Cholesterol-Modified Anti-Il6 siRNA Reduces the Severity of Acute Lung Injury in Mice.

Small interfering RNA (siRNA) holds significant therapeutic potential by silencing target genes through RNA interference. Current clinical applications of siRNA have been primarily limited to liver diseases, while achievements in delivery methods are expanding their applications to various organs, including the lungs. Cholesterol-conjugated siRNA emerges as a promising delivery approach due to its low toxicity and high efficiency. This study focuses on developing a cholesterol-conjugated anti-Il6 siRNA and the evaluation of its potency for the potential treatment of inflammatory diseases using the example of acute lung injury (ALI). The biological activities of different Il6-targeted siRNAs containing chemical modifications were evaluated in J774 cells in vitro. The lead cholesterol-conjugated anti-Il6 siRNA after intranasal instillation demonstrated dose-dependent therapeutic effects in a mouse model of ALI induced by lipopolysaccharide (LPS). The treatment significantly reduced Il6 mRNA levels, inflammatory cell infiltration, and the severity of lung inflammation. IL6 silencing by cholesterol-conjugated siRNA proves to be a promising strategy for treating inflammatory diseases, with potential applications beyond the lungs.

Tumor- and Fibroblast-Derived Cell-Free DNAs Differently Affect the Progression of B16 Melanoma In Vitro and In Vivo.

It is widely postulated that the majority of pathologically elevated extracellular or cell-free DNA (cfDNA) in cancer originates from tumor cells; however, evidence has emerged regarding the significant contributions of other cells from the tumor microenvironment. Here, the effect of cfDNA originating from murine B16 melanoma cells and L929 fibroblasts on B16 cells was investigated. It was found that cfDNAL929 increased the viability and migration properties of B16 cells in vitro and their invasiveness in vivo. In contrast, cfDNAB16 exhibited a negative effect on B16 cells, reducing their viability and migration in vitro, which in vivo led to decreased tumor size and metastasis number. It was shown that cell treatment with both cfDNAs resulted in an increase in the expression of genes encoding DNases and the oncogenes Braf, Kras, and Myc. cfDNAL929-treated cells were shown to experience oxidative stress. Gene expression changes in the case of cfDNAB16 treatment are well correlated with the observed decrease in proliferation and migration of B16 cells. The obtained data may indicate the possible involvement of fibroblast DNA in the tumor microenvironment in tumor progression and, potentially, in the formation of new tumor foci due to the transformation of normal cells.

Cholesterol-Conjugated Supramolecular Multimeric siRNAs: Effect of siRNA Length on Accumulation and Silencing In Vitro and In Vivo.

Conjugation of small interfering RNA (siRNA) with lipophilic molecules is one of the most promising approaches for delivering siRNA in vivo. The rate of molecular weight-dependent siRNA renal clearance is critical for the efficiency of this process. In this study, we prepared cholesterol-containing supramolecular complexes containing from three to eight antisense strands and examined their accumulation and silencing activity in vitro and in vivo. We have shown for the first time that such complexes with 2'F, 2'OMe, and LNA modifications exhibit interfering activity both in carrier-mediated and carrier-free modes. Silencing data from a xenograft tumor model show that 4 days after intravenous injection of cholesterol-containing monomers and supramolecular trimers, the levels of MDR1 mRNA in the tumor decreased by 85% and 68%, respectively. The in vivo accumulation data demonstrated that the formation of supramolecular structures with three or four antisense strands enhanced their accumulation in the liver. After addition of two PS modifications at the ends of antisense strands, 47% and 67% reductions of Ttr mRNA levels in the liver tissue were detected 7 days after administration of monomers and supramolecular trimers, respectively. Thus, we have obtained a new type of RNAi inducer that is convenient for synthesis and provides opportunities for modifications.

Bronchial Asthma, Airway Remodeling and Lung Fibrosis as Successive Steps of One Process.

