Bottom-up, high-throughput prototyping of extracellular vesicle mimetics using cell-free synthetic biology

使用无细胞合成生物学对细胞外囊泡模拟物进行自下而上的高通量原型设计

• 批准号: 10638114
• 负责人: Randy Carney
• 金额: $ 57.06万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2027-02-27
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10638114
• 关键词: Address; Animals; Autoimmune Responses; Benchmarking; Biodistribution; Biological; Biological Assay; Biological Models; Cell secretion; Cells; Cellular Assay; Chemicals; Clinic; Complex; Coupled; Disease; Dose; Drug Delivery Systems; Engineering; Evaluation; Exhibits; Experimental Autoimmune Encephalomyelitis; Future; Half-Life; Heterogeneity; Image; Immune; In Vitro; Integral Membrane Protein; Lead; Lipids; Liposomes; Mass Spectrum Analysis; Measurement; Membrane; Membrane Proteins; Mesenchymal; Mesenchymal Stem Cells; Methods; MicroRNAs; Molecular; Multiple Sclerosis; Neurons; Nucleic Acids; Organ; Performance; Physiologic pulse; Play; Printing; Production; Protein Biosynthesis; Proteins; Proteomics; Quality Control; RNA; Recombinants; Reproducibility; Research; Resolution; Role; Set protein; Standardization; Stromal Cells; System; Technology; Testing; Therapeutic; Therapeutic Effect; Toxic effect; Translating; Untranslated RNA; Vesicle; Wild Type Mouse; Work; analytical method; angiogenesis; cell type; clinical application; delivery vehicle; design; experimental study; extracellular vesicles; high throughput screening; immunoregulation; in vivo; in vivo Model; insight; intercellular communication; interest; mimetics; mouse model; nanolitre; nanoparticle; nervous system disorder; neuroprotection; pharmacokinetics and pharmacodynamics; prevent; prototype; public health relevance; reconstitution; regenerative; stem; superresolution microscopy; synthetic biology; transcriptome sequencing; unilamellar vesicle; uptake

PROJECT SUMMARY Bottom-up, high-throughput prototyping of extracellular vesicle mimetics using cell-free synthetic biology Cells secrete extracellular vesicles (EVs) that function as primary messengers of intercellular communication and are studied as promising drug-delivery vehicles and therapeutics. However, the clinical application of native EVs has been hindered by their low production yield, impurity, and inherent heterogeneity. Native EVs contain many biologically active components, such as RNAs and proteins, spread out over numerous subpopulations. This biological complexity is both the strength and the Achilles’ heel of native EVs. While various features of this complexity enable the beneficial therapeutic effects of EVs, it is not clear which plays a dominant role. However, the complex set of proteins and RNAs results in heterogeneous EVs that are challenging to study and use as a standardized treatment. Therefore, separating out and defining the critical biomolecular features from the overall heterogeneous set will allow us to perform quality control of EVs and to reproducibly produce or study EVs. A major bottleneck in finding the critical molecular parts of EVs is the lack of high-throughput methods. To overcome this difficulty, our team will create a synthetic biology-based, cell-free high-throughput discovery platform. The platform will be able to synthesize EV mimetics using a cell-free synthesis approach (Aim 1), coupled with high-throughput examination of EV mimetic potency in vitro (Aim 2). Select EV mimetics will also be investigated using an in vivo model system of neuroprotection and immune modulation (Aim 3). Throughout the study, we will use native mesenchymal stem/stromal cell EVs and neurological diseases as our model system to evaluate the platform. Our work will enable the high-throughput study of EVs for any disease and biological questions of interest. In addition, we will unveil new insights into EVs that address key debated topics in the EV field.

项目总结 使用无细胞合成生物学的自下而上、高通量的胞外囊泡模拟原型 细胞分泌细胞外小泡(EV)，作为细胞间通讯的主要信使 被认为是很有前途的药物输送载体和治疗药物。然而，国产西药的临床应用 电动汽车的低产量、杂质和固有的异质性阻碍了电动汽车的发展。本地电动汽车包含 许多生物活性成分，如RNA和蛋白质，分布在许多亚群中。 这种生物复杂性既是本土电动汽车的优势，也是其致命弱点。虽然这款产品的各种功能 复杂性使EVS具有有益的治疗效果，目前尚不清楚哪种作用占主导地位。然而， 一组复杂的蛋白质和RNA导致了不同种类的电动汽车，这对研究和作为一种 规范治疗。因此，从整体上分离和定义关键的生物分子特征 异质SET将使我们能够对电动汽车进行质量控制，并可重复生产或研究电动汽车。一个 寻找电动汽车关键分子部分的主要瓶颈是缺乏高通量的方法。至 克服这一困难，我们的团队将创造一个基于合成生物学的、无细胞的高通量发现 站台。该平台将能够使用无细胞合成方法合成电动汽车模拟物(目标1)， 再加上高通量的EV体外模拟效力检测(目标2)。选择电动汽车模拟也将 使用神经保护和免疫调节的体内模型系统进行研究(目标3)。贯穿始终 在这项研究中，我们将使用天然间充质干细胞/基质细胞EVS和神经疾病作为我们的模型系统 对该平台进行评估。我们的工作将使针对任何疾病和生物的电动汽车的高通量研究成为可能 感兴趣的问题。此外，我们将推出对电动汽车的新见解，以解决电动汽车中的关键辩论主题 菲尔德。