Systems and methods of isolation of extracellular vesicles

Abstract

Provided herein are methods of isolating extracellular vesicles (EVs) and kits for use with the methods disclosed herein. The methods include, at a first step, subjecting the sample to one or more cycles of phase separation using an aqueous two-phase system (ATPS). The ATPS includes a first phase comprising a first-phase polymer and a second phase comprising a second-phase polymer. The EVs are enriched in the first phase. The methods also include, at a second step, treating the first phase with the enriched EVs with one or more enzymes that degrades the first-phase polymer.

Description

TITLE: SYSTEMS AND METHODS OF ISOLATION OF EXTRACELLULAR VESICLESRELATED APPLICATIONS

[0001] The present application claims priority to United States Provisional Patent Application No. 63 / 605,235 entitled “Systems and Methods of Isolation of Extracellular Vesicles” filed on December 1 , 2023, the entire contents of which are hereby incorporated by reference herein in their entirety for all purposes.FIELD

[0002] The present disclosure relates to methods of isolating extracellular vesicles (EVs), specifically methods of isolating EVs from EV-containing samples using aqueous two-phase system fluids.BACKGROUND

[0003] The field of extracellular vesicles (EVs) is emerging across all biomedical research disciplines. This is because EVs are essentially cell fragments and can contain or express a portion of the proteome, transcriptome, and genome of the surrogate cell that released it. EVs are extremely heterogeneous due to the multiple possible mechanisms of their release by a surrogate cell, and also because all cell types can release EVs (1 ).

[0004] EVs are a clinically promising platform for biomarker development because of their release by all cell types, and their potential to contain the same biomarkers as the surrogate cell. They are also a highly promising type of medicine, as they can be made to contain various therapeutic cargo due to their lipid bilayer enclosure. However, much of the progress on EVs has been slow due to the methods that currently exist to isolate and purify them. These methods are either highly destructive (ultracentrifugation), costly and time consuming (size exclusion column technology), or low throughput (fluorescence- activated cell sorting (FACS)).

[0005] Improved systems and methods of isolating EVs are therefore needed.SUMMARY

[0006] It is demonstrated herein that adding enzymes to the EV-concentrating DEX phase to degrade DEX improves isolation of EVs from an EV-containing sample.

[0007] According to a broad aspect, a method of isolating (EVs from an EV- containing sample is described herein. The method includes:(i) subjecting the sample to one or more cycles of phase separation using an aqueous two-phase system (ATPS) comprising: (a) a first phase comprising a first- phase polymer; and (b) a second phase comprising a second-phase polymer, wherein the EVs are enriched in the first phase; and(ii) treating the first phase with the enriched EVs with one or more enzymes that degrades the first-phase polymer.

[0008] In at least one embodiment, the method also includes repeating steps (i) and (ii), shown above, for at least one time.

[0009] In at least one embodiment, the first phase polymer is dextran (DEX).

[0010] In at least one embodiment, the one or more enzymes that degrades the first-phase polymer is dextranase.

[0011] In at least one embodiment, the second phase polymer is polyethylene glycol (PEG).

[0012] In at least one embodiment, the method also includes treating the first phase with one or more enzymes that degrades the second phase polymer.

[0013] In at least one embodiment, the method also includes isolating a nucleic acid from the isolated EVs.

[0014] In at least one embodiment, the nucleic acid is DNA.

[0015] In at least one embodiment, the nucleic acid is RNA.

[0016] In at least one embodiment, the EV-containing sample is a body fluid.

[0017] In at least one embodiment, the body fluid is blood, plasma, serum, urine, sweat, semen, vaginal secretions, menstrual blood, sputum, or saliva.

[0018] In at least one embodiment, the EV-containing sample is tissue culture media.

