Optimized RNA Interference Therapeutics Combined with Interleukin-2 mRNA for Treating Hepatitis B Virus Infection. Optimized RNA Interference Therapeutics Combined with Interleukin-2 mRNA for Treating Hepatitis B Virus Infection

BACKGROUND & AIMS: This study aimed to develop a pan-genotypic and multifunctional small interfering RNA (siRNA) against hepatitis B virus (HBV) with an efficient delivery system for treating chronic hepatitis B (CHB), and explore combined RNA interference (RNAi) and immune modulatory modalities for better viral control. METHODS: Twenty synthetic siRNAs targeting consensus motifs distributed across the whole HBV genome were designed and evaluated. The lipid nanoparticle (LNP) formulation was optimized by adopting HO-PEG2000-DMG lipid and modifying the molar ratio of traditional polyethylene glycol (PEG) lipid in LNP prescriptions. The efficacy and safety of this formulation in delivering siHBV (tLNP/siHBV) along with the mouse IL-2 (mIL-2) mRNA (tLNP/siHBVIL2) were evaluated in the rAAV-HBV1.3 mouse model. RESULTS: A siRNA combination (terms “siHBV”) with a genotypic coverage of 98.55% was selected, chemically modified, and encapsulated within an optimized LNP (tLNP) of high efficacy and security to fabricate a therapeutic formulation for CHB. The results revealed that tLNP/siHBV significantly reduced the expression of viral antigens and DNA (up to 3log10 reduction) in dose- and time-dependent manners at single-dose or multi-dose frequencies, with satisfactory safety profiles. Further studies showed that tLNP/siHBVIL2 enables additive antigenic and immune control of the virus, via introducing potent HBsAg clearance through RNAi and triggering strong HBV-specific CD4+ and CD8+ T cell responses by expressed mIL-2 protein. CONCLUSIONS: By adopting tLNP as nucleic acid nanocarriers, the co-delivery of siHBV and mIL-2 mRNA enables synergistic antigenic and immune control of HBV, thus offering a promising translational therapeutic strategy for treating CHB. Overall design: HepG2 cells were incubated with tLNP/siNC or tLNP/siHBV at the dose of 50 nM for 16 h then applied for cDNA extraction and RNA sequencing. Mice were intravenously injected with tLNP/siNC or tLNP/siHBV at the dose of 1 mg/kg for 72 h, then liver samples were collected and snap frozen. RNA was extracted by TRIzol and cDNA libraries were prepared for following RNA sequencing, according to manufactures’ instructions.

研究背景与目的：本研究旨在开发一种泛基因型、多功能的抗乙型肝炎病毒（hepatitis B virus, HBV）小干扰RNA（small interfering RNA, siRNA），搭配高效递送系统用于治疗慢性乙型肝炎（chronic hepatitis B, CHB），同时探索联合RNA干扰（RNA interference, RNAi）与免疫调节策略以实现更优的病毒控制效果。
研究方法：设计并评价了二十条靶向分布于HBV全基因组保守基序的合成siRNA。本研究采用HO-PEG2000-DMG脂质，并调整脂质纳米粒（lipid nanoparticle, LNP）制剂中传统聚乙二醇（polyethylene glycol, PEG）脂质的摩尔比，以优化递送系统。在rAAV-HBV1.3小鼠模型中，评价了包载siHBV的优化LNP制剂（tLNP/siHBV）以及共递送小鼠白细胞介素2（mouse IL-2, mIL-2）mRNA的制剂（tLNP/siHBVIL2）的疗效与安全性。
研究结果：本研究筛选得到一组siRNA组合（命名为“siHBV”），其基因型覆盖率达98.55%，经化学修饰后包载于优化得到的高效安全LNP（tLNP）中，制备得到慢性乙型肝炎治疗制剂。结果显示，单剂或多剂给药时，tLNP/siHBV可呈剂量依赖性与时间依赖性方式显著降低病毒抗原与DNA的表达（最大降幅可达3log10），且安全性良好。进一步研究表明，tLNP/siHBVIL2可通过RNAi介导强效的乙肝表面抗原（hepatitis B surface antigen, HBsAg）清除，并通过表达的mIL-2蛋白触发强效的HBV特异性CD4+与CD8+ T细胞应答，从而实现对病毒的协同抗原与免疫控制。
研究结论：本研究以tLNP作为核酸纳米载体，共递送siHBV与mIL-2 mRNA可实现对HBV的协同抗原与免疫控制，为慢性乙型肝炎的治疗提供了极具转化潜力的治疗策略。
整体实验设计：将HepG2细胞以50 nM的剂量分别与tLNP/siNC或tLNP/siHBV共孵育16小时，随后提取cDNA并进行RNA测序。小鼠以1 mg/kg的剂量经静脉注射tLNP/siNC或tLNP/siHBV，72小时后采集肝脏样本并快速冷冻。采用TRIzol法提取RNA，并按照制造商说明书构建cDNA文库以用于后续RNA测序。