Therapeutic inhibition of peritoneal carcinomatosis by nanoparticle/siRNA-mediated knockdown of specific integrins and selectins

腹膜癌(PC)是胰腺癌和胃癌进展的一种常见形式，这在很大程度上导致了相当差的预后。因此迫切需要新的治疗策略，包括选择性抑制PC。在这方面，肿瘤细胞以及内皮/间皮/非恶性间质细胞可以作为治疗的靶点。整合素是由α-和β-亚基组成的异二聚体细胞黏附分子。某些整合素在肿瘤中上调，并可能在PC中发挥关键作用。Wicklein小组之前的研究结果表明，整合素α2、α3、α5、α6、αV、β1和β4是最有希望被开发的。同样，在异种移植中，E-和P-选择素被证明增加了血行转移和PC的形成。本项目旨在探索临床前小鼠模型中新的治疗干预策略，并对选定的选择素/整合素进行体外和体内的功能分析。对于特定的单个或联合靶基因抑制，我们将采用基于RNA分子(小干扰RNA，siRNAs)递送的RNA干扰(RNAi)介导的基因敲除。SiRNAs将被制成纳米颗粒，并在腹膜内应用。一个主要的挑战是开发在腹膜中具有高稳定性和活性的纳米颗粒制剂。从Aigner基团可获得含有低分子量聚乙烯亚胺(PEI)及其化学修饰的聚合物纳米颗粒。在本项目中，这些纳米粒子将首次被进一步开发和探索用于PC抑制。除了击倒整合素，这还将涉及靶向上游调控因子(PCDHB9，FBXO50)，并使用整合素抑制miR-31进行miRNA替代治疗。为了延长治疗时间，还将建立水凝胶作为NP的持续释放系统，并根据所需的释放动力学进行优化。因此，目标包括(I)鉴定和分析基于RNAi的PC抑制的最佳靶基因(整合素、选择素、整合素上游基因及其组合)，(Ii)探索肿瘤细胞(整合素)以及内皮/间皮/非恶性基质细胞(选择素、整合素)作为靶点，(Iii)开发优化的聚合物纳米颗粒和用于腹膜递送治疗性siRNAs/miRNAs的体外/体内分析系统，以及(Iv)使用优化的纳米颗粒系统和siRNAs在小鼠模型中进行临床前治疗研究。这将包括治疗前(在肿瘤细胞暴露之前，旨在消除PC的发病)、治疗后(在肿瘤细胞暴露之后，用于治疗已确定的腹膜内癌)和延长治疗方案，旨在同时针对两者。

Peritoneal carcinomatosis (PC) is a common form of pancreatic and gastric carcinoma progression, which substantially contributes to the rather poor prognosis. New therapeutic strategies, including the selective inhibition of PC, are thus desperately needed. In this regard, tumor cells as well as endothelial/mesothelial/non-malignant stroma cells can be explored as therapeutic target. Integrins are heterodimeric cell adhesion molecules consisting of α- and β-subunits. Certain integrins are upregulated in tumors and putatively play a pivotal role in PC. Previous results from the Wicklein group indicate that the integrins α2, α3, α5, α6, αV, β1 and β4 are most promising for being explored. Likewise, in xenografts E- and P-selectin were demonstrated to increase hematogenous metastasis and PC formation. This project aims at the exploration of novel therapeutic intervention strategies in preclinical mouse models and the functional analyses of selected selectins/integrins in vitro and in vivo. For specific single or combined target gene inhibition, we will employ RNA interference (RNAi)-mediated gene knockdown based on the delivery of RNA molecules (small interfering RNAs, siRNAs). siRNAs will be formulated in nanoparticles and applied intraperitoneally. A major challenge is the development of nanoparticle formulations with high stability and activity in the peritoneum. From the Aigner group, polymeric nanoparticles comprising low molecular weight polyethylenimines (PEI) and chemical modifications thereof are available. In this project, these nanoparticles will be further developed and explored for the first time for PC inhibition. Beyond knockdown of integrins, this will also involve targeting upstream regulators (PCDHB9, FBXO50), and using the integrin-inhibiting miR-31 for miRNA replacement therapy. For prolonged treatment duration, hydrogels as sustained NP release systems will be established as well and optimized with regard to desired release kinetics. Objectives thus include (i) the identification and analysis of optimal target genes (integrins, selectins, integrin upstream genes and combinations thereof) for RNAi-based inhibition of PC, (ii) exploring tumor cells (integrins) as well as endothelial/mesothelial/non-malignant stroma cells (selectins, integrins) as targets, (iii) the development and in vitro / in vivo analysis of optimized, polymeric nanoparticles and sustained release systems for peritoneal delivery of therapeutic siRNAs/miRNAs, and (iv) preclinical therapy studies in mouse models, using optimal nanoparticle systems and siRNAs. This will cover pre-treatment (prior to tumor cell exposure, aiming at the abolishment of the onset of PC), post-treatment (after tumor cell exposure, for treating established intraperitoneal carcinomas) and prolonged treatment protocols, aiming at both.