[0019] According to a broad aspect, a kit for isolating EVs from an EV-containing sample is described herein. The kit includes:(i) a first-phase polymer and a second-phase polymer of an aqueous two-phase system (ATPS);(ii) an enzyme capable of degrading the first-phase polymer; and, optionally(iii) an instruction manual, wherein the ATPS comprises: (a) a first phase comprising the first phase polymer; and (b) a second phase comprising the second-phase polymer, and wherein the EVs, when subject to phase separation using the ATPS, are enriched in the first phase.

[0020] In at least one embodiment, the first phase polymer is DEX.

[0021] In at least one embodiment, the enzyme capable of degrading the first- phase polymer is dextranase.

[0022] In at least one embodiment, the second phase polymer is polyethylene glycol (PEG).

[0023] In at least one embodiment, the kit also includes an enzyme capable of degrading the second-phase polymer.

[0024] In at least one embodiment, the kit also includes one or more reagents for isolating nucleic acids from the enriched EVs.

[0025] In at least one embodiment, the nucleic acids are RNA.

[0026] In at least one embodiment, the nucleic acids are DNA.

[0027] In at least one embodiment, the kit also includes one or more reagents for antibody labelling.

[0028] In at least one embodiment, the first polymer and the second polymer are provided in solid form.

[0029] In at least one embodiment, the first polymer and the second polymer are provided in solution form.

[0030] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The embodiments of the application will now be described in greater detail with reference to the attached drawings in which:

[0032] FIGs.lA and 1 B show EV isolation using ATPS. FIG. 1A is a schematic diagram of using a DEX-PEG ATPS for isolation of EVs. FIG. 1 B shows three nanoscale flow cytometry (nFC) scatterplot images of a sample before phase separation (left panel), PEG phase (middle panel) and DEX phase (right panel).

[0033] FIGs 2A and 2B are nFC scatterplot images and graphs showing more efficient isolation of EVs using ATPS. FIG. 2A shows nFC scatterplot images of sample before phase separation (left panel), DEX phase (middle panel) and EVs isolated by ultracentrifugation. FIG. 2B shows graphs showing comparison between ATPS and ultracentrifugation.

[0034] FIGs 3A and 3B show that second cycle ATPS improves EV enrichment in DEX phase. FIG. 3A is a schematic diagram of two rounds of phase separation by ATPS. FIG. 3B is a graph showing improved enrichment of EVs with a second round of phase separation.

[0035] FIG. 4 shows nFC scatterplot images of Dextranase at various concentriations and shows that dextranase (DEXnase) treatment does not impact EVs at different concentrations.

[0036] FIGs. 5A-5G show that dextranase treatment of DEX phase prevents formation of dextran-alcohol polymer and results in better quality RNA. FIG. 5A are figures showing dextranase treatment at various concentrations. FIG. 5B is a graph showing dextranase treatment reduces or prevents precipitate formation at various concentrations. FIG. 5C are nanodrop analyses of RNA isolated from EVs after dextranase treatment at various concentrations. FIG. 5D is a graph showing nanodrop analyses of RNA isolated from EVs after dextranase treatment at various concentrations. FIG. 5E are bioanalyzer analysis results of RNA isolated from EVs after dextranase treatment at various concentrations. FIG. 5F is a graph showing bioanalyzer analysis results of RNA isolated from EVs after dextranase treatment at various concentrations. FIG. 5G is a graph showing qPCR analysis on GAPDH copy number of RNA isolated from EVs after dextranase treatment at various concentrations.

[0037] FIGs. 6A and 6B show effects of concentration of dextranase on the volume of the DEX phase when a second round of phase separation is performed after dextranase treatment on the DEX phase from the first round of phase separation. FIG. 6A is a schematic diagram of the steps. FIG. 6B is a graph that shows increasing concentration of dextranase used decreases the volume of DEX phase from the second round of phase separation.

