阅读说明：本技术 用于针对循环无细胞dna纯化血液的方法和装置 (Method and apparatus for purifying blood against circulating cell-free DNA ) 是由 K·萨科夫 于 2018-09-17 设计创作，主要内容包括：本发明提供了单采装置以及将它们用于去除患者血液中基本上所有类型的无细胞DNA(cfDNA),包括核小体结合的cfDNA、外来体结合的cfDNA和未结合的cfDNA(包括双链DNA(dsDNA)、单链DNA(ssDNA)和寡核苷酸)),以限制循环cfDNA的负面作用并治疗各种疾病。(The present invention provides apheresis devices and their use to remove substantially all types of cell-free dna (cfDNA), including nucleosome-bound cfDNA, exosome-bound cfDNA and unbound cfDNA (including double-stranded dna (dsdna), single-stranded dna (ssdna) and oligonucleotides) from a patient's blood to limit the negative effects of circulating cfDNA and treat various diseases.)

1. An apparatus configured to perform apheresis, comprising one or more affinity matrices, wherein the one or more affinity matrices are capable of capturing nucleosome bound cell-free dna (cfDNA), exosome bound cfDNA and unbound cfDNA from blood or plasma of a subject.

2. The device of claim 1, wherein the unbound cfDNA comprises dsDNA, ssDNA, and oligonucleotides.

3. The device of claim 1 or claim 2, wherein the device comprises two or more affinity matrices.

4. The device of claim 3, wherein (i) the first one or more affinity matrices are capable of capturing nucleosome bound cell-free DNA (cfDNA) and/or exosome bound cfDNA, and (ii) the second one or more affinity matrices are capable of capturing unbound cfDNA, and wherein the first and second affinity matrices are arranged in any order within the device.

5. The device of claim 4, wherein (i) the first one or more affinity matrices comprise DNA binding proteins, anti-histone antibodies, anti-nucleosome antibodies, DNA intercalators, DNA binding polymers, anti-DNA antibodies, lectins, and any combination thereof, and (ii) the second one or more affinity matrices comprise DNA binding proteins, DNA intercalators, DNA binding polymers, anti-DNA antibodies, and any combination thereof.

6. The device of claims 4 and 5, wherein the DNA binding protein is a histone.

7. The device of claim 6, wherein the histone is H1 histone.

8. The device of any one of claims 5-7, wherein the DNA binding polymer is a polyamidoamine dendrimer.

9. The device of any one of claims 5-7, wherein the DNA binding polymer is a cationic polymer.

10. The device of claim 9, wherein the cationic polymer is poly-L-lysine or polyethyleneimine.

11. The device of claim 10, wherein the poly-L-lysine is hyperbranched poly-L-lysine.

12. The device of claim 10, wherein the polyethyleneimine is hyperbranched polyethyleneimine.

13. The device of any one of claims 5-12, wherein the DNA intercalator is Hoechst 33342.

14. The device of any one of claims 5-13, wherein the anti-histone antibody is an anti-histone H2A antibody.

15. The device of any one of claims 5-14, wherein the lectin is galanthus agglutinin (GNA), narcissus agglutinin (NPA), concanavalin a, a phytohemagglutinin, or a cyanobacterial antiviral protein.

16. The device of claim 15, wherein the lectin is galanthus lectin (GNA).

17. The device of any one of claims 3-16, wherein the two or more affinity matrices are arranged sequentially as two or more affinity columns.

18. The device of any one of claims 3-17, wherein the first affinity matrix in the sequence comprises a DNA-binding polymer or a DNA intercalator.

19. The device of claim 18, wherein the DNA binding polymer is a cationic polymer.

20. The device of claim 19, wherein the cationic polymer is poly-L-lysine or polyethyleneimine.

21. The device of claim 20, wherein the poly-L-lysine is hyperbranched poly-L-lysine.

22. The device of claim 20, wherein the polyethyleneimine is hyperbranched polyethyleneimine.

23. The device of claim 19, wherein the cationic polymer is a polyamidoamine dendrimer.

24. The device of claim 18, wherein the DNA intercalator is Hoechst 33342.

25. The apparatus of claim 17, comprising one of the following combinations, in any order:

(a) (ii) a DNA intercalator Hoechst33342 affinity column and (ii) an anti-DNA antibody affinity column; or

(b) (ii) an anti-nucleosome antibody affinity matrix (ANAM) column and (ii) an anti-DNA antibody affinity column; or

(c) (ii) an anti-nucleosome antibody affinity matrix (ANAM) column and (ii) a Polyamidoamine Dendrimer Affinity Matrix (PDAM) column; or

(d) (i) an anti-nucleosome antibody affinity matrix (ANAM) column and (ii) a hyperbranched poly-L-lysine affinity matrix (P LL AM) column, or

(e) (ii) an anti-histone H2A antibody affinity column, (ii) a lectin affinity column, and (iii) a histone H1 affinity column or a Polyamidoamine Dendrimer Affinity Matrix (PDAM) column or a hyperbranched poly-L-lysine matrix (P LL AM) column or a DNA intercalator Hoechst33342 affinity column.

26. The device of claim 1, wherein the device comprises a single affinity matrix.

27. The device of claim 26, wherein the affinity matrix comprises histone.

28. The device of claim 27, wherein the histone is H1 histone.

29. The device of claim 28, wherein the histone is H1.3 histone.

30. The device of claim 26, wherein the affinity matrix comprises a DNA-binding polymer.

31. The device of claim 30, wherein the DNA binding polymer is a polyamidoamine dendrimer.

32. The device of claim 30, wherein the DNA-binding polymer is hyperbranched poly-L-lysine.

33. The device of claim 26, wherein the affinity matrix comprises a DNA intercalator.

34. The device of claim 33, wherein the DNA intercalator is Hoechst 33342.

35. The device of claim 26, wherein the affinity matrix comprises anti-DNA antibodies.

36. The device of any one of claims 1-35, wherein the device captures at least 30mgcfDNA per single apheresis.

37. The device of any one of claims 1-35, wherein the device reduces blood cfDNA levels by at least 25% per single apheresis procedure.

38. The device of claim 37, wherein the device reduces blood cfDNA levels by at least 50% per apheresis procedure.

39. The device of claim 38, wherein the device reduces blood cfDNA levels by at least 75% per single harvest.

40. A method of reducing the level of cell-free dna (cfdna) in blood of a subject, the method comprising:

(a) performing an apheresis procedure comprising transferring the subject's blood or plasma to an apheresis device of any one of claims 1-39 to produce blood or plasma having reduced cfDNA levels; and

(b) returning blood or plasma with reduced cfDNA levels 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.

41. The method of claim 40, wherein the subject has a disease characterized by elevated cfDNA levels in blood.

42. The method of claim 40, wherein the subject has a disease selected from the group consisting of: neurodegenerative diseases, cancer, chemotherapy-related toxicity, radiation-induced toxicity, organ failure, organ injury, 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 diseases, eclampsia, infertility, pregnancy-related complications, blood coagulation disorders, and infections.

43. A method of treating a disease in a subject in need thereof, the method comprising:

(a) performing an apheresis procedure comprising transferring blood or plasma from the subject to an apheresis device of any one of claims 1-39 to produce blood or plasma having reduced cfDNA levels; and (c) and (d).

(b) Returning said blood or plasma with reduced levels of fDNA to said subject,

wherein the apheresis procedure reduces the level of nucleosome-bound cfDNA, exosome-bound cfDNA and unbound cfDNA in the subject's blood.

44. The method of claim 43, wherein the disease is characterized by elevated cfDNA levels in the blood.

45. The method of claim 43, wherein the disease is selected from the group consisting of: neurodegenerative diseases, cancer, chemotherapy-related toxicity, radiation-induced toxicity, organ failure, organ injury, 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), trauma, aging, diabetes, atherosclerosis, autoimmune diseases, eclampsia, infertility, pregnancy-related complications, blood coagulation disorders, and infections.

46. The method of any one of claims 40-45, further comprising monitoring the cfDNA level in the subject's blood.

47. The method of any one of claims 40-46, comprising continuing or repeating the apheresis procedure until the subject's blood cfDNA level is reduced by at least 25%.

48. The method of claim 47, comprising continuing or repeating the apheresis procedure until the cfDNA level is reduced by at least 50%.

49. The method of claim 48, comprising continuing or repeating the apheresis procedure until the cfDNA level is reduced by at least 75%.

50. The method of any one of claims 40-46, comprising continuing or repeating the apheresis procedure until at least 30mg of cfDNA is removed from the subject's blood.

