Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation

Abstract

Exosomes are attractive nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and of their ability to penetrate physiological barriers that are impermissible to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs (mRNAs), into cell-secreted exosomes leads to low yields. Here, we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs, and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides. Compared to bulk electroporation and to other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and more than a 10 3 -fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic PTEN-deficient glioma mouse models, mRNA-containing exosomes restored tumour-suppressor function, enhanced tumourgrowth inhibition, and increased animal survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.

Ral GTPases promote metastasis by controlling biogenesis and organ colonization of exosomes

“…Indeed, the absence of Ral (in both nematodes and mice), or the inhibition of PLD1 in 4T1 cells, drastically reduces the number of MVBs, which is accompanied by a decrease in secreted EVs. Similarly, a direct correlation between MVB density and levels of secreted EVs was recently suggested by studies showing that chemical or electric stimulation of MVB biogenesis results in increased EV secretion (Kanemoto et al, 2016;Yang et al, 2020). The formation of MVBs results from dramatic biochemical transformations of endosomes involving multiple protein and lipid switches (Huotari and Helenius, 2011;Scott et al, 2014).…”

Bone-targeted extracellular vesicles from mesenchymal stem cells for osteoporosis therapy

“…At last, Ale-EVs could be used as a bone-target delivery system. We can enrich MMSCsderived EVs with mRNAs or proteins that used to treat osteoporosis by genetic engineering technology [34][35][36], and strengthen the anti-osteoporosis effect of Ale-EVs. Besides, we also could load other drugs such as nucleic acid drugs [32,37], small molecule drugs [38] into the target-delivery system to achieve the purpose of combined treatment of osteoporosis…”

Extracellular Vesicles Function as Bioactive Molecular Transmitters in the Mammalian Oviduct: An Inspiration for Optimizing in Vitro Culture Systems and Improving Delivery of Exogenous Nucleic Acids during Preimplantation Embryonic Development

“…It is noteworthy that cellular nanoporation biochips have now been developed, enabling the large-scale generation of functional mRNA-encapsulating exosomes. Yang et al [115] cultured monolayers of source cells over the chip, which contained an array of nano-channels. These were subjected to transient electrical pulses that shuttled DNA plasmids from the buffer into the attached cells.…”