[0038] FIGs. 7A and 7B are graphs showing comparison of EV enrichment capacity (see FIG. 7A) and recovery efficiency (see FIG. 7B) between ATPS, with / without dextranase treatment, and three commercialized EV isolation kits on human plasma sample.DETAILED DESCRIPTION OF THE DISCLOSURE

[0039] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, 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 disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0040] The disclosure aims to use aqueous two-phase system (ATPS) to enhance the sensitivity and precision. The ATPS system is predominantly composed of water, with small amounts of incompatible polymers and / or salts, which separate into two distinct fluid phases at sufficient concentrations. The two phases of ATPS selectively attract different solute molecules based on their affinity partitioning behavior, which is influenced by surface chemistry, charge, and other properties. If the ATPS is selective properly, the resulting partitioning behavior can also gather EVs to just one of the two phases.

[0041] The most commonly used ATPS is composed of polyethylene glycol) (PEG) and dextran (DEX) in water, which separates into two distinct phases at specific concentrations. The PEG-rich phase contains most of the PEG and minimal amount of the DEX. Here the DEX-rich phase is referred to as the DEX phase and the PEG-rich phase is referred to as the PEG phase. Previous studies have used this DEX-PEG ATPS to enrich and separate EVs from the plasma of cancer patients, with all EVs observed to partition into the DEX phase (Shin et al., 2018).

[0042] However, there are challenges in extracting RNAs and DNAs from isolated EVs using the ATPS EV isolation method. Briefly, alcohols (isopropanol, ethanol, etc.) used in most RNA / DNA isolation methods (phenol-chloroform based approaches, column-based approaches, etc.) to precipitate RNA / DNA from aqueous solution also coprecipitate DEX in the system, resulting in a glob-like mass. This glob-like alcohol-DEX precipitation mass prevents RNA / DNA extraction and interferes with downstream analyses. There remains a need for improved nucleic acid isolation from EVs isolated using ATPS. The present disclosure provides a solution by adding enzymes to the EV- concentrating DEX phase to deplete DEX which then enables any alcohols added to effectively precipitate RNA / DNA from EVs.I. Definitions

[0043] In understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components,groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[0044] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0045] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0046] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0047] The term “consisting” and its derivatives, as used herein, are intended to be closed ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0048] Further, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0049] More specifically, the term “about” means plus or minus 0.1 to 20%, 5-20%, or 10-20%, 10%-15%, preferably 5-10%, most preferably about 5% of the number to which reference is being made.

[0050] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, a composition containing “a compound” includes a mixture of two ormore compounds. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0051] The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.

[0052] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0053] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.

[0054] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, examples of methods and materials are now described.II. Methods

[0055] It is disclosed herein that where ATPS is employed for isolating EVs from an EV-containing sample, and the phase where the EVs are enriched comprises a polymer, treating the phase where the EVs are enriched with an enzyme that degrades the polymer constituting the EV-containing phase improves the yield and quality of nucleic acids extracted from the enriched EVs. Without wishing to be bound by a theory, the polymer constituting the phase where the EVs are enriched can co-precipitate with the nucleic acids during extraction, resulting in low yield and / or quality of the extracted nucleic acids.

[0056] It is also disclosed herein that where more than one cycle of phase separation by ATPS is performed, treating the phase where the EVs are enriched with an enzyme that degrades the polymer constituting the EV-containing phase after the first cycle can result in more concentrated EVs in the EV-containing phase of the second ATPS cycle.

[0057] Accordingly, in at least one aspect, provided herein is a method of isolating nucleic acids from extracellular vesicles (EVs), the method comprising:(i) providing enriched EVs;(ii) treating the enriched EVs with one or more enzymes that degrades a first-phase polymer; and(iii) isolating nucleic acids from the EVs, wherein the enriched EVs are obtained from phase separation of an EV-containing sample using an aqueous-two phase system (ATPS) comprising: (a) a first phase comprising the first phase polymer; and (b) a second phase comprising a second-phase polymer, wherein EVs are enriched in the first phase.

