利用微环DNA载体在肌肉中持续表达HBV特异DART清除cccDNA

To cure chronic HBV infection requires the permanent removal of viral cccDNA. Dual-affinity re-targeting (DART) molecule, a novel rigid and compact format of bispecific antibody that bridges target cells and effector T cells, can induce robust redirected T-cell cytotoxicity to target cells. The peptides derived from the surface antigen (HBs) and core antigen (HBc) are able to presented onto cell surface by the major histocompatibility complex (MHC), where form the MHC/HBV peptides complexes (HBs/MHC and HBc/MHC). The HBs/MHC and HBc/MHC complexes differ from the excessive intact viral antigens in circulation and subviral particles, and they can distinguish the infected cells from the uninfected normal cells, by which they may serve as the attractive specific targets for immunotherapies. In this proposal, we are planning to design the HBs/MHC or HBc/MHC targeted DARTs to clear cccDNA indirectly via a redirected T-cell cytotoxicity on infected cells. However, apart from the high cost, the short half-life of DARTs inconveniences their clinical application, as they were often intravenously administrated with a pump. Therefore, we propose a convenient and affordable gene therapy strategy: durable producing HBV-specific DARTs in skeletal muscle with our established minicircle (MC)/MusFX system. In summary, the minicircle-mediated prolonged expression in muscle of DARTs may serve as an effective, convenient and affordable gene therapy for chronic HBV infection treatment.

根治慢性乙肝有待清除HBV cccDNA。DART(双亲和重定向分子)是一种结构紧凑的新型双特异性抗体，可重定向T细胞至靶细胞并介导T细胞对靶细胞的高效杀伤。HBV表面抗原(HBs)和核心抗原(HBc)胞内表达并加工成多肽后，可经MHC分子递呈至细胞表面，形成HBs/MHC或HBc/MHC复合物；它们有别于游离抗原，是感染细胞表面特有的靶标。本项目拟设计相应的DART，精准靶向感染细胞表面的HBs/MHC或HBc/MHC复合物，介导T细胞杀灭感染细胞，从而间接清除cccDNA。由于DART半衰期短、需连续给药，且成本高昂，本项目提出了体内持续表达DART的低成本基因治疗方案：即利用独特的微环DNA肌肉转染技术实现HBV特异DART在肌肉中的持续表达，达到“一次注射、长久治疗”的效果。总之，利用微环DNA在肌肉中持续表达DART有望成为高效方便、可负担的慢性乙肝基因治疗体系。

HBV共价闭合环状DNA (cccDNA)是病毒复制的原始模板，稳定隐藏于感染细胞核内，是HBV建立持续感染的分子基础。因此，根治慢性乙肝关键在于清除cccDNA。双特异性抗体(BsAb)是近年来兴起的代表性免疫治疗技术，可介导T细胞高效杀伤靶细胞（癌细胞或感染细胞）；其疗效优异，但价格昂贵，半衰期短、作用时间有限，造成给药不便，利用基因载体体内长期表达BsAb可克服这一缺陷。微环DNA(MC)是一种优化的非病毒基因载体，去除了真核来源的骨架DNA和由此带来的转基因沉默效应，使体内表达高效稳定。本项目共设计并构建了3个抗HBV双特异性抗体及相应的MC载体(MC.HBV-BsAb)；其中两个有很高的体外活性，能介导T细胞高效杀灭HBV阳性细胞，杀伤率高达90%以上。我们利用cccDNA小鼠模型（尾静脉高压注射技术将重组cccDNA注射至小鼠肝脏）发现，经MC.HBV-BsAb肌肉注射治疗后，小鼠体内病毒抗原和cccDNA都降低到极低水平，且未发现有严重的毒副作用。这些结果基本证明了利用微环DNA肌肉持续表达HBV双特异性抗体清除HBV感染细胞，从而间接清除cccDNA的设想，达到“一次注射，长期治疗”的效果。但cccDNA小鼠模型与HBV自然慢性感染状态仍有明显不同，MC.HBV-BsAb治疗慢性乙肝的方案仍有待深入研究。由于部分HBV相关肝癌细胞整合了病毒DNA片段且表达病毒抗原，MC.HBV-BsAb方案也可扩展应用于HBV相关肝癌的基因-免疫治疗。此外，本项目还初步研究了微环DNA表达干扰素lambda(MC.IFNλ3)抑制病毒表达和复制的可行性，由于IFNλ毒副作用低于现有干扰素药物(即IFNα)、微环DNA可长期表达，为开发新一代低毒长效抗乙肝干扰素疗法提供了新思路。