Engineering of Extracellular Vesicles for Oral Delivery of Nucleic Acid Therapies

口服给药是首选的服药方式，因为它方便、无痛、安全，并且药物可以由患者自己服用。然而，某些药物，包括核酸，被用作药物来操纵身体重要蛋白质的产生，目前不能口服施用。以核酸为基础的药物，其中一个例子是Covid-19疫苗，在胃酸等肠道的恶劣环境中不稳定。此外，由于它们非常大的尺寸，高效的肠壁屏障严重限制了生物制剂吸收到血液中。因此，核酸疗法目前需要由医疗保健专业人员通过注射施用。动物细胞产生并释放被称为细胞外囊泡（EV）的微小颗粒（比人类头发宽度小500-1000倍）。这些是膜结合颗粒，通过将各种生物分子货物从一个细胞转移到另一个细胞，在细胞间通讯中起着至关重要的作用。这种货物还包括核酸和蛋白质，使电动汽车成为理想的天然设计载体，可以将这些药物输送到整个肠壁。包括我们小组在内的先前研究表明，牛奶中存在的电动汽车能够有效地穿过肠道壁。因此，人们可以利用这些EV，它们从丰富、廉价和可持续的来源（牛奶）中分离出来，并能够口服递送核酸。然而，使用牛奶EV来实现核酸的口服递送的关键挑战涉及其异质性（具有不同生物功能的多种颗粒类型）和将大核酸加载到膜结合EV中。在这个项目中，我们将通过筛选这些EV穿过肠壁的能力来确定驱动肠道渗透的牛奶EV的关键成分。我们将在人类肠道壁的实验室模型（在塑料培养皿上生长的细胞）中进行这种筛选。我们将分析穿过肠壁的EV的组成，并将其与那些没有这种能力的EV进行比较。这将使我们能够确定哪些EV组分有助于它们穿过肠道壁。这些信息将同时使我们能够选择性地从牛奶中存在的EV混合物中分离出穿过肠壁的EV。然后将这些EV工程化以实现药物（核酸）加载。工程电动汽车将在人类肠道实验室模型和动物模型中测试其口服核酸的有效性。除了上述实验室研究外，该项目还将嵌入知识交流活动（研讨会，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。该设施将容纳最先进的电动汽车研究设备，并将免费提供给从事电动汽车研究的研究团体使用。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大学，即伦敦国王学院、阿斯顿大学和诺丁汉大学。此外，我们还与行业合作，特别是微孔技术公司，他们有能力扩大该项目中创建的新的廉价但强大的基于电动汽车的疗法的制造。学术团队将与工业合作伙伴密切合作，以实现项目的不同方面和项目的总体目标，即创造新的药物，改变许多疾病的管理，同时为患者提供负担得起的便利。

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.