RU2779220C2 - Method and device for blood purification from free-circulating dna - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: device for apheresis is proposed, containing one or more affine matrices capable of capturing free-circulating DNA (hereinafter – fcDNA) bound to nucleosomes, fcDNA bound to exosomes, and unbound fcDNA from subject’s blood or plasma, wherein the specified one or more affine matrices contain DNA-binding polymer, DNA-binding protein, an anti-histone antibody, an anti-nucleosome antibody, a DNA-intercalating agent, an anti-DNA antibody, or any combination thereof. A method for the reduction in a level of free-circulating DNA (fcDNA) in subject’s blood, and a method for the treatment of a diseases characterized by increased levels of circulating DNA in subject’s blood are proposed.

EFFECT: proposed group of inventions provides for a positive effect on the treatment of diseases characterized by increased levels of circulating fcDNA in blood.

24 cl, 9 dwg, 10 tbl, 21 ex

Description

LINK TO RELATED APPLICATIONS.

[001] This application claims priority over U.S. Provisional Application No. 62/559,222, filed September 18, 2017, which is incorporated herein by reference in its entirety.

FIELD OF TECHNOLOGY

[002] The invention discloses apheresis devices and their use for the removal of virtually all types of extracellular DNA (cfDNA) from the blood of patients, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including double-stranded DNA (dsDNA), single-stranded DNA (ssDNA ) and oligonucleotides), to limit the negative impact of circulating cfDNA and treat various diseases.

BACKGROUND OF THE INVENTION

[003] Circulating extracellular DNA (ecDNA), also called extracellular DNA (eDNA), is present in small amounts in the blood of healthy individuals.

[004] Elevated levels of circulating cfDNA are now widely recognized as a marker for a number of diseases and pathological conditions, including, but not limited to, cancer, metastatic cancer, acute organ failure, organ infarction (including myocardial infarction and ischemic stroke), hemorrhagic stroke, autoimmune disorders, graft-versus-host disease (GVHD), graft rejection, sepsis, systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), graft-versus-host disease (GVHD), traumatic injury, pro-inflammatory status in the elderly, diabetes, atherosclerosis, neurodegenerative diseases, autoimmune diseases, eclampsia, infertility, bleeding disorders, complications associated with pregnancy, and infections. Various subtypes of circulating extracellular DNA may play a significant role in the progression of certain diseases and pathological conditions.

[005] Systemic administration of a deoxyribonuclease (DNase) enzyme that specifically hydrolyzes circulating cfDNA has been proposed for the treatment of infertility (US Pat. No. 8,916,151); cardiovascular diseases (US patent No. 9642822); cancer; sepsis, graft-versus-host disease (GVHD); organ failure; diabetes; atherosclerosis; delayed-type hypersensitivity reactions (US patents No. 9248166; 8535663; 7612032; 8388951; 8431123).

[006] However, contrary to the results obtained in animal models, the results in real clinical conditions showed that the systemic application of the enzyme deoxyribonuclease (DNase) has a very limited effect on reducing the amount of circulating cfDNA.

[007] Hazout A. (PCT/IB 2013/056321) described 10 women with high circulating cfDNA levels (>80 ng/μl) who received DNasel 0.1 mg/kg intramuscularly twice daily for seven days, with there was only an average of 26% reduction in circulating cfDNA levels. Their observations are consistent with those of Davis et al., who were unable to demonstrate a reduction in circulating double-stranded DNA in patients with lupus nephritis receiving human recombinant deoxyribonuclease at 25 μg/kg as one intravenous and ten subcutaneous injections over a period of 19 days, despite to achieve catalytically effective plasma concentrations of deoxyribonuclease between 40-100 ng/ml. (Davis J.C. et al., Recombinant human Dnase I (rhDNase) in patients with lupus nephritis Lupus (1999) Vol 8 (1), pp. 68-76.)

[008] The most common type of circulating cfDNA is nucleosome-bound DNA. The nucleosome is a subunit of nuclear chromatin and consists of a central core formed by an octamer of histone proteins associated with a fragment of double-stranded DNA of approximately 147 base pairs. (Oudet P, Gross-Bellard M, Chambon P. Electron microscopic and biochemical evidence that chromatin structure is a repeating unit. Cell. 1975; 4: 281-300). Nucleosome-bound cfDNA can circulate in the blood as mononucleosomes or higher order structures such as oligonucleosomes or even chromatin fragments containing more than 50-100×10 ³ DNA base pairs. This particular type of circulating cfDNA originates from cells undergoing necrosis or apoptosis. Another source of circulating cfDNA is the formation of extracellular traps by neutrophils. Neutrophil extracellular traps (NETs) are extracellular strands of decondensed DNA isolated from activated neutrophils larger than 15×10 ³ DNA base pairs and organized into three-dimensional mesh structures 10-30 nm in size. The cfDNA that occurs during the formation of extracellular traps by neutrophils can be either free of particles or associated with particles. Extracellular traps for neutrophils also contain highly cytotoxic enzymes and cytotoxic proteins derived from the interior of neutrophils. (Sørensen, O. E. and Borregaard, N., Neutrophil extracellular traps - the dark side of neutrophils. J. Clin. Invest. 2016 May 2; 126(5): 1612-20). It has recently been shown that not only neutrophils but also macrophages can secrete NET-like structures. (Nat Med., 2018, 24(2): 232-238).

[009] Another important type of cfDNA associated with circulating particles is exosome-associated DNA. Exosomes are small membrane vesicles (30-100 nm) of exocytotic origin, secreted by most cell types, which can contain single-stranded DNA (ssDNA), mitochondrial DNA (mtDNA) and double-stranded (dsDNA) up to 2.5-10 × 10 ³ pairs in size. bases located in the internal space of the exosome or on the surface of its membrane. (Thakur, B.K. et al., Double-stranded DNA in exosomes: a novel biomarker in cancer detection, Cell Research (2014) 24: 766-769).

[010] A significant portion of circulating non-particle bound cfDNA is linear and circular double-stranded DNA and single-stranded DNA secreted by cancer cells, activated immune cells, and some other cell types. This type of cfDNA is typically 250-1000 base pairs or more in length and can be enriched with unique genome sequences (Kumar, P. et al., Normal and cancerous tissues release extrachromosomal circular DNA (eccDNA) into the circulation, (Mol. Cancer. Res ., June 20, 2017 DOI: 10.1158/1541-7786.MCR-17-0095.) Another important particle-free circulating cfDNA component is mitochondrial DNA (mtDNA) of various lengths.

[011] Another recently discovered type of particle-free circulating cfDNA is represented by ultrashort double-stranded DNA (dsDNA) oligonucleotides and single-stranded DNA (ssDNA) oligonucleotides with subnucleosomal length (i.e. typically less than ~147 base pairs). This particular cfDNA has been shown to be enriched in mitochondrial DNA (mtDNA), DNA of microbial origin, and mutated sequences from the human genome. (Burnham R., Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma, Scientific Reports 6, Article number: 27859 (2016), doi:10.1038/srep27859). Importantly, this type of circulating cfDNA also contains low molecular weight DNA fragments, which are similar to those that result from the breakdown of particle-bound DNA by the DNase I enzyme in patients' blood.

[012] Several attempts have been made to use in vitro removal technologies to purify a patient's blood from certain components of the circulating cfDNA pool. See for example US patent No. 9364601; U.S. Patent Application Publication No. 2007/0092509; Kusaoi et al., Ther. Apher. Dial, 2016, 20:348-353.

[013] There is a need for new extracorporeal methods for the treatment of diseases associated with high levels of circulating cfDNA in the blood, and for new more effective devices for implementing such methods.

BRIEF DESCRIPTION OF THE INVENTION

[014] As noted in the Background section above, there is a need for new extracorporeal methods for treating diseases associated with high levels of circulating cfDNA in the blood, and for new more efficient devices for implementing such methods. The present invention satisfies this and other needs by providing apheresis devices and related processes.

[015] In one aspect, the present invention provides an apheresis device comprising one or more affinity templates, wherein said one or more affinity templates are capable of capturing extracellular DNA (cfDNA) associated with nucleosome, exosome-bound cfDNA, and unbound cfDNA from the subject's blood or plasma.

[016] In some embodiments, unbound cfDNA includes double stranded dsDNA, single stranded ssDNA, and oligonucleotides.

[017] In some embodiments of the invention, the device contains two or more affine matrices. In some embodiments, (i) one or more affinity templates are capable of capturing nucleosome-bound extracellular DNA (cfDNA) and/or exosome-bound cfDNA and (ii) the next one or more affinity templates are capable of capturing unbound cfDNA, and wherein the first and second affinity matrices are located inside the device in any order. In some embodiments, (i) the one or more affinity templates comprise a DNA binding protein (e.g., histone [e.g., histone H1]), an anti-histone antibody (e.g., an anti-histone H2A antibody), an anti-nucleosome antibody (e.g., AN-1 , AN-44), DNA intercalating agent (e.g. Hoechst dye, such as e.g. Hoechst 33342), DNA binding polymer (e.g. cationic/basic polymer [e.g. polyethyleneimine, poly-L-lysine, poly-L- arginine, hexadimethrin bromide, amino-terminal (-NH2) polyamidoamine (PAMAM) dendrimer, polypropyleneimine (PPI) dendrimer], nonionic/neutral polymer [e.g., polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP), poly(4-vinylpyridine-N- oxide)], anionic/acidic polymer, linear polymer [e.g. polyethyleneimine, poly-L-lysine, poly-L-arginine], branched polymer [e.g. hyperbranched poly-L-lysine, hyperbranched polyethyleneimine], dendrimeric polymer [e.g. polyamidoamine (PAMAM) dendrimer, polypropy lenimin (PPI) dendrimer]), anti-DNA antibody (eg, mouse anti-ds+ss DNA monoclonal IgM antibody ([49/4A1], ab35576, Abeam), lectin (eg, Galanthus nivalis (GNA) lectin, lectin Narcissus Pseudonarcissus (NPA), Conconavalin A, phytohemaglutanin, or cyanovirin) and any combination thereof, and (ii) the following one or more affinity templates contain a DNA binding protein (e.g., a histone [e.g., histone HI]), a DNA intercalating agent (e.g. , a Hoechst dye such as e.g. Hoechst 33342), a DNA binding polymer (e.g. a cationic/base polymer [e.g. polyethyleneimine, poly-L-lysine, poly-L-arginine, hexadimethrin bromide, amino-terminal (-NH2 ) polyamidoamine (PAMAM) dendrimer, polypropyleneimine (PPI) dendrimer], non-ionic/neutral polymer [e.g., polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP), poly(4-vinylpyridine-N-oxide)], anionic/acidic polymer, linear polymer [e.g. polyethyleneimine, poly-L-lysine, poly-L-arginine], branched polymer [eg, hyperbranched poly-L-lysine, hyperbranched polyethyleneimine], dendrimeric polymer [eg, polyamidoamine (PAMAM) dendrimer, polypropyleneimine (PPI) dendrimer]), anti-DNA antibody (eg, mouse IgM monoclonal antibody against ds+ sc DNA ([49/4A1], ab35576, Abeam) and any combination thereof. In some embodiments, said two or more affinity templates are sequentially arranged as two or more affinity columns. In some embodiments, the first affinity template in the sequence contains a DNA binding polymer (e.g., an amino-terminal (-NH2) polyamidoamine (PAMAM) dendrimer, a polypropyleneimine (PPI) dendrimer, hyperbranched poly-L-lysine, or hyperbranched polyethyleneimine) or a DNA intercalating agent ( e.g. Hoechst 33342). In some embodiments, the affinity matrix is not a polyamidoamine (PAMAM) dendrimer.

[018] Non-limiting examples of useful column combinations (in any order) are: (a) (i) a Hoechst 33342 DNA intercalating affinity column and (ii) an anti-DNA antibody affinity column; or (b) (i) an affinity matrix column containing an anti-nucleosome antibody (ANAM), and (ii) an affinity matrix column containing anti-DNA antibodies; or (c) (i) an affinity matrix column containing an anti-nucleosome antibody (ANAM) and (ii) an affinity matrix column containing a polyamidoamine dendrimer (PDAM); or (d) (i) an affinity matrix containing anti-nucleosome antibody (ANAM) column and (ii) a hyperbranched poly-L-lysine (PLLAM) affinity matrix column; or (e) (i) anti-histone antibody H2A affinity column, (ii) lectin affinity column, and (iii) H1 histone affinity column or polyamidoamine dendrimer (PDAM) affinity matrix column or affinity matrix column containing hyperbranched poly-L-lysine (PLLAM) or an affinity matrix column containing Hoechst 33342 DNA intercalating agent.

[019] In some embodiments of the invention, the device according to the invention contains a single affinity matrix. Non-limiting examples of suitable matrices that can be used as a single affinity template include: affinity matrices containing a histone (e.g., histone H1, such as, for example, histone H1.3), affinity matrices containing a DNA binding polymer (e.g., cationic polymer, such as, for example, amino-terminal (-NH2) polyamidoamine (PAMAM) dendrimer or hyperbranched poly-L-lysine), affinity matrices containing a DNA intercalating agent (e.g. Hoechst 33342), affinity matrices containing an anti-DNA antibody (eg, mouse anti-ds+ss DNA IgM monoclonal antibody ([49/4A1], ab35576, Abcam). In some embodiments, the affinity template is not a polyamidoamine (PAMAM) dendrimer.

[020] In some embodiments, the device of the invention captures at least 30 mg of cfDNA in a single apheresis session.

[021] In some embodiments, the implementation of the device according to the invention reduces the level of cfDNA in the blood by at least 25% per apheresis procedure. In some embodiments, the device of the invention reduces blood levels of cfDNA by at least 50% in a single apheresis session. In some embodiments, the device of the invention reduces blood levels of cfDNA by at least 75% in a single apheresis session.

[022] In another aspect, the invention relates to a method for reducing the level of extracellular DNA (cfDNA) in the blood of a subject, including: (a) performing an apheresis procedure, including the withdrawal of blood or plasma from the subject into the apheresis device of the present invention to obtain blood or plasma with reduced levels of cfDNA; and (b) returning blood or plasma with reduced levels of cfDNA to the subject, wherein the apheresis procedure reduces the level of nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA in the subject's blood. In some embodiments, the subject has a disease characterized by elevated levels of cfDNA in the blood. In some embodiments, the subject has a disease selected from the group consisting of a neurodegenerative disease, cancer, chemotherapy-associated toxicity, radiation-induced toxicity (e.g., acute radiation syndrome), organ failure, organ damage, organ infarction, ischemia, acute vascular events, stroke, graft-versus-host disease (GVHD), graft rejection, sepsis, systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), traumatic injury, aging, diabetes, atherosclerosis, autoimmune disease, eclampsia, infertility, pregnancy-related complications, bleeding disorders and infections.

[023] In a further aspect, the invention relates to a method of treating a disease in a subject in need thereof, the method comprising: (a) performing an apheresis procedure comprising withdrawing blood or plasma from a subject into an apheresis device of the present invention to obtain blood or plasma with reduced levels of cfDNA; and (b) returning blood or plasma with reduced levels of cfDNA to the subject, wherein the apheresis procedure reduces the level of nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA in the subject's blood. In some embodiments, the subject has a disease characterized by elevated levels of cfDNA in the blood. Non-limiting examples of diseases that can be treated with the methods of the invention include, for example, neurodegenerative disease, cancer, chemotherapy-related toxicity, radiation-induced toxicity (e.g., acute radiation syndrome), organ failure, organ damage, organ infarction, ischemia, acute vascular disease, stroke, graft-versus-host disease (GVHD), graft rejection, sepsis, systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), traumatic injury, aging, diabetes, atherosclerosis, autoimmune disease, eclampsia, infertility , complications associated with pregnancy, bleeding disorders and infection.

