WO2008013473A1 - Disease treating method - Google Patents

Abstract

The invention relates to medicine, in particular to methods for treating patients by means of genetic material-containing preparations and can be used for treating disturbed hemopoiesis, associated with chromosome defects and with the subsequent death of stem blood cells during therapy by means of cytostatics or high radiation doses applied for treating diseases associated with defects of basic mechanisms for differentiating stem blood cells without separation towards specific hemopoietic elements which emerge during hemoblastosis due to malignant modifications of hemopoiesis cells which become incapable for normal differentiation and, thereby generate uncontrolled proliferation thereof, and also diseases associated with defects of stem cell chromosomes, the healing of which depends on the presence of genetically, phenotypically and physiologically healthy stem cells in an organism. The inventive method makes it possible to extend the field of application and consists in injecting a fragmented DNA into a patient organism, wherein said fragmented DNA is allogenic and consists of fragments, the length of which corresponds to 1-10 nucleosome units which compose, in combination with nuclear matrix proteins, the entire genome of the patient, wherein the quantity of the fragmented DNAis selected in such a way that it is equal or greater than the quantity of blood plasma DNA and tissue fluid of the patient, but not exceeds 1500 ng/ml.

Description

 A method of treating diseases

The invention relates to medicine, in particular, to methods for treating patients with preparations containing genetic material, and can be used to treat impaired hematopoiesis associated with chromosome defects and subsequent death of blood stem cells (CKK) during therapy with cytostatics or high doses of radiation, for treatment diseases associated with a violation of the basic mechanisms of CKK differentiation, without dividing to the side of specific hematopoietic elements that arise with rheoblastosis due to malignant Hematopoiesis cells that become incapable of normal differentiation, resulting in uncontrolled proliferation, as well as diseases associated with defects in CK chromosomes, the cure of which depends on the appearance of genetically, phenotypically, and physiologically healthy stem cells (CK) in the body.

A known treatment method based on the correction of point mutations in cells [U. S, No. 5,795,972, 08/18. 1998]. The method is characterized by a relatively low treatment efficiency and limited use, since according to this method, mutations must be accurately determined before treatment begins. Due to the fact that for no mutation there is reliable evidence that this particular mutation led to the death of CK or is the cause of malignant degeneration of hematopoietic cells, the development of this method requires rigorous and lengthy studies to identify specific mutations requiring correction, and, therefore, there is a need in creating methods that could be applied on the basis of already existing knowledge about the mechanisms of genome correction, without focusing on specific mutations that led to the death of CKK or their malignant Tween rebirth.

Also known are methods of treatment based on the local use of DNA (deoxyribonucleic acid) fragments for the treatment of precancerous conditions in the skin of patients [US, No.5, 955,059, 21.09. 1999; US No.5,470,577,28.11. 1995]. These methods also have limited applicability, since neither specific sequences nor DNA sources are identified in these patents. They suggest using both natural and synthetic DNA from “any suitable sources)), for example, salmon DNA from 200 to mononucleotides and nucleosides in length, including dimers, which is the most effective in accordance with the authors' tests. However, the effect and consequences of using DNA of a different nature on the human body is not yet fully studied, which determines a certain danger of the application of methods.

The closest in essence to the proposed one is a method based on the use of the Polydan preparation, which is a highly purified standardized mixture of sodium salts of DNA polychlorohydrates (deoxyribonucleic acid) and RNA (ribonucleic acid) obtained from sturgeon milk, the dose of which is administered subcutaneously or intramuscularly slowly within 1, 5 - 2 minutes with pre-heating the bottle with the drug in the hand to the patient’s body temperature [http://www.polidan.ru; reg. Ne 95 / 292.8; reg. Ns 000552 / 01-2001].

The disadvantage of the closest technical solution is the limited scope, since the active substance of the drug is isolated from the tissues of the xenogenic organism and contains both DNA and RNA molecules. Despite the fact that the active substance of the drug Polydan acts on the CKK genome in a manner similar to human DNA, namely, it mimics the presence of chromosome breaks by the presence of double-stranded ends of DNA fragments delivered to the nucleus, thereby activating CKK to terminal differentiation, and also creates conditions by finding partial homology between the DNA of the drug Polydan and the DNA of human chromosomes, for the implementation of acts of homologous recombination, and preservation of the physical length and structure of chromosomes sufficient for a chain of consecutive mi tozov, the substance of the drug Polydan, at the same time, introduces foreign sequences into the recipient genome. Such an action of the drug Polydan saves the hematopoietic function of CKK by restoring and maintaining the functional size of the chromosomes of these cells, and, apparently, does not generally affect the highly specialized properties of differentiated blood cells, while simultaneously activating them to terminal differentiation with its mimicking effect, nevertheless, such saved CKK can not be considered fully returned to their original genetic state, since any other direction of differentiation, where they take part chromosome loci that are recombined with foreign genetic material will inevitably lead to defects in this pathway of CKK development. The internal structure of chromosomes with such a recombination acquires completely unpredictable features, and the long-term prognosis of leukostimulation using foreign DNA preparations has not yet been studied.

The technical result, the achievement of which the invention is directed, is to expand the scope and treatment of impaired homopoiesis, associated with chromosome defects and subsequent death of blood stem cells (CKK), for example, during therapy with cytostatics or high doses of radiation, for the treatment of patients with diseases associated with a violation of the basic mechanisms of differentiation of CKK without dividing to the side of specific hematopoietic elements that occur with hemoblastoses due to malignant changes in hematopoietic cells that become incapable of normal differentiation, as a result of which their uncontrolled proliferation is observed, as well as f for the treatment of patients with diseases associated with defects in CK chromosomes, the cure of which depends on the appearance in the body of genetically, phenotypically and physiologically healthy stem cells (CK).

The claimed technical result is achieved by the fact that, in a method of treating diseases based on the introduction of fragmented DNA into a patient’s body, the fragmented DNA is allogeneic and consists of fragments whose length corresponds to 1-10 nucleosome units that comprise the patient’s complete genome in association with nuclear matrix proteins however, the amount of fragmented DNA is taken to be equal to or greater than the amount of DNA in the blood plasma and tissue fluids of the patient, but not more than 1500 n g / ml.

The method is based on the ability of CKK to respond to fragmented DNA preparations. DNA fragments delivered to the nuclear space of CKK, due to the natural mechanism of homologous recombination (GR), replace mutant regions of genes or restore the original physical length of chromosomes, which caused the loss of CK viability, and thereby non-viable or mutant CKK becomes viable. At the same time, CKK chromosomes acquire physical parameters that allow CKK to carry out a chain of sequential mitoses, and in the best case, the genome of the treated CKK is completely corrected and becomes identical to the genome of the initial healthy CK. At the same moment, fragments of extracellular DNA delivered to the nuclear space with their double-stranded ends create the illusion of the appearance of functional breaks in the CKK chromosomes, which are the inductor of their terminal differentiation. This mimicking effect of extracellular DNA fragments localized inside the CKK nucleus triggers the mechanisms of cell differentiation, activates and maintains the cometary division of CKK in a continuously active state. The drawing shows: in FIG. 1 - a characteristic of the effect of mouse and human DNA on the survival of mice after their γ-irradiation in a lethal dose, FIG. 2 and FIG. 3 is a characteristic of the radioprotective effect of allogeneic DNA upon intraperitoneal administration to mice before and after lethal γ-irradiation, FIG. 4a presents desected mouse spleens after γ - irradiation and therapy of extracellular fragmented human and mouse DNA. A large number of endogenous splenic colonies are observed (see Table 1) formed by descendants of rescued CKK. in FIG. 4,6 - desected mouse spleens are presented after γ - irradiation without therapy of extracellular fragmented human and mouse DNA. There is a practical absence (see Table 1) of colonies, which indicates the destruction of the mechanisms of recovery of CKK.

The proposed method of treatment is implemented as follows.

