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.

项目摘要 许多研究表明，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RNA介导。 细胞外囊泡（EV），其已知能够包封小RNA （microRNA和小片段mRNA）并将它们递送到受体细胞。最近的研究 表明全长mRNA可以通过细胞外囊泡在细胞间转移， 直接细胞间接触然而，控制mRNAs和 mRNA转移特异性仍然知之甚少。mRNA转移的效率是 也很差，这使得它很难用于治疗应用。我们最近发现 表达称为水泡性口炎病毒G蛋白（VSV-G）的单一病毒衍生蛋白， 哺乳动物细胞可以通过高度的蛋白质和核酸交换来促进细胞间的蛋白质和核酸交换。 融合微泡，我们称之为G蛋白外体（G蛋白外体）。我们证明 VSV-G可刺激：1）供体细胞质膜上囊泡向外出芽; 2)囊泡内化到受体细胞中; 3）从内部的内体有效释放货物 受体细胞; 4）Gectosomes在递送生物活性蛋白质方面的效率提高数百倍 比人造脂质体更好。由于VSV-G广泛的细胞嗜性， 蛋白质和RNA在哺乳动物细胞间的非特异性细胞间转移， 将是未来开发细胞或组织特异性递送mRNA作为一种治疗方法的挑战。 治疗方式受VSV-G研究结果的启发，我们搜索了其他VSV-G，如 蛋白质具有相似的功能，但具有更有限的细胞向性。我们发现了一种新的病毒 糖蛋白，CNV-G，来自Chandipura水疱病毒，与VSV-G在免疫学上具有许多相同的特性， 制造卵细胞外体与VSV-G不同，CNV-G外泌体显示出高度限制的细胞向性， 仅在神经元细胞中显著摄取。这个假设的核心是神经元- 特异性糖蛋白CNV-G可以被工程化以将功能性mRNA包封到 这些细胞外囊泡可用于将潜在的治疗性mRNA递送至神经元细胞。的 本申请的目的是表征CNV-G外膜体的特异性， 证明CNV-G gectosomes可以介导细胞mRNA转移到神经元， 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 这是将该系统用于开发mRNA如BDNF作为治疗药物的基础， 克服了向神经元递送治疗性mRNA的递送挑战。