A novel multitargeted self-assembling peptide-siRNA complex for simultaneous inhibition of SARS-CoV-2-host cell interaction and replication.

Effective therapeutics are necessary for managing severe COVID-19 disease despite the availability of vaccines. Small interfering RNA (siRNA) can silence viral genes and restrict SARS-CoV-2 replication. Cell-penetrating peptides is a robust method for siRNA delivery, enhancing siRNA stability and targeting specific receptors. We developed a peptide HE25 that blocks SARS-CoV-2 replication by various mechanisms, including the binding of multiple receptors involved in the virus's internalization, such as ACE2, integrins and NRP1. HE25 not only acts as a vehicle to deliver the SARS-CoV-2 RNA-dependent RNA polymerase siRNA into cells but also facilitates their internalization through endocytosis. Once inside endosomes, the siRNA is released into the cytoplasm through the Histidine-proton sponge effect and the selective cleavage of HE25 by cathepsin B. These mechanisms effectively inhibited the replication of the ancestral SARS-CoV-2 and the Omicron variant BA.5 in vitro. When HE25 was administered in vivo, either by intravenous injection or inhalation, it accumulated in lungs, veins and arteries, endothelium, or bronchial structure depending on the route. Furthermore, the siRNA/HE25 complex caused gene silencing in lung cells in vitro. The SARS-CoV-2 siRNA/HE25 complex is a promising therapeutic for COVID-19, and a similar strategy can be employed to combat future emerging viral diseases.

Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification.

Synthetic nanoparticles as lipid nanoparticles (LNPs) are widely used as drug delivery vesicles. However, they hold several drawbacks, including low biocompatibility and unfavorable immune responses. Naturally occurring extracellular vesicles (EVs) hold the potential as native, safe, and multifunctional nanovesicle carriers. However, loading of EVs with large biomolecules remains a challenge. Here, we present a controlled loading methodology using DNA-mediated and programmed fusion between EVs and messenger RNA (mRNA)-loaded liposomes. The fusion efficiency is characterized at the single-particle level by real-time microscopy through EV surface immobilization via lipidated biotin-DNA handles. Subsequently, fused EV-liposome particles (EVLs) can be collected by employing a DNA strand-replacement reaction. Transferring the fusion reaction to magnetic beads enables us to scale up the production of EVLs one million times. Finally, we demonstrated encapsulation of mCherry mRNA, transfection, and improved translation using the EVLs compared to liposomes or LNPs in HEK293-H cells. We envision this as an important tool for the EV-mediated delivery of RNA therapeutics.

Mitochondrial reactive oxygen species modify extracellular vesicles secretion rate.

Extracellular vesicle (EV) secretion rate is stimulated by hypoxia that causes increased reactive oxygen species (ROS) production by the mitochondrial electron transport chain (ETC) and hypoxia-induced factor (HIF)-1 signaling; however, their contribution to the increased EV secretion rate is unknown. We found that the EV marker secretion rate in our EV reporter cell line CD9truc-EGFP was unaffected by the HIF-1α stabilizer roxadustat; yet, ETC stimulation by dichloroacetic acid (DCA) significantly increased EV secretion. The DCA-induced EV secretion was blocked by the antioxidant TEMPO and rotenone, an inhibitor of the ETC's Complex I. Under hypoxic conditions, the limited oxygen reduction impedes the ETC's Complex III. To mimic this, we inhibited Complex III with antimycin A, which increased ROS-dependent EV secretion. The electron transport between Complex I and III is accomplished by coenzyme Q created by the mevalonate pathway and tyrosine metabolites. Blocking an early step in the mevalonate pathway using pitavastatin augmented the DCA-induced EV secretion, and 4-nitrobenzoate-an inhibitor of the condensation of the mevalonate pathway with tyrosine metabolites-increased ROS-dependent EV secretion. Our findings indicate that hypoxia-mimetics targeting the ETC modify EV secretion and that ROS produced by the ETC is a potent stimulus for EV secretion.