Polymeric Nanoparticles for siRNA Delivery to Bone Marrow Endothelium to Disrupt Tumor Cell Adhesion and Bone Metastasis Formation In Vivo

 描述(申请人提供)：转移仍然是癌症相关死亡的90%以上的原因，可以描述为一系列物理事件，包括肿瘤细胞从原发部位分离，侵入循环，通过血液转移到远处器官的微血管，以及通过内皮移位形成继发性肿瘤。骨是最常见的转移部位之一，它增加了发病率，5年生存率约为25%，中位生存期约为40个月。一旦肿瘤细胞在骨骼中定植，就会对化疗产生高度抗药性，有效的治疗方法仍然难以捉摸。骨髓内皮细胞(BMECs)表达黏附受体E-选择素(ES)和趋化基质细胞衍生因子-1(SDF-1)，分别与肿瘤细胞上的ES配体(ESL)和CXCR4结合。这种相互作用能够使肿瘤细胞与骨肿瘤形成所必需的BMECs黏附和归巢。下调或抑制ES和SDF-1对BMECs的作用为阻断骨转移的形成提供了一种潜在的解决方案。然而，ES和SDF-1对BMECs的直接和特异性抑制作用尚不清楚，封闭抗体可诱导内皮细胞(EC)激活，从而促进进一步的转移。因此，ES和SDF-1是RNA干扰(RNAi)联合治疗的有前景的候选药物，RNAi通过减少mRNA的表达来抑制传统上不可药物的靶点。利用siRNA的挑战在于需要安全、有效的递送方法，因为未经修饰的siRNA在血液中不稳定，具有免疫原性，并且不容易穿过细胞膜。在拟议的研究中，我们将开发和实现一个骨髓(BM)内皮靶向聚合物纳米颗粒(NP)siRNA传递平台，以阻断体内骨转移的形成。我们实验室开发的经证实的内皮特异性siRNA传递技术，称为7C1，现在将与靶向部分sialyl Lewis-X(SLeX)一起功能化，从而能够在体内靶向siRNA传递和沉默BMECs中的目标基因，而不会减少其他内皮细胞中的基因表达(目标1)。我们将进行一次筛选，以确定最有效的ES和SDF-1 siRNA候选被包裹在靶向7C1(t-7C1)中，验证t-7C1是否能够下调ES和SDF-1，并确定其在体外减少肿瘤细胞黏附和跨内皮细胞迁移的有效性(AIM II)。然后，我们将确定下调ES和SDF-1在体内BMECs上的最佳siRNA剂量，测试下调ES和SDF-1将扰乱肿瘤细胞黏附和骨转移小鼠肿瘤形成的中心假设，并确定我们的方法对正常干细胞和白细胞归巢到骨髓的潜在副作用(Aim III)。拟议的项目通过扰乱肿瘤细胞向骨骼的物理转移来瞄准转移，并提出了广泛的使siRNA递送技术能够沉默BMEC中导致一系列疾病的多个基因的技术。

 DESCRIPTION (provided by applicant): Metastasis remains responsible for over 90% of cancer-related deaths, and can be described as a series of physical events including tumor cell detachment from the primary site, invasion into the circulation, translocation through the blood to microvessels in distant organs, and transmigration through endothelium to form secondary tumors. One of the most common sites for metastasis is bone, which confers increased morbidity, a 5-year survival rate of ~25%, and median survival of ~40 months. Upon colonization in bone, tumor cells become highly resistant to chemotherapy, and effective treatments remain elusive. Bone marrow endothelial cells (BMECs) constitutively express the adhesion receptor E-selectin (ES) and the chemoattractant stromal cell-derived factor-1 (SDF-1), which bind to ES ligands (ESLs) and CXCR4 on tumor cells, respectively. Such interactions enable the adhesion and homing of tumor cells to BMECs necessary for tumor formation in bone. Down regulation or inhibition of ES and SDF-1 on BMECs provides a potential solution to disrupt bone metastasis formation. However, direct and specific inhibition of constitutive ES and SDF-1 on BMECs by small-molecules has been elusive, and blocking antibodies induce endothelial cell (EC) activation, which can promote further metastasis. Thus, ES and SDF-1 are promising candidates for combination RNA interference (RNAi) therapy, which inhibits traditionally undruggable targets by reducing mRNA expression. The challenge of utilizing siRNA is the need for safe, effective delivery methods, as unmodified siRNA is unstable in the bloodstream, immunogenic, and does not readily cross cell membranes. In the proposed research, we will develop and implement a bone marrow (BM) endothelium-targeted polymeric nanoparticle (NP) siRNA delivery platform to disrupt bone metastasis formation in vivo. Proven endothelium-specific siRNA delivery technology developed by our lab, termed 7C1, will now be functionalized with the targeting moiety Sialyl Lewis-X (SLeX) to enable targeted siRNA delivery and silencing of a target gene in BMECs in vivo, without reducing gene expression in other endothelium (Aim 1). We will conduct a screen to determine the most potent ES and SDF-1 siRNA candidates to encapsulate in targeted 7C1 (t-7C1), verify that t-7C1 can down regulate ES and SDF-1, and determine its efficacy in reducing tumor cell adhesion and transendothelial migration in vitro (Aim II). We will then determine the optimal siRNA dosage to down regulate ES and SDF-1 on BMECs in vivo, test the central hypothesis that down regulation of ES and SDF-1 will disrupt tumor cell adhesion and tumor formation in a mouse model of bone metastasis, and determine potential side effects of our approach on normal stem cell and leukocyte homing to BM (Aim III). The proposed project targets metastasis by disrupting the physical translocation of tumor cells to bone, and presents broadly enabling siRNA delivery technologies to silence multiple genes in BMECs that contribute to a range of diseases.