# Patent

## Title
Method for monitoring development of somatic mosaicism

## 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.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation of U.S. application Ser. No. 13/772,499 filed Feb. 21, 2013, 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 are incorporated by reference as if fully rewritten herein.


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 (EI 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; Amalich 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; Amalich 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. 2: Panel A shows a graph of the survival of NOD diabetic mice treated with different doses of DNASE1—50 mkg/kg, 500 mkg/kg, and control; Panel B 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. 3: 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). A—Doxorubicin+DNase; B—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. 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. 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 Pvull 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
Atheroscleros is/diabetes


Gene
of 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 of


transhydrogenase

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:














At DNASE course


Indicator
Prior DNASE 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 Sep. 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 (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. 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 (30 U, 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
Playing major role in cancer
Steeg P. S., Nat


24
G protein -
cell signalling. Linked with cell
Rew



coupled
transformation, supression of
Cancer, 2003,



receptor
apoptosis, hormone
v. 3, pp. 55-63.



family
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



Clone
Product
in oncogensis
Source







Clone
Snf2-coupled
Transcription activator.
Thaete c., Hum


43
CBP activator
Family members linked
Mol Genet, 1999,



protein
with synovial sarcoma
v. 8, pp. 585-91.



(SCRAP)
and leukaemia
Monroy M, A., J Biol




development
Chern,. 2001, v. 27 6,





pp. 40721-40726





Lee D. W., Cancer





Res., 2000,





v. 60, pp. 3612-3622.



















TABLE 3





Clone
Product
Participation in oncogensis
Source







Clone
SRY-box
Transcription modulator.
Graham J. D., J


51
containing
Expressed during
Mol endocrinol,



gene
embryogenesis.
1999, v. 22, pp. 295-304.




Family members linked
Lee C. J., J




with medulloblastomas,
Neurooncol,




gonadal tumors, highly
2002, v. 57, pp. 201-214.




metastatic melanoma.
Uehara S., Cancer





Genet Cytogenet, 1999,





v. 113., pp. 78-84.





Tani M., Genomics,





1997, v. 39, pp. 30-37



















TABLE 4





Clone
Product
Participation in oncogensis
Source







Clone
Protein-tyrosine
Family members playing
Hunter T., Philos


72
kinase
major role in cancer.
Trans R Soc Lond




Some PTK are cellular
B Biol Sci,




specific oncogenes
1998, v. 353., pp.




products.
583-605.





Scheijen B.,





Oncogene,





2002, v. 21., pp.





3314-3333.



















TABLE 5





Clone
Product
Participation in oncogensis
Source







Clone
Fibroblast
Family members playing
Chen W. T,


83
activation
role in cancer invasion
Enzyme Protein,



protein alpha;
and metastasis. The product
1996, v. 49.,



cell surface
is active in cancer stroma
pp. 59-71.



serine protease
and different carcinomas.
Scanlan M.





J., Proc





Nat Acad





Sci USA,





1994, v. 91,





pp. 5657-5661.





Mathew S.,





Genomics,





1995, v. 25,





pp. 335-337.



















TABLE 6





Clone
Product
Participation in oncogensis
Source







Clone
Brain testican
Proteoglycan with
Genini M.,


86

unknown function.
Int J Cancer,




Linked with neoplastic
1996, v. 66,




phenotype of embryonal
pp. 571-577.




rhabdomyosarcoma




cells.



















TABLE 7





Clone
Product
Participation in oncogensis
Source







Clone
KRAB domain,
Family members
Oguri T., Gene,


152
Zn-finger
are known
1998, v. 222,



proteins
as transcription
pp. 61-67




repressors.
Gou D. M.,




Linked with early
Biochim




embryogenesis,
Biophys Acta,




neuroblastoma,
2001, v. 1518,




Ewing sarcoma,
pp. 306-310




Tcell lymphoma,
Margolin




in progression
J. F., Proc




and chemoresistance
Nat Acad




in lung cancer.
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





Clone
Product
Participation in oncogensis
Source







Clone
Antigen linked
Antigen recognized
J. Immunol.