[0058] In another aspect, provided herein is a method of isolating nucleic acids from EVs, the method comprising:(i) providing a first phase comprising a first-phase polymer and EVs;(ii) treating the first phase with one or more enzymes that degrades the first-phase polymer; and(iii) isolating nucleic acids from the EVs, wherein the first phase, also referred to herein as an EV-enriching phase, is obtained from phase separation of an EV-containing sample using an ATPS comprising: (a) the first phase comprising the first phase polymer; and (b) a second phase comprising a second-phase polymer, and wherein EVs are enriched in the first phase.

[0059] In yet another aspect, provided herein is a method of isolating nucleic acids from EVs, the method comprising:(i) providing an EV-containing sample;(ii) subjecting the sample to phase separation using an ATPS comprising: (a) a first phase comprising a first-phase polymer; and (b) a second phase comprising a second-phase polymer, wherein the EVs are enriched in the first phase;(iii) treating the phase with the enriched EVs with one or more enzymes that degrades the first-phase polymer; and(iv) isolating nucleic acids from the EVs.ATPS

[0060] As used herein, the term “extracellular vesicle” means any vesicle secreted by a cell into the extracellular space and encompasses, for example, exosomes.

[0061] Enrichment or isolation of EVs by ATPS is well known in the art (See for example, Shin et al., 2015; U.S. Patent No. 11 ,016,009; the contents of which are incorporated by reference herein in their entirety). The two phases of ATPS selectively attract different solute molecules based on their affinity partitioning behavior, which is influenced by surface chemistry, charge, and other properties. The two phases of the ATPS used for isolating EVs can be two different polymers. The ATPS can be prepared, for example, by dissolving the polymers directly in an EV-containing sample. After the polymers are dissolved, the sample is then subject to centrifugation so that the two phases separate. As the phases separate, EVs are enriched in one phase, providing for isolation of EVs from the EV-containing sample.

[0062] As used herein, the term “first phase” refers to the phase where EVs are enriched and is one of the two phases of an ATPS. The other phase of the ATPS is referred to as “second phase”. As used herein, the term “first phase polymer” means the polymer constituting the first phase of the ATPS, and the term “second phase polymer” means the polymer constituting the second phase of the ATPS.

[0063] As used herein, the term “enriched EVs” refers to EVs that have been subject to phase separation by ATPS and as such are enriched in one of the two phases of the ATPS.

[0064] In a PEG-DEX ATPS, for example, EVs are enriched in the DEX phase.

[0065] Accordingly, in at least one embodiment, the first phase comprises dextran(DEX) and the second phase comprises polyethylene glycol (PEG).

[0066] ATPS other than DEX-PEG ATPS, such as FicollTM-PEG ATPS, can be used. Suitable combinations of polymers and their concentrations and their molecular weights that allow isolation of EVs can be determined by a person skilled in the art, for example, as disclosed in Shin et al., 2015.

[0067] More than one round of phase separation by ATPS can be performed. For example, an EV-containing DEX phase from a first round of ATPS separation may be subject to one or more additional rounds of phase separation by addition of fresh PEG phase. As demonstrated herein, subjecting the sample to an additional round of phase separation by ATPS improves enrichment of EVs in the DEX phase and may remove more contaminants.

[0068] Further, the addition of dextranase, for example, following additional rounds of phase separation may improve enrichment of EVs.

[0069] In at least one embodiment, the method further comprises subjecting the sample to at least one additional round of phase separation using ATPS. In at least one embodiment, the method further comprises subjecting the sample to one additional round of phase separation using ATPS. In at least one embodiment, the method further comprises subjecting the sample to two additional rounds of phase separation using ATPS.Enzyme Treatment

[0070] It is demonstrated herein that treating the EV-containing DEX phase with dextranase enables the extraction of nucleic acids from the enriched EVs. It is alsodemonstrated herein that where more than one cycle of ATPS is performed, treating the EV-containing DEX phase from the first ATPS cycle with dextranase results in a volume of the EV-containing DEX phase of the second ATPS cycle that is lower than where the DEX phase from the first ATPS cycle is not treated with dextranase. Accordingly, in at least one embodiment of the methods disclosed herein, treating the first phase comprising enriched EVs includes treating with one or more enzymes that degrade the first phase polymer.

