Immunoregulatory effect of microparticle delivered STING agonist in the control of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS)

虽然多发性硬化症治疗有效抑制复发缓解型多发性硬化症 （RRMS）疾病进展时，它们具有显著的副作用和毒性。我们还在寻找一种疾病 提供抗原特异性免疫耐受重建的特异性治疗。我们使用 微粒（MP）包封的STING激动剂cGAMP以增加其向吞噬细胞抗原的递送 在一些实施方案中，所述细胞包括呈递细胞（APC），特别是树突状细胞（DC）。一种新型的酸敏感聚合物--缩醛化葡聚糖 （Ace-DEX）由于其在细胞内酸性区室中的pH敏感性而允许调节MP分解。我们 初步的数据已经证明，包封2 '3'环GMP-AMP的MP的细胞内递送 二核苷酸（cGAMP）抑制实验性自身免疫性脑脊髓炎（EAE），MS的动物模型， 当在临床疾病的高峰期之前和高峰期施用时。环腺苷酸MPs治疗呼吸困难和 慢性EAE是通过STING诱导的IL-27和IL-10产生介导的，因为治疗效果是 在IL-27 ra-/-和IL-10-/-小鼠中消除，而在Ifnar-/-小鼠中维持部分疾病抑制。我们 中心假设是细胞内cGAMP MP递送将诱导产生IL-27和IL-10的致耐受性 调节产生IL-10的iT细胞的分化和扩增的DC。我们将表征cGAMP MP 致耐受性DC表型和功能，以及诱导的调节性CD 4 +IL-10+的抑制能力， 细胞，特别是其FoxP 3和1型iTreg（Tr 1）标志物的表达。我们建议， 来自DR 2+的cGAMP MP诱导的致耐受性DC的MHC II类DR 2锚定肽混合物（DR 2-APM） 复发缓解型多发性硬化（RRMS）患者可重建抗原特异性免疫耐受。 为了确定cAMP MP介导的疾病抑制的机制，我们将进行以下研究： 具体目的：1）证明cGAMP MP处理诱导EAE中的iTreg细胞扩增。的 拟议的研究将确定EAE的cGAMP MP治疗在多大程度上诱导EAE的扩展， 外周免疫器官及中枢神经系统炎性浸润中CD 4 + CD 25 +IL-27 R +IL-10+ T细胞亚群。研究 使用aCD 25 mAb将证实iTclad在治疗效果中的作用。FoxP 3 + Tencent的作用将是 使用瞬时诱导型FoxP 3 +Treg耗竭和具有选择性FoxP 3 +Treg IL-27 R耗竭的小鼠进行检测。 2)在人类体外研究中，我们将表征cGAMP MP诱导的致耐受性DC及其能力， 在RRMS患者中诱导iTlymphoma。具体实验将确定cGAMP MP处理的细胞的能力。 单核细胞和DC诱导iTreg扩增，测定cGAMP IL-27-和IL-10-的转录组。 诱导的Tlymphocyte和其抑制能力的重建。最后，共同施用cGAMP MP 用DR 2-APM对来自RRMS患者的DC进行刺激将测试它们诱导抗原特异性致耐受性DC的能力 和Tibet。该研究将提供将这种治疗方法转化为临床应用所需的临床前数据 RRMS患者

While multiple antiinflammatory therapies are effective in suppressing relapsing-remitting multiple sclerosis (RRMS) disease progression, they have significant side effects and toxicity. We are still seeking a disease specific treatment that would provide an antigen-specific immune tolerance reconstitution. We used microparticle (MP)-encapsulated STING agonist cGAMP to increase its delivery into phagocytic antigen presenting cells (APCs), particularly dendritic cells (DCs). A novel acid-sensitive polymer, acetalated dextran (Ace-DEX) allows regulated MP breakdown due to its pH sensitivity in intracellular acidic compartments. Our preliminary data have demonstrated that the intracellular delivery of MPs encapsulating 2’3’cyclic GMP-AMP dinucleotide (cGAMP) suppressed experimental autoimmune encephalomyelitis (EAE), an animal model of MS, when administered before and at the peak of clinical disease. The therapeutic effect of cGAMP MPs in RR and chronic EAE was mediated via STING-induced IL-27 and IL-10 production, since the therapeutic effect was abrogated in IL-27ra-/- and IL-10-/- mice, while partial disease suppression was maintained in Ifnar-/- mice. Our central hypothesis is that intracellular cGAMP MP delivery will induce IL-27 and IL-10-producing tolerogenic DCs that regulate the differentiation and expansion of IL-10-producing iTregs. We will characterize cGAMP MP tolerogenic DC phenotype and function, as well as suppressive capacity of the induced regulatory CD4+IL-10+ cells, in particular their expression of FoxP3 and type 1 iTreg (Tr1) markers. We propose that presentation of MHC class II DR2-anchored peptide mixtures (DR2-APMs) by cGAMP MP-induced tolerogenic DCs from DR2+ patients with relapsing remitting multiple sclerosis (RRMS) may reconstitute antigen-specific immune tolerance. In order to identify the mechanisms of cAMP MP-mediated disease suppression, we will pursue following specific aims: 1) Demonstrate that cGAMP MP treatment induces iTreg cell expansion in EAE. The proposed studies will determine to what extent cGAMP MP treatment of EAE induces the expansion of CD4+CD25+IL-27R+IL-10+ Tregs in the peripheral immune organs and the CNS inflammatory infiltrates. Studies using aCD25mAb will confirm the role of iTregs in the therapeutic effect. The role of FoxP3+Tregs will be examined using transient inducible FoxP3+Treg depletion and mice with selective FoxP3+Treg IL-27R depletion. 2) In human in-vitro studies, we will characterize cGAMP MP-induced tolerogenic DCs and their capacity to induce iTregs in RRMS patients. Specific experiments will determine the capacity of cGAMP MP-treated monocytes and DCs to induce iTreg expansion, determine the transcriptome of cGAMP MP IL-27- and IL-10- induced Tregs and the reconstitution of their suppressive capacity. Finally, co-administration of cGAMP MPs with DR2-APMs to DCs from RRMS patients will test their capacity to induce antigen-specific tolerogenic DCs and Tregs. The study will provide preclinical data required to translate this therapeutic approach into clinical use in patients with RRMS