Radiation-induced targeted extracellular vesicles -based gene delivery for glioma therapy

摘要 胶质母细胞瘤(GBM)是最常见的脑部原发恶性肿瘤，预后差。治疗性的 抑制免疫检查点分子可引起肿瘤免疫反应并改善长期疗效 在几种类型的癌症中存活。GBM细胞高度表达免疫检查点分子，包括 程序性死亡配体1(PD-L1)是适应性免疫系统的一个关键的“不要找到我”的信号，以及 CD47是先天免疫系统的信号，也是获得性免疫的调节器 回应。虽然放射治疗已被证明可以中和免疫抑制的GBM 微环境通过增强通常被抑制的肿瘤相关抗原的呈递， 促进CD8+T细胞募集，辐射进一步增强肿瘤细胞表面PD-L1的表达 微环境。不幸的是，检查点抑制剂对GBM的影响很小。一个 有效的GBM治疗需要一个到达脑部肿瘤的递送系统，而全身治疗有限 效果。内源性小囊泡被称为细胞外小泡(EVS)，作为一种递送方式具有很大的前景 由于其独特的属性，包括低免疫原性和天生的稳定性。然而， 由于对未经修饰的EV的靶向性差，将EV静脉输送到大脑仍然是一个主要挑战， 可以通过表面改性来改善其性能。我们的初步结果表明，环状(Arg-Gly-Asp- 与肿瘤血管内皮细胞上的整合素αvβ3具有高亲和力的D-酪氨酸赖氨酸)多肽可能是 结合在EVS表面(来自FDA批准的正常神经前体细胞)，导致 静脉给药后改善EV在脑肿瘤中的积聚。此外，在最近 研究表明，短时间的放射治疗可以促进肿瘤的积累和 肿瘤相关巨噬细胞依赖的纳米治疗药物在肿瘤内的分布 结果显示，经放射治疗的胶质母细胞瘤对靶向EV的摄取增强。在这份提案中， 我们将利用目标电动汽车的这些独特特性，为它们加载小干扰 针对PD-L1和CD47的RNA(SiRNAs)以在胶质母细胞瘤部位实现增强的免疫反应， 被辐射启动的。我们将评估放射治疗是否会促进胶质母细胞瘤的增强。 通过BBB摄取这些靶向EV向GBM传递siRNAs，增加CD8T细胞的细胞毒作用 在同基因移植的GBM小鼠模型中，该药物具有抑制肿瘤生长和延长动物存活的活性。

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor with poor prognosis. Therapeutic suppression of immune checkpoint molecules elicits a tumor immune response and improves long-term survival in several types of cancers. GBM cells highly express immune checkpoint molecules including programmed death-ligand 1 (PD-L1) a critical “don’t find me” signal to the adaptive immune system, and CD47, a “don’t eat me” signal to the innate immune system as well as a regulator of the adaptive immune response. Although radiation therapy has been shown to counteracts the immunosuppressive GBM microenvironment by enhancing the presentation of normally suppressed tumor-associated antigens, promoting CD8+ T cell recruitment, radiation enhances even further the expression of PD-L1 on tumor and microenvironment. Unfortunately, little effect has been observed with checkpoint inhibitors against GBM. An effective GBM therapy requires a delivery system that reaches the tumor in the brain, with limited systemic effect. Endogenous small vesicles known as extracellular vesicles (EVs) hold a great promise as a delivery vehicle given their unique properties including low immunogenicity and innate stability. However, intravenous delivery of EVs to the brain remains a major challenge due to poor targeting of unmodified EVs, which can be improved by surface modification. Our preliminary results shows that The cyclo(Arg-Gly-Asp- D-Tyr-Lys) peptide, which exhibits high affinity to integrin αvβ3 on tumor vascular endothelial cells, could be conjugated on EVs surface (derived from FDA-approved normal neural progenitor cells), resulting in improved EV accumulation in brain tumors after intravenous administration. Furthermore, building on recent studies showing that short bursts of radiation therapy can prime tumors for enhanced accumulation and intratumoral distribution of nanotherapeutics in tumor-associated macrophages-dependent fashion, we showed that glioblastomas primed with radiation had an enhanced uptake of targeted EVs. In this proposal, we will take advantage of these unique characteristics of targeted EVs and load them with small interfering RNAs (siRNAs) against PD-L1 and CD47 to achieve enhanced immune response at the glioblastoma site, primed with radiation. We will evaluate whether radiation therapy will prime glioblastomas for enhanced uptake of these targeted EV across the BBB to deliver siRNAs to GBM, to increase CD8 T cells cytotoxic activity, thus halting tumor growth and prolonging animal survival in a syngeneic graft GBM mouse model.