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The emergence of new influenza virus strains presents a continuous challenge for global public health. mRNA technology offers a promising platform for rapidly developing therapeutics, particularly monoclonal antibodies, that can protect against viral infections. In this study, we engineered mRNA constructs encoding two types of antibodies: secreted antibodies specific to the hemagglutinin of the influenza A virus, based on previously characterized Fi6 antibodies, and intracellular Fab fragments targeting the nucleoprotein of the influenza B virus, derived from the 2/3 antibodies. The administration of mRNA constructs in vitro resulted in the successful synthesis of functional antibodies, which exhibited antiviral activity against influenza viruses. This study confirms the feasibility of using mRNA technology to develop therapeutic antibodies against influenza virus infections. The findings pave the way for future clinical applications of mRNA-based therapeutics, enhancing preparedness for emerging viral threats., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Oral delivery of small interfering RNAs (siRNAs) draws significant attention, but the gastrointestinal tract (GIT) has many biological barriers that limit the drugs' bioavailability. The aim of this work was to investigate the potential of micro- and nano-sized CaCO 3 and PLA carriers for oral delivery of siRNA and reveal a relationship between the physicochemical features of these carriers and their biodistribution., Research Design and Methods: In vitro stability of carriers was investigated in simulated gastric and intestinal fluids. Toxicity and cellular uptake were investigated on Caco-2 cells. The biodistribution profiles of the developed CaCO 3 and PLA carriers were examined using different visualization methods, including SPECT, fluorescence imaging, radiometry, and histological analysis. The delivery efficiency of siRNA loaded carriers was investigated both in vitro and in vivo ., Results: Micro-sized carriers were accumulated in the stomach and later localized in the colon tissues. The nanoscale particles (100-250 nm) were distributed in the colon tissues. nPLA was also detected in small intestine. The developed carriers can prevent siRNA from premature degradation in GIT media., Conclusion: Our results reveal how the physicochemical properties of the particles, including their size and material type can affect their biodistribution profile and oral delivery of siRNA.

In the present study, a highly effective carrier system has been developed for the delivery of antiviral siRNA mixtures. The developed hybrid microcarriers, made of biodegradable polymers and SiO 2 nanostructures, more efficiently mediate cellular uptake of siRNA than commercially available liposome-based reagents and polyethyleneimine (PEI); they also demonstrate low in vitro toxicity and protection of siRNA from RNase degradation. A series of siRNA designs (targeting the most conserved regions of three influenza A virus (IAV) genes: NP, NS, and PA) were screened in vitro using RT-qPCR, ELISA analysis, and hemagglutination assay. Based on the results of screening, the three most effective siRNAs (PA-1630, NP-717, and NS-777) were selected for in situ encapsulation into hybrid microcarriers. It was revealed that pre-treatment of cells with a mixture of PA-1630, NP-717, and NS-777 siRNAs, delivered by hybrid microcarriers, provided stronger inhibition of viral M1 mRNA expression and control of NP protein level, after viral infection, than single pre-treatment by any of three encapsulated siRNAs used in the study. Moreover, the effective inhibition of replication in several IAV subtypes (H1N1, H1N1pdm, H5N2, and H7N9) using a cocktail of the three selected siRNAs, delivered by our hybrid capsules to the cells, was achieved. In conclusion, we have developed a proof-of-principle which shows that our hybrid microcarrier technology (utilizing a therapeutic siRNA cocktail) may represent a promising approach in anti-influenza therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

The diagnostic oligonucleotide microarray for subtyping of human and animal influenza A viruses (IAVs) was developed. We proposed a simple method of the fluorescent labeling of genomic segments of all known IAVs subtypes, the composition of the hybridization buffer, as well as the software of the data processing. 48 IAVs strains of different subtypes were analyzed using our microarray. All of them were identified, while 45 of 48 strains were unambiguously subtyped.