Bronchial asthma is a heterogeneous disease characterized by persistent respiratory system inflammation, airway hyperreactivity, and airflow obstruction. Airway remodeling, defined as changes in airway wall structure such as extensive epithelial damage, airway smooth muscle hypertrophy, collagen deposition, and subepithelial fibrosis, is a key feature of asthma. Lung fibrosis is a common occurrence in the pathogenesis of fatal and long-term asthma, and it is associated with disease severity and resistance to therapy. It can thus be regarded as an irreversible consequence of asthma-induced airway inflammation and remodeling. Asthma heterogeneity presents several diagnostic challenges, particularly in distinguishing between chronic asthma and other pulmonary diseases characterized by disruption of normal lung architecture and functions, such as chronic obstructive pulmonary disease. The search for instruments that can predict the development of irreversible structural changes in the lungs, such as chronic components of airway remodeling and fibrosis, is particularly difficult. To overcome these challenges, significant efforts are being directed toward the discovery and investigation of molecular characteristics and biomarkers capable of distinguishing between different types of asthma as well as between asthma and other pulmonary disorders with similar structural characteristics. The main features of bronchial asthma etiology, pathogenesis, and morphological characteristics as well as asthma-associated airway remodeling and lung fibrosis as successive stages of one process will be discussed in this review. The most common murine models and biomarkers of asthma progression and post-asthmatic fibrosis will also be covered. The molecular mechanisms and key cellular players of the asthmatic process described and systematized in this review are intended to help in the search for new molecular markers and promising therapeutic targets for asthma prediction and therapy.

The Effect of Cell-Free DNA from Blood Serum of Mice with Metastatic Melanoma on Enhancement of Oncogenic Properties of Melanoma Cells.

Currently, a significant increase in the levels of circulating cell-free DNA (cfDNA) in the blood of patients is considered as a generally recognized marker of the development of oncological diseases. Although the tumor-associated cfDNA has been well studied, its biological functions remain unclear. In this work, we investigated the effect of cfDNA isolated from the blood serum of the mice with B16-F10 metastatic melanoma on the properties of the B16-F10 melanoma cells in vitro. It was found that the profile of cfDNA isolated from the blood serum of mice with melanoma differs significantly from the cfDNA isolated from the blood serum of healthy mice, and is similar to the genomic DNA of B16 cells with regards to abundance of oncogenes and mobile genetic elements (MGE). It was shown that the cfDNA of mice with melanoma penetrated into B16 cells, resulting in the increase in abundance of oncogenes and MGE fragments, and caused 5-fold increase of the mRNA level of the secreted DNase Dnase1l3 and a slight increase of the mRNA level of the Jun, Fos, Ras, and Myc oncogenes. cfDNA of the healthy mice caused increase of the mRNA level of intracellular regulatory DNase EndoG and 4-fold increase of the mRNA level of Fos and Ras oncogenes, which are well-known triggers of a large number of signal cascades, from apoptosis inhibition to increased tumor cell proliferation. Thus, it is obvious that the circulating cfDNA of tumor origin is able to penetrate into the cells and, despite the fact that no changes were found in the level of viability and migration activity of the tumor cells, cfDNA, even with a single exposure, can cause changes at the cellular level that increase oncogenicity of the recipient cells.

Influence of the Composition of Cationic Liposomes on the Performance of Cargo Immunostimulatory RNA.

In this study, the impact of different delivery systems on the cytokine-inducing, antiproliferative, and antitumor activities of short immunostimulatory double-stranded RNA (isRNA) was investigated. The delivery systems, consisting of the polycationic amphiphile 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20 tetraazahexacosan tetrahydrochloride (2X3), and the lipid-helper dioleoylphosphatidylethanolamine (DOPE), were equipped with polyethylene glycol lipoconjugates differing in molecular weight and structure. The main findings of this work are as follows: (i) significant activation of MCP-1 and INF-α, ß, and γ production in CBA mice occurs under the action of isRNA complexes with liposomes containing lipoconjugates with long PEG chains, while activation of MCP-1 and INF-γ, but not INF-α or ß, was observed under the action of isRNA lipoplexes containing lipoconjugates with short PEG chains; (ii) a pronounced antiproliferative effect on B16 melanoma cells in vitro, as well as an antitumor and hepatoprotective effect in vivo, was induced by isRNA pre-complexes with non-pegylated liposomes, while complexes containing lipoconjugates with long-chain liposomes were inactive; (iii) the antitumor activity of isRNA correlated with the efficiency of its accumulation in the cells and did not explicitly depend on the activation of cytokine and interferon production. Thus, the structure of the delivery system plays a vital role in determining the response to isRNA and allows for the choice of a delivery system depending on the desired effect.

