Oleg Svyatoslavovich Taranov

The final part of our series of studies reports the results of a pilot study of a clinical case (patient K.), indicating that therapy enabling reconstruction of the genome of hematopoietic stem cells (HSCs) reduces the frequency of SNPs in exons of the genes associated with clonal hematopoiesis of uncertain potential in a patient with a neuroendocrine tumor of the small intestine in the terminal stage of progression. The fundamental principles of the therapy were as follows. Control samples of peripheral blood mononuclear cells (baseline) were collected from the patient. CD34+ HSCs were then mobilized and collected. The HSC genome was modified using a technology enabling ex vivo correction of the nucleotide sequences of DNA chromosomes of poorly differentiated hematopoietic progenitor cells. For this purpose, the collected leukocyte suspension enriched with CD34+ HSCs was treated with fragmented deproteinized genomic DNA obtained from approximately 100 young healthy women in labor (hDNA ^gr ). The patient was reinfused intravenously with the treated cells. Five days before reinfusion, the patient received immunosuppressive therapy. Samples of peripheral blood mononuclear cells were also collected 4, 8, 12, and 27 months after the therapeutic intervention. Full-exome sequencing of DNA isolated from selected cell samples was performed. A group of commonly accepted clonal hematopoiesis genes was selected as a criterion demonstrating changes in the genome. The following multiplex panels were used at the initial time point as well as time points after 4, 8, and 12 months: MGIEasy Exome Capture V5 Probe set, Roche KAPA HyperExome, Nanodigmbio NEXome Plus Panel v1.0, covering the entire exome and non-coding regions of the analyzed genes adjacent to exons with ~100× coverage. A ~1000× coated panel (Nanodigmbio NanOnco Plus Panel v3.0), including the genes with mutant alleles identified at the initial time point, was used to verify the identified mutations and more accurately determine their proportions in readings at the last stage of the analysis (27 months after the therapy). Whole-exome sequencing of hDNA ^gr (Roche KAPA HyperExome), which is used to reconstruct the genome of HSCs whose fragments collectively constitute the complete human genome, was also performed. Although the analysis of readings of whole-exome sequencing of DNA isolated from blood samples after treatment did not reveal any currently known signs of clonal hematopoiesis with uncertain potential in the patient K., we found a significant, within the framework of the selected criterion, decrease in the frequency range of SNPs to 18 % by 27 months of follow-up in 15 % cases of the heterozygous alleles ( DNMT1 , SF3B1 ( 1–2 )). Subsequent analysis of the frequency of occurrence of controlled SNPs and the corresponding data on the depth of their sequencing, performed using the All-FIT algorithm, indicates that by the 27th month of follow-up, the proportion of cells carrying the basic set of SNPs significantly decreases by 12 % (confidence interval (CI) = 5–16 %). In other words, in a mixture of cells heterozygous for this SNP, a population of cells carrying a homozygote without an SNP appears. Our findings may indicate that a significant proportion of cells underwent correction of the SNP to an alternative allele, and this correction was associated with the treatment of the original HSCs with hDNA ^gr . The detected changes in the group of clonal hematopoiesis genes suggest the possibility of correcting other loci throughout the HSCs genome. Therefore, the clinical manifestations in the development of the disease show a pronounced positive trend, which has persisted for four years, until present. It is assumed that the positive clinical outcome after therapy with reconstructed hematopoietic stem cells is associated with an increase in the regenerative potential of HSCs, which resulted from the genetic correction of unfavorable mutations in the HSCs genome.

