Investigation of the internal structure of lipid nanoparticles for gene delivery

用于基因传递的脂质纳米粒子的内部结构研究

• 批准号: ST/Y000587/1
• 负责人: Arwen Tyler
• 金额: $ 2.41万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=ST%2FY000587%2F1
• 关键词: Investigation; internal; structure; lipid; nanoparticles

Lipid nanoparticles (LNPs) are very small self-assembled particles of approximately 100 nanometres (0.1 micrometres) in diameter, which are able to enclose and protect biologically active molecules such as nucleic acids (RNA or DNA). They are able to deliver their payload intact to the inside of cells, which has led to their use for COVID-19 mRNA vaccines, with spectacular success. However, how it all works is still something of a 'black-art', due to a lack of detailed understanding at a molecular level of the factors which control each stage of this very complex process. It is now well known that four types of lipid are needed for the LNP, with the most critical of these being an ionizable lipid (CIL), which changes its charge state upon being acidified inside compartments (endosomes) within cells, destabilising the endosome wall, and allowing the mRNA to escape into the cell interior, where it can carry out its work.Although the Astrazeneca team have extensive knowledge of LNPs, and have synthesized and screened hundreds of lipids, the relationship between LNP lipid composition, LNP structure, and its biological activity is not well understood. This information is needed to help rationale the design of the optimal lipids to enable more efficient and safer intracellular delivery of mRNAs and other nucleic acids for vaccines and other advanced treatments such as gene editing. In this project we aim to take advantage of the state of the art facilities at Rutherford Appleton Laboratories to generate high quality structural data from LNPs formulated from a set of promising lipid candidates developed by Astrazeneca. The techniques we will use are Small Angle X-ray Scattering(SAXS) and Small Angle Neutron Scattering (SANS), and cryo- Transmission Electron Microscopy (Cryo-TEM). Synchrotron SAXS at Diamond Light Source is a very powerful way of probing the self-assembled structures, identifying and characterising liquid-crystalline or other types of ordering within the LNPs. SANS at the neutron scattering facility ISIS has the unique ability to determine where the different lipids are located within the LNPs. This approach takes advantage of a unique feature of neutron scattering, whereby molecules can be 'highlighted' by substituting deuterium for hydrogen in the chemical structure of the synthesized molecule. Cryo-TEM is able to give additional structural detail, particularly with regard to the release of mRNA inside cells. The experimental work will be complemented by computer simulation studies of the interaction of mRNA with the lipids.

脂质纳米颗粒（LNP）是直径约为100纳米（0.1微米）的非常小的自组装颗粒，能够包裹和保护生物活性分子，如核酸（RNA或DNA）。它们能够将其有效载荷完整地传递到细胞内部，这导致它们用于COVID-19 mRNA疫苗，并取得了巨大的成功。然而，这一切是如何工作的仍然是一个“黑艺术”的东西，由于缺乏详细的了解，在分子水平上的因素，控制这个非常复杂的过程的每个阶段。现在众所周知，LNP需要四种类型的脂质，其中最关键的是可电离脂质（CIL），其在隔室内被酸化后改变其电荷状态细胞内的内体（内体），使内体壁不稳定，并允许mRNA逃逸到细胞内部，在那里它可以执行其工作。尽管阿斯利康团队对LNP有广泛的了解，虽然已经合成和筛选了数百种脂质，但是LNP脂质组成、LNP结构和其生物活性之间的关系还没有很好地理解。需要这些信息来帮助合理设计最佳脂质，以实现更有效和更安全的mRNA和其他核酸的细胞内递送，用于疫苗和其他先进治疗，如基因编辑。在这个项目中，我们的目标是利用卢瑟福阿普尔顿实验室的最先进的设施，从阿斯利康开发的一组有前途的脂质候选物配制的LNP产生高质量的结构数据。我们将使用的技术是小角X射线散射（SAXS）和小角中子散射（SANS），以及低温透射电子显微镜（Cryo-TEM）。金刚石光源的同步加速器SAXS是探测自组装结构，识别和表征LNP内液晶或其他类型有序的非常强大的方法。中子散射设施ISIS的SANS具有确定不同脂质位于LNP内的独特能力。这种方法利用了中子散射的一个独特特征，通过在合成分子的化学结构中用氘取代氢，可以“突出显示”分子。Cryo-TEM能够提供额外的结构细节，特别是关于细胞内mRNA的释放。实验工作将通过mRNA与脂质相互作用的计算机模拟研究来补充。