Fruit exosome-like particles for therapeutic delivery of extracellular miRNAs

描述（由申请人提供）：本提案的总体目标是开发外泌体样纳米颗粒作为递送载体，用于靶向递送细胞外RNA（exRNA），主要集中于在小鼠肿瘤模型中递送时调节基因表达并诱导免疫应答的miRNA。基于exRNA的治疗剂的开发必须克服挑战，包括递送、潜在脱靶效应、安全性、毒性、大规模生产的成本、消除对患者的潜在生物危害和组织特异性的问题。与人工合成的纳米颗粒或脂质体的情况不同，天然来源的纳米颗粒大小的外泌体从许多不同类型的细胞中释放。miRNA和RNA可以被封装在天然来源的外泌体中，并且RNA是稳定的，并且许多miRNA可以同时被天然封装在这些外泌体中。最近，我们和其他小组发表的数据表明，哺乳动物外泌体可以在体内递送难溶性药物、化疗药物和siRNA。这种模式的限制是需要大规模生产外泌体，以及潜在的生物安全性。我们的初步数据表明，外泌体样纳米颗粒可以从可食用植物（包括葡萄）的组织中大量分离出来。我们已经证明，来自葡萄的外泌体样纳米颗粒由小RNA、蛋白质和脂质组成。由葡萄外泌体样衍生脂质组装的可食用植物衍生纳米载体（EPNV）能够包封siRNA。通过EPNV共同递送叶酸与化疗药物紫杉醇导致肿瘤组织靶向效率提高。通过使EPNV穿过适当孔径的过滤器来减小EPNV的尺寸显著增强了EPNV向脑的易位，并且口服施用EPNV导致其向肝的迁移。基于这些初步数据和现有文献，我们提出：（1）确定ENV是否可以通过口服给药递送治疗性miR 17以靶向肝脏中的转移性结肠肿瘤，随后诱导NK细胞活化以杀死转移性肿瘤细胞;（2）确定包封在EPNV中的miR 155是否促进髓源性抑制细胞（MDSC）的分化在小鼠乳腺/脑肿瘤模型中将miRNA 17或miRNA 155导入成熟树突状细胞;（3）确定通过ENV递送的治疗剂量的miRNA 17或miRNA 155是否在小鼠肿瘤模型中不诱导副作用;和（4）确定是否可以经济地大规模产生ENV以用于临床环境。使用装载有期望的治疗性细胞外RNA（exRNA）（包括微小RNA）的基于EPNV的递送系统在体内靶向炎性细胞和肿瘤细胞的证明将不仅是癌症治疗中的重要一步，而且还提供了治疗癌症的有力工具。 鉴定包封在驻留或循环微泡中的每种小RNA对受体细胞的作用和影响。我们将在我们的研究中通过敲除和敲入策略来实现这一点。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop exosome-like nanoparticles as a delivery vehicle for targetable delivery of extracellular RNA (exRNA) primarily focusing on miRNAs which modulate gene expression and induce immune responses upon delivery in mouse tumor models. Development of exRNA-based therapeutics must overcome challenges including issues of delivery, potential off-target effects, safety, toxicity, the cost of large scale production, elimination of potential biohazards to the environmen and tissue specificity. Unlike the situation with artificially synthesized nanoparticles or liposomes, naturally derived nanoparticle-size exosomes are released from many different types of cells. miRNAs and RNAs can be encapsulated in naturally derived exosomes and the RNAs are stable, and a number of miRNAs can be naturally encapsulated simultaneously in these exosomes. Recently, data published by ours and other groups have demonstrated that mammalian exosomes can deliver poorly soluble agents, chemotherapeutic drugs, and siRNA in vivo. A limitation of this mode is the need for large-scale production of exosomes, and potential biosafety. Our preliminary data show that exosome-like nanoparticles can be isolated in large quantities from the tissue of edible plants, including grapes. We have demonstrated that exosome-like nanoparticles from grapes are composed of small RNAs, proteins, and lipids. An edible plant-derived nano-vector (EPNV) assembled from grape exosome-like derived lipids is capable of encapsulating siRNAs. Co-delivery of folic acid, with the chemotherapy drug paclitaxel by EPNVs results in an increased efficiency in tumor tissue targeting. Reducing the size of EPNVs by passing them through an appropriate pore size filter significantly enhances EPNV translocation to the brain and oral administration of the EPNVs results in their migration to the liver. Based on these preliminary data and the current literature, we propose to: (1) Determine whether an EPNV can deliver therapeutic miR17 via oral administration to target metastatic colon tumor in the liver, subsequently inducing NK cell activation to kill metastatic tumor cells; (2) Determine whether miR155 encapsulated in EPNV promotes differentiation of myeloid derived suppressor cells (MDSCs) into mature dendritic cells in mouse breast/brain tumor models; (3) Determine whether a therapeutic dose of miRNA17 or miRNA155 delivered by EPNV induces no side-effects in mouse tumor models; and (4) Determine whether EPNV can be produced in large scale amounts economically for use in clinical settings. Demonstration of using a EPNV-based delivery system loaded with a desired therapeutic extracellular RNA (exRNA) including microRNA to target to inflammatory cells and tumor cells in vivo would not only be a significant step forward in the treatment of cancer, but also provide a powerful tool for identifying the role and effects of each small RNA encapsulated in resident or circulating microvesicles on the recipient cells. We will accomplish this in our study by knock-out and knock-in strategies.