51. The method of any one of claims 40-50, wherein the apheresis procedure is repeated two or more times.

52. The method of any one of claims 40-51, wherein the blood for the apheresis procedure is derived from the portal vein.

53. The method of any one of claims 40-52, wherein the unbound cfDNA comprises dsDNA, ssDNA, and oligonucleotides.

54. The method of any one of claims 40-53, wherein the subject is a human.

Technical Field

The present invention provides apheresis devices and their use for removing substantially all types of cell free DNA (cfDNA), including nucleosome bound cfDNA, exosome bound cfDNA and unbound cfDNA (including double stranded DNA (dsdna), single stranded DNA (ssdna) and oligonucleotides) in a patient's blood to limit the negative effects of circulating cfDNA and to treat various diseases.

Background

Circulating extracellular DNA (edna), also known as cell free DNA (cfDNA), is present in small amounts in the blood of healthy individuals.

Elevated levels of circulating cfDNA are now widely accepted as markers 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 diseases, 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 states in the elderly (proinflammatory states in induced virals), diabetes, atherosclerosis, neurodegenerative diseases, autoimmune diseases, eclampsia, infertility, coagulation disorders, pregnancy related complications and infections. Different subtypes of circulating cell-free DNA may play an important role in the progression of certain diseases and pathological states.

Systemic administration using deoxyribonuclease (dnase) enzyme that specifically hydrolyzes circulating cfDNA to treat infertility has been suggested (U.S. patent No. 8916151); cardiovascular disorders (U.S. patent No. 9,642,822); cancer; sepsis, graft versus host disease (GBHD); organ failure; diabetes mellitus; atherosclerosis; delayed type hypersensitivity (U.S. patent nos. 9,248,166, 8,535,663, 7,612,032, 8,388,951, 8,431,123).

However, in contrast to earlier animal models, data in the actual clinical setting show that systemic application of deoxyribonuclease (dnase) has limited effect on reducing the amount of circulating cfDNA.

Hazout, A. (PCT/IB 2013/056321) have described 10 women with high circulating cfDNA levels (>80 ng/. mu.l) who received 0.1mg/kg DNase I treatment by the intramuscular route twice daily for 7 consecutive days and observed only an average decrease in circulating cfDNA levels of 26%. their observations are consistent with Davis et al, who, despite achieving catalytically effective DNase concentrations in plasma of 40-100ng/ml, failed to demonstrate a decrease in circulating levels of α -DS DNA in lupus nephritis patients receiving a 25g/kg dose of human Recombinant DNase over a 19 day period (as a total of 1 intravenous and 10 subcutaneous injections) (Davis J.C. et al, rebinant human DNase with lung neuritis L upkis (1999), Vol 8 (1), pp 68-76

The most abundant circulating cfDNA types are represented by nucleosome-bound DNA nucleosomes are subunits of chromatin consisting of a central core protein (which is formed by an octamer representing the core histones) and double stranded DNA of about 147 base pairs (Oudet P, Gross-Bellard M, Chamton P.Electron microscopy and biochemical development of which is a nucleic acid construct a reproducing unit. cell.1975; 4:281-³The form of chromatin fragments of one base pair DNA circulates. This particular type of circulating cfDNA originates from cells that undergo necrosis or apoptosis. Another source of circulating cfDNA is neutrophils NETosis. Neutrophil Extracellular Traps (NET) are extracellular strands of de-densified DNA that are expelled from activated neutrophils, with over 15x 10³A length of DNA of base pairs, organized into a 3D network structure of 10-30 nm. cfDNA from NETosis may be particle-free or particle-bound. NET also contains highly cytotoxic enzymes and cytotoxic proteins derived from the inner space of neutrophils. (S0rensen, O.E. and Borregaard, N., neutrophile extracellular tracks-the dark side of neutrophiles.J. Clin. invest.2016 May 2; 126(5): 1612-20). It has recently been shown that not only neutrophils but also macrophages may produce NET-like structures (Nat Med.,2018,24(2): 232-.

Another important type of circulating particle-bound cfDNA is exosome-bound DNA. Exosomes are small membrane vesicles (30-100nm) of secreted cell origin secreted by most cell types, which may be internal to the exosome orThe outer space contains single-stranded DNA (ssDNA), mitochondrial DNA (mtDNA) and 2.5-10x 10³Double stranded of base pairs (dsDNA). (Thakur, B.K., et al, Double-stranded DNA in exosomes: a novel biomarker inhibitor detection, Cell Research (2014)24: 766-.

A large portion of the circulating cfDNA free of particles is represented by linear and circular dsDNA and ssDNA secreted by cancer cells, activated immune cells, and certain other cell types. cfDNA of this type is typically 250-1000 base pairs or longer in length and may be enriched in unique genomic sequences. (Kumar, P. et al, Normal and cancer release enzyme extrachromosomal circular DNA (eccDNA) interaction, mol. cancer. Res., June 20,2017 DOI:10.1158/1541-7786. MCR-17-0095). Another important component of circulating cfDNA without particles is mitochondrial dna (mtdna) of varying length.

Another recently discovered type of particle-free circulating cfDNA is represented by ultrashort double-stranded dna (dsdna) oligonucleotides and single-stranded dna (ssdna) oligonucleotides having a sub-nucleosomal (sub-nucleosomal) length (i.e., typically less than about 147 base pairs). This particular cfDNA has been shown to be abundant in mitochondrial DNA (mtdna), microbial origin, DNA-deprived, and mutated human genomic sequences. (Burnham P., Single-stranded DNA library preparation of noncoverche origin and diversity of ultrashort cell-free DNA in plasma, scientific reports 6, particle number:27859(2016), doi:10.1038/srep 27859). Importantly, this type of circulating cfDNA also contains low molecular weight DNA fragments that are similar to those that occur after dnase I degrades particle-bound DNA in the patient's blood.

Several attempts have been made to purify patient blood from certain components of the circulating cfDNA pool using extracorporeal clearance techniques. See, for example, U.S. patent nos. 9,364,601; U.S. patent application publication No. 2007/0092509; kusaoi et al, the ther. Dial,2016,20: 348-.

New in vitro methods are needed to treat diseases associated with high circulating levels of blood cfDNA and new more effective devices are needed to implement such methods.

Summary of The Invention

As described in the background section above, there is a need for new in vitro methods to treat diseases associated with high levels of circulating blood cfDNA, and new more efficient devices to implement such methods. The present invention meets this and other needs by providing an extraction device and associated processes.

In one aspect, the invention provides an apparatus configured to perform apheresis comprising one or more affinity matrices, wherein the one or more affinity matrices are capable of capturing nucleosome-bound cell-free dna (cfDNA), exosome-bound cfDNA and unbound cfDNA from blood or plasma of a subject.

In some embodiments, the unbound cfDNA comprises dsDNA, ssDNA, and oligonucleotides.