For its implementation in the patient’s body, by known methods and methods (intravenously, intramuscularly, by intradermal injection, subcutaneously, intraperitoneally, by application to various mucous membranes, by mouth (with or without preliminary neutralization of the acidic environment of the stomach, or using a special insoluble membrane in the stomach ), through the rectum, intravaginally, introkulyarno, through the nose, by introocular injection or inhalation are introduced specially treated fragments of allogeneic DNA obtained from genetically and physiologically healthy donors.The amount of drug administered should be such that the concentration of fragmented DNA in the blood and tissue fluids of the patient does not exceed the maximum allowable amount of 1500 ng / ml In the case of treatment with CKK ex vivo for subsequent autotransplantation, CKK should be processed fragmented DNA preparation in a concentration not exceeding 30 μg / ml, while fragmented DNA, being introduced into the patient’s body, is delivered to actively dividing cells of the body, including and to CKK, using a natural delivery mechanism (blood flow, specific receptors located on the surface of actively dividing cells). Next, the fragmented extracellular DNA delivered to the intracellular space is deposited in the interchromosomal compartment of the nucleus and is subjected to intranuclear processing. In this case, DNA fragments, being deposited in the interchromosomal compartment of the nucleus, due to the natural mechanism of homologous recombination (GR), replace mutant chromosome regions of the mutation in which CKK is not viable. As a result of these events, a reversible hollow or partial restoration of the functions of CKK, activation of the mitotic process and the entry of CKK on the path of differentiation. In the indicated process of exposure of exogenous fragmented DNA to CKK, the property of CKK (and other types of CK, including ESCs) is important in the course of several (1-3) first mitoses to completely rearrange its genome, leading it to a cometated state. At the same time, numerous chromatin breaks occur in the CKK genome, which are an integral part of the genome rearrangement process. If at this moment homologous DNA of the required size in complex with the proteins of the nuclear matrix is located in the CK nucleus, then it becomes available for integration into the genome by the type of homological recombination, while the activity of this process due to the appearance of numerous physiological, functional breaks in chromatin increases many times. In addition, the activating effect of DNA fragments on the differentiation of CKK is associated with their mimicking effect. Fragments of extracellular DNA delivered to the nuclear space introduce a large number of double-stranded ends into the cell nucleus, which the cell perceives in the Jacques their own functional chromosome breaks, which are formed and quickly repair during their terminal differentiation. This mimicking effect of extracellular DNA fragments localized inside the CKK nucleus triggers the mechanisms of cellular differentiation, activates and maintains the cometary division of CKK in a continuously active state. Thus, when a fragmented allogeneic DNA product obtained from physiologically and genetically healthy donors is introduced into the body, it is absorbed by CKK during division by the natural delivery mechanism inherent in actively dividing cells (receptor-mediated pinocytosis). The fragmented DNA delivered to the nuclear compartment — the interchromosomal space — is deposited and enters into the process of homologous exchange with the corresponding chromosome loci at the moment of the CKK genome rearrangement and the numerous temporary breaks in chromatin associated with this. Due to the natural mechanism of homologous recombination, DNA fragments deposited in the interchromosomal space replace mutant chromatin regions, mutations in which led to non-viability of CKK, and can also replace any mutant loci of CK genes with non-mutant ones. As a result of these events, the CKK genome undergoes partial or complete correction, which leads to the restoration of the mitotic potential of CKK or to the complete restoration of the damaged CKK genome. At the very moment delivered Extractive DNA fragments bring a large number of double-stranded ends into the nucleus of the cell, which the cell perceives as its own functional breaks in the chromosomes that form and quickly repair during their terminal differentiation. This mimicking effect of extra cellular DNA fragments localized inside the CKK nucleus triggers the mechanisms of cellular differentiation, activates and maintains the cometated CKK division in a continuously active state.

The effect achieved by using the present invention can be justified by the following theoretical data.

According to the modern classification, stem cells include moripotipotent morula cells, blastocyst pluripotent cells, from which all body tissues are formed in the body, and multipotent regional germ cells [Orkip SH, Morrison SJ. Stem-sll com. Ibid. 2002. V. 418. #. 6893. P. 25-27]. Regional or tissue-specific stem cells persist throughout the life of the body, self-sustain and provide a constant replenishment of lost cell populations. Tissue-specific CKs contribute to the regeneration and maintenance of numerous, if not all mammalian tissues, including blood, liver, intestines, skeletal muscle, and the central nervous system. Until recently, it was believed that tissue-specific CK are determined in certain types of cells and distributed in the tissues from which they originated. Nevertheless, in recent years, a large number of works have appeared that testify to the much greater plasticity of regional CK [Veda F. V. Teshpau A., Еbelt Y., Сhulze M., Beaun T. Аtivаtiop оf myogenic differentiation ratwaups and adult bope mаrrоw-deverеdеd STEM SELLS. MoII. CeII. Viologu. 2005. V. 25. Na 21. P. 9509-9519; Kues WA, Retersep W., Musegades W., Carpath JW, Niemapp H. Isolatop of murpe apd rorsip fetal stell sommotomis paid. Viologu of rrrodastiop. V. 72. 1020-1028. 2005]. For example, it was confirmed that CK neurons isolated from mouse brain tissues differentiate into blood cells, skeletal muscles and endothelial cells, that CK human adipose tissue cells differentiate into bone, muscle and cartilage cells, and also that mammalian skin CK dermis differentiate into skeletal cells muscles, neurons, glial and fat cells. It has been shown that some rare cell populations, the so-called multipotent progenitor cells, are able to differentiate into derivatives of all three germ layers. Also, the ability to differentiate into cells of non-related tissues was demonstrated for bone marrow CK initially involved in the formation and maintenance of a blood cell population. The discovery of the phenomenon of stem cell plasticity and the possibility of their differentiation into tissue cells of ecto-, meso-, and endodermal origin, as well as the ability of CK to migrate from the bone marrow and replenish stem cell pools of various tissues, allowed us to formulate a concept about the general regenerative mechanism of tissues, where the main role is played by Ck. This served as the basis for the emergence and rapid development of a new direction - regenerative medicine, based on the use of CK cells in order to restore the cellular composition of organs and tissues damaged due to diseases, exposure to chemical agents, injuries or age-related degenerative changes. One of the main and most frequently encountered negative types of effects on the regenerative processes of the body is radiation and chemo - therapies used in oncological practice. Due to the use of hard radiation or cytostatics of a different nature, along with cancer cells, all actively proliferating cells die, including stem cells. In particular, the consequence of this effect is a partial or complete violation of hematopoiesis, which leads to a negative prognosis. As you know, postembryonic hematopoiesis is a process of physiological blood regeneration, which compensates for the physiological destruction of differentiated cells. Particularly strongly regenerating potential of blood is affected by high-dose chemotherapy, which without a compensating effect gives a high percentage of toxic mortality. Currently, the high-dose chemotherapy (VHT) method has been widely used in conjunction with transplantation of autologous hematopoietic progenitor cells. The only source of hematopoiesis recovery is hematopoietic CK located in the bone marrow. For the autotransplantation procedure, stem cells (CD34 ⁺ ) are isolated from bone marrow or peripheral blood, frozen in the presence of a cryostabilizer (DMSO) and used to restore hematopoietic function of bone marrow damaged by radiation or high doses of cytostatics [Blum KG, Tomas ED A révéufutoutol Hematoroieticcell trapslaptatiop. AMERISAP SOCIETO FOR FORD APD MARROW TRAPSRLAPTATIOP. 2000. P. 1-12]. The proposed method of treatment is fundamentally different from the method based on the effect of mediator, colony stimulating molecules, which are growth factors that stimulate the activity of stem cells. Under CKK activity follows to understand the increase in the proliferative potential of CKK, the preservation of the ability to differentiate and develop specific types of cells, and it is these qualities of CKK that regulate growth factors. Growth factors include colony stimulating factors (CSF), interleukins, and inhibitory factors. All of them are glycoproteins with a molecular weight of about 20 CD and act both as circulating hormones and as local mediators that regulate hematopoiesis and differentiation of specific cell types. The action of growth factors extends to CKK, colony forming units (CFU), comet and mature cells. So, for example, with a decrease in the number of red blood cells and, accordingly, a decrease in the partial pressure of oxygen (PO ₂ ) is a signal for the production of erythropoietin. Erythropoietin acts on CFU-E sensitive to it, stimulating from proliferation and differentiation. Interleukins act on pluripotent CK, most CFU and sometimes terminally differentiated cells. Inhibiting factors give the opposite effect - inhibit hematopoiesis. Their deficiency may be one of the causes of leukemia, characterized by a significant increase in the number of white blood cells in the blood. A leukemia-inhibiting factor that inhibits the proliferation and differentiation of monocytes - macrophages was isolated. All of these effects are regulatory and are determined by mediator molecules of growth factors that act on various cellular mechanisms that trigger or regulate chromatin replication and transcription of various proteins that mediate the metabolism of proliferating and differentiating CKK. At the same time, growth factors are used to restore hematopoietic blood function.

