The development and application of therapeutic oligonucleotides, such as siRNA, miRNA, ASOs and aptamers, is a rapidly growing field in biomedicine. These molecules are undergoing extensive preclinical and clinical testing, and the market for synthetic RNA drugs is expanding. However, several challenges remain, including targeted delivery and high costs associated with development, screening and production. One significant advance has been the creation of GalNAc-conjugates, which selectively target ASGPR and deliver oligonucleotides to hepatocytes. Although these conjugates have shown promising results, their widespread use is limited by the lack of effective synthesis methods. Thus, the development of new methods for the synthesis of ligand-oligonucleotide conjugates is an important task to which this study is devoted. In this study, we created a library of siRNA conjugates with the GalNAc L-96 ligand to suppress the expression of the PCSK9 gene associated with elevated LDL and an increased risk of developing cardiovascular diseases. The selection of the most effective siRNA molecules was carried out using an algorithm previously developed by our research group, which considers thermodynamic stability, predicted specificity and effectiveness. To experimentally confirm the effectiveness of conjugates, an in vitro model based on the cultivation of hepatocyte cells was developed. Optimization of the conjugate synthesis process has significantly reduced the cost of manufacturing technology, which creates the potential for efficient scaling of synthesis for transfer and application in the pharmaceutical industry. The results of the study showed that the development of the siRNA sequence optimized in silico resulted in a significant increase in the inhibitory effect of the GalNAc-siRNA conjugate compared to a compound similar to a commercial drug.

BACKGROUND:We have recently reported the preclinical characterization of a recombinant birch pollen allergy vaccine based on a fusion protein, AB-PreS, consisting of allergen-derived peptides fused to the hepatitis B virus (HBV)-derived PreS surface protein as immunological carrier. OBJECTIVE:To investigate in a clinical study the usefulness of the AB-PreS vaccine, termed GNR-127, for allergen-specific immunotherapy of birch pollen allergy. METHODS:Stage 1 of the study, an open-label Phase I study, determined among three doses (20 μg: n = 6; 40 μg: n = 6; 80 μg: n = 6) administered as Aluminum-hydroxide-adsorbed vaccines subcutaneously five times in monthly intervals, the highest tolerated doses. In the following Stage 2, randomized patients (placebo: n = 45; 40 μg: n = 43; 80 μg: n = 44) received in a single-blind, placebo-controlled Phase II study monthly up to five pre-seasonal injections. Safety monitoring followed the Medical Dictionary for Regulatory Activities (MedDRA); efficacy parameters were determined according to the European Academy for Allergy and Clinical Immunology (EAACI) guidelines; allergen-specific antibodies and basophil sensitivity were determined by ELISA and basophil activation testing, respectively. RESULTS:A dose-dependent reduction of the combined symptom medication score (CSMS) (40 μg: 17.41%; 80 μg: 24.13%) over placebo was observed which was significant for the 80 μg group and accompanied by the strongest induction of Bet v 1-specific IgG (median 20-fold increase) and a blunting of the seasonally boosted IgE response and basophil sensitivity. The vaccine showed an acceptable safety profile despite high administered doses. CONCLUSION:A pre-seasonal course of 5 monthly injections of GNR-127 80 μg improved symptoms of birch pollen allergy accompanied by a robust Bet v 1-specific IgG response, paving the road for a Phase III efficacy study. TRIAL REGISTRATION:ClinicalTrials.gov (Identifier: NCT07155499).

AIMS: To evaluate the antiallergic activity of an aqueous dispersion of fullerene C60 (ADF) and its safety in preclinical studies. MATERIALS AND METHODS: Food allergy was induced in BALB/c mice by ovalbumin. ADF was administered intragastrically in different regimens. Specific IgE, Th2 cytokines, body weight, diarrhea, and jejunal histology were assessed. Allergenicity was tested in mice and guinea pigs using standard hypersensitivity assays, and immunotoxicity was evaluated in humoral, cellular, and phagocytic immunity models. RESULTS: ADF reduced OVA-specific IgE, IL-4 and IL-5, alleviated diarrhea, and supported weight gain. Histology showed decreased eosinophilia in the jejunum. No allergenic or immunotoxic effects were detected in preclinical tests.

In recent years, understanding of the key role of epithelial cytokines (IL-25, IL-33, and TSLP) in the development of respiratory tract inflammatory diseases has significantly expanded. These molecules, released by airway epithelium in response to allergens, pathogens, and damaging agents, act as alarmins, initiating a cascade of the Th2-mediated inflammation that leads to the IgE-mediated reactions, eosinophilic infiltration, and tissue remodeling. Beyond classical allergic processes, epithelial cytokines play a crucial role in the virus-induced exacerbations of respiratory diseases and pathogenesis of chronic obstructive pulmonary disease (COPD). Monoclonal antibody therapeutics targeting these cytokines have demonstrated efficacy in treating severe asthma. This review summarizes current data on contribution of TSLP, IL-33, and IL-25 to the respiratory tract inflammation and discusses the prospects of their targeted inhibition to improve therapeutic efficacy.

