Preservative compositions for nucleic acids and biological samples and methods of use - Patents.com

Abstract

The present disclosure is directed to nucleic acid and cell preservation compositions.A method for preserving nucleic acid and / or cells in blood or other biological samples, and a kit for preserving nucleic acid and / or cells in blood or other biological samples are also described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of, and priority to, U.S. Provisional Patent Application No. 63 / 222,394, filed July 15, 2021, which is incorporated by reference in its entirety.

[0002] The present disclosure relates to compositions, methods, and kits for preserving nucleic acids and / or cells in blood or other biological samples. [Background technology]

[0003] Many nucleic acid-based tests are used to analyze variations in DNA and RNA sequence, structure or expression for various diagnostic purposes. In fact, nucleic acids are a common test target in non-invasive biomedical research. However, after a biological sample is collected, the nucleic acids in that sample, e.g., RNA and DNA, whether cellular / genomic or acellular, begin to degrade. In addition, gene induction and gene transcript degradation begin to occur within minutes of blood or other biological sample collection, making it difficult to accurately analyze the gene expression of the sample at the time of collection. Furthermore, generally, the fresher the blood or other biological sample, the better the quality of the nucleic acid of that sample. This presents the problem of when the nucleic acid of a subject's blood or biological sample is analyzed. It is often the case that blood and other biological samples are collected in different locations and at very different times from the location and time at which they are analyzed. For this reason, after blood or other biological samples are collected, they need to be stored and transported before they are analyzed. Due to the rapid degradation of nucleic acids that occurs in blood and other biological samples after they are collected, there is a need for compositions and methods that preserve the nucleic acids present in the sample to ensure that the nucleic acid of the sample is of high quality at the time it is analyzed.

[0004] In the case of cell-free nucleic acid in blood or other biological samples, different locations and timings of collection and analysis present the additional problem of cell lysis.Cell lysis in collected samples can lead to contamination of cell-free nucleic acid profile with cellular nucleic acid, making it difficult to accurately analyze cell-free nucleic acid in blood or biological samples.Cell lysis begins to occur immediately after blood or other biological samples are collected.This presents a problem when samples need to be stored for a long period of time before analysis.Therefore, there is an additional need to store blood and other biological samples so that cell-free profile of nucleic acid is maintained.

[0005] Similarly, for diagnostic applications based on the detection or analysis of cells, such as, for example, circulating tumor cells, in a biological sample, the preservation of those cells in an intact form is important. Summary of the Invention

[0006] The present disclosure is directed, in various aspects, to nucleic acid and cell preservative compositions, kits containing those compositions, and methods of using the compositions and kits.

[0007] In a first aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: a. optionally one or more osmotic agents; b. one or more enzyme inhibitors; c. optionally one or more metabolic inhibitors; d. optionally one or more cell surface remodeling polymers; e. optionally, one or more agents selected from the group consisting of hydroxyethyl starch, polymers of N-vinylpyrrolidone (NVP), ficoll, protein colloids, non-protein synthetic colloids, ethylenediol, propylene glycol, water soluble polymers, and carboxymethylcellulose, or a salt of any thereof; e. optionally polypropylene glycol (PPG); The invention relates to nucleic acid and cell preservative compositions in which at least one cell surface remodeling polymer (d) or drug (e) is present, and optionally one or more osmotic agents and one or more enzyme inhibitors are present in a combined amount sufficient to produce a hypertonic solution.

[0008] In a second aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: a. one or more enzyme inhibitors; b. optionally one or more metabolic inhibitors; c. optionally one or more cell surface remodeling polymers; d. optionally, one or more agents selected from the group consisting of hydroxyethyl starch, polymers of N-vinylpyrrolidone (NVP), ficoll, protein colloids, non-protein synthetic colloids, ethylenediol, propylene glycol, water soluble polymers, and carboxymethylcellulose, or a salt of any thereof; e. optionally polypropylene glycol (PPG); The invention relates to nucleic acid and cell preservative compositions in which at least one cell surface remodeling polymer (c) or agent (d) is present and one or more enzyme inhibitors are present in an amount sufficient to produce a hypertonic solution.

[0009] In a third aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: a. one or more enzyme inhibitors; b. optionally one or more metabolic inhibitors; c. one or more cell surface remodeling polymers; d. optionally polypropylene glycol (PPG); Contemplated are nucleic acid and cell preservative compositions in which one or more enzyme inhibitors are present in an amount sufficient to produce a hypertonic solution.

[0010] In a fourth aspect, the present disclosure is directed to a combination of a preservative composition of the present disclosure and a biological sample.

[0011] In a fifth aspect, the present disclosure is directed to a method for preserving nucleic acids and / or cells in a biological sample comprising combining the biological sample with a preservative composition of the present disclosure.

[0012] In a sixth aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: a. a preservative composition of the present disclosure; and b. Optionally, instructions for use of the preservation composition.

[0013] In a seventh aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: a. a blood or other biological sample collection tube, optionally containing an anticoagulant; b. a syringe containing the preservative composition of the present disclosure; c. Optionally, a needle that can be attached to the syringe.

[0014] In an eighth aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: a. a blood or other biological sample collection tube, optionally containing an anticoagulant; b. A kit for preserving nucleic acids and / or cells in a biological sample comprising: a sealed ampoule containing a preservative of the present disclosure, the ampoule having a removable closure, and the ampoule configured to receive a dispensing means upon removal of the closure by a user.

[0015] In some embodiments, the biological sample is derived from a bodily fluid, hi some embodiments, the bodily fluid is blood.