[024] In some embodiments of any of the above methods of the invention, the method further comprises monitoring the level of cfDNA in the subject's blood.

[025] In some embodiments of any of the above methods of the invention, the method includes continuing or repeating the apheresis procedure until the level of cfDNA is reduced by at least 25%. In some embodiments of any of the above methods of the invention, the method includes continuing or repeating the apheresis procedure until the level of cfDNA is reduced by at least 50%. In some embodiments of any of the above methods of the invention, the method includes continuing or repeating the apheresis procedure until the level of cfDNA is reduced by at least 75%.

[026] In some embodiments of any of the above methods of the invention, the method includes continuing or repeating the apheresis procedure until at least 30 mg of cfDNA has been removed from the subject's blood.

[027] In some embodiments of any of the above methods of the invention, the apheresis procedure is repeated two or more times.

[028] In some embodiments of any of the above methods of the invention, blood for the apheresis procedure is obtained from the portal vein.

[029] In some embodiments of any of the above methods of the invention, unbound cfDNA includes dsDNA, ssDNA, and oligonucleotides.

[030] In some embodiments of any of the above methods of the invention, the subject is a human.

[031] These and other aspects of the present invention will be apparent to those skilled in the art from the following description, claims, and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[032] Figure 1 shows the electrophoretic profile of cfDNA isolated from the blood plasma of a patient with metastatic cancer.

[033] Figures 2A and 2B show tumors excised from mice injected with DNA according to Example 3, where blood was purified with an anti-histone antibody affinity matrix and a lectin affinity matrix from Galanthus nivalis (snowdrop). Figure 2A shows tumors excised from control mice. Figure 2B shows tumors excised from mice injected with DNA from a patient with NSCLC T3N2M+ purified from circulating cfDNA associated with nucleosomes and exosomes.

[034] Figure 3 shows an electrophoretic profile of circulating cfDNA from the plasma of a metastatic cancer patient and a stroke patient.

[035] Figure 4 shows an electrophoretic profile of circulating cfDNA from the plasma of a patient with systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS).

[036] Figure 5 shows an electrophoretic profile of circulating cfDNA used in cell culture experiments.

[037] Figure 6 shows an electrophoretic profile of circulating cfDNA, DNase I Western blotting and quantification of DNase I activity and circulating cfDNA.

[038] Figure 7 shows an electrophoretic profile of circulating cfDNA from the plasma of a septic patient.

[039] Figure 8 shows the results of 1% agarose gel electrophoresis of model cfDNA-enriched plasma before and after the volume adsorption test. Lane 1 - model plasma enriched with cfDNA prior to incubation; lane-2 - model plasma enriched with cfDNA after incubation with ethanolamine-sepharose FF control; lane 3 - cfDNA enriched model plasma after incubation with PDAM; lane-4 - model plasma enriched with cfDNA after incubation with PLLAM; lane-5 - model plasma enriched with cfDNA after incubation with an affinity matrix containing histone H1.3.

[040] Figure 9 shows the results of 1% agarose gel electrophoresis in the plasma of a patient diagnosed with odontogenic sepsis before and after a volume adsorption test. Lane-1 - plasma of a patient with odontogenic sepsis after incubation with ethanolamine-sepharose FF-control; lane-2 control-distilled water; lane 3 - plasma of a patient with odontogenic sepsis after incubation with an affinity matrix containing histone H1.3; track-4 - control-distilled water; lane-5 - plasma of a patient with odontogenic sepsis after incubation with PDAM; lane-6 - plasma of a patient with odontogenic sepsis after incubation with PLLAM.

DETAILED DESCRIPTION OF THE INVENTION

Terms and Definitions

[041] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains.

[042] The singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "method" includes one or more methods and/or steps of the type described herein and/or that will become apparent to those skilled in the art upon reading this disclosure.

[043] The term "about" or "approximately" includes being within a statistically significant range of a value. Such a range may be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of the given value or range. The tolerance covered by the terms "about" or "approximately" depends on the particular system under study and can be easily estimated by a person skilled in the art.

[044] As used herein, the term "device" refers to any assembly known in the art to enable the purification of liquid solutions, such as, without limitation, for example, any hollow vessel, column, column matrix, filter, membrane, semi-permeable material, bead (eg microbead or nanobead) or tube. The terms "column" and "cartridge" are used interchangeably herein in the context of an apheresis device.

[045] The term "affinity matrix" as used herein refers to (i) a solid support in which a ligand is immobilized (e.g., an cfDNA binding molecule), or (ii) a solid support formed by the ligand itself (e.g., insoluble in water DNA-binding polymer).

[046] The term "DNA-binding protein" refers to proteins that bind to single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). DNA-binding proteins can bind DNA in a sequence-specific manner (eg, transcription factors and nucleases) or in a non-sequence-specific manner (eg, polymerases and histones). Members of the H1 family of linker histones are a key component of chromatin and bind to the nucleosome nucleus at DNA entry and exit sites.

[047] As used herein, the terms "circulating DNA", "extracellular DNA (ecDNA)," "extracellular DNA (eDNA)", and "circulating extracellular DNA (eDNA)" are used interchangeably to refer to DNA present in blood or plasma, located outside circulating cells of hematopoietic and non-hematopoietic origin.

[048] Nucleosome-associated cfDNA is DNA that is associated with a nucleosome. The nucleosome is a subunit of nuclear chromatin. Nucleosome-bound cfDNA can circulate in the blood as mononucleosomes or higher order structures such as oligonucleosomes or even chromatin fragments containing more than 50-100×10 ³ DNA base pairs. Circulating nucleosome-bound cfDNA can originate from cells undergoing necrosis or apoptosis, as well as from the formation of neutrophil extracellular traps (NETs).

[049] Exosome-associated cfDNA is cfDNA that is associated with or present in exosomes. Exosomes are small membrane vesicles (about 30-100 nm) of exocytotic origin, secreted by most cell types, which may contain single-stranded DNA (ssDNA), mitochondrial DNA (mtDNA), and double-stranded DNA (dsDNA) in the interior or exterior space of the exosome.

[050] The terms “unbound cfDNA” or “particle-free cfDNA” or “particle-free cfDNA” refers to cfDNA that is not associated with exosomes or nucleosomes and includes double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), linear or circular, and oligonucleotides, including ultrashort DNA molecules of subnucleosomal size (usually less than 147 base pairs).

[051] As used herein, the terms "subject" and "patient" are used interchangeably and refer to animals, including mammals such as humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc. ) and experimental animal models. In certain embodiments, the subject is a human patient, including both sexes in adult and pediatric populations.

[052] In the context of the present invention, as it refers to any of the disease states referred to in this description, the terms "treat", "treatment" and the like mean the alleviation or weakening of at least one symptom associated with such a condition, or slowing down or reversing the progression of such a condition. In the meaning of the present invention, the term "treat" also means stopping, delaying the onset (ie, the period before the clinical manifestation of the disease) and/or reducing the risk of developing or worsening the disease. The terms "treat", "treatment" and the like in relation to a condition, disorder or condition may also include (1) preventing or delaying the onset of at least one clinical or subclinical symptom of the condition, disorder or condition developing in a subject that may be affected or is predisposed to the condition, disorder, or condition, but is not yet experiencing or showing clinical or subclinical symptoms of the condition, disorder, or condition; or (2) suppression of the condition, disorder or condition, i.e. cessation, reduction or delay in the development of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) alleviating the disease, that is, causing regression of the condition, disorder, or condition, or at least one of its clinical or subclinical symptoms.

[053] The practice of the present invention uses, unless otherwise indicated, conventional methods of statistical analysis, molecular biology (including recombinant methods), microbiology, cell biology, conjugation chemistry, and biochemistry, which are known to those skilled in the art. Such tools and techniques are described in detail in, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Hermanson (2013) Bioconjugate Techniques' 3rd ed., Academic Press; Niemeyer (2004) Bio conjugation Protocols: Strategies and Methods, Springer Science & Business Media and Hermanson et al. (1992) Immobilized Affinity Ligand Techniques, Academic Press. Additional techniques are described, for example, in US Patent No. 7,912,698 and US Patent Applications No. 2011/0202322 and 2011/0307437.

Devices and Methods according to the invention

[054] As noted in the Background section, there is a great need in the art to develop new methods and devices to reduce the level of virtually all types of circulating cfDNA in the blood. The present disclosure addresses this and other needs by providing apheresis devices and methods wherein the apheresis device reduces virtually all types of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides) .

[055] The use of extracorporeal removal technologies can provide an effective solution to remove cfDNA from the circulation and, accordingly, reduce the level and negative effects of circulating cfDNA. Therapeutic apheresis is an extracorporeal treatment that removes blood components from patients; it is used to treat conditions in which a pathogenic substance or component in the blood causes disease: see, for example, Ward M.D., Conventional Apheresis Therapies: A Review Journal of Clinical Apheresis 26: 230 238 (2011).

[056] Unexpectedly, as shown herein, in vitro removal of virtually all types of circulating cfDNA has a positive effect on the treatment of diseases characterized by elevated levels of circulating cfDNA in the blood.

[057] The present disclosure provides a method for treating diseases characterized by elevated circulating levels of cfDNA by removing substantially all types of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides) from a subject's blood to reduce negative effects of circulating cfDNA.

[058] Without wishing to be bound by theory, in certain diseases in which circulating cfDNA levels are elevated, different types of circulating cfDNA may work together to trigger different molecular mechanisms, each leading to disease progression and patient mortality; different types of circulating cfDNA acting together can generate synergistic toxicity, that is, the toxic (negative) effect of two or more types of circulating cfDNA is greater than the sum of the negative effects of each type of cfDNA taken separately.

[059] The inventors found that the removal of virtually all types of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including double-stranded DNA [dsDNA], single-stranded DNA [ssDNA], and oligonucleotides) from the blood of patients with elevated levels of circulating cfDNA can effectively reduce or even completely abolish the pathogenic effects mediated by said circulating cfDNA. Removal of virtually all types of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (dsDNA, ssDNA, and oligonucleotides) appears to be critical for reducing cfDNA-mediated pathogenic effects.

[060] The inventors also unexpectedly found that the removal of almost all types of circulating cfDNA can lead to the reactivation of endogenous deoxyribonucleases.

[061] It is further described that multiple affinity templates, or combinations thereof, are capable of effectively capturing substantially all types of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides) from the blood of patients in need thereof. Examples of affinity matrices used in the apheresis devices and methods of the invention include (i) matrices containing a DNA binding protein (e.g., histone [e.g., histone H1]), (ii) matrices containing an anti-histone antibody (e.g., antibody against histone H2A), anti-nucleosome antibody (e.g., AN-1, AN-44), (iii) matrices containing a DNA intercalating agent (e.g., a Hoechst dye, such as, for example, Hoechst 33342), (iv) matrices, containing a DNA-binding polymer (e.g., cationic/basic polymer [e.g., polyethyleneimine, poly-L-lysine, poly-L-arginine, hexadimethrin bromide, terminal amino-terminal (-NH2) polyamidoamine (dendrimer, polypropyleneimine) (PPI) dendrimer ], nonionic/neutral polymer [e.g. polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP), poly(4-vinylpyridine-N-oxide)], anionic/acidic polymer, linear polymer [e.g. polyethyleneimine, poly-L-lysine, poly -L-arginine], branched polymer [for example, hyperbranched poly- L-lysine, hyperbranched polyethyleneimine], dendrimeric polymer [eg, polyamidoamine dendrimer (PAMAM), polypropyleneimine dendrimer (PPI); see Kaur et al., J NanopartRes., 2016, 18:146]; see, e.g., U.S. Patent No. 7,713,701 and Morozov et al., General Reanimatology, 2016, 12:6 for additional examples), (v) matrices containing anti-DNA antibody, (vi) matrices containing lectin (e.g., Galanthus nivalis (GNA) lectin, lectin Narcissus Pseudonarcissus (NPA), conconavalin A, phytohemagluttanin or cyanovirin) and any combination thereof. In some embodiments, two or more affinity matrices are sequentially arranged as two or more affinity columns. In some embodiments, the first affinity template in the sequence comprises a DNA binding polymer (e.g., amino-terminal (-NH2) polyamidoamine (PAMAM) dendrimer, polypropyleneimine (PPI) dendrimer, hyperbranched poly-L-lysine, or hyperbranched) polyethyleneimine, or a DNA intercalating agent (e.g. Hoechst 33342).

[062] Affinity matrices and apheresis devices containing such matrices are described herein. The apheresis device according to the present invention can be made in accordance with the experience of a person skilled in the art, for example, as described in the US patent application. no. 2017/0035955 (Eliaz Issac, Published February 9, 2017). In one possible embodiment of the apheresis device, the affinity matrices are placed in various affinity apheresis columns or cartridges. The apheresis device may comprise a filter cartridge and one or more affinity columns having an inlet and outlet, in which the device is capable of capturing nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides) from a patient's blood or plasma. . In some embodiments, the device comprises two or more affinity apheresis columns in sequence. The inlet and outlet can be positioned relative to the affinity matrices such that blood entering the inlet must contact the affinity matrices before exiting through the outlet. Preferably, the geometry of the device is designed to maximize contact of blood (or plasma) with affinity matrices during passage through the device. A variety of such designs are known in the art. For example, the device may be a hollow cylinder filled with affinity ligand immobilized on beads, having an inlet at one end and an outlet at the opposite end. Other devices such as arrays of microtubules can also be constructed. All such variations of the geometry and volume of the container and the affine matrices it contains can be designed according to known principles. When preparing an affinity matrix column, the affinity matrix may be loaded to a column volume of at least 50%, 60%, 70%, 75%, 80%, 85%, or 90%. A suitable buffer (eg PBS, in particular cold PBS) can be used to equilibrate the column.

[063] In one aspect, a histone-based affinity matrix is provided comprising cellulose beads and recombinant human H1.3 histone, wherein the recombinant human H1.3 histone is immobilized on cellulose beads and wherein the beads are 50 to 350 micrometers in size. In some embodiments, the beads are 100 to 250 micrometers in size.

[064] In some embodiments, a histone-based affinity matrix is prepared by a method comprising a) oxidizing 100 to 250 micrometer cellulose beads to form activated cellulose beads; b) washing the activated cellulose granules; c) preparation of a concentrated solution of recombinant human histone H1.3; d) incubation of the activated cellulose beads with a concentrated solution of recombinant human histone H1.3; and e) blocking any free CHO groups on the activated cellulose granules.

[065] In some embodiments, the method further comprises f) washing the activated cellulose beads with buffer.

[066] In some embodiments, in step a), the cellulose beads are in an aqueous suspension and oxidized with NaIO. In some embodiments, in step b), the activated cellulose beads are washed with sodium bicarbonate, hydrochloric acid and water. In some embodiments, step c) comprises dialyzing a solution of recombinant human H1.3 histone and concentrating the dialyzed solution in 0.1M NaHCO3 at pH 7-9. In some embodiments, the dialyzed solution is concentrated in 0.1M NaHCO3 at pH 8. In some embodiments, step d) is incubated for 3-5 hours at 15-30°C. In some embodiments, in step d) the incubation is carried out for 4 hours at room temperature. In some embodiments, in step e), the blocking step comprises adding 1M ethanolamine to the activated cellulose beads and reacting for 30 minutes to 2 hours at 15-30°C. In some embodiments, in step f), the activated cellulose beads are washed with TBS buffer.

