US20170056482A1 - Method for treating systemic dna mutation disease - Google Patents

Method for treating systemic dna mutation disease Download PDF

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US20170056482A1
US20170056482A1 US15/287,447 US201615287447A US2017056482A1 US 20170056482 A1 US20170056482 A1 US 20170056482A1 US 201615287447 A US201615287447 A US 201615287447A US 2017056482 A1 US2017056482 A1 US 2017056482A1
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Dmitry Dmitrievich Genkin
Georgy Viktorovich Tets
Viktor Veniaminovich Tets
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CLS Therapeutics Ltd
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    • A61K38/00—Medicinal preparations containing peptides
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
    • C12Y301/21001—Deoxyribonuclease I (3.1.21.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates generally to medical treatment of the systemic DNA mutation diseases accompanied with development of somatic mosaicism and elevation of blood extracellular DNA and, more particularly, to a treatment of diabetes mellitus and atherosclerosis.
  • Mosaicism refers to a mixture of cells of different genetic composition in one individual. When DNA mutation is detectable in number, but not all somatic cells in one individual, it is called somatic mosaicism. Development of somatic mosaicism has been recently recognized as important mechanism of systemic DNA mutation diseases progression (Gott Kunststoff B et al., Selection and mutation in the “new” genetics: an emerging hypothesis, Hum Genet. 2010 March; 127(5): 491-501.) Importance of somatic mosaicism involving disease-causing mutations has been reported for variety of monogenic (reviewed by Youssoufian H., Nature Reviews Genetics 3, 748-758, October 2002) and more recently for multifactor DNA mutation diseases: cardiac rhythm disorders (M. H.
  • the systemic DNA mutation diseases represent very distinct subsets of human pathology different in etiology and pathogenesis and accordingly has fundamentally different, usually palliative treatment modalities-cholesterol lowering therapy for atherosclerosis (New Concepts and Paradigms in Cardiovascular Medicine: The Noninvasive Management of Coronary Artery Disease, K. Lance Gould, THE AMERICAN JOURNAL OF MEDICINE, Volume 104, Jun. 22, 1998, pp. 2-17) and insulin therapy or insulin sensitization therapy for diabetes mellitus (Pharmacological Management of Diabetes: Recent Progress and Future Perspective in Daily Drug Treatment, Gerard Emilien et al., Pharmacol. Ther. Vol. 81, No. 1, pp. 37-51, 1999).
  • Circulating extracellular nucleic acids were discovered more than 60 years ago (Anker P Circulating DNA in plasma or serum, Clin Chim Acta. 2001 November; 313(1-2): 143-6). However until now elevated levels of extracellular blood DNA in systemic DNA mutation diseases, and in particular in atherosclerosis and diabetes mellitus were considered only as useful diagnostic and research tool (El Tarhouny S. A. et al., Assessment of cell-free DNA with microvascular complication of type II diabetes mellitus, using PCR and ELISA. Nucleosides Nucleotides Nucleic Acids.
  • Circulating extracellular nucleic acids have never been considered as potential therapeutic target in systemic DNA mutation diseases. Accordingly, no therapeutic method was developed which targets extracellular blood DNA in systemic DNA mutation diseases. Thus it makes impossible to take any technical solution as prototype.
  • DNASE Deoxyribonuclease
  • Extracellular blood DNA number average molecular weight is a way of determining the molecular weight of a polymer.
  • the number average molecular weight is the ordinary arithmetic mean or average of the molecular weights of the individual DNA macromolecules. It is determined by measuring the molecular weight of n polymer molecules, summing the weights, and dividing by n.
  • the number average molecular weight of extracellular blood DNA can be determined by gel electrophoresis. The shift of extracellular blood DNA bands to low-MW areas reflect decrease number average molecular weight and in fact reflects enzymatic cleavage of extracellular blood DNA.
  • DNA mutation disease refers to diseases where specific DNA mutation has been identified as single leading cause (monogenic or single gene disorders) or multifactor disorders resulting from mutations in multiple genes, often coupled with environmental causes.
  • Systemic disease is one that affects a number of organs and tissues, or affects the body as a whole.
  • the object of this invention is to develop high-performance and low-toxic method for treatment of systemic DNA mutation diseases accompanied with development of somatic mosaicism and elevation of blood extracellular DNA and, more particularly, to a treatment of diabetes mellitus and atherosclerosis.
  • this task is resolved by introducing a treatment agent into a circulating blood system of a patient diagnosed with systemic DNA mutation disease when said treatment agent destroys extracellular DNA in said blood of said patient and wherein said treatment agent used to destroy said extracellular DNA is a DNASE enzyme.
  • said agent must be administered in doses and regimens which sufficient to decrease number average molecular weight of circulating extracellular blood DNA in the blood of said patient; such decrease of number average molecular weight might be measured by gel electrophoresis of extracellular blood DNA fraction from the blood of said patient.
  • the method according the invention can be effectively applied for treatment of diabetes mellitus and atherosclerosis.
  • a DNASE enzyme may be further applied in a dose and regime that results in a DNA hydrolytic activity measured in blood plasma that exceeding 1.5 Kunitz units per 1 ml of blood plasma for more than 12 hours within a period of 24 hours.
  • the present invention suggests that systemic DNA mutation disease can be treated by reducing of circulating extracellular blood DNA levels.
  • the extracellular blood DNA of patients with systemic DNA mutation disease contains the unique quantitative and qualitative repertoire of genes, which non-randomly represents human genome and contains genetic elements involved in to the development of the disease. It was shown that extracellular blood DNA might promote the development of somatic mosaicism and systemic DNA mutation disease.
  • FIG. 1 NF-kappa B expression in (A) cultured aortic endothelial cells treated with extracellular blood DNA fraction from patient suffering from systemic atherosclerosis; (B) cultured aortic endothelial cells treated with extracellular blood DNA fraction from patient suffering from systemic atherosclerosis plus 1 mkg/ml of DNASE1; and (C) control cells.
  • FIG. 2A-2B show a graph of the survival of NOD diabetic mice treated with different doses of DNASE1—50 mkg/kg, 500 mkg/kg, and control;
  • FIG. 2B shows average molecular weight of extracellular blood plasma DNA (as measured by electrophoresis) in blood of NOD diabetic mice treated with 50 mkg/kg DNASE 1 (A), 500 mkg/kg DNASE1 (B), and control (C).
  • FIG. 3A-3B Results of immunochemical staining of mice tumor's histological slices after administration of 5 day course of Doxorubicin therapy (2 mg/kg every day intravenous) and DNase 1 (0.5 mg/kg four times a day during 5 days).
  • 3 A Doxorubicin+DNase
  • 3 B Doxorubicin.
  • the inventive method is realized as follows:
  • bovine pancreatic DNASE Sigma, specific activity 2 400 Kunitz units/mg; Samson-Med, specific activity 1 500 Kunitz units/mg
  • recombinant human DNASE 1 Gentech, specific activity 1000 U/mg
  • Extracellular DNA from blood plasma was isolated as follows: fresh plasma (no more than 3-4 hours after sampling) with anticoagulant (sodium citrate) was centrifuged on Ficoll-Plaque Plus (Amersham-Pharmacia) during 20 minutes at 1500 g. at room temperature. 1 ⁇ 2 of plasma was detached, not affecting the rest of cells on the Ficoll pillow, and further centrifuged at 10000 g. during 30 min for separation from cell fragments and debris. Supernatant was detached, without affecting of the sediment, and was toped up to 1% of sarkosil, 50 MM tris-HCl, pH 7.6, 20 MM EDTA, 400 MM NaCl, and then mixed with equal volume of phenol-chloroform(1:1) mixture.
  • anticoagulant sodium citrate
  • the sediment of nucleic acids was dissolved in of 10 MM tris-HCl buffer, pH 7, 6 with 5 MM EDTA, and inflicted to the CsCl gradient (1M, 2.5M, 5.7M) in test-tube for rotor SW60Ti.
  • the volume of DNA solution was 2 ml, volume of each step of CsCl was 1 ml.
  • Presence of DNA in fractions was determined by agar electrophoresis and DNA was visualized by ethidium bromide staining. The amount of DNA was determined with spectrophotometer (Beckman DU70) in cuvetts (100 mkl) at wavelength of 220-230 nm.
  • NOD mice were obtained from ⁇ Pushcino>> animal breeding house.
  • Mononuclear cells were isolated from the whole blood samples using Ficoll-Paquee (Becton Dickinson). Mitogenic stimulation of the separated lymphocytes (1 ⁇ 10 6 /ml) was initiated with 1 mg/ml phytohemagglutinin (PHA) in RPMI 1640 media supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), 20% nutrient medium HL-1 and 5% BSA at 5% CO 2 at 37° C. for 24 h. Following wash the cells were then seeded in 96-well round-bottomed plates at cell density of 2 ⁇ 10 4 cells per well in selection medium to determine cloning efficiency.
  • PHA phytohemagglutinin
  • the cells were plated in 200 ml of the RPMI medium containing 1 mg/ml 6-thioguanine, 0.125 mg/ml PHA, 20% HL-1 and 5% BSA supplemented with interleukin-2 (BD Biosciences, 10 BRMP units/mi).
  • Four 96-well plates were seeded for each patient. After 5 days of culture, the colonies on mutant selection plates were scored for growth using an inverted microscope.
  • inventive treatment suppresses spread of HPRT ( ⁇ ) mutation and suppresses the development of somatic mosaicism.
  • the extracellular blood plasma DNA was purified from blood of patient ENV as specified in methods section.
  • Mononuclear cells were isolated from the whole blood samples of patients KNP, PGP, BAI and FW as specified in Example 1.
  • the mitogenic stimulation and selective cloning were performed as specified in Example 1 with modification as follows: during mitogenic activation stage lymphocyte cultures of patients KNP, PGP, BAI and FW were supplemented with 50 mkg/ml of extracellular blood plasma DNA purified from patient ENV. After 5 days of culture, the colonies on mutant selection plates were scored for growth using an inverted microscope. The results of selective T-lymphocyte cloning are presented in the table below:
  • extracellular blood DNA promotes the development of somatic mosaicism.
  • Type 2 Diabetes and Systemic Atherosclerosis Treatment of Atherosclerosis.
  • the probes of patient's extracellular blood DNA were taken before initiation and on day 35 of therapy.
  • the extracellular DNA was cloned by the method which allows to get non amplified plasmid libraries of blood extracellular DNA with representativeness up to one million of clones with the average size of 300-500 base pairs.
  • the DNA which has been isolated using the protocol specified in Materials and Methods section was treated with Proteinase K (Sigma) at 65° C. and subjected to additional phenol-chloroform extraction step with further overnight precipitation by ethanol.
  • the DNA fraction was than treated by Eco RI restrictase or by Pfu polymerase (Stratagene) in presence of 300 mkM of all desoxynucleotidetriphosphates for sticky-ends elimination.
  • the completed DNA was phosphorylated by polynucleotidkinase T4 and ligated to pBluescript plasmid (Stratagene), which had been digested with EcoRI or PvulI and dephosphorylated by phosphatase CIP (Fermentas).
  • the process of ligation was conducted with Rapid Legation Kit (Roche).
  • the ligated library was transformed into DH12S cells (Life Technologies) by electroporation ( E. Coli porator; BioRad). 12-20 electroporation covets were used for the transformation of one library.
  • the library serial dilutions were cloned on 1.5% agar and LB media supplemented with ampicillin. In both cases the libraries represented 2-3 ⁇ 10 6 clones.
  • GAD2 glutamate 2 2 One of the main pancreatic decarboxylase 2 autoantigens.
  • Phospholipase C, epsilon 2 Induces expression of receptors of low-density lipoproteins
  • BAI3 brain-specific 1 Angiogenesis inhibitor angiogenesis inhibitor Nicotinamide nucleotide 1 Involved in detoxification transhydrogenase of reactive oxygen species and insulin secretion.
  • 17 kD fetal brain protein 1 UNCLEAR CRTL1 cartilage linking 1 Involved in morphogenetic protein 1 process in heart and large vessels Transient receptor potential 1 UNCLEAR cation channel
  • extracellular blood DNA from patient having diabetes mellitus and systemic atherosclerosis contains significant non-random presence of human disease-relevant unique genomic DNA.
  • Extracellular blood plasma DNA sampled from patients blood at day 35 following start of DNASE therapy was assayed by gel electrophoresis and cloning. Analysis of 50 clones randomly chosen from the library obtained from the extracellular blood plasma DNA of patient on the day 35 after the beginning of treatment has shown that more than 90% of revealed clone sequences are short fragments of repetitive human DNA with the dominance of alpha-satellite DNA.
  • Endothelial NF-kappa B signaling orchestrates proinflammatory gene expression at the arterial wall and promotes the pathogenesis of atherosclerosis.
  • Endothelial NF-kappa B signaling orchestrates proinflammatory gene expression at the arterial wall and promotes the pathogenesis of atherosclerosis.
  • Blood plasma was obtained from vascular surgery clinic of St. Russia Medical Academy from the patient undergoing femoro-femoral bypass surgery due to severe atherosclerotic arterial occlusion.
  • the extracellular blood DNA was purified as described in Materials and Methods section.
  • the aortic endothelial cells (C-006-5C; Invitogen) were plated at density of 5-8 ⁇ 10 2 cells/mm 2 in multiwell (12 ⁇ ) cell culture plates in Clonetics® EGM®-2MV media (Lonza Cologne AG) and incubated for 48 h. at 37° C. and 5% CO 2 . Following 24 h of growth the culture media was supplemented with 50 mkg/ml of patient extracellular blood DNA fraction or 50 mkg/ml of patient extracellular blood DNA fraction plus human recombinant DNASE-1 (Genentech) at 1 mkg/ml concentration.
  • Extracellular blood DNA fraction from patient suffering from systemic atherosclerosis increases the expression of NF-kappa B in cultured aortic endothelial cells and treatment with DNASE ameliorate this effect.
  • Non-obese diabetic mice exhibit a susceptibility to spontaneous development of autoimmune insulin dependent diabetes mellitus.
  • 60 NOD mice were recruited to the study at the age of 14 weeks when all of them became hyperglycemic.
  • the recombinant human DNASE 1 (Gentech) at 50 mkg/kg and 500 mkg/kg was administered intramuscularly twice daily for 21 day. 2 mice from each group were sacrificed at the last treatment day to perform evaluation of extracellular blood plasma DNA. The efficacies of DNASE were assessed based on the survival rate at day 35. The results of experiments are presented at the FIG. 2 .
  • a 46-years-old patient with 3 years history of type 2 diabetes mellitus was admitted to the internal therapy clinic of St. Louis Medical Academy. Patient failed to achieve proper glucose control using oral hypoglycemic agents including those of thiazolidinediones, biguanides and sulfonylureas.