[0016] In some embodiments, the nucleic acid is cell-free ("cf") DNA. In other embodiments of the disclosure, the nucleic acid is cellular (i.e., genomic or "g") DNA.

[0017] In some embodiments, the nucleic acid is cell-free ("cf") RNA. In other embodiments of the disclosure, the nucleic acid is cellular (i.e., genomic or "g") RNA.

[0018] In some embodiments, the cells are stem cells, bone cells, blood cells (e.g., red blood cells and / or white blood cells), muscle cells, fat cells, skin cells, neuronal cells, endothelial cells, sex cells, pancreatic cells, cancer cells, tumor cells, or circulating tumor cells. In some embodiments, the cells are laboratory-derived or engineered cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] definition Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. In case of conflict, the present specification, including definitions, shall control.

[0020] Throughout this application, and its various embodiments and aspects, the word "comprise", or variations such as "comprises" or "comprising", will be understood to include a stated integer or group of integers, but not to exclude any other integer or group of integers.

[0021] The terms "including" or "includes" are used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0022] Any examples following the term "eg" or "for example" are not intended to be exhaustive or limiting of the disclosure.

[0023] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0024] The articles "a", "an" and "the" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article.

[0025] All ranges disclosed herein should be understood to encompass all subranges subsumed therein. For example, a specified range of "1 to 10" should be considered to include every subrange between (and including) a minimum value of 1 and a maximum value of 10, i.e., every subrange beginning with a minimum value that is 1 or greater, e.g., 1 to 6.1, and ending with a maximum value that is 10 or less, e.g., 5.5 to 10. When used in the context of weight percent of a component or v / v of a mixture, the term "about" means + / - 10% of the recited number.

[0026] Each embodiment of the present disclosure may be used alone or in combination with one or more other embodiments of the present disclosure.

[0027] Exemplary methods and materials are described herein, although it should be understood that methods and materials similar or equivalent to those described herein can also be used in the practice or testing of various aspects and embodiments of the disclosure. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0028] In order to make this disclosure easier to understand, certain terms are defined first.These definitions should be read in light of the remaining parts of this disclosure as understood by those skilled in the art.Unless otherwise defined, all technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art.Additional definitions are described throughout the detailed description.

[0029] As used herein, the term "osmotic agent" refers to an agent that produces a hypertonic, isotonic, or hypotonic solution. Additionally, as used herein, enzyme inhibitors may additionally function as osmotic agents. Examples of osmotic agents include, but are not limited to, for example, sodium, potassium, magnesium, and calcium salts, lactate Ringer's solution, acetate Ringer's solution, amino acids, sorbitol, glycerol, mannitol, sugars such as sucrose or glucose, tartaric acid, and glucaric acid, or any salt thereof. Examples of enzyme inhibitors that may additionally function as osmotic agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), dithiothreitol (DTT), ethyleneglycol-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), citric acid, oxalate, aurintricarboxylic acid (ATA), tartaric acid, glucaric acid, or any salt thereof, including but not limited to sodium and potassium salts. Without wishing to be bound by theory, osmotic agents play a role in changing the osmotic pressure in blood or other biological samples, for example, by releasing water from cells present in blood or other biological samples to eliminate imbalance.This, for example, causes cells to shrink, thereby making them more resistant to cell lysis, which would otherwise contaminate the cell-free nucleic acid of the biological sample with cellular nucleic acid or make the cells less suitable for assay and analysis.Furthermore, plasma expanders are believed to enhance this effect.

[0030] As used herein, the term "hypertonic solution" refers to a solution having a solute concentration higher than physiological concentration. Examples of hypertonic solutions include, but are not limited to, about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25% NaCl solutions (by weight).

[0031] As used herein, the term "hypotonic solution" refers to a solution having a solute concentration lower than physiological concentration. Examples of hypotonic solutions include, but are not limited to, about 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, and 0.45% NaCl solutions (by weight).

[0032] As used herein, the term "isotonic solution" refers to a solution having a solute concentration approximately equal to physiological concentration. Examples of isotonic solutions include, but are not limited to, about 0.5%, 0.7%, and 1% NaCl solutions (by weight).

[0033] As used herein, the term "enzyme inhibitor" refers to an agent that, alone or in the preservative composition of the present disclosure, forms a complex with a metal ion such as calcium, magnesium, manganese, or zinc, and these complexes are believed to reduce blood clotting, inhibit nucleases, and / or reduce enzymatic cell lysis. Examples of enzyme inhibitors of the present disclosure include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), dithiothreitol (DTT), ethyleneglycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), citric acid, oxalate, aurintricarboxylic acid (ATA), tartaric acid, glucaric acid, or any salt thereof, including but not limited to sodium and potassium salts. Without wishing to be bound by theory, inhibition of nucleases prevents or reduces the degradation of cell-free nucleic acids in a biological sample. Examples of enzymes inhibited by the enzyme inhibitors of the present disclosure include, but are not limited to, lysostaphin, zymolase, proteases, glycanases, or other enzymes known to induce cell lysis, thereby acting to preserve cells in blood or other biological samples.

[0034] As used herein, the term "metabolic inhibitor" refers to an agent that inhibits cellular processes, such as cellular respiration, cellular metabolism, and metabolic functions, either alone or in the preservative composition of the present disclosure, and this inhibition is believed to reduce the degradation of cell-free nucleic acid. Without wishing to be bound by theory, it is believed that the metabolic inhibitor of the present disclosure slows cell growth by inhibiting cellular metabolic functions and inhibiting bacterial growth, thereby reducing the degradation of cell-free nucleic acid. Examples of metabolic inhibitors of the present disclosure include, but are not limited to, sodium azide, thimerosal, proclin, or chlorhexidine.