[0071] For example, the one or more enzymes can be added directly to the EV- containing first phase after phase separation. When more than one cycle of phase separation by ATPS is performed, the one or more enzymes can be added to the first phase that contains EVs after each cycle of phase separation, or after any cycle of phase separation.

[0072] Enriched EVs can also be collected from the first phase containing the EVs, for example by centrifugation followed by removal of supernatant, and the EVs can be resuspended in a different solution for enzyme treatment, optionally at a later time. In such case, the one or more enzymes are added to the different solution comprising the enriched EVs.

[0073] Selection of the enzyme is based on the polymers used in the ATPS. The enzyme breaks down the polymer of the phase where the EVs are enriched. For example, where DEX-PEG ATPS is used, the one or more enzymes are dextran-degrading enzymes.

[0074] It should be understood that after enzyme treatment, a small concentration of PEG in the DEX-rich phase may remain intact. Generally, the presence of PEG does not interfere with post RNA / DNA extraction processes. However, in some cases, PEG removal may be required. PEG removal may be by biodegradation, such as but not limited to, aerobic biodegradation, anaerobic biodegradation, and / or extracellular one-electron oxidation.

[0075] In at least one embodiment, the one or more enzymes are dextran- degrading enzymes. In at least one embodiment, the one or more enzymes are dextranase.

[0076] The term “dextranase”, as used herein, means an enzyme that catalyzes hydrolysis of a-1 , 6 glycosidic bonds, a-1 , 3 glycosidic bonds in dextran, and encompasses dextranases from any organism.

[0077] In at least one embodiment, the dextranase is a dextranase from Chaetomium erraticum. In at least one embodiment, the dextranase is a dextranase from Penicillium sp.

[0078] The amount of enzyme to be used depends on the enzymatic activity per unit volume of the source of the enzyme and can be determined by routine experimentation. For example, various volumes of the enzyme can be added and the degree of reduction of formation of dextran-alcohol precipitate can be measured at each volume to determine a suitable amount. If, for example, the downstream application requires nucleic acid extraction from the isolated EVs, quality of the extracted nucleic acid can be measured at each volume of enzyme used to determine a suitable amount.

[0079] After phase separation, the first phase can be further treated with an additional enzyme that degrades the polymer of the second phase. For example, if DEX- PEG ATPS is used, the DEX phase can also be treated with an enzyme that degrades PEG. Such a treatment can reduce or remove any small amount of the second phase polymer present in the first phase and may be beneficial for downstream applications.

[0080] In at least one embodiment, the method further comprises one or more cycles, or rounds, of phase separation before enzyme treatment. For example, after a single round of ATPS-EV isolation is performed, the EVs can be partitioned into, for example, the DEX phase. In at least one embodiment, at this point, the EV-containing DEX phase may be removed, and an enzyme (e.g. dextranase) can be added to the DEX phase to remove the dextran. The remaining concentrated EV solution can be put into another DEX-PEG ATPS to concentrate the EV even more in a new DEX phase.

[0081] In at least one embodiment, the methods described herein include enzyme treatment to degrade polymers after a first round of ATPS EV isolation. In these embodiments, enzyme treatment after a first round of ATPS EV isolation enables a second round of ATPS EV isolation that further increases the concentration of EVs. Serial phase separations may offer a valuable solution to reduce the volume of the DEX phase (first phase) containing EVs. In other words, this process may provide for an increased enrichment yield. Each set of phase separations can be strategically designed to concentrate EVs to just 10% of the original volume, resulting in a 10 times increase in EVs' concentration. This approach may also be beneficial for samples with extremely low EV concentrations, as serial separation effectively prepares a sample with a sufficient number of particles for subsequent analysis.