Identification of Novel Core Genes Involved in Malignant Transformation of Inflamed Colon Tissue Using a Computational Biology Approach and Verification in Murine Models.

Inflammatory bowel disease (IBD) is a complex and multifactorial systemic disorder of the gastrointestinal tract and is strongly associated with the development of colorectal cancer. Despite extensive studies of IBD pathogenesis, the molecular mechanism of colitis-driven tumorigenesis is not yet fully understood. In the current animal-based study, we report a comprehensive bioinformatics analysis of multiple transcriptomics datasets from the colon tissue of mice with acute colitis and colitis-associated cancer (CAC). We performed intersection of differentially expressed genes (DEGs), their functional annotation, reconstruction, and topology analysis of gene association networks, which, when combined with the text mining approach, revealed that a set of key overexpressed genes involved in the regulation of colitis (C3, Tyrobp, Mmp3, Mmp9, Timp1) and CAC (Timp1, Adam8, Mmp7, Mmp13) occupied hub positions within explored colitis- and CAC-related regulomes. Further validation of obtained data in murine models of dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS-stimulated CAC fully confirmed the association of revealed hub genes with inflammatory and malignant lesions of colon tissue and demonstrated that genes encoding matrix metalloproteinases (acute colitis: Mmp3, Mmp9; CAC: Mmp7, Mmp13) can be used as a novel prognostic signature for colorectal neoplasia in IBD. Finally, using publicly available transcriptomics data, translational bridge interconnecting of listed colitis/CAC-associated core genes with the pathogenesis of ulcerative colitis, Crohn's disease, and colorectal cancer in humans was identified. Taken together, a set of key genes playing a core function in colon inflammation and CAC was revealed, which can serve both as promising molecular markers and therapeutic targets to control IBD and IBD-associated colorectal neoplasia.

Protective effect of soloxolone derivatives in carrageenan- and LPS-driven acute inflammation: Pharmacological profiling and their effects on key inflammation-related processes.

The anti-inflammatory potential of three cyanoenone-containing triterpenoids, including soloxolone methyl (SM), soloxolone (S) and its novel derivative bearing at the C-30 amidoxime moiety (SAO), was studied in murine models of acute inflammation. It was found that the compounds effectively suppressed the development of carrageenan-induced paw edema and peritonitis as well as lipopolysaccharide (LPS)-driven acute lung injury (ALI) with therapeutic outcomes comparable with that of the reference drugs indomethacin and dexamethasone. Non-immunogenic carrageenan-stimulated inflammation was more sensitive to the transformation of C-30 of SM compared with immunogenic LPS-induced inflammation: the anti-inflammatory properties of the studied compounds against carrageenan-induced paw edema and peritonitis decreased in the order of SAO > S > > SM, whereas the efficiency of these triterpenoids against LPS-driven ALI was similar (SAO ≈ S ≈ SM). Further studies demonstrated that soloxolone derivatives significantly inhibited a range of immune-related processes, including granulocyte influx and the expression of key pro-inflammatory cytokines and chemokines in the inflamed sites as well as the functional activity of macrophages. Moreover, SM was found to prevent inflammation-associated apoptosis of A549 pneumocytes and effectively inhibited the protease activity of thrombin (IC50 = 10.3 µM) tightly associated with rodent inflammatome. Taken together, our findings demonstrate that soloxolone derivatives can be considered as novel promising anti-inflammatory drug candidates with multi-targeted mechanism of action.

siRNA-Mediated Timp1 Silencing Inhibited the Inflammatory Phenotype during Acute Lung Injury.

Acute lung injury is a complex cascade process that develops in response to various damaging factors, which can lead to acute respiratory distress syndrome. Within this study, based on bioinformatics reanalysis of available full-transcriptome data of acute lung injury induced in mice and humans by various factors, we selected a set of genes that could serve as good targets for suppressing inflammation in the lung tissue, evaluated their expression in the cells of different origins during LPS-induced inflammation, and chose the tissue inhibitor of metalloproteinase Timp1 as a promising target for suppressing inflammation. We designed an effective chemically modified anti-TIMP1 siRNA and showed that Timp1 silencing correlates with a decrease in the pro-inflammatory cytokine IL6 secretion in cultured macrophage cells and reduces the severity of LPS-induced acute lung injury in a mouse model.