[0035] As used herein, the term "plasma expander" refers to an agent that produces a hyperoncotic or hypertonic solution. Examples of plasma expanders include, but are not limited to, glycerol, starch, protein colloids (e.g., albumin, ovalbumin, and gelatin), and non-protein colloids (e.g., hydroxyethyl starch). Without wishing to be bound by theory, plasma expanders also serve to increase the osmotic pressure in plasma or other biological samples, forcing cells to release water to eliminate imbalances. This causes cells to shrink, thereby making them more resistant to cell lysis, which would otherwise contaminate the cell-free nucleic acid of the biological sample with cellular nucleic acid or make the cells less suitable for assay and analysis.

[0036] As used herein, the term "cell surface remodeling polymer" refers to a polymer that interacts with the cell surface in blood or other biological samples (e.g., by binding to cell surface receptors or by reacting with specific functional groups on the cell surface) via covalent, hydrophobic, or electrostatic interactions. Such interactions are believed to cause cells in blood or other biological samples to sediment in some cases. Without wishing to be bound by theory, it is believed that the sedimentation of cells in the biological sample and / or the interaction of the polymer with the cell surface prevents or reduces cell lysis and the subsequent release of cellular nucleic acid into the sample (which may, for example, contaminate acellular nucleic acid or intact cells in the sample). The nucleic acid and / or cells can then be isolated and analyzed via conventional methods known in the art. In some embodiments of the present disclosure, the "cell surface remodeling polymer" is a surfactant. Examples of cell surface remodeling polymers include, but are not limited to, copolymers of N-vinylpyrrolidone (NVP) and boronic acid, arginylglyclaspartic acid (RGD) tripeptide polymer derivatives, mung bean phytohemagglutinin, poloxamers, and synthetic glycopeptides characterized by one or more ligands for the mannose 6-phosphate receptor (e.g., glycopeptides having multiple serine-O-mannose-6-phosphate (M6Pn) residues). For examples of glycopeptides with repeating ligands for the mannose 6-phosphate receptor, see Banik, Steven; Pedram, Kayvon; Wisnovsky, Simon; Riley, Nicholas; Bertozzi, Carolyn (2019): Lysosome Targeting Chimeras (LYTACs) for the Degradation of Secreted and Membrane Proteins. ChemRxiv. Preprint. https: / / doi.org / 10.26434 / chemrxiv.7927061.v2.

[0037] As used herein, "Ficoll" refers to a water-soluble high molecular weight sucrose polymer formed from the polymerization of sucrose and epichlorohydrin. For example, Ficoll 400 and Ficoll 70.

[0038] As used herein, "poloxamer" refers to a water-soluble triblock copolymer having a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene. Examples of poloxamers include, but are not limited to, poloxamer p188 and poloxamer p407.

[0039] As used herein, "protein colloid" refers to a mixture in which one or more proteins are dispersed in a solution. Examples of protein colloids include, but are not limited to, albumin, ovalbumin, or gelatin. Albumin may be provided, for example, as human serum albumin (HSA), bovine serum albumin (BSA), or ovalbumin. Examples of gelatin include, but are not limited to, urea-bound gelatin (e.g., Haemaccel®), succinylated gelatin (e.g., Gelofusine®), and oxypolygelatin.

[0040] As used herein, the term "non-protein colloid" refers to a mixture of one or more large molecules or ultrafine particles dispersed in a solution. Examples of non-protein colloids include, but are not limited to, branched natural polymers of amylopectin, such as hydroxyethyl starch (HES), and polysaccharides such as dextrans, e.g., dextran 40 and / or dextran 70.

[0041] As used herein, "water-soluble polymer" refers to a polymer that is soluble in an aqueous solution. Examples of water-soluble polymers include, but are not limited to, polyacrylamides, polyacrylates, polydextrose, polyglycines, polyethyleneimines, polylysines, polyethylene glycols, polyvinylpyrrolidones, polyvinyl alcohols, polyacrylic acids, polymers of N-(2-hydroxypropyl)methacrylamides, polymers of divinyl ether-maleic anhydride, polyoxazolines, polyphosphates, polyphosphazenes, xanthan gums, pectins, chitosan derivatives, dextran, carrageenans, guar gums, cellulose ethers, sodium carboxymethylcellulose, hydroxypropylcellulose, hypromellose, hyaluronic acid, albumin, starch, or starch-based derivatives. For further non-limiting examples of water-soluble polymers of the present disclosure, see Betageri, GV, Kadajji, VG, Polymers, 2011, 3, pp. 1972-2009.

[0042] As used herein, the term "nucleic acid" includes both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). RNA and / or DNA may be linear or branched, single-stranded or double-stranded, or fragmented. RNA and DNA may be cellular RNA (i.e., genomic RNA), cellular DNA (i.e., genomic DNA), cell-free RNA, cell-free DNA, or combinations thereof. Nucleic acids are found in biological samples, particularly blood samples.

[0043] As used herein, the term "biological sample" refers to a sample obtained from a biological source, including laboratory-derived or laboratory-modified cells, that contains nucleic acids and / or cells. A biological sample may be a cell sample, a culture sample, or a tissue sample. Additionally, a biological sample may be derived from a bodily fluid, such as blood, plasma, serum, urine, saliva, stool, breast milk, tears, sweat, cerebrospinal fluid, synovial fluid, semen, vaginal fluid, peritoneal fluid, amniotic fluid, or cell culture medium.