[0082] It can be appreciated that it is not necessary that enzyme treatment be performed immediately following phase separation. For example, after phase separation, the EV-containing phase may be collected and stored. Enzyme treatment can then be performed at a later time.

[0083] EVs can also be collected from the EV-containing phase, for example by centrifugation followed by removal of supernatant, and the EVs can be resuspended in a different solution for enzyme treatment, optionally at a later time. The EVs can also be stored as a pellet and resuspended for enzyme treatment at a later time. EVs may also remain in the DEX phase and stored in a fridge or ultra-low temperature freezer.EV-Containing Sample

[0084] The methods disclosed herein can be used with any suitable EV-containing sample.

[0085] For example, the EV-containing sample can be a biological sample, such as but not limited to a body fluid. EV-containing body fluids include but are not limited to blood, plasma, serum, urine, sweat, semen, vaginal secretions, menstrual blood, sputum, and saliva.

[0086] The EV-containing sample can be a non-biological sample. For example, EVs can be released by cultured cells or tissues. Media in which such cultured cells and / or tissues are grown can be collected and for use with the method disclosed herein.Nucleic Acids

[0087] In at least one embodiment, the EVs isolated using the methods disclosed herein are present in a polymer-free solution and, therefore may be used for future applications that require polymer-free solutions. One example of such as use may be used nucleic acid extraction and / or isolation.

[0088] Any nucleic acid associated with EVs can be extracted from the EVs isolated using the methods disclosed herein. For example, the nucleic acid can be DNA or RNA, and can be double-stranded or single-stranded. After enzyme treatment, EVs can be lysed, and nucleic acids can be precipitated with an alcohol such as isopropanol and ethanol. Protocols to purify DNA and / or RNA from other cellular or biological components are well known in the art (for example in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in: Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001 ; the contents of which are incorporated herein by reference in their entirety). Commercial nucleic acid extraction kits can also be used with the EVs after enzyme treatment.

[0089] In at least one embodiment, the nucleic acid is DNA. In at least one embodiment, the nucleic acid is RNA.

[0090] The nucleic acid isolated from the EVs isolated using the methods disclosed herein can be used in various downstream applications and analyses, such as but not limited to RT-qPCR and sequencing, such as but not limited to RNA sequencing or DNA sequencing.III. Kits

[0091] In a further aspect, provided herein is a kit for isolating nucleic acids from EVs, the kit comprising:(i) a first-phase polymer and a second-phase polymer of an ATPS; and(ii) an enzyme capable of degrading the first-phase polymer; wherein the ATPS comprises: (a) a first phase comprising the first phase polymer; and (b) a second phase comprising the second-phase polymer, and wherein the EVs, when subject to phase separation using the ATPS, are enriched in the first phase.

[0092] In at least one embodiment, the kit further comprises an instruction manual.

[0093] The kits provided herein can be used with the methods disclosed herein.

[0094] The first polymer and the second polymer can be in any suitable form. For example, the first polymer and the second polymer can be provided in solid form in the kit, which can be resuspended in a buffer or dissolved directed in an EV-containing sample by a user. The first polymer and the second polymer can be provided in solution form in the kit. The first polymer and the second polymer can be provided as separate components in the kit. The first polymer and the second polymer can be provided premixed in the kit.

[0095] In at least one embodiment, the first polymer is DEX, and the second polymer is PEG.

[0096] In at least one embodiment, the enzyme is a dextranase.

[0097] The kit can comprise other components and / or reagents. For example, the kit can comprise reagents for extraction or isolation of nucleic acids from the enriched EVs after enzyme treatment using for example a method that involves alcohol precipitation. The kit can also comprise components such as columns and collection tubes.

[0098] In at least one embodiment, the kit further comprises one or more reagents for isolating nucleic acids from the enriched EVs.

