的作用。预期在肺癌联合基因治疗的关键环节有所突破，为肺癌联合基因治疗的临床应用奠定实验基础。

The carcinogenesis and progression of lung cancer is a result of multi-gene, multi-factor and multiple step process. Combined gene therapy may provide a new direction for lung cancer gene therapy. However, the choice of target gene and gene delivery vector remains a critical challenge in combined gene therapy. In our previous studies, it has been shown that the mutant CD40L (CD40L-M) can reduce potential side-effects associated with soluble CD40L, but it has better anti-cancer effect. What is more important is that in our preliminary experiment, we found a synergistic anti-cancer effect of CD40L-M and klotho gene, which is characterized as a new anti-aging gene, and a potential anti-cancer agent. Recombinant adeno-associated virus (AAV) vectors are one of the most attractive viral vectors. Recent research shows that phosphorylation of capsid proteins at tyrosine residues is a prerequisite for ubiquitination and degradation of AAV, leading to inefficient intracellular transduction. Our initial research has proven that self-complementary double strained AAV serotypes 5 and 6 (scAAV5 and scAAV6) achieved high-efficiency transduction to lung cancer cells. Based on the previous studies, this project is to further improve AAV by site-directed mutagenesis of surface-exposed tyrosine residues for phenylalanine on viral capsid so as to build a new generation of AAV gene-delivery system which will be capable of targeted delivery of CD40L-M and klotho combination gene by sc-Y719F-AAV5 and sc-Y731F-AAV6. The efficient and safe antineoplastic effects will be assessed in vitro and in vivo by exhibiting anti-proliferative and pro-apoptotic effects and activating host immune systems. Meanwhile, we expect our research to provide an experimental basis for further studies and lead to breakthroughs in combined gene therapy for lung cancer.