In some embodiments, the device of the invention comprises two or more affinity matrices. In some embodiments, (i) a first affinity matrix or matrices are capable of capturing nucleosome bound cell-free dna (cfDNA) and/or exosome bound cfDNA, and (ii) a second affinity matrix or matrices are capable of capturing unbound cfDNA, and wherein the first and second affinity matrices are arranged in any order within the device. In some embodiments, (i) the first one or more affinity matrices comprise a DNA binding protein (e.g., a histone [ e.g., H1 histone)]) Anti-histone antibodies (e.g., anti-histone H2A antibodies), anti-nucleosome antibodies (e.g., AN-1, AN-44), DNA intercalators (e.g., Hoechst dyes such as Hoechst33342), DNA binding polymers (e.g., cationic/basic polymers [ e.g., polyethyleneimine, poly-L-lysine, poly-L-arginine, hexadimethrine bromide, amino-terminated (-NH)₂) Polyamidoamine (PAMAM) dendrimers, polypropyleneimine (PPI) dendrimers of]Nonionic/neutral polymers [ e.g., polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP), poly (4-vinylpyridine-N-oxide)]Anionic/acidic polymers, linear polymers [ e.g. polyethyleneimine, poly-L-lysine, poly-L-arginine]Branched polymers [ e.g. hyperbranched poly-L-lysine, hyperbranched polyethyleneimine]Dendritic polymer[ e.g., Polyamidoamine (PAMAM) dendrimers, Polypropyleneimine (PPI) dendrimers]) anti-DNA antibodies (e.g., mouse monoclonal IgM anti-ds + ss DNA antibodies ([49/4a1), ab35576, Abeam), lectins (e.g., galanthamine (galanthas nivalis L actin, GNA), Narcissus (Narcissus Pseudonarcissus L actin, NPA), concanavalin a, phytohemagglutinin, or cyanobacterial antiviral protein (cyanovirin)), and (ii) a second one or more affinity matrices comprising a DNA binding protein (e.g., a histone [ e.g., H1 histone ]]DNA intercalators (e.g., Hoechst dyes such as Hoechst33342), DNA binding polymers (e.g., cationic/basic polymers [ e.g., polyethyleneimine, poly-L-lysine, poly-L-arginine, hexadimethrine bromide, Polyamidoamine (PAMAM) amino terminated (-NH)₂) The dendrimer of (a), the polypropyleneimine (PPI) dendrimer), the nonionic/neutral polymer [ e.g., polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP), poly (4-vinylpyridine-N-oxide)]Anionic/acidic polymers, linear polymers [ e.g. polyethyleneimine, poly-L-lysine, poly-L-arginine]Branched polymers [ e.g. hyperbranched poly-L-lysine, hyperbranched polyethyleneimine]Dendrimers [ e.g., Polyamidoamine (PAMAM) dendrimers, polypropyleneimine (PPI) dendrimers]) anti-DNA antibodies (e.g., mouse monoclonal IgM anti-ds + ss DNA antibody ([49/4a1), ab35576, Abeam), and any combination thereof. In some embodiments, the two or more affinity matrices are arranged sequentially as two or more affinity columns. In some embodiments, the first affinity matrix in the sequence comprises a DNA binding polymer (e.g., amino-terminated (-NH)₂) Polyamidoamine (PAMAM) dendrimers, polypropyleneimine (PPI) dendrimers, hyperbranched poly-L-lysine or hyperbranched polyethyleneimine) or DNA intercalators (e.g., Hoechst 33342).

Non-limiting examples of useful column combinations (in any order) are (a) (i) a DNA intercalator Hoechst33342 affinity column and (ii) an anti-DNA antibody affinity column, or (b) (i) an anti-nucleosome antibody affinity matrix (ANAM) column and (ii) an anti-DNA antibody affinity column, or (c) (i) an anti-nucleosome antibody affinity matrix (ANAM) column and (ii) a Polyamidoamine Dendrimer Affinity Matrix (PDAM) column, or (d) (i) an anti-nucleosome antibody affinity matrix (ANAM) column and (ii) a hyperbranched poly-L-lysine affinity matrix (P LL AM) column, or (e) (i) an anti-histone H2A antibody affinity column, (ii) a lectin affinity column, and (iii) a histone H1 affinity column or a Polyamidoamine Dendrimer Affinity Matrix (PDAM) column or a hyperbranched poly-L-lysine affinity matrix (P LL) column or a DNA intercalator Hoechst affinity column.

In some embodiments, the device of the invention comprises a single affinity matrix. Non-limiting examples of useful matrices that can be used as a single affinity matrix include affinity matrices comprising histones (e.g., histone H1, such as histone H1.3), affinity matrices comprising DNA binding polymers (e.g., cationic polymers, such as amino-terminated (-NH-) and affinity matrices comprising a single affinity matrix₂) Polyamidoamine (PAMAM) dendrimers or hyperbranched poly-L-lysine), affinity matrix comprising a DNA intercalator (e.g., Hoechst33342), affinity matrix comprising an anti-DNA antibody (e.g., mouse monoclonal IgM anti-ds + ss DNA antibody ([49/4A1)]Ab35576, Abeam)). In certain embodiments, the affinity matrix is not a Polyamidoamine (PAMAM) dendrimer.

In some embodiments, the devices of the invention capture at least 30mg of cfDNA per single apheresis.

In some embodiments, the devices of the invention reduce blood levels of cfDNA by at least 25% per single apheresis procedure. In some embodiments, the devices of the invention reduce blood levels of cfDNA by at least 50% per single apheresis procedure. In some embodiments, the devices of the invention reduce blood levels of cfDNA by at least 75% per single apheresis procedure.

In another aspect, the invention provides a method of reducing the level of cell-free DNA in a subject's blood, the method comprising (a) performing an apheresis procedure comprising transferring blood or plasma from the subject into an apheresis device of the invention to produce blood or plasma having a reduced cfDNA level; and (b) returning blood or plasma with a reduced level 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 cfDNA levels in blood. In some embodiments, the subject has a disease selected from the group consisting of: neurodegenerative diseases, cancer, chemotherapy-related toxicity, radiation-induced toxicity (e.g., acute radiation syndrome), organ failure, organ injury, 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 diseases, eclampsia, infertility, pregnancy-related complications, blood coagulation disorders, and infections.

In a further aspect, the invention provides a method of treating a disease in a subject in need thereof, the method comprising (a) performing an apheresis procedure comprising transferring blood or plasma from the subject into an apheresis device of the invention to produce blood or plasma having a reduced cfDNA level; and (b) returning blood or plasma with a reduced level 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 cfDNA levels in blood. Non-limiting examples of diseases that can be treated by the methods of the invention include, for example, neurodegenerative diseases, cancer, chemotherapy-related toxicity, radiation-induced toxicity (e.g., acute radiation syndrome), organ failure, organ injury, 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 diseases, eclampsia, infertility, pregnancy-related complications, blood coagulation disorders, and infections.

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

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

In some embodiments of any of the above methods of the invention, the method comprises continuing or repeating the apheresis procedure until at least 30mg of cfDNA is removed from the blood of the subject.

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

In some embodiments of any of the above methods of the invention, the blood used for the apheresis procedure is derived from the portal vein.

In some embodiments of any of the above methods of the invention, the unbound cfDNA comprises dsDNA, ssDNA, and oligonucleotides.

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

These and other aspects of the invention will be apparent to those of ordinary skill in the art in view of the following description, claims and drawings.

Brief Description of Drawings

Figure 1 shows an electropherogram of circulating cfDNA from plasma of a metastatic cancer patient.

FIGS. 2A and 2B show tumors excised from mice treated with DNA of example 3, in which blood was purified with an affinity matrix with anti-histone antibodies and an affinity matrix with lectin from snowdrop (snowdrop). fig. 2A shows tumors excised from control mice.fig. 2B shows tumors excised from mice treated with DNA from NSC L C T3N2M + patients purified from nucleosomes and exosome-bound circulating cfDNA.

Figure 3 shows an electropherogram of circulating cfDNA from plasma of metastatic cancer patients and stroke patients.

Figure 4 shows an electropherogram of circulating cfDNA from plasma of patients with Systemic Inflammatory Response Syndrome (SIRS) and multiple dysfunction syndrome (MODS).

Fig. 5 shows an electropherogram of circulating cfDNA used in cell culture experiments.

Figure 6 shows electropherograms of circulating cfDNA, dnase I western blots, and dnase I activity and quantification of circulating cfDNA.

Figure 7 shows an electropherogram of circulating cfDNA from sepsis patient plasma.

Figure 8 shows the results of 1% agarose gel electrophoresis of cfDNA enriched model plasma before and after the volume adsorption test, lane 1 is cfDNA enriched model plasma before incubation, lane 2 is cfDNA enriched model plasma after incubation with ethanolamine sepharose FF control, lane 3 is cfDNA enriched model plasma after incubation with PDAM, lane 4 is cfDNA enriched model plasma after incubation with P LL AM, lane 5 is cfDNA enriched model plasma after incubation with H1.3 affinity matrix.

FIG. 9 shows the results of 1% agarose gel electrophoresis of plasma from patients diagnosed with dental related sepsis before and after the volume adsorption test, lane 1 is plasma from dental related sepsis patients incubated with ethanolamine agarose gel FF control, lane 2 is distilled water blank line, lane 3 is plasma from dental sepsis patients incubated with H1.3 affinity matrix, lane 4 is distilled water blank line, lane 5 is plasma from dental sepsis patients incubated with PDAM, lane 6 is plasma from dental sepsis patients incubated with P LL AM.

Detailed Description

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

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

The term "about" or "approximately" is included within a statistically significant range of values. Such a range may be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, more preferably within 10%, even more preferably within 5%. The allowable variations encompassed by the terms "about" or "approximately" depend on the particular system under study and can be readily understood by one of ordinary skill in the art.

As used herein, the term "device" refers to any component known in the art that enables purification of a liquid solution, such as, but not limited to, any hollow vessel, column matrix, filter, membrane, semipermeable material, bead (e.g., microbead or nanobead), or tube. The terms "column" and "cylinder/barrel" are used interchangeably in the context of the apheresis device herein.