190
with melanoma
by autologous tumor
166(4),




infiltrating lymphocytes.
2871-2877, 2001



















TABLE 9





Clone
Product
Participation in oncogensis
Source







Clone
N-cadherin
Cell adhesion molecule with
Hazan R. B.,


167

major role in cancer growth,
J Cell Biol,




invasion and metastasis.
2000, v. 148,





pp. 779-790.





Li G., Cancer





Res,





2001, v. 61,





pp. 3819-3825.





Tran N. L,





J Biol Chern,





2002, v. 277,





pp. 32905-32914.



















TABLE 10





Clone
Product
Participation in oncogensis
Source







Clone
FAFI: Fas
Phosphoprotein known
Jensen H. H.,


197
associated factor
to be the proapoptosis
Int J Biochem



1
factor.
Cell Biol,





2001, v. 33,





pp. 577-589.





Ryu S. W.,





Biochem Biophys





Res Commun,



















TABLE 11





Clone
Product
Participation in oncogensis
Source







Clone
Interleukin 7
Proposed as essential
Trinder P.,


114

paracrineautocrine
Int J Oneol,




growth factor for
1999, v. 14,




variety of cancers.
pp. 23-31.





Cosenza L., Cell





Signalling,





2002, v. 14,





pp. 317-325.



















TABLE 12





Clone
Product
Participation in oncogensis
Source







Clone
DEAD Box RNA
Family members involved
Iggo R. D.,


208
helicase-like
to RNA methabolism. Linked
Mol Cell Biol,



protein
to exponential cell growth
1991, v. 11,




in cancer.
pp. 1326-1333.





Causevic M.,





Oncogene,





2001, v. 20,





pp. 7734-7743.



















TABLE 13





Clone
Product
Participation in oncogensis
Source







Clone
Lipin 1
One of tumor cells'
Brachat A. et.


97

response regulators on
al., Oncogene,




cytotoxic compounds.
2002, v. 21,





pp. 8361-8371



















TABLE 14





Clone
Product
Participation in oncogensis
Source







Clone
Dynein
Takes part in transport of p53
Bull J. H.,


121

protein, is hypersecreted at
et. al., Br




cancer of prostate and
J Cancer,




hepatocellular cancer.
2001, v. 84,





pp. 1512-1519.





Giannakakou





P., et. al.,





Nat Cell Biol,





2000, v. 2,





pp. 709-717





Jiang 1, et.





al., Gene,





2001, v. 281,





pp. 103-113.



















TABLE 15





Clone
Product
Participation in oncogensis
Source







Clone
Ramp protein
Linked with human embrional
Cheung W. M.,


178

carcinoma cells'
et. al., J




development.
Biol 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, v 8, 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
LIMC4-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
Ribosomal 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
L1 PA3 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 mk1 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 mk1 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 mk1 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 mk1 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 (number of
50 day (number of



alive\number
alive\number


Group
of dead in group)
of 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 mk1 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 mk1 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 mk1 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 mk1 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

1. A method for monitoring development of somatic mosaicism in a subject diagnosed with cancer, said method comprising:
(a) isolating extracellular blood DNA from the blood of said subject at two or more different time points,
(b) creating a separate DNA library from the extracellular blood DNA isolated at each time point in step (a) by cloning said extracellular blood DNA, wherein each said DNA library has at least 106 clones,
(c) sequencing multiple DNA clones from each library created in step (b), and
(d) determining quantitative and qualitative differences between the sequenced clones from the libraries representing different time points.

2. The method of claim 1, wherein the development of somatic mosaicism is a spread of cancer promoting mutations.

3. The method of claim 2, wherein the development of somatic mosaicism is a spread of metastasis promoting mutations.

4. The method of claim 1, wherein the development of somatic mosaicism is a spread of mutations promoting resistance to cancer chemotherapy.

5. The method of claim 1, wherein cloning in step (b) is performed without prior amplification.