In the case of treatment according to the proposed method, the damaged CK chromatin is restored and thereby the stem cell becomes able to carry out full mitosis and respond to the influence of growth factors. It can be said briefly that if there is nothing to stimulate, then no growth factors will help. At the same moment, in the treatment according to the proposed method, fragments of extracellular DNA delivered to the nuclear space with their double-stranded ends create the illusion of the appearance of functional breaks in the CKK chromosomes, which are the inductor of their terminal differentiation. This mimicking effect of extracellular DNA fragments localized inside the CKK nucleus triggers the mechanisms of cellular differentiation, activates and maintains the cometary division of CKK in a continuously active state. The mechanism of occurrence and maintenance of mutations in CK associated with the use of mutagens, the aging process, in our opinion, is as follows.

Genomic DNA of any organism is subject to mutagenic effects of environmental factors, such as ionizing radiation, chemical mutagens, as well as internal factors, including errors in cell repair systems. The well-known reparative mechanism of the cell eliminates DNA damage or incorrect nucleotide substitutions and covalently connects single-stranded and double-stranded breaks (OCR, DTSR) after mutational events [Noijmakers J. H. Genome maintenance mesophisms forreceptor sapser // Natury. 2001. V. 411. P. 366- 374]. In the case when damage affects both DNA strands and has a noticeable extent, the repair of such sites becomes problematic and damage often leads to mutations. DSBs are an example of lesions that are almost impossible to repair with the known repair mechanism, since they affect both the chromosomal integrity and the integrity of the DNA molecule itself. A fast mechanism for repairing double-stranded breaks is known, including the use of KU70 / KU-86 proteins and nuclear DNA-dependent protein kinase, which combine the ends of the DNA and restore genomic integrity [Pfeifer P. The mutagenic potential of DNA double-strapd repair // Toxicol. Lettt. 1998. 96. 97. V. 119. P. 1 19-129]. It is not possible to recover nucleotides or short DNA fragments that were lost during simultaneous multiple breaks of a DNA molecule. And as a consequence of such losses after the restoration of the integrity of the chromosome, chromosomal aberrations occur, and DCRs at the same time act as a source of mutations.

In modern oncological practice, chemical cytostatics and hard γ radiation are widely used, which violate the integrity of double-stranded DNA due to the alkylation effect in the first case, and the direct action of rays and the action of free radicals resulting from ionization in the second. Multiple double-stranded breaks occurring in DNA during this kind of exposure lead to a stop of the synthetic processes in the nucleus and to disruption of the cell cycle. Ultimately, a self-destruction program is activated, and the cell either undergoes apoptosis or profound genetic changes occur in it. It is such DNA damage that occurs in all cells of the body, and especially in actively proliferating ones, leading either to the death of these cells or to the emergence of mutant descendants. Unfortunately, along with cancer cells, other actively dividing cells of the body, such as epithelial cells, white blood cells, stem cells of red and white blood sprouts, hair follicle cells, and others, die or mutate. The most dangerous is the destruction of blood cells supporting the body's active immune response. A number of works have hypothesized the existence of a natural mechanism that can affect the genetic component of cells in multicellular organisms using extracellular genomic DNA from biological fluids as an external genomic standard [Yakubov, LA, RetrovaN.A., PorovaKA, FamilyV DV, Nikolip, VP , Oskipa, IN RoIe оf expracellulаr DNA in maintaining оf stаbilitu аd varіаbilituf оllulаr gepomes. Doklada Biochemistr ap Biorshus 382, 31-34, (2002); Yakubov JA, Porova NA, Nikolip DV, Symepov, SS Vogashev apd IN Os'kipa. Extreceiluiar genomic DNA processes mixtures agaipst radiopath apd chemil mutageps // Gepom Biol. 2003. V.5. R.Z.]. According to the proposed mechanism, DNA-binding receptors located on the cell surface deliver fragments of genomic DNA from the external environment to the nucleus, where they replace the corresponding homologous fragments in cellular genomic DNA through the mechanism of homologous recombination.

Next, we will consider the question of how mutated DNA fragments from cells susceptible to mutagenic effects can be used in the progressive development of mutational changes in the body.

Elements of DNA circulation in the body, such as extracellular DNA of the bloodstream, DNA transport to the nucleus, and somatic homologous recombination can be the essence of a mechanism that can remove any fixed or newly formed mutations from the cellular genome. The effectiveness of this process fully depends on the quality and properties of the external DNA standard. On the one hand, this mechanism can remove mutations, replacing them with exogenous non-mutant sequences; on the other hand, this mechanism can introduce mutations into the cell, replacing healthy cell sequences with mutant exogenous homologous fragments. The latter is especially applicable to the situation that arises in the body immediately after exposure to a strong mutagenic agent. It is known that chemical mutagens as well as hard radiation induce apoptosis, as a result of which DNA fragments with a large number of various defects, including lethal ones, are released into the bloodstream [Leop SA, Sharigo V., Skloff DM, Yaros MJ. Fgee DNA Ip thе serum оf sapperrattiepts аnd thе effеt оf thеraru // Sapser Res. - 1977. Vol. 37. P. 646-650; Nikolip VP, Calide VI, Matiepko NA, Gruntenko EV Сis-Diаmmiпеpediсhlororlаtipum (II) а а and аtotheptal сhemotheterareutiс agept agaipst peorlastiс lesiops іp thе liveg. J. Slipal Nemat. Opt. 1979. VoI. 9. - P.198-207].

The DNA transport mechanism delivers all these fragments to cells, and GH incorporates them into the cellular genome. Further, cells that received DNA fragments carrying lethal mutations die, and sequentially secrete their genomic DNA containing mutant sequences into the environment. Cells that received DNA that does not carry lethal mutations survive, and can further constitute the starting population for breeding, for example, a cancer clone. Among apoptotic DNA fragments, those that are formed as a result of repair without homology may be present. Such fragments will contain combined DNA fragments from different regions of the genome from one or different chromosomes. These “chimeric” fragments will be inserted into the genome of the recipient cell, taking into account the homology of the terminal regions of the fragment. Since these ends can belong to different parts of the chromosome or to different chromosomes, recombination events can lead to different chromosome aberrations. It is assumed that mutant DNA fragments of cells exposed to a strong mutagen will be re-delivered from dead cells to living cells. These fragments will induce the same mutations and chromosomal aberrations in living cells, and during numerous rounds of cell death, these fragments will propagate the defect that occurred during the initial exposure to the mutagen.

According to the existing theoretical and experimental assumptions on which our reasoning is based, if healthy DNA fragments that make up the whole genome homologous to the recipient’s genome are present in the bloodstream of an organism exposed to a strong mutagen or high dose of radiation, then they will compete with the DNA circulation system with fragments ejected into the blood as a result of the events described above after the action of a mutagen. Healthy fragments will be a substrate for reparative homologous recombination in the nucleus and their participation in reparative processes will lead to the correction of mutant loci and, as a result, to the appearance of a healthy population of the corresponding group of cells. The fragmented DNA preparation contains a pool of healthy fragments of specially processed human DNA, due to which there is a direct effect on the damaged CK genome leading to recovery

AND chromosome integrity and restoration of the ability of CKK to carry out chains of sequential mitoses with subsequent differentiation.

The proposed method of treatment based on the effect on CKK can be used for any genetic disorder, correcting it in the CKK pool, which are then transplanted into the same organism. The effect achieved by using the present invention can be justified by the following theoretical data.