As used herein, the term "affinity matrix" refers to (i) a solid support to which ligands (e.g., cfDNA-binding molecules) are immobilized, or (ii) a solid support (e.g., a water-insoluble DNA-binding polymer) formed from the ligands themselves.

The term "DNA binding protein" refers to a protein that binds to single-stranded DNA (ssdna) or double-stranded DNA (dsdna). DNA binding proteins can bind DNA in a sequence-specific manner (e.g., transcription factors and nucleases) or a non-sequence-specific manner (e.g., polymerases and histones). The linker histone H1 family member is a key component of chromatin and binds to the nucleosome core particle around the site of DNA entry and exit.

As used herein, the terms "circulating DNA", "cell-free DNA (cfdna)", "circulating cell-free DNA (cfdna)", "extracellular DNA (edna)", and "circulating extracellular DNA (edna)", which are used interchangeably, refer to DNA present in blood or plasma that is located outside circulating cells of hematopoietic and non-hematopoietic origin.

Nucleosome-bound cfDNA is DNA that binds to nucleosomes. Nucleosomes are subunits of chromatin. Nucleosome-bound cfDNA may circulate in the blood as mononucleosomes or higher order structures, such as oligonucleosomes, or even contain more than 50-100x 10³Chromatin fragments of one base pair DNA. Circulating nucleosome bound cfDNA can be derived from cells undergoing necrosis or apoptosis and the neutrophil NETosis.

Exosome-bound cfDNA is cfDNA that binds to or is present in an exosome. Exosomes are small membrane vesicles (about 30-100nm) of extracellular origin secreted by most cell types, which may contain single-stranded dna (ssdna), mitochondrial dna (mtdna), and double-stranded dna (dsdna) in the internal or external space of the exosome.

The term "unbound cfDNA" or "particle-free cfDNA" (particulate free cfDNA) "refers to cfDNA that does not bind to exosomes or nucleosomes, including double-stranded DNA (dsdna), single-stranded DNA (ssdna), linear or circular oligonucleotides, including ultra-short DNA molecules of sub-nuclear size (typically less than 147 base pairs).

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 refers to a human patient, including both sexes in the adult and pediatric population.

In the description of the present invention, to the extent that it is associated with any disease condition described herein, the terms "treat", "treating" and the like are intended to alleviate or alleviate at least one symptom associated with such condition, or slow or reverse the progression of such condition. Within the meaning of the present invention, the term "treatment" also means preventing, delaying onset (i.e. the period before clinical manifestation of the disease) and/or reducing the risk of developing or worsening the disease. The terms "treat", "treating", and the like with respect to a state, disorder, or condition may also include (1) preventing or delaying the occurrence of at least one clinical or subclinical symptom of a state, disorder, or condition developing in a subject who may be suffering from or susceptible to the state, disorder, or condition, but has not yet experienced or exhibited clinical or subclinical symptoms of the state, disorder, or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or its recurrence (in the case of maintenance therapy) or at least one clinical or subclinical symptom thereof; or (3) ameliorating the disease, i.e., causing regression of at least one of the states, disorders or conditions or clinical or subclinical symptoms thereof.

Unless otherwise indicated, the practice of the present invention employs conventional Techniques of statistical analysis, Molecular biology (including recombinant Techniques), microbiology, Cell biology, joint chemistry and biochemistry within the skill of the art. such tools and Techniques are described in detail in, for example, Sambrook et al (2001) Molecular Cloning: A L laboratory Manual, 3 rd edition Cold Spring Harbor L organism Press: Cold Spring Harbor, New York, Ausubel et al, editor (2005) Current Protocols in Molecular biology, John Wiley, and Sons, Inc., Hobond, NJ, Bonifacino et al, editor (2005) Current Protocols in Cell biology, John Wileyans, Inc., Home, J, Inc., Press et al, Inc. and moisture, Inc., moisture, Inc. and moisture, Inc. in the U.S. Pat. No. 5, Inc. 7, Inc. and moisture, Inc. of the application, Inc. and moisture, Inc. of the invention, Inc. the application, Inc. of the invention is incorporated, Inc. in the aforementioned, Inc. and applications, Inc. of the present application, Inc. and application, Inc. for the invention.

Apparatus and method of the invention

As discussed in the background section, there is a significant need in the art to develop new methods and devices to reduce the levels of essentially all types of circulating cfDNA in blood. The present disclosure meets this and other needs by providing an apheresis device and methods that reduce the levels of essentially all types of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides).

The use of in vitro removal techniques can provide an effective solution to remove cfDNA from the circulation and correspondingly reduce the level and negative impact of circulating cfDNA. Therapeutic apheresis is an extracorporeal treatment that removes blood components from a patient; for the treatment of conditions in which disease development is caused by pathogenic substances or components in the blood, see, for example, Ward M.D., modified Apheresis therapeutics: A Review Journal of Clinical Apheresis 26: 230-.

Surprisingly, as described herein, the in vitro removal of substantially all types of circulating cfDNA has a positive impact on the treatment of diseases characterized by elevated levels of circulating cfDNA in blood.

The present disclosure provides methods of 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 the blood of a subject to reduce the negative effects of circulating cfDNA.

Without wishing to be bound by theory, in certain diseases where the level of circulating cfDNA is elevated, different types of circulating cfDNA may act synergistically by triggering different molecular pathways, each leading to disease progression and patient death; the different types of circulating cfDNA acting together may produce synergistic toxicity, i.e., the toxic (negative) effects of two or more types of circulating cfDNA are greater than the sum of the negative effects of each type of cfDNA acting alone.

The present inventors have found that removal of substantially 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 eliminate the pathogenic effects mediated by the circulating cfDNA.

The inventors have also surprisingly observed that removal of substantially all types of circulating cfDNA may result in reactivation of endogenous dnases.

It is further described herein that several affinity matrices, or combinations thereof, are capable of effectively capturing substantially all types of cfDNA from the blood of a patient in need thereof, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides)₂) Polyamidoamine (PAMAM) dendrimers, polypropyleneimine (PPI) dendrimers of]Nonionic/neutral polymers [ e.g., polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP), poly (4-vinylpyridine-N-oxide)]Anionic/acidic polymers, linear polymers [ e.g. polyethyleneimine, poly-L-lysine, poly-L-arginine]Branched polymers [ e.g. hyperbranched poly-L-lysine, hyperbranched polyethyleneimine]Dendrimers [ e.g., Polyamidoamine (PAMAM) dendrimers, polypropyleneimine (PPI) dendrimers](ii) a For further examples, see, e.g., U.S. Pat. No. 7,713,701 and Morozov et al, General research, 2016,12:6), (v) a substrate comprising an anti-DNA antibody, (vi) a substrate comprising a lectin (e.g., Boehringer Mannheim, BiotechSuch as galanthus agglutinin (GNA), narcissus agglutinin (NPA), concanavalin a, phytohemagglutinin or cyanobacterial antiviral protein), and any combinations thereof. In some embodiments, two or more affinity matrices are arranged sequentially as two or more affinity columns. In some embodiments, the first affinity matrix in the sequence comprises a DNA binding polymer (e.g., amino-terminated (-NH)₂) Polyamidoamine (PAMAM) dendrimers, polypropyleneimine (PPI) dendrimers, hyperbranched poly-L-lysine or hyperbranched polyethyleneimine) or DNA intercalators (e.g., Hoechst 33342).

Affinity matrices and apheresis devices comprising such matrices are described herein. The apheresis device of the present invention may be configured according to the knowledge of one of ordinary skill in the art, for example, as described in U.S. patent application No. 2017/0035955 (EliazIssac, published 2017, 2/9). In one possible embodiment of the apheresis device, the affinity matrix is placed in various affinity columns or cartridges. The apheresis device may comprise a filter cartridge and one or more affinity columns having an inlet and an outlet, wherein 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 columns in sequence. The inlet and outlet may be positioned relative to the affinity matrix such that blood entering the inlet must contact the affinity matrix before exiting through the outlet. Preferably, the geometry of the device is designed to maximize contact of the blood (or plasma) with the affinity matrix 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 ligands immobilized on beads, with the inlet at one end and the outlet at the opposite end. Other devices, such as micro-tube arrays, may also be constructed. All such variations of the vessel geometry and volume and the affinity matrix contained therein can be designed according to known principles. In preparing the affinity matrix column, the affinity matrix may be loaded to at least 50%, 60%, 70%, 75%, 80%, 85% or 90% of the column volume. The column can be equilibrated using a suitable buffer (e.g., PBS, particularly cold PBS).

