FMSG: Enabling Technologies for Biomanufacturing EV-Based Therapeutics

细胞外囊泡（EVs）是细胞制造的颗粒，提供细胞间信息和物质传递的自然机制。人们对大规模生产电动汽车越来越感兴趣，因为电动汽车能够从产生细胞传递信号，并有可能用作药物分子的载体。目前正在开发EV疗法，用于治疗多种疾病，包括代谢紊乱、癌症和神经变性。尽管几家处于早期发展阶段的电动汽车生物技术公司令人兴奋，但大规模生产具有可调性能的纯电动汽车的技术仍处于起步阶段。该项目的长期目标是实现“设计电动汽车”的生产，这种汽车可以用所需的货物分子包装，用可调的表面配体装饰，并从特定的生产细胞类型中高产地分泌。当前项目的总体目标是确定可以控制治疗性ev的生产、含量和体内贩运的细胞过程。向公众提供这类新药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。该项目还将为受训者提供独特的教育机会，让他们与行业科学家进行合作研究。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项目和由pi领导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的装载和递送上，小rna是很有前途的药物分子，但不能通过基于纳米颗粒的载体有效地递送到受体细胞。研究的核心假设是，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异性ev的产量和ev向受体细胞递送RNA的效率。首先，它们将通过调节内质网接触部位的特定分子来促进mirna靶向细胞外囊泡。其次，他们将通过改造电动汽车，使其具有有效的内体逃逸和巨噬细胞逃逸特性，来优化向目标细胞的货物递送。第三，他们将开发一个可扩展的平台，通过将诱导多能干细胞（iPSCs）分化为3D悬浮培养的生产细胞来制造组织特异性电动汽车。拟议的研究具有创新性，因为它采用多学科方法解决了治疗性电动汽车商业化生产的几个关键障碍：货物装载、货物交付和可扩展制造。由间充质干细胞分泌的ev被认为是克服原代间充质干细胞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。

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.