  • Patient was switched to 0.3 IU/kg of NPH insulin monotherapy (21 IU daily) and discharged from clinics under the supervision of ambulatory endocrinologist.
  • Three month later patient was readmitted to the clinic since glycosylated hemoglobin (HbA1) level was still too high (above 10%) with evolving microalbuminuria and decrease in vision sharpness despite daily insulin dose was adjusted up to 1.2 U/kg (84 U/day) during ambulatory period.
  • HbA1 glycosylated hemoglobin
  • Atherosclerosis is a systemic disease that is accompanied by formation of specific atherosclerotic plaques in large and middle sized artery walls. Depends on the localization, stage and size of atherosclerotic plaques the disease has different clinical signs (angina, stroke and so on). The signs especially associated with organ dysfunction caused by systemic atherosclerosis are cured by drug therapy or by surgical operation. There is no cure for Atherosclerosis by drug therapy methods as for any systemic disease.
  • HMG CoA 3-hydroxy-3-methylglutaryl-CoA reductase reductase
  • Human recombinant DNase I (Gentech) was used. ⁇ cells of human embryonic pancreas and endothelial cells of human aorta were used for primary cell culture formation. DNA isolated from plasma of patient with severe form of diabetes mellitus type 2 that was complicated by atherosclerosis (0.0025 mkg on 1 ml of culture media) was added to one of the experimental series in cell culture 24 hours after passage and DNA extracted from the blood of the same patient but treated by DNase (1 mkg/ml; 37 C; 30 minutes) was added to the second series of cell culture. The number of viable cells was counted using the trypan blue uptake technique in a 24 hours.
  • Fresh blood plasma (not more than 3-4 hours after isolation) with anticoagulant (sodium citrate) addition was centrifuged on Ficoll-PlaquePlus (Amersham-Pharmacia) step at 1500 g for 20 minutes at room temperature.
  • Plasma (1 ⁇ 2 of all amount) was neatly isolated avoiding touching cells sediment on ficoll step and was centrifuged at 10 000 g for 30 minutes to eliminate cells and debris.
  • Supernatant was taken away not touching sediment and up to 1% of sarcosile, up to 50 mM of tris HCl pH 7.6, up to 20 mM EDTA, up to 400 mM NaCl and equal volume of phenolchloroform mixture 1:1 were added.
  • Received emulsion was incubated at 65° C.
  • phenol-chloroform was separated by centrifuging at 5000 g during 20 minutes at room temperature. Deproteinization by phenol-chloroform method was identically repeated for three times after what water phase was processed by chloroform and after it by diethyl ether.
  • nucleic acids were separated by centrifuging at 10 000 g during 30 minutes. Sediment of nucleic acids was dissolved in buffer that consisted of 10 mM tris-HCl, pH 7.6, 5 mM EDT A and was inflicted on step made from chlorine cesium gradient (1M, 2.5M 5.7) in centrifuge test tube for SW60Ti rotor. DNA volume was 2 ml, volume of each CsCl step was 1 ml. Ultracentrifuging was done in L80-80 (Beckman) centrifuge for 3 hours at 250000 g. DNA was isolated according to fractions from the step's surface 5.7M. Fractions were dialized during 12 hours.
  • DNA presence in fractions was defined by agarose electrophoresis with DNA visualization by ethidium bromide.
  • DNA amount was spectrophotometric ally estimated (Beckman DU 470) in cuvette with volume 100 mkl, using 220-320 nm spectrum. Average runout of DNA was 10-20 ng according to 1 ml of plasma.
  • DNA was deproteinized with the use of proteinase K (Sigma) at 65° C. for tightly-bound proteins elimination. After deproteinization DNA was processed by phenol-chloroform at 65° C. and sedimented by 2.5 volumes of ethanol during night. After it DNA was processed by EcoRI restrictase during 3 hours or by Pfu polymerase (Stratagene) at the presence of 300 mkM of all desoxynucleothydethreephosphates for “sticky” edges elimination. Completed DNA was phosphorylated by polynucleotide kinase T4 (30U, 2 hours).
  • Received samples/preparations were ligated in Bluescript (Stratagene), plasmid digested by EcoR1 or PvuII accordingly and dephosphorylated by alkaline phosphatase CIP (Fermentas) during 1 hour.
  • 1 mkg of vector and 0.1-0.5 mkg of serum DNA were usually used for ligation.
  • Ligation was done by Rapid Ligation Kit (Roche) use for 10 hours at 16° C. Volume of ligase mixture was 50 mkl.
  • Ligated library was transformed into DH12S (Life Technologies) cells with electroporator (BioRad) use. For transformation of one library 12-20 electroporation cuvettes were used.
  • Dilutions of the library at concentrations 10 ⁇ 4 , 10 ⁇ 5 and 10 ⁇ 6 were plated for control on dishes with 1.5% agar and LB media, supplemented with 100 mkg ⁇ ml ampicillin. In both cases library's representativeness was approximately 2-3 ⁇ 10 6 clones.
  • Theoretically set of DNA sequences that circulate in plasma should correspond to set of genome's DNA sequences.
  • apoptosis is accompanied by quantitative and nonspecific DNA degradation before its exit out of the cell, so the most wide spread DNA in plasma should be repetitive elements of genome in proportion that correspond to nonspecific degradation of DNA.
  • Such elements are L1 repeats, satellite DNA, Alu, MER, MIR, THE repeats and some others. Quantity of unique sequences should be small in accordance to their small percent in human genome they may be not detected in cloning DNA without PCR.
  • Source Clone KRAB Family members are known as Oguri T., 152 domain, transcription repressers. Gene, 1998, v. 222, Zn-finger Linked with early pp. 61-67 proteins embryogenesis, Gou D. M., Biochim neuroblastoma, Ewing Biophys Acta, sarcoma, Tcell lymphoma, in 2001, v. 1518, pp. progression and 306-310 chemoresistance in lung Margolin J. F., Proc cancer. Nat Acad Sci USA, 1994, v/91, pp. 4509-4513. Bellefroid E. J., EMBO J, 1993, v. 12, pp. 1363-1374 Gonzales-Lamuno D., Pediatr Pathol Mol Med, 2002, v. 21, pp. 531-540. Marilee J. W., Gene, 1994, v. 152, pp. 227-232.
  • alpha-satellite DNA (30 clones). It is possible to say that that alpha-satellite DNA was the only highly repeated element from human genome, which behaves exactly as repeat in this material. The rest of highly repetitive elements were presented in material as one or several clones (L1 variant and MLT26), or were not found among patterns (MER, Alu, THE, MIR, ⁇ -satellite). Based on today's knowledge one can assume that plasma blood composition for the most part should repeat composition of genome DNA, so listed repeats should be represented in the major part of clones while unique and moderately repeated consequences in analysis of such a small number of clones should not be recognized at all.
  • duplicones that were recently unknown, also support the evidence of special way of plasma DNA formation in patients with malignant tumor.
  • duplicones were found in human genome less then two years ago.
  • Known duplicones (Eichler E. E., et al., Genome Res, 1998, v8, pp. 791-808; Ji Y, et al., Genome Res, 2000, v. 10, pp. 597-610; Pujana M. A., et al., Genome Res, 2001, v. 11, pp.
  • 98-111 are extensive regions of DNA that were multi-plied for several times in the frame of one chromosome (unlike other repeats that are randomly allocated in genome). Duplicones' formation and expansion is connected with different genetic syndromes (for example Prader-Willi/An-gelmane syndrome), with multigenic families' evolution such as MHS (Shiina T., et al., Proc Nat Acad Sci USA, 1999, v. 96, pp. 13282-13287) and with chromosome instability in tumors.
  • MHS Melt al., Proc Nat Acad Sci USA, 1999, v. 96, pp. 13282-1328
  • Blood plasma DNA of oncological patient is highly enriched with unique genes.
  • 55 out of 96 analyzed clones contain fragments of genome's unique sequences.
  • 14 out of 15 sequences with identified in functions refer to process of tumor progression and maintenance of “malignant phenotype”.
  • blood plasma DNA of healthy people and oncological patients for the most part contain unique fragments of human genome.
  • unique sequences of blood plasma DNA correspond to genes which products take part in the formation and maintenance of tumor cell's “malignant phenotype”.
  • DNA circulating in patient's blood can be messenger of horizontal genetic information transfer during the course of oncological diseases, assisting to accumulation and spreading of genes that are necessary for “malignant phenotype” formation and maintenance within population of tumor cells.
  • Somatic mosaicism is a condition that is a result of genetically non-identical somatic cells' presence in organism. Modern vision presents that many non tumor and noninfectious (so called somatic) human diseases (for example atherosclerosis, diabetes, nonspecific chronic lung diseases and so on), including aging process, are connected with appearance and spreading (expansion) in the process of individual development of somatic cells' clones that have mutant genes.
  • somatic tumor and noninfectious
  • somatic mosaicism's appearance There are two alternative models of somatic mosaicism's appearance. The first is appearance of somatic mosaicism as a result of numerous “de novo” mutations in polyclonal cellular pool. The second model is clonal expansion of mutant cells' clone (Khrapko K., et al., Muation Res., 2003, v. 522, pp. 13-19).
  • SCRAP Snf2-related CBP activator protein
  • RAMP RA-regulated nuclear matrix-associated protein
  • NFKB 1 nuclear factor of kappa light polypeptide gene enhancer
  • Doxorubicin causes expression of P-glycoprotein in tumor tissue that is one of the main MDR (Multi drug Resistance) phenotype mediators. Immunohistochemical staining of mice's tumor histological cuts are listed below.
  • mice were subjected to course of 5 day therapy with Doxorubicin (2 mg/kg intravenously daily) or Doxorubicin+DNase I (0.5 mg/kg four times a day during 5 days)
  • Treatment has begun on the 3 rd day after tumor's transplantation. Tissue preparations were executed on the 8th day of tumor's transplantation. Multifocal expression of P-glycoprotein was observed in tumor tissue after 5 days of therapy ( FIG. 3 ).
  • mice mice (control).
  • mice intravenously treated with Doxorubicin chemotherapy from 3 rd up to 8 th day at 2 mg/kg dose daily.
  • mice intravenously treated with Doxorubicin chemotherapy from 3 rd up to 8 th day at 2 mg/kg dose daily+intravenous administration of DNA fraction from mice previously subjected to Doxorubicin chemotherapy (0.05 mkg of DNA in 200 mkl of phosphate buffer at day 1 and day 3 after initiation of treatment)+intraperitoneal administration of DNase I at 0.5 mg/kg dose for 4 times a day at the first and second days of treatment.
  • mice with transplanted LLC tumor participated in the experiment.
  • mice transplanted with low metastatic LLC strain +intravenous administration of total DNA fraction from mice with transplanted highly metastatic strain (0.05 mkg of DNA in 200 mkl of phosphate buffer on the 7 th and 8 th day after transplantation)+intraperitoneal administration of DNase I at 1 mg/kg dose two times daily at 7 th and 8 th day after transplantation.
  • N Number of metastatic foci in lungs was estimated on the 15 th day after transplantation (N).
  • Received data indicates that blood plasma DNA from mice with highly metastatic LLC strain intensify metastasizing of low metastatic LLC strain.
  • mice mice (control).
  • mice were treated with intraperitoneal administration of DNase at 1 mg/kg dose two times a day starting from 3 up to 7 day after tumor transplantation.
  • mice were treated with intraperitoneal administration of DNase at 1 mg/kg dose two times a day starting from 3 up to 10 day after tumor transplantation.
  • mice were treated with intraperitoneal administration of DNase at 1 mg/kg dose two times a day starting from 3 up to 15 day after tumor transplantation.
  • mice were treated with intraperitoneal administration of DNase at 1 mg/kg dose two times a day starting from 3 up to 18 day after tumor transplantation.
  • Results of experiment were estimated according to animals' survival on the 30 and 50 day after tumor transplantation.
  • DNase I does not cause direct cytotoxic effect on tumor cells (in our in vitro experiments at concentration of 100 mkg/ml) and experimental data confirm that antitumor effect is connected with destruction of DNA in blood plasma and DNase's therapeutic effect increases with increasing of its treatment course duration.
  • mice 100 mice were transplanted with highly metastatic LLC strain. On the 9th day after transplantation, animals were euthanized and total blood plasma was taken. After isolation total fraction of blood plasma DNA was stored at ⁇ 20° C. in phosphate buffer.
  • mice with transplanted low metastatic LLC strain participated in the experiment.
  • mice transplanted with low metastatic LLC strain Group 1-6 mice transplanted with low metastatic LLC strain.
  • mice transplanted with low metastatic LLC strain+two intravenous injections of total DNA fraction from mice transplanted with highly metastatic strain on the 7th and 8th day after transplantation 0.05 mkg of DNA was dissolved in 500 mkl of fresh blood plasma before injection.
  • the sample was photochemically disinfected (1 mkM of methylene blue was added with following irradiation by red light during 10 minutes (60 000 Lux).
  • mice transplanted with low metastatic LLC strain+two intravenous injections of total DNA fraction from mice transplanted with highly metastatic strain on the 7th and 8th day after transplantation 0.05 mkg of DNA was dissolved in 500 mkl of fresh blood plasma before injection.
  • the sample was passed through the column containing DEAE-cellulose for two times before administration.
  • mice transplanted with low metastatic LLC strain+two intravenous injections of total DNA fraction from mice transplanted with highly metastatic strain on the 7th and 8th day after transplantation 0.05 mkg of DNA was dissolved in 500 mkl of fresh heparinized blood before injection. 1 mkg of fragment A of Ricin toxin was added to the sample before administration and sample was incubated at 370 C for 1 hour.
  • Ricin toxin is representative of RIP toxins family (proteins that inactivate ribosomes) which are used for immunotoxin's creation. Besides their ability to inactivate ribosomes these proteins can deadenylate DNA.
  • catalytic subunit A of RIP II type should by delivered to cell by B subunit. Without B subunit A chain is not toxic but can be used for blood plasma DNA's inactivation due to its polynucleotide-adenylglicozidase activity.
  • mice transplanted with low metastatic LLC strain+two intravenous injections of total DNA fraction from mice transplanted with highly metastatic strain on the 7th and 8th day after transplantation 0.05 mkg of DNA was dissolved in 500 mkl of fresh heparinized blood before injection.
  • Total DNA fraction was enzymatically methylated before administration (I. Muiznieks et. al., FEBS Letters, 1994, v. 344, pp. 251-254).
  • Received data indicates that all used methods inhibited ability of blood plasma DNA of mice with highly metastatic LLC tumor strain to increase metastasizing process of low metastatic LLC tumor strain.