In one aspect, a histone affinity matrix is provided comprising cellulose beads and recombinant human histone H1.3, wherein the recombinant human histone H1.3 is immobilized on the cellulose beads, and wherein the size of the beads is between 50 and 350 microns. In some embodiments, the beads are between 100 and 250 microns in size.

In some embodiments, the histone affinity matrix is prepared by a method comprising the steps of a) oxidizing cellulose beads having a size between 100 and 250 microns to produce activated cellulose beads;

b) washing the activated cellulose beads;

c) preparing a concentrated solution of recombinant human histone H1.3;

d) incubating 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 beads.

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

In some embodiments, in step a), the cellulose beads are in 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 the solution of recombinant human histone H1.3 and concentrating the dialyzed solution in 0.1M sodium bicarbonate at pH 7-9. In some embodiments, the dialyzed solution is subjected to 0.1M NaHCO at pH8₃Concentrating. In some embodiments, in step d), the incubation is performed at 15-30 ℃ for 3-5 hours. In some embodiments, in step d), the incubation is performed at room temperature for 4 hours. In some embodiments, in step e), the blocking step comprises adding 1M ethanolamine to the activated cellulose beads and reacting at 15-30 ℃ for 30 minutes to 2 hours. In some embodiments, in step f), the activated cellulose beads are washed with TBS buffer.

Also provided is a column comprising the histone affinity matrix of any of the aspects and embodiments described above.

In another aspect, a lectin affinity matrix produced according to a method comprising the steps of

A) Reacting the lectin with the activated agarose beads to produce lectin-coupled agarose; and

b) the lectin-coupled agarose was washed with buffer.

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

Also provided is a column comprising the lectin affinity matrix of any of the aspects and embodiments described above.

In yet another aspect, a Polyamidoamine Dendrimer Affinity Matrix (PDAM) is provided, which is prepared by a method comprising the steps of

a) Washing the cellulose beads 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 Polyamidoamine (PAMAM) dendrimers to produce PDAM beads, and removing the PAMAM dendrimers that have not reacted with the activated cellulose beads; and

d) blocking unconverted epoxy groups on the PDAM beads.

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

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 having an ethylenediamine core. In some embodiments, in step c), the suspension is carried out at 20-30 ℃ for 3-6 hours. In some embodiments, in step c), the suspension is performed at 24 ℃ for 5 hours.

Also provided are columns comprising the aforementioned PAMAM Dendrimer Affinity Matrix (PDAM). In some embodiments, the column is a PTFE column and the polyamidoamine dendrimer affinity matrix is sterilized.

In another aspect, an anti-DNA antibody affinity matrix is provided, which is prepared by a method comprising the steps of:

A) preparing activated agarose beads by cross-linking N-hydroxysuccinimide with the agarose beads; b) with a solution containing NaHCO₃Washing the activated agarose beads with a coupling buffer of NaCl; c) adding antibodies against double-stranded and single-stranded DNA to the coupling buffer; d) incubating a coupling buffer comprising an antibody with activated agarose beads to produce an anti-DNA antibody affinity matrix; and

e) the anti-DNA antibody affinity matrix is washed with coupling buffer and acetate buffer.

In some embodiments, the agarose beads have an average size of 90 microns. In some embodiments, the coupling buffer comprises 0.2M NaHCO₃And 0.5M NaCl, and pH 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 0.1M acetate buffer at ph 4.0.

Also provided is a column comprising the above anti-DNA antibody affinity matrix.

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

In another aspect, an anti-nucleosome antibody affinity matrix (ANAM) is provided, prepared by a method comprising the steps of:

a) preparing activated agarose beads by cross-linking N-hydroxysuccinimide with agarose beads; b) with a catalyst comprising NaHCO₃And NaCl, c) adding to the coupling buffer an antibody that binds to nucleosomes, wherein the antibody is prepared in an MR L/Mp (-) +/+ mouse;

d) incubating a coupling buffer comprising the antibody with the activated agarose beads to produce an anti-nucleosome antibody affinity matrix; and e) washing the anti-nucleosome antibody affinity matrix with a coupling buffer and an acetate buffer.

In some embodiments, the matrix binds to nucleosome bound circulating cfDNA and the matrix does not bind to unbound cfDNA including dsDNA, ssDNA, and oligonucleotides.

Also provided is a column comprising the anti-nucleosome antibody affinity matrix (ANAM) described above.

In yet another aspect, there is provided a DNA intercalator Hoechst3342 affinity matrix prepared by a method comprising the steps of

a) Oxidizing cellulose beads;

b) washing the oxidized cellulose beads;

c) reacting the washed oxidized cellulose beads with a solution comprising Hoechst33342 and N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC) to give Hoechst33342 immobilized cellulose beads; and

d) the Hoechst33342 immobilized cellulose beads were washed.

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 beads are washed with 1M sodium bicarbonate, 0.1M hydrochloric acid, and water. In some embodiments of step c), the solution is a pH buffered solution. In some embodiments in step d), the washing is performed at least three times.

In another aspect, there is provided a hyperbranched poly-L-lysine affinity matrix (P LL AM) prepared by a process comprising the steps of

a) L-lysine monohydrochloride is dissolved in water and neutralized by KOH to obtain L-lysine solution;

b) heating L-lysine solution to produce a solution comprising hyperbranched poly-L-lysine, c) removing L-lysine and salts from the solution comprising hyperbranched poly-L-lysine, d) fractionating the solution comprising hyperbranched poly-L-lysine to obtain a fraction comprising hyperbranched poly-L-lysine having an average molecular weight of 21,000 to 32,000, e) dialyzing and freeze-drying the fraction comprising hyperbranched poly-L-lysine having an average molecular weight of 21,000 to 32,000 to produce a lyophilizate;

f) dissolving the lyophilized extract in distilled water, and dissolving with NaHC0₃Dialyzing to obtain a solution containing HBP L, and g) suspending a solution containing hyperbranched poly-L-lysine in NaHC0₃The cyanogen bromide activated agarose 4B in (1) was incubated together to prepare a hyperbranched poly-L-lysine affinity matrix.

In some embodiments, the L-lysine solution is heated to 150 ℃ under a stream of nitrogen for 48 hours in step b) in some embodiments, the solution comprising hyperbranched poly-L-lysine is dialyzed against water in step c).

Also provided is a column comprising the hyperbranched poly L-lysine affinity matrix (P LL AM) described above.

In yet a further aspect, there is provided an apparatus configured to perform apheresis, the apparatus comprising one or more affinity columns comprising an affinity matrix and configured to remove substantially all types of cfDNA from blood or plasma of a patient. In some embodiments, the device comprises 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 affinity columns have an inlet and an outlet.

In some embodiments, the device comprises two or more of the following affinity columns in any order:

a) a column comprising a DNA binding protein (e.g., histone) affinity matrix;

b) a column comprising a lectin (e.g., galanthus agglutinin (GNA), narcissus agglutinin (NPA), concanavalin a, phytohemagglutinin, or a cyanobacterial antiviral protein) affinity matrix;

c) comprising a DNA binding polymer (e.g., a cationic polymer such as amino-terminated (-NH)₂) PAMAM dendrimer, hyperbranched poly-L-lysine, or hyperbranched polyethyleneimine) affinity matrix;

d) a column comprising an anti-DNA antibody affinity matrix;

e) a column comprising a DNA intercalator (e.g., Hoechst 3342) affinity matrix;

f) a column comprising an anti-nucleosome antibody affinity matrix (ANAM); and

g) a column comprising an anti-histone antibody affinity matrix.

In some embodiments, the device comprises one of the following combinations of columns in any order:

(a) (ii) a DNA intercalator Hoechst33342 affinity column and (ii) an anti-DNA antibody affinity column; or

(b) (ii) an anti-nucleosome antibody affinity matrix (ANAM) column and (ii) an anti-DNA antibody affinity column; or

(c) (ii) an anti-nucleosome antibody affinity matrix (ANAM) column and (ii) a Polyamidoamine Dendrimer Affinity Matrix (PDAM) column; or

(d) (i) an anti-nucleosome antibody affinity matrix (ANAM) column and (ii) a hyperbranched poly-L-lysine affinity matrix (P LL AM) column, or

(e) (ii) an anti-histone H2A antibody affinity column, (ii) a lectin affinity column, and (iii) a histone H1 affinity column or a Polyamidoamine Dendrimer Affinity Matrix (PDAM) column or a hyperbranched poly-L-lysine affinity matrix (P LL AM) column or a DNA intercalator Hoechst33342 affinity column.

