Dual-Omics Approach Unveils Novel Perspective on the Quality Control of Genetically Engineered Exosomes.

Exosomes, nanoscale vesicles derived from human cells, offer great promise for targeted drug delivery. However, their inherent diversity and genetic modifications present challenges in terms of ensuring quality in clinical use. To explore solutions, we employed advanced gene fusion and transfection techniques in human 293T cells to generate two distinct sets of genetically engineered samples. We used dual-omics analysis, combining transcriptomics and proteomics, to comprehensively assess exosome quality by comparing with controls. Transcriptomic profiling showed increased levels of engineering scaffolds in the modified groups, confirming the success of genetic manipulation. Through transcriptomic analysis, we identified 15 RNA species, including 2008 miRNAs and 13,897 mRNAs, loaded onto exosomes, with no significant differences in miRNA or mRNA levels between the control and engineered exosomes. Proteomics analysis identified changes introduced through genetic engineering and over 1330 endogenous exosome-associated proteins, indicating the complex nature of the samples. Further pathway analysis showed enrichment in a small subset of cellular signaling pathways, aiding in our understanding of the potential biological impacts on recipient cells. Detection of over 100 cow proteins highlighted the effectiveness of LC-MS for identifying potential contaminants. Our findings establish a dual-omics framework for the quality control of engineered exosome products, facilitating their clinical translation and therapeutic applications in nanomedicine.

A Novel Dual-Reporter System Reveals Distinct Characteristics of Exosome-Mediated Protein Secretion in Human Cells.

BACKGROUND: Exosomes, a special subtype of extracellular vesicles derived from human cells, serve as vital mediators of intercellular communication by transporting diverse bioactive cargos, including proteins and enzymes. However, the underlying mechanisms governing exosome secretion and regulation remain poorly understood. In this study, we employed a dual-reporter system consisting of bioluminescent Gaussia luciferase and fluorescent proteins to investigate the dynamics and regulation of exosome secretion in cultured human cells. RESULTS: Our results demonstrated that the engineered dual-reporters effectively monitored both exosome-mediated and ER-Golgi-mediated secretory pathways in a specific and quantitative manner. Notably, we observed distinct characteristics of exosome-mediated protein secretion, including significantly lower capacity and different dynamics compared to the ER-Golgi pathway. This phenomenon was observed in human kidney 293T cells and liver HepG2 cells, emphasizing the conserved nature of exosome-mediated secretion across cell types. Furthermore, we investigated the impact of brefeldin A (BFA), an inhibitor of ER-to-Golgi membrane trafficking, on protein secretion. Interestingly, BFA inhibited protein secretion via the ER-Golgi pathway while stimulating exosome-mediated protein secretion under same experimental conditions. CONCLUSIONS: Collectively, our study highlights the utility of the dual-reporter system for real-time monitoring and quantitative analysis of protein secretion through conventional ER-Golgi and unconventional exosome pathways. Moreover, our findings unveil distinct features of exosome-mediated protein secretion, shedding light on its differential capacity, dynamics, and regulatory mechanisms compared to ER-Golgi-mediated proteins in human cells.