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Abstract

The invention is directed to treatment of systemic DNA mutation diseases accompanied with development of somatic mosaicism and elevation of blood extracellular DNA. The inventive method comprises introducing a DNASE enzyme into the systemic blood circulation of a patient in doses and regimens which are sufficient to decrease average molecular weight of circulating extracellular blood DNA in the blood of said patient.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a Divisional of U.S. application Ser. No. 14/309,363, filed Jun. 19, 2014, which is a Continuation of U.S. application Ser. No. 13/772,499 filed Feb. 21, 2013, now U.S. Pat. No. 8,796,004, which is a Continuation of U.S. application Ser. No. 12/835,029 filed Jul. 13, 2010, now U.S. Pat. No. 8,388,951, which is a Continuation-in-Part of U.S. application Ser. No. 10/564,609 filed Jan. 12, 2006, which is a U.S. national phase application under 35 U.S.C. §371 of International Patent Application No. PCT/RU2004/000260, filed Jul. 1, 2004 (published in Russian on Jan. 20, 2005 as WO 2005/004789), which claims priority of Russian Federation Patent Application No. RU2004108057, filed Mar. 12, 2004 and International Patent Application No. PCT/RU2003/000304 filed Jul. 14, 2003, all of which applications are incorporated by reference herein in their entirety.
    • SEQUENCE LISTING
  • The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 16, 2014, is named 243736.000083_SL.txt and is 59,899 bytes in size.
    • BACKGROUND OF THE INVENTION
  • 1. Field of the Invention
  • The present invention relates generally to medical treatment of the systemic DNA mutation diseases accompanied with development of somatic mosaicism and elevation of blood extracellular DNA and, more particularly, to a treatment of diabetes mellitus and atherosclerosis.
  • 2. Description of the Related Art
  • Mosaicism refers to a mixture of cells of different genetic composition in one individual. When DNA mutation is detectable in number, but not all somatic cells in one individual, it is called somatic mosaicism. Development of somatic mosaicism has been recently recognized as important mechanism of systemic DNA mutation diseases progression (Gottlieb B et al., Selection and mutation in the “new” genetics: an emerging hypothesis, Hum Genet. 2010 March; 127(5): 491-501.) Importance of somatic mosaicism involving disease-causing mutations has been reported for variety of monogenic (reviewed by Youssoufian H., Nature Reviews Genetics 3, 748-758, October 2002) and more recently for multifactor DNA mutation diseases: cardiac rhythm disorders (M. H. Gollob et al., Somatic mutations in the connexin 40 gene (GJA5) in atrial fibrillation, N. Eng. J. Med. 354 (2006), pp. 2677-2688.); atherosclerosis (S. De Flora et al., Mutagenesis and cardiovascular diseases. Molecular mechanisms, risk factors, and protective factors, Mutat. Res. 621 (2007), pp. 5-17), systemic vascular disorders (B. Gottlieb et al., BAK1 gene variation and abdominal aortic aneurysms, Hum. Mutat. 30 (2009), pp. 1043-1047); immune deficiencies (Wada T. et al., Somatic mosaicism in primary immune deficiencies, Curr Opin Allergy Clin Immunol. 2008 December; 8(6): 510-4); Alzheimer disease (Beck J A et al., Somatic and germline mosaicism in sporadic early-onset Alzheimer's disease. Hum Mol Genet. 2004 Jun. 15; 13(12): 1219-24.); diabetes mellitus (Emma L. Edghill et al, Origin of de novo KCNJ11 mutations and risk of neonatal diabetes for subsequent siblings. The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 5 1773-1777).
  • According to current knowledge the systemic DNA mutation diseases represent very distinct subsets of human pathology different in etiology and pathogenesis and accordingly has fundamentally different, usually palliative treatment modalities-cholesterol lowering therapy for atherosclerosis (New Concepts and Paradigms in Cardiovascular Medicine: The Noninvasive Management of Coronary Artery Disease, K. Lance Gould, THE AMERICAN JOURNAL OF MEDICINE, Volume 104, Jun. 22, 1998, pp. 2-17) and insulin therapy or insulin sensitization therapy for diabetes mellitus (Pharmacological Management of Diabetes: Recent Progress and Future Perspective in Daily Drug Treatment, Gerard Emilien et al., Pharmacol. Ther. Vol. 81, No. 1, pp. 37-51, 1999).
  • More recently the gene therapy was recognized as potential tool for disease specific intervention which may target the function of certain specific disease involved genes and provide more efficient cure based on repair of existing genetic defects in atherosclerosis (Ishisaki A, et al., Novel ideas of gene therapy for atherosclerosis: modulation of cellular signal transduction of TGF-beta family. Curr Pharm Des. 2006; 12(7): 877-86; Harris J D, et al. ApoE gene therapy to treat hyperlipidemia and atherosclerosis. Curr Opin Mol Ther. 2006 August; 8(4): 275-87; Hayden et al. Gene therapy method for reducing risk of atherosclerosis, U.S. Pat. No. 6,784,162) and diabetes mellitus (G B Parsons, Ectopic expression of glucagon-like peptide 1 for gene therapy of type II diabetes, Gene Therapy (2007) 14, 38-48; L. Chan, In vivo gene therapy for diabetes mellitus, Trends in Molecular Medicine, Volume 9, Issue 10, October 2003, Pages 430-435; M. During, Compositions for gene therapy of diabetes, EP1889914).
  • However no cure exists which may target the evolution of disease causing DNA mutations leading to development of somatic mosaicism. Accordingly, the development of new effective, non-toxic method that may suppress the development of somatic mosaicism and consequently be effective cure for systemic DNA mutation disease is an extremely important task.
  • Circulating extracellular nucleic acids were discovered more than 60 years ago (Anker P Circulating DNA in plasma or serum, Clin Chim Acta. 2001 November; 313(1-2): 143-6). However until now elevated levels of extracellular blood DNA in systemic DNA mutation diseases, and in particular in atherosclerosis and diabetes mellitus were considered only as useful diagnostic and research tool (El Tarhouny S. A. et al., Assessment of cell-free DNA with microvascular complication of type II diabetes mellitus, using PCR and ELISA. Nucleosides Nucleotides Nucleic Acids. 2010 March; 29(3): 228-36; Langford M P et al., Plasma levels of cell-free apoptotic DNA ladders and gamma-glutamyltranspeptidase (GGT) in diabetic children. Exp Biol Med (Maywood). 2007 October; 232(9): 1160-9; Arnalich F. et al., Prognostic value of cell-free plasma DNA in patients with cardiac arrest outside the hospital: an observational cohort study, Critical Care 2010, 14; Arnalich F. Association of cell-free plasma DNA with preoperative mortality in patients with suspected acute mesenteric ischemia, Clinica Chimica Acta, in press; Zhong S, Presence of mitochondrial tRNA (Leu (UUR)) A to G 3243 mutation in DNA extracted from serum and plasma of patients with type 2 diabetes mellitus 2000 June; 53(6): 466-9.)
  • Circulating extracellular nucleic acids have never been considered as potential therapeutic target in systemic DNA mutation diseases. Accordingly, no therapeutic method was developed which targets extracellular blood DNA in systemic DNA mutation diseases. Thus it makes impossible to take any technical solution as prototype.
  • As used in this application, the following terms are meant to have the following corresponding definitions.
  • Deoxyribonuclease (DNASE) is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone.
  • Extracellular blood DNA number average molecular weight—the number average molecular weight is a way of determining the molecular weight of a polymer. The number average molecular weight is the ordinary arithmetic mean or average of the molecular weights of the individual DNA macromolecules. It is determined by measuring the molecular weight of n polymer molecules, summing the weights, and dividing by n. The number average molecular weight of extracellular blood DNA can be determined by gel electrophoresis. The shift of extracellular blood DNA bands to low-MW areas reflect decrease number average molecular weight and in fact reflects enzymatic cleavage of extracellular blood DNA.
  • DNA mutation disease refers to diseases where specific DNA mutation has been identified as single leading cause (monogenic or single gene disorders) or multifactor disorders resulting from mutations in multiple genes, often coupled with environmental causes.
  • Systemic disease is one that affects a number of organs and tissues, or affects the body as a whole.
    • SUMMARY OF THE INVENTION
  • The object of this invention is to develop high-performance and low-toxic method for treatment of systemic DNA mutation diseases accompanied with development of somatic mosaicism and elevation of blood extracellular DNA and, more particularly, to a treatment of diabetes mellitus and atherosclerosis.
  • According to the invention this task is resolved by introducing a treatment agent into a circulating blood system of a patient diagnosed with systemic DNA mutation disease when said treatment agent destroys extracellular DNA in said blood of said patient and wherein said treatment agent used to destroy said extracellular DNA is a DNASE enzyme. In one of preferred embodiments said agent must be administered in doses and regimens which sufficient to decrease number average molecular weight of circulating extracellular blood DNA in the blood of said patient; such decrease of number average molecular weight might be measured by gel electrophoresis of extracellular blood DNA fraction from the blood of said patient. In one of preferred embodiments the method according the invention can be effectively applied for treatment of diabetes mellitus and atherosclerosis. A DNASE enzyme may be further applied in a dose and regime that results in a DNA hydrolytic activity measured in blood plasma that exceeding 1.5 Kunitz units per 1 ml of blood plasma for more than 12 hours within a period of 24 hours.
  • The present invention suggests that systemic DNA mutation disease can be treated by reducing of circulating extracellular blood DNA levels.
  • Development of systemic DNA mutation disease in humans is accompanied by quantitative and/or qualitative change of blood extracellular DNA.
  • There are no analysis of blood extracellular DNA spectrum and its biological role in systemic DNA mutation disease prior to this invention. A search of the prior art reveals no published data concerning an analysis of blood extracellular DNA spectrum in systemic DNA mutation disease performed by direct cloning and without use of polymerase chain reaction (PCR). PCR can pervert a pattern of blood extracellular DNA because of specificity of primers used for amplification. There is no available knowledge about genetic repertoire of extracellular blood DNA in patients suffering from systemic DNA mutation disease and about biological role of extracellular blood DNA in course of these diseases. Nothing is known about potential therapeutic value of extracellular blood DNA enzymatic destruction for treatment of systemic DNA mutation disease; so, taking into account all aforesaid, the invention complies with requirements of “novelty” criteria (N).
  • As the applicant established by direct cloning and sequencing of extracellular blood DNA without PCR (Polymerase Chain Reaction), the extracellular blood DNA of patients with systemic DNA mutation disease contains the unique quantitative and qualitative repertoire of genes, which non-randomly represents human genome and contains genetic elements involved in to the development of the disease. It was shown that extracellular blood DNA might promote the development of somatic mosaicism and systemic DNA mutation disease.
  • It was established that enzymatic destruction of extracellular blood DNA by DNASE enzyme when applied in certain surprisingly high specific doses has significant therapeutic effect on the course of systemic DNA mutation disease.
  • Aforesaid new characteristics of the claimed invention are based on new ideas about mechanism of development of systemic DNA mutation disease. In this way the claimed method conformances to requirements of “invention step” criteria (IS).
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • The advantages and features of the present invention have been explained by detailed description of embodiments with references to drawings:
  • FIG. 1: NF-kappa B expression in (A) cultured aortic endothelial cells treated with extracellular blood DNA fraction from patient suffering from systemic atherosclerosis; (B) cultured aortic endothelial cells treated with extracellular blood DNA fraction from patient suffering from systemic atherosclerosis plus 1 mkg/ml of DNASE1; and (C) control cells.
  • FIG. 2A-2B: FIG. 2A shows a graph of the survival of NOD diabetic mice treated with different doses of DNASE1—50 mkg/kg, 500 mkg/kg, and control; FIG. 2B shows average molecular weight of extracellular blood plasma DNA (as measured by electrophoresis) in blood of NOD diabetic mice treated with 50 mkg/kg DNASE 1 (A), 500 mkg/kg DNASE1 (B), and control (C).
  • FIG. 3A-3B: Results of immunochemical staining of mice tumor's histological slices after administration of 5 day course of Doxorubicin therapy (2 mg/kg every day intravenous) and DNase 1 (0.5 mg/kg four times a day during 5 days). 3A—Doxorubicin+DNase; 3B—Doxorubicin.
    •  DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • The inventive method is realized as follows:
  • Materials and Methods:
  • The following agents, which destroy extracellular blood DNA, were used: bovine pancreatic DNASE (Sigma, specific activity 2 400 Kunitz units/mg; Samson-Med, specific activity 1 500 Kunitz units/mg), recombinant human DNASE 1 (Gentech, specific activity 1000 U/mg).
  • Extracellular DNA from blood plasma was isolated as follows: fresh plasma (no more than 3-4 hours after sampling) with anticoagulant (sodium citrate) was centrifuged on Ficoll-Plaque Plus (Amersham-Pharmacia) during 20 minutes at 1500 g. at room temperature. ½ of plasma was detached, not affecting the rest of cells on the Ficoll pillow, and further centrifuged at 10000 g. during 30 min for separation from cell fragments and debris. Supernatant was detached, without affecting of the sediment, and was toped up to 1% of sarkosil, 50 MM tris-HCl, pH 7.6, 20 MM EDTA, 400 MM NaCl, and then mixed with equal volume of phenol-chloroform(1:1) mixture. The prepared emulsion was incubated during 2 hours at t=65° C., then phenol-chloroform mixture was separated by centrifuging (500 g during 20 minutes, room temperature). The procedure of deproteinization with phenol-chlorophorm mixture was repeated 3 times, and then the water phase was processed with chloroform and diethyl ether. Separation from organic solvents was made by centrifugation at 5000 g during 15 minutes. Then equal volume of izopropanol was added to resulting aqueous phase and the mixture was incubated overnight at 0° C. After sedimentation the nucleic acids were separated by centrifugation at 10000 g during 30 minutes. The sediment of nucleic acids was dissolved in of 10 MM tris-HCl buffer, pH 7, 6 with 5 MM EDTA, and inflicted to the CsCl gradient (1M, 2.5M, 5.7M) in test-tube for rotor SW60Ti. The volume of DNA solution was 2 ml, volume of each step of CsCl was 1 ml. Ultracentrifugation was conducted in L80-80 (Beckman) centrifuge during 3 hours at 250000 g. DNA was collected from the surface of each gradient step into fractions. These fractions were dialyzed during 12 hours (t=4° C.) and pooled. Presence of DNA in fractions was determined by agar electrophoresis and DNA was visualized by ethidium bromide staining. The amount of DNA was determined with spectrophotometer (Beckman DU70) in cuvetts (100 mkl) at wavelength of 220-230 nm.
  • NOD mice were obtained from <<Pushcino>> animal breeding house.
