Engineering of Extracellular Vesicles for Oral Delivery of Nucleic Acid Therapies

口服给药是首选的用药方式，因为它方便、无痛、安全，而且患者可以自行给药。然而，某些药物，包括被用作操纵身体产生重要蛋白质的药物的核酸，目前不能口服。以核酸为基础的药物，新冠肺炎疫苗就是一个例子，在胃酸等恶劣的肠道环境中是不稳定的。此外，由于它们的体积非常大，高效的肠壁屏障严重限制了生物制剂进入血液的吸收。因此，核酸疗法目前需要由医疗保健专业人员注射给药。以前试图开发口服核酸输送技术的研究努力并不成功。动物细胞产生和释放被称为细胞外小泡(EVS)的微小颗粒(比人类头发宽度小500-1000倍)。它们是膜结合的颗粒，通过将各种生物分子货物从一个细胞转移到另一个细胞，在细胞间的通讯中发挥着至关重要的作用。这些货物还包括核酸和蛋白质，这使得电动汽车成为理想的、自然设计的载体，可以通过肠壁运送这些药物。之前的研究，包括我们小组的研究，已经表明牛奶中存在的电动汽车能够有效地穿过肠壁。因此，人们可以利用这些电动汽车，这些电动汽车是从丰富、廉价和可持续的来源(牛奶)中分离出来的，并能够口头输送核酸。然而，使用牛奶电动汽车能够口头递送核酸的关键挑战涉及它们的异质性(具有不同生物功能的多种颗粒类型)以及将大核酸装载到膜结合电动汽车中。在这个项目中，我们将通过筛选牛奶电动汽车穿过肠壁的能力来确定推动肠道渗透的牛奶电动汽车的关键成分。我们将在人体肠壁(生长在塑料培养皿上的细胞)的实验室模型中进行这一筛选。我们将分析穿过肠壁的电动汽车的组成，并将其与那些没有这种能力的电动汽车进行比较。这将使我们能够确定哪些EV组件促进了它们通过肠壁的运输。与此同时，这些信息将使我们能够有选择地将穿过肠壁的电动汽车与牛奶中存在的电动汽车的混合物隔离开来。然后，这些电动汽车将被改造成能够装载药物(核酸)。改造后的电动汽车将在人体肠道和动物的实验室模型中进行测试，以测试其口服核酸的有效性。除了上述详细的实验室研究外，该项目还嵌入了知识交流活动(讲习班、培训和研讨会)，并将在伦敦国王学院建立一个电动汽车研究的开放获取研究设施。这个设施将容纳最先进的电动汽车研究设备，并将免费供从事电动汽车研究的社区使用。为了实现该项目的愿景和总体目标，我们拥有一支强大的多学科研究团队，他们来自伦敦和米德兰兹郡的研究机构，即伦敦国王学院、阿斯顿大学和诺丁汉大学。此外，我们还与业界合作，特别是微孔技术公司，他们有能力扩大在该项目中创造的基于电动汽车的新疗法的制造，这种疗法价格低廉，但功能强大。学术团队将与行业合作伙伴密切合作，以交付项目的不同方面和总体项目目标，即创造将改变许多疾病的管理的新药，同时为患者提供负担得起和方便的药物。

Oral administration is the preferred way of taking medicines because it is convenient, painless, safe and the medicine can be self-administered by the patient. However, certain drugs, including nucleic acids, which are used as drugs to manipulate the production of important proteins by the body, currently cannot be administered orally. Nucleic acid-based drugs, an example of which is the Covid-19 vaccine, are unstable in the harsh environment of the gut, such as stomach acid. Additionally, because of their very large size, the highly efficient gut wall barrier severely limits the absorption of biologics into the bloodstream. Nucleic acid therapies therefore currently require administration by injection by a healthcare professional. Previous research efforts attempting to develop technologies for oral delivery of nucleic acids have not been successful.Animal cells produce and release tiny particles (500-1000 times smaller than human hair width) called extracellular vesicles (EVs). These are membrane-bound particles and play a crucial role in cell-cell communication by transferring various biological molecule cargoes from one cell to another. This cargo also includes nucleic acids and proteins, making EVs ideal, naturally designed carriers to deliver these drugs across the gut wall. Previous research, including by our group, has shown that EVs present in cow milk are capable of efficiently crossing the gut wall. One could hence utilise these EVs, which are isolated from an abundant, inexpensive and sustainable source (milk) and to enable oral delivery of nucleic acids. However, the key challenge with the use of milk EVs to enable oral delivery of nucleic acids relates to their heterogenous nature (multiple particle types with different biological function) and the loading of large nucleic acids into membrane-bound EVs. In this project we will identify the key components of milk EVs that drive intestinal permeation by screening the ability of these EVs to cross the gut wall. We will conduct this screening in laboratory models of the human gut wall (cells grown on plastic dishes). We will analyse the composition of EVs which cross the gut wall and compare it with those that do not have this ability. This will enable us to establish which EV components facilitate their transport across the gut wall. This information will at the same time enable us to selectively isolate EVs that cross the gut wall from a mixture of EVs present in milk. These EVs will then be engineered to enable drug (nucleic acid) loading. Engineered EVs will be tested in laboratory models of the human intestine, as well as animals for their efficacy for oral delivery of nucleic acids.In addition to laboratory research detailed above, the project embeds knowledge-exchange activities (workshops, training and seminars) and will also establish an open access research facility for EV research at King's College London. This facility will house state-of-the-art equipment for studying EVs and will be available for free use to the research community working on EVs. To deliver on the project's vision and overall objective, we have a strong, multidisciplinary team of researchers from London and Midlands institutions, namely King's College London, Aston University and the University of Nottingham. Additionally, we have incorporated collaboration with industry, specifically Micropore Technologies, who have capability to scale up the manufacturing of the new inexpensive but powerful EV-based therapies created in this project. The academic team will work closely together, as well as with the industrial partner, to deliver on different aspects of the project and the overall project objective, which is to create new medicines that would transform the management of many diseases, while being affordable and convenient for patients.