Evgenii Vladimirovich Levites

Introduction: GcMAF ^anti is a Gc protein-derived macrophage-activating factor that enhances macrophage phagocytic activity and stimulates the production of anti-inflammatory cytokines. The aim of this study was to describe the clinical picture of monotherapy in the treatment of systemic lupus erythematosus. Case Presentation: The patient (female, 37 years old) was diagnosed with systemic lupus erythematosus according to the EULAR/ACR 2019 diagnostic criteria. The patient had elevated titers of antinuclear antibodies, recurrent episodes of fever, a low C4 level, and a high level of anti-dsDNA antibodies. The activity of systemic lupus erythematosus was low, 5 points on the SLEDAI-2K scale. The patient was transitioned to GcMAF ^anti monotherapy after two years of standard therapy with hydroxychloroquine and prednisolone. A total of four courses of subcutaneous GcMAF ^anti injections were administered, each consisting of 12 injections over 24–36 days (a single 2–5 μg injection every 2–3 days), with 2–4-month intervals between treatment courses. GcMAF ^anti monotherapy did not reverse the positive clinical changes in the patient achieved by standard therapy with hydroxychloroquine and prednisolone. No febrile episodes were observed during therapy and for 2–3 months following treatment; neither patient-reported complaints nor adverse events were reported during the same period. The result of GcMAF ^anti monotherapy was 2- 4 points on the SLEDAI-2K scale. Indicators of immunobiological markers of systemic lupus erythematosus indicated stabilization of the disease with GcMAF ^anti monotherapy. Conclusion: The results obtained in this case report of systemic lupus erythematosus treatment using the novel agent GcMAF ^anti suggest that clinical studies are required to further analyze the effect of GcMAF ^anti on systemic lupus erythematosus.

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.

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.

The article presents an analysis of the published data on the structure and functions of vitamin D₃-binding protein (DBP), which is controlled by a single gene with three clearly defined alleles Gc1-F, Gc1-S, and Gc2. The article presents information on various structural and functional variants of DBP, which are determined genetically and can undergo post-translational modification by glycosylation. One of the derivatives of vitamin D₃-binding protein is its partially deglycosylated form, which exhibits properties of a specific activator of macrophages and is designated as the GcMAF factor. The article pays special attention to rare forms of DBP, detected by the isofocusing method. An assumption is made about the nature of rare forms of DBP and the mechanism of occurrence of changes underlying the appearance of these forms.

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.

Vitamin D₃ transporter (DBP) is a multifunctional protein. Site-specific deglycosylation results in its conversion to group-specific component protein-derived macrophage activating factor (GcMAF), which is capable of activating macrophages. It has been shown that depending on precursor conversion conditions, the resulting GcMAF activates mouse peritoneal macrophages towards synthesis of either pro- (IL-1β, TNF-α—M1 phenotype) or anti-inflammatory (TGF-β, IL-10—M2 phenotype) cytokines. The condition for the transition of the direction of the inflammatory response of macrophages when exposed to GcMAF is the initial glycosylated state of the population of DBP molecules and the associated effective deglycosylation of DBP by β-galactosidase. In vivo experiments with GcMAF exhibiting anti-inflammatory properties on models of induced arthritis in mice and cystitis in rats indicate a significant anti-inflammatory effect of the macrophage activator. The feasibility of unidirectional induction of anti-inflammatory properties of macrophages allows creation of combined therapeutic platforms where M2 macrophages are among the key therapeutic components.

(1) Background: Multi-year research into the synergistic effect of cyclophosphamide and complex composite double-stranded DNA preparation (DNAmix) has made it possible to develop the novel Karanahan technology for treating malignant tumors, which is based on the eradication of cancer stem cells; (2) Methods: Pilot clinical trials for Karanahan technology were conducted in 12 patients with advanced stage IV breast cancer; (3) Results: The findings indicate that the analyzed treatment regimen ensures a local positive therapeutic response, significantly increases patient survival time, and activates the adaptive antitumor immune response. Adjustment of the basic regimen of Karanahan technology has been developed, which consists of increasing the dose of the DNAmix complex composite double stranded DNA preparation and its lymphotropic administration to the main lymph depots simultaneously with intratumoral injection; (4) Conclusions: Karanahan technology has a promising therapeutic potential in the treatment of inoperable stage IV breast cancer.

