FMSG: Enabling Technologies for Biomanufacturing EV-Based Therapeutics

细胞外小泡(EVS)是细胞制造的颗粒，为细胞之间的信息和物质转移提供了一种自然的机制。人们对大规模生产可用作治疗药物的电动汽车越来越感兴趣，因为它们能够从生产细胞传递信号，以及它们作为药物分子输送载体的潜在用途。EV疗法正在被开发用于治疗包括代谢紊乱、癌症和神经变性在内的各种疾病。尽管几家早期电动汽车生物技术公司令人兴奋，但大量生产性能可调的纯电动汽车的技术仍处于初级阶段。该项目的长期目标是使“设计师电动汽车”的生产能够与所需的货物分子包装在一起，用可调节的表面配体装饰，并从特定类型的生产者细胞中以高产量分泌。当前项目的总体目标是确定可以工程设计的细胞过程，以控制治疗性电动汽车的生产、内容和体内贩运。将这种新型药物提供给公众，有可能改善美国和世界各地数百万患者的健康和生活质量。该项目还将为学员提供与行业科学家进行合作研究的独特教育机会。其他更广泛的影响将通过让本科生和高中生参与电动汽车研究，以及通过将该项目与范德比尔特电动汽车研究计划和由PI牵头的生物工程课程相结合来实现。研究人员将把他们的研究重点放在小RNA的装载和传递上，这是一种有希望的药物分子，使用现有的基于纳米颗粒的载体无法有效地传递到受体细胞。中心假设是，开发组织特异性生产细胞，调节EV相关蛋白和RNA的表达，将使研究人员能够最大限度地提高特定EV的产品产量，并提高EV向受体细胞运送RNA的效率。首先，他们将通过调节ER接触部位的特定分子来促进miRNAs对细胞外小泡的靶向。其次，他们将通过设计电动汽车来优化向目标细胞的货物输送，以实现高效的内体逃逸和巨噬细胞逃避特性。第三，他们将开发一个可扩展的平台，通过在3D悬浮培养中将诱导多能干细胞(IPSCs)分化为产生细胞来制造组织特异性电动汽车。这项拟议的研究具有创新性，因为它应用了多学科的方法来解决治疗性电动汽车商业化生产的几个关键障碍：货物装载、货物交付和可扩展的制造。MSCs分泌的EVS被认为是克服原代MSCs移植潜在风险的可行选择。通过开发工具和策略来定制和最大限度地利用IPSC衍生的MSCs生产电动汽车，研究人员将建立一个灵活的电动汽车制造平台，可以扩展到其他相关的器官和组织系统。该奖项反映了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。

Extracellular vesicles (EVs) are cell-made particles that provide a natural mechanism of information and material transfer between cells. There is growing interest in large-scale production of EVs that can be used as therapeutics due to their ability to communicate signals from producer cells and their potential use as carriers for delivery of drug molecules. EV therapeutics are being developed for treatment of a wide range of diseases including metabolic disorders, cancer, and neurodegeneration. Despite the excitement generated by several early-stage EV biotech companies, the technology to produce mass quantities of purified EVs with tunable properties is still in its infancy. The long-term goal of this project is to enable production of "designer EVs" that can be packaged with desired cargo molecules, decorated with tunable surface ligands, and secreted in high yield from specific producer cell types. The overall objective of the current project is to identify cellular processes that can be engineered to control the production, content, and in vivo trafficking of therapeutic EVs. Making this new class of drugs available to the public has potential to improve the health and quality of life of millions of patients in the US and around the world. The project will also provide the unique educational opportunity for trainees to engage in collaborative research with industry scientists. Other broader impacts will be accomplished through engaging undergraduate and high-school students in EV research, and through integration of the project with the Vanderbilt Program for EV Research and bioengineering courses led by the PIs. The investigators will focus their research program on loading and delivery of small RNAs, which are promising drug molecules that are not delivered efficiently to recipient cells using established nanoparticle-based carriers. The central hypothesis is that developing tissue-specific producer cells with tuned expression of EV-associated proteins and RNAs will enable researchers to maximize the product yield of specific EVs and the efficiency of RNA delivery from EVs to recipient cells. First, they will boost targeting of miRNAs to extracellular vesicles through modulating specific molecules at ER-contact sites. Second, they will optimize cargo delivery to target cells by engineering EVs for efficient endosomal escape and macrophage evasion properties. Third, they will develop a scalable platform for manufacturing tissue-specific EVs by differentiating induced pluripotent stem cells (iPSCs) to producer cells in 3D suspension cultures. The proposed research is innovative because it applies a multidisciplinary approach to address several critical barriers to commercial production of therapeutic EVs: cargo loading, cargo delivery, and scalable manufacturing. EVs secreted by MSCs are recognized as a viable alternative to overcome the potential risks from transplantation of primary MSCs. By developing the tools and strategies to customize and maximize EV production from iPSC-derived MSCs, the researchers will establish a flexible EV manufacturing platform that can expand to other relevant organ and tissue systems.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.