[0044] As used herein, the term "preservative" refers to a composition that is added to a biological sample to inhibit, prevent, or delay degradation of nucleic acids and / or cell lysis in the sample.

[0045] As used herein, the term "treated biological sample" refers to a biological sample that has been combined with a preservative composition of the present disclosure.

[0046] As used herein, the term "cell" refers to any cell that can be found in blood or other biological samples. Cell types include, but are not limited to, stem cells, bone cells, blood cells (e.g., red blood cells or white blood cells), muscle cells, adipocytes, skin cells, neural cells, endothelial cells, sex cells, pancreatic cells, cancer cells, tumor cells, circulating tumor cells (CTCs), and laboratory-derived and / or modified cells.

[0047] Preservative Compositions of the Present Disclosure The compositions of the present disclosure are useful for preserving and stabilizing nucleic acids and / or cells in biological samples. When the preservative compositions of the present disclosure are added to biological samples containing nucleic acids and / or cells, degradation of nucleic acids and / or cell lysis in the sample is reduced, delayed, or prevented compared to unprocessed biological samples, allowing for subsequent isolation and more accurate analysis of the nucleic acids and / or cells in the sample via conventional techniques known in the art, particularly high-throughput techniques. Furthermore, the preservative compositions of the present disclosure inhibit, delay, or reduce cell lysis, allowing the cell-free nucleic acids in the sample to remain more consistent in quantity and characteristics over an extended period of time. The reduction of cell lysis in processed biological samples according to the present disclosure also reduces the release of nucleases, thereby further preventing or reducing degradation of nucleic acids and / or cells in the sample. Nucleic acids that can be preserved by the compositions of the present disclosure include RNA, DNA, or combinations thereof. RNA and DNA can be cellular or acellular, or combinations thereof, i.e., cellular RNA, cellular DNA, cell-free RNA, cell-free DNA, or combinations thereof. Preferably, DNA and / or RNA is cell-free DNA and / or cell-free RNA.The cell that is reduced in lysis using the compositions and methods of the present disclosure can be, but is not limited to, stem cells, bone cells, blood cells, muscle cells, adipocytes, skin cells, nerve cells, endothelial cells, sex cells, pancreatic cells, cancer cells, tumor cells, circulating tumor cells, and laboratory-derived or modified cells.

[0048] In a first aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: a. optionally one or more osmotic agents; b. one or more enzyme inhibitors; c. optionally one or more metabolic inhibitors; d. optionally one or more cell surface remodeling polymers; e. optionally, one or more agents selected from the group consisting of hydroxyethyl starch, polymers of N-vinylpyrrolidone (NVP), ficoll, protein colloids, non-protein synthetic colloids, ethylenediol, propylene glycol, water soluble polymers, and carboxymethylcellulose, or a salt of any thereof; f. optionally polypropylene glycol (PPG); The invention relates to nucleic acid and cell preservative compositions in which at least one cell surface remodeling polymer (d) or drug (e) is present, and optionally one or more osmotic agents and one or more enzyme inhibitors are present in a combined amount sufficient to produce a hypertonic solution.

[0049] In a second aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: a. one or more enzyme inhibitors; b. optionally one or more metabolic inhibitors; c. optionally one or more cell surface remodeling polymers; d. optionally, one or more agents selected from the group consisting of hydroxyethyl starch, polymers of N-vinylpyrrolidone (NVP), ficoll, protein colloids, non-protein synthetic colloids, ethylenediol, propylene glycol, water soluble polymers, and carboxymethylcellulose, or a salt of any thereof; e. optionally polypropylene glycol (PPG); The invention relates to nucleic acid and cell preservative compositions in which at least one cell surface remodeling polymer (c) or agent (d) is present and one or more enzyme inhibitors are present in an amount sufficient to produce a hypertonic solution.

[0050] In a third aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: a. one or more enzyme inhibitors; b. optionally one or more metabolic inhibitors; c. one or more cell surface remodeling polymers; d. optionally polypropylene glycol (PPG); Contemplated are nucleic acid and cell preservative compositions in which one or more enzyme inhibitors are present in an amount sufficient to produce a hypertonic solution.

[0051] In some embodiments of the first aspect of the present disclosure, one or more of the enzyme inhibitors additionally function as an osmotic agent.

[0052] In some embodiments of the first aspect and other aspects of the present disclosure (including but not limited to the second through eighth aspects referred to herein), the optional one or more osmotic agents are not present, but one or more enzyme inhibitors that may function as osmotic agents are present.

[0053] In some embodiments of the first and other aspects of the disclosure, no plasma expander is present.

[0054] In some embodiments of the first aspect and other aspects of the disclosure, the one or more enzyme inhibitors are present in the preservative compositions of the disclosure in an amount of about 0.5% to about 30% by weight of the composition, in some aspects in an amount of about 0.5% to about 5% by weight, and in other aspects in an amount of about 1% to about 30% by weight. In some embodiments, the enzyme inhibitor is present in an amount of about 1% to about 20% by weight of the composition. In other embodiments, the enzyme inhibitor is present in an amount of about 1% to about 10% by weight of the composition.

[0055] In some embodiments of the first and other aspects of the disclosure, the one or more enzyme inhibitors are selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), dithiothreitol (DTT), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), citric acid, oxalate, aurintricarboxylic acid (ATA), tartaric acid, and glucaric acid, or any salt thereof, including, but not limited to, monovalent, divalent, trivalent, or tetravalent sodium and potassium salts, or mixtures thereof.