    • Example 1 DNASE Treatment Suppresses the Development of Somatic Mosaicism
  • Frequency of HPRT gene's mutations in blood T-lymphocytes was studied as the model of development of somatic mosaicism in vivo. The human HPRT gene, mapped to chromosome Xq26, codes for a constitutively expressed, but non-essential, enzyme involved in purine metabolism. Mutant peripheral blood T-lymphocytes which do not express a functional HPRT gene product can be enumerated and clonally expanded by selective growth in the normally toxic purine analog 6-thioguanine in the presence of specific mitogens and growth factors. In normal, unexposed individuals the frequency of 6-thioguanine resistant T-lymphocytes is typically 10−6 to 10−5 (R. J. Albertini, J. A. Nicklas, J. P. O'Neill, S. H. Robison, In vivo somatic mutations in humans: measurement and analysis, Annu. Rev. Genet. 24 1990. 305-326.) Molecular analyses of the mutant, HPRT deficient, clones have demonstrated that 85% of the gene inactivating mutations observed in unexposed adults arises by localized HPRT gene alterations—single base changes, small deletions or insertions and frame shift.
  • Selective lymphocyte cloning was performed using peripheral blood of 8 female patients with different forms of advanced cancer who got surgical resection at Kostushko municipal Hospital (St. Petersburg) and immunomodulation therapy at neoadjuvant setting (Neovir, 250 mg IM once every 2 days for 3 weeks). Following surgical resection 4 patients were further treated by IV infusions of bovine pancreatic DNASE (Samson) according the following schedule: 2000 mkg/kg×4 times daily for 21 day. Following completion of treatment the patients were assayed for HPRT (−) mutation in blood lymphocytes.
  • Mononuclear cells were isolated from the whole blood samples using Ficoll-Paquee (Becton Dickinson). Mitogenic stimulation of the separated lymphocytes (1×106/ml) was initiated with 1 mg/ml phytohemagglutinin (PHA) in RPMI 1640 media supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), 20% nutrient medium HL-1 and 5% BSA at 5% CO2 at 37° C. for 24 h. Following wash the cells were then seeded in 96-well round-bottomed plates at cell density of 2×104 cells per well in selection medium to determine cloning efficiency. The cells were plated in 200 ml of the RPMI medium containing 1 mg/ml 6-thioguanine, 0.125 mg/ml PHA, 20% HL-1 and 5% BSA supplemented with interleukin-2 (BD Biosciences, 10 BRMP units/mi). Four 96-well plates were seeded for each patient. After 5 days of culture, the colonies on mutant selection plates were scored for growth using an inverted microscope.
  • The results of selective T-lymphocyte cloning are presented in the table below:
  • Total number of HPRT wells
    Patient Treatment wells (growth positive)
    KNP DNASE 384 7
    PGP DNASE 384 12
    BAI DNASE 384 2
    FVV DNASE 384 11
    SLS NO 384 47
    GAN NO 384 22
    PMI NO 384 31
    ENV NO 384 55
  • Thus, inventive treatment suppresses spread of HPRT (−) mutation and suppresses the development of somatic mosaicism.
    • Example 2 Extracellular Blood DNA Promotes the Development of Somatic Mosaicism
  • The extracellular blood plasma DNA was purified from blood of patient ENV as specified in methods section. Mononuclear cells were isolated from the whole blood samples of patients KNP, PGP, BAI and FW as specified in Example 1. The mitogenic stimulation and selective cloning were performed as specified in Example 1 with modification as follows: during mitogenic activation stage lymphocyte cultures of patients KNP, PGP, BAI and FW were supplemented with 50 mkg/ml of extracellular blood plasma DNA purified from patient ENV. After 5 days of culture, the colonies on mutant selection plates were scored for growth using an inverted microscope. The results of selective T-lymphocyte cloning are presented in the table below:
  • Total number of HPRT wells
    Patient wells (growth positive)
    KNP 384 18
    PGP 384 15
    BAI 384 21
    FVV 384 31
  • Thus, extracellular blood DNA promotes the development of somatic mosaicism.
    • Example 3 Sequencing of Extracellular Blood DNA from the Patient Suffering from
  • Type 2 Diabetes and Systemic Atherosclerosis. Treatment of Atherosclerosis.
  • A 54-year-old man has been admitted to the Cardiothoracic surgery department of Kostushko municipal hospital (St. Petersburg) in severe condition complaining on intensive pain in abdomen, diarrhea, intensive pain in legs that appear during walking, loss of weight. Diabetes mellitus type 2 was diagnosed 12 years ago and glybencamide was prescribed. Pain in epigastrium after food intake appeared 15 months ago. Antacids were prescribed but pain continued to increase and steatorrhea appeared in the last 3 months. Because of intensive pain syndrome anorexia has developed in a couple of days prior admittance. Considerable exhaustion (body weight was 44 kg; body weight loss was 28 kg for the last 5 months) and absence of arterial pulsation on legs were found out during examination. No organic changes were observed during gastroduodenoscopy and colonoscopy. Electrocardiographic data was not changed pathologically. Moderate increase of cholesterol level and low-density lipoprotein fraction was observed in blood analysis. Glycated hemoglobin′ level was 11%. Partial occlusion of aorta below renal artery (70%), partial occlusion of iliac arteries (90%), total occlusion of upper and lower mesenteric artery were observed on aortography.
  • The probes of patient's extracellular blood DNA were taken before initiation and on day 35 of therapy. The extracellular DNA was cloned by the method which allows to get non amplified plasmid libraries of blood extracellular DNA with representativeness up to one million of clones with the average size of 300-500 base pairs. The DNA which has been isolated using the protocol specified in Materials and Methods section was treated with Proteinase K (Sigma) at 65° C. and subjected to additional phenol-chloroform extraction step with further overnight precipitation by ethanol. The DNA fraction was than treated by Eco RI restrictase or by Pfu polymerase (Stratagene) in presence of 300 mkM of all desoxynucleotidetriphosphates for sticky-ends elimination. The completed DNA was phosphorylated by polynucleotidkinase T4 and ligated to pBluescript plasmid (Stratagene), which had been digested with EcoRI or PvulI and dephosphorylated by phosphatase CIP (Fermentas). The process of ligation was conducted with Rapid Legation Kit (Roche). The ligated library was transformed into DH12S cells (Life Technologies) by electroporation (E. Coli porator; BioRad). 12-20 electroporation covets were used for the transformation of one library. The library serial dilutions were cloned on 1.5% agar and LB media supplemented with ampicillin. In both cases the libraries represented 2-3×106 clones.
  • Analysis of 75 randomly selected clones with the size 300-1000 base pairs from the “before treatment” library revealed 56 clones containing unique human DNA sequences as presented at the table below:
  • Potential role in
    Number of Atherosclerosis/diabetes
    Gene clones mellitus
    Neutral endopeptidase 2 At atherosclerosis its
    activity is increased in
    endothelial cells,
    nonstriated muscle cells,
    stromal cells of artery'
    intima. Decreasing of its
    activity can decrease lipids
    accumulation in vessels
    wall.
    Muskelin 1 1 Works as mediator of cell
    response on
    thrombospondin 1.
    Thrombospondin 1 -
    mediated processes are
    pathophysiological
    components of
    atherosclerotic affection of
    artery wall.
    Nf-kappaB 3 At hyperglycemia and
    atherosclerosis activity is
    increased in cells of artery
    wall.
    E-selectin 3 High level of expression is
    a risk factor of angiopathy
    development at diabetes
    type
    2.
    GAD2: glutamate 2 2 One of the main pancreatic
    decarboxylase
    2 autoantigens.
    Phospholipase C, epsilon 2 Induces expression of
    receptors of low-density
    lipoproteins
    BAI3: brain-specific 1 Angiogenesis inhibitor
    angiogenesis inhibitor
    Nicotinamide nucleotide
    1 Involved in detoxification
    transhydrogenase of reactive oxygen species
    and insulin secretion.
    17 kD fetal brain protein 1 UNCLEAR
    CRTL1: cartilage linking 1 Involved in morphogenetic
    protein
    1 process in heart and large
    vessels
    Transient receptor potential 1 UNCLEAR
    cation channel
  • Thus, extracellular blood DNA from patient having diabetes mellitus and systemic atherosclerosis contains significant non-random presence of human disease-relevant unique genomic DNA.
  • Patient was considered as not eligible for surgery so, conservative therapy was chosen. Intensive IV nutrition was started. Insulin and anti-aggregation therapy have being started. Under patient consent daily intravenous infusions of bovine pancreatic DNASE (Samson) at daily dose of 800 mg (1 200 000 Kuntz units) divided to 4 two-hour deliveries were started. Week after start of DNASE therapy pain syndrome disappeared and patient was allowed to take light dietetic food orally. 20 days after start of DNASE treatment patient was switched to full value oral nutrition. General state was improved, body weight has increased. 45 days following initiation of DNASE treatment patient was reexamined by angiography. 20% decrease of aorta occlusion and 35% decrease of iliac artery occlusion level as well as appearance of blood circulation in upper and lower mesenteric was observed. Patient was considered as eligible for revascularization surgery.
  • Extracellular blood plasma DNA sampled from patients blood at day 35 following start of DNASE therapy was assayed by gel electrophoresis and cloning. Analysis of 50 clones randomly chosen from the library obtained from the extracellular blood plasma DNA of patient on the day 35 after the beginning of treatment has shown that more than 90% of revealed clone sequences are short fragments of repetitive human DNA with the dominance of alpha-satellite DNA.
    • Example 4 Influence of Extracellular Blood DNA from the Patient with Systemic Atherosclerosis on Disease Causing Protein Expression in Aortic Endothelial Cells
  • Endothelial NF-kappa B signaling orchestrates proinflammatory gene expression at the arterial wall and promotes the pathogenesis of atherosclerosis. Here we assayed the influence of extracellular blood DNA from the patient diagnosed with systemic atherosclerosis on NF kappa B expression in primary aorta endothelial cell culture. Blood plasma was obtained from vascular surgery clinic of St. Petersburg Medical Academy from the patient undergoing femoro-femoral bypass surgery due to severe atherosclerotic arterial occlusion.
  • The extracellular blood DNA was purified as described in Materials and Methods section. The aortic endothelial cells (C-006-5C; Invitogen) were plated at density of 5-8×102 cells/mm2 in multiwell (12×) cell culture plates in Clonetics® EGM®-2MV media (Lonza Cologne AG) and incubated for 48 h. at 37° C. and 5% CO2. Following 24 h of growth the culture media was supplemented with 50 mkg/ml of patient extracellular blood DNA fraction or 50 mkg/ml of patient extracellular blood DNA fraction plus human recombinant DNASE-1 (Genentech) at 1 mkg/ml concentration.
  • After 24 h. culturing the explants were lysed in buffer containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM phenylmethylsulfonylfluoride, 5 mg/ml aprotinin, 0.5% Nonidet P-40 (Sigma-Aldrich) for 1 hour at 4° C. The lysates were centrifuged for 10 min at 20,000 rpm. The supernatants were diluted with reducing sample buffer and were separated by electrophoresis on a 10% SDS-PAGE gel (20 mkg protein per lane loaded). The proteins were transferred onto Hybond-C-nitrocellulose membrane (Amersham Italia, Milan, Italy. For immunoblot analysis, the membranes were incubated with the NF-.kappa.B antibodies (Stressgen). The bands were detected using the chemiluminescence system.
  • The results are presented at the FIG. 1. Extracellular blood DNA fraction from patient suffering from systemic atherosclerosis increases the expression of NF-kappa B in cultured aortic endothelial cells and treatment with DNASE ameliorate this effect.
    • Example 5 Treatment of Diabetes Mellitus
  • Non-obese diabetic (NOD) mice exhibit a susceptibility to spontaneous development of autoimmune insulin dependent diabetes mellitus. 60 NOD mice were recruited to the study at the age of 14 weeks when all of them became hyperglycemic. The recombinant human DNASE 1 (Gentech) at 50 mkg/kg and 500 mkg/kg was administered intramuscularly twice daily for 21 day. 2 mice from each group were sacrificed at the last treatment day to perform evaluation of extracellular blood plasma DNA. The efficacies of DNASE were assessed based on the survival rate at day 35. The results of experiments are presented at the FIG. 2. There is evident increase in survival of mice treated with 500 mkg/kg DNASE 1; such survival is accompanied with decrease of average molecular weight of extracellular blood plasma DNA (as measured by electrophoresis) in blood of NOD diabetic mice. Thus, high doses of DNASE according to inventive treatment are able to decrease the quantities of circulating extracellular blood plasma DNA and are effective against systemic DNA mutation disease-diabetes mellitus.
    • Example 6 Treatment of Diabetes Mellitus
  • A 46-years-old patient with 3 years history of type 2 diabetes mellitus was admitted to the internal therapy clinic of St. Petersburg Medical Academy. Patient failed to achieve proper glucose control using oral hypoglycemic agents including those of thiazolidinediones, biguanides and sulfonylureas. Patient was switched to 0.3 IU/kg of NPH insulin monotherapy (21 IU daily) and discharged from clinics under the supervision of ambulatory endocrinologist. Three month later patient was readmitted to the clinic since glycosylated hemoglobin (HbA1) level was still too high (above 10%) with evolving microalbuminuria and decrease in vision sharpness despite daily insulin dose was adjusted up to 1.2 U/kg (84 U/day) during ambulatory period. Under patient consent he was assigned for intramuscular injections of bovine pancreatic DNASE (Samson) twice daily at 200 mg/day dose for 4 months and again discharged from clinics. At 4 month after initiation of treatment patient were reexamined in clinics outpatient department. Significant improvement in insulin sensitivity, improvement of glycemia control and normalization of kidney function has been reported by patient ambulatory endocrinologist and confirmed by laboratory examination in clinic. The effect of DNASE treatment on patient disease indicators is presented in table below:
  • Prior At DNASE
    DNASE course
    Indicator treatment completion
    Insulin requirement  1.2 IU/kg 0.6 IU/kg
    HbA1 13.2% 7.2%
    24 h. albuminuria 275 mg  60 mg

    Thus, the inventive treatment is effective in diabetes mellitus.
    • Example 7
  • Atherosclerosis is a systemic disease that is accompanied by formation of specific atherosclerotic plaques in large and middle sized artery walls. Depends on the localization, stage and size of atherosclerotic plaques the disease has different clinical signs (angina, stroke and so on). The signs especially associated with organ dysfunction caused by systemic atherosclerosis are cured by drug therapy or by surgical operation. There is no cure for Atherosclerosis by drug therapy methods as for any systemic disease. An established method of prevention that delays the disease progression is therapy with inhibitors of 3 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) reductase (Lovastatin, Parvastatine e.t.c.) leading to the inhibition of endogenous cholesterol synthesis and increasing of clearance of low density lipoproteins of blood plasma and it attenuates atherosclerosis development (New Concepts and Paradigms in Cardiovascular Medicine: The Noninvasive Management of Coronary Artery Disease, K. Lance Gould, THE AMERICAN JOURNAL OF MEDICINE, Volume 104, Jun. 22, 1998, 2s-17s). Disadvantages of such treatment are adverse effects (A safety look at currently available statins, Moghadasian M H, Expert Opin Drug Saf 2002 September 1 pp. 269-74) and limited efficacy (Statins: balancing benefits, efficacy and safety, Clearfield M B, Expert Opin Pharmacother, 2002, May 3 pp. 469-77).
