Inhalation Delivery of Exosome and microRNA Therapy for Lung Fibrosis

项目摘要 特发性肺纤维化(Ipf)是一种最终致命的疾病，其唯一的治疗方法是肺。 移植。IPF的特征是在肺内形成纤维化病变，最终导致 结疤和进行性肺功能丧失。尽管有一些新的治疗方法，IPF患者仍然有 确诊后中位存活率仅为3-5年。显然，新的治疗方法正在 需要治疗这种毁灭性的疾病。干细胞疗法是一种很有前途的方法。在 在过去的8年里，我们的实验室一直在开发肺球形细胞(Lscs)作为一种新的治疗来源。 肺细胞和FDA批准LSC治疗IPF患者的临床试验正在进行中。 尽管如此，基于干细胞的治疗面临着几个重要的限制。活细胞需要 在使用前仔细保存和处理，细胞移植带有一定的 免疫原性和致瘤性风险。重要的是，活干细胞不能输送到肺部。 通过吸入，这是将治疗药物输送到肺部的最方便和有效的途径。 最近，我们和其他人进行了一项新颖而令人兴奋的观察，许多成体干细胞 发挥它们的有益作用主要是通过分泌再生因子来继续促进 内源性修复。在构成这项申请的基础的初步研究中，我们有 发现培养的LSCs的分泌物与LSCs一样有效，如果不是更有效的话 在疾病的啮齿动物模型中减轻和解决IPF的作用。在寻求活跃的过程中 在LSC分泌物中，我们发现LSC分泌大量的外切体(30-150 由多种细胞类型分泌的NM囊泡)。我们已经证明外体来源于LSCs (LSC-Exo)对受损的肺具有治疗和再生作用，表明这些纳米结构 在IPF的啮齿动物模型中，对LSC分泌物的修复性反应起主要作用。这是众所周知的 Exosome携带的microRNAs(MiRs)货物可能在细胞-细胞中发挥重要作用 沟通和组织修复，事实上，我们发现LSC-Exo高度富含miR- 30A和let-7。在这项拟议的研究中，我们计划确定安全性和有效性以及媒介 肺纤维化啮齿动物模型肺修复所需的LSC-Exo有效剂量，以确定主要 肺组织中LSC-Exo的受体细胞，并确定相关的分子靶点(S)的Exosomal 中介修复和恢复。我们假设带有LSC-Exo的关键MIR，如miR-30a和 利用scRNA-Seq WE产生的数据，let-7是转化生长因子-β信号通路的介体 最终将确定这些外体中进一步丰富的miR是否实现最佳的肺修复。 从干细胞衍生的无细胞或无生命疗法的发展有可能 使当前的再生医学实践发生革命性变化。

PROJECT ABSTRACT Idiopathic pulmonary fibrosis (IPF) is an ultimately fatal disease whose only curative treatment is lung transplant. IPF is characterized by formation of fibrotic lesions in the lung, eventually resulting in scarring and progressive loss of lung function. Despite some newer treatments, IPF patients still have a median survival rate of only 3-5 years once diagnosed. Clearly, new therapeutic approaches are needed to treat this devastating disease. A promising avenue of approach is stem cell therapy. In the past 8 years, our lab has been developing lung spheroid cells (LSCs) as a novel source of therapeutic lung cells, and FDA approval of clinical trials with LSC treatment of patients with IPF are being pursued. Nonetheless, stem cell-based therapy faces several important limitations. Live cells need to be carefully preserved and processed before usage, and cell transplantation carries certain immunogenicity and tumorigenicity risks. Importantly, live stem cells cannot be delivered to the lung via inhalation, which is the most convenient and effective route to deliver therapeutics to the lung. Recently, we and others have made the novel and exciting observation that many adult stem cells exert their beneficial effects mainly through secretion of regenerative factors that go on to promote endogenous repair. In the preliminary studies that form the basis for this application, we have discovered that secretions from cultured LSCs are just as, if not more, effective than the LSCs themselves in attenuating and resolving IPF in rodent models of the disease. In the quest for active components in the LSC secretions, we found that LSCs secrete large numbers of exosomes (30-150 nm vesicles secreted by numerous cell types). We have shown that exosomes derived from LSCs (LSC-Exo) are therapeutic and regenerative to the injured lung, suggesting these nanostructures are largely responsible for the reparative response to LSC secretions in rodent models of IPF. It is known that exosomes carry microRNAs (miRs) cargoes that could play important roles in cell-cell communication and tissue repair, and indeed we found that LSC-Exo are highly enriched with miR- 30a and Let-7. In this proposed study, we plan to determine safety and efficacy as well as medium effective dose of LSC-Exo required for lung repair in rodent models of IPF, to determine the major recipient cells of LSC-Exo in the lung, and determine that the relevant molecular target(s) of exosomal mediated repair and recovery. We hypothesize that key miRs withing LSC-Exo such as miR-30a and Let-7 are mediators of the TGF-beta signaling pathway, using the data produced by scRNA-Seq we will finally determine whether further miR enrichment in these exosomes achieves optimal lung repair. The development of cell-free or non-living therapeutics derived from stem cells has the potential to revolutionize current regenerative medicine practice.