Technology of genomic balancing of chromatin of autologous hematopoietic stem cells for gene therapy of fatal immune-mediated diseases of civilization, extended life expectancy and sudden human death prevention.

A biotechnology for personalized ex vivo gene therapy based on molecular genomic balancing of hematopoietic stem cell (HSC) chromatin with nucleosome monomers of human genomic DNA (hDNAnmr) has been developed and implemented in the clinic to change (to "correct") mutant chromosome loci genomes of dominant HSC clones that form mono- and oligoclonal hematopoiesis during aging and major (oncological, cardiovascular, neurodegenerative and autoimmune) fatal immune-mediated diseases of civilization. A fundamentally new biotechnological approach has been applied to the delivery of genetic material into eukaryotic stem and progenitor cells by establishing an artificial "recombinogenic situation" in them to induce homologous recombination (equivalent replacement) of mutant DNA regions with healthy hDNAnmr. In experimental preclinical trials, the effectiveness of genomic balancing technology has been proven to reduce the risk of sudden death in old animals and to increase the lifespan of outbred mice by 30% and Wistar rats by 57%. The improvement in their quality of life, compared with the control, is explained by an increase in the telomeric regions of the HSCs and HPCs chromosomes by 1.5-2 times. The potential of the technology to slow down the hereditary neurodegenerative diseases on the model of amyotrophic lateral sclerosis is shown. The effectiveness of this technology in clinical practice is presented on the example of a terminal patient with stage 4 neuroendocrine cancer. This technology used in the treatment of a number of oncological, neurodegenerative, autoimmune and hereditary diseases with clonal hematopoiesis is able to arrest the progression of the disease, prevent its recurrence, prolong the active life of a person, increase the average life expectancy and prevent sudden death.

[Cell analogs of viral proteins].

Horizontal transfer of genes between viruses and their hosts played an important role in the evolution of various eukaryotes including contemporary mammals as well as the pathogens themselves. Elements of viruses of various types can be found in the genome of animals. Endogenous retroviral elements composing up to 8% of human genome length not only determine its high flexibility and rapid adaptation potential. Many of virus genes such as Fv1, Lv1, Lv2 being analogues of capsid and other proteins determine effective suppression of viral replication after cell penetration by the causative agent. Introduction of these elements into genome of a wide variety of animals from fish to primates could have taken place against the background of global natural cataclysms of viral origin. Integration of retrovirus genes coding surface glycoproteins with immunosuppressing domains into genetic apparatus of animals served as an impetus to the development of viviparity and spread ofplacental mammals. Their cell analogs syncytins perform a dual function: take direct part in the formation of syncytiotrophoblast layer of placenta and ensure tolerance of immune system of mother to embryo. The acquisition of cell genes by viruses also played an important role in their evolution: various interleukins and other modulators of immune response introduced into viral genome from cell genetic apparatus became one of the most important factors of pathogenicity of a wide variety of causative agents including poxviruses, cytomegalovirus, Epstein-Barr virus and many others. Evolutionary pathways of the virus and host are thus inseparable from each other, and character of one of these directions is largely dictated by the vector of another.

[Mechanisms of retroviral immunosuppressive domain-induced immune modulation].

Immunosuppressive domains (ISD) of viral envelope glycoproteins provide highly pathogenic phenotypes of various retroviruses. ISD interaction with immune cells leads to an inhibition of a response. In the 1980s it was shown that the fragment of ISD comprising of 17 amino acids (named CKS-17) is carrying out such immune modulation. However the underlying mechanisms were not known. The years of thorough research allowed to identify the regulation of Ras-Raf-MEK-MAPK and PI3K-AKT-mTOR cellular pathways as a result of ISD interaction with immune cells. By the way, this leads to decrease of secretion of stimulatory cytokines (e.g., IL-12) and increase of inhibitory, anti-inflammatory ones (e.g., IL-10). One of the receptor tyrosine kinases inducing signal in these pathways acts as the primary target of ISD while other key regulators--cAMP and diacylglycerol (DAG), act as secondary messengers of signal transduction. Immunosuppressive-like domains can be found not only in retroviruses; the presence of ISD within Ebola viral envelope glycoproteins caused extremely hard clinical course of virus-induced hemorrhagic fever. A number of retroviral-origin fragments encoding ISD can be found in the human genome. These regions are expressed in the placenta within genes of syncytins providing a tolerance of mother's immune system to an embryo. The present review is devoted to molecular aspects of retroviral ISD-induced modulation of host immune system.

