Neuron Specific mRNA Transfer With Fusogenic Microvesicles

项目摘要 大量研究表明，RNA可以在特定类型的 哺乳动物细胞。这种交换大多限于不可翻译的RNA，并由 细胞外小泡(EV)，已知能够包裹小RNA (microRNAs和小片段mRNAs)，并将它们递送到受体细胞。最新研究 提示全长的mRNAs可以通过细胞外小泡或 细胞与细胞的直接接触。然而，调控mRNA和mRNAs细胞间转移的机制 目前对mRNAs转移的特异性仍知之甚少。信使核糖核酸转移效率为 也很差，这使得治疗应用变得困难。我们最近发现， 水疱性口炎病毒G蛋白(VSV-G)在大肠杆菌中的表达 哺乳动物细胞可以通过高度的 融合微泡，我们称之为壁体(G蛋白外体)。我们演示了 VSV-G可刺激：1)供体细胞质膜上的小泡向外萌发； 2)囊泡内化到受体细胞；3)从内吞体内有效地释放货物 受体细胞；4)壁体在运送生物活性蛋白方面的效率提高了数百倍 而不是人工脂质体。由于VSV-G广泛的细胞嗜性，壁球体能介导 蛋白质和RNA在哺乳动物细胞之间的非特异性细胞间转移，这是 对于未来发展细胞或组织特异性递送作为一种 治疗方式。受我们对VSV-G的发现的启发，我们搜索了其他VSV-G 蛋白质具有相似的功能，但具有更有限的细胞趋向性。我们发现了一种新的病毒 囊泡病毒囊泡病毒的糖蛋白CNV-G与VSV-G有许多共同的性质 制作壁小体。与VSV-G不同，CNV-G壁体表现出高度受限的细胞嗜性 仅在神经细胞中显著摄取。这一提议的中心假设是神经元- 特定的糖蛋白CNV-G可以被改造成将功能mRNAs封装到 细胞外小泡用于将潜在的治疗性mRNAs输送到神经细胞。这个 本应用的目的是鉴定CNV-G gecosome的特异性和 CNV-G凝集体可介导细胞内mRNAs向神经元的转移 细胞和重新编程细胞蛋白质含量。神经元特异性mRNA转移的方法将出台 将该系统用于开发BDNF等信使核糖核酸作为治疗药物的基础 克服向神经元递送治疗性RNA的交付挑战。

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.