Macrophages are the immune cells of high‐immunological plasticity, which can exert both pro‐ and anti‐inflammatory activity, as well as repolarize their phenotype to the opposite or neutral one. In this regard, M2 macrophages of the tumor‐associated stroma (TAS) are a promising therapeutic target in treating malignant neoplasms. Using FACS assay, we have estimated the CD11b+/Ly‐6G+/Ly‐6C+ fraction of macrophages from the peritoneum and TAS in intact healthy mice and those with developed Lewis carcinoma, both untreated and treated according to Karanahan technology in combination with group‐specific macrophage activator (GcMAF‐RF). As well, the pattern of pro‐ and anti‐inflammatory cytokines mRNA expression in different groups of experimental and tumor‐bearing animals was assessed. It was found that: (i) exposure of intact mice to GcMAF‐RF results in the increased number of CD11b+/Ly‐6C+ peritoneal macrophages and, at the same time, the expression pattern of cytokines in peritoneal macrophages switches from that characteristic of the mixed M1/M2 phenotype to that characteristic of the neutral M0 one; (ii) combination of Karanahan technology and GcMAF‐RF treatment results in M0/M1 repolarization of TAS macrophages; (iii) in tumor‐bearing mice, the response of peritoneal macrophages to such a treatment is associated with the induction of anti‐inflammatory reaction, which is opposite to that in TAS macrophages.

Group-specific component macrophage-activating factor (GcMAF) is the vitamin D₃-binding protein (DBP) deglycosylated at Thr⁴²⁰. The protein is believed to exhibit a wide range of therapeutic properties associated with the activation of macrophagal immunity. An original method for GcMAF production, DBP conversion to GcMAF, and the analysis of the activating potency of GcMAF was developed in this study. Data unveiling the molecular causes of macrophage activation were obtained. GcMAF was found to interact with three CLEC10A derivatives having molecular weights of 29 kDa, 63 kDa, and 65 kDa. GcMAF interacts with high-molecular-weight derivatives via Ca²⁺-dependent receptor engagement. Binding to the 65 kDa or 63 kDa derivative determines the pro- and anti-inflammatory direction of cytokine mRNA expression: 65 kDa—pro-inflammatory (TNF-α, IL-1β) and 63 kDa—anti-inflammatory (TGF-β, IL-10). No Ca²⁺ ions are required for the interaction with the canonical 29 kDa CLEC10A. Both forms, DBP protein and GcMAF, bind to the 29 kDa CLEC10A. This interaction is characterized by the stochastic mRNA synthesis of the analyzed cytokines. Ex vivo experiments have demonstrated that when there is an excess of GcMAF ligand, CLEC10A forms aggregate, and the mRNA synthesis of analyzed cytokines is inhibited. A schematic diagram of the presumable mechanism of interaction between the CLEC10A derivatives and GcMAF is provided. The principles and elements of standardizing the GcMAF preparation are elaborated.

It is well-established that double-stranded RNA (dsRNA) exhibits noticeable radioprotective and radiotherapeutic effects. The experiments conducted in this study directly demonstrated that dsRNA was delivered into the cell in its native form and that it induced hematopoietic progenitor proliferation. The 68 bp synthetic dsRNA labeled with 6-carboxyfluorescein (FAM) was internalized into mouse hematopoietic progenitors, c-Kit+ (a marker of long-term hematopoietic stem cells) cells and CD34+ (a marker of short-term hematopoietic stem cells and multipotent progenitors) cells. Treating bone marrow cells with dsRNA stimulated the growth of colonies, mainly cells of the granulocyte–macrophage lineage. A total of 0.8% of Krebs-2 cells internalized FAM-dsRNA and were simultaneously CD34+ cells. dsRNA in its native state was delivered into the cell, where it was present without any signs of processing. dsRNA binding to a cell was independent of cell charge. dsRNA internalization was related to the receptor-mediated process that requires energy from ATP. Synthetic dsRNA did not degrade in the bloodstream for at least 2 h. Hematopoietic precursors that had captured dsRNA reinfused into the bloodstream and populated the bone marrow and spleen. This study, for the first time, directly proved that synthetic dsRNA is internalized into a eukaryotic cell via a natural mechanism.