低强度超声激励微泡靶向释放外泌体介导miRNA抑制PD-L1表达促进抗肝肿瘤T细胞免疫的作用研究

Recent reports showed that an increasing number of antibodies directed against immune checkpoint programmed death receptor (PD-1)/PD-1 ligand (PD-L1) holds a great promise to be developed as novel immunotherapies for Hepatocellular carcinoma（HCC）. However, this strategy can induce systemic antibody immune-related adverse events (IRAE). In addition, the novel PD-1/PD-L1-based immunotherapy bear an unaffordable cost as an obstacle for a large number of low-income patients. The goal of this proposal is to examine our new concept and hypothesis that low intensity ultrasound (LIUS) guided, HCC-focused, endogenous exosome-delivered novel PD-L1 targeted microRNAs to HCC cells can be used as a novel therapy to knock-down PD-L1 expression in HCC cells, remove PD-1/PD-L1 immune checkpoint pathway-mediated inhibition of T cell activation, enhance anti-HCC T cell immune cytotoxic responses, and suppress HCC growth. Based on our mostly recent publications on low intensity ultrasound (LIUS)-guided HCC therapy, LIUS upregulation of exosome biogenesis and docking mediators, and LIUS-increased exosome delivery of microRNAs, this conceptually and technically innovative proposal is to use patients' endogenous exosomes as a new type of carrier, in a LIUS guided, HCC-focused, highly efficient, precision and highly safe and less side effect manner, to deliver PD-L1 specifically targeted miRNA to HCC, inhibit PD-L1 expression in HCC, and promote anti-HCC T cell immune responses. The success of this project will be highly significant and will have a high impact in the field in improving the efficacy and safety of anti-tumor/cancer immune therapies, which will demonstrate the novel proof-of-principle for cancer-focused immunotherapies for HCC and other malignancies.

以细胞程序性死亡受体-1（PD-1）/ 细胞程序性死亡-配体1（PD-L1）为靶点的肿瘤免疫治疗有望解决肝细胞癌高转移率和高复发率的难题。但全身抗体治疗缺乏靶向性且可能引发自身超敏反应等严重毒副作用。因此，如何提高靶向性，减少副作用是肿瘤免疫治疗亟待解决的问题。自身源性外泌体具有低免疫原性、生物相容性好等独特优势是理想的靶向基因/药物运送载体。低强度超声激励微泡是一种新型基因/药物递送方式，具有主动寻靶功能，有效提高肿瘤局部基因/药物治疗浓度及治疗效果，降低副作用。本项目拟在前期研究证实低强度超声激励微泡增效外泌体释放及对接的基础上，运用自身源性外泌体为载体，靶向输送抑制PD-L1的特异性miRNA，使其安全高效、精准可控地导入肝癌组织中，抑制PD-L1表达，促进T细胞活化抗肝肿瘤免疫反应。该研究对提高肝肿瘤免疫治疗的安全性及有效性具有重要意义，并为肿瘤免疫靶向治疗技术提供新的思路。

细胞程序性死亡受体-1(PD-1)/ 细胞程序性死亡-配体1(PD-L1)为靶点的肿瘤免疫治疗有望解决肝细胞癌高转移率和高复发率的难题。但全身抗体治疗缺乏靶向性且可 能引发自身超敏反应等严重毒副作用。因此，如何提高靶向性，减少副作用是肿瘤免疫治 疗亟待解决的问题。自身源性外泌体具有低免疫原性、生物相容性好等独特优势是理想的 靶向基因/药物运送载体。低强度超声激励微泡是一种新型基因/药物递送方式，具有主动 寻靶功能，有效提高肿瘤局部基因/药物治疗浓度及治疗效果，降低副作用。本项目明确具有抑制PD-L1的miRNA20b和miRNA494-3p能够抑制PD-L1表达，促进抗肿瘤T细胞作用增效的病理生理相关关系。探讨了参数优化最佳的低强度超声激励微泡辐照参数和外泌体释放途径。明确参数优化的低强度超声激励微泡可增强外泌体携带miRNA20b和miRNA494-3p靶向释放能力，抑制PD-L1表达，增强T细胞对肿瘤细胞的杀伤作用。该研究对提高肝肿瘤免疫治疗的安 全性及有效性具有重要意义，并为肿瘤免疫靶向治疗技术提供新的思路。