Pulmonary Fibrosis as a Result of Acute Lung Inflammation: Molecular Mechanisms, Relevant In Vivo Models, Prognostic and Therapeutic Approaches.

Pulmonary fibrosis is a chronic progressive lung disease that steadily leads to lung architecture disruption and respiratory failure. The development of pulmonary fibrosis is mostly the result of previous acute lung inflammation, caused by a wide variety of etiological factors, not resolved over time and causing the deposition of fibrotic tissue in the lungs. Despite a long history of study and good coverage of the problem in the scientific literature, the effective therapeutic approaches for pulmonary fibrosis treatment are currently lacking. Thus, the study of the molecular mechanisms underlying the transition from acute lung inflammation to pulmonary fibrosis, and the search for new molecular markers and promising therapeutic targets to prevent pulmonary fibrosis development, remain highly relevant tasks. This review focuses on the etiology, pathogenesis, morphological characteristics and outcomes of acute lung inflammation as a precursor of pulmonary fibrosis; the pathomorphological changes in the lungs during fibrosis development; the known molecular mechanisms and key players of the signaling pathways mediating acute lung inflammation and pulmonary fibrosis, as well as the characteristics of the most common in vivo models of these processes. Moreover, the prognostic markers of acute lung injury severity and pulmonary fibrosis development as well as approved and potential therapeutic approaches suppressing the transition from acute lung inflammation to fibrosis are discussed.

Bovine Pancreatic RNase A: An Insight into the Mechanism of Antitumor Activity In Vitro and In Vivo.

In this investigation, we extensively studied the mechanism of antitumor activity of bovine pancreatic RNase A. Using confocal microscopy, we show that after RNase A penetration into HeLa and B16 cells, a part of the enzyme remains unbound with the ribonuclease inhibitor (RI), resulting in the decrease in cytosolic RNAs in both types of cells and rRNAs in the nucleoli of HeLa cells. Molecular docking indicates the ability of RNase A to form a complex with Ku70/Ku80 heterodimer, and microscopy data confirm its localization mostly inside the nucleus, which may underlie the mechanism of RNase A penetration into cells and its intracellular traffic. RNase A reduced migration and invasion of tumor cells in vitro. In vivo, in the metastatic model of melanoma, RNase A suppressed metastases in the lungs and changed the expression of EMT markers in the tissue adjacent to metastatic foci; this increased Cdh1 and decreased Tjp1, Fn and Vim, disrupting the favorable tumor microenvironment. A similar pattern was observed for all genes except for Fn in metastatic foci, indicating a decrease in the invasive potential of tumor cells. Bioinformatic analysis of RNase-A-susceptible miRNAs and their regulatory networks showed that the main processes modulated by RNase A in the tumor microenvironment are the regulation of cell adhesion and junction, cell cycle regulation and pathways associated with EMT and tumor progression.

Novel Epoxides of Soloxolone Methyl: An Effect of the Formation of Oxirane Ring and Stereoisomerism on Cytotoxic Profile, Anti-Metastatic and Anti-Inflammatory Activities In Vitro and In Vivo.

It is known that epoxide-bearing compounds display pronounced pharmacological activities, and the epoxidation of natural metabolites can be a promising strategy to improve their bioactivity. Here, we report the design, synthesis and evaluation of biological properties of αO-SM and ßO-SM, novel epoxides of soloxolone methyl (SM), a cyanoenone-bearing derivative of 18ßH-glycyrrhetinic acid. We demonstrated that the replacement of a double-bound within the cyanoenone pharmacophore group of SM with α- and ß-epoxide moieties did not abrogate the high antitumor and anti-inflammatory potentials of the triterpenoid. It was found that novel SM epoxides induced the death of tumor cells at low micromolar concentrations (IC50(24h) = 0.7-4.1 µM) via the induction of mitochondrial-mediated apoptosis, reinforced intracellular accumulation of doxorubicin in B16 melanoma cells, probably by direct interaction with key drug efflux pumps (P-glycoprotein, MRP1, MXR1), and the suppressed pro-metastatic phenotype of B16 cells, effectively inhibiting their metastasis in a murine model. Moreover, αO-SM and ßO-SM hampered macrophage functionality in vitro (motility, NO production) and significantly suppressed carrageenan-induced peritonitis in vivo. Furthermore, the effect of the stereoisomerism of SM epoxides on the mentioned bioactivities and toxic profiles of these compounds in vivo were evaluated. Considering the comparable antitumor and anti-inflammatory effects of SM epoxides with SM and reference drugs (dacarbazine, dexamethasone), αO-SM and ßO-SM can be considered novel promising antitumor and anti-inflammatory drug candidates.