Each cell of the body has genomic DNA, in which information is encoded both about all the proteins of all body cells and about the spatial organization of genes in the nucleus necessary for the correct spatio-temporal expression of these proteins. As a result, genomic DNA is a matrix of the body’s life that includes information about the body as a whole, as well as information about its development. Initially, genomic DNA is identical in all cells of the body. However, as the body grows, the genomic DNA of each cell undergoes mutational influences caused by environmental factors and errors that occur during cell replication and repair. It is known that repair mechanisms replace incorrect or defective DNA bases and again close the ends of the DNA molecule after one- and two-link breaks (DCB) of chains immediately after mutational events. For this, they can use a second DNA strand as a template. In cases where the lesion sites are very long and affect both DNA strands, repair becomes problematic, and a mutation may result from the violation. Thus, mutagenic environmental factors and errors in cell repair and replication are sources of somatic mutations in cells. The most important for the regenerative potential of the body are pluripotent CK. Defects in the genome structure of these cells entail the appearance of defective tissue and the formation of pathology. The only pathogenetically substantiated approach to the treatment of diseases associated with genetic disorders, including the structure of the CK genome, is to change, in one way or another, the genome of these cells of the individual’s body and restore the original genetic homeostasis characteristic of a healthy cell.

Numerous methods of gene therapy have been developed in modern experimental medicine, but all of them have serious shortcomings and are ineffective in the framework of a complex eukaryotic organism. Classical methods of gene therapy (gepetargetipg, gene targeting) aimed at correcting chromosomal mutations using viral constructs able to efficiently introduce working copies of genes into the cellular genome. Nevertheless, providing random insertion of the gene and regulatory regions, these approaches are associated with a number of problems including the dependence of gene expression on the site of integration, silencing of the introduced viral genome, and insertional mutagenesis associated with carcinogenesis [Khop DB, Sadelain M., Glorioso J. C. Ossurreps Of leukaemia fallowipg hepe teruof of X-lipid SCID. Natuge Rev. Sapser, 477-88 (2003); Resops, DA, Niephuis, A.W. Gepe théraru of hemoglobip disogeders. Nematol. Re 2, 348-355 (2003)]. An attractive alternative to these methods could be the correction or substitution of a mutant gene in the chromosome by natural homologous recombination with a DNA matrix that does not contain mutations delivered to the cell, if it were not for the extremely low efficiency of the method found in early attempts to use it [Yapez, RJ, Porteg, ACG. Theareutic Gepe Targetig. Gepe thearu. 5, 149-159 (1998); Rothstеip RJ. Operationalgepe disshtiop ip west. Methods Epzumol. 101: 202-11 (1983)]. Historically, a gene substitution strategy (heteropercept) developed to overcome the problem of insertional mutagenesis on yeast and later on mammalian cells included the use of linear recombinant DNA in which the two ends are homologous to the regions flanking the replaced gene, and the gene itself is replaced by a selectable marker [Thomas KR, Sarehi. Sit-direction mutagepsis bu gepe targetg ip mousem-derived couplings. CeII. 1987, 51 (3): 503-12]. This type of gene targeting is called “ends-out” because the ends of the construct correspond to two diverging (from the target gene) chromosomal DNA sequences. At the same time, the ends of “ends-out” gene targeting are recombinogenic and facilitate the replacement of the gene sequence between the terminal homologies. Recently, it was shown that in yeast when replacing an entire gene with a heterologous sequence or replacing a single mutant base pair with this method, the process is initiated by two independent "strapd ipvasiops" (chain insertion), which implies the search for homology and recombination events carried out by two terminal segments of the therapeutic construct [Lapgstop LD, Symingtom LS. Gepe targetg ip ueast is iptiad bu two ipredept strapd ipasiops. Rros NM Acad Sсi USA. 2004, 101 (43): 15392-7]. A similar mechanism was previously proposed to explain the “ends-out” of gene targeting in mammalian cells [Li J, Red LR, Vac MD. The Meshapism of the Mammaliap of the HepéReplacement is the support with the Formatiop of the Lopg Regiops Oi Heterodullech DNA Associated with the Twocsipg-OVER EVPTS. MoI CeII Biol 2001 Jap; 21 (2): 501-10]. Somatic GR, judging by the fact that it has survived for billions of years from protozoa to mammals, should obviously fulfill the absolutely necessary and universal function in all the kingdoms of the living, and perform it effectively. Since somatic homologous recombination (GR) in unicellular yeast is the main mechanism for the precision repair of DCR, its effectiveness must be very high to ensure the salvation of the individual. Early experiments with microinjections of plasmids into the cell nucleus led to the discovery that plasmids were always integrated into the cell genome in the form of one concatemer consisting of all (often more than one hundred) injected plasmids assembled into a single molecule tail to head, and that the result was surprisingly effective the work of the GR mechanism [Saresshi, M. Gepertipg n \ ice with target mutatiops. NATURE MEDICIPE 7, 1086-1090 (2001)]. The reason for the low efficiency of classical gene targeting involving the use of GR in mammalian cells may be a mismatch between the task of GR and its properties and capabilities selected by evolution to perform its own, albeit unknown function. The data described in the literature allow us to identify some factors that are critical for the operation of the GR mechanism.

Important for the effective functioning of the GR mechanism is the linear size of the DNA fragments delivered to the location of the recombination complex. This describes a 100-fold exponential increase in the effectiveness of gene targeting with an increase in the length of homology in the corrective design from 2 bp to 14.5 bp. and at the same time, the achieved efficiency, 10 ^{~ 5} events of gene correction per cell, remained nevertheless insufficient for the application of the method without selection [Depg, C. app. thе target vestоr apd thе target losus. MoI CeII Biol. 12.3365-3371 (1992)]. A striking contrast to the above data is presented by the results on the use of short fragments of homologous DNA (SFHR, smallest homologous receptacle) for correction of a mutant gene with a correction efficiency of 1-20% per cell [Group D., Brusia E., Noveli G. A. Colosi , Dallarissola B., Sangiuolo F., Gopsz K. Sewekse-sresifiс modifiсtiop оf genomic DNA bu small DNA fragmepts. CHn. Ipvest. 112, 637-641 (2003)]. The gene targeting efficiency obtained in these experiments may mean that GR prefers to work with either short fragments or forms from short fragments by ligating DNA fragments with ligase IV of the type head-tail multimeric fragments that are most effective in use in recombination events.

A prerequisite for obtaining a recombinant product in mammalian cells is the length of the terminal homology of the fragments, which is about 200 bp (base pairs) [Un Y., Lukasovish T., Weldmap A.S. Multirle ratthouses for DNA of DNA double-strapd breaks ip mamaliap chromosomes. MoI CeII Biol. 19, 8353-60 (1999); Nassif N., Engels W. DNA homologous protein for mit mitotis gift rerair ip Drorshila. Roc. Nail, Acad. Sсi. USA 1993 Feb 15; 90 (4): 1262-6].

Of fundamental importance for the implementation of GH is the linear form of the delivered therapeutic DNA as a source of double-stranded ends. The ring forms of therapeutic plasmids practically do not participate in homologous exchange. The generation of DSBs and the formation of double-stranded ends of the DNA molecule, locally activates and enhances chromosomal and extrachromosomal recombination and gene targeting [Kucherlätti RS, Eves EM, Song KY, Morms BS, Smiths Otomépelimés bémépém iprut DNA. Rros NatlАсadSсi USA. 81, 3153-7 (1984)]. This principle was used to induce the correction of the IL2R gene, a point mutation in exon 5 of which causes a deadly hereditary disease associated with the X chromosome, severe combined immunodeficiency (lack of combined immunity, SCID). By transfecting cells with genetic constructs expressing synthetic zinc-finger nuclease capable of specifically cleaving a sequence near a mutation in the IL2R gene in the genomic DNA of cells and a plasmid containing a non-mutant fragment of the same gene that overlaps the SCID region of the mutation showed a high, up to 20% level of gene correction [Umоv FD, Miller JC, Lea YL, Beausеjоur CM, Рosk JM, Аustustus S, Jamеsop AC, Рroteus MH, Gregoru PD, Holmes MC. Nighlfficient epogepous humap geporistiop uspig desigpid zip-fipuser. NATURE 435, 646-51 (2005)].

It can be assumed that, in principle, the ability of GRs to very efficiently carry out gene correction is associated with the initiating start of the appearance in the nuclear space of free double-stranded DNA ends, whether it is the ends of a broken chromosome formed as a result of DCR, or the double-stranded ends of DNA fragments delivered to the nucleus and which are typical “ends-out” »Structures. Any unmodified fragment of genomic DNA, having two ends homologous to two sequences of chromosomal DNA and the middle part is able to replace the genomic fragment and accordingly correct the existing mutation, is a typical "ends-out" design. This means that the ends of the fragment will initiate a search for homology and GR with genomic DNA, leading to the replacement of the genomic DNA fragment by the central part of the fragment delivered from external to the cell medium. Under conditions of using genetically correct exogenous DNA, these events will lead to the correction of a genetic defect [Yakubov L., Porova N., Nikolin V., Semepov D., Bogasheva S., Oskina I. Excellulogenomic DNA processes mixtures agadicompactiagi Gepote Biologu, 2003.5: RE].