[0056] In some embodiments of the first aspect and other aspects of the disclosure, the one or more optional metabolic inhibitors are present in the preservative composition of the disclosure in an amount of about 0.01% to about 10% by weight of the composition. In some embodiments, the optional metabolic inhibitor is present in an amount of about 0.01% to about 5% by weight of the composition. In some embodiments, the optional metabolic inhibitor is present in an amount of about 0.01% to about 2% by weight of the composition.

[0057] In some embodiments of the first and other aspects of the disclosure, the one or more optional metabolic inhibitors is sodium azide, thimerosal, proclin, or chlorhexidine.

[0058] In some embodiments of the first and other aspects of the present disclosure, the one or more optional agents is Ficoll. In some embodiments, the Ficoll is Ficoll-400.

[0059] Without wishing to be bound by theory, Ficoll acts as a crowding agent, driving cells out of solution, thereby preventing or reducing cell lysis and subsequent cell degradation or release of cellular nucleic acid into the sample that may otherwise contaminate, for example, cell-free nucleic acid within the sample. The nucleic acid and / or cells can then be isolated and more accurately analyzed via conventional methods known in the art.

[0060] In some embodiments of the first and other aspects of the disclosure, Ficoll is present in an amount of about 10% to about 50% by weight of the composition, hi some embodiments, the one or more agents are present in an amount of about 10% to about 40%, or about 15% to about 35%, or about 20% to about 30% by weight of the composition.

[0061] In some embodiments of the first and other aspects of the disclosure, the one or more cell surface remodeling polymers are selected from the group consisting of copolymers of N-vinylpyrrolidone (NVP) and boronic acid, arginylglycylaspartic acid (RGD) tripeptide polymer derivatives, mung bean phytohemagglutinin, and synthetic glycopeptides with repeating ligands for the mannose 6-phosphate receptor. In some embodiments of the first and other aspects of the disclosure, one or more of the cell surface remodeling polymers are surfactants.

[0062] In some embodiments of the first and other aspects of the disclosure, the cell surface remodeling polymer is a poloxamer. In some embodiments, the cell surface remodeling polymer is poloxamer p188. In some embodiments, the cell surface remodeling polymer is poloxamer p407. In some embodiments, the cell surface remodeling polymer is a combination of poloxamer p188 and poloxamer p407.

[0063] In some embodiments of the first aspect and other aspects of the disclosure, the one or more cell surface remodeling polymers are present in the compositions of the disclosure in an amount of about 10% to about 40% by weight of the composition. In some embodiments, when the cell surface remodeling polymer is a poloxamer, the poloxamer is present in an amount of about 10% to about 40% by weight, about 10% to about 35% by weight, about 10% to about 25% by weight, about 10% to about 20% by weight, about 15% to about 20% by weight, about 15% by weight, or about 30% by weight. In some embodiments, when the cell surface remodeling polymer is a combination of poloxamer p188 and poloxamer p407, the combination is present in an amount of about 10% to about 40% by weight, or about 30% by weight of the composition. In some embodiments, when the cell surface remodeling polymer is a combination of poloxamer p188 and poloxamer p407, each poloxamer is present in an amount of 15% by weight of the composition.

[0064] In some embodiments of the first and other aspects of the disclosure, the one or more cell surface remodeling polymers are present and the optional agent is absent.

[0065] In some embodiments of the first and other aspects of the disclosure, one or more optional agents are present and one or more cell surface remodeling polymers are also present.

[0066] In some embodiments of the first and other aspects of the disclosure, the optional polypropylene glycol (PPG) is present in an amount of about 0.1 to about 10% by weight of the composition. In some embodiments, the optional PPG is present in an amount of about 5% to about 10%, or about 1% to about 5%, or about 0.1% to about 1% by weight of the composition.

[0067] In some embodiments of the first and other aspects of the disclosure, one or more components of the preservative composition of the present disclosure may fulfill the role or function of one or more other components of the preservative composition.

[0068] In some embodiments of aspects of the present disclosure, one or more components of the preservative composition may fulfill the role or function of one or more other components of the preservative composition. For example, tartaric acid or glucaric acid or EDTA or salts thereof may be present in the compositions of the present disclosure as an enzyme inhibitor, an osmotic agent, or both.

[0069] In some embodiments, the present disclosure provides: a. 1.57% by weight of EDTA or a salt thereof; b. 15.00% by weight of poloxamer p188; c. 15.00% by weight of poloxamer p407, and EDTA or a salt thereof is present in an amount sufficient to produce a hypertonic solution.

[0070] In some embodiments, the present disclosure provides: a. 2.5% by weight of EDTA or a salt thereof; b. 15.00% by weight of poloxamer p188; c. 15.00% by weight of poloxamer p407, and EDTA or a salt thereof is present in an amount sufficient to produce a hypertonic solution.

[0071] The preservative composition according to various aspects of the present disclosure may be in the form of a lyophilized powder, granules, tablets, or a solution (e.g., the preservative composition is reconstituted in a suitable vehicle). The lyophilized powder, granules, and / or tablets may be added directly to the biological sample or may be reconstituted before being added to the biological sample. The lyophilized powder, granules, and / or tablets may be reconstituted, for example, by dissolving the composition in a suitable vehicle. Suitable vehicles include, but are not limited to, water, saline, Ringer's solution, fixed oils of vegetable origin, mono- and diglycerides of fatty acids, ethanol, glycerin, and propylene glycol. Alternatively, the biological sample may be added directly to the lyophilized powder, granules, tablets, or reconstituted composition (i.e., a solution). In some embodiments, when the biological sample is derived from a bodily fluid, the bodily fluid may serve as an acceptable vehicle for solubilizing the preservative composition. For example, the lyophilized powder, granules, and / or tablet form of the preservative composition may be combined with and thereby solubilized by the bodily fluid. In some embodiments, the collection tube or container contains the preservative composition as a lyophilized powder, granules, tablets or solution before the biological sample is collected into the tube or container.