Asthma and Post-Asthmatic Fibrosis: A Search for New Promising Molecular Markers of Transition from Acute Inflammation to Pulmonary Fibrosis.

Asthma is a heterogeneous pulmonary disorder, the progression and chronization of which leads to airway remodeling and fibrogenesis. To understand the molecular mechanisms of pulmonary fibrosis development, key genes forming the asthma-specific regulome and involved in lung fibrosis formation were revealed using a comprehensive bioinformatics analysis. The bioinformatics data were validated using a murine model of ovalbumin (OVA)-induced asthma and post-asthmatic fibrosis. The performed analysis revealed a range of well-known pro-fibrotic markers (Cat, Ccl2, Ccl4, Ccr2, Col1a1, Cxcl12, Igf1, Muc5ac/Muc5b, Spp1, Timp1) and a set of novel genes (C3, C3ar1, Col4a1, Col4a2, Cyp2e1, Fn1, Thbs1, Tyrobp) mediating fibrotic changes in lungs already at the stage of acute/subacute asthma-driven inflammation. The validation of genes related to non-allergic bleomycin-induced pulmonary fibrosis on asthmatic/fibrotic lungs allowed us to identify new universal genes (Col4a1 and Col4a2) associated with the development of lung fibrosis regardless of its etiology. The similarities revealed in the expression profiles of nodal fibrotic genes between asthma-driven fibrosis in mice and nascent idiopathic pulmonary fibrosis in humans suggest a tight association of identified genes with the early stages of airway remodeling and can be considered as promising predictors and early markers of pulmonary fibrosis.

Core genes involved in the regulation of acute lung injury and their association with COVID-19 and tumor progression: A bioinformatics and experimental study.

Acute lung injury (ALI) is a specific form of lung damage caused by different infectious and non-infectious agents, including SARS-CoV-2, leading to severe respiratory and systemic inflammation. To gain deeper insight into the molecular mechanisms behind ALI and to identify core elements of the regulatory network associated with this pathology, key genes involved in the regulation of the acute lung inflammatory response (Il6, Ccl2, Cat, Serpine1, Eln, Timp1, Ptx3, Socs3) were revealed using comprehensive bioinformatics analysis of whole-genome microarray datasets, functional annotation of differentially expressed genes (DEGs), reconstruction of protein-protein interaction networks and text mining. The bioinformatics data were validated using a murine model of LPS-induced ALI; changes in the gene expression patterns were assessed during ALI progression and prevention by anti-inflammatory therapy with dexamethasone and the semisynthetic triterpenoid soloxolone methyl (SM), two agents with different mechanisms of action. Analysis showed that 7 of 8 revealed ALI-related genes were susceptible to LPS challenge (up-regulation: Il6, Ccl2, Cat, Serpine1, Eln, Timp1, Socs3; down-regulation: Cat) and their expression was reversed by the pre-treatment of mice with both anti-inflammatory agents. Furthermore, ALI-associated nodal genes were analysed with respect to SARS-CoV-2 infection and lung cancers. The overlap with DEGs identified in postmortem lung tissues from COVID-19 patients revealed genes (Saa1, Rsad2, Ifi44, Rtp4, Mmp8) that (a) showed a high degree centrality in the COVID-19-related regulatory network, (b) were up-regulated in murine lungs after LPS administration, and (c) were susceptible to anti-inflammatory therapy. Analysis of ALI-associated key genes using The Cancer Genome Atlas showed their correlation with poor survival in patients with lung neoplasias (Ptx3, Timp1, Serpine1, Plaur). Taken together, a number of key genes playing a core function in the regulation of lung inflammation were found, which can serve both as promising therapeutic targets and molecular markers to control lung ailments, including COVID-19-associated ALI.

Mesyl phosphoramidate backbone modified antisense oligonucleotides targeting miR-21 with enhanced in vivo therapeutic potency.