INTRODUCTION. The development of new effective drugs based on granulocyte-macrophage colony-stimulating factor (GM-CSF) remains a relevant objective: the study of the drug’s biological effects opens new prospects for its clinical use in the treatment of a wide range of diseases – from oncological and hematological disorders to neurodegenerative conditions. Developing a new lyophilized recombinant human GM-CSF (rhGM-CSF) based on Es с herichia coli BL21/pET-GST-6His-GM producer strain warrants an assessment of its specific activity and toxicity. AIM. This study aimed to examine the specific proliferative activity ( in vitro ) and hematopoietic stimulatory effect ( in vivo ), as well as acute and subchronic toxicity of a new lyophilized rhGM-CSF preparation for subcutaneous administration. MATERIALS AND METHODS. Lyophilized rhGM-CSF (strain E. coli BL21/pET-GST-6His-GM) produced by State Research Center of Virology and Biotechnology “Vector” (Russia) was used in the study. The proliferative activity was assessed in the human erythroleukemia TF-1 cell culture by XTT assay. Hematopoietic stimulatory activity was assessed in male CBA/Calac mice subjected to cyclophosphamide-induced myelosuppression (200 mg/kg, intraperitoneally). The rhGM-CSF preparation was administered at a dose of 90 μg/kg subcutaneously daily for 4 days. A leukogram was calculated on Day 5. The acute toxicity of the preparation was assessed by a single subcutaneous administration to male and female ICR mice at 500 and 1,000 μg/kg, subchronic toxicity – by four subcutaneous administrations at 90 μg/kg. On Days 1 and 7, body weight, temperature, complete blood count, biochemical markers and internal organ histology (heart, lungs, liver, kidneys, etc.) were evaluated. RESULTS. The rhGM-CSF preparation demonstrated pronounced proliferative activity on TF-1 cells (ED ₅₀ =0.48 ng/mL). In mice with myelosuppression, the preparation resulted in a 2.1-fold increase in the number of segmented neutrophils compared to the control group. Single and repeated administration did not cause death of animals, did not affect body weight or temperature, nor did it result in significant changes of hematological or biochemical parameters or macro- and microscopic anatomy of organs. CONCLUSIONS. The new lyophilized rhGM-CSF preparation has demonstrated high proliferative and hematopoietic stimulatory activity, as well as insignificant acute and subchronic toxicity. The obtained results substantiate further pharmaceutical development of the preparation as an effective and safe hematopoietic stimulatory agent.

Approaches to delivering gene editing tools in the form of ribonucleoproteins may provide a safety advantage over the delivery of nucleic acids encoding ribonucleoproteins. Virus-based vectors are widely used as a delivery platform. However, the persistence of viral exogenous nucleic acids can cause increased genotoxicity. Virus-like particles (VLPs) do not contain an expression cassette and can act as a platform for the delivery of ready-made ribonucleoprotein complexes. The absence of nucleic acids in VLPs eliminates the risk of insertional mutagenesis compared to widely used lentiviruses or adeno-associated viruses. Therefore, we used VLPs to deliver the ribonucleoprotein complex MmCas12m–TadDE to disrupt the HIV-1 gag gene start codon. We detected VLP morphogenesis using electron microscopy. We confirmed the incorporation of MmCas12m–TadDE into VLPs. We achieved an editing efficiency of about 9% in some cases with minimal off-target effects, which confirms the prospect of using VLPs as a platform for delivering genomic editing tools.