[067] Also provided is a column containing a histone-based affinity matrix according to any of the above aspects and embodiments.

[068] In another aspect, there is provided a lectin-based affinity matrix prepared according to a method comprising a) reacting a lectin with activated agarose beads to form lectin-bound agarose; and b) washing the lectin-bound agarose with buffer.

[069] In some embodiments, the lectin is derived from Galanthus nivalis (snowdrop). In some embodiments, the activated agarose beads are agarose-activated CNBr beads. In some embodiments, the buffer is PBS, such as sterile cold PBS at pH 7.2-7.4.

[070] Also provided is a column containing a lectin-based affinity matrix according to any of the above aspects and embodiments.

[071] In yet another aspect, there is provided a polyamidoamine dendrimer (PDAM) affinity matrix prepared by a method comprising a) washing cellulose granules with ethanol and water; b) incubating the washed cellulose beads with (±)-epichlorohydrin and NaOH to obtain activated cellulose beads; c) reacting the activated cellulose beads with a polyamidoamine (PAMAM) dendrimer to form PDAM beads and removing the PAMAM dendrimer that did not react with the activated cellulose beads; and d) blocking the unconverted epoxy groups on the PDAM beads.

[072] In some embodiments, the method further comprises e) washing the PDAM beads with 0.1 M phosphate buffer and water.

[073] In some embodiments, in step a), the cellulose beads are washed with 98% ethanol and distilled water. In some embodiments, in step b), the washed cellulose beads are incubated with a mixture of (±)-epichlorohydrin and 2.5M NaOH. In some embodiments, in step c), the activated cellulose beads are suspended with a 20% solution of PAMAM dendrimer with an ethylenediamine core. In some embodiments, in step c), the suspension is carried out at 20-30° C. for 3-6 hours. In some embodiments, in step c), the suspension is carried out at 24° C. for 5 hours.

[074] Also provided is a column containing the PAMAM dendrimer (PDAM) affinity matrix described above. In some embodiments, the column is a PTFE (polytetrafluoroethylene) column and the polyamidoamine dendrimer affinity matrix is sterilized.

[075] In another aspect, an affinity matrix containing anti-DNA antibodies is provided, obtained by a method including a) obtaining activated agarose beads by cross-linking N-hydroxysuccinimide with agarose beads; b) washing the activated agarose beads with binding buffer containing NaHCO ₃ and NaCl; c) adding the anti-double-stranded and single-stranded DNA antibody to the binding buffer; d) incubating the binding buffer containing the antibody with activated agarose beads to obtain an affinity matrix based on the anti-DNA antibody; and f) washing the anti-DNA antibody affinity template with binding buffer and acetate buffer.

[076] In some embodiments, the implementation of the agarose beads have an average size of 90 micrometers. In some embodiments, the binding buffer contains 0.2 M NaHCO ₃ and 0.5 M NaCl and has a pH of 8.3. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a mouse antibody. In some embodiments, the washing step is performed at least three times. In some embodiments, the acetate buffer is 0.1 M acetate buffer at pH 4.0.

[077] Also provided is a column containing an affinity matrix based on an anti-DNA antibody described above.

[078] In some embodiments, the column is prepared by incubating the anti-DNA antibody affinity template with sterile Tris-HCl buffer. In some embodiments, sterile Tris-HCl buffer has a pH of 7.4.

[079] In another aspect, there is provided an anti-nucleosome antibody (ANAM) affinity matrix prepared by a method comprising a) obtaining activated agarose beads by crosslinking N-hydroxysuccinimide to agarose beads; b) washing the activated agarose beads with binding buffer containing NaHCO ₃ and NaCl; c) adding to the binding buffer an antibody that binds to nucleosomes, where the antibody is obtained from an MRL/Mp(-)+/+ mouse; d) incubating the binding buffer containing the antibody with activated agarose beads to obtain an antinucleosome antibody affinity matrix; and f) washing the antinucleosome antibody affinity template with binding buffer and acetate buffer.

[080] In some embodiments, the template binds to nucleosome-bound circulating cfDNA and the template does not bind to unbound cfDNA, which includes dsDNA, ssDNA, and oligonucleotides.

[081] Also provided is a column containing an anti-nucleosome antibody affinity array (ANAM) as described above.

[082] In yet another aspect, there is provided an affinity matrix based on the DNA intercalating agent Hoechst 3342 obtained by a method comprising a) oxidizing cellulose beads; b) washing the oxidized cellulose granules; c) reacting the washed oxidized cellulose beads with a solution containing Hoechst 33342 and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) to form Hoechst 33342 immobilized cellulose beads; and d) washing the pellets with Hoechst 33342 immobilized cellulose.

[083] In some embodiments, in step a), the cellulose beads are oxidized with NaIO for 3-5 hours. In some embodiments, in step b), the oxidized cellulose pellets are washed with 1 M sodium bicarbonate, 0.1 M hydrochloric acid and water. In some embodiments, in step c), the solution is a pH buffer solution. In some embodiments, step d) washes at least three times.

[084] In another aspect, there is provided a poly-L-lysine hyperbranched affinity matrix (PLLAM) obtained by a method comprising a) dissolving L-lysine monohydrochloride in water and neutralizing with KOH to form an L-lysine solution; b) heating the solution of L-lysine to obtain a solution containing hyperbranched poly-L-lysine; c) removing L-lysine and salt from a solution containing hyperbranched poly-L-lysine; d) fractionating the solution containing hyperbranched poly-L-lysine to obtain a fraction containing hyperbranched poly-L-lysine with an average molecular weight of 21,000 to 32,000; e) dialysis and lyophilization of a fraction containing hyperbranched poly-L-lysine with an average molecular weight of 21,000 to 32,000 to obtain a lyophilisate; f) dissolving the lyophilizate in distilled water and dialysis against NaHCO ₃ to obtain a solution containing HBPL; and g) incubating the solution containing hyperbranched poly-L-lysine with sepharose 4B activated by cyanore and bromide suspended in NaHCO ₃ to obtain a hyperbranched poly-L-lysine affinity matrix.

[085] In some embodiments, in step b), the L-lysine solution is heated to 150° C. for 48 hours under nitrogen flow. In some embodiments, in step c), the hyperbranched poly-L-lysine containing solution is dialyzed against water. In some embodiments, step d) fractionation is performed using a size exclusion column. In some embodiments, in step d), fractionation is performed on a gel filtration column.

[086] Also provided is a column containing the hyperbranched poly-L-lysine affinity matrix (PLLAM) described above.

[087] In yet another aspect, an apheresis device is provided, comprising one or more affinity columns containing an affinity matrix, and configured to remove substantially all types of cfDNA from a patient's blood or plasma. In some embodiments, the implementation of the device contains two or more affinity columns in sequence. In some embodiments, the device further comprises a filter cartridge. In some embodiments, the filter cartridge has an inlet and an outlet. In some embodiments, one or more affine columns have an input and an output.

[088] In some embodiments, the device comprises two or more of the following affinity columns, in any order: a) a column containing a DNA-binding protein (eg, histone) affinity template; b) a column containing a lectin affinity matrix (eg, Galanthus nivalis (GNA) lectin, Narcissus Pseudonarcissus (NPA) lectin, conconavalin A, phytohemaggluttanin, or cyanovirin); c) a column containing a DNA-binding polymer affinity template (eg, a cationic polymer such as, for example, amino-terminal (-NH2) PAMAM dendrimer, hyperbranched poly-L-lysine, or hyperbranched polyethyleneimine); d) a column containing an anti-DNA antibody affinity template; f) a column containing an affinity template for a DNA intercalating agent (eg Hoechst 3342); f) a column containing an anti-nucleosome antibody affinity array (ANAM); and g) a column containing an anti-histone antibody affinity matrix.

[089] In some embodiments, the device comprises one of the following combinations of columns, in any order: (a) (i) an affinity column containing the Hoechst 33342 DNA intercalating agent, and (ii) an affinity column containing an anti-DNA antibody; or (b) (i) an anti-nucleosome antibody affinity array (ANAM) column and (ii) an anti-DNA antibody affinity array column; or (c) (i) an anti-nucleosome antibody affinity matrix (ANAM) column and (ii) a polyamidoamine dendrimer (PDAM) affinity matrix column; or (d) (i) an anti-nucleosome antibody affinity matrix (ANAM) column and (ii) a hyperbranched poly-L-lysine (PLLAM) affinity matrix column; or (e) (i) an anti-histone H2A affinity column, (ii) a lectin affinity column, and (iii) an H1 histone affinity column, or a polyamidoamine dendrimer (PDAM) containing affinity matrix column, or an affinity matrix containing hyperbranched poly-L-lysine (PLLAM) or an affinity matrix column containing Hoechst 33342 DNA intercalating agent.

[090] In another aspect, an apheresis device is provided, comprising a filter cartridge and one or more affinity columns having an inlet and outlet, in which the device is capable of capturing substantially all types of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA ( including dsDNA, ssDNA and oligonucleotides) from a patient's blood or plasma.

[091] In some embodiments, the implementation of the device contains two or more affinity columns in sequence. In some embodiments, the first affinity column in the sequence contains a DNA binding polymer or a DNA intercalating agent.

[092] In some embodiments, the implementation of the device contains a column containing a histone-based affinity matrix at the entrance or before a column containing a lectin-based affinity matrix. In some embodiments, the device comprises a column containing a histone-based affinity matrix before a column containing a lectin-based affinity matrix or before a column containing a PAMAM-based affinity matrix. In some embodiments, the device comprises a column containing an anti-DNA antibody affinity matrix before a column containing a Hoechst 3342 affinity matrix. based on RAMAM.

[093] In some embodiments, the apheresis device captures at least 30 mg of cfDNA in a single apheresis session. In some embodiments, the affinity column comprises a molecule that effectively captures one or more cfDNA fractions: nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA, including dsDNA, ssDNA, and oligonucleotides. In some embodiments, the immobilized molecule is selected from the group consisting of: a DNA binding antibody, a DNA intercalating agent, a DNA binding protein, a DNA binding polymer, a lectin, an anti-nucleosome antibody, and an anti-histone antibody.

[094] In some embodiments, the implementation of the DNA-binding protein is a histone H1 (eg, histone H1.3).

[095] In some embodiments, the DNA-binding polymer is a cationic polymer. In some embodiments, the cationic polymer is poly-L-lysine. In some embodiments, the poly-L-lysine is a hyperbranched poly-L-lysine. In some embodiments, the implementation of the cationic polymer is a polyethyleneimine. In some embodiments, the polyethyleneimine is a hyperbranched polyethyleneimine. In some embodiments, the cationic polymer is an amino-terminal (-NH2) polyamide amine (PAMAM) dendrimer.

[096] In some embodiments of the above apheresis device, the apheresis device comprises two consecutive affinity columns, in which one column captures nucleosome-bound cfDNA and exosome-bound cfDNA, and the other column captures unbound cfDNA, including dsDNA, ssDNA, and oligonucleotides. In some embodiments, the affinity matrix contains simultaneously immobilized molecules from a combination of two or more groups of molecules: a DNA binding antibody, a DNA intercalating agent, a DNA binding protein, a DNA binding polymer, a lectin, an anti-nucleosome antibody, or an anti-histone antibody.

[097] In another aspect, a method for reducing the level of cfDNA in a patient's blood is provided. The method includes (a) performing an apheresis procedure comprising withdrawing blood or plasma from a patient into an apheresis device to obtain purified blood or plasma with reduced levels of cfDNA; and (b) returning the purified blood or plasma to the patient. The apheresis procedure reduces the level of virtually all types of cfDNA in the patient's blood, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides).

[098] In some embodiments, the method is effective for treating one or more diseases selected from: multiple organ failure, neurodegenerative disease (e.g., Alzheimer's disease), cancer, sepsis, septic kidney injury, radiation-induced toxicity (e.g., acute radiation syndrome) , and chemotherapy-induced toxicity.

[099] In some embodiments, the patient has a disease selected from the group consisting of cancer, metastatic cancer, acute organ failure, organ infarction, hemorrhagic stroke, graft-versus-host disease (GVHD), transplant rejection, sepsis, systemic inflammatory disease response (SIRS), multiple organ dysfunction syndrome (MODS), radiation-induced toxicity (eg, acute radiation syndrome), chemotherapy-associated toxicity, traumatic injury, pro-inflammatory status in the elderly, diabetes, atherosclerosis, neurodegenerative diseases, autoimmune diseases, eclampsia, infertility, bleeding disorders and infections.

[0100] In some embodiments, the method is effective for treating a disorder in a patient, wherein the disorder is selected from cancer, metastatic cancer, acute organ failure, organ infarction (including myocardial infarction and ischemic stroke, hemorrhagic stroke), autoimmune disorders, graft versus host disease ( GVHD), graft rejection, sepsis, systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), graft versus host disease (GVHD), traumatic injury, pro-inflammatory status in the elderly, diabetes, atherosclerosis, neurodegenerative disease, autoimmune disease, eclampsia, infertility, clotting disorders, pregnancy-related complications, and infections. In some embodiments, the patient is in need of treatment for the disorder.

[0101] In yet another aspect, a method for treating multiple organ failure syndrome (MODS) in a patient is provided. The method includes (a) performing an apheresis procedure including withdrawing blood or plasma from a patient into an apheresis device to obtain purified blood or plasma; and (b) returning the purified blood or plasma with reduced levels of cfDNA to the patient. The apheresis procedure reduces the level of virtually all types of cfDNA in the patient's blood, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides). In some embodiments, the patient is in need of MODS treatment.

[0102] In another aspect, a method for treating a neurodegenerative disease in a patient is provided. The method includes (a) performing an apheresis procedure comprising withdrawing blood or plasma from a patient into an apheresis device to obtain purified blood or plasma with reduced levels of cfDNA; and (b) returning the purified blood or plasma to the patient. The apheresis procedure reduces the level of virtually all types of cfDNA in the patient's blood, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides). In some embodiments, the patient is in need of treatment for a neurodegenerative disease.

[0103] In another aspect, a method for treating Alzheimer's disease in a patient is provided. The method includes (a) performing an apheresis procedure comprising withdrawing blood or plasma from a patient into an apheresis device to obtain purified blood or plasma with reduced levels of cfDNA; and (b) returning the purified blood or plasma to the patient. The apheresis procedure reduces the level of virtually all types of cfDNA in the patient's blood, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides). In some embodiments, the patient is in need of treatment for Alzheimer's disease.

[0104] In another aspect, a method for treating cancer in a patient is provided. The method includes (a) performing an apheresis procedure comprising withdrawing blood or plasma from a patient into an apheresis device to obtain purified blood or plasma with reduced levels of cfDNA; and (b) returning the purified blood to the patient. The apheresis procedure reduces the level of virtually all types of cfDNA in the patient's blood, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides). In some embodiments, the patient is in need of cancer treatment.

[0105] In another aspect, a method for treating sepsis in a patient is provided. The method includes (a) performing an apheresis procedure comprising withdrawing blood or plasma from a patient into an apheresis device to obtain purified blood or plasma with reduced levels of cfDNA; and (b) returning the purified blood or plasma to the patient. The apheresis procedure reduces the level of virtually all types of cfDNA in the patient's blood, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides). In some embodiments, the patient is in need of treatment for sepsis.