[Effect of exogenous dna injection on leukopoietic repair and antitumor action of cyclophosphamide].

The cytostatic drug cyclophosphamide (CPA) in high dosage suppressed hemopoiesis by causing multiple double-strand breaks to occur in hemopoietic cell DNA and leading to mutation, chromosomal abberations and finally cell death. We tested fragmented DNA drugs for an ability of CPA to protect murine leukopoiesis on an assumption that once exogenous fragmented DNA had infiltrated into a cell, it might integrate with chromosomal DNA through homologous recombinations thus repairing damaged segments. DNA drugs did promote repair of leukocyte count in murine peripheral blood with leukopoiesis being suppressed by CPA administration. The levels of DNA derived from murine organs and human placenta were higher than those from salmon roe. Tumor growth was significantly inhibited following injection of placental DNA into mice bearing intramuscularly transplanted lymphosarcoma. Antitumor effect of combined CPA and DNA treatment was much higher than after CPA alone.

[Effect of exogenous DNA on the growth of transplantable tumors].

Using transplantable Ehrlich ascites tumor, hepatoma HA-1 and Lewis carcinoma it was shown, that preparations of fragmented genomic DNA can more or less effectively inhibit such tumors as well as the growth of their metastases. Such effects were produced by DNA preparations derived from tissues of mice, both syngeneic or allogeneic to tumor-bearer, as well as from human tissues.

Topological state of DNA in polytene chromosomes.

A new microfluorometric method was developed for measuring two topological characteristics of DNA in isolated nuclei, chromosomes and other DNA containing structures: (1) the relative amount of the topologically non-closed DNA (tncDNA) and (2) the supercoiling density of the topologically closed unconstrained DNA (tcDNA). The method was applied to isolated polytene nuclei and chromosomes of Chironomus thummi. The relative amount of tncDNA was found to be 0.21. Evidence in favour of the tncDNA localization in transcriptionally active loci (puffs) of the polytene chromosomes is presented. The supercoiling density of tcDNA localized, presumably, in inactive loci (bands) of the polytene chromosomes is about -0.001.

Fluorescence polarization study of tertiary structure of DNA within bacteriophage lambda.

The fluorescence polarization of acridine orange-stained, oriented lambda phages was measured. The parameters of DNA packing within the phage head cos2 theta and cos4 theta were calculated (theta, angle between the direction of a small segment of DNA and the phage axis). It is shown that simple models of lambda phage DNA tertiary structure are not consistent with calculated values. A new model is proposed.

Fluorescence study of secondary structure of DNA within bacteriophage lambda.

Bromoacetaldehyde (BAA) was used to study the secondary structure of DNA in lambda-phage particles. It was determined that about 1% of the adenines in the intraphage lambda-DNA reacts readily with BAA, thus, they are placed in DNA sites with disturbed complementary interactions. These adenines are close to the tryptophan residues of the phage protein. Fluorescence emission of epsilon A in the intraphage DNA is dramatically quenched. This, apparently, indicates the interaction between epsilon A and Trp- and/or Tyr- and/or Met-residues of phage protein.

[Packing of binding stained DNA strands in bacteriophage lambda]. / Upakovka sviazyvaiushchikh krasiteli uchastkov DNK v bakteriofage liambda.

Distribution of stainable DNA strands in phage lambda has been studied by polarized fluorescence. The effect of tight DNA-packing on fluorescence depolarization of complex dye-DNA was calculated. It is shown that stainable DNA in the phage is not concentrated in the central region. The arrangement of acridine orange molecules on the surface layers of the packed DNA is the most probable one.

[Binding of acridine orange and ethidium bromide to DNA in phage lambda]. / Vzaimodeistvie krasitelei akridinovogo oranzhevogo i étidiuma bromida s DNK v fage lambda.

Binding of acridine orange and ethidium bromide to phage lambda C1857 has been studied by fluorescence and absorption measurements. It is shown that these dyes are strongly bound to DNA in the phage and are not bound to phage proteins. 50% of interphage DNA is stainable.