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细胞的自我破坏性免疫反应导致胰岛素产生不足而发展起来的。虽然已知在T1D实验系统中有许多因素可以促进有利的免疫细胞反应，但由于对旁观者细胞、组织和器官的剂量不受控制，这些药物的全身静脉注射通常会导致显著的有害脱靶效应。这个项目的重点是在体内靶向递送促耐受性因子和胰岛素抗原，以颗粒形式，针对一个关键的免疫细胞类型，树突状细胞（dc）。树突状细胞是一种吞噬性抗原呈递细胞，对维持和启动对外来抗原的免疫和对自身抗原的耐受至关重要。这使得DCs成为以颗粒形式提供的靶向递送剂的理想受体。此外，某些免疫调节剂对DC的外源调节已被证明可诱导促耐受性DC表型并改善T1D。用dc靶向微粒（MPs）接种疫苗有望纠正T1D自身免疫反应，关键是，不需要dc细胞治疗所需的昂贵的体外操作。这使其具有广泛应用的潜力。微米大小的可生物降解聚合物颗粒具有可吞噬性，这有效地促进了包裹因子在非吞噬细胞上传递到dc的细胞内部位。这些可吞噬的颗粒可以通过靶向DC受体的表面固定化分子进一步靶向DC。较大的（但仍然小到可以注射），不可吞噬的可生物降解聚合物颗粒在皮下注射部位的局部细胞外环境中提供被包裹因子的可控释放。包裹在这些大颗粒中的因子由生物活性因子组成，树突状细胞具有同源的细胞表面受体。本提案的目的是设计一种皮下注射双MP疫苗系统，该系统由i。可吞噬的dc靶向MPs将抗原和免疫调节因子（胰岛素和维生素D3）递送到细胞内部位；和ii)。非吞噬性MPs递送细胞外因子（GM- CSF和TGF-b1），用于DC募集和耐受诱导。我们期望在非肥胖糖尿病（NOD）小鼠中影响促耐受性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系统中多种成分的组合将比单成分配方更有效地提供强大、持久的抗原特异性免疫抑制，无论是MP还是可溶性形式。目的1是制定双MP系统，通过表征DC表型（激活，未成熟或耐受性）和t细胞反应（刺激，Th1, Th2， Treg或Th17）在体外进行测试。目的2是评估双MP制剂在体内预防和逆转NOD小鼠糖尿病的能力。这种新颖和创新的方法有望纠正T1D的自身免疫反应，并代表了一个简单的，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.