Another significant point, apparently affecting the efficiency of homologous recombination and correction of defective chromosome loci, is the effect of “genome opening” observed in CK and other differentiating cells when they enter the pathway of differentiation [Farzaphe F ., Zalin R., Brill D., Shall S. DNA strapd breaks apd ADP-ribosul trapheras astiviop duriпg cell diffeptiatiop. NATURE. V. 300. P.362-366; Johpstope A.R., Williams GT. RoIe of DNA BREAKS APD ADP-ribosi! trapsfеrе аsеtivitu іp eukaguotis differeptiаtiop demopstrаsted іп humap lіmrhosutes. NATURE. V. 300. P. 368-370; Vatolip AY, Okarkipa EV, Matveeva NM, Shilov AG, Baiborod SI, Rhilimopepko VV, Zhdapova NS, Serov O. L Scheduled repturbatiopédépépédefépépédefép MoI Reród. Develor. V. 47. P. 1-10. 1997]. In the study of ribosyltransferase activity, the phenomenon of the appearance of numerous single-stranded chromatin ruptures in differentiating cells was described, the appearance of which involves the involvement of genetic material in the reorganization, which is an indispensable and integral part of differentiation and sequential change in gene expression. In different tissues of one organism (one person), different organization of genetic material is revealed after chromatin reorganization as a result of single-stranded (or double-stranded) breaks. The number of breaks per haploid genome of differentiating cells in different studies is estimated at several hundred (100-300 breaks per halide genome). At the same time, it was shown that this phenomenon is not a defect in the repair system, but a necessary mandatory step in the process of terminal cell differentiation. So when inducing about 600 additional breaks at the time of the largest number of immanent breaks in the cell using hard cell irradiation, the repair rate of these 600 additional breaks was the same as for cells that are not in the stage of differentiation. At the same time, the number of breaks dropped to the initial level. Important to explain the essence of the invention is the discovered fact that at the time of terminal differentiation and the occurrence of chromatin breaks, the number of rearrangements in the genome increases very significantly. It has been shown that the level of sister chromatid exchanges is 5-6 times higher in the pre-implantation period, that is, at a time when chromatin reorganization is observed with the identification of functional chromatin ruptures. During the first three mitoses, when a dramatic reorganization of the genome is detected, other recombination events also occur, such as shuffling of minisatellites and lengthening of the chain of simple repeats. The proposed mechanism for this kind of exchange in the genome is homologous recombination. However, in all these events, the first step is the appearance of functional breaks in chromatin DNA.

It can be assumed that in the presence of therapeutic fragmented DNA in the nuclear space at the time of chromatin reorganization into the first three mitosis of CK, homologous recombination takes place between extracellular exogenous fragments of therapeutic DNA and accessible sections of CK chromatin, in which defective loci are restored. This leads to the rescue of CK from death. In this case, the initial properties of the stem cell are restored to carry out a chain of sequential mitoses and determine the direction of terminal differentiation. At the same moment, fragments of extracellular DNA delivered to the nuclear space with their double-stranded ends create the illusion of the appearance of functional breaks in the CKK chromosomes, which are the inductor of their terminal differentiation. This mimicking effect of extracellular DNA fragments localized inside the CKK nucleus triggers the mechanisms of cell differentiation, activates and maintains the cometary division of CKK in a continuously active state.

The stated technical problem is achieved by the fact that it is based on the developed theoretical concept of extracellular DNA turnover in the body, confirmed by numerous experiments. The main provisions of the concept of extracellular DNA turnover in a eukaryotic organism are formulated below.

Extracellular fragmented genomic DNA containing mutant sequences in proportions corresponding to the occurrence of specific mutations in cells is formed as a result of programmed cell death or apoptosis of body cells and is always present in the intercellular space and blood plasma [Apker P, Mulshahu H, Chen XQ, Stroun M. Detestsop оf сirculatipg tumoig DNA ipе blеd (рlasma / serum) оf sapper paratepts. Sapser Metastasis Rev. 18, 65-73 (1999); Giasopa M.V. Ruben G. S., Iszkowski K. Roots T., Porteg D., Sorenson G. CeII-free DNA ip humap blodplasma: lepth mesurepts ip ratepets with rapeseptsapress aphethrops // rap. 1998. V. 17. P. 89-97]. Cells are capable of capturing the resulting DNA or chromatin fragments by a receptor-mediated mechanism and transporting them to the cell nuclei [Yakubov, LA, Deweva, EA, Zarutova VF, Yevapova EM, Rutе, AS, Yurskhepoko, Vlеkеlсhеpеmism IPvoltage of sresifiсrespertors? Roc. Nail. Acad. Sсi. USA 86, 6454-6458, (1989); Shestova OE, Andreeva A.Yu., Vlasov V.V. Yakubov L.A. Transport of complexes of oligonucleotides with proteins of the cell surface into the cell nucleus // DAN. 1999. T. 368. S. 264-267]. The delivered fragments recombine with genomic DNA in the nuclei by the mechanism of homologous recombination (GR) and are capable of initiating this process themselves. It can be assumed that together these three processes constitute a mechanism capable of controlling the genetics of somatic cells at the body level, correcting mutations or inducing genetic changes in cells using extracellular DNA from tissue fluids and blood plasma as an external genomic standard. The work of such a mechanism could serve as the basis for the genomic unity of body cells. This mechanism could eliminate the emerged and established mutations, as well as introduce mutations into the cells using the mutant sequences of the extracellular DNA standard. In that case, if the mechanism could use the standard of DNA, practically devoid of harmful mutations, its work would be exclusively the elimination of mutations in cells of the body. Since allelic genes usually have a high degree of homology and often differ only in relatively small regions of altered sequences, another aspect of the proposed mechanism could be the replacement of allelic gene sequences in the chromosome with alternative ones, similar to replacing mutant sequences with non-mutant ones.

The stated technical task is also achieved by the fact that the complete genome of the individual is introduced into the body in the form of a set of polynucleotide molecules of a certain length, which are fragments of genomic DNA, circulates and is captured by CK, enters the nucleus, where it is deposited and homologous recombination with the genomic DNA of the cell during reorganization of the CK genome accompanied by the emergence of physiological single-chain breaks. Thus, it becomes possible to correct genetic defects present in CK by introducing into the body a complete genome in the form of polynucleotide molecules of a certain length, which are fragments of genomic DNA that does not have genetic defects.

In the claimed method of treatment, a fragmented homologous DNA preparation is used, which is represented by a set of fragments making up the whole genome of a physiologically and genetically healthy individual and in association with proteins of the nuclear matrix. The size of exogenous fragments corresponds to the size of fragments circulating in the patient’s blood plasma that arise during apoptotic nucleolysis (1-30 nucleosome units). The prototypes use xenogenic DNA material mixed with RNA molecules. This DNA is not related, is not in association with the proteins of the nuclear matrix and does not correspond to the size of the body’s natural apoptotic DNA of 1-30 nucleosome units. Given the distinctive properties of the proposed method of treatment based on the properties of CKK to respond to the effect of the fragmented DNA preparation were investigated on a laboratory line of mice (ip vivo). The inventive method of treatment using the property of CKK to respond to the effect of a fragmented DNA preparation was studied on experimentally isolated human table cells (ex vivo). The restoration of hematopoiesis, the rescue of CKK and, as a result, the rescue of mice from death after lethal irradiation with hard radiation was studied in mice (in the ip vivo system). A series of experiments was conducted to study the protective effect of the fragmented DNA preparation at various doses. There are early classical experiments in which mortally irradiated mice (having lost their own hematopoietic cells) were injected with a suspension of bone marrow cells or fractions of enriched CKK. In this case, cell colonies appeared in the spleen, descendants of donor CKK. Each CKK in the spleen forms one colony and is called the colony forming unit of the spleen. Colony stimulating factors and interleukins modulate proliferative activity. Counting colonies allows you to judge the number of stem cells delivered to the bloodstream by infusion. Thus, it was found that 10 ⁵ bone marrow cells account for 50 CK, from the spleen 3.5 and among white blood cells - 1.4 CK. The study of purified CKK in an electron microscope suggests that CKK in its ultrastructure is close to small dark lymphocytes.