[0106] In another aspect, a method for treating kidney injury in a patient is provided. The method includes (a) performing an apheresis procedure comprising withdrawing blood or plasma from a patient into an apheresis device to obtain purified blood or plasma with reduced levels of cfDNA; and (b) returning the purified blood or plasma to the patient. The apheresis procedure reduces the level of virtually all types of cfDNA in the patient's blood, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides). In some embodiments, the patient is in need of treatment for a kidney injury.

[0107] In another aspect, a method for treating chemotherapy-associated toxicity in a patient is provided. The method includes (a) performing an apheresis procedure comprising withdrawing blood or plasma from a patient into an apheresis device to obtain purified blood or plasma with reduced levels of cfDNA; and (b) returning the purified blood or plasma to the patient. The apheresis procedure reduces the level of virtually all types of cfDNA in the patient's blood, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides). In some embodiments, the patient is in need of treatment for toxicity associated with chemotherapy.

[0108] In another aspect, a method for treating radiation-induced toxicity (eg, acute radiation syndrome) in a patient is provided. The method includes (a) performing an apheresis procedure comprising withdrawing blood or plasma from a patient into an apheresis device to obtain purified blood or plasma with reduced levels of cfDNA; and (b) returning the purified blood or plasma to the patient. The apheresis procedure reduces the level of virtually all types of cfDNA in the patient's blood, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides). In some embodiments, the patient is in need of treatment for radiation-induced toxicity.

[0109] In some embodiments of any of the above methods, blood is drained from the patient's portal vein.

[IT] In some embodiments of the foregoing, the purified blood has reduced levels of cfDNA compared to blood levels of cfDNA in the patient prior to the apheresis procedure.

[0111] In some embodiments, the purified blood has reduced levels of all nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA, including dsDNA, ssDNA, and oligonucleotides. In some embodiments, the method further includes periodically monitoring the level of circulating cfDNA in the patient's blood and continuing the apheresis procedure to reduce the level of circulating cfDNA by at least 25% before completing the apheresis procedure. In some embodiments, the method further includes periodically monitoring the level of circulating cfDNA in the patient's blood and continuing the apheresis procedure on the patient to reduce circulating levels of cfDNA by at least 50% before completing the apheresis procedure. In some embodiments, the method further comprises periodically monitoring the level of circulating cfDNA in the patient's blood and continuing the apheresis procedure on the patient to reduce circulating cfDNA levels by at least 75% before completing the apheresis procedure.

[0112] In some embodiments of any of the above, at least 30 mg of cfDNA is removed from the patient's blood during one or more consecutive apheresis procedures.

[0113] In some embodiments of the above, the steps of the method are repeated or scheduled. The method steps may be performed twice a day, every day, every two days, every three days, every four days, every five days, every six days, every week, every eight days, every nine days, every 10 days, every 11 days. , every 12 days, etc. Blood samples may be taken from the patient and tested for cfDNA levels to assess the frequency of treatments.

[0114] The arrangement of affinity columns in the sequence can allow the capture of virtually all types of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides), from a patient's blood or plasma.

[0115] Various sequences described herein and other combinations may be used. In some embodiments, the device comprises a column containing a histone-based affinity matrix before a column containing a lectin-based affinity matrix. In some embodiments, the device comprises a column containing a histone-based affinity matrix before a column containing a lectin affinity matrix or before a column containing a polyamidoamine dendrimer (PDAM) affinity matrix. In some embodiments, the device comprises a column containing an affinity matrix containing anti-DNA antibodies before a column containing an affinity matrix based on Hoechst 3342. In some embodiments, the device contains a column containing an affinity matrix based on anti-nucleosome antibodies (ANAM) before a column containing an affinity matrix based on a polyamidoamine dendrimer (PDAM).

[0116] Part of the various aspects described in the application is (a) performing an apheresis procedure, including drawing blood or plasma from a patient into an apheresis device to obtain purified blood or plasma; and (b) returning the purified blood or plasma with reduced levels of cfDNA to the patient.

[0117] The apheresis device may contain an affinity matrix containing a histone. The histone-based affinity matrix may contain recombinant human H1.3 histone. A histone-based affinity matrix may be part of an apheresis affinity column. The beads used as a carrier in a column containing a histone-based affinity matrix may be cellulose beads that are oxidized with an oxidizing agent prior to binding to the histone. The beads may be, for example, Sepharose beads. Alternatively, carriers other than beads (hollow fibre, membrane, tubes, etc.) can be used. The affinity matrix carrier can be made from other organic and inorganic compounds known to the person skilled in the art, e.g. PMMA), poly(glycidyl methacrylate) (PGMA), poly(hydroxy methacrylate), polystyrene (PS), polytetrafluoroethylene (PTFE), polyacrylamide, polyacrolein, acrylonitrile butadiene styrene (ABS), polyacrylonitrile (PAN), eupergit®, polyethylene glycol (PEG) ), hyperfluorocarbon, agarose (e.g. cross-linked agarose), alginate, carrageenan, chitin, starch, cellulose, nitrocellulose, Sepharose®, glass, silica, kieselguhr, zirconia, alumina, iron oxide, porous carbon and mixtures and/or derivatives of said solid carriers; and protonated and deprotonated forms of this spacer material.

[0118] Beads or other carriers may be coated with DNA-binding proteins. DNA binding proteins such as histones or anti-DNA antibodies can be immobilized by chemically linking them to a solid insoluble carrier such as poly acharide beads. For example, agarose beads are activated using cyanogen bromide and the cfDNA capture protein is incubated with activated agarose to allow binding to occur. The unconjugated material is removed by washing with buffer and the protein-bound agarose is packaged in the target apheresis device/affinity cartridge. There are many different ways to chemically bind proteins to various matrices of insoluble carriers. These and other matrix materials, and methods for their binding to proteins known to those skilled in the art, can be used in any of the methods and devices described herein.

[0119] For example, attachment of the cfDNA capturing molecule to a solid support may be by an amine, thiol, imide (e.g., water-soluble carbodiimide), or other chemical attachment method known to those skilled in the art for attaching a polypeptide or oligonucleotide to a solid support.

[0120] The size of the beads may be, for example, 30 to 200 microns, 40 to 180 microns, 45 to 165 microns, 60 to 150 microns. Various oxidizing agents can be used, such as sodium metaperiodate (NaIO). Alternatively, the primary hydroxyl group of cellulose can be selectively converted to give 6-deoxy-6-carboxycellulose by piperidinoammonium salt mediated oxidation (TEMPO) or oxidized with chlorite. See, for example, Eyle, S. and Thielemans, W., Surface modifucation of cellulose nanocrystals, Nanoscale, 2014, 6, 7764, DOI: 10.1039/c4nr01756k). Also, the cellulose (or agarose) carrier can be oxidized with other compounds known to the person skilled in the art, such as chromic acid, chromium pyridine trioxide, dimethyl sulfoxide. (See for example Peng, L. et al. Evaluation of activation methods with cellulose beads for immunosorbent purification of immunoglobulins, J. Biotechnology, 5 (1987) 255-265). The oxidized beads are then incubated with a sufficiently purified and concentrated histone protein solution such as recombinant human H1.3 histone. The reaction can be stopped and then washed with buffer to remove soluble protein contaminants. Alternatively, the primary hydroxyl group of cellulose can be selectively converted to give 6-deoxy-6-carboxycellulose by piperidinoammonium salt mediated oxidation (TEMPO) or oxidized with chlorite. See for example Eyle, S. and Thielemans, W., Surface modifucation of cellulose nanocrystals, Nanoscale, 2014, 6, 7764, DOI: 10.1039/c4nr01756k. Also, the cellulose (or agarose) carrier can be oxidized with other compounds known to the person skilled in the art, such as chromic acid, chromium pyridine trioxide, dimethyl sulfoxide. See for example Peng, L. et al. Evaluation of activation methods with cellulose beads for immunosorbent purification of immunoglobulins, J. Biotechnology, 5 (1987) 255-265).

[0121] The apheresis device may contain an affinity matrix containing a histone. The histone-based affinity matrix may contain recombinant human H1.3 histone. A histone-based affinity matrix may be part of an apheresis affinity column. The beads used as a carrier in an affinity column containing a histone-based affinity matrix may be cellulose beads that are oxidized with an oxidizing agent prior to binding to the histone. The beads may be, for example, Sepharose beads. The beads may be coated with streptavidin. The size of the beads may be, for example, 30 to 200 microns, 40 to 180 microns, 45 to 165 microns, 60 to 150 microns. Various oxidizing agents can be used, such as sodium metaperiodate (NaIO). Alternatively, the primary hydroxyl group of cellulose can be selectively converted to give 6-deoxy-6-carboxycellulose by piperidino-ammonium salt mediated oxidation (TEMPO). See, for example, Eyle, S. and Thielemans, W., Surface modifucation of cellulose nanocrystals, Nanoscale, 2014, 6, 7764, DOI: 10.1039/c4nr01756k). Also, the cellulose (or agarose) carrier can be oxidized with other compounds known to the person skilled in the art, such as chromic acid, chromium pyridine trioxide, dimethyl sulfoxide. See for example Peng, L. et al. Evaluation of activation methods with cellulose beads for immunosorbent purification of immunoglobulins, J. Biotechnology, 5 (1987) 255-265). The oxidized beads are then incubated with a sufficiently purified and concentrated histone protein solution such as recombinant human H1.3 histone. The reaction can be stopped and then washed with buffer to remove soluble protein contaminants.

[0122] A histone-based affinity matrix can be obtained by a method including a) oxidizing cellulose beads ranging in size from 100 to 250 micrometers to obtain activated cellulose beads; b) washing the activated cellulose granules; c) preparation of a concentrated solution of recombinant human histone H1.3; d) incubation of activated cellulose beads with a concentrated solution of recombinant human histone H1.3; and e) blocking any free CHO groups on the activated cellulose granules.

[0123] The above process may further include: f) washing the activated cellulose beads with buffer.

[0124] Any oxidizing agent can be used in step a). One example of an oxidizing agent is NaIO. Any washing method can be undertaken in step b). For example, activated cellulose granules are washed with sodium bicarbonate, hydrochloric acid and water. In step c), dialysis or other methods may be used. For example, a solution of recombinant human histone H1.3 is dialyzed, and the dialyzed solution is concentrated in 0.1 M NaHCO ₃ at pH 7-9 or at pH 8. In step d), incubation can be carried out for 3-5 hours at 15-30° C or for 4 hours at room temperature. In step e), the blocking step includes adding 1M ethanolamine to the activated cellulose beads and reacting for 30 minutes to 2 hours at 15-30°C. In step f) the activated cellulose beads can be washed with TBS buffer.

[0125] The beads may be loaded into a column, such as, for example, a polytetrafluoroethylene (PTFE) column. Other examples of columns may have a wall of polycarbonate, polyethylene, polyvinyl chloride, polypropylene, polyethersulfone, polyester, or other polymeric material approved by the FDA or EMEA for making devices for extracorporeal processing of blood or a blood component.

[0126] The column or cartridge device can also be made from a non-toxic material that provides rigid support for the affinity matrix inside. Typically, the material is a plastic composition such as polycarbonate, polyethylene, polyvinyl chloride, polypropylene, polyethersulfone, polyester, polystyrene, or other similar material approved by regulators such as the FDA or EMEA for making devices for extracorporeal processing of blood or a blood component. In some embodiments, there is an internal filter at the bottom of the column (cartridge) to prevent the affine matrix from exiting the device. In some embodiments, the top of the device also has an internal filter to contain the affine matrix within the device. In some embodiments, these filters are made of plastic and/or cellulose material and have pores that allow the passage of a liquid, such as plasma, but not a particulate material, such as an affinity matrix.

[0127] When preparing a column containing a histone-based affinity matrix, the histone-based affinity matrix may be loaded at least 50%, 60%, 70%, 75%, 80%, 85%, or 90% of the column volume. Phosphate buffer, in particular cold phosphate buffer, can be used to equilibrate the column. Other suitable buffers may also be used to equilibrate the column.

[0128] The apheresis device may contain a lectin-based affinity matrix. Non-limiting examples of useful lectins include, for example, Galanthus nivalis (snowdrop) lectin (GNA), Narcissus Pseudonarcissus (daffodil) lectin (NPA), Conconavalin A, phytohemaggluttanin, and cyanovirin. In one embodiment, the lectin can be associated with the agarose matrix by overnight incubation at neutral or slightly alkaline pH. After such an incubation, a vigorous wash with buffer at a pH of about 7.0-7.5 can be undertaken to remove unbound lectin.

[0129] A lectin-based affinity matrix can be prepared according to a method comprising a) reacting a lectin with activated agarose beads to produce lectin-bound agarose; and b) washing the lectin-bound agarose with buffer.

[0130] The apheresis device may comprise an affinity matrix based on a polyamidoamine (PAMAM) dendrimer (PDAM) or a polypropyleneimine (PPI) dendrimer. See, for example, Kaur et al., J Nanopart Res., 2016, 18: 146. Dendrimers are unique nanometer-sized synthetic polymers with a highly branched structure and spherical shape. Among dendrimers, polyamidoamine (PAMAM) has received the most attention as a potential agent for gene transfection and delivery because these macromolecules bind DNA at physiological pH. PAMAM dendrimers consist of an alkyldiamine core and tertiary amine branches. They are available in ten generations (G0-10) with 5 different core types and 10 functional surface groups. DNA and polyamidamine (PAMAM) dendrimers form complexes based on electrostatic interactions between negatively charged nucleic acid phosphate groups and protonated (positively charged) amino groups of polymers. The formation of complexes with high molecular weight and high density depends mainly on the concentration of DNA with an increase due to an increase in the charge ratio of the dendrimer-DNA. (Shcharbin, D. et al., Practical Guide to Studying Dendrimers. Book, iSmithers Rapra Publishing: Shawbury, Shrewsbury, Shropshire, UK, 2010. 120 p. ISBN: 978-1-84735-444-0).

[0131] PAMAM-dendrimer-based affinity matrix obtained by a method comprising a) washing the cellulose granules with ethanol and water; b) incubating the washed cellulose beads with (±)-epichlorohydrin and NaOH to obtain activated cellulose beads; c) reacting the activated cellulose beads with the PAMAM dendrimer to form PAMAM beads and removing the PAMAM dendrimer that did not react with the activated cellulose beads; and d) blocking the unconverted epoxy groups on the PAMAM beads.

[0132] The beads may be loaded into a column, such as a polytetrafluoroethylene (PTFE) column. Other examples of columns may have a wall of polycarbonate, polyethylene, polyvinyl chloride, polypropylene, polyethersulfone, polyester, or other polymeric material approved by the FDA or EMEA for making devices for extracorporeal processing of blood or a blood component.

[0133] An apheresis device containing a PAMAM dendrimer-based affinity matrix may be more efficient at removing cfDNA or alternatively may remove cfDNA more completely or alternatively may remove more total cfDNA in a particular blood sample than using an apheresis device comprising a histone-based affinity matrix and a lectin-based affinity matrix.

[0134] In certain embodiments, the apheresis device may comprise both a PAMAM dendrimer-based affinity matrix, a histone-based affinity matrix, and a lectin-based affinity matrix.