[0072] In some embodiments, the preservative composition of the present disclosure is in the form of an aqueous solution. The aqueous solution may be combined with the biological sample, or the biological sample may be combined with the aqueous solution.

[0073] Combination of a preservative composition with a biological sample and a method for preserving nucleic acids and / or cells of the biological sample In a fourth aspect, the present disclosure is directed to a combination of a preservative composition of the present disclosure and a biological sample.

[0074] In a fifth aspect, the present disclosure is directed to a method for preserving nucleic acids and / or cells in a biological sample comprising combining the biological sample with a preservative composition of the present disclosure.

[0075] In some embodiments of the fourth and fifth aspects, the biological sample is a cell or tissue sample.

[0076] In some embodiments of the fourth and fifth aspects, the biological sample is derived from a bodily fluid. In some embodiments, the bodily fluid is blood, plasma, serum, urine, saliva, stool, breast milk, tears, sweat, cerebrospinal fluid, synovial fluid, semen, vaginal fluid, peritoneal fluid, or amniotic fluid. In a preferred embodiment, the biological fluid is blood, e.g., whole blood or a fraction thereof. The biological sample may contain cells or may be acellular.

[0077] In some embodiments of the fourth and fifth aspects, the biological sample comprises stem cells, bone cells, blood cells, muscle cells, adipocytes, skin cells, neuronal cells, endothelial cells, sex cells, pancreatic cells, cancer cells, tumor cells, or circulating tumor cells.

[0078] In some embodiments of the fourth and fifth aspects, the biological sample comprises the nucleic acid selected from RNA, DNA, or a combination thereof.In some embodiments, the nucleic acid is cell-free RNA, cell-free DNA, or a combination thereof.In some embodiments, the nucleic acid is cellular RNA, cellular DNA, or a combination thereof.

[0079] Biological samples can be combined with the preservative composition of the present disclosure in many ways. For example, the biological sample can be collected in a suitable container, and then the preservative composition can be added to the container, for example, by a syringe or pipette. The preservative composition can alternatively be added to a suitable container for collecting the biological sample before collecting the biological sample. In some embodiments, the preservative composition is added to the biological sample. In some embodiments, the biological sample is added to the preservative composition.

[0080] The disclosure of these various aspects also contemplates methods in which the components of the preservative composition are added to the biological sample simultaneously or separately. Thus, in some embodiments, the disclosure is directed to a method of preserving nucleic acids and / or cells in a biological sample, the method comprising contacting the biological sample with the components of the preservative composition of the disclosure in any order or simultaneously. In some embodiments, a container suitable for collecting a biological sample already contains one or more of the components of the preservative composition, and the remaining components are added to the collected biological sample sequentially or simultaneously. For example, a blood collection tube that already contains a suitable enzyme inhibitor (e.g., tartaric acid, or EDTA or its salt, or glucaric acid) may be used to collect the biological sample. Following collection of the biological sample, the remaining components may be added to the biological sample. In another embodiment, the components of the preservative composition are added to the biological sample sequentially or simultaneously after the biological sample is collected. In some embodiments, all of the necessary components of the preservative composition, and optionally the optional components, are present in the container before the container is used to collect the sample.

[0081] In some embodiments, the container used for sample collection contains the preservative composition in lyophilized powder form. In some embodiments, the container used for sample collection contains the preservative composition in granular form. In some embodiments, the container used for sample collection contains the preservative composition in tablet form. In some embodiments, the container used for sample collection contains the preservative composition and a suitable vehicle. In some embodiments, the container used for sample collection contains the preservative composition as an aqueous solution. In another embodiment, the container used is for blood sample collection and further comprises an anticoagulant. Examples of anticoagulants include, but are not limited to, EDTA (which may also function as an enzyme inhibitor), sodium citrate, citrate-theophylline-adenosine-dipyridamole (CTAD), lithium heparin, sodium heparin, sodium fluoride, acid-citrate-dextrose (ACD), and sodium polyantholsulfonate. In some embodiments, the suitable container is an evacuated blood sampling tube.

[0082] The amount of preservative composition that can be combined with a biological sample can be determined by one of skill in the art through routine experimentation. In some embodiments, the ratio of the preservative composition to the biological sample can be about 1:10 to about 1:1 v / v. In some embodiments, the ratio of the preservative composition to the biological sample is about 1:8 to about 1:2 v / v. In some embodiments, the ratio of the preservative composition to the biological sample is about 1:6 to about 1:3 v / v. In some embodiments, the ratio of the preservative composition to the biological sample is about 1:5 to about 1:4 v / v.

[0083] After the biological sample is collected and contacted with the preservative composition of the present disclosure, nucleic acid and / or cells can be isolated from the biological sample for analysis using methods known to those skilled in the art. Such methods can include extraction, centrifugation, and chromatographic methods. Those skilled in the art will recognize that there are many methods that can be used to isolate nucleic acid and / or cells from biological sample.