The design of modified oligonucleotides that combine in one molecule several therapeutically beneficial properties still poses a major challenge. Recently a new type of modified mesyl phosphoramidate (or µ-) oligonucleotide was described that demonstrates high affinity to RNA, exceptional nuclease resistance, efficient recruitment of RNase H, and potent inhibition of key carcinogenesis processes in vitro. Herein, using a xenograft mouse tumor model, it was demonstrated that microRNA miR-21-targeted µ-oligonucleotides administered in complex with folate-containing liposomes dramatically inhibit primary tumor growth via long-term down-regulation of miR-21 in tumors and increase in biosynthesis of miR-21-regulated tumor suppressor proteins. This antitumoral effect is superior to the effect of the corresponding phosphorothioate. Peritumoral administration of µ-oligonucleotide results in its rapid distribution and efficient accumulation in the tumor. Blood biochemistry and morphometric studies of internal organs revealed no pronounced toxicity of µ-oligonucleotides. This new oligonucleotide class provides a powerful tool for antisense technology.

Antitumour Activity of the Ribonuclease Binase from Bacillus pumilus in the RLS40 Tumour Model Is Associated with the Reorganisation of the miRNA Network and Reversion of Cancer-Related Cascades to Normal Functioning.

The important role of miRNA in cell proliferation and differentiation has raised interest in exogenous ribonucleases (RNases) as tools to control tumour-associated intracellular and extracellular miRNAs. In this work, we evaluated the effects of the RNase binase from Bacillus pumilus on small non-coding regulatory RNAs in the context of mouse RLS40 lymphosarcoma inhibition. In vitro binase exhibited cytotoxicity towards RLS40 cells via apoptosis induction through caspase-3/caspase-7 activation and decreased the levels of miR-21a, let-7g, miR-31 and miR-155. Intraperitoneal injections of binase in RLS40-bearing mice resulted in the retardation of primary tumour growth by up to 60% and inhibition of metastasis in the liver by up to 86%, with a decrease in reactive inflammatory infiltration and mitosis in tumour tissue. In the blood serum of binase-treated mice, decreases in the levels of most studied miRNAs were observed, excluding let-7g, while in tumour tissue, the levels of oncomirs miR-21, miR-10b, miR-31 and miR-155, and the oncosuppressor let-7g, were upregulated. Analysis of binase-susceptible miRNAs and their regulatory networks showed that the main modulated events were transcription and translation control, the cell cycle, cell proliferation, adhesion and invasion, apoptosis and autophagy, as well as some other tumour-related cascades, with an impact on the observed antitumour effects.

Immunostimulating RNA Delivered by P1500 PEGylated Cationic Liposomes Limits Influenza Infection in C57Bl/6 Mice.

The emergence of highly pathogenic viruses and a high speed of infection spread put forward the problem of the development of novel antivirals and their delivery vehicles. In this study, we investigated the antiviral effect of the previously identified immunostimulatory 19-bp dsRNA (isRNA) with 3'-nucleotide overhangs, which stimulates interferon α synthesis when delivered using cationic liposomes consisting of 1,26-bis(cholest-5-en-3ß-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride and lipid-helper dioleoylphosphatidylethanolamine and its PEGylated formulation P1500 in vitro and in vivo. In vitro data showed that isRNA/2X3-DOPE complexes protected L929 cells from encephalomyocarditis virus infection, while isRNA/P1500 complexes were not active, which correlates with their lower transfection activity in cell culture. Comparison of the interferon-inducing activity of isRNA in BALB/c, CBA and C57Bl/6 mice showed that PEGylated liposomes significantly enhance the interferon-inducing activity of isRNA in vivo. The antiviral efficacy of the isRNA in vivo was considerably affected by the delivery system. The cationic liposomes 2X3-DOPE did not enhance the antiviral properties of isRNA in vivo. Similar liposomes equipped with a PEGylated lipoconjugate provided a pronounced anti-influenza effect of the isRNA in vivo. Administration of isRNA to C57Bl/6 led to a decrease in virus titers in the lungs and a significant decrease in the severity of the infection. Administration of a similar formulation to BALB/c mice caused only a mild antiviral effect at the initial stages of the infection. The data show that isRNA in combination with the PEGylated delivery system can be considered an effective means of suppressing influenza A infection.