In another aspect, an apheresis device is provided that comprises a filter cartridge and one or more affinity columns having an inlet and an outlet, wherein 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.

In some embodiments, the device comprises two or more affinity columns in sequential order. In some embodiments, the first affinity column in the sequence comprises a DNA-binding polymer or a DNA intercalator.

In some embodiments, the device comprises a column comprising a histone affinity matrix upstream of or before a column comprising a lectin affinity matrix. In some embodiments, the device comprises a column comprising a histone affinity matrix upstream of or before a column comprising a lectin affinity matrix, the column comprising the lectin affinity matrix being upstream of or before a column comprising a PAMAM affinity matrix. In some embodiments, the device comprises a column comprising an anti-DNA antibody affinity matrix upstream of or before a column comprising Hoechst3342 affinity matrix. In some embodiments, the device comprises a column comprising an anti-nucleosome antibody affinity matrix upstream of or before a column comprising a PAMAM affinity matrix.

In some embodiments, the apheresis device captures at least 30mg cfDNA per single apheresis procedure. In some embodiments, the affinity column comprises an immobilized portion effective to capture one or more of nucleosome bound cfDNA, exosome bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides). In some embodiments, the immobilization moiety is selected from the group consisting of a DNA binding antibody, a DNA intercalator, a DNA binding protein, a DNA binding polymer, a lectin, an anti-nucleosome antibody, and an anti-histone antibody.

In some embodiments, the DNA binding protein is histone H1 (e.g., H1.3).

In some embodiments, the DNA binding polymer is a cationic polymer, in some embodiments, the cationic polymer is poly-L-lysine, in some embodiments, poly-L-lysine is hyperbranched poly-L-lysine, in some embodiments, the cationic polymer is polyethyleneimine₂) Polyamidoamine (PAMAM) dendrimers.

In some embodiments described above, the apheresis device comprises two sequential affinity columns, one of which captures nucleosome bound DNA and exosome bound DNA, the other of which captures unbound cfDNA, including dsDNA, ssDNA and oligonucleotides. In some embodiments, the immobilization moiety is selected from the group consisting of a combination of two or more of a DNA binding antibody, a DNA intercalator, a DNA binding protein, a DNA binding polymer, a lectin, an anti-nucleosome antibody, or an anti-histone antibody.

In another aspect, a method of reducing cfDNA levels in blood of a patient is provided. The method comprises (a) performing an apheresis procedure comprising shunting blood or plasma from the patient into an apheresis device to produce purified blood or plasma having reduced cfDNA levels; and (b) returning the purified blood or plasma to the patient. The apheresis procedure reduces the levels of essentially all types of cfDNA (including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides)) in the patient's blood.

In some embodiments, the methods are effective to treat one or more of the following diseases: multiple organ failure, neurodegenerative diseases (e.g., alzheimer's disease), cancer, sepsis, septic kidney injury, radiation-induced toxicity (e.g., acute radiation syndrome), and chemotherapy-related toxicity.

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), graft rejection, sepsis, Systemic Inflammatory Response Syndrome (SIRS), Multiple Organ Dysfunction Syndrome (MODS), radiation-induced toxicity (e.g., acute radiation syndrome), chemotherapy-related toxicity, traumatic injury, proinflammatory state in elderly, diabetes, atherosclerosis, neurodegenerative disease, autoimmune disease, eclampsia, infertility, coagulation disorders, and infections.

In some embodiments, the method is effective to treat a condition in a patient, wherein the condition is selected from the group consisting of cancer, metastatic cancer, acute organ failure, organ infarction (including myocardial infarction and ischemic stroke, hemorrhagic stroke, autoimmune disease, Graft Versus Host Disease (GVHD), graft rejection, sepsis, Systemic Inflammatory Response Syndrome (SIRS); multiple organ dysfunction syndrome; Graft Versus Host Disease (GVHD), traumatic injury, pro-inflammatory states in the elderly, diabetes, atherosclerosis, neurodegenerative disease, autoimmune disease, eclampsia, infertility, coagulation disorders, pregnancy related complications, and infections.

In another aspect, a method of treating Multiple Organ Dysfunction Syndrome (MODS) in a patient is provided. The method comprises (a) performing an apheresis procedure comprising diverting blood or plasma from a patient into an apheresis device to produce purified blood or plasma; and (b) returning the purified blood or plasma with reduced cfDNA levels to the patient. The apheresis procedure reduces the levels of essentially all types of cfDNA (including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides)) in the patient's blood. In some embodiments, the patient is in need of treatment for MODS.

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

In another aspect, a method of treating alzheimer's disease in a patient is provided. The method comprises (a) performing an apheresis procedure comprising shunting blood or plasma from the patient into an apheresis device to produce purified blood or plasma having reduced cfDNA levels; and (b) returning the purified blood or plasma to the patient. The apheresis procedure reduces the level of essentially 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.

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

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

In another aspect, a method of treating renal injury in a patient is provided. The method comprises (a) performing an apheresis procedure comprising shunting blood or plasma from the patient into an apheresis device to produce purified blood or plasma having reduced cfDNA levels; and (b) returning the purified blood or plasma to the patient. The apheresis procedure reduces the levels of essentially all types of cfDNA (including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides)) in the patient's blood. In some embodiments, the patient is in need of treatment for a renal injury.

In another aspect, methods of treating chemotherapy-related toxicity in a patient are provided. The method comprises (a) performing an apheresis procedure comprising shunting blood or plasma from the patient into an apheresis device to produce purified blood or plasma having reduced cfDNA levels; and (b) returning the purified blood or plasma to the patient. The apheresis procedure reduces the levels of essentially all types of cfDNA (including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides)) in the patient's blood. In some embodiments, the patient is in need of treatment for chemotherapy-related toxicity.

In another aspect, a method for treating radiation-induced toxicity (e.g., acute radiation syndrome) in a patient is provided. The method comprises (a) performing an apheresis procedure comprising shunting blood or plasma from the patient into an apheresis device to produce purified blood or plasma having reduced cfDNA levels; and (b) returning the purified blood or plasma to the patient. The apheresis procedure reduces the levels of essentially all types of cfDNA (including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides)) in the patient's blood. In some embodiments, the patient is in need of treatment for radiation-induced toxicity.

In some embodiments of any of the above methods, the blood is diverted from the portal vein of the patient.

In some embodiments of the foregoing, the cfDNA level in the purified blood is reduced compared to the cfDNA level in the patient's blood prior to the apheresis procedure.

In some embodiments, the level of all nucleosome bound cfDNA, exosome bound cfDNA and unbound cfDNA (including dsDNA, ssDNA and oligonucleotides) is reduced in the purified blood. In some embodiments, the method further comprises periodically monitoring the level of circulating cfDNA in the patient's blood and continuing the apheresis procedure to reduce the circulating level of cfDNA by at least 25% before ending 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 the circulating cfDNA level by at least 50% before ending 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 the level of circulating cfDNA by at least 75% before ending the apheresis procedure.

In some embodiments of any of the above, at least 30mg cfDNA is removed from the patient's blood in one or several sequential apheresis procedures.

In some of the embodiments described above, the method steps are repeated, or performed on a timed schedule. Method steps may be performed twice daily, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once weekly, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, etc. Blood samples can be taken from patients and tested for cfDNA levels to assess the frequency of performing a treatment method.

The sequential arrangement of the affinity columns may allow capture of 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.

Various orders are described herein, and any order may be used. In some embodiments, the device comprises a column comprising a histone affinity matrix upstream of or before a column comprising a lectin affinity matrix. In some embodiments, the device comprises a column comprising a histone affinity matrix upstream of or before a column comprising a lectin affinity matrix, the column comprising the lectin affinity matrix being upstream of or before a column comprising a Polyamidoamine Dendrimer Affinity Matrix (PDAM). In some embodiments, the device comprises a column comprising an anti-DNA antibody affinity matrix upstream of or before a column comprising Hoechst3342 affinity matrix. In some embodiments, the device comprises a column comprising an anti-nucleosome antibody affinity matrix (ANAM) upstream of or before a column comprising a Polyamidoamine Dendrimer Affinity Matrix (PDAM).

As part of the various aspects described throughout this application, further comprising (a) performing an apheresis procedure comprising diverting blood or plasma from a patient into an apheresis device to produce purified blood or plasma; and (b) returning the purified blood or plasma with reduced cfDNA levels to the patient.

