Exosome RNA-Therapeutics to Promote CNS Myelination

描述(申请人提供)：多发性硬化症(MS)是青壮年最常见的致残性神经系统疾病。这是一种无法治愈的自身免疫性疾病，包括少突胶质细胞丢失、脱髓鞘和受损大脑无法重新髓鞘形成。随着年龄和MS的进展，再髓鞘形成能力下降，少突胶质前体细胞(OPC)被积极阻止分化为髓鞘细胞。目前的治疗方法只针对通过免疫抑制来减少脱髓鞘，免疫抑制会降低免疫能力。重要的是，目前还没有任何治疗方法可以防止不可避免的髓鞘再生减少。该项目旨在消除这一空白。OPC向髓鞘细胞的分化受某些在MS皮损中减少的microRNAs(MiRNAs)的调控。值得注意的是，年轻大鼠的血液可以促进衰老大脑的髓鞘再生。这项工作的基本原理来自于试点数据显示，血液来源的外体，即细胞释放的小囊泡，含有触发重新髓鞘形成的miRNAs。来自幼年和环境富集型(EE)大鼠血清的非免疫原性外体miRNAs可促进髓鞘再生。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。这项应用的总体目标是开发包含miRNAs的外切体，作为治疗大脑重新髓鞘形成的药物。该项目有四个目标：目标1：建立干扰素-γ刺激的树突状细胞(DC)作为模拟EE-exosome的外切体的有效来源，从而在切片培养中无损伤或免疫反应地增强重新髓鞘形成。目的：建立一种高效的靶向miRNA载体负载DC外切体的方法，并与EE-exosome和干扰素刺激的DC exosome相比，建立其在切片培养中无损伤或无免疫反应地增强再髓鞘形成的能力。目的：建立一种无损伤、无免疫反应的大鼠溶血磷脂脱髓鞘模型，确定静脉(然后鼻腔)注射(AIMS 1-2)外切体促进髓鞘再生的最佳效果。目的：确定静脉(然后鼻腔)注射(AIMS 1-2)外切体促进实验性自身免疫性脑脊髓炎(EAE)小鼠模型髓鞘再生的最佳效果，而不损伤或增强免疫反应。结果指标将是：1)OPC、分化的少突胶质细胞和致密髓鞘水平，靶向miRNAs和靶水平；2)毒性，通过细胞死亡、肝和肾功能分析以及肿瘤形成来衡量；3)通过血清和脑标记物分析免疫激活；以及4)疾病严重程度的临床指标。这项工作是创新的，因为它使用自然产生的信号分子和载体来传递EE样的增强髓鞘形成。这项拟议的研究具有重要意义，因为它将建立基于EE的包含miRNA的外切体作为一种新的治疗方法的临床前可行性和实用性，以在不影响免疫的情况下对MS受损的脑进行髓鞘修复。

DESCRIPTION (provided by applicant): 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.