BACKGROUND: The emergence of the coronavirus (SARS-CoV-2) caused a pandemic that led to an increase in morbidity and mortality among infected individuals. The SARS-CoV-2 variants circulating during that period, including Delta, were characterized by a broad spectrum of pathological effects at both the tissue and cellular levels. In 2025, the Omicron variant predominates, producing milder forms of respiratory conditions. Among the key research questions related to SARS-CoV-2, considerable attention has been devoted to its pathogenicity, which is characterized by high virulence and the ability to induce systemic pathological processes. Transmission electron microscopy is used to visualize virus–cell receptor interactions and to study the destructive impact of the pathogen on host cells. The findings of such studies form the basis for understanding the course and pathogenesis of the disease and contribute to vaccine development. AIM: The work aimed to investigate SARS-CoV-2 virion reproduction and the trends of cytopathic effects in Vero E6 cell culture infected with Delta and Omicron variants. METHODS: Ultrastructural analysis was performed on Vero E6 cell cultures infected with the Delta and Omicron SARS-CoV-2 variants. Infected cell cultures were fixed at three time points: 6, 18, and 24 hours after viral inoculation. RESULTS: Most samples in both groups (Delta and Omicron) retained near-normal morphology 6 hours post-inoculation and exhibited numerous transport vesicles. At 18 hours, Delta and Omicron groups showed similar morphological changes, including vacuolization of the endoplasmic reticulum and Golgi complex. The Omicron group demonstrated altered mitochondrial morphology. Marked destructive changes were observed 24 hours post-inoculation. Both groups displayed cytoplasmic condensation, extensive organelle vacuolization, and numerous vesicles containing viral particles. Analysis of virion reproduction trends revealed an increase in viral particle numbers at 18 hours, followed by a decline in viral virion replication by 24 hours. CONCLUSION: The study suggests similar mechanisms of cytopathic pattern formation in Vero E6 cells infected with the Delta and Omicron SARS-CoV-2 variants, along with active virion reproduction in cell culture between 6 and 18 hours post-infection.

Laboratory animals allow modeling infectious diseases to study pathogenesis and conduct drug trials. The creation of animal models that most accurately reproduce COVID-19 is promising for assessing the effectiveness of drugs and vaccines against the SARS-CoV-2 virus. The aim is to study the histological changes in the lungs and the other indicators of the infectious process in modeling COVID-19 in guinea pigs as a type of laboratory animals susceptible to SARSCoV-2 virus.Material and methods. The experiments were carried out on 12 guinea pigs of both sexes infected with SARS-CoV-2 alpha variant. The dynamics of the viral load was determined in nasal washes by RT-PCR; SARS-CoV-2 antibody content in the blood serum were measured by ELISA; titers of neutralizing antibodies were estimated in the neutralization reaction in Vero E6 cell culture. As controls, 12 lung autopsies of patients infected with SARS-CoV-2 (confirmed by PCR analysis) and those who died as a result of pneumonia were studied. Structural changes in the lungs of autopsy and experimental samples were studied on paraffin sections stained with haematoxylin and eosin.Results. After intranasal infection with SARS-CoV-2 alpha variant at a dose of 4 lg TCID₅₀ (the dose infecting 50 % of the cell culture and causing cytopathic effect in 50 % of the cell monolayer), a low level of viral load with high rates of elimination was detected in the nasal cavity and lungs of guinea pigs. On days 3 and 5, the viral load in nasal swab samples was significantly higher in males (p < 0.05). After 7 days, viral RNA was not detected in nasal swabs. Antibodies to SARSCoV-2 were detected in the blood serum of guinea pigs on the 15th day. With a neutralizing antibody titer of 1:100 for all animals, the positivity rate in females was 2 times higher. Histologically confirmed severe viral pneumonia was recorded in dead animals and at the end of the experiment, similar to changes in the autopsy material of patients with COVID-19.Conclusions. Histological changes in the lungs of guinea pigs during modeling of SARS-CoV-2 infection indicate the susceptibility of this animal species to the studied pathogen with reproduction of the disease in patients, which allows us to consider guinea pigs as a promising animal model for the study and prevention of COVID-19.