[0135] The apheresis device may contain an affinity matrix based on an anti-DNA antibody. Anti-DNA antibodies constitute a subgroup of antinuclear antibodies that bind single-stranded DNA, double-stranded DNA, or both (anti-ds+ss DNA antibody). They may be, for example, IgM antibodies or any of the subclasses of IgG antibodies. Antibodies that bind exclusively to single-stranded DNA can bind its constituent bases, nucleosides, nucleotides, oligonucleotides, and the ribose phosphate backbone, all of which are present in individual DNA strands. Antibodies that bind double-stranded DNA can bind to the ribose phosphate backbone, base pairs (deoxyguanosine-deoxycytidine and deoxyadenosine-deoxythymidine), or specific double helix conformations (Bevra Hannahs Hahn, Antibodies to DNA. N Engl J Med 1998; 338: 1359- 1368). Anti-DNA antibodies can also bind supramolecular structures containing DNA, such as nucleosomes and chromatin.

[0136] An anti-DNA antibody affinity matrix can be obtained by activating the agarose beads, for example with N-hydroxysuccinimide (NHS). The activated beads can then be incubated with an antibody or other reagent that has an affinity for DNA. Excess antibodies/reagents are then removed by washing.

[0137] An anti-nucleosome antibody affinity matrix (ANAM) obtained by a method comprising a) obtaining activated agarose beads by crosslinking N-hydroxysuccinimide to agarose beads; b) washing the activated agarose beads with binding buffer containing NaHCO ₃ and NaCl; c) adding to the binding buffer an antibody that binds to nucleosomes, where the antibody is derived from an MRL/Mp (-)+/+ mouse; d) incubating the binding buffer containing the antibody with activated agarose beads to obtain an affinity matrix based on antinucleosome antibodies; and f) washing the antinucleosome antibody affinity template with binding buffer and acetate buffer.

[0138] The apheresis device may contain an affinity matrix based on a DNA intercalator. There are several ways that molecules can interact with DNA. Ligands can interact with DNA by covalent bonding, electrostatic bonding, or intercalation. Intercalation occurs when ligands of the appropriate size and chemical nature are placed between DNA base pairs. DNA binding agents tend to interact non-covalently with the host DNA molecule in two main ways: (i) threading intercalation into grooves, stabilized by a mixture of hydrophobic, electrostatic, and hydrogen interactions, and (ii) classical intercalation via intercalation association, in which a flat, heteroaromatic moiety is inserted between pairs bases of DNA. The most commonly studied intercalating binding is a non-covalent stacking interaction resulting from the insertion of a planar heterocyclic aromatic ring between the base pairs of the DNA double helix. See http://nptel.ac.in/courses/104103018/35. Hoechst 33342 is a bisbenzimide derivative that binds to AT-rich sequences in the minor groove of double-stranded DNA. The heterocyclic fragment in this dye is important for efficient interaction with the DNA double helix, which makes the Hoechst-DNA complex more stable.

[0139] A DNA intercalator affinity template can be obtained by oxidizing (activating) beads such as cellulose beads (carrier) reacting with a compound (linker) such as N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC ) that binds the DNA intercalator (DNA-binding fragment, i.e. Hoechst 33342) to the carrier surface. The beads are then washed.[0140] Affinity matrix based on Hoechst 3342 obtained by a method comprising a) oxidation of cellulose beads; b) washing the oxidized cellulose beads; c) reacting the washed oxidized cellulose beads with a solution containing Hoechst 33342 and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) to form beads with Hoechst 33342 immobilized thereon; and d) washing the beads with Hoechst 33342 immobilized on them.

[0141] The apheresis device may comprise a hyperbranched poly-L-lysine affinity matrix. Affine matrix based

hyperbranched poly-L-lysine can be obtained by a method comprising a) dissolving L-lysine monohydrochloride in water and neutralizing with KOH to obtain a solution of L-lysine; b) heating the solution of L-lysine to obtain a solution containing poly-L-lysine; c) removing L-lysine and salt from the solution containing poly-L-lysine; d) fractionating the solution containing poly-L-lysine to obtain a fraction containing poly-L-lysine with an average molecular weight of 21,000 to 32,000; e) dialyzing and lyophilizing a fraction containing poly-L-lysine with an average molecular weight of 21,000 to 32,000 to obtain a lyophilizate; f) dissolving the lyophilizate in distilled water and dialysis against NaHCO ₃ to obtain a solution containing HBPL; and g) incubating the HBPL containing solution with cyanogen bromide activated Sepharose 4B suspended in NaHCO ₃ .

[0142] In some embodiments, the apheresis device may comprise all or any of the following: a DNA intercalator affinity matrix, a Hoechst 33342 affinity matrix, an anti-DNA antibody affinity matrix, a PAMAM affinity matrix, a histone affinity matrix, a lectin-based affinity matrix; and a poly-L-lysine-based affinity matrix.

[0143] Various apheresis procedures and treatments are described in the application. The various methods and procedures include (a) performing an apheresis procedure including withdrawing blood or plasma from a patient into an apheresis device to obtain purified blood or plasma; and (b) returning the purified blood or plasma with reduced levels of cfDNA to the patient. Any vein can be selected for optimal blood drainage. For example, blood may be drained from a patient's portal vein. Alternatively, blood may be drawn from the patient's femoral vein or jugular vein.

[0144] In various treatment options, the apheresis procedure may be performed more than once or even twice, for example, on the 1st and 3rd day. In the treatment of kidney damage, the level of kidney damage can be assessed by measuring serum. Blood creatinine and blood urea nitrogen levels using Roche Reflotron Plus (Roche Diagnostics) before each apheresis procedure.

[0145] Circulating cfDNA can be extracted from plasma samples using the conventional THP (Triton-Heat-Phenol) method (Breitbach et al., PLoS ONE, 2014, 9 (3): e87838). The extracted cfDNA can be quantified using various assays such as the PicoGreen assay (Molecular Probes, The Netherlands) following the manufacturer's instructions. Well-known DNA stains can be used to visualize cfDNA on agarose gel as described in the examples below, including, for example, ethidium bromide (Sigma-Aldrich), Diamond™ (Promega), SYBR® Gold Nucleic Acid Gel (Molecular Probes). The dyes can be used as a gel stain, pretreatment agent, or can be preloaded directly into the sample loading buffer.

[0146] In various embodiments, the apheresis procedure further comprises separating blood into plasma. The plasma portion can then be directed to one or more affinity templates to remove the cfDNA.

EXAMPLES

[0147] The present invention is also described and demonstrated using the following examples. However, the use of these and other examples anywhere in the description is illustrative only and in no way limits the scope and meaning of the invention or any exemplary term. Likewise, the invention is not limited to any specific preferred embodiments described herein. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this description, and such variations may be made without departing from the spirit or scope of the invention. Therefore, the invention is to be limited only by the terms of the appended claims together with the full scope of the equivalents to which the said claims are entitled.

Example 1. Obtaining an affinity matrix based on histone H1 and an affinity column.

[0148] An H1 histone affinity matrix and affinity column were prepared as follows: cellulose beads (bead size 100-250 micrometers, Sigma-Aldrich) were oxidized with sodium metaperiodate. For this, an aqueous suspension of beads (3 g, 5 ml) and NaIO (0.1 g, 0.5 mmol) in 10 ml of water was shaken at room temperature for 4 hours. The activated beads were collected and washed with 1 M sodium bicarbonate, 0.1 M hydrochloric acid and 200 ml of water. A solution of recombinant human histone H1.3 (≥98% purity, Institute of Bioorganic Chemistry, Moscow) was subjected to dialysis and concentrated (10 ml; 5 mg/ml) in 0.1 M NaHCO ₃ (pH 8). The solution was then incubated with oxidized beads (5 ml) at room temperature for 4 hours with stirring. After incubation, 1 M ethanolamine (1.5 ml) was added to a suspension of activated beads (15 ml) to block free CHO groups; The reaction was continued for 1 hour at room temperature. The resulting histone H1-immobilized cellulose beads were washed three times with TBS buffer to remove soluble protein contaminants and to obtain an affinity matrix based on histone H1. Polycarbonate columns with a volume of 4–30 ml were loaded (up to 70–90% by volume) with a cellulose matrix with immobilized histone H1.

Example 2: Purification of the blood of a cancer patient from various types of circulating cfDNA

[0149] Separation of particle-bound cfDNA type (i.e., nucleosome-bound cfDNA and exosome-bound cfDNA) from unbound circulating cfDNA was performed as follows: plasma from a cancer patient with advanced gastric adenocarcinoma and multiple lung and liver metastases ( T4N2M1) was prepared by collecting blood in citrate-treated tubes and centrifuging for 10 minutes at 2000 g using a refrigerated centrifuge and collecting the supernatant.

[0150] Nucleosome-bound cfDNA and exosome-bound cfDNA were removed using two sequentially coupled affinity columns containing an anti-histone antibody affinity matrix and a lectin-based affinity matrix, as described in WO 2007/049286 A1 and US Pat. No. 9,364,601, respectively.

[0151] Briefly, an anti-histone affinity matrix and column was prepared as follows: 0.5 ml (1 vol.) 1.3 ml. The column was equilibrated with 2 ml (4 volumes) phosphate buffered saline (PBS). 1 ml (volume) of a 100 μg/ml biotinylated anti-histone antibody solution (H2A.X; Santa Cruz Biotechnologies) was added to the column and allowed to enter the gel bed. The bottom and top caps were replaced sequentially and incubated for 2 hours at room temperature. After incubation, the column was washed with 2 ml (4 volumes) of cold phosphate buffered saline (PBS).

[0152] The lectin affinity matrix was prepared as follows: 2 ml (1 volume) Galanthus nivalis (snowdrop) lectin, i.e. GNA solution (Sigma-Aldrich), at a concentration of 10 mg/ml in 0.1 M NaHCO ₃ , pH 9.5 was added to 2 ml (1 vol) CNBr activated agarose beads (cyanogen bromide activated sepharose 6MB, 6% agarose, macrobeads 200-300 µm diameter, Sigma-Aldrich) and allowed to react overnight in the cold at pH 7.4-8.0. When the reaction was complete, the lectin-bound agarose was thoroughly washed with sterile cold phosphate-buffered saline (PBS) at pH 7.2-7.4. The resulting affinity matrix based on lectin was transferred into a polystyrene column 0.6 × 6 cm in size.

[0153] To purify cfDNA bound to nucleosomes, 1.0 ml of plasma was applied to the first affinity column (containing an affinity matrix based on the anti-histone antibody H2A) and passed through the column. The plasma was then loaded into a second affinity (exosome-binding) column containing a lectin-based affinity template [GNA] and passed through the column.

[0154] Alternatively, the same amount of patient plasma was passed through one single H1 histone affinity column prepared as described in Example 1 (cellulose beads bound to immobilized H1.3 histone).

[0155] All plasma samples were analyzed by gel electrophoresis with fluorescent DNA staining before apheresis and after completion of apheresis.

[0156] Electrophoretic profile of circulating cfDNA from the plasma of a cancer patient before removal of nucleosome-bound DNA and exosomes (lane A), after sequential affinity purification on columns containing an affinity matrix based on anti-histone antibodies H2A and an affinity matrix based on lectin (lane B ) and purification on a column containing an affinity matrix based on histone H1.3 (lane C) is shown in figure 1.

[0157] Although nucleosome-bound circulating cfDNA and exosomes were removed from plasma, the sample shown in the middle lane (lane B) still contained significant amounts of circulating cfDNA, visualized within the molecular range of 100-1000 bp. As shown in the right lane, DNA was not visualized in the plasma sample after passing through a column containing histone H1.3 affinity matrix. Thus, apheresis/purification of patient plasma through a DNA-binding protein (H1.3 histone) affinity matrix column can remove most, nearly all, or all of nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA, including dsDNA, ssDNA and oligonucleotides from the patient's blood.

Example 3: Circulating cfDNA Purified of Nucleosome and Exosome Bound cfDNA Accelerates Tumor Growth

[0158] 60 ml of plasma from a patient with metastatic non-small cell lung carcinoma (NSCLC T3N2M+) was collected over consecutive days and purified from nucleosome-bound cfDNA and exosome-bound cfDNA using successive anti-histone antibody affinity columns H2.A and lectin as described in Example 2 (anti-histone antibody affinity matrix and lectin affinity matrix from Galanthus nivalis (snowdrop)). For the preparation of affinity columns, polycarbonate columns sized 2.0×7.0 cm were used. Each column was loaded at 70-80% of the column volume with the corresponding affinity matrix. The remaining circulating cfDNA after passing through the columns was extracted from purified plasma using classical phenol/chloroform extraction and ethanol precipitation (Stirling, D. et al, DNA extraction from plasma and serum, In: Methods in Molecular Biology, vol. 226: PCR Protocols, Second Edition, Ed. by J.M.C. Bartlett and D. Stirling, Humana Press Inc., Totowa, NJ, 2003, 556 pages). The dried extracted cfDNA was stored at -70°C. The total amount of residual cfDNA isolated from the patient's plasma after purification from cfDNA associated with nucleosomes and cfDNA associated with exosomes was 9.7 μg. The cfDNA was redissolved in PBS and used for animal experiments as described below.

[0159] The effect on tumor growth of cfDNA that was not bound to nucleosomes and exosomes was studied using the Panc02/C57/BL6 orthotopic model (Jiang YJ, Lee CL, Wang Q, et al. Establishment of an orthotopic pancreatic cancer mouse model World Journal of Gastroenterology: WJG 2014; 20(28): 9476-9485). 1×10 ⁶ Panc02 cells suspended in ice martigel were injected into the tail of the pancreas of each animal (day 0). 24 mice with tumors were divided into 3 groups of 8 mice each. Control mice received single daily injections of PBS (100 μl; retro orbital sinus venosus) for 10 days from day 10 to day 20. Group 1 mice were injected daily with 100 ng of cfDNA from a cancer patient, purified as described above. Group 2 mice were injected with 100 ng UltraPure™ salmon sperm DNA (Life Technologies) with a mean size of ≤2000 base pairs as a non-specific control using the same schedule and injection method.

[0160] Table 1 below summarizes the results of the effect of DNA injections on tumor mass in animals compared to the control group. Tumor weight was measured at the end of the study on day 23.

[0161] Figure 2A shows tumors excised from control mice. Figure 2B shows tumors excised from mice treated with cfDNA from NSCLC T3N2M+ patient plasma purified from cfDNA associated with nucleosomes and exosomes. Tumors in the control group were significantly smaller, dense, well separated from neighboring organs, and had no necrosis or hemorrhages.

[0162] Circulating cfDNA from the plasma of a cancer patient, purified from circulating cfDNA associated with the nucleosome and exosome, retains significant oncogenic properties. Thus, it may be beneficial to reduce levels of all circulating cfDNA: nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA, including dsDNA, ssDNA, and oligonucleotides.

