Engineering Exosome for Pancreatic Cancer Targeting Therapies

胰腺导管腺癌(PDAC)是最致命的癌症之一，在癌症中排名第四。 在美国发生了相关死亡事件。胰腺癌的治疗在很大程度上受到缺乏 有效的交付系统。外切体正在成为一种很有前途的纳米载体，用于药物/基因的传递 这些自然产生的纳米级细胞外小泡的独特性质以及它们天生的 蛋白质、脂质和DNA/RNA在细胞间穿梭。然而，存在重大挑战，包括他们无法 小鼠单核巨噬细胞系统对靶肿瘤细胞及其高清除率的研究 肝和脾。我们的长期目标是开发具有低免疫原性、高免疫原性和高免疫原性的创新纳米载体 生物相容性、更高的稳定性、更长的循环时间和高活性的肿瘤细胞靶向。使用小说 外体工程技术，我们最近发现：(A)掺入肿瘤归巢多肽(RGD) 在胞外体表面标记CD9(ExoCD9-RgD)导致与整合素v3-的特异性结合和摄取 表达癌细胞，以及(B)过表达CD47的外切体，这是一个“不要吃我”的信号，通过其最小的自身- 多肽(CD47p110-130)与巨噬细胞上的信号调节蛋白(Sirp，CD172a)相互作用 降低外切体对肝、脾的清除。这些发现导致了我们的中心假设，即 通过CD9工程，外切体上的RGD和CD47p110-130将允许外切体在体内靶向PDAC，而 允许外显体逃避MPS的许可。为了实现这一假设，我们通过以下方式开发了“智能外显体” 在胞外体表面共显示RGD和CD47p110-130(ExoSmart)。这导致增强的受体结合， 从而增加3D间质丰富的PDAC球状肿瘤的蓄积和细胞毒治疗效果 模特们。有了这些强有力的初步数据，我们建议追求三个具体目标来描述ExoSmart 并验证了ExoSmart在药物输送中的应用。(1)评价共表达CD9-的SMART外切体 RGD和CD9-CD47p110-130(ExoSmart)，用于使用人PDAC基质进行独家主动PDAC靶向治疗 丰富的3D球体模型，包括体外和体内。(2)验证ExoSmart PDAC靶向的有效性 基因工程小鼠PDAC肿瘤模型(KPC)和临床相关患者的化疗 来源异种移植(PDX)小鼠胰腺癌模型。(3)将ExoSmart扩展到个性化PDAC 通过优化CD9和肿瘤靶向多肽的多个插入位点进行靶向，以获得最佳的PDAC靶向。 总而言之，我们提议的研究将通过开发创新的纳米载体来广泛影响该领域 优化的货物和表面，以实现精确的PDAC目标。这个项目具有很大的翻译潜力 癌症治疗，同时为利用新型多肽工程外切体的未来工作提供坚实的基础 战略。

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the deadliest cancers and ranks fourth in cancer- related deaths in the United States. Therapies for pancreatic cancer are largely hindered by the lack of an effective delivery system. Exosomes are emerging as a promising type of nanocarrier for drug/gene delivery due to the unique properties of these naturally derived, nanoscale extracellular vesicles and their innate ability to shuttle proteins, lipids and DNA/RNA between cells. However, major challenges exist, including their inability to target tumor cells and their high proportion of clearance by the mononuclear phagocyte system (MPS) of the liver and spleen. Our long-term goal is to develop innovative nanocarriers with low immunogenicity, high biocompatibility, increased stability, longer circulation times, and highly active tumor cell targeting. Using novel exosomal engineering techniques, we recently found that (a) incorporation of a tumor-homing peptide (RGD) onto exosomal surface marker CD9 (ExoCD9-RGD) results in specific binding to and uptake by integrin v3- expressing cancer cells, and (b) exosomes overexpressing CD47, a “don’t eat me” signal, via its minimal self- peptide (CD47p110-130), interact with signal regulatory proteins (SIRP, CD172A) on macrophages to significantly reduce liver and spleen clearance of exosomes. These findings lead to our central hypothesis that displaying RGD and CD47p110-130 on exosomes through CD9 engineering will permit exosomes to target PDAC in vivo while allowing exosomes to evade MPS clearance. Toward this hypothesis, we have developed “smart exosomes” by co-displaying RGD and CD47p110-130 on the exosome surface (ExoSmart). This results in enhanced receptor binding, thereby increasing accumulation and cytotoxic therapeutic effects in 3D stroma-rich PDAC spheroid tumor models. With these strong preliminary data, we propose to pursue three Specific Aims to characterize ExoSmart and validate the application of ExoSmart in drug delivery. (1) To evaluate smart exosomes co-expressing CD9- RGD and CD9-CD47p110-130 (ExoSmart) for exclusive active PDAC targeting therapy using human PDAC stroma- rich 3D spheroid models, both in vitro and in vivo. (2) To validate the efficacy of ExoSmart PDAC targeting chemotherapies in a genetically engineered mouse PDAC tumor model (KPC) and clinically relevant patient- derived xenografted (PDX) pancreatic cancer mice models. (3) To extend ExoSmart to personalized PDAC targeting by optimizing multiple insertion sites of CD9 and tumor-targeting peptides for optimal PDAC targeting. Collectively, our proposed research will broadly impact the field by developing innovative nanocarriers with optimized cargos and surfaces for precision PDAC targeting. This project holds great translational potential for cancer therapy while providing a solid basis for future work utilizing novel peptide-engineered exosome strategies.