[0084] Nucleic acids and / or cells preserved using the preservative composition of the present disclosure can be isolated from the treated biological sample after long-term storage under various temperature conditions. In some embodiments, the biological sample contacted with the preservative composition of the present disclosure can be preserved for at least 1 day, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks, either under ambient conditions or at low temperature. In some embodiments, the composition of the present disclosure allows for preservation of the biological sample (i.e., the nucleic acids and / or cells in the biological sample) for long periods of time at temperatures ranging from about -20°C to about 30°C. In some embodiments, the preservative composition can preserve the biological sample (i.e., the nucleic acids and / or cells in the biological sample) for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks at ambient temperature. In some embodiments, the preservative composition can preserve the biological sample for at least 2 weeks at ambient temperature. In some embodiments, the preservative composition of the present disclosure can preserve the biological sample (i.e., the nucleic acids and / or cells in the biological sample) for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks at 4°C. In some embodiments, the preservative compositions of the present disclosure are capable of preserving a biological sample (i.e., nucleic acids and / or cells in a biological sample) for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks at −20° C. Nucleic acids (RNA and DNA) preserved using the compositions and methods of the present disclosure exhibit good yield, purity, and integrity for RNA amplifiability.

[0085] Kit for preserving nucleic acids and / or cells in a biological sample The preservative composition according to the present disclosure may be provided as part of a kit that is received by a user. The kit allows the preservative composition of the present disclosure to be easily combined with a biological sample, so that the nucleic acids and / or cells present in the biological sample are preserved for an extended period of time, for example, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks. The preservative composition may be provided to be combined with the biological sample after the biological sample is collected. Alternatively, the preservative composition is provided to be combined with the biological sample at the time the biological sample is collected.

[0086] In some embodiments, the preservative composition is provided as an aqueous solution in the dispensing means. In some embodiments, the dispensing means is a syringe. In some embodiments, the amount of preservative in the dispensing means is a predetermined amount such that the ratio of the preservative composition combined with the biological sample can preserve the nucleic acid and / or cells of the sample for an extended period of time. The kit may further comprise a needle that can be attached to said syringe. In some embodiments, the kit is for preserving nucleic acid and / or cells in a blood sample, and further comprises a blood collection tube that optionally contains an anticoagulant. The amount of the optional anticoagulant can be predetermined so that the collected blood sample before the cells or nucleic acid are isolated shows reduced or minimal clotting. Those skilled in the art can easily determine these amounts using conventional methods.

[0087] In some embodiments, the preservative composition is provided in a sealed ampoule, the ampoule including a removable closure, the ampoule configured to receive a dispensing means when the closure is removed by a user. In some embodiments, the dispensing means is a pipette or a syringe. In some embodiments, the kit is for preserving nucleic acids and / or cells in a blood sample and further includes a blood collection tube containing an anticoagulant.

[0088] In additional embodiments, the kit is intended to preserve nucleic acids and / or cells in a blood sample and optionally includes a blood collection tube containing a predetermined amount of an anticoagulant of the present disclosure, and a predetermined amount of a preservative composition.

[0089] In a sixth aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: a. a preservative composition disclosed herein; and b. optionally, instructions for use of the preservative composition.

[0090] In a seventh aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: a. a blood or other biological sample collection tube, optionally containing an anticoagulant; b. a syringe containing a preservative composition of the present disclosure; c. Optionally, a needle that can be attached to the syringe.

[0091] In an eighth aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: a. a blood or other biological sample collection tube, optionally containing an anticoagulant; b. A kit for preserving nucleic acids and / or cells in a biological sample comprising: a sealed ampoule containing a preservative of the present disclosure, the ampoule having a removable closure, and the ampoule configured to receive a dispensing means upon removal of the closure by a user.

[0092] In some embodiments of the sixth to eighth aspects, the biological sample is a cell or tissue sample.

[0093] In some embodiments of the sixth to eighth aspects, the biological sample is derived from a bodily fluid. In some embodiments, the bodily fluid is blood, plasma, serum, urine, saliva, stool, breast milk, tears, sweat, cerebrospinal fluid, synovial fluid, semen, vaginal fluid, peritoneal fluid, or amniotic fluid. In some embodiments, the bodily fluid is whole blood or a fraction thereof. The biological sample may contain cells or may be acellular.

[0094] In some embodiments of the sixth to eighth aspects, the biological sample comprises stem cells, bone cells, blood cells, muscle cells, adipocytes, skin cells, neuronal cells, endothelial cells, sex cells, pancreatic cells, cancer cells, tumor cells, circulating tumor cells, or combinations thereof.

[0095] In some embodiments of the sixth to eighth aspects, the biological sample comprises a nucleic acid selected from RNA, DNA, or a combination thereof. In some embodiments, the nucleic acid is cell-free RNA, cell-free DNA, or a combination thereof. In some embodiments, the nucleic acid is cellular RNA, cellular DNA, or a combination thereof.

[0096] Equivalent The above description and the following examples detail certain embodiments of the present disclosure and explain the best way of carrying out the present disclosure contemplated by the inventors. However, it should be understood that no matter how detailed the above contents are in text, the present disclosure can be carried out in many ways, and the present disclosure should be interpreted according to the attached embodiments and any equivalents thereof.

[0097] Although the present disclosure has been described with respect to various applications, methods, compounds, and compositions, it will be understood that various changes and modifications can be made without departing from the disclosure herein. The following examples are provided to better illustrate the present disclosure and are not intended to limit the scope of the teachings presented herein. Although the present disclosure has been described with respect to these exemplary embodiments, those skilled in the art will readily understand that many variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the present disclosure. EXAMPLES

[0098] Example 1 - Composition The preservative compositions of the present disclosure are shown in Table 1. [Table 1]

[0099] Example 2 - Plasma Volume Analysis Blood samples from various donors are collected in blood sampling tubes to evaluate the plasma volume of samples treated with the preservative composition according to the present disclosure. The preservative composition is tested by adding the blood sample to a tube containing 2 mL of the preservative composition.