Example 4: Obtaining an affinity matrix with a polyamidoamine dendrimer and an affinity column based on it

[0163] A PAMAM dendrimer (PDAM) affinity matrix and a column containing a PDAM affinity matrix were prepared according to Wang (Wang, Y., et al., New method for the preparation of adsorbent with high adsorption capacity, Chinese Science Bulletin 2005, Vol.50, No. 21, pp 2432-2435) as follows. Cellulose beads (Macroporous Bead Cellulose MT 500, particle size 100–250 μm, Iontosorb, Czech Republic) were washed twice with 98% ethanol and distilled water. 1 gram of beads were incubated with a mixture of 1.0 ml of (±)-epichlorohydrin (Sigma-Aldrich) and 3.0 ml of 2.5 M NaOH. The activation reaction was carried out at 40°C for 2.5 h in a shaker. The activated beads were thoroughly washed with distilled water. The content of epoxy groups was determined as about 0.31 mmol/g dry granules by titration with sodium thiosulfate with hydrogen chloride. 4.0 ml of the prepared wet activated cellulose granules were suspended with 9.0 ml of a 20% solution of amino-terminated PAMAM dendrimer (-NH2) (ethylenediamine core, generation 3.0, Sigma-Alrich) and shaken at 24°C in within 5 hours. After modification, unreacted PAMAM was removed by washing with distilled water, and the remaining unreacted epoxy groups on the beads were blocked by reaction with ethylamine. The functionalized affinity template was then washed with 0.1 M phosphate buffer and MilliQ water. 2.0-20.0 ml of the prepared affinity matrix was placed in a pyrogen-free column of polytetrafluoroethylene (PTFE) (0.5-3.0) cm×(1.0-10.0) cm to load 70-90% of the column volume. The prepared affinity column was sterilized by autoclaving at 121°С for 30 min.

Example 5: Purification of the blood plasma of a cancer patient and a stroke patient from various types of circulating cfDNA.

[0164] 1.0 ml aliquots of plasma samples from a patient with ischemic stroke (24 hours after the onset of stroke), and a cancer patient with advanced gastric adenocarcinoma with multiple lung and liver metastases (T4N2M1) were purified sequentially using affinity columns based on anti -histone antibody H2.A and lectin as described in example 2 (affinity matrix with anti-histone antibodies H2A and affinity matrix with lectin from Galanthus nivalis (snowdrop)) or only through an affinity column with a polyamidoamine dendrimer with a size of 0.6 × 10.0 see prepared as described in example 4 (affinity matrix based on cellulose beads associated with PAMAM dendrimer).

[0165] All plasma samples were analyzed by gel electrophoresis with DNA fluorescent dye staining before and after purification.

[0166] The electrophoretic profile of circulating cfDNA from the plasma of these patients before removal of nucleosome-bound cfDNA and exosomes, after purification on anti-histone antibody and lectin affinity columns, and after purification with a polyamidoamine dendrimer affinity column, is shown in Figure 3.

[0167] Sequential purification of the plasma of a cancer patient using affinity columns with anti-histone antibodies and lectin removed most of the cfDNA associated with the particles; however, visible amounts of nucleosome-bound cfDNA and circulating cfDNA of mononucleosome and subnucleosome size (less than 147 bp in length) remained in plasma. Plasma purification using a polyamidoamine a dendrimer (PAMAM) affinity column results in the complete removal of circulating cfDNA from the cancer patient's plasma. In stroke patients, affinity purification with a polyamidoamine dendrimer affinity column (used as a one-step process) results in sufficient removal of virtually all types of circulating cfDNA from plasma such that they are not detectable.

[0168] Thus, a patient's plasma can be purified from essentially all types of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides) using an affinity matrix containing DNA- binding polymer.

Example 6: Plasma cfDNA purified from nucleosome-bound cfDNA and exosomes has procoagulant activity

[0169] US Pat. No. 9,642,822 discloses that circulating high molecular weight nucleosome-bound cfDNA, extracellular neutrophil traps (NETs), has procoagulant activity in patients with advanced cancer and acute vascular conditions. Blood plasma from a stroke patient (24 hours from onset) and a cancer patient with advanced gastric adenocarcinoma with multiple lung and liver metastases (T4N2M1) were collected and purified sequentially through both lectin and anti-histone antibody affinity columns (prepared, as described in Example 2 (H2A anti-histone antibody affinity matrix and Galanthus nivalis lectin affinity matrix

[snowdrop]) or through a 1.0 x 5.0 cm polyamidoamine dendrimer affinity column (prepared as described in Example 4). Further, purified and untreated control plasma samples were defibrinated by rotation at 3000 g for 20 minutes and filtered through a 0.22 μm filter. The samples were divided into aliquots in 1.0 ml plastic tubes, shaken in a water bath at 50°C for 25 minutes and centrifuged at 10,000 g (10 minutes). The resulting supernatants were stored at -80°C and then tested in the thrombin formation assay as follows: a mixture of 25 µl of diluted (1:9) thromboplastin (Sigma), 25 µl of 0.9% NaCl and 50 µl of 1:1 diluted defibrinated plasma ( all reagents were diluted in 0.9% NaCl).

[0170] All reagents in the thrombin formation assay were diluted in 0.9% NaCl. A mixture of 25 µl of thromboplastin, 25 µl of 0.9% NaCl and 50 µl of diluted defibrinated plasma at a 1:1 dilution was added to the wells of the microtiter plate and preheated to 37°C for 10 minutes. Then 50 µl of 1 mM spectrozyme, chromogenic substrate for thrombin and 50 µl of 30 mM calcium chloride. Plates were read in an automated ELISA plate reader (Victor, Perkin Elmer) at 1000 sec. and 405 nm at room temperature. All measurements were performed in triplicate. In this test, the OD value is proportional to the plasma procoagulant activity (thrombin generation).

[0171] The results are shown in Table 2. Thus, not only cfDNA associated with nucleosomes and exosomes, but also unbound cfDNA has procoagulant activity in cancer and acute vascular events. Thus, reducing the levels of all types of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides) is advantageous.

Example 7 Preparation of Affinity Matrix and Column Based on Anti-DNA Antibody

[0172] An anti-DNA antibody affinity matrix and affinity column were prepared as follows: 5 ml N-hydroxysuccinimide (NHS) activated 4% highly cross-linked agarose beads, average bead size 90 micrometers (NHS-activated Sepharose 4 Fast Flow, GE Healthcare Life Sciences). The activated matrix was washed twice with cold (2-4° C.) binding buffer (0.2 M NaHCO ₃ , 0.5 M NaCl, pH 8.3). 1000 μg of high affinity mouse anti-ds and ss DNA IgM monoclonal antibody ([49/4A1], ab35576, Abeam) was dialyzed against binding buffer and then coupled according to the manufacturer's instructions to NHS-activated Sepharose. Three cycles of washing with binding buffer followed by washing with 0.1 M acetate buffer (pH 4.0) was used to remove excess unbound antibody. ,four).

Example 8: Preparation of DNA Intercalator Affinity Matrix and Column Based on DNA Intercalator

[0173] Affinity matrix based on Hoechst 33342 and the corresponding affinity column were prepared as follows: cellulose beads (bead size 100-250 micrometers, Sigma-Aldrich) were oxidized with sodium metaperiodate. For this, an aqueous suspension of beads (3 g, 5 ml) and NaIO (0.1 g, 0.5 mmol) in 10 ml of water was shaken at room temperature for 4 hours. The activated beads were collected and washed with 1 M sodium bicarbonate, 0.1 M hydrochloric acid and 200 ml of water. 450 mg activated cellulose beads were mixed with 1000 ml pH buffered solution containing 0.047 mg/ml Hoechst 33342 (Sigma-Aldrich) and 0.4 mg/ml N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and reacted with constant shaking speed for 1 hour at 32°C. Beads with immobilized Hoechst 33342 were washed three times with deionized water to remove unreacted dye. The prepared affinity matrix based on the DNA intercalator was placed into a 4 ml plastic (polycarbonate) column. The column was stored at 4°C.

Example 9 Separation of different types of circulating cfDNA from a patient with systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS).

[0174] Plasma samples were obtained from a patient admitted to the intensive care unit (ICU) diagnosed with systemic inflammatory response syndrome (SIRS) with multiple organ failure (multiple organ dysfunction syndrome, MODS) secondary to acute pancreatitis. Therapeutic plasma exchange (TPE) was carried out as a rescue therapy. 1 ml aliquots of patient plasma obtained by plasma exchange were purified through affinity columns with both lectin and anti-histone antibody as described in Example 2 (anti-histone antibody affinity matrix and lectin affinity matrix from Galanthus nivalis (snowdrop )) or through an affinity column with an intercalator as described in example 8 (cellulose beads coupled to Hoechst 33342, DNA intercalator affinity matrix). All plasma samples were analyzed by gel electrophoresis with DNA fluorescent staining before and after purification.

[0175] As shown in Figure 4, the SIRS patient's plasma contained significant amounts of circulating cfDNA, which gave a strong fluorescent signal after staining with a fluorescent DNA dye. Plasma purification with anti-histone antibody and lectin affinity columns removed nucleosome-bound cfDNA, however, a certain amount of nucleosome-bound circulating cfDNA and circulating subnucleosomal cfDNA (less than 147 base pairs long) remained in the plasma. Affinity purification with a Hoechst 33342 affinity column removed circulating subnucleosome cfDNA, but some nucleosome bound circulating cfDNA was still present. Therefore, the inventors tested sequential purification with different columns: a 1 ml aliquot of patient plasma was purified sequentially through an affinity column with a Hoechst 33342 intercalator, followed by purification on an affinity column containing an anti-DNA antibody template in the manner described in Example 2 for the sequential use of affinity columns based on anti-histone and lectin-based antibodies. Thereafter, the plasma was examined by gel electrophoresis with fluorescent DNA staining and no circulating cfDNA was detected.

[0176] Thus, purification using the affinity template combinations of the present invention can remove virtually all types of cfDNA in a patient's blood, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides) from blood, or the patient's plasma.

Example 10 Circulating plasma cfDNA purified from nucleosome-associated cfDNA and exosome-associated cfDNA has pro-inflammatory activity and contributes to organ dysfunction in sepsis.

[0177] Plasma was obtained from a patient admitted to the intensive care unit (ICU) diagnosed with systemic inflammatory response syndrome with multiple organ dysfunction syndrome (MODS) secondary to acute pancreatitis. Therapeutic plasma exchange was performed as a salvage therapy. 100 ml of collected patient plasma was purified twice through both lectin and anti-histone antibody affinity columns (as described in Example 2, i.e. through anti-histone antibody affinity matrix and lectin affinity matrix from Galanthus nivalis

[snowdrop]) twice to ensure complete purification of cfDNA associated with nucleosomes and exosomes. The remaining circulating cfDNA was extracted from plasma purified from nucleosomes and exosomes as described in Example 3. The total amount of residual DNA (isolated from patient plasma, previously purified from circulating cfDNA bound to nucleosomes and exosomes) was about 50 μg. Isolated DNA resuspended in phosphate buffered saline (PBS) at pH 7.2 and used for animal experiment as described below.

[0178] Eight 10-week-old C57/BL6 male mice were intravenously injected with 1 μg of the extracted cfDNA three times 1 hour apart. Animals were euthanized 4 hours after the last DNA injection for blood collection.

[0179] Plasma creatinine levels were measured by enzymatic assay. Fluorescent magnetic bead immunoassay (Bio-Rad Laboratories, USA) was used to determine plasma TNF-a, IFN-g, and IL-12. The results are shown in Table 3 below.

[0180] Thus, plasma cfDNA purified from cfDNA associated with nucleosomes and exosomes has a strong pro-inflammatory activity and contributes to organ dysfunction.

Example 11 Circulating patient plasma cfDNA purified of nucleosome- and exosome-bound cfDNA but not purified of unbound cfDNA is responsible for TLR9 activation.

[0181] Activation of TLR9 receptors has recently been recognized as an important component in the development of a systemic inflammatory response, organ failure, cancer invasion and metastasis, neuronal damage in stroke, an autoimmune condition, eclampsia, and age-related immune dysregulation leading to an age-related pro-inflammatory status.

[0182] The patient was a 33-year-old male with acute myeloid leukemia and received an HLA-matched bone marrow transplant (BMT) followed by standard immunosuppression and antibiotic prophylaxis. Approximately 1 month after TDC, the patient developed an erythematous rash consistent with grade III GVHD and severe diarrhea. Plasma samples were obtained on admission and purified sequentially through both lectin and anti-histone antibody affinity columns (as described in Example 2, i.e., through anti-histone antibody affinity matrix and lectin affinity matrix from Galanthus nivalis

[snowdrop]) or purified using a histone H1.3 affinity column prepared as described in Example 1 (affinity matrix based on cellulose beads bound to histone H1.3).

[0183] hEK-Blue™ hTLR9 reporter cells (Invivogen) were washed with medium to separate them from the culture flask and the cells were resuspended to the cell density specified in the manufacturer's protocol. 180 µl of the cell suspension per well was stimulated for 24 h (37°C, 5% CO) with 60 µl of untreated patient plasma, plasma purified through affinity columns with anti-histone antibodies and plasma lectin, purified through a histone H1.3 affinity column (per stage). After incubation, secreted embryonic alkaline phosphatase (SEAP) analysis was performed using Quanti-Blue detection medium as described in the manufacturer's instructions. Absorbance detection at 650 nm was measured using a microplate reader.

[0184] TLR9 activation was quantified by reading the optical density (OD) at 620 nm. (N=3.) Results are shown in Table 4. Surprisingly, removal of circulating exosome- and nucleosome-bound cfDNA prevented TLR9 activation by patient plasma to a rather limited extent, while removing virtually all types of cfDNA, including particle-bound and unbound with dsDNA particles, ssDNA and oligonucleotides, almost completely prevented TLR9 activation by the patient's plasma.

Example 12 Preparation of a Hyperbranched Poly-L-Lysine Affinity Matrix (PLLAM) and Affinity Column.

[0185] Cationic polyamino acids such as poly-E-lysine (PLL) are known to be effective for condensing plasmid DNA into compact nanostructures and are used for DNA binding in vitro and in vivo.

[0186] A cationic DNA-binding polymer, namely hyperbranched poly-E-lysine (HBPL), was prepared as described by Kadlecova, Z. et al, A comparative study on the in vitro cytotoxicity of linear, dendritic and hyperbranched polylysine analogs, Biomacromolecules, v. 13 (2012) pp 3127-3137): 27.45 g of L-lysine monohydrochloride (reagent level>98%, Sigma-Aldrich, USA) was dissolved in 55 ml of Milli-Q water and neutralized with 8.4 g of KOH. The solution was then heated to 150° C. for 48 h under nitrogen flow. Then, to remove excess salt and remaining L-lysine, the polymerization product was dialyzed with a Milli-Q anti-water dialysis membrane tube (Snakeskin Dialysis Tubing, Thermo Fisher Scientific, Switzerland, cut-off molecular weight: 3000 g/mol). The dialysis product was dried and later fractionated on a Sephadex G75 gel filtration column (GE Healthcare Life Science, Switzerland): 50 ml of a 2 mg/ml solution of HBPL in 0.01 M HCl was loaded into the column and subsequently eluted with 0.01 M HCl. Fractions of 20 ml were collected and lyophilized. A fraction with an average molecular weight of 21,000-32,000 (as determined by size exclusion chromatography) was collected and lyophilized. The lyophilized fraction was dissolved in bidistilled water, dialyzed against 0.1 M NaHCO ₃ and used for further preparation of the affinity matrix. An agarose matrix that contains immobilized HBPL was prepared in the usual manner as follows: Sepharose 4B activated with cyanogen bromide (wet weight 10 g, Sigma) was suspended in 10 ml of 0.1 M NaHCO ₃ mixed with 10 ml of fraction 21000-32000 HBPL ( 5 mg/ml in 0.1 M NaHCO ₃ ) and stirred for 24 h at 4°C. 750 ml 0.1 M NaHCO3, 750 ml 0.5 M NaCl, pH 9.2. The column was equilibrated with 0.05 M Tris-HCl buffer, pH 7.5. The prepared affinity column with hyperbranched poly-L-lysine affinity matrix (PLLAM) was stored at 4°C.

Example 13 Separation of different types of circulating cfDNA from a patient with a neurodegenerative disease.