[0099] In at least one embodiment, the kit is for isolation of RNA from EVs.EXAMPLES

[0100] Example 1

[0101] In this example, EV-containing cell culture conditioned media (CM) was processed through one-step PEG / DEX ATPS. DEX phase was collected and aliquoted. Dextranase from Chaetomium erraticum (Sigma) was mixed with EV containing DEX phase at multiple concentrations, volume by volume (i.e. 0%, 0.01 %, 0.05%, 0.1 %, 0.5%, 1 %, 5%, 10%). After incubating at 37 °C for 30 minutes, dextranase treated samples were then processed to downstream nFC analysis, RNA isolation, etc.

[0102] Example 2

[0103] In this example, EVs were generated from PC3-zsGreen cells by incubating the cells in serum-free growth media for 72 hours. PC3-zsGreen is a transgenic cell line with cytosolic expression of green fluorescent protein, zsGreenl . EVs generated from this cell line could be measured by nanoscale flow cytometry (nFC) analysis based on green fluorescence signal. EV concentrations in CM pre-ATPS, PEG phase and DEX phase post-ATPS, and ultracentrifugation processed CM EVs were measured with nFC analysis. Total EV counts were calculated based on volume of samples to compare EV recovery of both methods.

[0104] EVs were enriched in the DEX phase according to the methodology shown in FIG. 1A. FIG. 1 B shows nanoscale flow cytometry (nFC) scatterplot images of a sample before phase separation (left panel), PEG phase (middle panel) and DEX phase (right panel).

[0105] ATPS has greater purification capability than ultracentrifugation (see FIGs. 2A-2B). Adding a second step ATPS improved EV enrichment in DEX phase (see FIG. 3A for the methodology used here and FIG. 3B fora graph showing improved enrichment of EVs with a second round of phase separation).

[0106] Example 3

[0107] In this example, EVs from PC3-zsGreen cell culture CM were isolated in DEX phase through PEG / DEX ATPS. DEX phase was treated with dextranase at multiple concentrations, v / v, at 37 °C for 30 minutes. EV concentration before and after dextranasetreatment was measured with nFC analysis. FIG. 4 shows that dextranase concentration does not impact EV isolation.

[0108] Example 4

[0109] In this example, dextranase treated DEX phase containing PC3-zsGreen EVs were processed through Trizol-chloroform RNA isolation. White dextran-isopropanol precipitates were generated in samples with no or little amount of dextranase treatment (concentrations lower than 0.5% v / v). RNA isolation was carried on. Precipitate volume was measured after diluting the pellet in water. FIGs. 5A-5B show that volume of dextranalcohol polymer negatively correlated with dextranase input.

[0110] Example s

[0111] In this example, EV RNA was isolated from DEX phase treated with dextranase at multiple concentrations. Dextran-isopropanol precipitates formed in DEX phase treated with less than 0.5% dextranase. RNA samples with / without precipitates were processed through downstream nanodrop, bioanalyzer analysis, and qPCR to validate RNA yield and quality. RNA yield is much lower in samples with precipitates. Quality of RNA improved with dextranase treatment (see FIGs. 5C-5G).

[0112] Example 6

[0113] Besides improving RNA yield, dextranase could also improve other aspects of EV isolation with ATPS. In this example, a DEX phase was treated after one-step ATPS with dextranase at multiple concentrations. These samples were then processed in a second round of ATPS. This was performed to look for optimal dextranase treatment concentration to control dextran content for repeatable ATPS. FIGs. 6A and 6B show effects of concentration of dextranase on the volume of the DEX phase when a second round of phase separation is performed after dextranase treatment on the DEX phase from the first round of phase separation.

[0114] FIGs. 7A and 7B are graphs showing comparison of EV enrichment capacity (7A) and recovery efficiency (7B) between ATPS, with / without dextranase treatment, and three commercialized EV isolation kits on human plasma samples.

[0115] Example ?

[0116] In another example, copper ions and PEG-EDTA may be used to control dextranase enzymatic activity for repeatable ATPS. Additional options such as alginate beads and activated carbon may also be used.