    • Influence of DNase Therapy on the Viability of Pancreatic Beta-Cells and Endothelium of Aorta
  • Human recombinant DNase I (Gentech) was used. β cells of human embryonic pancreas and endothelial cells of human aorta were used for primary cell culture formation. DNA isolated from plasma of patient with severe form of diabetes mellitus type 2 that was complicated by atherosclerosis (0.0025 mkg on 1 ml of culture media) was added to one of the experimental series in cell culture 24 hours after passage and DNA extracted from the blood of the same patient but treated by DNase (1 mkg/ml; 37 C; 30 minutes) was added to the second series of cell culture. The number of viable cells was counted using the trypan blue uptake technique in a 24 hours.
  • Results of the experiment are presented in table 4:
  • TABLE 4
    Percentage of viable cells 48 hours after their
    cultivation (in percents).
    DNA of DNA of patent
    Cells Control patient treated by DNse
    β cells 73% 43% 61%
    Endotelium 62% 35% 55%
  • Thus extracellular blood plasma DNA of patient with severe form of diabetes mellitus type 2 and atherosclerosis negatively influence both the normal pancreatic β-cells and the normal endothelial cells. Destruction of the patient' blood extracellular DNA prevents development of negative influence according to the claimed method.
    • Example 8 Isolation of Free Circulating DNA from Blood Plasma
  • Fresh blood plasma (not more than 3-4 hours after isolation) with anticoagulant (sodium citrate) addition was centrifuged on Ficoll-PlaquePlus (Amersham-Pharmacia) step at 1500 g for 20 minutes at room temperature. Plasma (½ of all amount) was neatly isolated avoiding touching cells sediment on ficoll step and was centrifuged at 10 000 g for 30 minutes to eliminate cells and debris. Supernatant was taken away not touching sediment and up to 1% of sarcosile, up to 50 mM of tris HCl pH 7.6, up to 20 mM EDTA, up to 400 mM NaCl and equal volume of phenolchloroform mixture 1:1 were added. Received emulsion was incubated at 65° C. for 2 hours than phenol-chloroform was separated by centrifuging at 5000 g during 20 minutes at room temperature. Deproteinization by phenol-chloroform method was identically repeated for three times after what water phase was processed by chloroform and after it by diethyl ether.
  • Organic solvents' separation was done by centrifuging at 5000 g during 15 minutes. Equal part of isopropanol was added to water phase and incubated during night at 0° C.
  • After sedimentation nucleic acids were separated by centrifuging at 10 000 g during 30 minutes. Sediment of nucleic acids was dissolved in buffer that consisted of 10 mM tris-HCl, pH 7.6, 5 mM EDT A and was inflicted on step made from chlorine cesium gradient (1M, 2.5M 5.7) in centrifuge test tube for SW60Ti rotor. DNA volume was 2 ml, volume of each CsCl step was 1 ml. Ultracentrifuging was done in L80-80 (Beckman) centrifuge for 3 hours at 250000 g. DNA was isolated according to fractions from the step's surface 5.7M. Fractions were dialized during 12 hours. mM tris-HCl, pH 7.6, 1 mM EDTA at 4° C. will be added. DNA presence in fractions was defined by agarose electrophoresis with DNA visualization by ethidium bromide. DNA amount was spectrophotometric ally estimated (Beckman DU 470) in cuvette with volume 100 mkl, using 220-320 nm spectrum. Average runout of DNA was 10-20 ng according to 1 ml of plasma.
  • Cloning and sequencing of blood plasma DNA.
  • We have developed new method of DNA isolation and cloning from blood plasma, that allows to construct not amplified plasmide library of such DNA with representativeness up to million clones with average size 300-500 base pair isolated from 50 ml of blood, even taking into account significant amount of elevated liposaccharides level and non identified mixtures that troubled purification of nucleic acids. So representative analysis can be done with less amount of plasma pattern-10-20 ml depending on pathological contaminates' presence.
  • Isolated according to above-mentioned method DNA was deproteinized with the use of proteinase K (Sigma) at 65° C. for tightly-bound proteins elimination. After deproteinization DNA was processed by phenol-chloroform at 65° C. and sedimented by 2.5 volumes of ethanol during night. After it DNA was processed by EcoRI restrictase during 3 hours or by Pfu polymerase (Stratagene) at the presence of 300 mkM of all desoxynucleothydethreephosphates for “sticky” edges elimination. Completed DNA was phosphorylated by polynucleotide kinase T4 (30U, 2 hours). Received samples/preparations were ligated in Bluescript (Stratagene), plasmid digested by EcoR1 or PvuII accordingly and dephosphorylated by alkaline phosphatase CIP (Fermentas) during 1 hour. 1 mkg of vector and 0.1-0.5 mkg of serum DNA were usually used for ligation. Ligation was done by Rapid Ligation Kit (Roche) use for 10 hours at 16° C. Volume of ligase mixture was 50 mkl. Ligated library was transformed into DH12S (Life Technologies) cells with electroporator (BioRad) use. For transformation of one library 12-20 electroporation cuvettes were used. Dilutions of the library at concentrations 10−4, 10−5 and 10−6 were plated for control on dishes with 1.5% agar and LB media, supplemented with 100 mkg\ml ampicillin. In both cases library's representativeness was approximately 2-3×106 clones.
  • Theoretically set of DNA sequences that circulate in plasma should correspond to set of genome's DNA sequences. Usually cells apoptosis is accompanied by quantitative and nonspecific DNA degradation before its exit out of the cell, so the most wide spread DNA in plasma should be repetitive elements of genome in proportion that correspond to nonspecific degradation of DNA.
  • Such elements are L1 repeats, satellite DNA, Alu, MER, MIR, THE repeats and some others. Quantity of unique sequences should be small in accordance to their small percent in human genome they may be not detected in cloning DNA without PCR.
  • Blood plasma DNA library of oncological patient with clinically advanced tumor stage.
  • We have constructed blood plasma DNA library of patient with diagnosed advanced stage mesothelioma. Representativeness of library was 8.5×105 clones, that is a good result, taking into account rather small amount of DNA (5 μg) received after purification from non character for healthy donors liposaccharides that were in extremely high concentrations at plasma of patient.
  • We have got the unexpected results after analysis of 96 clones with length from 300 up to 1000 base pairs. (It is necessary to mention that only one clone was not identified as human DNA. For all others correspondent information from HumanGenBank that identifies DNA of these clones as human DNA was received.) As mentioned above, according to data from literature it is logically to assume that there will be a lot of highly repetitive elements in DNA samples.
  • But at least 55 out of 96 clones presented unique sequences of human DNA. Taking into account real ratio of repetitive and unique elements of human genome (95% to 5%) it is obvious that blood plasma DNA repertoire of this patient differs a lot from human genomic DNA repertoire. In this sample an abrupt enrichment by unique DNA sequences is observed.
  • For 15 out of 55 unique DNA fragments that were identified during sequencing of 96 clones from the library of blood plasma DNA, functions or product of correspondent gene were identified. Tables 1-15 present list of these sequences and information about their participation in formation and maintenance of “malignant phenotype”.
  • TABLE 1
    Clone Product Participation in oncogensis Source
    Clone Member of G Playing major role in cancer cell Steeg P. S., Nat
    24 protein - coupled signalling. Linked with cell Rew
    receptor family transformation, supression of Cancer, 2003, v. 3, pp.
    apoptosis, hormone 55-63.
    independence and metastasis Raj G. V, J
    Urology, 2002,
    v. 167, pp. 1458-1463.
    Hoff A. O.,
    Neoplasia, 1999
    v. 1, pp. 485-491.
  • TABLE 2
    Participation in
    Clone Product oncogensis Source
    Clone Snf2-coupled Transcription activator. Thaete c., Hum
    43 CBP activator Family members linked Mol Genet, 1999, v. 8,
    protein with synovial sarcoma pp. 585-91.
    (SCRAP) and leukaemia Monroy M, A., J BioI
    development Chern,. 2001, v. 27 6,
    pp. 40721-40726
    Lee D. W., Cancer
    Res., 2000,
    v. 60, pp. 3612-3622.
  • TABLE 3
    Participation in
    Clone Product oncogensis Source
    Clone SRY-box Transcription modulator. Graham J. D., J
    51 containing Expressed during Mol endocrinol,
    gene embryogenesis. 1999, v. 22, pp.
    Family members linked 295-304.
    with medulloblastomas, Lee C. J., J
    gonadal tumors, highly Neurooncol,
    metastatic melanoma. 2002, v. 57, pp.
    201-214.
    Uehara S., Cancer
    Genet Cytogenet, 1999,
    v. 113., pp. 78-84.
    Tani M., Genomics,
    1997, v. 39, pp. 30-37
  • TABLE 4
    Participation in
    Clone Product oncogensis Source
    Clone Protein-tyrosine Family members Hunter T., Philos
    72 kinase playing major role Trans R Soc Lond
    in cancer. B BioI Sci,
    Some PTK are 1998, v. 353., pp.
    cellular specific 583-605.
    oncogenes products. Scheijen B., Oncogene,
    2002, v. 21., pp.
    3314-3333.
  • TABLE 5
    Participation in
    Clone Product oncogensis Source
    Clone Fibroblast Family members playing Chen W. T.,
    83 activation role in cancer invasion Enzyme
    protein alpha; and metastasis. Protein, 1996, v. 49.,
    cell surface The product is active in pp. 59-71.
    serine protease cancer stroma and Scanlan M. J., Proc
    different carcinomas. Nat Acad Sci USA,
    1994, v. 91,
    pp. 5657-5661.
    Mathew S.,
    Genomics,
    1995, v. 25, pp.
    335-337.
  • TABLE 6
    Participation in
    Clone Product oncogensis Source
    Clone Brain testican Proteoglycan with unknown Genini M.,
    86 function. lnt J Cancer,
    Linked with neoplastic 1996, v. 66, pp.
    phenotype of embryonal 571-577.
    rhabdomyosarcoma
    cells.
  • TABLE 7
    Participation in
    Clone Product oncogensis Source
    Clone KRAB Family members are known as Oguri T.,
    152 domain, transcription repressers. Gene, 1998, v. 222,
    Zn-finger Linked with early pp. 61-67
    proteins embryogenesis, Gou D. M., Biochim
    neuroblastoma, Ewing Biophys Acta,
    sarcoma, Tcell lymphoma, in 2001, v. 1518, pp.
    progression and 306-310
    chemoresistance in lung Margolin J. F., Proc
    cancer. Nat Acad Sci USA,
    1994, v/91, pp.
    4509-4513.
    Bellefroid
    E. J., EMBO J,
    1993, v. 12, pp.
    1363-1374
    Gonzales-Lamuno
    D., Pediatr Pathol
    Mol Med,
    2002, v. 21, pp.
    531-540.
    Marilee J. W., Gene,
    1994, v. 152, pp.
    227-232.
  • TABLE 8
    Participation in
    Clone Product oncogensis Source
    Clone Antigen linked Antigen recognized by J. ImmunoI. 166(4),
    190 with melanoma autologous tumor 2871-2877, 2001
    infiltrating lymphocytes.
  • TABLE 9
    Participation in
    Clone Product oncogensis Source
    Clone N-cadherin Cell adhesion molecule with Kazan R. B., J Cell
    167 major role in cancer growth, BioI,
    invasion and metastasis. 2000, v. 148, pp.
    779-790.
    Li G., Cancer Res,
    2001, v. 61, pp.
    3819-3825.
    Tran N. L., J BioI
    Chern,
    2002, v. 277, pp.
    32905-32914.
  • TABLE 10
    Participation in
    Clone Product oncogensis Source
    Clone FAF1: Fas Phosphoprotein known Jensen H. H., Int J
    197 associated to be the proapoptosis Biochem Cell BioI,
    factor 1 factor. 2001, v. 33, pp. 577-589.
    Ryu S. W., Biochem
    Biophys Res
    Commun,
  • TABLE 11
    Participation in
    Clone Product oncogensis Source
    Clone Interleukin
    7 Proposed as essential Trinder P., lnt J
    114 paracrineautocrine Oneol,
    growth factor for variety of 1999, v. 14, pp.
    cancers. 23-31.
    Cosenza L., Cell
    Signalling,
    2002, v. 14, pp.
    317-325.
  • TABLE 12
    Participation in
    Clone Product oncogensis Source
    Clone DEAD Family members Iggo R. D., Mol Cell
    208 Box RNA involved to RNA BioI,
    helicase-like methabolism. Linked 1991, v. 11, pp.
    protein to exponential cell 1326-1333.
    growth in cancer. Causevic M., Oncogene,
    2001, v. 20, pp.
    7734-7743.
  • TABLE 13
    Participation in
    Clone Product oncogensis Source
    Clone Lipin
    1 One of tumor cells' response Brachat A. et. al.,
    97 regulators on cytotoxic Oncogene,
    compounds. 2002, v. 21, pp.
    8361-8371
  • TABLE 14
    Participation in
    Clone Product oncogensis Source
    Clone Dynein Takes part in transport of p53 Bull J. H., et. aI., Br J
    121 protein, is hypersecreted at Cancer, 2001, v. 84, pp.
    cancer of prostate and 1512-1519.
    hepatocellular cancer. Giannakakou P.,
    et. aI.,
    Nat Cell BioI, 2000, v. 2,
    pp. 709-717
    Jiang 1, et. aI., Gene,
    2001, v. 281, pp.
    103-113.
  • TABLE 15
    Participation in
    Clone Product oncogensis Source
    Clone Ramp Linked with human embrional Cheung W. M.,
    178 protein carcinoma cells' development. et. aI.,
    J BioI Chern, 2001,
    v. 276, pp. 17083-17091
  • In this way 14 out of 15 sequences with identified function or protein that encodes different products (protein kinases, growth factors, proteinases, adhesive molecules and regulatory nuclear proteins) are described in literature as related to “malignant phenotype” formation and maintenance. Only product of 197th clone identified as pro-apop-totic factor is not clearly linked with malignant progression. Though there is data concerning relationship between high apoptotic activity of tumor with its progression (Nishimura R., et al., J Surg Oncol, 1999, v. 71, pp. 226-234) and possible role of apoptotic inductors in formation and maintenance of immunosupression in malignant growth (O'Connel J., et al., Dis Esophagus, 1999, v. 12, pp. 83-89).
