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: 10451377
• 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/10451377
• 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 membrane; Cells; Cellular Tropism; 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; Neuraxis; 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; 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.

项目概要 大量研究表明，RNA 可以在特定类型的细胞之间转移。 哺乳动物细胞。这种交换主要限于不可翻译的 RNA，并由 细胞外囊泡 (EV)，已知能够封装小 RNA （microRNA 和小片段 mRNA）并将其递送至受体细胞。最近的研究 表明全长 mRNA 可以通过细胞外囊泡或 细胞与细胞的直接接触。然而，控制 mRNA 和细胞间转移的机制 mRNA 转移的特异性仍然知之甚少。 mRNA转移效率为 也很差，这使得其难以用于治疗应用。我们最近发现 水疱性口炎病毒 G 蛋白 (VSV-G) 的单一病毒衍生蛋白在 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 融合微泡，我们称之为基因胞体（G 蛋白胞外体）。我们展示了 VSV-G 可以刺激： 1) 供体细胞质膜上的囊泡向外出芽； 2)囊泡内化到受体细胞中； 3) 内体内有效的货物释放 受体细胞； 4) 外泌体传递生物活性蛋白的效率提高了数百倍 比人工脂质体。由于 VSV-G 具有广泛的细胞趋向性，gectosomes 可以介导 哺乳动物细胞之间蛋白质和 RNA 的细胞间转移相当非特异性， 对于未来开发细胞或组织特异性递送 mRNA 作为载体将是一个挑战 治疗方式。受我们对 VSV-G 的研究结果的启发，我们搜索了其他 VSV-G，例如 具有相似功能但具有更有限的细胞趋向性的蛋白质。我们发现了一种新病毒 糖蛋白 CNV-G，来自金迪普拉水泡病毒，与 VSV-G 具有许多相同的特性 制作胚胎体。与 VSV-G 不同，CNV-G 基因胞体表现出高度受限的细胞趋向性 仅在神经元细胞中显着摄取。该提案的中心假设是神经元 可以设计特定的糖蛋白 CNV-G 将功能性 mRNA 封装到 细胞外囊泡用于将潜在的治疗性 mRNA 传递到神经元细胞。这 本应用的目的是表征 CNV-G 基因组的特异性和 证明 CNV-G gectosome 可以介导细胞 mRNA 向神经元的转移 细胞并重新编程细胞蛋白质含量。神经元特异性 mRNA 转移的方法将奠定 为利用该系统开发 BDNF 等 mRNA 作为治疗药物的基础 克服将治疗性 mRNA 递送至神经元的递送挑战。