[0187] Circulating cfDNA in patients with neurodegenerative disorders can cross the blood-brain barrier (BBB) and cause neuronal cell death. The use of the deoxyribonuclease enzyme may prevent this effect. WO2016190780. To investigate the effect of various subtypes of circulating cfDNA on neuronal cell death and to find out whether the simultaneous purification of blood from nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA, including dsDNA, ssDNA, and oligonucleotides, can prevent neuronal cell death, the following experiments were performed.

[0188] To obtain neuronal cultures, cerebral cortices were removed from (E) 15-17 day old Sprague Dawley rat embryos. Cortical explants were dissected into pieces of about 200-400 μm ² using fine needles and dissociated using a papain dissociation system (Worthington Biochemicals) according to the manufacturer's instructions and then stored on ice-cold minimum essential medium (Gibco). Neurons were plated on glass coverslips 13 mm in diameter coated first with poly-D-lysine (10 µg/mL in PBS) and then with laminin (10 µg/mL in PBS) (Gibco) and cultured at 37°C in a humidified atmosphere 8 % CO ₂ (v/v) for 24 hours in neurobasal medium with 1% (v/v) antimycotic antibiotic (Gibco).

[0189] After the initial culture period, the cell culture medium was diluted two-fold (v/v) with one of the following plasma samples, followed by cultivation for an additional 24 hours: (a) healthy 20-year-old donor plasma, (b) plasma of a patient with rapidly progressive Alzheimer's disease (AD), (c) plasma from the same AD patient treated for 6 hours with DNase I (Pulmozyme, Genentech) at a concentration of 5 µg/mL, (d) plasma from the same AD patient, skipped through affinity columns with both lectin and anti-histone antibodies (as described in Example 2, i.e. through affinity matrix with anti-histone antibodies and affinity matrix with lectin from Galanthus nivalis [snowdrop]) and (f) plasma of the same patient after passing through an affinity column with histone H1.3 (the matrix was prepared as described in example 1, that is, an affinity matrix based on cellulose beads associated with histone H1.3 placed in a polycarbonate column 0.8 × 9 cm (up to 80 % of column volume) . Approximately 2.0 ml were passed through the respective columns. plasma.

[0190] An electrophoretic profile of circulating cfDNA from plasma samples used in cell culture experiments is shown in Figure 5.

[0191] In the plasma of a healthy donor, only a small limited amount of nucleosome-bound circulating cfDNA in the form of mononucleosomes was found. High levels of nucleosome-bound circulating cfDNA in the form of mono- and oligonucleosomes have been found in the plasma of a patient with AD. Treatment of AD patient plasma with DNase I resulted in a decrease in cfDNA content in the oligonucleosome and mononucleosome fractions, but with a significant increase in cfDNA in the subnucleosome fraction (~less than 147 base pairs in length). AD patient plasma purified with anti-H2A and lectin affinity columns contained no nucleosome-bound circulating cfDNA, but only sub-nucleosomal (i.e., unbound) cfDNA. AD patient plasma purified with an H1.3 histone affinity column (in one step) contained no circulating cfDNA.

[0192] Induction of the apoptotic cell death marker caspase 3 was determined in dissociated cortical neurons cultured for 24 hours after the addition of plasma samples. Cells were fixed in 4% (w/v) paraformaldehyde (PFA) and incubated for 1 hour with cleaved anti-caspase 3 antibody (Abeam) diluted 1:500 in PBS. Cells were washed and incubated for 1 hour with Alexa Fluor 488 goat anti-rabbit polyclonal antibodies (hwitrogen in PBS followed by washing and counting.

[0193] The results are shown in Table 5. Thus, purification of blood from essentially all types of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides), prevents neuronal cell death significantly to a greater extent. better than simple purification from nucleosome-bound cfDNA and exosome-bound cfDNA, and even better than cleavage of circulating cfDNA in plasma by the enzyme DNase I, probably due to the release of by-products of enzymatic DNA cleavage or the low sensitivity of circulating cfDNA to DNase I.

Example 14 Endogenous Deoxyribonuclease Reactivation

[0194] The enzyme deoxyribonuclease (DNase) is the main enzyme responsible for the degradation of high molecular weight DNA in the circulation. Numerous studies show that deoxyribonuclease activity is suppressed under certain conditions, including an increase in circulating cfDNA in the blood, such as cancer, metastatic cancer, autoimmune disease, sepsis, infertility (Tamkovich SN, Circulating DNA and DNase activity in human blood. Ann N Y Acad Sci. 2006 Sep;1075:191-6;Martinez-Valle, DNase 1 activity in patients with systemic lupus erythematosus: relationship with epidemiological, clinical, immunological and therapeutic features.Lupus.2009 Apr;18(5):418-23;EP20070827224; Travis J Gould, Cellular and Biochemical Properties of Cell-Free DNA: A Prognostic Marker in Severe Sepsis Patients, Blood 2011, 118: 2169).

[0195] The following experiment was performed to evaluate how a decrease in the level of nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA, including dsDNA, ssDNA, and oligonucleotides, affects DNase I activity in plasma, the following experiment was performed. CfDNA was measured in plasma using the method described Goldstein (Goldshtein, H. et al., A rapid direct fluorescent assay for cell-free DNA quantification in biological fluids, Annals of Clinical Biochemistry, Vol 46, Issue 6, pp. 488^194). SYBR® Gold Nucleic Acid Gel Stain (Invitrogen) was first diluted 1:1000 in DMSO and then 1:8 in PBS. 10 μl plasma samples were loaded into 96-well plates. 40 µl of diluted SYBR Gold was added to each well (final dilution 1:10,000) and fluorescence was measured with a 96-well fluorometer at an emission wavelength of 535 nm and an excitation wavelength of 485 nm.

[0196] Western blotting of DNase I was performed on plasma samples separated using 10% SDS-PAGE gels transferred to polyvinylidene difluoride (PVDF) blotting membranes and incubated with goat anti-human DNase I antibodies (Santa Cruz Biotechnology). Binding was visualized using SuperSignal chemiluminescent substrate (Pierce) after incubation with HRP-conjugated goat IgG.

[0197] Serum deoxyribonuclease activity was measured using ORG590 (Orgentec) according to the manufacturer's protocol. Detection was performed using a microplate photometer (Multiscan FC) at 450 nm with a wavelength correction of 620 nm.

[0198] Blood samples were taken from a 56-year-old patient with breast cancer, multiple lung, liver and mediastinal metastases (T4N3M1). A 5 ml aliquot of plasma was subjected to multiple passages through a 1 ml polycarbonate column (0.5 × 5 cm) containing 0.5 ml of an affinity matrix based on histone H1.3: assessment of the electrophoretic profile of circulating cfDNA, western blotting of DNase and quantification deoxyribonuclease activity was checked after each passage of plasma through the column. The results are summarized in figure 6.

[0199] Electrophoretic evaluation of the profile of circulating cfDNA showed a continuous decrease in the content of all fractions of cfDNA as the number of passages through the columns increased. This observation was confirmed by direct quantification of circulating cfDNA in plasma. Comparable amounts of the DNase I enzyme detected by Western blotting were initially present in the patient's plasma. However, the enzymatic activity of DNase I was strongly suppressed and became significant only after a 4-fold passage through the columns, when the amount of circulating cfDNA was approximately halved.

[0200] Thus, apheresis in accordance with the present invention, in which total circulating levels of cfDNA are reduced by at least 50%, can reactivate endogenous DNase I enzyme activity, which is useful for patients who require a decrease in circulating cfDNA levels.

[0201] Considering the highest recorded levels of circulating cfDNA of approximately 5000 ng/ml (which are reported for some patients with advanced cancer, septic disorders, and patients with trauma), an affinity column or combination of affinity columns with a binding capacity of 30 mg could would provide nearly complete purification of patient plasma from all nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA, including dsDNA, ssDNA, and oligonucleotides.

Example 15 Preparation of an affinity column containing an anti-nucleosome antibody (ANAM) affinity template

[0202] A mouse monoclonal nucleosome-specific antibody was generated using mouse MRL/Mp (-)+/+ according to the method described in M. J. Losman (Monoclonal autoantibodies to subnucleosomes from a MRL/Mp (-)+/+ mouse Oligoclonality of the antibody response and recognition of a determinant composed of histones H2A, H2B, and DNA J Immunol March 1, 1992, 148 (5) 1561-1569). Prepared monoclonal (IgG) antibodies (mAbs), referred to herein as AN-1 and AN-44, respectively, were selected based on their ability to selectively bind nucleosomes, but not nucleosome components such as core histones or DNA. (Kees Kramers, Specificity of monoclonal anti-nucleosome auto-antibodies derived from lupus mice, Journal of Autoimmunity, V. 9, Issue 6, 1996, P. 723-729). The relative affinities of AN-1 and AN-44 for nucleosomes and the histone and non-histone components of the nucleosome are summarized in Table 6 below.

[0203] A 1 ml HiTrap column containing NHS activated Sepharose High Performance Sepharose (GE Healthcare) was used to prepare the affinity matrix and cartridge. 200 μg of AN-1 was bound according to the manufacturer's procedure with NHS-activated Sepharose.

[0204] Based on the affinity data presented in the table above, it is clear that the affinity template ANAM binds only nucleosome-bound cfDNA, but not unbound circulating cfDNA, including dsDNA, ssDNA, and oligonucleotides.

[0205] Thus, to ensure binding of unbound cfDNA, including dsDNA, ssDNA, and oligonucleotides, two sequentially linked columns were used in the animal experiment. One anti-nucleosome antibody affinity matrix (ANAM) column was prepared as described above in this example. A second column with a polyamidoamine dendrimer affinity matrix was prepared as described in Example 4.

Example 16: Apheresis Procedure

[0206] Indwelling venous catheters were inserted into the femoral vein and jugular vein of experimental rats under general anesthesia (i.e. injection of 0.8 mg xylazine and 4 mg ketamine). Catheters were flushed three times a week with heparinized saline during the study. Before each apheresis session, a heparin bolus (90 IU/100 g body weight (body weight)) was administered. The extracorporeal system was completely filled with heparinized saline, after which the ends of the catheters were connected to the extracorporeal system.

[0207] For animal apheresis experiments, the affinity columns described herein were provided with inlets and outlets for further incorporation of these prepared affinity columns into the second (plasma) circuit of the extracorporeal/apheresis system.

[0208] In the primary circuit of the system, blood was evacuated (rotary mini-peristaltic pump, Fisher Scientific) from the animal (femoral vein) through a plasma separator (Saxonia medical, Radeberg, Germany) and returned to the animal via a venous catheter. The separated plasma entered the second circuit (via a second rotating peristaltic mini-pump) and passed through one or more affinity cartridges in accordance with specific examples of the apheresis procedures described in this specification. In the first circuit of the system, blood was evacuated (rotating mini-peristaltic pump, Fisher Scientific) from the animal (femoral vein) through a plasma separator (Saxonia medical, Radeberg, Germany) and returned to the animal with an inserted jugular venous catheter. The separated plasma entered the second circuit (supported by a second peristaltic mini-pump Rotary) and passed through the affinity cartridges (according to the specific examples of the apheresis procedures described in this description) and returned to the animal through a polymer line also connected to a catheter inserted into the jugular vein.

Example 17: Use of apheresis for the treatment of sepsis and septic kidney disease

[0209] Classical models of sepsis induction by cecal ligation and puncture (CLP) were used. Female Sprague-Dawley (SD) rats weighing 350-400 g were used. Animals were anesthetized with sodium pentobarbital (50 mg/kg ip).

[0210] A median abdominal incision of about 1.5 cm was made. The mesentery of the caecum was opened to expose the caecum. The caecum was then ligated between the terminal and the ileocecal valve to maintain bowel continuity. Then the caecum was perforated with a single through puncture with a 21-gauge needle in the central segment of the ligation. The connected segment was gently pressed so that a small amount of feces was squeezed out onto the surface of the intestine. The caecum was returned to the abdominal cavity. The surgical wound was sutured in layers with an absorbable suture for the muscle layer and surgical staples for the skin. After surgery, rats were given 10 ml/kg of warm 0.9% sodium chloride for injection and after recovery, the animals were randomly divided into groups (groups 1-3; 6 animals in each group) according to the treatment.

[0211] Apheresis treatment was performed as described in Example 16. The apheresis procedure was performed twice: on day 1 (24 hours after CLP) and on day 3 (72 hours after CLP). 6 rats received an apheresis procedure using an anti-nucleosomal antibody (ANAM) affinity matrix (ANAM) affinity matrix column/cartridge prepared as described in Example 15 and 6 rats received an apheresis procedure using a column/cartridge containing a PAMAM dendrimer affinity matrix (PDAM) prepared as described in Example 4. Six rats (negative control group) received an apheresis procedure with a cartridge loaded with an appropriate volume of unmodified matrix material. The level of acute kidney injury (renal function) was assessed by measuring the levels of creatinine and blood urea nitrogen (BUN) using Roche Reflotron Plus (Roche Diagnostics) before each apheresis procedure. Circulating cfDNA was extracted from 100 µl plasma samples using the conventional THP (Triton-Heat-Phenol) method (Breitbach et al., PLoS ONE, 2014, 9 (3): e87838). DNA was quantified using the PicoGreen test (Molecular Probes, Netherlands) following the manufacturer's instructions. Changes in the level of cfDNA were expressed as a percentage of the baseline, i.e. to the level before the first apheresis procedure. For the negative control group, the columns/cartridges contained the appropriate amount of unmodified matrix material (Macroporous Bead Cellulose MT 500, particle size 100-250 µm, Iontosorb, Czech Republic, washed twice with 98% ethanol and bidistilled water). The survival rate (120 hours after CLP) was adopted as the main parameter of the effectiveness of the treatment of sepsis. Animal distribution and results are shown in Table 7 below.

[0212] The results show that the PDAM apheresis device is capable of capturing virtually all types of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides), and provided better therapeutic efficacy and reduced levels more effectively. circulating cfDNA in sepsis and septic kidney injury.

Example 18: Use of apheresis for the treatment of chemotherapial toxicity

[0213] Eighteen female Sprague-Dawley (SD) rats weighing 300-350 g were prepared for the apheresis procedure as described in Example 16. The animals received a single intravenous bolus injection of paclitaxel (Taxol, Bristol-Myers Squibb SrL) at a dose of 10 mg/day. kg. The apheresis procedure was started 4 hours after paclitaxel injection and continued for 12 hours; 6 rats received an apheresis procedure using an anti-nucleosome antibody affinity matrix (ANAM) column/cartridge, and 6 rats received an apheresis procedure using a hyperbranched poly-L-lysine (PLLAM) affinity matrix (PLLAM) column/cartridge. 6 rats (negative control) received the apheresis procedure with a cartridge that was loaded with the appropriate volume of unmodified matrix (Sepharose 4B).

[0214] Circulating cfDNA levels were quantified and presented as described in Example 17 (cfDNA level expressed as a percentage of baseline). Survival (24 hours post-bolus) was adopted as the primary measure of treatment success. Animal distribution and results are shown in Table 8 below.

[0215] The results show that the PLLAM apheresis device, which is capable of capturing virtually all types of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides), provides better protection/therapeutic efficacy and is more effective reduces the level of circulating cfDNA in animals poisoned with a chemotherapeutic drug.