Every year, the scientific community continues to drive advances in healthcare, opening up new perspectives in the treatment and management of various diseases. Despite vast strides being made in the quality of life and longevity, we still face an equally significant growth in the burden of oncological pathologies. Although current trends lean towards preventive and personalized medicine, numerous hurdles remain to be cleared to develop robust strategies in the field of oncology. Among all types of tumors, one of the prominent positions is occupied by hepatocellular carcinoma (HCC), which is one of the most widespread primary cancers with a high mortality rate. Conventional approaches to HCC therapy, such as surgery or chemotherapy, rarely provide steady performance due to the highly polymorphous nature of the cancerous process. In this study, we suggest an alternative methodological framework for designing potent siRNAs targeting genes implicated in hepatocellular carcinoma, implementing RNA interference mediated by synthetic small interfering RNAs (siRNAs) against mRNAs of ITGB1 and CD47 genes. Products of these genes are renowned drivers of tumor progression. We have developed a software algorithm for the design of unmodified and modified siRNAs, carried out solid-phase synthesis of the most promising molecules, and proved their capability to perform a more than 50-fold suppression of expression of the target genes in vitro.

Th2 cytokines (IL-4, IL-5, and IL-13) play an important role in the development of allergies, including allergic rhinitis (AR). IL-13 promotes mucus hyperproduction in the airway and IL-5 recruits eosinophils to the nasal mucosa, leading to increased inflammation and tissue damage. Drugs based on monoclonal antibodies that block the activity of these cytokines are being developed for the treatment of allergic diseases. However, studies of drugs that target IL-13 alone (such as Tralokinumab and Lebrikizumab) were not successful. Given that IL-5 and IL-13 have different roles in AR, simultaneous inhibition of both cytokines may be a promising approach. New methods of regulating gene activity, such as RNA interference (RNAi), offer new perspectives for the development of drugs. This study describes a complex consisting of siRNA that inhibit the activity of Il5 and Il13 genes and a currier peptide LTP (cationic dendrimeric peptide). The effects of this complex on the allergic inflammation in the murine AR model was studied. Suppression of Il5 expression decreased nasal hyperreactivity and reduced the number of goblet cells in the respiratory epithelium of AR-induced mice. Inhibiting the Il13 gene had a more beneficial effect than suppression Il5 alone, further contributing to reducing the number of cells infiltration the nasal cavity. When both Il5 and Il13 were suppressed simultaneously, the result was similar that of Il13 inhibition alone. Likely, IL-13 plays a more significant role in the development of allergic rhinitis than IL-5. As a result, the possibility of using RNAi for anti-cytokine therapy for AR has been demonstrated. However, dual inactivation of IL-5 and IL-13 by siRNA does not provide any advantages over inactivating IL-13 alone in the current mouse model of AR. However, the lack of success of anti-IL-13 therapy in clinical practice indicates the promise of an approach based on the dual blocking of IL-5 and IL-13.

BACKGROUND: The incidence of food allergy in adults and children is very high. The key approach in the therapy of food allergy can be quality drug selection. New synthetic derivatives of fullerene, a high-tech hydrocarbon, can become such drugs. A unique biocompatible method for obtaining a stable aqueous dispersion of fullerene C60 (ADF-C60) as the main component for drugs for the treatment of allergic diseases has been developed. In this article, reproductive toxicity of the drug ADF-C60, currently in preclinical safety studies, was studied in female white outbred rats. Due to its properties, the ADF-C60 is promising for treatment of food allergy and can presumably be used during pregnancy. AIM: Preclinical study of reproductive toxicity of an ADF-C60 in the antenatal and postnatal periods of rat offspring development with oral administration to the mother generation during pregnancy. MATERIALS AND METHODS: Female rats were orally administered the ADF-C60 in 1-, 5-, and 50-fold therapeutic doses recommended for humans during the whole pregnancy. The general parameters of embryotoxic and teratogenic effects of the ADF-C60 before and after birth were studied. RESULTS: No evidence of embryotoxic and teratogenic effects of the ADF-C60 was found during the study of gestational parameters and general fetuses development. No adverse effects of the ADF-C60 were revealed during observation of postnatal development of offspring. CONCLUSION: ADF-C60 for the treatment of food allergy has no reproductive toxicity at doses up to 50 times higher than the therapeutic dose. After the conducted experiments it is possible to assume a significant probability of using the ADF-C60 during pregnancy, but additional preclinical and clinical studies are required to substantiate the obtained conclusion.

