Mesenchymal stem cell-derived extracellular vesicles as a cell-free therapy for ARDS.

急性呼吸窘迫综合征(ARDS)是一种严重的临床综合征，影响所有重症监护患者的20%。不幸的是，大约30%-50%的人会死于这种疾病，幸存者的生活质量可能会因为随之而来的慢性肺部问题而严重下降。目前还没有有效的治疗方法，因此迫切需要新的药物。ARDS的特点是肺部过度和不受控制的炎症，导致免疫细胞的不适当渗透，肺充满水，无法维持其主要功能-呼吸。最近，越来越多的临床医生和基础科学家开始关注基于细胞的治疗。基于细胞治疗的最有希望的候选细胞之一是被称为间充质干细胞(MSCs)的细胞，它代表一种成人干细胞(可以很容易地从成人患者或健康志愿者的组织中分离出来)，因此，不存在伦理问题。MSCs可以在实验室条件下很容易地繁殖和操纵，并具有产生多种具有免疫调节特性的介体的能力。然而，对MSC应用的长期影响(例如肿瘤的发展)的安全性以及后勤挑战的担忧可能会限制其成功的临床翻译。现在越来越多的证据表明，MSCs主要通过分泌不同的生物活性介质来发挥作用。因此，研究人员越来越多地考虑将MSC细胞产品(不能复制从而形成肿瘤)作为全细胞治疗的替代方案。在本项目中，我们将研究MSCs释放的小膜包裹的细胞外小泡的治疗潜力。这些小泡是MSCs分泌体的关键成分，通过高速离心法较容易从MSC条件培养液中分离出来。以前，我们和其他人已经证明，这些囊泡能够在各种ARDS临床前模型中概括MSCs的治疗效果。此外，在我们最近的工作中，我们发现这些小泡含有负责维持细胞生物能量(线粒体)的细胞器。在这个项目中，我们将使用ARDS的小动物模型来研究这些囊泡的治疗潜力，优化给药的最佳途径和策略，了解它们在组织中的生物分布，并调查功能活跃的线粒体是否对治疗效果重要，以及哪些组织是EVS中转移的线粒体的主要受体。这些研究将由体外EV有丝分裂转移到原代人肺细胞的研究进一步补充。这些数据是英国药品和医疗保健产品监管机构和欧洲药品管理局等监管机构对新疗法临床翻译的基本要求。该项目的主要预期成果将是为临床开发建立明确的战略，并了解骨髓间充质干细胞来源的细胞外小泡的具体作用机制(基于线粒体转移)。更直接的是，这些数据将为在更大的ARDS动物模型中进行进一步的临床前研究提供信息，这些研究需要获得关于MSCs EVS安全性、有效性和作用机制的必要数据。总的来说，在小动物和大动物模型中的数据将为ARDS患者的MSC胞外囊泡的早期临床试验提供信息。

Acute Respiratory Distress Syndrome (ARDS) is a severe clinical syndrom that affects 20% of all critically ill patients in intensive care. Unfortunately, around 30-50% of these people will die from the condition and the quality of life of the survivers can be seriously reduced due to the consequent chronic lung problems.There is no effective treatments for this condition at this time and therefore new medicines are urgently needed. ARDS is characterised by an excessive and disregulated inflammation in the lung that leads to inappropriate infiltration of immune cells, lungs fill with water and fail to maintain its main function - breathing. In order to breath these patients require mechanical ventilation.Recently more and more clinicians and basic scientists attention is drawn to cell-based therapies. One of the most promising candidate for a cell based therapy are cells termed Mesenchymal Stem Cells (MSCs), which represent one kind of adult stem cells (can be easily isolated from tissues of adult patients or healthy volunteers), because of this, there is no ethical issues. MSCs could easily be propagated and manipulated under laboratory conditions and have capacity to produce multiple mediators with immuno-modulatory properties. However, concerns regarding safety of long-term effects of MSC administration (e.g. development of tumors) as well as logistical challenges may limit their successful clinical translation. Accumulating evidence now suggests that MSCs act primarily through secretion of different biologically active mediators. Therefore researchers are increasingly considering MSC cell products (which can not replicate and therefore form tumors) as an alternative to the whole cell therapy.In this project we will investigate the therapeutic potential of small membrane wrapped "exracellular vesicles" released by MSCs. These vesicles represent the key component of MSCs secretome and are relatively easy to isolate from MSC conditioned medium by high speed centrifugation. Previously we and others had demonstrated that these vesicles were capable to recapitulate therapeutic effects of MSCs in various pre-clinical models of ARDS. Furthemore, in our recent work we had found that these vesicles contain organelles responsible for maintaining cell bioenergetics (mitochondria). This is very exciting finding as it is known that mitochondrial function is impaired in acute inflammatory conditions such as ARDS.In this project we will use small animal model of ARDS to investigate the therapeutic potential of these vesicles, optimise best route and strategy of administration, gain isights into their bio-distribution in the tissues and also investigate whether or not functionally active mitochondria are important for the therapeutic effect and which tissues are major recipients of the mitochondria transferred in the EVs. These studies will be further complemented by the investigation of the EV mitochndrial transfer to primary human lung cells in vitro. These data are an essential requirement by regulatory bodies such as UK Medicines and Healthcare Products Regulatory Agency and European Medicines Agency for clincal translation of new therapies. The principle anticipated output of this project will be the establishment of a defined strategy for clinical development and knowledge of the specific mechanism of action (based on mitochondrial transfer) of the MSC derived extracellular vesicles. More immediately these data will inform further pre-clinical studies in larger animal models of ARDS required to obtain neccessary data on safety, efficacy and mechanism of action of MSCs EVs. Collectively data in small and large animal models will inform early phase clinical trials for MSC extracellular vesicles in patients with ARDS.