The study of the cellular composition of the spleen colonies revealed two differentiation lines of the studied CK. One line gives rise multipotent cell - the ancestor of granulocytic, erythrocyte, monocytic and megakaryocytic hematopoiesis series (CFU-GEM). The second line gives rise to a multipotent cell - the ancestor of lymphopoiesis (CFU-L).

According to the theoretical concept considered in this application, in the experiments performed, along with the integration of live CK into the lymphoid organs and the formation of colonies in the spleen, another effect leading to the same results could be the effect of extracellular DNA located in the introduced CKK sample. This DNA was genetically consistent with the healthy CKK genome and its effect on damaged inherent CK mice could restore CK viability through the homologous recombination mechanism described in the present invention. We tested this assumption under similar conditions, using either DNA extracted from the mouse (allogeneic) or a fragmented human placenta DNA preparation as a therapeutic agent.

Materials and methods.

In experiments, three-month-old female mice of the CBA / Las line of vivarium wiring of the Institute of Cytology and Genetics of the SB RAS were used. The animals were kept in plastic cages of 9-10 animals each with free access to food and water. Mice received granulated feed PK120-1 (Laboratsnab, Moscow). Mice were irradiated on the IGU-1 γ-unit (Cs ¹³⁷ ). Conducted 4 experiments.

In the first three experiments, various doses of nucleic acids and DNA of various origins (isolated from mouse organs, human placenta) were tested. In addition, the time of administration relative to radiation (before / after) was varied. The life span of the mice in each group was recorded.

The 4th experiment examined the effect of human placenta DNA on hematopoietic stem cells when administered to mice irradiated with a sublethal dose of gamma rays. Since the bone marrow is a radiosensitive tissue, when exposed to gamma rays, CKK die. It was assumed that exogenous DNA, penetrating the cell and being involved in reparative processes, allows CKK to survive. The surviving CKKs migrate and colonize lymphoid organs such as lymph nodes, spleen, thymus, etc. This contributes to the further restoration of blood formation and the preservation of the life of mice. Experiment 1.

Mice were irradiated with 9.6 Gy at a dose rate of 1.4 Gy / min. 10 mice (labeled) 30 mg prior to irradiation were injected intraperitoneally with 1 mg of DNA extracted from organs CBA mice (group 2). The remaining mice after irradiation were randomly divided into 3 groups.

Group 1, 30 min after irradiation and the next 2 days, was injected with a physiological saline solution of 0.2 ml intraperitoneally.

Group 3 was injected with 1 mg DNA of CBA mice in 0.2 ml of nat. solution 30 minutes after irradiation and in the next 2 days - 0.5 mg each.

Group 4 mice were similarly injected with DNA from human placenta.

In FIG. 1 shows data on the effect of mouse and human DNA on the survival of mice after their γ-irradiation in a lethal dose.

Experiment 2.

Mice were irradiated on a gamma-ray setup (Cs ¹³⁷ ) with a dose of 9.5 Gy at a dose rate of 1.3 Gy / min. One group of mice (n = 10) was injected intraperitoneally with DNA from the organs of mice of the CBA line: 20 mg before irradiation, 1-2 mg, 30 min after irradiation, and also 24 hours and 48 hours after irradiation - Vg of the initial dose . The control group of mice was injected with saline / b 0.5 ml (according to the DNA scheme). In FIG. Figure 2 shows the radioprotective effects of allogeneic DNA upon intraperitoneal administration to mice before and after lethal γ-irradiation. Experiment 3.

CBA mice ($) were irradiated on a gamma unit with a dose of 9.5 Gy (dose rate 1.3 Gy / min). Control mice (n = 10) 10 min before and 30 min after irradiation, as well as the next 2 days, were injected intraperitoneally with 0.2 ml of physiological saline. A human placenta DNA preparation was administered 20 min before irradiation, 1 mg each; 30 minutes after irradiation, as well as 24 hours and 48 hours after irradiation, 0.5 mg each. Similarly, the DNA of mice was introduced - either only before irradiation, or only after irradiation. In FIG. Figure 3 shows the radioprotective effect of allogeneic DNA upon intraperitoneal administration to mice before and after lethal γ-irradiation. Experiment 4.

Mice (n = 20) of the CBA line were irradiated with a gamma unit at a dose of 800 P (8.21 Gray). An experimental group of mice (n = 10) was injected with i.v. 1000 mg of fragmented human placenta DNA 30 minutes after irradiation another 500 μg, on the 2nd day - 250 μg, and on the 3rd day after radiation 170 mcg of DNA. Similarly to control mice (n = 10) was introduced physical. 0.2 ml solution. On the 10th day, the animals were killed and the number of splenic hematopoietic colonies was determined. Table 1.

Despite the small sample size and high variability of the trait in the experimental group, the differences between these samples are significant with a probability of> 0.95. On average, the number of splenic colonies increased 18 times with the introduction of intravenous DNA compared with the control. But this process is individual: it either proceeds or is not specific to each individual. A positive response to DNA injection was observed in ~ 50% of mice (Fig. 4).

Thus, the introduction of allogeneic DNA and xenogenic DNA of human placenta into mice allows animals to survive at lethal doses of radiation, that is, a specially processed fragmented DNA composed of nuclear matrix proteins has a pronounced radioprotective effect. Such survival of experimental animals is associated with the preservation of the effect of DNA preparations of CKK viability as a result of endogenous colony formation in the spleen of mice. The scatter in individual sensitivity in both the survival experiments and the KOE-C counting experiment seems to be related to whether therapeutic DNA was available to blood stem cells at the moment when functional breaks occurred in the cell manifesting the transition of CK into phase of terminal differentiation, go no.

The use of the property of CKK to respond to the effect of a fragmented DNA preparation, as defined in the claimed method of treatment, was studied on experimentally isolated human table cells (ex vivo). Extremely convenient objects for studying the healing and healing properties of a fragmented DNA preparation are bone marrow stem cells from patients with hemoblastoses damaged as a result of bone marrow leukemia and high-dose programmed chemotherapy with cytostatic drugs. This is dictated by the fact that CK are able, on the one hand, to give rise to all sprouts of hematopoiesis, and on the other hand, to maintain the constancy of their quantitative and qualitative composition without an influx of cells from outside. Moreover, the mechanism of self-reproduction and differentiation of cells of this group is at the same time a mechanism for regulating kinetic equilibrium in the entire pool of hematopoietic cells. With hemoblastoses, there is a deep violation of the basic mechanisms of CK cell homeostasis, the reasons for which are determined both by the presence of primary genetic defects of CK that cause the development of malignant hemoblastoses, and secondary damage to their genetic apparatus associated with the use of high doses of cytostatic drugs for the treatment of this group of pathologies.

Based on the foregoing, the goal of this study was formulated as a study of the modulating effect of the fragmented human DNA preparation on the proliferative activity of mononuclear cells and the differentiation of hematopoietic stem cells from the bone marrow of hemoblastoses subjected to program chemotherapy.

The main objective of this stage of the work was to evaluate ex vivo the effect of human exogenous DNA on various functional parameters of bone marrow cells in patients with hemoblastoses subjected to program chemotherapy. The program of the research phase included the study of the effect of the fragmented DNA preparation on the proliferative activity of bone marrow mononuclear cells and the generation of hematopoietic colonies by hematopoietic stem cells.

Research results.

The object of the study was bone marrow mononuclear cells (MHK). The MHK fraction was obtained from bone marrow aspirate by density gradient ficoll-verographin separation method (p = 1.078 g / cm ³ ). The study group consisted of 10 patients with hemoblastoses who received second-line PCT courses.

Bone marrow was taken by trepanobiopsy, which was performed during a routine diagnostic examination of patients after undergoing programmed high-dose therapy with cytostatic drugs. Marrow trepanobiopsy was performed in a specialized department hematology and bone marrow transplantation of the Clinic of Immunopathology of the Institute of Clinical Immunology SB RAMS (Table 2).