Hair loss (alopecia) is a common disorder caused by an interruption in the body’s cycle of hair production. This pathology negatively affects the psychoemotional state of patients and significantly reduces their quality of life. The currently available medical treatments (including minoxidil therapy) are effective in arresting the progression of the disease; however, they allow only partial regrowth of hair at best. A significant clinical result occurs only with regular drug use. There is still great interest in finding new drugs for the treatment of alopecia. In this study, we aimed to examine the effect of an aqueous dispersion of unmodified fullerene C60 (ADF) on hair growth. ADF, produced by a unique technology, is biocompatible and non-toxic. Nu/nu mice were subcutaneously injected (2 μg/animal) every two days for a period of 11 days with ADF and, for control purposes, with phosphate-buffered saline (PBS). It was shown that ADF stimulated hair growth. Histological analysis of the nu/nu mice skin areas showed that animals treated with ADF had significantly more (about twice as many) hair follicles in the anagen phase compared to mice treated with PBS. The effect on hair growth persisted even after discontinuation of ADF administration. Analysis of gene expression demonstrated that ADF affected the Wnt-signaling pathway, increased the expression of the Wnt10b (wingless-type Mouse Mammary Tumor Virus integration site family, member 10B) factor, angiogenetic factors, and downregulated tumor necrosis factor-alpha levels. We propose that the mechanism of ADF action is likely related to its ability to attract macrophages to the hair follicle microenvironment and promote their polarization to the M2 phenotype. In addition, using molecular modeling, we tried to substantiate our hypothesis about the interaction of ADF with the adenosine A2A receptor, which may cause a decrease in tumor necrosis factor-alpha production. Thus, ADF may become a promising drug for the development of new approaches to the treatment of alopecia associated with immune disorders.

Allergic rhinitis (AR) occupies a leading position among the causes of morbidity throughout the world, to date, it has been diagnosed in 400 million people. In the formation and progression of AR, a significant role is assigned to cytokines associated with the second type of immune response, in particular, IL-4, IL-5, IL-9, IL-13, IL-25, IL-33, thymic stromal lymphopoietin (TSLP). This literature review provides information on the influence of the listed mediators on the structural cells of the nasal cavity and blood immune cells (T- and B-lymphocytes, eosinophils, macrophages, dendritic cells), and discusses their association with the manifestation of AR symptoms and the severity of the disease. The results of studies aimed at establishing the level of IL-4, IL-5, IL-9, IL-13, IL-25, IL-33 and TSLP in biological fluids (blood serum, nasal lavage) and their expression in nasal epithelial cells in patients with AR compared to healthy people are assessed.

Natural polyphenols are promising compounds for the pharmacological control of oxidative stress in various diseases. However, low bioavailability and rapid metabolism of polyphenols in a form of glycosides or aglycones have stimulated the search for the vehicles that would provide their efficient delivery to the systemic circulation. Conjugation of polyphenols with cationic amphiphilic peptides yields compounds with a strong antioxidant activity and ability to pass through biological barriers. Due to a broad range of biological activities characteristic of polyphenols and peptides, their conjugates can be used in the antioxidant therapy, including the treatment of viral, oncological, and neurodegenerative diseases. In this work, we synthesized linear and dendrimeric cationic amphiphilic peptides that were then conjugated with gallic acid (GA). GA is a non-toxic natural phenolic acid and an important functional element of many flavonoids with a high antioxidant activity. The obtained GA-peptide conjugates showed the antioxidant (antiradical) activity that exceeded 2-3 times the antioxidant activity of ascorbic acid. GA attachment had no effect on the toxicity and hemolytic activity of the peptides. GA-modified peptides stimulated the transmembrane transfer of the pGL3 plasmid encoding luciferase reporter gene, although GA attachment at the N-terminus of peptides reduced their transfection activity. Several synthesized conjugates demonstrated the antibacterial activity in the model of Escherichia coli Dh5α growth inhibition.