Introduction. RNA-containing viruses, especially influenza viruses, are of high epidemiological significance. The manifestation of COVID-19 has led to the registration of coinfection cases, the pathogenesis of which is poorly studied. The Vero (E6) cell line is widely used to study the morphogenesis of various viruses, including influenza and coronavirus. The aim of the work is to study the ultrastructure of Vero (E6) cells and the reproduction of viral particles during monoinfection with the influenza A virus and coinfection of this virus with two SARS-CoV-2 genovariants in dynamics 6, 18 and 24 hours after inoculation. Materials and methods. The Vero (E6) cell line model was used for in vitro study of the viral infection effects and an analysis of the dynamics of changes in the number of intracellular viral particles. The study involved 4 experimental groups: Vero (E6) cells mono-infected with the influenza virus strain A/H1N1 pmd09 at a dose of 0.1 MOI; Vero (E6) cells co-infected with the influenza virus strain A/H1N1 pmd09 and Delta strain of SARS-CoV-2 at a total dose of 0.1 MOI; Vero (E6) cells co-infected with the influenza virus strain A/H1N1 pmd09 and Omicron strain of SARS-CoV-2 at a total dose of 0.1 MOI. In each study group, cells were monitored at time points of 6, 18, and 24 hours. Results. After 6 h, no pathological structures were detected in all groups, except for virus-containing transport vesicles. After 18 h, vacuolization of the ER of varying degree was noted in all the studied groups. After 24 h, ultrastructural changes, namely vacuolization of organelles and/or compaction of the cytoplasm, were encountered in all groups comparatively more frequently than at 6 h and 18 h time points. . The dynamics of the number of viral particles increased significantly by 24 h time point in the monoinfection group. However, none of the coinfection groups demonstrated a tendency for the number of viral particles to change, since no statistically significant differences were found between the 6 h, 18 h, and 24 h stages. Conclusion. The results obtained suggested that the interaction between A/H1N1 pmd09 and SARS-CoV-2 viruses contributed to an overall decrease in the formation of new virions in Vero (E6) cells in both cases of coinfection.

Background: At present, there are no universal markers of tumor stem cells known, including for B-lymphomas. Previously, we have shown that Epstein–Barr virus-induced B-cell lymphoma culture contains cells capable of internalizing TAMRA-labeled DNA. These cells form sphere-forming centers and are essential for the development of xenografts genetically identical to the initial culture. Objective: To analyze the stem characteristics of cells that internalize DNA. Methods: Sorting and RNA sequencing of two subpopulations (TAMRA + and TAMRA–) of Epstein–Barr virus-induced B-cell lymphoma culture and a series of quantitative real-time reverse transcription PCR were performed. Results: TAMRA + cells were shown to have increased synthesis of mRNA of genes associated with the maintenance of a poorly differentiated state ( SOX2, NANOG, POU5F1, CYP26A1 ), self-renewal ( FZD5, FZD7, TCF3, LEF1 ) and epithelial–mesenchymal transition ( MMP2, ITGB7 ). Transcriptomic analysis revealed that in TAMRA + cells, the synthesis of mitochondrial genes, as well as caspases and some apoptosis inhibitors, is reduced. TAMRA + cells possess clonogenic properties, increased level of synthesis of mRNA for key genes associated with self-renewal and poorly differentiated state maintenance. Conclusions: Internalization of the TAMRA-DNA probe is the marker of B-lymphoma cancer stem cells and can be used to detect tumor stem cells and develop new approaches to targeted treatment of B-lymphoma.

Introduction. Monitoring and research on arthropod-borne microorganisms is important. Recently, with the development of next-generation sequencing methods, many previously unknown viruses have been identified in insects. Aim of the study. Isolation of viruses from mosquitoes sampled in the Republic of Sakha (Yakutia), followed by the study of a new for Russia negevirus isolated from mosquitoes of the species Ochlerotatus caspius, including determination of its complete nucleotide sequence, phylogenetic and virological characteristics. Materials and methods. Dezidougou virus isolation was performed on C6/36 (Aedes albopictus) cell culture. Electron microscopy was performed using a JEM 1400 electron microscope. Nucleotide sequence screening was performed by NGS on a high-throughput sequencer MiSeq, Illumina (USA). Full genome nucleotide sequence was determined by Sanger sequencing. Phylogenetic analysis was performed using GenBank database, using Vector NTI Advance 11 and MEGA 11 programs. Results. The virus isolated from mosquitoes replicated efficiently in C6/36 cells, causing their death. However, it did not replicate in the mammalian cell cultures used. The isolated virus did not cause pathologic manifestations in suckling mice when infected intracerebrally. Electron microscopic examination of the purified virus-containing suspension showed the presence of spherical viral particles with a diameter of 45‒55 nm. The results of full genome sequencing identified it as belonging to Dezidougou virus, first isolated in Côte d’Ivoire. The nucleotide sequence of the genome of Yakutsk 2023 strain of Dezidougou virus was deposited in GenBank (PP975071.1). Conclusion. Dezidougou virus of genus Negevirus was isolated and characterized for the first time in the Russian Federation. Further studies on the prevalence of negeviruses, their virological features, potential importance for public health and their impact on vector competence of vectors are important and promising.