After a biopsy, the bone marrow was suspended in RPMI-1640 medium with 5% fetal calf serum (ETS). To obtain the MHK fraction, the resulting suspension was layered on a density gradient of ficoll-verographin, then centrifuged at 400 G for 30 min. The interphase layer containing MHK was collected and washed three times with RPMI-1640 medium with 5% ETS centrifugation at 200 G for 10 min. After the last centrifugation, the supernatant was replaced with RPMI-1640 medium containing 20% ETS. The final concentration of MHK was adjusted to 1 x 10 ⁵ viable cells in 1 ml of culture medium.

Bone marrow MHKs obtained from a part of patients were cryopreserved using programmed cooling of cells to –8O ⁰ C in the presence of a cryostabilizer (dimethyl sulfoxide). Subsequently, cryopreserved cells were stored at a temperature of -80 ° C until use, while the shelf life did not exceed 2 months. Before use, the cells were thawed and washed three times by centrifugation using RPMI-1640 medium with 5% ETS. After resuspension of the pellet, the concentration of viable mononuclear cells (MHK), as in the case of native bone marrow cells, was adjusted to 1 * 10 ⁵ cells in 1 ml of complete culture medium.

Table 2 Characterization of bone marrow MHK

Let us evaluate the effect of the preparation of exogenous fragmented human DNA on the proliferative activity of bone marrow mononuclear cells.

A model for studying the effect of exogenous DNA on the mitotic activity of MHK is an assessment of the intensity of their proliferation. The proliferative activity of bone marrow mononuclear cells was evaluated in tests of spontaneous and induced proliferative activity using standard techniques. Anti-CD3 monoclonal antibodies were used as an inducer of bone marrow MHK proliferation. Cells incubated with fragmented DNA (experiment) and cells not incubated with exogenous fragmented DNA (control) were examined in triplets. The proliferation activity was tested on the 3rd day of cultivation in 96 well plates for immunological reactions. The intensity of cell proliferation was evaluated by the incorporation of NZ-thymidine into the DNA of proliferating cells. Proliferation activity was expressed as pulses per minute (cpm). The results of studying the effect of exogenous fragmented DNA on spontaneous and stimulated anti-CD3 monoclonal antibody proliferation are presented in table. 3 and 4. As follows from the data obtained, after 1 hour of preincubation with DNA, a significant decrease in the mitotic activity of bone marrow mononuclear cells is observed (p <0.05). It is important to note that this phenomenon is registered not only under conditions of additional stimulation of the proliferative activity of anti-CD3 by monoclonal antibodies, but also observed in relation to the spontaneous mitotic activity of MHK enriched in hematopoietic precursors. Based on the calculated influence index (IV), characterizing the change in the intensity of cell proliferation in the "experiment" relative to the "control", the level of mitotic activity of bone marrow mononuclear cells after processing of exogenous DNA decreases by about 4 times and amounts to no more than 30% (14-35%) from its initial level in patients with hemoblastosis.

Table 3. The effect of exogenous fragmented DNA on spontaneous proliferation of bone marrow MHK.

Note. ^* Diagnosis: MM- multiple myeloma; * Cells: CB - freshly isolated, PM- thawed cryopreserved; * IB: DNA influence index.

Table 4. The effect of exogenous fragmented DNA on the stimulated proliferation of bone marrow MHK.

Note. * Diagnosis: MM-multiple myeloma; ^* Cells: CB- freshly isolated, PM-thawed cryopreserved; * IB: DNA influence index. Thus, our results indicate an active, clearly pronounced effect of exogenous DNA on the proliferative status of bone marrow mononuclear cells, including hematopoietic stem cells.

Turning to the discussion of the obtained result, we will proceed from the well-known self-maintenance mechanism of the CK pool, which causes a certain number of resting stem cells per unit time to begin differentiation without dividing in the direction of specific hematopoietic elements - committed precursors of erythroid, ganulocyte-macrophage and other hematopoietic growths. Differentiation, in turn, causes “excitation” in the same (per unit time) number of resting cells. The “excited” cells pass from the G ₀ phase to the mitotic cycle and are not capable of differentiation until they complete the division. After division, daughter cells enter the G ₀ phase again _, replenishing a group of cells potentially capable of both differentiation and return to the mitotic cycle. These basic mechanisms are violated in hemoblastoses, as a result of which there is uncontrolled proliferation of malignantly modified hematopoietic cells, which are not capable of normal differentiation. At the same time, the amount of labeled thymidine included in the cells characterizes the fraction of dividing cells at a given time, that is, in fact, reflects the number of active - “excited” stem cells that are in the mitotic cycle and are not capable of differentiation.

Therefore, a significant decrease in the mitotic activity of bone marrow mononuclear cells during their processing of exogenous DNA suggests a change (increase) in the proportion of dormant stem cells (G ₀ -phase of the mitotic cycle), which become capable of differentiation towards committed specific hematopoietic elements. Such a calming of CK is associated with the effect of the Panagenum preparation on the genetic loci of the chromosomes of cells that underwent cancerous degeneration. Moreover, as was shown in our other invention [Application for invention N ^Q 2006100561], the mechanism of this phenomenon can be associated with the replacement of mutant chromosome loci with non-mutant ones during which malignant metaplasized cells lose fully or partially the status of malignant degenerate.

Verification of this assumption can be carried out on the basis of assessing changes in the differentiation of hematopoietic stem cells contained in the mononuclear fraction of the bone marrow. Let us evaluate the effect of exogenous fragmented human DNA on the differentiation of hematopoietic stem cells in bone marrow.

Models for studying the differentiation of hematopoietic stem cells can be the method of formation of erythroid and granulocyte macrophage colonies on a semi-liquid medium [Рluzпiik D.H., Sсhs L. Те сlopiпg оf received “mast” sell ip sizing sulture. J. Cell. al. Phisiol. - 1965.-V.66.-P.319; Vadleu T.R., Metzlf D. The Gruwth of the Toe Bose Tag Sell IP Vitro. - Aust. J. exp. Biol. those. Sci.-1966.- V.44.- P.287]. Using chromosomal markers, it has been shown that cells of one colony develop from a single precursor cell. At the same time, the possibility of obtaining secondary colonies of this type by transferring the primary erythroid or granulocyte-macrophage colony to a new medium indicates that the source of colony development in vitro is committed stem hematopoietic cells [Metcalf D., Foster R. Behperulotherpodrum marrow colopies. Roc. Soc. exp Biol. - 1967.- V.126.- P.758].

Thus, the number of growing colonies on a semi-liquid medium, on the one hand, characterizes the total number of stem cells capable of differentiating in the direction of specific specific hematopoietic elements, and on the other hand, characterizes the direction of such differentiation, which can be judged by the ratio of different types of hematopoietic colonies. The change in mitotic activity due to the fragmented DNA preparation can have a different effect on the ability to differentiate stem cells in the direction of erythroid and granulocyte-macrophage hematopoiesis. This, in particular, is determined by the fact that, in their natural mitotic activity, committed precursors of erythropoiesis significantly differ from other committed precursors. If in the total pool of precursors, DNA synthesizes only 10% of stem cells, then among the committed erythropoietin-sensitive elements their share is more than 75%. The presence of the main part of erythropoietin-sensitive cells in the mitotic cycle suggests that a change in the proliferative activity of bone marrow mononuclear cells under the action of exogenous DNA can primarily affect the increase in the number of committed stem cells differentiating in the direction of erythropoiesis [LG Lactomycephylocephalitis .- In: Sumros. on normal and malignant Sell Grooth: New York.- 1969.- P.10]. Let us evaluate the effect of exogenous fragmented human DNA on the formation of erythroid colonies.

For the cultivation and counting of functionally active hematopoietic stem cells capable of differentiation in the erythroid direction, we used semi-liquid media prepared on the basis of methyl cellulose with the addition of erythropoietin (EP), recombinant interleukin 3 (IL-3), as well as a number of additional inducers of erythroid proliferation and differentiation cells [Goldberg E. D., Digay AM, Shakhov V. M. // Methods of tissue culture in hematology: Iz-in Tomsk state. u-ta.- Tomsk, 1992. -272s].

During this stage of the study, only cryopreserved bone marrow mononuclear cells were used. Thawed and washed, as described above, were divided into 2 aliquots. In the experimental series, bone marrow mononuclear cells (MHKs) were preincubated for 1 hour at 37 ° C with exogenous DNA, which was added to the incubation mixture at a dose of 100 μg / ml. In the control, cell culture medium was added instead of DNA in the appropriate amount.

