可避免转基因产物细胞免疫毒性的重组腺相关病毒载体的构建及评价

A longstanding goal for the gene therapy of genetic diseases is the development of a gene transfer vector that can maintain sustained gene expression in the absence of an immune response. Although recombinant adeno-associated vector (rAAV) remains one of the most promising candidates for therapeutic gene transfer because of its safety and effectiveness, the immune toxicity discovered in clinic trial recently also represents one of the last major roadblocks to the development of a successful therapeutic platform based on rAAV-mediated gene transfer. There is no agreement among many views about its mechanisms till now. Due to rAAV's poor transduction of professional APCs (professional antigen presenting cell), especially the matured DCs (dendritic cells), the roles of DCs in rAAV related immunology were neglected before. Based on recent findings that rAAV can transducer DCs under specific conditions and the facts that DCs are the most powerful APCs, we suggest that transgene products endogenously expressed by matured DCs are responsible for direct antigen presentation onto MHCI and priming of CD8+ T cells. Molecular engineering of viral-vector cassettes with microRNA(miRNA) targets is the latest attempt at regulating expression in gene therapy. As we know that miRs are selectively expressed depending on the develop state of DCs, it's reasonable to to construct a novel rAAV vector to avoid or attenuate the immune potential by including miR targets into expression cassettes of the original rAAV vector. Experiments of gene expression, antigen presentation, T cell priming, CTL-mediated killing of rAAV-transduced target cell in vitro or in vivo will be performed to evaluate the effectiveness of this proposed strategy. We hope a novel rAAV vector will be obtained that not only avoid cellular immune toxicity but also mediate long term transgene expression, after this project is completed, a safe and reliable therapeutic gene transfer platform based on rAAV vector will be supplied.

对于遗传疾病的基因治疗，转基因表达长期稳定、宿主免疫反应轻微的转基因载体，是学界孜孜以求的目标。近期发现被公认为安全性最高的重组腺相关病毒（rAAV）载体也存在细胞免疫毒性风险。因其发生机制尚不明确，目前还没有适当的应对策略。本课题提出减少免疫系统抗原递呈细胞，特别是成熟树突状细胞（DC）内转基因表达，是规避转基因产物细胞免疫毒性的有效方法。尽管rAAV转导DC的效率很低，但最新研究发现在一定条件下仍然可以发生。本课题拟将在成熟DC内特异性高表达的miRNA的结合序列引入常规rAAV载体，构建新型rAAV载体。通过在不同来源及发育阶段的DC等细胞内的转基因表达、抗原递呈、T细胞激活、靶细胞杀伤力等系列研究，证明新型rAAV载体可有效降低转基因产物细胞免疫风险，并通过体内实验证明新型rAAV载体甚至可长期表达抗原性极高的转基因产物，为今后rAAV基因药物的临床研究提供安全保障。

重组腺相关病毒（rAAV）因具有无致病性、低免疫原性、能介导外源基因长期表达、宿主范围广泛等优点，迅速成为最有发展前景的基因治疗载体，但临床研究发现的细胞免疫毒性成为制约其临床疗效的关键。在总结前人报道和自身实验数据的基础上，本课题组认为体内最强大的抗原呈递细胞树突状细胞（DC）应该在rAAV基因载体引起的细胞免疫毒性上发挥主导作用。.本课题利用miRNA可以细胞选择性阻断基因表达的特点，选取成熟树突状细胞 (mDC) 内特异性高表达的miRNA，构建含miRNA完全互补结合序列的rAAV载体，选择性阻断转基因抗原在mDC内的表达、加工和呈递，抑制mDC对细胞毒性T淋巴细胞（CTL细胞）的激活，将为避免或减轻rAAV细胞免疫毒性反应的发生提供新的解决方法。.实验结果：.（1）miR-155在mDC中特异性高表达，含miR-155结合序列（miR-155T）的新型rAAV转基因表达可以选择性的在mDC中沉默；.（2）新型rAAV可以抑制DC的抗原呈递作用；.（3）新型rAAV可以抑制DC对T细胞的激活作用。