Despite the end of the COVID-19 pandemic, there still remain risks of new aggressive strains of coronavirus. As the human population increases progressively, it is mandatory to ensure both preventive measures and an immediate response to emerging infectious threats. Another essential component for rapidly restraining a new possible pandemic is the development of new anticoronaviral therapeutics. In the present study, the anticoronaviral capabilities of Gc protein-derived macrophage-activating factor (GcMAF) are characterized. It is demonstrated that the administration of GcMAF to Syrian hamsters infected with SARS-CoV-2 within the first phase of infection (six days postinfection) is accompanied by (i) a statistically significant reduction in the viral load of the lung tissue and (ii) the switching of the inflammatory status of the lung tissue to a neutral one in terms of mRNA expression levels of the groups of pro/anti-inflammatory cytokines and chemokines. The potential mechanism for this antiviral action and the containment of the inflammatory response by the drug associated with the engagement of terminal N-acetylgalactosamine GcMAF and C-type lectin domain containing 10A expressed at the surface of lung-infiltrating macrophages and pneumocytes, which simultaneously express angiotensin-converting enzyme 2, is discussed.

In this part of the study, the first component of the concept of “natural genome reconstruction” is being proven. It was shown with mouse and human model organisms that CD34+ hematopoietic bone marrow progenitors take up fragments of extracellular double-stranded DNA through a natural mechanism. It is known that the process of internalization of extracellular DNA fragments involves glycocalyx structures, which include glycoproteins/protein glycans, glycosylphosphatidylinositol-anchored proteins and scavenger receptors. The bioinformatic analysis conducted indicates that the main surface marker proteins of hematopoietic stem cells belong to the indicated groups of factors and contain specific DNA binding sites, including a heparin-binding domain and clusters of positively charged amino acid residues. A direct interaction of CD34 and CD84 (SLAMF5) glycoproteins, markers of hematopoietic stem cells, with double-stranded DNA fragments was demonstrated using an electrophoretic mobility shift assay system. In cells negative for CD34, which also internalize fragments, concatemerization of the fragments delivered into the cell occurs. In this case, up to five oligonucleotide monomers containing 9 telomeric TTAGGG repeats are stitched together into one structure. Extracellular fragments delivered to hematopoietic stem cells initiate division of the original hematopoietic stem cell in such a way that one of the daughter cells becomes committed to terminal differentiation, and the second retains its low-differentiated status. After treatment of bone marrow cells with hDNA^gr, the number of CD34+ cells in the colonies increases to 3 % (humans as the model organism). At the same time, treatment with hDNA^gr induces proliferation of blood stem cells and their immediate descendants and stimulates colony formation (mouse, rat and humans as the model organisms). Most often, the granulocyte-macrophage lineage of hematopoiesis is activated as a result of processing extracellular double-stranded DNA. The commitment process is manifested by the appearance and repair of pangenomic single-strand breaks. The transition time in the direction of differentiation (the time it takes for pangenomic single-strand breaks to appear and to be repaired) is about 7 days. It is assumed that at the moment of initiation of pangenomic single-strand breaks, a “recombinogenic situation” ensues in the cell and molecular repair and recombination mechanisms are activated. In all experiments with individual molecules, recombinant human angiogenin was used as a comparison factor. In all other experiments, one of the experimental groups consisted of hematopoietic stem cells treated with angiogenin.