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

非技术概述开发安全有效的核酸输送手段变得越来越重要，特别是在新城疫大流行的情况下。能够为我们的免疫系统提供抗击新冠肺炎的工具的信使核糖核酸疫苗，在帮助我们抗击这种毁灭性病毒方面至关重要。该项目支持专注于创造更好的递送剂的基础研究，这些递送剂可以更好地保护和携带重要的核酸，如信使核糖核酸，从而使其能够发挥作用。我们探索使用外切体作为潜在的递送工具。外切体本质上是从我们自己的细胞衍生出来的小隔间。正因为如此，他们拥有关键部件，使他们能够进入其他牢房并运送货物。虽然外切体具有这种独特的优势，但将靶向治疗加载到它们是具有挑战性的。为了解决这一问题，研究小组将开发能够温和地结合带负电荷的核酸的小分子带电蛋白质，从而帮助改善外切体的负载。该项目不仅将培训下一代研究生和研究人员使用这些新方法，还将帮助激励高中生，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。国际和平协会和联合国际在培训妇女和代表性不足的少数民族方面都有很好的记录，并计划继续招募和培训她们，作为这项工作的一部分，目标是发展和多样化未来的科学力量。技术摘要核酸拥有关键的指令，用于控制生物过程，在基础细胞和有机体研究、生物技术和医学方面具有切实的影响，特别是鉴于新冠肺炎的疫苗交付。需要开发一种用于广泛递送核酸的模块化平台，而外切体最近已被确定为有前途的递送剂。膜衍生脂质和重组蛋白之间的组装机制尚不清楚，但为优化和探索基于外切体的递送载体的基本材料性质提供了机会。该研究小组建议开发一种工程蛋白-外切体系统或脂蛋白体，能够：1)将siRNA高效地包裹到外切体中；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传递。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。课程将侧重于混合的面授和虚拟课程，重点是以小组为基础的项目，以促进主动学习。在外展方面，Montclare博士将参与科学与工程应用研究创新(ARISE)计划，并让纽约市当地的10年级和11年级学生参与增压蛋白质的计算设计。拉巴尼博士将作为Wyss Sumer Research Fellowship的一部分实施一项生物化学培训，涉及外体的准备、表征和用于转基因，以及记录方案视频和在线杂志俱乐部讨论。这将使学生在大流行期间和之后的研究经验民主化。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.