Collaborative Research: Development of an exosome based lipoproteoplex (E-LPP) for siRNA delivery

开发安全有效的核酸输送手段变得越来越重要，特别是在COVID大流行的背景下。mRNA疫苗可以为我们的免疫系统提供对抗COVID-19的工具，对于帮助我们对抗这种破坏性病毒至关重要。该项目支持基础研究，重点是创造更好的递送剂，更好地保护和携带重要的核酸，如mRNA，使其发挥作用。我们探索使用外泌体作为潜在的运载工具。外泌体本质上是来自我们自身细胞的小隔间。正因为如此，它们具有允许它们进入其他细胞并运送货物的关键成分。虽然外泌体具有这种独特的优势，但将它们装载目标治疗是具有挑战性的。为了解决这个问题，研究小组将开发一种小的、带电的蛋白质，这种蛋白质可以轻轻地附着在带负电荷的核酸上，从而有助于改善外泌体的负载。该项目不仅将培养下一代研究生和研究人员使用这些新方法，而且还有助于激励高中生，因为他们将能够进行有意义的研究体验，无论是虚拟的还是面对面的。PI和联合PI在培训妇女和代表性不足的少数民族方面都有良好的记录，并计划继续招募和培训她们，作为这项工作的一部分，目标是发展未来的科学劳动力并使其多样化。核酸具有控制生物过程的关键指令，在基础细胞和生物体研究、生物技术和医学方面具有实际意义，特别是在提供Covid-19疫苗方面。有必要开发一种模块化的平台来广泛递送核酸，外泌体最近被确定为有前途的递送剂。膜源性脂质和重组蛋白之间的组装机制尚不清楚，但这为优化和探索基于外泌体的递送载体的基本材料特性提供了机会。研究小组建议开发一种工程蛋白-外泌体系统或脂蛋白复合物，能够：1)高效地将siRNA包封到外泌体中；2)通过增加内体逃逸增加siRNA在细胞内的释放；3)以良好的安全性进行高效的siRNA递送。在教育和推广方面，该团队的目标是专注于经过验证的虚拟平台，为学生提供安全、有意义的研究体验。课程将侧重于面对面和虚拟课程的混合，强调以小组为基础的项目，以促进主动学习。为了推广，Montclare博士将参加科学与工程应用研究创新（ARISE）计划，并与当地纽约市10年级和11年级的学生一起参与增压蛋白的计算设计。Rabbani博士将作为Wyss Sumer研究奖学金的一部分，实施一项生物化学培训，包括外泌体的制备，表征和转染的使用，以及记录协议视频和在线期刊俱乐部讨论。这将使学生在大流行期间和以后的研究经验民主化。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

NON-TECHNICAL SUMMARY The development of safe and effective means to deliver nucleic acids has become increasingly important, especially in light of the COVID pandemic. mRNA vaccines that can provide our immune system the tools to fight COVID-19 are crucial in helping us fight this devastative virus. This project supports fundamental research focused on creating better delivery agents that can better protect and carry the important nucleic acids like mRNA so it can do its job. We explore the use of exosomes as potential delivery vehicles. Exosomes are essentially little compartments that are derived from our own cells. Because of this, they have key components that allow them to enter other cells and deliver cargo. While exosomes have this unique advantage, loading them with the target therapeutic is challenging. To address this, the research team will develop small, charged proteins that can gently hold onto the negatively charged nucleic acids, which then helps improve the loading of exosomes. This project will not only train the next generation graduate student and researcher in these new approaches, but also help inspire high school students as they will be able to carry out meaningful research experience both virtually and in person. Both the PI and co-PI have a strong record of training women and underrepresented minorities and plan to continue to recruit and train them as part of this work with the goal of developing and diversifying the future scientific workforce.TECHNICAL SUMMARY Nucleic acids possess critical instructions for controlling biological processes with tangible implications in fundamental cell and organismal studies, biotechnology and medicine especially in light of delivery of vaccines for Covid-19. There is a need to develop a modular platform for the widespread delivery of nucleic acids, and exosomes have recently been identified as promising delivery agents. The mechanisms of assembly between membrane-derived lipids and recombinant proteins is poorly understood but provides an opportunity to optimize and explore the fundamental material properties of exosome-based delivery vehicles. The research team proposes to develop an engineered protein-exosome system or lipoproteoplex capable of: 1) encapsulating siRNA at a high efficiency into the exosome; 2) increasing siRNA release once inside the cell through increased endosomal escape; 3) performing efficient siRNA delivery with a favorable safety profile. For education and outreach, the team aims to focus on verified virtual platforms to provide students safe, meaningful research experiences. Courses will focus on hybrid in-person and virtual lessons with an emphasis on group-based projects to facilitate active learning. For outreach, Dr. Montclare will participate in the Applied Research Innovations in Science and Engineering (ARISE) program and engage local NYC 10th and 11th grade students in computational design of supercharged proteins. Dr. Rabbani will implement as part of the Wyss Sumer Research Fellowship, a biochemistry training that involves exosome preparation, characterization and use for transfection as well as recording protocol video and journal club discussion online. This will democratize research experience for students during the pandemic and beyond.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.