Abstract
Exosomes represent a promising delivery tool for nucleic acid-based pharmaceuticals. They are highly suitable for transporting therapeutic miRNAs to tumor cells, due to their natural membrane components. Further, exosomes are capable of effectively protecting nucleic acids against ribonucleases and enable the delivery of their content through cell membranes. However, no suitable production host for miRNA containing exosomes of non-tumorigenic origin has yet been identified. In this study we engineered an immortalised human amniocyte cell line (CAP® cells), whose exosomes were enriched and characterised. The cell line modifications not only enabled the production of GFP-labelled but also pro-apoptotic miRNA containing exosomes without negative influence on host cell growth. Furthermore, we demonstrated that pro-apoptotic miRNA containing CAP exosomes are taken up by ovarian cancer cells. Strikingly, delivery of functional exosomal miRNA led to downregulation of several reported target genes in the treated tumor cells. In summary, we revealed CAP cells of non-tumorigenic origin as a novel and efficient exosome production host with the potential to produce functional miRNA-loaded exosomes.
Highlights
Exosomes are small membrane vesicles of 50–150 nm in size, which originate from the endosomal pathway by fusion of intracellular multivesicular bodies (MVB) with the plasma membrane and are released into the extracellular space [1,2]
For the evaluation of CAP cells as production hosts for exosomes, the parental CAP cell line was engineered by using the pStbl-bsd-CMV-CD63-tGFP vector to overexpress the tetraspanin CD63 fused to GFP
The respective genomic human pre-miRNA sequences were PCR amplified from isolated CAP cell genomic DNA, subcloned into the miRNASelectTM pEGP-miR cloning and expression vector and used for the generation of CAP cell lines overexpressing the respective miRNA
Summary
Exosomes are small membrane vesicles of 50–150 nm in size, which originate from the endosomal pathway by fusion of intracellular multivesicular bodies (MVB) with the plasma membrane and are released into the extracellular space [1,2]. In order to exert their functions, exosomes can fuse with the plasma membrane of a recipient cell to release their content into the cytosol, undergo endocytosis or bind to membrane receptors to activate signalling pathways [9,10].
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