After pre-incubation of MHK without washing, in the amount of 1 x 10 viable CD34 + cells were introduced into a semi-liquid medium for cultivation and

5 were placed in 24-well tablets at a dose of 0.25 ml per well (0.25 x 10 cells / ml).

Cells incubated with fragmented DNA (experiment) and cells not incubated with DNA (control) were examined in tetraplets (4 wells) containing a total of 1 x

10 bone marrow mononuclear cells. The plates were cultured in a CO2 incubator (37 ° C) at 100% humidity for 8-14 days. The number of erythroid colonies was estimated in dynamics and recorded on days 8 and 14 of cultivation. Erythroid colonies (PFU-E) included cell aggregates formed during cultivation that contained more than 100 erythroid elements of various degrees of maturity or a group (up to 50 cells) of small adjacent cells with morphological features characteristic of erythroid elements.

The results of counting the number of erythroid colonies on days 8 and 14, in the form of a range of fluctuations in the number of grown colonies in the tetraplet wells for each patient examined, are presented in tables 5, 6. Table 5. The effect of exogenously fragmented human DNA on the number of erythroid colonies on the 8th day of bone marrow MHK cultivation.

Note. * Diagnosis: LGM-lymphogranulomatosis.

Table b. The effect of exogenously fragmented human DNA on the number of erythroid colonies on the 14th day of bone marrow MHK cultivation.

Note. * Diagnosis: LGM-lymphogranulomatosis.

As follows from the results of the study, quantitatively characterized in the tables, after the preincubation of MHK with exogenous DNA, a significant, statistically significant increase in the number of generated erythroid colonies is recorded (p <0.001). In this case, the most demonstrative increase in the number of erythroid colonies after treatment with the preparation of fragmented DNA is determined in the early stages - on the 8th day of cultivation, when the increase in the number of forming erythroid colonies in the experiment exceeds the number of those in the control by 3-4 or more times.

Thus, the texture of the obtained data to a certain extent confirms the assumption about the stimulating effect of exogenous DNA on the ability of CKK to differentiate in the erythroid direction. At the same time, given that a sharp increase in the number of erythroid colonies in patients after undergoing high-dose chemotherapy occurs in the early stages of cultivation, it can be assumed that the action of exogenous DNA is not limited to the effect on the mitotic cycle, but is also determined by other mechanisms.

One of such mechanisms may be the participation of exogenous DNA in the repair of the stem cell genetic apparatus damaged by cytostatic drugs. Based on the results of the study, the participation of exogenous DNA in the repair of the stem cell genome can be assumed in at least 2 out of 5 studied samples of bone marrow mononuclear cells (40%), when against the background of the almost complete absence of erythroid colony formation in the “control” after preincubation with fragmented DNA, the ability of stem cells to differentiate into erythroid progenitors is restored. Thus, when using the model of erythroid colony formation, the effect of the preparation of fragmented human DNA on damaged hematopoietic precursors in 40% of cases is not quantitative, but qualitative, which is highly likely mediated by the inclusion of exogenous DNA in the genetic apparatus of the stem cell.

Other indirect signs of the inclusion of exogenous DNA in the stem cell genome may include morphophysiological features of erythroid colonies formed after pre-incubation with human fragments of human DNA. So, in the process of recording the results of the study, it was noted that in the “experienced” wells of the plates, in comparison with the “control” wells, immature - BFU-E erythroid colonies (70 - 90%) begin to prevail. The formation of the latter is characteristic of young highly active stem cells, atypical of the initial stem precursors obtained from patients with hemoblastoses after conducting program therapy with cytostatic drugs (<5 - 10%).

A drawback of the erythroid colony formation model is the impossibility of analyzing complex competitive relationships between different blood-forming sprouts, during which bone marrow stem cells can not only significantly change the nature of their differentiation, but also the severity of the response to various regulatory and recovery effects. Given this circumstance, at the next stage of the study of the effects of the fragmented DNA preparation, we were a more complex model for studying the functional state of stem cells was used — a model for the simultaneous cultivation of erythroid and granulocyte-macrophage colonies.

Let us evaluate the effect of exogenous fragmented human DNA on the simultaneous formation of erythroid and granulocyte macrophage colonies.

For the simultaneous study of the differentiation of committed stem precursors into erythroid and granulocyte macrophage elements, the complete commercial methylcellulose medium MetoCult ^® H 4434 (Stem CeII Teslogologu, Sapada, Cat N ° 04434) was used. Complete methylcellulose medium H 4434 contains a wider range of growth factors. In addition to erythropoietin (EPO) and interleukin-3 (IL-3), the complete medium contains stem cell growth factor (SCF), interleukin-b (IL-6), granulocyte macrophage cell growth factor (GM-CSF) and other biologically active components supporting the proliferation and differentiation of committed stem precursors.

The studies were conducted in 4 series of experiments, on MHK isolated from the bone marrow of patients with hemablastoses, after courses of programmed cytostatic therapy. The technique of the experiment and the concentration of bone marrow mononuclear cells in the “experience”, that is, after 1 hour of pre-incubation of cells with the preparation of human fragmented DNA and “control” were identical to those in the study of differentiation of committed precursors into erythroid cells (see the previous section). The only difference was that in addition to thawed cryopreserved cells, freshly isolated MHKs were also used to study the formation of erythroid and granulocyte-macrophage colonies.

Granulocyte-macrophage (CFU-GM), erythroid (CFU-E) and mixed-type colonies, i.e. colonies containing cell elements of both types (CFU-HEMM), were identified and counted under a microscope at the end of a 14-day incubation at 37 ° C in a humid atmosphere containing 5% CO2.

The results of counting the number of committed stem cells forming colonies of various types are summarized in table 7. Significant deviations in the number of formed hematopoietic colonies after processing of exogenous DNA mononuclear cells are marked with superscripts “+” and “-”, respectively marking cases of increase and decrease in indicators in the “experience” relative to the value of “control”.

Table 7. The effect of exogenous fragmented human DNA on the differentiation of bone marrow MHK into erythroid and granulocyte macrophage colonies.

Note. * Diagnosis: MM- multiple myeloma, ALL- acute lymphocytic leukemia; * Cells: CB - freshly isolated, PM - thawed cryopreserved.

As follows from the data obtained, in the presence of exogenous DNA, a significant 2-3-fold increase in the total number of formed colonies, both erythroid and granulocyte-macrophage type, is observed only when thawed cryopreserved bone marrow cells are used, characterized by an initially low number of colony-forming cells. This confirms our earlier assumption about the possible participation of exogenous DNA in the process of repairing defects that are the result not only of PCT, but also of the procedure for cryopreservation of bone marrow stem precursors. The latter, given the widespread use of cryopreserved stem cells in the treatment of not only hematological, but also many other diseases, is of not only scientific, but also of great practical interest. In this regard, the fragmented exogenous human DNA in combination with the proteins of the nuclear matrix can be considered as a new promising drug for an increase in the number of functionally active stem cells after cryopreservation, which has obvious patentability.

The effects of the preparation of human fragmented DNA on freshly isolated stem cells are more difficult to interpret. At the same time, as can be seen from the results of the study, the exogenous DNA of the studied drug undoubtedly affects the differentiation of stem cells. This is manifested in the selective, competitive increase / decrease of committed precursors involved in the formation of erythroid (PFU-E) or granulocyte-macrophage (CFU-GM) colonies, as well as an increase in the number of immature stem precursors forming mixed-type hematopoietic colonies (CFU-HEMM) .

Any cellular differentiation is a profound change in the entire genome of a cell, in which a pathway for the specific development of a pluripotent cell is selected.

The revealed phenomena can be considered as indirect evidence of the active participation of extracellular exogenous DNA in the genesis of blood stem cells.

Claims

Claim

A method of treating diseases based on the introduction of fragmented DNA into the patient’s body, characterized in that the fragmented DNA is allogeneic and consists of fragments whose length corresponds to 1-10 nucpeosomal units that make up the patient’s complete genome in association with nuclear matrix proteins, while the amount of fragmented DNA is taken to be equal to or greater than the amount of DNA in the blood plasma and tissue fluids of the patient, but not more than 1500 ng / ml.