Exosome RNA-Therapeutics to Promote CNS Myelination

多发性硬化症（MS）是青年期最常见的致残性神经系统疾病。它是一种无法治愈的自身免疫性疾病，涉及少突胶质细胞丢失、脱髓鞘和无法使受损大脑髓鞘再生。再髓鞘化随着年龄和MS进展而下降，其中少突胶质细胞前体细胞（OPC）被积极地阻止分化成髓鞘形成细胞。目前的治疗仅针对通过免疫抑制减少脱髓鞘，免疫抑制降低免疫能力。重要的是，没有治疗方法可以防止髓鞘再生不可避免的下降。该项目旨在消除这种空白。OPC分化为髓鞘形成细胞受某些在MS病变中减少的microRNA（miRNAs）调节。值得注意的是，年轻大鼠的血液增强了老年大脑的髓鞘再生。这项工作的基本原理来自试点数据，这些数据显示血液来源的外泌体，即从细胞释放的小泡，含有触发髓鞘再生的miRNA。来自年轻和环境富集（EE）大鼠血清的非免疫原性外泌体miRNA增强髓鞘再生。我们研究的长期目标是在了解EE如何保护大脑的基础上开发神经治疗学。该申请的总体目标是开发含有miRNA的外泌体作为脑髓鞘再生的治疗药物。中心假设是这些外泌体将增强脑的髓鞘再生，并且可以被制作成MS的疗法。该项目具有四个目的：目的1：建立干扰素-γ（IFN-）刺激的树突状细胞（DC）作为外泌体的有效来源，其模拟EE-外泌体以在切片培养物中增强髓鞘再生而没有损伤或免疫反应。目标2：建立用制造的靶向miRNA种类加载DC外泌体的有效手段，并建立它们与EE-外泌体和IFN-刺激的DC外泌体相比在切片培养物中增强髓鞘再生而无损伤或免疫反应的能力γ。目标3：在MS脱髓鞘的大鼠溶血素模型中建立静脉内（然后经鼻）递送（目的1-2）外泌体促进髓鞘再生而无损伤或免疫反应的最佳功效。目标4：在MS [实验性自身免疫性脑脊髓炎（EAE）]小鼠模型中，确定静脉内（然后经鼻）给药（目的1-2）外泌体促进髓鞘再生的最佳功效，而无损伤或增强的免疫反应。结果指标将是：1）OPC、分化的少突胶质细胞和致密髓磷脂水平、靶向的miRNA和靶水平; 2）毒性，通过细胞死亡、肝和肾功能测定以及肿瘤发生来测量; 3）免疫活化，通过血清和脑标志物来测定;以及4）疾病严重程度的临床测量。这项工作是创新的，因为它使用天然存在的信号分子和载体来传递EE样的髓鞘形成增强。所提出的研究是重要的，因为它将建立基于EE的含有miRNA的外泌体作为一种新的治疗剂的临床前可行性和实用性，以使MS受损的大脑髓鞘再生而不损害免疫。

Multiple sclerosis (MS) is the most common disabling neurological disease of young adulthood. It is an incurable autoimmune disorder involving oligodendrocyte loss, demyelination, and an inability to remyelinate damaged brain. Remyelination declines with age and MS progression, where oligodendrocyte precursor cells (OPCs) are actively prevented from differentiating into myelinating cells. Current therapies are only directed at reducing demyelination via immune suppression which reduces immune competence. Importantly, no therapies exist to prevent the inevitable decline in remyelination. This project is designed to remove this void. OPC differentiation into myelinating cells is regulated by certain microRNAs (miRNAs) that are reduced in MS lesions. Notably, blood from young rats enhances remyelination of aged brain. The rationale for this work comes from pilot data showing that blood-derived exosomes, small vesicles released from cells, contain miRNAs that trigger remyelination. Non-immunogenic exosomal miRNAs from the serum of young and environmentally enriched (EE) rats enhanced remyelination. The long-term goal of our research is to develop neurotherapeutics based on understanding how EE protects brain. The overall goal of this application is to develop exosomes containing miRNAs as a therapeutic for brain remyelination. The central hypothesis is that these exosomes will enhance remyelination of brain and can be crafted into a therapy for MS. The project has four Aims: AIM 1: Establish interferon-gamma (IFN-γ) -stimulated dendritic cells (DCs) as an effective source of exosomes that mimic EE-exosomes to enhance remyelination without injury or immune reaction in slice cultures. AIM 2: Establish an efficient means of loading DC exosomes with fabricated targeted miRNA species and establish their ability to enhance remyelination without injury or immune reaction in slice cultures compared to EE-exosomes and IFN-γ-stimulated DC exosomes. AIM 3: Establish the optimal efficacy of intravenously (then nasally) delivered (Aims 1-2) exosomes to promote remyelination without injury or immune reaction in a rat lysolecithin model of MS demyelination. AIM 4: Establish the optimal efficacy of intravenously (then nasally) administered (Aims 1-2) exosomes to promote remyelination in a mouse model of MS [experimental autoimmune encephalomyelitis (EAE)] without injury or enhanced immune reaction. Outcome measures will be: 1) OPC, differentiated oligodendrocyte, and compact myelin levels, targeted miRNAs & target levels; 2) toxicity, measured by cell death, liver and renal function assays, plus oncogenesis; 3) immune activation assayed by serum and brain markers; and 4) clinical measures of disease severity. The work is innovative because it uses naturally occurring signaling molecules and vehicles for delivery of EE-like enhancement of myelination. The proposed research is significant because it will establish the preclinical feasibility and utility of EE-based miRNA-containing exosomes as a novel therapeutic to remyelinate MS- damaged brain without immune compromise.