Dendritic Cell-Targeting Microparticles for Subcellularly-Targeted Delivery of In

描述(由申请人提供)：1型糖尿病(T1D)是由于对产生胰岛素的β细胞的自毁免疫反应而产生的胰岛素不足所致。尽管在T1D的实验系统中，已知有许多因素可以促进有利的免疫细胞反应，但由于旁观者细胞、组织和器官的不受控制的剂量，全身静脉注射这些药物往往会导致显著的有害的非靶点效应。该项目专注于体内靶向递送促耐受因子和胰岛素抗原，以颗粒形式靶向一种关键的免疫细胞类型--树突状细胞(DC)。树突状细胞是一种具有吞噬功能的抗原提呈细胞，对维持和启动对前驱抗原的免疫和对自身抗原的耐受至关重要。这使得DC成为以颗粒形式提供的药物的靶向递送的理想接收者。此外，某些免疫调节剂对DC的外源性调节可诱导出支持耐受的DC表型，并改善T1D。用DC靶向微粒(MPS)接种有望纠正T1D自身免疫反应，关键是不需要基于DC的细胞治疗所需的昂贵的体外操作。这使其具有广泛使用的潜力。微米级可生物降解的聚合物颗粒是可吞噬的，这有效地促进了包裹的因子通过非吞噬细胞向树突状细胞内的位置输送。通过表面固定针对DC受体的分子，这些可吞噬的颗粒可以进一步靶向DC。较大的(但仍小到足以注射)，不可吞噬的可生物降解的聚合物颗粒提供被包裹的因子到皮下注射部位的局部细胞外环境的受控释放。包裹在这些大颗粒中的因子由生物活性因子组成，树突状细胞对这些生物活性因子具有同源的细胞表面受体。这项建议的目标是设计一种皮下注射的双MP疫苗系统，该系统由i.可吞噬的树突状细胞靶向MPS，将抗原和免疫调节因子(胰岛素和维生素D3)递送到细胞内位置；非吞噬的MPS提供细胞外因子(GM-CSF和转化生长因子-β1)，用于DC募集和耐受诱导。我们期望在非肥胖型糖尿病(NOD)小鼠中实现支持耐受的DC表型，促进调节性T细胞的诱导，抑制自身反应性T细胞，并预防和逆转糖尿病。我们假设，双MP系统中多组分的组合将比单组分制剂更有效地提供强大、持久的抗原特异性免疫抑制，无论是以MPS形式还是以可溶性形式。目的1是建立双重MP系统，通过对DC表型(活化、未成熟或耐受)和T细胞反应(刺激、Th1、Th2、Treg或Th17)进行体外测试。目的2评价双MP制剂在NOD小鼠体内预防和逆转糖尿病的能力。这一新颖和创新的方法有望纠正T1D的自身免疫反应，并代表了一种简单的、临床可翻译的系统。

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) develops as a result of insufficient insulin being produced due to a self-destructive immune response against insulin producing beta cells. Although a number of factors are known to promote advantageous immune cell responses in experimental systems for T1D, systemic intravenous delivery of these agents often results in significant harmful off-target effects due to the uncontrolled dosing of bystander cells, tissues and organs. This project focuses on the targeted in vivo delivery of pro-tolerance factors and insulin antigen, in particulate form, targeted to a key immune cell type, dendritic cells (DCs). Dendritic cells, critical for maintenance and initiation of immunity to foregn antigens and tolerance to self-antigens, are phagocytic, antigen presenting cells. This makes DCs an ideal recipient for the targeted delivery of agents provided in particulate form. Moreover, exogenous conditioning of DCs with certain immunomodulatory agents has been shown to induce a pro-tolerance DC phenotype as well as ameliorate T1D. Vaccination with DC-targeting microparticles (MPs) holds promise to correct T1D autoimmune responses, critically, without the costly ex vivo manipulations required of DC-based cellular therapy. This enables the potential for widespread use. Micron-sized biodegradable polymeric particles are phagocytosable, which effectively promotes delivery of encapsulated factors to intracellular sites of DCs over non-phagocytes. These phagocytosable particles can be further targeted to DCs by surface immobilizing molecules targeting DC receptors. Larger (but still small enough to be injectable), non-phagocytosable biodegradable polymeric particles provide controlled release of encapsulated factors to the local extracellular environment at the subcutaneous injection site. Encapsulated factors in these large particles consist of bioactive factors for which DCs have the cognate cell-surface receptors. The objective of this proposal is to engineer a subcutaneously injectable dual MP vaccine system consisting of i.) Phagocytosable DC-targeting MPs delivering antigen and immunomodulatory factor (insulin and vitamin D3) to intracellular sites; and ii.) Non-phagocytosable MPs to deliver, extracellular, factors (GM- CSF and TGF-b1) for DC recruitment and tolerance induction. We expect to effect a pro-tolerogenic DC phenotype and promote induction of regulatory T-cells, suppression of auto-reactive T-cells, and prevent and reverse diabetes in non-obese diabetic (NOD) mice. We hypothesize that the combination of the multiple components in the dual MP system will more effectively provide robust, durable antigen-specific immune suppression than single-component formulations, either in MPs or in soluble form. Aim 1 is to formulate the dual MP system, test it in vitro by characterizing DC phenotype (activated, immature or tolerogenic) and T-cell response (stimulation, Th1, Th2, Treg, or Th17). Aim 2 is to evaluate the ability of the dual MP formulation in vivo, aiming to prevent and reverse diabetes in NOD mice. This novel and innovative approach holds promise for correcting autoimmune responses in T1D and represents a simple, clinically translatable system.