The apheresis device may comprise a histone affinity matrix. The histone affinity matrix may comprise recombinant human histone H1.3. The histone affinity matrix may be part of an affinity column. The beads used as carriers in the histone affinity matrix column may be cellulose beads, which are oxidized by an oxidizing agent prior to coupling with histone. For example, the bead may be a sepharose bead. Alternatively, forms of support other than beads (hollow fibers, membranes, tubes, etc.) may be used. The support of the affinity matrix may be made of other organic and inorganic compounds known to the person skilled in the art, such as polyvinylpyrrolidone (PVP), Polysulfone (PS), Polyethersulfone (PES), Polyarylethersulfone (PAES), polyacrylate, poly (methyl methacrylate) (PMMA), poly (glycidyl methacrylate) (PGMA), poly (hydroxy methacrylate), Polystyrene (PS), Polytetrafluoroethylene (PTFE), polyacrylamide, polyacrolein, acrylonitrile-butadiene-styrene (ABS), Polyacrylonitrile (PAN), Polyurethane (PU), poly (hydroxy methacrylate), poly (acrylonitrile-co-styrene) (pp), poly (,Polyethylene glycol (PEG), super-fluorinated carbon, agarose (i.e., cross-linked agarose), alginate, carrageenan, chitin, starch, cellulose, nitrocellulose, chitosan, and the like,Mixtures and/or derivatives of glass, silica, diatomaceous earth, zirconia, alumina, iron oxide, porous carbon and the solid support; and protonated and deprotonated forms of the separation material.

Beads may be coated with DNA binding proteins. DNA binding proteins such as histones or anti-DNA antibodies can be immobilized by chemically coupling them to a solid insoluble carrier matrix such as polysaccharide beads. For example, agarose beads are activated using cyanogen bromide, and proteins that capture cfDNA are incubated with the activated agarose to allow coupling to occur. Unconjugated material is removed by washing with buffer and protein-bound agarose is loaded into the target apheresis device/affinity cartridge. There are many different methods for chemically coupling proteins to various insoluble support matrices. These and other matrix materials and protein coupling methods known to those skilled in the art may be used in any of the methods and devices described herein.

For example, the cfDNA-capturing molecule can be attached to a solid support by an amine, thiol, imide (i.e., water-soluble carbodiimide), or other chemical attachment methods known to those skilled in the art to attach the polypeptide or oligonucleotide to the solid support.

For example, the size of the beads may be in the range of 30 to 200 microns, 40 to 180 microns, 45 to 165 microns, 60 to 150 microns the primary hydroxyl groups of cellulose may be selectively converted to 6-deoxy-6-carboxy cellulose using a number of oxidizing agents such as sodium metaperiodate (NaIO) or by oxidation mediated by piperidinoxoammonium salt (TEMPO) or by chlorite see, for example, ethylene, s. and Thielemans, w.,. Surface modification of cellulose and cellulose, nanoscales, 2014,6,7764, DOI:10.1039/C4nr01756 k. furthermore, cellulose (or agarose) carriers may be oxidized with other compounds known to the skilled person (e.g. chromic acid, chromium-pyridine, dimethyl sulfoxide) (see, for example, peing, L et al. Evaluation of activation and cellulose, e.g. oxidation of cellulose may be sufficiently removed by oxidation of cellulose by a solution such as a hydroxyl group, such as a phosphate buffer, cellulose, e.g. oxidation of cellulose, by a phosphate buffer, cellulose, or agarose, e.g. a phosphate.

The single-sampling device may comprise a histone affinity matrix the histone affinity matrix may comprise recombinant human histone H1.3. the histone affinity matrix may be part of an affinity column the beads used in the histone affinity matrix column may be cellulose beads oxidised with an oxidising agent the beads may be agarose gel beads for example the beads may be coated with streptavidin for example the beads may be in the size range 30 to 200 microns, 40 to 180 microns, 45 to 165 microns, 60 to 150 microns the primary hydroxyl groups of cellulose may be selectively converted to 6-deoxy-6-carboxy cellulose using a number of oxidising agents such as sodium meta-periodate (NaIO) or, alternatively, the oxidation mediated by the piperidinoxoammonium salt (TEMPO) may be used the primary hydroxyl groups of cellulose may be selectively converted to 6-deoxy-6-carboxy cellulose see for example, Eyle, s and Thielemans, w, Surface modification of cellulose nanocrystays, nanoscales, trioxide, 6,7764, DOI:10.1039/C4nr, 01 k, the fibre may be further purified by further incubation with a soluble protein oxidising agent such as a chromophorin (e.g. protein) with a chelating agent such as a chelating agent, e.g. a chelating agent, e.7, a chelating agent, e.g. a chelating agent, e.g. for purification method for example, a chelating agent, for example, a chelating agent, for example, for purification for example.

The histone affinity matrix is prepared by a method comprising the steps of:

a) oxidizing cellulose beads having a size between 100 microns and 250 microns to produce activated cellulose beads;

b) washing the activated cellulose beads;

c) preparing a concentrated solution of recombinant human histone H1.3;

d) incubating 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 beads.

The above method may further comprise: f) washing the activated cellulose beads with a buffer.

Any oxidizing agent may be used in step a). An exemplary oxidizing agent is NaIO. Any manner of washing may be performed in step b). For example, activated cellulose beads are washed with sodium bicarbonate, hydrochloric acid and water. Dialysis or other methods may be used in step c). For example, a solution of recombinant human histone H1.3 is dialyzed, and the dialyzed solution is subjected to 0.1M NaHCO at pH 7-9 or pH8₃Concentrating. In step d), the incubation may be carried out at 15-30 ℃ for 3-5 hours, or at room temperature for 4 hours. In step e), the blocking step comprises adding 1M ethanolamine to the activated cellulose beads and reacting at 15-30 ℃ for 30 minutes to 2 hours. In step f), the activated cellulose beads may be washed with TBS buffer.

The beads can be loaded onto a column, such as a Polytetrafluoroethylene (PTFE) column. Other exemplary columns may have walls made of polycarbonate, polyethylene, polyvinyl chloride, polypropylene, polyethersulfone, polyester, or other polymeric materials approved by the FDA or EMEA for use in the manufacture of extracorporeal treatment devices for blood or blood components.

The column or cartridge device may also be made of a material that is non-toxic and provides rigid support for the affinity matrix therein. Typically, the material will be a plastic composition such as polycarbonate, polyethylene, polyvinyl chloride, polypropylene, polyethersulfone, polyester, polystyrene, or other similar material approved by regulatory agencies (such as the FDA or EMEA) for use in the manufacture of blood or blood component extracorporeal treatment devices. In some embodiments, there is an internal filter at the bottom of the column (cartridge) to prevent the affinity matrix from leaving the device. In some embodiments, there is also an internal filter on the top of the device to contain the affinity matrix within the device. In some embodiments, these filters are composed of plastic and/or cellulosic materials and have pores that allow passage of fluids such as plasma, but not passage of particulate materials such as affinity matrices.

In preparing a histone affinity matrix column, the histone affinity matrix may be loaded to at least 50%, 60%, 70%, 75%, 80%, 85% or 90% of the column volume. PBS, especially cold PBS, can be used to equilibrate the column. Other suitable buffers may also be used to equilibrate the column.

The apheresis device may include a lectin affinity matrix. Non-limiting examples of useful lectins include, for example, Galanthus (Galanthus nivalis) (snowdrop) lectin (GNA), narcissus (narcissus) lectin (NPA), concanavalin a (concanavalin a), phytohemagglutinin (phytohemaglutatin), or cyanobacterial antiviral protein (cyanovirin). In one embodiment, lectins can be coupled to an agarose affinity matrix by incubation overnight at a neutral to slightly basic pH. After such incubation, sufficient washing with a buffer at a pH of approximately 7.0 to 7.5 may be performed to remove unbound lectin.

The lectin affinity matrix may be prepared according to a method comprising the steps of:

a) reacting the lectin with the activated agarose beads to produce lectin-coupled agarose; and

b) the lectin-coupled agarose was washed with buffer.

