Development of delivery methods for combination microRNA treatment of alcohol-associated liver disease

项目总结 酒精相关性肝病(AALD)是一个主要的、日益严重的健康问题，治疗选择有限。 AALD(以前称为酒精性肝病)的自然病史包括脂肪肝、酒精性肝病 肝炎与终末期肝硬变前纤维化的发展。MicroRNAs(MiRNAs)代表了一种新的 由于它们能够同时影响多条与纤维化相关的途径，因此被列为治疗药物的一类。其中 可能的靶点，miR-155参与Kupffer细胞(KCs)介导的炎症反应，从而影响 多个其他肝细胞中的纤维化事件。趋化因子受体CXCR4及其同源配体基质细胞 衍生因子-1在急性酒精性肝病的发病机制中发挥着重要而复杂的作用，包括与 肝损伤后的初始免疫反应及其在控制肝纤维化进展中的作用 对肝星状细胞(HSCs)和胶原生成的激活作用。这个项目的目标是开发 基于自组装纳米粒子(多链)的集成miRNA递送平台，可递送抗miR- 155激活KCs，同时抑制肝脏HSCs激活的CXCR4信号转导。交付平台 是基于创新的CXCR4抑制剂，基于Cyclam修饰的低分子聚乙烯亚胺S (C-PEI)，有效地封装和系统地递送miRNA。我们的目标是检验这一假设 C-PEI/miRNA由于两者的促纤维化信号减弱而导致联合作用的增强 肝巨噬细胞和产生基质的肝星状细胞。我们将从三个方面具体完成总体目标。 目标。在目标1中，我们将优化C-PEI-miRNA纳米粒的配方，该纳米粒可在肝脏中传递抗miR-155 纤维化症。根据我们初步研究中鼓励的抗纤维化活性，我们假设Polyplex 用甘露糖和稳定的胆固醇部分进行修饰将导致miRNA的有效传递 激活的KCs，而过量的游离C-PEI将靶向激活的HSCs。在目标2中，我们将测试体内治疗 胆管结扎(BDL)模型和小鼠慢性酒精模型的实验研究 暴露在CCl4中的政府。目的是对慢性阻塞性肺疾病的治疗效果进行综合评价 优化的C-PEI-Chol/抗miR-155复合体在不同模型和多阶段纤维化中的应用。这个 小鼠模型的发现将在培养的人类精切肝脏切片中得到验证。在《目标3》中，我们将 确定miR-155和CXCR4联合抑制的抗纤维化作用机制。虽然我们的 初步数据与KCs中miR-155的下调以及激活的HSCs中CXCR4的抑制一致， C-PEI/anti-miR-155复合体的确切作用机制尚不清楚。因此，中国的研究 这一目标旨在确定复合体的基本作用机制。总体而言，这个项目 将有助于从根本上理解CXCR4和miR-155的作用和治疗潜力 抑制AALD，并将创新性地解决靶向递送药物/miRNA组合作为抗纤维化药物的问题 治疗。

PROJECT SUMMARY Alcohol-associated liver disease (AALD) is a major and growing health concern with limited treatment options. The natural history of AALD (formerly known as alcoholic liver disease) includes fatty liver disease, alcoholic hepatitis and the development of fibrosis preceding end-stage cirrhosis. MicroRNAs (miRNAs) represent a new class of therapeutics due to their ability to simultaneously affect multiple fibrosis-associated pathways. Among possible targets, miR-155 is involved in inflammatory responses mediated by Kupffer cells (KCs) that affect fibrogenic events in multiple other hepatic cells. Chemokine receptor CXCR4 and its cognate ligand stromal cell- derived factor-1 play important and complex roles in the pathogenesis of AALD, including the coordination of the initial immune reaction upon liver injury and later in controlling the progression of liver fibrosis through its activating effect on hepatic stellate cells (HSCs) and collagen production. The goal of this project is to develop integrated miRNA delivery platform based on self-assembled nanoparticles (polyplexes) that deliver anti-miR- 155 to activated KCs and in parallel inhibit CXCR4 signaling in activated HSCs in the liver. The delivery platform is based on innovative CXCR4 inhibitors based on cyclam-modified low molecular weight poly(ethylenimine)s (C-PEI) that efficiently encapsulate and systemically deliver miRNA. The objective is to test the hypothesis that C-PEI/miRNA will lead to enhanced combination effect due to attenuation of profibrogenic signaling of both hepatic macrophages and the matrix-producing HSCs. We will accomplish the overall objectives in three specific aims. In Aim 1, we will optimize formulation of the C-PEI-miRNA nanoparticles that deliver anti-miR-155 in liver fibrosis. Based on encouraging antifibrotic activity in our preliminary studies, we hypothesize that polyplex modification with mannose and with stabilizing cholesterol moieties will result in efficient delivery of the miRNA to activated KCs, while excess free C-PEI will target activated HSCs. In Aim 2, we will test the in vivo therapeutic efficacy of the polyplexes in the bile-duct ligation (BDL) model and mouse models of chronic alcohol administration with exposure to CCl4. The goal is to conduct comprehensive evaluation of therapeutic efficacy of the optimized C-PEI-Chol/anti-miR-155 polyplexes in different models and multiple stages of fibrosis. The findings from the mouse models will be validated in cultured human precision-cut liver slices. In Aim 3, we will determine the mechanism of antifibrotic activity of the combined miR-155 and CXCR4 inhibition. Although our preliminary data are consistent with miR-155 downregulation in KCs and inhibition of CXCR4 in activated HSCs, the precise mechanism of action of the C-PEI/anti-miR-155 polyplexes is not known. Therefore, the studies in this aim will be designed to ascertain the underlying mechanisms of action of the polyplexes. Overall, this project will contribute to the fundamental understanding of the role and therapeutic potential of CXCR4 and miR-155 inhibition in AALD and will innovatively address targeted delivery of drug/miRNA combinations as an anti-fibrotic treatment.