  • The most significant presence from repetitive elements in this material was alpha-satellite DNA (30 clones). It is possible to say that that alpha-satellite DNA was the only highly repeated element from human genome, which behaves exactly as repeat in this material. The rest of highly repetitive elements were presented in material as one or several clones (L1 variant and MLT26), or were not found among patterns (MER, Alu, THE, MIR, β-satellite). Based on today's knowledge one can assume that plasma blood composition for the most part should repeat composition of genome DNA, so listed repeats should be represented in the major part of clones while unique and moderately repeated consequences in analysis of such a small number of clones should not be recognized at all. Received result clearly indicates on special way of plasma DNA formation in oncological patients. Another unexpected result is that of finding in the material of two new moderately repeated sequences—duplicones, that were recently unknown, also support the evidence of special way of plasma DNA formation in patients with malignant tumor. For the first time duplicones were found in human genome less then two years ago. Known duplicones (Eichler E. E., et al., Genome Res, 1998, v8, pp. 791-808; Ji Y, et al., Genome Res, 2000, v. 10, pp. 597-610; Pujana M. A., et al., Genome Res, 2001, v. 11, pp. 98-111) are extensive regions of DNA that were multi-plied for several times in the frame of one chromosome (unlike other repeats that are randomly allocated in genome). Duplicones' formation and expansion is connected with different genetic syndromes (for example Prader-Willi/An-gelmane syndrome), with multigenic families' evolution such as MHS (Shiina T., et al., Proc Nat Acad Sci USA, 1999, v. 96, pp. 13282-13287) and with chromosome instability in tumors.
  • It is necessary to mention that analysis of clones received from blood plasma DNA of patient has given us unexpected results.
  • Blood plasma DNA of oncological patient is highly enriched with unique genes. 55 out of 96 analyzed clones contain fragments of genome's unique sequences. 14 out of 15 sequences with identified in functions refer to process of tumor progression and maintenance of “malignant phenotype”.
  • Strong impoverishment of the most wide spread human repeat such as MER, Alu, THE, MIR, β-satellites is found in plasma DNA material.
  • Finding of two consequences with previously unknown duplicones' characteristics indicates on dupli-cones' representativeness in such DNA samples.
  • DNA library of healthy donor's blood plasma.
  • For method's value proof of blood plasma DNA cloning and sequencing for identification of genome's unique genetic consequences we have constructed DNA library of healthy donor's blood plasma. It is known that plasma of clinically healthy people also contains DNA but in significantly less amount than plasma of oncological patients (Shapiro B., et al., Cancer, 1983, v. 51, pp. 2116-2120).
  • Representativeness of library was near 8×105 clones. We have got interesting result after analysis of 70 clones with length from 300 up to 1000 base pairs. We found out that 58 out of 70 analyzed clones are unique DNA sequences of human genome. After searching the HumanGenBank, we identified the function or product of correspondent gene for 14 out of 58 unique DNA fragments.
  • Only 12 clones contained fragments of repetitive sequences, herewith without of alpha-satellite DNA dominance.
  • So, it was unexpectedly found out that blood plasma DNA of healthy people and oncological patients for the most part contain unique fragments of human genome. In the case of oncological pathology unique sequences of blood plasma DNA correspond to genes which products take part in the formation and maintenance of tumor cell's “malignant phenotype”.
  • Basing on this unexpected discovery we have suggested that DNA circulating in patient's blood can be messenger of horizontal genetic information transfer during the course of oncological diseases, assisting to accumulation and spreading of genes that are necessary for “malignant phenotype” formation and maintenance within population of tumor cells.
  • Somatic mosaicism is a condition that is a result of genetically non-identical somatic cells' presence in organism. Modern vision presents that many non tumor and noninfectious (so called somatic) human diseases (for example atherosclerosis, diabetes, nonspecific chronic lung diseases and so on), including aging process, are connected with appearance and spreading (expansion) in the process of individual development of somatic cells' clones that have mutant genes. (Youssoufian H., et al., Nature Rew. Genet., 2002, v. 3, pp. 748-758; J. Vijg, Mutation Res., 2000, v. 447, pp. 117-135; R. Erickson, Mutation Res., 2003, v. 543, pp. 125-136; Andreassi M., Mutation Res., 2003, v. 543, pp. 67-87; Anderson G., et al., Trends in Pharmacological Sci., 2003., v. 24, pp. 71-76).
  • Bright example of such process is progression of mitochondrial heteroplasmia (expansion of mutant mitochondrial DNA) at different diseases and in the aging process (E. Jazin et al., Proc Nat Acad Sci USA, 1996, v. 93, pp. 12382-12387; Michikawa Y. et al., Science, 1999, v. 286, pp. 774-779; Calloway C. et al., Am J Hum Gen, 2000, v. 66, pp. 1384-1397).
  • There are two alternative models of somatic mosaicism's appearance. The first is appearance of somatic mosaicism as a result of numerous “de novo” mutations in polyclonal cellular pool. The second model is clonal expansion of mutant cells' clone (Khrapko K., et al., Muation Res., 2003, v. 522, pp. 13-19).
  • In the process of work above the invention we have found that DNA circulating in the blood of healthy people play significant role in somatic mosaicism's development and its binding, destruction or inactivation inhibits development of somatic mosaicism. Binding, destruction or inactivation of circulating in blood plasma DNA provides treatment effect at diseases which appearance is connected with somatic mosaicism's development.
  • Examples that are mentioned later indicates role of circulating in blood of oncological patients DNA in the development of tumor's resistance to chemotherapy, development of metastasing process, in sepsis development and in some other pathological conditions. High therapeutic effect of blood plasma DNA's binding, destruction or inactivation is found.
    • Example 9
  • DNA Clones' Sequences, Received from Free Circulating Blood Plasma DNA of Patient with Malignant Mesothelioma
  • Clone 1
  • Duplicon, chromosome 15 and Y
  • Sequence No 1.
  • Clone 3
  • Unique, chromosome 2.
  • Sequence No 2
  • Clone 8
  • MLT2B repeat
  • Sequence No 3
  • Clone 9
  • Centromeric satellite DNA
  • Sequence No 4
  • Clone 10
  • MLT2B repeat
  • Sequence No 5
  • Clone 20
  • L1MC4-like (LINE-element)
  • Sequence No 6
  • Clone 15
  • Alpha-satellite DNA
  • Sequence No 7
  • Clones 18, 21
  • Alpha-satellite DNA
  • Sequence No 8
  • Clone 24
  • Unique, family of G protein-bound proteins, chromosome 6.
  • Sequence No 9
  • Clone 25
  • Unique, chromosome 3.
  • Sequence No 10
  • Clone 26
  • SatB1/Vimentin/nuclear matrix binding DNA
  • Sequence No 11
  • Sequence 33
  • Duplicon specific to the chromosome 10
  • Sequence No 12
  • Clone 32
  • alpha-satellite DNA
  • Clone 35
  • LTR repeat
  • Sequence No 13
  • Clone 36
  • Unique, chromosome 18
  • Sequence No 14
  • Clone 37
  • Unique, chromosome 4
  • Sequence No 15
  • Clone 41
  • Sequence No 16
  • Clone 43
  • Snf2-related CBP activator protein (SCRAP)
  • Unique, chromosome 16
  • Sequence No 17
  • Clone 45
  • Unique, chromosome 3
  • Sequence No 18
  • Clone 47
  • Alpha-satellite DNA
  • Clone 51
  • SRY-box containing gene.
  • Sequence No 19
  • Clone 52
  • Repeat
  • Sequence No 20
  • Clone 53, 55
  • Alpha-satellite DNA
  • Sequence No 21
  • Clone 56
  • Centromeric repeat
  • Sequence No 22
  • Clone 60
  • Gene repeated on several chromosomes, contains MER5A repeat.
  • Sequence No 23
  • Clone 62
  • Repeat
  • Sequence No 24
  • Clone 65
  • Unique, chromosome 2
  • Sequence No 25
  • Clone 71
  • Unique, chromosome 2
  • Sequence No 26
  • Clone 72
  • Unique, chromosome 8
  • Sequence No 27
  • Clone 73
  • Unique
  • Sequence No 28
  • Clone 78
  • Transposon Tigger fragment
  • Sequence No 29
  • Clone 81
  • Sequence No 30
  • Repeat (LINE)
  • Clone 82
  • Unique, chromosome 1
  • Sequence No 31
  • Clone 83
  • Unique, Fibroblast activation protein alpha; cell surface serine protease
  • Chromosome 2
  • Sequence No 32
  • Clone 79
  • Alpha-satellite DNA
  • Clone 86
  • Unique, gene highly similar to brain testican, chromosome 4.
  • Sequence No 33
  • Clone 90
  • Unique, chromosome X
  • Sequence No 34
  • Clone 93
  • Unique, chromosome 9
  • Sequence No 35
  • Clones 89 and 92
  • Alpha-satellite DNA
  • Clone 96
  • Fragment LINE.
  • Sequence No 36
  • Clone 97
  • Chromosome 2 unique, Lipin
  • Clone 98
  • Unique, chromosome X
  • Sequence No 38
  • Clone 102
  • Chromosome 17 unique
  • Sequence No 39
  • Clone 99
  • Alpha-satellite DNA
  • Clone 105
  • Unique, chromosome 13
  • Sequence No 40
  • Clone N106
  • Chromosome 9 unique
  • Sequence No 41
  • Clone 107
  • Unique, chromosome 8
  • Sequence No 42
  • Clone N 111
  • Unique, chromosome 12
  • Sequence No 43
  • Clone N 112
  • Chromosome 5 unique
  • Sequence No 44
  • Clone 114
  • Chromosome 8 unique; Interleukin 7
  • Sequence No 45
  • Clone 116
  • Chromosome 1 unique
  • Sequence No 46
  • Clone 121
  • Chromosome 5 unique; Dynein
  • Sequence No 47
  • Clone 115; 119; 120
  • Alpha-satellite DNA
  • Clone 125
  • Chromosome 9 unique
  • Sequence No 48
  • Clone 127
  • Unique chromosome 20
  • Sequence No 49
  • Clone 130
  • Unique, chromosome is not determined.
  • Sequence No 50
  • Clone 124
  • SatB1/Vimentin/nuclear matrix binding DNA
  • Clone 133
  • Alpha-satellite DNA
  • Clone 137
  • MLT1A2 repeat
  • Sequence No 51
  • Clone 140
  • Unique, chromosome 2; zinc finger protein, sub-family 1A
  • Sequence No 52
  • Clone 141
  • Chromosome 2 unique
  • Sequence No 53
  • Clone 143
  • Fragment of Alu-repeat
  • Sequence No 54
  • Clone 144
  • Chromosome 2 unique
  • Sequence No 55
  • Clone 146
  • Chromosome 4 unique
  • Sequence No 56
  • Clone 139 and 142
  • Alpha-satellite DNA
  • Clone 148
  • Repeat ( chromosomes 1, 2 and 4)
  • Sequence No 57
  • Clone 152
  • Unique, chromosome 16; KRAB-Domain, zinc finger protein
  • Sequence No 58
  • Clone 154
  • Chromosome 9 unique
  • Sequence No 59
  • Clone 161
  • Fragment LINE
  • Sequence No 60
  • Clone 151
  • Chromosome 5 unique
  • Sequence No 61
  • Clone 150
  • Chromosome 1 unique
  • Sequence No 62
  • Clone 153
  • Chromosome 11 unique
  • Sequence No 63
  • Clone 159
  • Chromosome 6 unique
  • Sequence No 64
  • Clone 163
  • Alpha satellite DNA
  • Sequence No 65
  • Clone 166
  • Chromosome 12 unique
  • Sequence No 66
  • Clone 167
  • Unique, chromosome 18, CDH2; cadherin 2, type 1, N-cadherin
  • Sequence No 67
  • Clones 169, 170
  • Chromosome 18 unique
  • Sequence No 68
  • Clone 178
  • Unique chromosome 1; RAMP: RA-regulated nuclear matrix-associated protein
  • Sequence No 69
  • Sequence No 69
  • Clone 180
  • Unique, chromosome 20
  • Sequence No 70
  • Clone 181
  • Unique chromosome 18
  • Sequence No 71
  • Clone 185
  • Alpha-satellite DNA
  • Sequence No 72
  • Clone 187
  • Mer repeat
  • Sequence No 73
  • Clone 188
  • HSATII repeat
  • Sequence No 74
  • Clone 189
  • Chromosome 9 unique
  • Sequence No 75
  • Clone 190
  • Chromosome 1 unique; melanoma antigen recognized by T cells 2
  • Sequence No 76
  • Clone 195
  • Chromosome 10 unique
  • Sequence No 77
  • Clone 196
  • Chromosome X unique
  • Sequence No 78
  • Clone 197
  • Chromosome 1 unique, FAF 1: Fas (TNFRSF6) associated factor 1
  • Sequence No 79
  • Clone 200
  • Chromosome 8 unique
  • Sequence No 80
  • Clone 202
  • Unique chromosome 13
  • Sequence No 81
  • Clone 205
  • Alpha satellite DNA
  • Sequence No 82
  • Clone 206
  • Repeat
  • Sequence No 83
  • Clone 208
  • Unique chromosome 8; Human DEAD box RNA helicase-like protein
  • Sequence No 84
    • Example 10 DNA Clones Sequences Received from Free Circulating Blood Plasma DNA of Healthy Donor
  • Clone 1
  • Chromosome 5 unique
  • Sequence No 85
  • Clone 9
  • Unique chromosome 21
  • Sequence No 86
  • Clone 7
  • Unique chromosome 3
  • Sequence No 87
  • Clone 8
  • Chromosome 4 unique
  • Sequence No 88
  • Clone 10
  • 18S RNA gene
  • Sequence No 89
  • Clone 11
  • Alu repeat
  • Sequence No 90
  • Clone 13
  • Unique chromosome 3
  • Sequence No 91
  • Clone 15
  • Unique chromosome 1
  • Sequence No 92
  • Clone 16
  • Unique chromosome 3, neutral endopeptidase
  • Sequence No 93
  • Clone 17
  • Chromosome 8 unique
  • Sequence No 94
  • Clone 18
  • Chromosome 1 unique
  • Sequence No 95
  • Clone 21
  • Unique chromosome 19; Zinc Finger protein
  • Sequence No 96
  • Clone 22
  • Unique chromosome 18
  • Sequence No 97
  • Clone 23
  • Unique chromosome 7, muskelin 1
  • Sequence No 98
  • Clone 25
  • Unique chromosome 11
  • Sequence No 99
  • Clone 27
  • Repeat
  • Sequence No 100
  • Clone 29
  • Unique chromosome 6
  • Sequence No 101
  • Clone 30
  • Unique chromosome 14
  • Sequence No 102
  • Clone 31
  • Unique chromosome 17
  • Sequence No 103
  • Clone 32
  • MER4B repeat
  • Sequence No 104
  • Clone 33
  • Chromosome 1 unique
  • Sequence 105
  • Clone 34
  • Unique chromosome 2
  • Sequence 106
  • Clone 35
  • Repeat
  • Sequence 107
  • Clone 36
  • Chromosome 1 unique
  • Sequence No 108
  • Clone 37
  • HERVH repeat
  • Sequence No 109
  • Clone 41
  • Chromosome X unique
  • Sequence No 110
  • Clone 42
  • Chromosome 6 unique
  • Sequence No 111
  • Clone 43
  • Unique chromosome 22; KREMEN1
  • Sequence No 112
  • Clone 44
  • Unique chromosome 14
  • Sequence No 113
  • Clone 45
  • Unique
  • Sequence No 114
  • Clone 46
  • Chromosome 20 unique
  • Sequence No 115
  • Clone 47
  • Nf-kappaB
  • Sequence No 116
  • Clone 38
  • Unique chromosome 16
  • Sequence No 117
  • Clone 48
  • Chromosome 6 unique
  • Sequence No 118
  • Clone 53
  • Unique
  • Sequence No 119
  • Clone 51
  • Chromosome 5 que
  • Sequence No 120
  • Clone 59
  • Unique chromosome 4, NFKB 1: nuclear factor of kappa light polypeptide gene enhancer
  • Sequence No 121
  • Clone 61
  • Repeat
  • Sequence No 122
  • Clone 62
  • L1 repeat
  • Sequence No 123
  • Clone 64
  • Duplicon chromosome 7
  • Sequence No 124
  • Clone 65
  • Ribosomal DNA
  • Sequence No 125
  • Clone 66
  • Rbosomal DNA
  • Sequence No 126
  • Clone 75
  • Repeat
  • Sequence No 127
  • Clone 76
  • Chromosome 4 unique
  • Sequence No 128
  • Clone 83
  • Chromosome 4 unique
  • Sequence No 129
  • Clone 85
  • Unique chromosome 2; phospholipase C, epsilon
  • Sequence No 130
  • Clone 87
  • L1PA3 repeat
  • Sequence No 131
  • Clone 86
  • Unique chromosome 5; CRTL 1: cartilage linking protein 1
  • Sequence No 132
  • Clone 89
  • Alu repeat
  • Sequence No 133
  • KOH 92
  • Unique chromosome 6
  • Sequence No 134
  • Clone 100
  • Unique, chromosome 6
  • Sequence No 135
  • Clone 105
  • AluSx repeat
  • Sequence No 136
  • Clone 111
  • Alphoid repetitive DNA
  • Sequence No 137
  • Clone 112
  • Chromosome 9 unique
  • Sequence No 138
  • Clone 113
  • Chromosome 22 unique
  • Sequence No 139
  • Clone 114
  • AluSx repeat
  • Sequence No 140
  • Clone 116
  • Unique chromosome 9; 17 kD fetal brain protein
  • Sequence No 141
  • Clone 123
  • Unique chromosome 5
  • Sequence No 142
  • Clone 124
  • Unique chromosome 13
  • Sequence No 143
  • Clone 126
  • Unique chromosome 8
  • Sequence No 144
  • Clone 130
  • Unique chromosome 1
  • Sequence No 145
  • Clone 131
  • Unique chromosome 4
  • Sequence No 146
  • Clone 136
  • Unique chromosome 8
  • Sequence No 147
  • Clone 141
  • Unique chromosome 2
  • Sequence No 148
  • Clone 146
  • Unique chromosome 16
  • Sequence No 149
  • Clone 147
  • Unique chromosome 5; nicotinamide nucleotide transhydrogenase
  • Sequence No 150
  • Clone 149
  • Unique chromosome 9
  • Sequence No 151
  • Clone 151
  • Unique chromosome 16
  • Sequence No 152
  • Clone 152
  • Unique chromosome 6, BA13: brain-specific angiogenesis inhibitor 3
  • Sequence No 153
  • Clone 153
  • Unique chromosome 9, GAD2: glutamate decarboxylase 2
  • Sequence No 154
  • Clone 155
  • Unique chromosome 9
  • Sequence No 155
    • Example 11 Dynamics of P-Glycoprotein Expression in Erlich Carcinoma in Mice that Receive Doxorubicin Therapy and DNase I Effect
  • Treatment by Doxorubicin causes expression of P-glycoprotein in tumor tissue that is one of the main MDR (Multi drug Resistance) phenotype mediators. Immunohistochemical staining of mice's tumor histological cuts are listed below.
