Exosomes are natural nanocontainers actively secreted by the body’s cells and transmitting molecular signals of various types to recipient cells. Cellular mechanisms of exosomes’ biogenesis involve specific sorting of RNA for incorporation into them. As a result, the molecular composition of exosomes is closely related to the donor cell’s functional state, and this makes exosomes an important diagnostic and prognostic marker in a number of diseases (primarily oncological). The ability of exosomes to transport biologically active molecules and to protect the cargo from degradation makes them nearly ideal candidates as delivery carriers of RNA in therapeutic or prophylactic regimes. Potential of exosomal surface functionalization enables improved targeting to specific organs, tissues and cells. However, the development of an effective technology for RNA’s loading into exosomes cannot be considered resolved. This review is focused on experimental data on the use of exosomes as vehicles for the delivery of therapeutic and prophylactic RNAs. We briefly consider the biogenesis and functions of exosomes, focusing on those biological properties that make them formidable candidates in the race to develop effective delivery carriers. Furthermore, we describe various techniques of cargo loading into exosomes. Prospects of exosomes application as therapeutic delivery system for siRNAs, miRNAs, and long RNAs are considered.

In this paper, we compared the transfection efficiency and cytotoxicity of methylglycol-chitosan (MG-CS) and diethylaminoethyl-chitosan (DEAE-CS_I and DEAE-CS_II with degrees of substitution of 1.2 and 0.57, respectively) to that of Lipofectamine (used as a reference transfection vector). MG-CS contains quaternary amines to improve DNA binding, whereas the DEAE-CS exhibits pH buffering capability that would ostensibly enhance transfection efficiency by promoting endosomal escape. Gel retardation assays showed that both DEAE-CS and MG-CS bound to DNA at a polysaccharide:DNA mass ratio of 2:1. In Calu-3 cells, the DNA transfection activity was significantly better with MG-CS than with DEAE-CS, and the efficiency improved with increasing polysaccharide:DNA ratios. By contrast, the efficiency of DEAE-CS_I and DEAE-CS_II was independent of the polysaccharide:DNA ratio. Conversely, in the transfection-recalcitrant JAWSII cells, both Lipofectamine and MG-CS showed significantly lower DNA transfection activity than in Calu-3 cells, whereas the efficiency of DEAE-CS_I and DEAE-CS_II was similar in both cell lines. The toxicity of DEAE-CS increased with increasing concentrations of the polymer and its degree of substitution, whereas MG-CS demonstrated negligible cytotoxicity, even at the highest concentration studied. Overall, MG-CS proved to be a more efficient and less toxic transfection agent when compared to DEAE-CS.

Secretion of extracellular vesicles (EVs) is a fundamental property of living cells. EVs are known to transfer biological signals between cells and thus regulate the functional state of recipient cells. Such vesicles mediate the intercellular transport of many biologically active molecules (proteins, nucleic acids, specific lipids) and participate in regulation of key physiological processes. In addition, EVs are involved in the pathogenesis of multiple diseases: infectious, neurodegenerative, and oncological. The current EV classification into microvesicles, apoptotic bodies, and exosomes is based on their size, pathways of cellular biogenesis, and molecular composition. This review is focused on analysis of the role of EVs (mainly exosomes) in the pathogenesis of viral infection. We briefly characterize the biogenesis and molecular composition of various EV types. Then, we consider EV-mediated pro- and anti-viral mechanisms. EV secretion by infected cells can be an important factor of virus spread in target cell populations, or a protective factor limiting viral invasion. The data discussed in this review, on the effect of EV secretion by infected cells on processes in neighboring cells and on immune cells, are of high significance in the search for new therapeutic approaches and for design of new generations of vaccines.

Background: Exosomes are involved in intercellular communication and can transfer regulatory molecules between cells. Consequently, they can participate in host immune response regulation. For the influenza A virus (IAV), there is very limited information on changes in exosome composition during cell infection shedding light on the potential role of these extracellular membrane vesicles. Thus, the aim of our work was to study changes in exosomal composition following IAV infection of cells, as well as to evaluate their effect on uninfected cells. Methods: To characterize changes in the composition of cellular miRNAs and mRNAs of exosomes during IAV infection of A549 cells, NGS was used, as well as PCR to identify viral genes. Naïve A549 cells were stimulated with infected-cell-secreted exosomes for studying their activity. Changes in the expression of genes associated with the cell’s immune response were shown using PCR. The effect of exosomes on IAV replication was shown in MDCK cells using In-Cell ELISA and PCR of the supernatants. Results: A change in the miRNA composition (miR-21-3p, miR-26a-5p, miR-23a-5p, miR-548c-5p) and mRNA composition (RPL13A, MKNK2, TRIB3) of exosomes under the influence of the IAV was shown. Many RNAs were involved in the regulation of the immune response of the cell, mainly by suppressing it. After exosome stimulation of naïve cells, a significant decrease in the expression of genes involved in the immune response was shown (RIG1, IFIT1, MDA5, COX2, NFκB, AnxA1, PKR, IL6, IL18). When infecting MDCK cells, a significant decrease in nucleoprotein levels was observed in the presence of exosomes secreted by mock-infected cells. Viral levels in supernatants also decreased. Conclusions: Exosomes secreted by IAV-infected cells could reduce the immune response of neighboring intact cells, leading to more effective IAV replication. This may be associated both with regulatory functions of cellular miRNAs and mRNAs carried by exosomes, or with the presence of viral mRNAs encoding proteins with an immunosuppressive function.

Influenza A virus (IAV) is a segmented negative-sense RNA virus that causes seasonal epidemics and periodic pandemics in humans. Two regions (nucleotide positions 82–148 and 497–564) in the positive-sense RNA of the NS segment fold into a multi-branch loop or hairpin structures. We studied 25,384 NS segment positive-sense RNA unique sequences of human and non-human IAVs in order to predict secondary RNA structures of the 82–148 and 497–564 regions using RNAfold software, and determined their host- and lineage-specific distributions. Hairpins prevailed in avian and avian-origin human IAVs, including H1N1pdm1918 and H5N1. In human and swine IAV hairpins distribution varied between evolutionary lineages. These results suggest a possible functional role for these RNA secondary structures and the need for experimental evaluation of these structures in the influenza life cycle.