Example 19 Purification/apheresis of cfDNA plasma with one cartridge that captures nucleosomal and exosomal cfDNA and another cartridge that captures unbound cfDNA, including dsDNA, ssDNA and oligonucleotides

[0216] To measure the level of cfDNA in plasma, cfDNA was isolated from 500 μl of plasma samples using a modified HTP method (Xue, X., et al. Optimizing the yield and utility of circulating cell-free DNA from plasma and serum, Clinica Chimica Acta, V.404 (2009), pp.100-104) and quantified using the PicoGreen assay (Molecular Probes, The Netherlands) according to the manufacturer's instructions.

[0217] When cfDNA was not detected in a sample using PicoGreen analysis, the absence of cfDNA in the samples was further confirmed by DNA agarose gel electrophoresis in the manner described in the description above.

[0218] For apheresis/purification procedures, plasma samples were applied to and passed through appropriate affinity columns.

[0219] A 2.0 ml plasma sample from a 67-year-old patient with septic shock was purified sequentially through an ANAM affinity column (as described in Example 15) and a lectin affinity column (which captures exosome-bound cfDNA) prepared as described in Example 2 (galanthus nivalis [snowdrop] lectin affinity array).

[0220] The initial level of cfDNA in the patient's plasma was 1150 ng/ml with a significant presence of almost all types of cfDNA visualized by DNA agarose gel electrophoresis (see figure 7, lane A). The plasma level of cfDNA after the first pass through the combination of affinity columns based on ANAM and lectin decreased to 350 ng/ml. Partially purified patient plasma was additionally subjected to a second run through the same combination of fresh columns based on ANAM and lectin. Plasma cfDNA levels remained unchanged after the second pass with visible amounts of cfDNA of non-nucleosomal origin up to 750 kDa molecular weight visualized by DNA agarose gel electrophoresis with DNA fluorescent dye staining (see Figure 7, lane C).

[0221] The experiment clarified that the inability of the ANAM and lectin based affinity columns to completely clear the patient's plasma of cfDNA is not related to the overall binding capacity of the combination of AMAM and lectin based affinity columns, but rather to their inability to capture cfDNA of non-nucleosomal and non-exosomal origin. To confirm this, the inventors further ran the sample through an anti-DNA antibody affinity column prepared as described in Example 7 (agarose-based matrix coupled to a high affinity mouse monoclonal IgM antibody binding ds+ssDNA). After one purification cycle, the level of cfDNA in the patient's plasma became undetectable when measured by the PicoGreen assay. This observation was also confirmed by the absence of visible DNA material after DNA agarose gel electrophoresis.

[0222] Thus, the use of two consecutive affinity columns/cartridges, in which one column/cartridge captures DNA bound to the nucleosome and exosomal DNA, and the other column/cartridge captures unbound cfDNA, including dsDNA, ssDNA, and oligonucleotides, is very efficient for purification. /apheresis of the patient's blood from all types of circulating cfDNA.

[0223] Another 2 ml plasma sample from the same patient was purified sequentially through a DNA intercalator affinity column (prepared as described in Example 8, i.e. cellulose beads conjugated with Hoechst 33342) and an anti-DNA antibody affinity column ( prepared as described in Example 7, i.e. an agarose matrix linked to high affinity mouse monoclonal anti-DNA IgM). The plasma level of cfDNA after the first run through the combination of DNA-intercalator and anti-DNA antibody affinity columns decreased to 475 ng/ml, and cfDNA of different origin was visualized by DNA agarose gel electrophoresis (Figure 7, lane B). This partially purified patient plasma was further subjected to a second run through the same combination of fresh DNA intercalator and anti-DNA antibody affinity columns. The plasma level of cfDNA after the second run in the patient's plasma became undetectable when measured by the PicoGreen test. This observation was also confirmed by the absence of visible DNA material after DNA agarose gel electrophoresis with DNA fluorescent dye staining.

[0224] Thus, the use of a combination of columns/cartridges containing templates that bind virtually all types of cfDNA (including nucleosome-bound cfDNA, exosome-bound cfDNA and unbound cfDNA [including dsDNA, ssDNA and oligonucleotides]) according to the invention allows you to capture an unusually large the amount of cfDNA.

Example 20 Purification/Apheresis of Portal Vein Plasma to Purify Blood of cfDNA in Rats with Acute Pancreatitis

[0225] Six male Sprague-Dawley rats weighing 250-350 grams were used in the experiment. All surgical procedures were performed on a heated operating table under general anesthesia with intraperitoneal injections of 0.8 mg xylazine and 4 mg ketamine.

[0226] Acute pancreatitis was called as follows. During laparotomy, Vater's papilla was cannulated transduodally using a 24G Abbocath®-T i.v. infusion cannula. Prior to controlled pressure infusion of 0.5 ml of sterilized glycodeoxycholic acid in buffered glycylglycine-NaOH (10 mmol/L, pH 8.0, 37° C.), the common bile duct was clamped and bile and pancreatic fluid were allowed to drain through the cannula. Immediately after the infusion, the hepatoduodenal flow of bile was restored by removing the clamp. The puncture hole in the duodenum was carefully closed using an 8.0 polypropylene serous suture.

[0227] After abdominal closure in rats 1, 2, and 3, chronic venous catheters were inserted into the femoral vein and jugular vein as described in Example 16.

[0228] Rats 4, 5 and 6 had a portal vein catheter implanted in the hepatic portal vein in the caudal region of the liver as described by Strubbe (Strubbe J.H. et al, Hepatic-portal and cardiac infusion of CCK-8 and glucagon induce different effects on feeding Physiol Behav 46: 643-646, 1989).

[0229] The apheresis procedure was performed as described in Example 16 using a PDAM affinity cartridge prepared as described in Example 4 and fitted with polypropylene inlet and outlet fittings. The apheresis procedure was performed daily for 1-3 days with a duration of 12 hours for each apheresis procedure.

[0230] Survival (96 hours after induction of pancreatitis) was adopted as the main parameter of treatment efficacy. For quantification of rat cfDNA and cfDNA of bacterial origin (i.e., bacterial load), total cfDNA was isolated from 200 µl rat plasma samples using a QIAamp DNA mini kit according to the manufacturer's instructions. cfDNA concentration in plasma samples was measured by quantitative polymerase chain reaction (PNR) using ABI PRISM 7700 sequence detector (Applied Biosystems) and TaqMan Universal PCR Master Mix (Applied Biosystems) according to the manufacturer's protocol. To quantify cfDNA of bacterial origin, specific primers were developed for conserved regions of bacterial 16S rDNA: forward primer, 5'-TCCTACGGGAGGC AGCAGT-3', reverse primer 5'-GGACT ACC AGGGTATCTAATCCTGTT-3', and probe (6-FAM)-5' -CGTATTACCGCGGCTGCTGGCAC-3' - (TAMRA) (see: Mangala, A.; Nadkarni, A. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology, 2002, vol. 148 , pp.257-266). The TaqMan Gene Expression Assay rat p-actin Rn00667869_m1 (Applied Biosystems) was used to amplify rat genomic cfDNA.

[0231] Survival and results of each PCR (Ct, i.e. cycle threshold value) for the gene (rat 3-actin and bacterial 16S rDNA in blood plasma collected from the jugular vein) are shown in Table 9.

[0232] The results show that diverting/drawing blood for apheresis into the apheresis device according to the invention from the portal vein resulted in better (compared to diverting blood from the femoral, i.e. non-regional vein) survival and more efficient blood purification from cfDNA (including cfDNA of bacterial origin) in rats with acute pancreatitis.

[0233] Thus, in a clinical setting, when the pathological process leading to the release of cfDNA (tumor growth, septic or aseptic inflammation, release of bacterial DNA) occurs from areas / regions drained mainly by the portal vein (esophagus, stomach, intestines, spleen , pancreas, gallbladder, abdomen) may be useful to drain blood for the portal vein apheresis procedure.

Example 21 Comparison of removal of cfDNA from patient plasma with an H1 histone affinity matrix, a PAMAM dendrimer affinity matrix, and a poly-L-lysine (PLLAM) affinity matrix.

[0234] Poly-L-lysine affinity matrix (PLLAM) was prepared as described in Example 12. PAMAM dendrimer (PDAM) affinity matrix was prepared as described in Example 4. Histone H1 affinity matrix was prepared as indicated in example 1.

[0235] Model cfDNA enriched plasma was generated by mixing healthy volunteer plasma with marker DNA (1 kbp plus DNA Ladder, Invitrogen) to a final cfDNA concentration of 10 μg/mL. The adsorption capacity of poly-L-lysine affinity matrix (PLLAM), PAMAM dendrimer affinity matrix (PDAM), and histone H1 affinity matrix to model plasma enriched with artificial lkbp plus Ladder DNA was tested by adsorption with volume ratio of affinity matrix: plasma 1:5 (100 μl of affinity matrix was mixed with 500 μl of model plasma) for 1 hour at 37°C with slow rotation. Ethanolamine Sepharose FF was used as a control. Plasma samples were analyzed by 1% agarose gel electrophoresis using the E-Gel Invitrogen system before incubation and after affinity matrix precipitation. cfDNA was isolated from patient plasma using a QIAamp DNA Blood Mini Kit, Quagen and quantified using a Qubit 3.0 fluorimeter. The same affinity matrices were incubated with the plasma of a patient diagnosed with odontogenic sepsis with an affinity matrix:plasma ratio of 1:10 (100 μl of the affinity matrix was mixed with 1 ml of the patient's plasma) using the same incubation conditions. After one hour, the affinity matrix was removed by centrifugation. Plasma samples were analyzed by 1% agarose gel electrophoresis using the E-Gel Invitrogen system before incubation and after affinity matrix precipitation. Ethanolamine Sepharose FF was used as a control. cfDNA was isolated from patient plasma using a QIAamp DNA Blood Mini Kit, Quagen and quantified using a Qubit 3.0 fluorimeter. Quantification data for cfDNA are shown in Table 10 below.

[0236] It can be seen that the poly-L-lysine affinity matrix (PLLAM), the PAMAM dendrimer (PDAM) affinity matrix, and the histone H1 affinity matrix have equal ability to remove model cfDNA from cfDNA enriched model plasma, but the H1 histone affinity template is significantly superior to others in removing cfDNA from patient plasma. These results were confirmed by electrophoretic analysis of the samples (see Figures 8 and 9).

[0237] A bulk adsorption test with a model plasma with a poly-L-lysine affinity matrix (PLLAM), a PAMAM dendrimer (PDAM) affinity matrix, and a histone H1 affinity matrix gave almost the same electrophoretic pattern with a small amount of cfDNA .

[0238] A volume adsorption test with patient plasma with poly-E-lysine affinity matrix (PLLAM), PAMAM dendrimer (PDAM) affinity matrix, and histone H1 affinity matrix showed a different electrophoretic pattern with marginal cfDNA content after incubation with an affinity matrix based on histone H1, but a significant content of cfDNA after incubation with an affinity matrix based on poly-E-lysine and an affinity matrix based on PAMAM-dendrimer.

[0239] The present invention should not be limited in scope to the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. In addition, it should be understood that all values are approximate and are provided for description.

[0240] Patents, patent applications, publications, product descriptions, and protocols are cited throughout the specification of this application, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

Claims (30)

1. An apheresis device comprising one or more affinity templates, wherein said one or more affinity templates are capable of capturing extracellular DNA (ecDNA) associated with nucleosomes, cfDNA associated with exosomes, and unbound cfDNA from the blood or plasma of a subject, and wherein said one or more affinity templates comprise a DNA binding polymer, a DNA binding protein, an anti-histone antibody, an anti-nucleosome antibody, a DNA intercalating agent, an anti-DNA antibody, or any combination thereof. 2. The device of claim 1, wherein the unbound cfDNA contains dsDNA, ssDNA, and oligonucleotides. 3. The device according to claim 1 or claim 2, where the device contains two or more affine matrices. 4. The device of claim 3, wherein (i) the first one or more affinity templates are capable of capturing extracellular DNA (ecDNA) associated with nucleosomes and/or cfDNA associated with exosomes, and (ii) the second one or more affinity templates are capable of to capture unbound cfDNA, and said first and second affinity templates are located within the device in any order. 5. The device of claim 4, wherein (i) the first one or more affinity templates comprise a DNA binding protein, an anti-histone antibody, an anti-nucleosomal antibody, a DNA intercalating agent, a DNA binding polymer, an anti-DNA antibody , a lectin, or any combination thereof, and (ii) the second one or more affinity templates contain a DNA binding protein, a DNA intercalating agent, a DNA binding polymer, an anti-DNA antibody, or any combination thereof. 6. The device according to claim. 1, where the device contains one affine matrix. 7. The device of claim 6 wherein said affinity template contains a DNA binding protein. 8. The device of claim 7 wherein the DNA binding protein contains a histone. 9. The device of claim 8 wherein the histone is an H1 histone. 10. The device of claim 9 wherein the histone is H1.3 histone. 11. A method for reducing the level of extracellular DNA (cfDNA) in the blood of a subject, including: (a) performing an apheresis procedure comprising withdrawing blood or plasma from a subject into an apheresis device according to any one of paragraphs. 1-10 to obtain blood or plasma with reduced levels of cfDNA; and (b) returning blood or plasma with reduced levels of cfDNA to the subject, wherein the apheresis procedure reduces the level of nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA in the subject's blood. 12. The method of claim 11, wherein the disease in the subject is characterized by elevated levels of cfDNA in the blood. 13. The method of claim 11, wherein the subject is suffering from a disease selected from the group consisting of a neurodegenerative disease, cancer, chemotherapy-related toxicity, radiation-induced toxicity, organ failure, organ damage, organ infarction, ischemia, acute vascular event , stroke, graft versus host disease (GVHD), graft rejection, sepsis, systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), traumatic injury, aging, diabetes, atherosclerosis, autoimmune disorder, eclampsia, infertility, related complications with pregnancy, bleeding disorders and infections. 14. The method according to any one of paragraphs. 11-13, further comprising monitoring the level of cfDNA in the subject's blood. 15. The method according to any one of paragraphs. 11-14, which includes continuing or repeating the apheresis procedure until the level of cfDNA in the blood of the subject decreases by at least 25%. 16. The method of claim 15, comprising continuing or repeating the apheresis procedure until the level of cfDNA is reduced by at least 50%. 17. The method of claim 16, comprising continuing or repeating the apheresis procedure until the level of cfDNA is reduced by at least 75%. 18. The method according to any one of paragraphs. 11-17, wherein the unbound cfDNA includes dsDNA, ssDNA, and oligonucleotides. 19. A method for treating a disease characterized by elevated levels of circulating DNA in the blood in a subject in need, comprising: (a) performing an apheresis procedure comprising withdrawing blood or plasma from a subject into an apheresis device according to any one of paragraphs. 1-10 to obtain blood or plasma with reduced levels of cfDNA; and (b) returning blood or plasma with reduced levels of cfDNA to the subject, wherein the apheresis procedure reduces the level of nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA in the subject's blood. 20. The method of claim 19, further comprising monitoring the level of cfDNA in the subject's blood. 21. The method according to any one of paragraphs. 19-20, which includes continuing or repeating the apheresis procedure until the level of cfDNA in the blood of the subject decreases by at least 25%. 22. The method of claim 21, comprising continuing or repeating the apheresis procedure until the level of cfDNA is reduced by at least 50%. 23. The method of claim 22, comprising continuing or repeating the apheresis procedure until the level of cfDNA is reduced by at least 75%. 24. The method according to any one of paragraphs. 19-23, wherein the unbound cfDNA includes dsDNA, ssDNA, and oligonucleotides.

Images