  • Mice were subjected to course of 5 day therapy with Doxorubicin (2 mg/kg intravenously daily) or Doxorubicin+DNase I (0.5 mg/kg four times a day during 5 days)
  • Treatment has begun on the 3rd day after tumor's transplantation. Tissue preparations were executed on the 8th day of tumor's transplantation. Multifocal expression of P-glycoprotein was observed in tumor tissue after 5 days of therapy (FIG. 3).
  • Total level of P-glycoprotein expression and amount of P-glycoprotein positive nodules in tumor tissue was much lower at the case of combined treatment by Doxorubicin+DNase (FIG. 3). So treatment by DNase delays development of multidrug resistant phenotype in tumor that is caused by antitumor antibiotic Doxorubicin use.
    • Example 12 Influence of Plasma DNA of C57B1 Mice with LLC Tumor after Chemotherapeutical Treatment by Doxorubicin, on LLC Tumor Growth and Metastasizes Development at C57B1 Mice Receiving Doxorubicin Therapy and Effect of DNase I
  • LLC tumor was replanted to 30 C57B1 mice. Twenty mice were treated with Doxorubicin at 2 mg/kg dose daily for 5 days, starting day 3 after transplantation. Ten mice were treated with Cyclophosphamide at 15 mg/kg dose intraperitoneally for once on the 3rd day after replantation. Such treatment scheme does not lead to animal's recovery but leads to 50% tumor inhibition at day 8 in doxorubicin treated animals and 30% tumor inhibition at day 8 in Cyclophosphamide treated animals. On the next day after end of chemotherapy course animals were euthanized and total blood plasma from both mice groups was taken. After isolation total fraction of blood plasma DNA was stored at −20° C. in phosphate buffer.
  • Five groups of mice that were transplanted with LLC tumor participated in experiment.
  • Group 1-7 mice (control).
  • Group 2-6 mice intravenously treated with Doxorubicin chemotherapy from 3rd up to 8th day at 2 mg/kg dose daily.
  • Group 3-6 mice intravenously treated with Doxorubicin chemotherapy from 3rd up to 8th day at 2 mg/kg dose daily+intravenous administration of DNA fraction from mice previously subjected to Doxorubicin chemotherapy (0.05 mkg of DNA in 200 mkl of phosphate buffer at day 1 and day 3 after initiation of treatment)
  • Group 4-6 mice intravenously treated with Doxorubicin chemotherapy from 3rd up to 8th day at 2 mg/kg dose daily+intravenous administration of DNA fraction from mice previously subjected to cyclophosphamide chemotherapy (0.05 mkg of DNA in 200 mkl of phosphate buffer at day 1 and day 3 after initiation of treatment)
  • Group 5-6 mice intravenously treated with Doxorubicin chemotherapy from 3rd up to 8th day at 2 mg/kg dose daily+intravenous administration of DNA fraction from mice previously subjected to Doxorubicin chemotherapy (0.05 mkg of DNA in 200 mkl of phosphate buffer at day 1 and day 3 after initiation of treatment)+intraperitoneal administration of DNase I at 0.5 mg/kg dose for 4 times a day at the first and second days of treatment.
  • Tumor's size on the 8th day after transplantation.
  • Group Tumor's size
    1 127 +/− 13
    2 67 +/− 7
    3 115 +/− 20
    4  75 +/− 11
    5 82 +/− 9
  • So administration of blood plasma DNA from mice subjected to chemotherapy lead to tumor's resistance to chemotherapeutic treatment. DNase's administration prevents appearance of this effect.
    • Example 13 Influence of Blood Plasma DNA from C57B1 Mice with Highly Metastatic LLC Strain on Metastasizing of Low Metastatic LLC Tumor Strain in C57B1 Mice and Effect of DNase I
  • LLC tumor was transplanted to 30 C57B1 mice. Twenty mice were transplanted with highly metastatic strain and 10 mice were transplanted with low metastatic strain. On the 9th day animals were euthanized and total blood plasma of both mice groups was collected. After isolation the total fraction of blood plasma DNA was stored at −20° C. in phosphate buffer.
  • Five groups of mice with transplanted LLC tumor participated in the experiment.
  • 1 Group-6 mice transplanted with low metastatic LLC strain.
  • 2 Group-6 mice transplanted with low metastatic LLC strain+intravenous administration of total DNA fraction from mice with transplanted highly metastatic strain (0.05 mkg of DNA in 200 mkl of phosphate buffer on the 7th and 8th day after transplantation).
  • 3 Group-6 mice transplanted with low metastatic LLC strain+intravenous administration of total DNA fraction from mice with transplanted low metastatic strain (0.05 mkg of DNA in 200 mkl of phosphate buffer on the 7th and 8th day after transplantation)
  • 4 Group-6 mice transplanted with low metastatic LLC strain+intravenous administration of total DNA fraction from mice with transplanted highly metastatic strain (0.05 mkg of DNA in 200 mkl of phosphate buffer on the 7th and 8th day after transplantation)+intraperitoneal administration of DNase I at 1 mg/kg dose two times daily at 7th and 8th day after transplantation.
  • 5 Group-6 mice transplanted with highly metastatic LLC strain.
  • Number of metastatic foci in lungs was estimated on the 15th day after transplantation (N).
  • Experiments' results are presented in the table.
  • Group N
    1 12, 0
    2 24, 1
    3 14, 6
    4 11, 6
    5 33, 6
  • Received data indicates that blood plasma DNA from mice with highly metastatic LLC strain intensify metastasizing of low metastatic LLC strain.
  • DNase administration prevents appearance of this effect.
    • Example 14 DNase I Influence on Life Span of C57B1 Mice Transplanted with LLC Tumor (Highly Metastatic Strain)
  • Five groups of LLC transplanted mice participated in the experiment.
  • Group 1-7 mice (control).
  • Group 2-6 mice were treated with intraperitoneal administration of DNase at 1 mg/kg dose two times a day starting from 3 up to 7 day after tumor transplantation.
  • Group 3-6 mice were treated with intraperitoneal administration of DNase at 1 mg/kg dose two times a day starting from 3 up to 10 day after tumor transplantation.
  • Group 4-6 mice were treated with intraperitoneal administration of DNase at 1 mg/kg dose two times a day starting from 3 up to 15 day after tumor transplantation.
  • Group 5-6 mice were treated with intraperitoneal administration of DNase at 1 mg/kg dose two times a day starting from 3 up to 18 day after tumor transplantation.
  • Results of experiment were estimated according to animals' survival on the 30 and 50 day after tumor transplantation.
  • 30 day 50 day
    (number of (number of
    alive\number of alive\number of
    Group dead in group) dead in group)
    1 0-7 0-7
    2 0-6 0-6
    3 3-6 0-6
    4 5-1 3-3
    5 6-0 6-0
  • The significant inhibition of tumor's growth was observed at the last day of DNase treatment in the 2nd and 3rd groups, but tumor's growth renewed after DNase withdrawal and to the 25th day size of tumor in this groups and in control has equalized.
  • The most longitudinal course of DNase treatment (from 3rd up to 18th day-group number 6) has lead to maximal survival. Inhibition of tumor growth was more than 95% at day 18.
  • In all experiments single and multiple injection of up to 2.5 mg/kg of human DNase I (maximal dose that was used in experiments) had no toxic effect on animals.
  • So, DNase I does not cause direct cytotoxic effect on tumor cells (in our in vitro experiments at concentration of 100 mkg/ml) and experimental data confirm that antitumor effect is connected with destruction of DNA in blood plasma and DNase's therapeutic effect increases with increasing of its treatment course duration.
    • Example 15 Influence of Different Methods of Blood Plasma DNA's Destruction, Inactivation, and Binding on Ability of Blood Plasma DNA from C57B1 Mice with Transplanted Highly Metastatic LLC Strain to Intensify Metastasizing of Low Metastatic LLC Tumor Strain in C57B1 Mice
  • 100 mice were transplanted with highly metastatic LLC strain. On the 9th day after transplantation, animals were euthanized and total blood plasma was taken. After isolation total fraction of blood plasma DNA was stored at −20° C. in phosphate buffer.
  • Six groups of mice with transplanted low metastatic LLC strain participated in the experiment.
  • Group 1-6 mice transplanted with low metastatic LLC strain.
  • Group 2-6 mice transplanted with low metastatic LLC strain+two intravenous injections of total DNA fraction from mice transplanted with highly metastatic strain on the 7th and 8th day after transplantation (0.05 mkg of DNA was dissolved in 500 mkl of fresh heparinized blood before injection).
  • Group 3-6 mice transplanted with low metastatic LLC strain+two intravenous injections of total DNA fraction from mice transplanted with highly metastatic strain on the 7th and 8th day after transplantation (0.05 mkg of DNA was dissolved in 500 mkl of fresh blood plasma before injection). Before administration the sample was photochemically disinfected (1 mkM of methylene blue was added with following irradiation by red light during 10 minutes (60 000 Lux).
  • Group 4-6 mice transplanted with low metastatic LLC strain+two intravenous injections of total DNA fraction from mice transplanted with highly metastatic strain on the 7th and 8th day after transplantation (0.05 mkg of DNA was dissolved in 500 mkl of fresh blood plasma before injection). The sample was passed through the column containing DEAE-cellulose for two times before administration.
  • Group 5-6 mice transplanted with low metastatic LLC strain+two intravenous injections of total DNA fraction from mice transplanted with highly metastatic strain on the 7th and 8th day after transplantation (0.05 mkg of DNA was dissolved in 500 mkl of fresh heparinized blood before injection. 1 mkg of fragment A of Ricin toxin was added to the sample before administration and sample was incubated at 370 C for 1 hour. Ricin toxin is representative of RIP toxins family (proteins that inactivate ribosomes) which are used for immunotoxin's creation. Besides their ability to inactivate ribosomes these proteins can deadenylate DNA. For realization of toxic effect catalytic subunit A of RIP II type should by delivered to cell by B subunit. Without B subunit A chain is not toxic but can be used for blood plasma DNA's inactivation due to its polynucleotide-adenylglicozidase activity.
  • Group 6-6 mice transplanted with low metastatic LLC strain+two intravenous injections of total DNA fraction from mice transplanted with highly metastatic strain on the 7th and 8th day after transplantation (0.05 mkg of DNA was dissolved in 500 mkl of fresh heparinized blood before injection. Total DNA fraction was enzymatically methylated before administration (I. Muiznieks et. al., FEBS Letters, 1994, v. 344, pp. 251-254).
  • Number of metastaic nodules in lungs was estimated on the 15th day after transplantation.
  • Results of the experiments are presented in the table.
  • Group Ncp.
    1 12, 0
    2 22, 5
    3 14, 1
    4 15, 5
    5 15, 1
    6 12, 3
  • Received data indicates that all used methods inhibited ability of blood plasma DNA of mice with highly metastatic LLC tumor strain to increase metastasizing process of low metastatic LLC tumor strain.
