Immature hematopoietic progenitors are a constant source for renewal of hemocyte populations and the basic component of the tissue and cell repair apparatus. A unique property of these cells of internalizing extracellular double-stranded DNA has been previously shown. The leukostimulatory effect demonstrated in our pioneering studies was considered to be due to the feature of this cell. In the present research, we have analyzed the effects of DNA genome reconstructor preparation (DNA^gr), DNAmix, and human recombinant angiogenin on both hematopoietic stem cells and multipotent progenitors. Treatment with bone marrow cells of experimental mice with these preparations stimulates colony formation by hematopoietic stem cells and proliferation of multipotent descendants. The main lineage responsible for this is the granulocyte-macrophage hematopoietic lineage. Using fluorescent microscopy as well as FACS assay, co-localization of primitive c-Kit- and Sca-1-positive progenitors and the TAMRA-labeled double-stranded DNA has been shown. Human recombinant angiogenin was used as a reference agent. Cells with specific markers were quantified in intact bone marrow and colonies grown in the presence of inducers. Quantitative analysis revealed that a total of 14,000 fragment copies of 500 bp, which is 0.2% of the haploid genome, can be delivered into early progenitors. Extracellular double-stranded DNA fragments stimulated the colony formation in early hematopoietic progenitors from the bone marrow, which assumed their effect on cells in G0. The observed number of Sca1+/c-Kit+ cells in colonies testifies to the possibility of both symmetrical and asymmetrical division of the initial hematopoietic stem cell and its progeny.

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.

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 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.