BACKGROUND: Allergens from the pollen of wormwood (Latin: Artemisia absinthium) are among the most dangerous in terms of allergization in central Russia, Europe and a number of other regions. The use of allergoids created using glutaraldehyde instead of native allergen extracts is due to the reduced risk of developing allergic reactions while maintaining immunogenicity and eliminating the highly toxic element formalin from the production process, which makes allergoids safer for use in allergen-specific immunotherapy. AIM: Obtaining an allergoid from wormwood pollen by treatment with glutaraldehyde to evaluate its properties prior to preclinical testing. MATERIALS AND METHODS: Purified extract of wormwood pollen, isolated by defatting and water-salt extraction, was dissolved in phosphate-buffered saline (PBS) pH 7.5, after which it was polymerized in 0.1 % glutaraldehyde solution. Stabilization was performed with sodium borohydride solution. The extract and allergoid of wormwood pollen were studied using chromatographic analysis and competitive enzyme-linked immunosorbent assay (ELISA). Then, intraperitoneal immunization with the obtained allergoid was carried out in CBA × C57Bl/6 (F1) mice. RESULTS: After evaluation of the specific allergenic activity using competitive immune assay ELISA with biotinylated extract of wormwood pollen allergen, it was shown that the 50 % inhibition point for the allergoid is determined at a drug concentration of 0.03 mg/ml, and for the allergen – already at 0.0008 mg/ml. Consequently, the allergenicity of wormwood pollen allergoid is significantly reduced compared to that of the original extract. In addition, the immunogenic activity of the obtained allergoid was confirmed in mice: the average optical density of the ELISA reaction, indicating the amount of IgG, in the group immunized with the allergoid was more than at least 1.5 times higher than the values for the allergen extract. CONCLUSIONS: During the work, an extract containing allergens of wormwood pollen was obtained. An allergoid was obtained on its basis by polymerization with glutaraldehyde. The obtained allergoid has a high molecular weight (the range shifted to 44–66 kDa), while it has low allergenic activity, in comparison with the original extract, and also retains immunogenicity. In this regard, this allergoid can become the basis for obtaining new ASIT treatments.

Роль триады цитокинов, продуцируемых респираторным эпителием, в патогенезе аллергического ринита 1 Федеральное го сударственное бюджетное учреждение «Государственный научный центр «Институт иммунологии» Федерального медико-

Atopic dermatitis is a highly heterogeneous disease that usually requires a tailored approach to therapy. Recent studies have described a variety of biomarkers linked to diverse pathophysiological aspects of atopic dermatitis. Furthermore, they have provided encouraging evidence supporting high clinical importance of these biomarkers as promising tools for personalized diagnosis, assessment of disease severity, and monitoring of treatment efficacy. This review aims to provide a comprehensive summary of research progress on atopic dermatitis biomarkers, their diagnostic and prognostic value, as well as the benefits they might potentially offer not only for determining the disease endotype, but also for developing novel management strategies. The implementation of easily accessible and simple methods to measure atopic dermatitis biomarkers into clinical practice is an ambitious task for allergologists and dermatologists. A detailed overview of currently available data will allow us to identify the most important trajectories for future research in this area that will promote the concept of personalized atopic dermatitis therapy, reduce the disease burden, and improve the quality of life of patients.

Allergic rhinitis (AR) is an inflammatory disease of the upper respiratory tract (nasal mucosa). AR affects up to 40% of the world’s population; in the Russian Federation, the incidence is 18% to 30%, depending on the region. Despite the fact that AR is not a severe pathology, it causes significant economic burden. Another threat associated with this disease is that in 40% of cases, patients with AR eventually develop a more severe disabling pathology – AD. Widespread prevalence and significant economic disadvantages caused by AR determine the importance of developing new ways of prevention and control of this disease, as the existing methods of therapy are insufficient. However, the search for new ways of therapy is impossible without a detailed investigation of the molecular mechanisms of AR pathogenesis. For a long time it was considered that this allergic inflammation is formed by Th2-dependent mechanism with involvement of Th2-lymphocytes, B-cells and eosinophils and pro-inflammatory cytokines: IL-4, IL-5 and IL-13. However, experimental evidence has now accumulated on the role of epithelial cells of the respiratory tract and the proinflammatory cytokines they secrete (IL-25, IL-33 and TSLP) in the pathogenesis of AR and AD. IL-25 has been shown to induce the production of IL-4, IL-5 and IL-13, directing a Th2-type immune response. At the same time, mice with inactivated IL-25 developed barely any Th2-immune response. Inactivation of IL-33 significantly reduces inflammation (mediated by eosinophils) of the respiratory tract. Mice knockout for the cytokine receptor TSLP did not develop nasal hyperreactivity in response to allergen, but the level of nasal mucosal inflammation remained high. Currently, work is actively progressing on the development of new drugs capable of specifically blocking the activity of the listed cytokines; first of all, drugs based on neutralizing monoclonal antibodies. However, there are other technologies that can be used to regulate the activity of genes, such as the technology based on the RNA interference. It can be used to suppress the expression of any gene with a known nucleotide sequence, including genes encoding pro-inflammatory cytokines.Considering the above, the aim of this work was to design synthetic miRNA molecules and study their ability to specifically block the expression of genes encoding proinflammatory cytokines IL-25 and TSLP in experiments in vitro.