    • INDUSTRIAL APPLICABILITY
  • For the realization the methods there were used well-known materials and equipment manufactured in plant conditions and according to aforesaid the invention conformances to requirements of “industrial applicability” criteria (IA).

Claims (6)

1. A method for suppressing spread of mutated DNA sequences within the body and development of somatic mosaicism in a subject in need thereof, wherein said method comprises administering a DNase enzyme to said subject, wherein said DNase enzyme is administered in a dose and regimen which is sufficient to decrease the average molecular weight of extracellular blood DNA in the blood of said subject.
2-4. (canceled)
5. The method according to claim 1, wherein said DNase enzyme is administered in a dose and regimen that results in a DNA hydrolytic activity measured in blood plasma that exceeds 1.5 Kunitz units per 1 ml of blood plasma for more than 12 hours within a period of 24 hours.
6. The method of claim 1, wherein the subject has a cancer and the development of somatic mosaicism results from a spread of mutated DNA sequences promoting cancer development.
7. The method of claim 6, wherein the subject has a metastatic cancer and the development of somatic mosaicism results from a spread of mutated DNA sequences promoting metastasis development.
8. The method of claim 1, wherein the subject has a cancer and is receiving a chemotherapy and the development of somatic mosaicism results from a spread of mutated DNA sequences promoting resistance to said cancer chemotherapy.
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Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8710012B2 (en) 2003-07-14 2014-04-29 Cls Therapeutics Limited Method for treating oncological diseases
US8431123B2 (en) 2003-07-14 2013-04-30 Cls Therapeutics Limited Method for treating systemic bacterial, fungal and protozoan infection
EP1880733A4 (en) * 2005-04-25 2009-08-12 Genkin Dmitry Dmitrievich Method for increasing longevity of a human being and animals
EP2095825B8 (en) * 2006-11-28 2012-03-28 CLS Therapeutics Limited Method for treating human diseases associated with an increased deoxyribonucleic acid content in extracellular spaces of tissues and a medicinal preparation for carrying out said method
WO2012166611A2 (en) 2011-05-27 2012-12-06 Immune Disease Institute, Inc. Methods for treating and preventing neutrophil-derived net toxicity and thrombosis
US10617743B2 (en) 2014-06-19 2020-04-14 Cls Therapeutics Limited Method to improve safety and efficacy of anti-cancer therapy
NZ733912A (en) 2015-01-04 2023-04-28 Protalix Ltd Modified dnase and uses thereof
JP6745873B2 (en) * 2015-05-22 2020-08-26 ドミトリエヴィッチ ゲンキン，ドミトリー Extracellular DNA as a therapeutic target in neurodegeneration
EP3351263A1 (en) 2017-01-20 2018-07-25 Universitätsklinikum Hamburg-Eppendorf Pharmaceutical preparation for treating or preventing tissue adhesion
WO2018197547A1 (en) 2017-04-25 2018-11-01 Lipoxen Technologies Limited Methods of treating diseases related to net formation with parenteral administration of polysialylated dnase i
WO2019036719A2 (en) 2017-08-18 2019-02-21 Neutrolis Therapeutics, Inc. Engineered dnase enzymes and use in therapy
PL3685165T3 (en) 2017-09-18 2022-08-08 Santersus Ag Method and device for purification of blood from circulating cell free dna
EP3740581A4 (en) 2018-01-16 2021-10-27 CLS Therapeutics Limited Treatment of diseases by liver expression of an enzyme which has a deoxyribonuclease (dnase) activity
US11058724B2 (en) 2018-10-08 2021-07-13 Neutrolis, Inc. Methods of using DNASE1-like 3 in therapy
US10988746B2 (en) 2018-10-08 2021-04-27 Neutrolis, Inc. Manufacturing and engineering of DNASE enzymes for therapy
CN112996911A (en) 2018-10-08 2021-06-18 纽特雷斯股份有限公司 Engineering of deoxyribonucleases for manufacture and therapy
US11352613B2 (en) 2019-02-04 2022-06-07 Neutrolis, Inc. Engineered human extracellular DNASE enzymes
US11475981B2 (en) 2020-02-18 2022-10-18 Tempus Labs, Inc. Methods and systems for dynamic variant thresholding in a liquid biopsy assay
US11211147B2 (en) 2020-02-18 2021-12-28 Tempus Labs, Inc. Estimation of circulating tumor fraction using off-target reads of targeted-panel sequencing
US11211144B2 (en) 2020-02-18 2021-12-28 Tempus Labs, Inc. Methods and systems for refining copy number variation in a liquid biopsy assay
WO2022074656A1 (en) 2020-10-07 2022-04-14 Protalix Ltd. Long-acting dnase
WO2023126672A1 (en) 2021-12-27 2023-07-06 Santersus Ag Method and device for removal of circulating cell free dna

Family Cites Families (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3224942A (en) 1962-01-15 1965-12-21 Rorer Inc William H Anti-inflammatory, anti-edema and anti-thrombi drug
US3324002A (en) 1962-09-17 1967-06-06 Armour Pharma Anti-inflammatory preparations containing proteolytic enzymes and adrenal glucocorticoids
JPS5826822A (en) 1981-08-10 1983-02-17 Kaken Pharmaceut Co Ltd Preventing and pemedy for liver and kidney diseases
JPS61293927A (en) 1986-06-18 1986-12-24 Kaken Pharmaceut Co Ltd Preventive and remedy for hepatic disease
US4985243A (en) 1988-01-20 1991-01-15 Ml Technology Ventures, L.P. Composition and method for protecting against diseases caused by microorganisms
ATE358176T1 (en) * 1988-12-23 2007-04-15 Genentech Inc HUMAN DNASE
US5464817A (en) * 1990-04-11 1995-11-07 Brigham And Women's Hospital Method for reducing the viscosity of pathological mucoid airway contents in the respiratory tract comprising administering actin-binding compounds with or without DNASE I
DE4024530A1 (en) 1990-08-02 1992-02-06 Klinger & Co Dr Control of viral disease using bovine pancreatic nuclease(s) - for treatment of e.g. rabies, herpes, Epstein-Barr virus and cow-pox
FR2666096B1 (en) 1990-08-21 1993-12-31 Sfri Sa NEW PROCESS FOR DETERMINING DEOXYRIBONUCLEIC ACID PRESENT IN EXTRACELLULAR POSITION IN A MEDIUM.
EP0599850B1 (en) 1991-08-16 1996-01-10 Vical, Inc. Composition and method for treating cystic fibrosis
US5484589A (en) * 1992-04-20 1996-01-16 Rufeld, Inc. Anti-viral methods using RNAse and DNAse
NZ299257A (en) 1992-06-08 2000-08-25 Genentech Inc Treating purulent material (eg pulmonary disease) accumulation in patients using deamidated DNase
WO1995000170A1 (en) 1993-06-24 1995-01-05 Rufeld, Inc. Inhibition of human immunodeficiency virus reproduction by deoxyribonuclease i
CH686982A5 (en) * 1993-12-16 1996-08-15 Maurice Stroun Method for diagnosis of cancers.
RU2099080C1 (en) 1994-02-11 1997-12-20 Дмитрий Иванович Финько Method for preventing from cancer development
JP2986215B2 (en) 1994-03-04 1999-12-06 ジェネンテック インコーポレイテッド Pharmaceutical preparation containing DNase
US5830744A (en) 1995-06-06 1998-11-03 Human Genome Sciences, Inc. Gene encoding human Dnase
WO1995032219A1 (en) * 1994-05-20 1995-11-30 Hisamitsu Pharmaceutical Co., Inc. Protein or polypeptide, process for producing the same, and intermediate compound tehrefor
DE69736276T2 (en) 1996-02-05 2007-08-30 Genentech Inc., San Francisco HUMAN DNASE RESISTANT AGAINST ACTININHIBITORS
AU2324997A (en) 1996-03-15 1997-10-01 Penn State Research Foundation, The Detection of extracellular tumor-associated nucleic acid in blood plasma or ser um using nucleic acid amplification assays
US6391607B1 (en) 1996-06-14 2002-05-21 Genentech, Inc. Human DNase I hyperactive variants
US5855920A (en) 1996-12-13 1999-01-05 Chein; Edmund Y. M. Total hormone replacement therapy
US6455250B1 (en) * 1997-12-11 2002-09-24 The Regents Of The University Of California Endonuclease compositions and methods of use
CA2337517A1 (en) 1998-07-16 2000-01-27 Research Development Foundation Novel dna-cleaving antitumor agents
US6465177B1 (en) * 1998-10-26 2002-10-15 John Wayne Cancer Institute Detection of loss of heterozygosity in tumor and serum of melanoma patients
JP2002530436A (en) 1998-11-25 2002-09-17 ジェネティカ インコーポレイテッド Method and reagent for enhancing proliferation ability and preventing replication senescence
JP4522632B2 (en) * 1999-08-17 2010-08-11 靖一 田沼 Novel deoxyribonuclease, gene encoding it and use thereof
ES2185549T3 (en) * 1999-09-27 2003-05-01 Ciba Sc Holding Ag CARMESI RED COLOR INKS (MAGENTA) CONTAINING COPPER COMPLEX AZOIC COLORANTS BASED ON 1-NAFTOL-DI- OR -TRISULPHONIC ACIDS.
US6428785B1 (en) * 1999-10-28 2002-08-06 Immunolytics Inc. Method and composition for treating prostate cancer
UA78486C2 (en) 1999-12-10 2007-04-10 Хемджен Корпорейшн Formulation for oral use in fowls and animals for treating or decreasing risk of infections of digestive tract (variants), use of composition (variants), method for treating or decreasing risk of infections of digestive tract (variants)
WO2001074905A1 (en) 2000-04-03 2001-10-11 Antisoma Research Limited Compounds for targeting
RU2202109C1 (en) 2001-08-03 2003-04-10 Научно-исследовательский институт неврологии РАН Method for detecting degree of disorders in rheological blood properties
RU2207876C1 (en) 2001-11-08 2003-07-10 Ткаченко Виталий Васильевич Method for complex- individualized impact upon body at slow viral infection and method for preparing laboratory animal for testing the method of such impact
US7402724B2 (en) 2002-01-04 2008-07-22 Mayo Foundation For Medical Education And Research Longevity and PAPP-A
DK1549337T3 (en) 2002-02-18 2014-11-24 Ockham Biotech Ltd COMBINATION OF DNASE I AND GLYCOSAMINOGLYCANES FOR USE IN EXTRACELLULAR DNA CLEARANCE
DE10221194B4 (en) 2002-05-13 2005-02-10 Fryda, Waltraud, Dr.med. Hyaluronidase containing skin care product
US20040001817A1 (en) * 2002-05-14 2004-01-01 Giampapa Vincent C. Anti-aging nutritional supplement
ATE360817T1 (en) 2002-12-18 2007-05-15 Coy Johannes F Dr COMPOUNDS AND METHODS FOR DETECTING CARCINOMAS AND THEIR PRECURSORS
RU2239442C1 (en) 2003-05-05 2004-11-10 Красноярская государственная медицинская академия Method for prophylaxis of intrauterine infection in children
RU2269359C2 (en) 2004-03-12 2006-02-10 Дмитрий Дмитриевич Генкин Method for prophylaxis of oncological diseases, or infections mordibidized by bacteria or fungi and protozoa, or arteriosclerosis, or diabetes mellitus, or diseases mediated by delayed hyperresponsiveness reaction, or diseases mediated by somatic cell gene mutations (variants)
WO2005007187A1 (en) 2003-07-14 2005-01-27 Viktor Veniaminovich Tets Method for treating oncological, virulent and somatic diseases, method for controlling treatment efficiency, pharmaceutical agents and composition for carrying out said treatment
RU2269358C2 (en) 2003-07-14 2006-02-10 Дмитрий Дмитриевич Генкин Method for treatment of generalized infections mordibidized by bacteria or fungi and protozoa, or arteriosclerosis, or diabetes mellitus, or diseases mediated by delayed hyperresponsiveness reaction, or diseases mediated by somatic cell gene mutations
WO2005004904A1 (en) 2003-07-14 2005-01-20 Dmitry Dmitrievich Genkin Method for treating diseases associated with modifications of qualitative and/ quantitative composition of blood extracellular dna (variants)
US8431123B2 (en) * 2003-07-14 2013-04-30 Cls Therapeutics Limited Method for treating systemic bacterial, fungal and protozoan infection
US8710012B2 (en) * 2003-07-14 2014-04-29 Cls Therapeutics Limited Method for treating oncological diseases
RU2269356C2 (en) 2003-07-14 2006-02-10 Дмитрий Дмитриевич Генкин Method for treating oncological patients
RU2239404C1 (en) 2003-09-17 2004-11-10 Бессонов Дмитрий Михайлович Method for preventing human body aging
RU2267329C2 (en) 2004-03-12 2006-01-10 Дмитрий Дмитриевич Генкин Method for treating diseases accompanied with alterations of qualitative and/or quantitative composition of blood extra-cellular dna (variants)
RU2269357C2 (en) 2004-03-12 2006-02-10 Дмитрий Дмитриевич Генкин Method for deteriorating the onset of aging-associated sick states
WO2005115444A2 (en) 2004-04-14 2005-12-08 Avirid, Inc. Compositions with modified nucleases targeted to viral nucleic acids and methods of use for prevention and treatment of viral diseases
JP3989936B2 (en) 2005-04-07 2007-10-10 進 須永 Antitumor agent and novel DNase
EP1880733A4 (en) 2005-04-25 2009-08-12 Genkin Dmitry Dmitrievich Method for increasing longevity of a human being and animals
WO2007010533A2 (en) 2005-07-18 2007-01-25 Protalix Ltd. Mucosal or enteral administration of biologically active macromolecules
RU2308968C2 (en) 2005-07-19 2007-10-27 Дмитрий Дмитриевич Генкин Method for treating human diseases accompanied with increased dna content in extracellular spaces of tissues and medicinal preparation for its realization
WO2008047364A2 (en) * 2006-10-18 2008-04-24 Periness Ltd. Method and pharmacological composition for the diagnosis and treatment of male sub-fertility
EP2095825B8 (en) 2006-11-28 2012-03-28 CLS Therapeutics Limited Method for treating human diseases associated with an increased deoxyribonucleic acid content in extracellular spaces of tissues and a medicinal preparation for carrying out said method
GB0922006D0 (en) * 2009-12-17 2010-02-03 Genome Res Ltd Diagnostic
US20130243794A1 (en) 2010-12-03 2013-09-19 Beth Israel Deaconess Medical Center, Inc. Methods for predicting and treating infection-induced illnesses and predicting the severity of infection-induced illnesses
AU2013298144B2 (en) 2012-08-03 2018-03-08 Ferring B.V. Cell-free DNA as a therapeutic target for female infertility and diagnostic marker

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