重组型腺相关病毒载体调控树突状细胞先天性免疫反应的分子机制

Recombinant adeno-associated viral vectors (rAAV) have been used in a number of gene therapy clinical trials. Inflammatory responses have been reported in those trials that were triggered by the rAAV capsid. These responses induced cytotoxic T cell activity and eventually reduced gene therapy efficacy. Due to the lack of information about the immune cells targeted by rAAV, previous research focused on the adaptive immune responses. On the other hand, the mechanism of innate immune responses is largely unclear. In recent years, we and others documented that rAAV can efficiently transduce dendritic cells. Furthermore, our pilot experiment suggested that rAAV transduction can significantly enhance the phosphorylation and nuclear translocation of glucocorticoid receptor (GR), a well-known innate immune regulator. Thus, the current proposal aims to explore the detailed molecular mechanisms using dendritic cells. First, through techniques such as sub-cellular co-localization, molecular cloning, etc, we will study various rAAV capsid domains to demonstrate their ability of GR activation. The capsid will then be modified accordingly through site-specific mutagenesis, in order to further increase the GR activator function. Second, we plan to reveal the effect of rAAV-activated GR on the expression of its downstream target genes, both in vitro and in vivo, using high-throughput techniques such as Chip assays. In addition, the cross talk between GR and other immune-related signal pathways will also be investigated. To summarize, our proposed study will provide not only the answers of fundamental virological questions, but also the pre-clinical data for future gene therapy clinical trials.

临床数据表明重组型腺相关病毒载体（rAAV）衣壳蛋白能引起炎症反应和杀伤性T细胞反应，显著降低基因治疗疗效。由于不清楚rAAV靶向哪些免疫细胞，过去的研究集中于获得性免疫应答，而对先天性免疫反应知之甚少。近年来，我们以及他人的工作成果均表明rAAV能够高效感染树突状细胞。进一步，我们的预试验结果提示rAAV能显著提高免疫调控因子糖皮质激素受体（GR）的磷酸化与核定位。因此，本项目将以树突状细胞为研究对象，探索通过GR信号通路抑制rAAV先天性免疫反应的详细分子机制。首先，我们将通过共定位、分子克隆等方法研究衣壳蛋白不同结构域激活GR的作用。并以此改造病毒载体，使之更有效地激活GR。其次，我们将在体内外实验中通过芯片技术等高通量方法阐明由rAAV激活的GR对下游靶基因表达的影响，以及与其它免疫相关信号通路的对话。综上，我们的研究将为临床用药降低rAAV免疫应答提供理论基础和实验依据。

重组腺相关病毒载体介导的基因治疗近年来在临床上有多个成功案例，获得了美国食品药品监督管理局（FDA）的上市批准。然而，先天性免疫反应严重限制了重组腺相关病毒载体的临床应用。在重组腺相关病毒载体转导细胞过程中，载体与宿主细胞膜受体结合，通过受体介导的内吞作用被内在化进入细胞内含体。在内含体的酸性环境下，重组腺相关病毒载体外壳蛋白发生构想改变，暴露出磷脂酶结构域，从而逃离被内含体、溶酶体降解的命运，转而进入细胞质并进一步进入细胞核表达转基因。然而，尚不清楚重组腺相关病毒载体外壳蛋白能否在激活内含体中Toll样受体以及细胞质中的糖皮质激素受体。本项目评估了Toll样受体和糖皮质激素受体通路抑制重组载体先天性免疫反应的作用，并初步探索了分子机制。我们发现Toll样受体激动剂（腺嘌呤类似物CL264和咪唑喹诺酮化合物R848）和糖皮质激素受体激动剂（地塞米松和可的松）都能引起树突状细胞和B、T细胞的广泛激活，但Toll样受体激动剂并没有改变先天性免疫反应的水平。进一步研究表明，在没有诱导单核细胞来源的树突细胞的情况下，重组载体激活B细胞和树突状细胞亚群，但不会影响先天性免疫反应以及针对转基因蛋白的抗体形成。此外，我们发现重组载体中的残留内毒素能够激活宿主先天性免疫反应，并显著改变Toll样受体和糖皮质激素受体下游靶基因表达水平。据此，我们设计了新型重组腺相关病毒载体外壳蛋白，改进了生产步骤，为低免疫原性的重组腺相关病毒载体开发提供了理论与实验依据。