Neuron Specific mRNA Transfer With Fusogenic Microvesicles

使用融合微泡进行神经元特异性 mRNA 转移

• 批准号: 10578732
• 负责人: XUEDONG LIU
• 金额: $ 7.83万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10578732
• 关键词: Address; Affect; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Animal Model; Biochemical; Biological Assay; Biological Models; Brain; Brain region; Brain-Derived Neurotrophic Factor; Cell Separation; Cell membrane; Cells; Cellular Tropism; Central Nervous System; Development; Disease; Effectiveness; Encapsulated; Endosomes; Engineering; Fluorescent in Situ Hybridization; Foundations; Future; GTP-Binding Proteins; Glycoproteins; Goals; Half-Life; Human; In Vitro; Length; Liposomes; Mammalian Cell; Mediating; Messenger RNA; Methods; MicroRNAs; Modality; Mus; Neurodegenerative Disorders; Neurons; Nucleic Acids; Parkinson Disease; Peripheral Nervous System Diseases; Persons; Pharmaceutical Preparations; Pharmacotherapy; Property; Proteins; RNA; Reporter; Reporting; Role; Small RNA; Specificity; Symptoms; System; Technology; Testing; Therapeutic; Tissues; Translating; Tropism; Vesicle; Vesiculovirus; Viral; Virus; blood-brain barrier penetration; cell type; design; effective therapy; extracellular vesicles; improved; innovation; mRNA delivery; microvesicles; nervous system disorder; neuron loss; neurotropic; novel strategies; postnatal; programs; reconstitution; therapeutic protein; uptake; vesicular stomatitis virus G protein

Project Summary Numerous studies have shown that RNAs can be transferred between specific types of mammalian cells. Such exchanges are mostly limited to non-translatable RNA and mediated by extracellular vesicles (EVs), which are known to be capable of encapsulating small RNAs (microRNAs and small pieces mRNAs) and delivering them to the recipient cells. Recent studies suggest that full-length mRNAs can be transferred intercellularly via by extracellular vesicles or direct cell-cell contact. However, the mechanisms governing intercellular transfer of mRNAs and the specificity of mRNAs transfer are still poorly understood. The efficiency of mRNA transfer is also poor, which makes it difficult for therapeutic applications. We recently discovered that expression of a single virus-derived protein called vesicular stomatitis virus G protein (VSV-G) in mammalian cells can promote intercellular exchange of proteins and nucleic acids via highly fusogenic microvesicles, which we call gectosomes (G protein ectosomes). We demonstrated that VSV-G can stimulate: 1) outward budding of vesicles at the plasma membrane of donor cells; 2) internalization of vesicles into recipient cells; 3) efficient cargo release from endosomes inside recipient cells; 4) gectosomes are hundreds fold more efficient in delivering bioactive proteins than artificial liposomes. Due to the broad cellular tropism of VSV-G, gectosomes can mediate intercellular transfer of proteins and RNAs rather nonspecifically between mammalian cells, which would be a challenge for future development of cell or tissue specific delivery of mRNAs as a therapeutic modality. Inspired by our findings with VSV-G, we searched for other VSV-G like proteins for similar functions but with more restricted cellular tropism. We discovered a new viral glycoprotein, CNV-G, from Chandipura vesiculovirus that shares many properties with VSV-G in making gectosomes. Unlike VSV-G, CNV-G gectosomes show highly restricted cell tropism with significant uptake only in neuronal cells. The central hypothesis of this proposal is that neuron- specific glycoprotein CNV-G can be engineered to encapsulate functional mRNAs into extracellular vesicles to be used to deliver potential therapeutic mRNAs to neuronal cells. The objective of this application is to characterize the specificity of CNV-G gectosomes and demonstrate that CNV-G gectosomes can mediate the transfer of cellular mRNAs to neuronal cells and reprogram cellular protein contents. Methods for neuron-specific mRNA transfer will lay the foundation to co-opt this system for developing mRNA such as BDNF as therapeutic drugs to overcome the delivery challenges for delivering therapeutic mRNAs to neurons.

项目摘要 大量研究表明，RNA可以在特定类型的特定类型之间转移 哺乳动物细胞。这样的交换主要限于不可转移的RNA，并由 细胞外囊泡（EV），已知能够封装小RNA （microRNA和小块mRNA）并将其输送到受体细胞。最近的研究 表明可以通过细胞外囊泡或 直接细胞接触。但是，管理mRNA和细胞间转移的机制和 mRNA转移的特异性仍然鲜为人知。 mRNA传递的效率为 也很差，这使得治疗应用困难。我们最近发现 单个病毒衍生的蛋白质的表达称为囊泡气孔病毒G蛋白（VSV-G） 哺乳动物细胞可以通过高度促进蛋白质和核酸的细胞间交换 融合型微泡，我们称之为牛to（G蛋白质过体）。我们证明了 VSV-G可以刺激：1）在供体细胞的质膜上的囊泡向外萌芽； 2）将囊泡在受体细胞中内部化； 3）从内部内体释放有效的货物 受体细胞； 4）甲登胸小体在输送生物活性蛋白方面的效率更高 比人造脂质体。由于VSV-G的宽细胞向向趋势，甲甲室可以介导 蛋白质和RNA的细胞间转移，而不是在哺乳动物细胞之间非特定的，这 对于未来的细胞或组织特异性传递作为一个的挑战，将是一个挑战 治疗方式。受VSV-G的发现的启发，我们搜索了其他VSV-G 蛋白质的类似功能，但具有更受限制的细胞向潮流。我们发现了一种新病毒 来自Chandipura Vesiculovirus的糖蛋白CNV-G，该病毒与VSV-G共有许多特性 制作三十体。与VSV-G不同，CNV-G g型体显示出高度受限的细胞向向主义， 仅在神经元细胞中大量摄取。该提议的中心假设是神经元 可以设计特定的糖蛋白CNV-G以将功能mRNA封装到 细胞外囊泡用于向神经元细胞传递潜在的治疗mRNA。这 该应用的目的是表征CNV-G g型体的特异性和 证明CNV-G贡献体可以介导细胞mRNA向神经元的转移 细胞和重编程细胞蛋白含量。神经元特异性mRNA转移的方法将放置 选择该系统用于开发mRNA（例如BDNF）作为治疗药物的基础 克服向神经元传递治疗性mRNA的分娩挑战。