Neuron Specific mRNA Transfer With Fusogenic Microvesicles

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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.