[0100] The combination of the preservative composition and blood sample is then centrifuged at 425g for about 15 minutes at room temperature, resulting in the formation of a pellet in the collection tube.Then, without disturbing the separated components, the upper plasma layer (supernatant) is transferred to a separate collection tube using a pipette.The transferred supernatant is then centrifuged again at 4°C and 16,000g for about 15 minutes to remove any inadvertently transferred cell debris or sediment, and the volume of the remaining plasma is measured.The measured volume is referred to herein as "plasma volume".

[0101] Without wishing to be bound by theory, it is anticipated that "plasma volume" will be an important factor in facilitating the use of embodiments of the present disclosure in high throughput applications. The use of automation and robotics in these applications will require a consistent plasma volume, ideally between 3-6 mL.

[0102] The "plasma volumes" of mixtures processed according to the above procedure and stored in tubes containing compositions 1-5, 8-9, and 11-12 were observed to be in the range of 4.3-4.9 mL after 2 days, 4.1-5.2 mL after 7 days, 4.2-5.0 mL after 14 days, and 4.5-5.1 mL after 21 days, all within the desired range.

[0103] Example 3 - Analysis of the integrity of isolated cfDNA and RNA Blood samples from various donors are collected in blood sample collection tubes to evaluate the ability of the present disclosure's embodiment of the preservative composition to preserve cfDNA and RNA.The preservative composition is tested by adding the blood sample to the tube containing 2mL of the preservative composition.

[0104] cfDNA and RNA are isolated from the sample using extraction and separation techniques known in the art.One of these cfDNA extraction methods involves using MagMAX™ Cell-Free DNA Isolation Kit.One of these RNA extraction methods involves using the procedure based on Beckman Coulter's RNAdvance Blood Kit.

[0105] The isolated cfDNA and RNA are analyzed on day 1 and various subsequent days, with blood being drawn on day 0. Nucleic acid integrity is analyzed to assess the properties of the preservative composition.

[0106] cfDNA Integrity The integrity of cfDNA is analyzed by qPCR of long and short DNA fragments and characterized by the ratio of long to short fragments (222bp / 90bp). The resulting ratio is referred to herein as the DNA Integrity Number (DIN). The DIN is an objective metric of cfDNA quality. If the DIN is <0.5, the cfDNA is considered pure (i.e., the plasma is not contaminated by gDNA (cellular or genomic DNA)).

[0107] Prepare a 10-fold dilution series of gDNA (1 ng / µL to 0.01 ng / µL) for the standard curve. Prepare the forward and reverse primer mix at a concentration of 5 µM by mixing 5 µL of 100 µM forward primer, 5 µL of 100 µM reverse primer with 90 µL of nuclease-free water.

[0108] Prepare two separate mixtures using 1 μL of each standard or cfDNA sample and 8 μL of nuclease-free water for one reaction in mixture #1, and 1 μL of primer mixture and 10 μL of 2X PowerTrack SYBR Green Master Mix (ThermoFisher Scientific) for one reaction in mixture #2. Add 9 μL of mixture #1 and 11 μL of mixture #2 to the wells of a 96-well optical plate to perform real-time PCR. The thermal cycling conditions are described in the table below. [Table 2]

[0109] RNA Integrity The integrity of RNA from samples is analyzed by BioAnalyzer using agarose gel electrophoresis and characterized by the RNA Integrity Number (RIN). The RIN is an objective measure of total RNA quality that ranges from 10 (highly intact RNA) to 1 (fully degraded RNA).

[0110] Characteristics of cfDNA in blood samples stored using the compositions in Table 1 : The integrity (i.e., DIN) of cfDNA extracted from blood samples stored in tubes containing compositions 1-5, 8-9, and 11-12 was generally high. The ratios of long to short fragments (222bp / 90bp) from qPCR assays were observed to be within the ranges of <0.1-0.5 after 2 days, <0.1-0.2 after 7 days, <0.1-0.2 after 14 days, and <0.1-0.21 after 21 days.

[0111] Characteristics of RNA in blood samples preserved using the compositions of Table 1 : The RINs of RNA isolated from blood samples stored in tubes containing compositions 1-5, 8-9, and 11-12 were generally high as they were observed to have RINs ranging from 9.3 to 8.8 on day 2, 8.8 to 8.4 on day 3, 8.2 to 7.6 on day 5, 8.0 to 6.9 on day 7, and 7.4 to 5.2 on day 10.

[0112] Any of the individual embodiments recited herein may be used individually or in combination in one or more embodiments of the present invention.

Claims

Claim 1 A preservative composition comprising: a. Optionally, one or more osmotic agents; b. One or more enzyme inhibitors; c. Optionally, one or more metabolic inhibitors; d. Optionally, one or more cell surface remodeling polymers; e. Optionally, one or more agents selected from the group consisting of hydroxyethyl starch, polymers of N-vinylpyrrolidone (NVP), ficoll, protein colloids, non-protein synthetic colloids, ethylene glycol, propylene glycol, water-soluble polymers, and carboxymethyl cellulose, or salts thereof; f. Optionally, polypropylene glycol (PPG), wherein at least one cell surface remodeling polymer (d) or agent (e) is present, and wherein said optionally one or more osmotic agents and said one or more enzyme inhibitors are present in a combined amount sufficient to produce a hypertonic solution.