The apheresis device may include a Polyamidoamine (PAMAM) dendrimer affinity matrix (PDAM) or a polypropyleneimine (PPI) dendrimer affinity matrix. See, e.g., Kaur et al, J Nanopart Res, 2016,18: 146. Dendrimers are unique nano-sized synthetic polymers with highly branched structures and spherical shapes. Among dendrimers, Polyamidoamines (PAMAMs) are of most interest as potential transfection agents for gene delivery, since these macromolecules bind DNA at physiological pH. Polyamidoamine dendrimers consist of an alkyldiamine core and tertiary amine branches. They have 10 generations (GO-10), 5 different core types and 10 functional surface groups. DNA and Polyamidoamine (PAMAM) dendrimers form complexes based on electrostatic interactions between the negatively charged phosphate groups of nucleic acids and the protonated (positively charged) amino groups of the polymers. The formation of high molecular weight, high density complexes is mainly dependent on the DNA concentration, enhanced by increasing the dendrimer-DNA charge ratio. (Shcharbin, D. et al, Practical Guide to study Dendrimers. book, iSmithersScapra Publishing: Shawbury, Shrewsbury, Shropshire, UK,2010. page 120 ISBN: 978-1-84735-.

A PAMAM dendrimer affinity matrix was prepared by a method comprising the steps of:

a) washing the cellulose beads 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 PAMAM dendrimers to produce PAMAM beads and removing PAMAM dendrimers that have not reacted with the activated cellulose beads; and

d) blocking unconverted epoxy groups on PAMAM beads.

The beads can be loaded onto a column, such as a Polytetrafluoroethylene (PTFE) column. Other exemplary columns may have walls made of polycarbonate, polyethylene, polyvinyl chloride, polypropylene, polyethersulfone, polyester, or other polymeric materials approved by the FDA or EMEA for use in the manufacture of extracorporeal treatment devices for blood or blood components.

An apheresis device comprising a PAMAM dendrimer affinity matrix may remove cfDNA more efficiently, or alternatively may remove cfDNA more completely, or alternatively may remove a greater total amount of cfDNA in a particular blood sample, than using an apheresis device comprising a histone affinity matrix and a lectin affinity matrix.

In certain embodiments, the apheresis device may comprise all of a PAMAM dendrimer affinity matrix, a histone affinity matrix, and a lectin affinity matrix.

The apheresis device may include an anti-DNA antibody affinity matrix. anti-DNA antibodies constitute a subset of anti-nuclear antibodies that bind single-stranded DNA, double-stranded DNA, or both (anti-ds + ss DNA antibodies). They may be, for example, IgM antibodies or IgG antibodies of any subclass. Antibodies that bind only to single-stranded DNA can bind to their constituent bases, nucleosides, nucleotides, oligonucleotides, and phosphoribosyl backbones, all of which are exposed in a single strand of DNA. Antibodies that bind double-stranded DNA can bind to a ribose-phosphate backbone, base pairs (deoxyguanosine-deoxycytidine and deoxyadenosine-deoxythymidine) or a specific conformation of the double helix (BevraHannahshahn, Antibodies to DNA. N Engl J Med 1998; 338: 1359-. Antibodies directed against DNA may also bind to DNA-containing supramolecular structures (e.g., nucleosomes and chromatin).

The anti-DNA antibody affinity matrix can be prepared by, for example, activating agarose beads with N-hydroxysuccinimide (NHS). The activated beads can then be incubated with antibodies or other reagents having affinity for DNA. Excess antibody/reagent is then removed by washing.

The anti-nucleosome antibody affinity matrix (ANAM) is prepared by a method comprising a) preparing activated agarose beads by cross-linking N-hydroxysuccinimide with agarose beads, b) washing the activated agarose beads with a coupling buffer comprising NaHCO3 and NaCl, c) adding to the coupling buffer an antibody that binds to nucleosome, wherein the antibody is prepared in an MR L/Mp (-) +/+ mouse, d) incubating the coupling buffer comprising the antibody with activated agarose beads to produce an anti-nucleosome antibody affinity matrix, and

e) the anti-nucleosome antibody affinity matrix was washed with coupling buffer and acetate buffer.

The apheresis device may comprise a DNA intercalator affinity matrix. Molecules can interact with DNA in several ways. Ligands can interact with DNA by covalent binding, electrostatic binding, or intercalation. Intercalation occurs when a ligand of appropriate size and chemical nature fits itself between the base pairs of DNA. DNA binding agents tend to interact non-covalently with host DNA molecules in two general ways: (i) threading insertion in a groove-binding fashion stabilized by a mixture of hydrophobic, electrostatic and hydrogen bonding interactions, and (ii) classical insertion by intercalation association, in which planar heteroaromatic moieties slide between DNA base pairs. The most commonly studied intercalation binding is a non-covalent stacking interaction resulting from the insertion of planar heterocyclic aromatic rings between base pairs of the DNA double helix. See http:// nptel. ac.in/courses/104103018/35. Hoechst33342 is a bisbenzimide derivative that binds to an AT-rich sequence in the minor groove of double-stranded DNA. The heterocyclic moiety in this dye is important for efficient interaction with the DNA duplex, and thus for greater stability of the Hoechst-DNA complex.

The DNA intercalator affinity matrix may be prepared by oxidizing (activating) the bead, such as a cellulose bead (carrier) reacting the cellulose bead (carrier) with a compound (linker), such as a cellulose bead (carrier) linking a DNA intercalator (DNA binding moiety, i.e. Hoechst33342) with N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC) on the surface of the carrier. The beads were then washed.

Preparing a Hoechst3342 affinity matrix by a method comprising the steps of:

a) oxidizing cellulose beads;

b) washing the oxidized cellulose beads;

c) reacting the washed oxidized cellulose beads with a solution comprising Hoechst33342 and N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC) to give Hoechst33342 immobilized cellulose beads; and

d) the Hoechst33342 immobilized cellulose beads were washed.

The apheresis device may include hyperbranched poly-L-lysine affinity matrix the hyperbranched poly-L-lysine affinity matrix may be prepared by a method comprising the steps of a) dissolving L-lysine monohydrochloride in water, and neutralizing with KOH to obtain L-lysine solution;

b) heating L-lysine solution to obtain solution containing poly-L-lysine;

c) removing L-lysine and salts from the solution containing poly L-lysine;

d) fractionating the solution comprising poly-L-lysine to obtain a fraction comprising poly-L-lysine having an average molecular weight of 21,000 to 32,000;

e) dialyzing and lyophilizing a fraction comprising poly-L-lysine having an average molecular weight of 21,000 to 32,000 to obtain a lyophilizate;

f) dissolving the lyophilized extract in distilled water, and dissolving with NaHCO₃Dialyzing to obtain a solution containing HBP L, and

g) the solution containing HBP L is suspended in NaHCO₃Sepharose4B activated with cyanogen bromide in (1).

In certain embodiments, the apheresis device may comprise all or any number of DNA intercalator affinity matrices, Hoechst33342 affinity matrices, anti-DNA affinity matrices, PAMAM affinity matrices, histone affinity matrices, lectin affinity matrices, and poly-L-lysine affinity matrices.

Various apheresis procedures and treatment methods are described throughout the application. Various methods and procedures include (a) performing an apheresis procedure comprising diverting blood or plasma from a patient into an apheresis device to produce purified blood or plasma; and (b) returning the purified blood or plasma with reduced cfDNA levels to the patient. Any vein can be selected for optimal shunting of blood. For example, blood may be diverted from the patient's portal vein. Alternatively, blood may be diverted from the femoral or jugular vein of the patient.

In various embodiments of treatment, the apheresis procedure may be performed more than once, or even twice, for example on days 1 and 3. If renal injury is treated, the level of renal injury can be assessed by measuring serum creatinine and Blood Urine Nitrogen (BUN) levels with Roche reflex Plus (Roche diagnostics) prior to each apheresis procedure.

Circulating cfDNA can be extracted from plasma samples using the conventional THP (Triton-Heat-phenol) method (Breitbach et al, P L oS ONE,2014,9(3): e 87838.) the extracted cfDNA can be quantified using various assays (such as the PicoGreen assay (Molecular Probes, Netherlands)) according to the manufacturer's instructions^TMNucleic acid dyes (Promega),Gold nucleic acid gel dyes (Molecular Probes). The dye can be used as a gel coloring agent and a precasting agent, and can also be directly preloaded on a sample for loadingIn a buffer.

In various embodiments, performing the apheresis procedure further comprises separating the blood into plasma. The plasma fraction can then be shunted to one or more affinity matrices to remove cfDNA.

Examples

The invention is further described and illustrated by the following examples. However, the use of these and other examples anywhere in this specification is illustrative only and in no way limits the scope and meaning of the invention or any exemplary terms. Likewise, the present invention is not limited to any particular preferred embodiment described herein. Indeed, many modifications and variations of the present invention may be apparent to those skilled in the art upon reading this specification, and such variations may be made without departing from the spirit or scope of the invention. Accordingly, the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.