[0117] The use of copper ions was reported to inhibit dextranase enzymatic activity in literature: Pittrof, S. L., Kaufhold, L., Fischer, A., & Wefers, D. (2021 ). Products Released from Structurally Different Dextrans by Bacterial and Fungal Dextranases. Foods (Basel, Switzerland), 10(2), 244.

[0118] CuCI2may be used to stop dextranase activity after treatment.

[0119] While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0120] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers and / or biomarker sequences (e.g. protein and / or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely.

[0121] The scope of the claims should not be limited by the preferred embodiments and examples, but should be given the broadest interpretation consistent with the description as a whole.CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATIONNomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) Enzyme Nomenclature: Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes. Academic Press; Cambridge, MA, USA: 1965.Khalikova E, Susi P, Korpela T. Microbial dextran-hydrolyzing enzymes: fundamentals and applications. Microbiol Mol Biol Rev. 2005 Jun;69(2):306-25.Barzkar N, Babich 0, Das R, Sukhikh S, Tamadoni Jahromi S, Sohail M. Marine Bacterial Dextranases: Fundamentals and Applications. Molecules. 2022 Aug 28;27(17):5533.Shin H, Park YH, Kim Y-G, Lee JY, Park J (2018) Aqueous two-phase system to isolate extracellular vesicles from urine for prostate cancer diagnosis. PLoS ONE 13(3): e0194818.

Claims

CLAIMSWhat is claimed is:1 . A method of isolating extracellular vesicles (EVs) from an EV-containing sample, the method comprising:(i) subjecting the sample to one or more cycles of phase separation using an aqueous two-phase system (ATPS) comprising: (a) a first phase comprising a first- phase polymer; and (b) a second phase comprising a second-phase polymer, wherein the EVs are enriched in the first phase; and(ii) treating the first phase with the enriched EVs with one or more enzymes that degrades the first-phase polymer.

2. The method of claim 1 , further comprising repeating steps (i) and (ii) for at least one time.

3. The method of claim 1 or claim 2, wherein the first phase polymer is dextran (DEX).

4. The method of claim 3, wherein the one or more enzymes that degrades the first- phase polymer is a dextranase.

5. The method of any one of claims 1 to 4, wherein the second phase polymer is polyethylene glycol (PEG).

6. The method of any one of claims 1 to 5, further comprising treating the first phase with one or more enzymes that degrades the second phase polymer.

7. The method of any one of claims 1 to 6, further comprising isolating a nucleic acid from the isolated EVs.

8. The method of claim 7, wherein the nucleic acid is DNA.

9. The method of claim 7, wherein the nucleic acid is RNA.

10. The method of any one of claims 1 to 9, wherein the EV-containing sample is a body fluid.11 . The method of claim 10, wherein the body fluid is blood, plasma, serum, urine, sweat, semen, vaginal secretions, menstrual blood, sputum, or saliva.

12. The method of any one of claims 1 to 9, wherein the EV-containing sample is tissue culture media.

13. A kit for isolating extracellular vesicles (EVs) from an EV-containing sample, the kit comprising(i) a first-phase polymer and a second-phase polymer of an aqueous two-phase system (ATPS);(ii) an enzyme capable of degrading the first-phase polymer; and optionally(iii) an instruction manual, wherein the ATPS comprises: (a) a first phase comprising the first phase polymer; and (b) a second phase comprising the second-phase polymer, and wherein the EVs, when subject to phase separation using the ATPS, are enriched in the first phase.

14. The kit of claim 13, wherein the first phase polymer is dextran (DEX).

15. The kit of claim 13 or 14, wherein the enzyme capable of degrading the first- phase polymer is dextranase.

15. The kit of any one of claims 13 to 15, wherein the second phase polymer is polyethylene glycol (PEG).

16. The kit of any one of claims 13 to 15, further comprising an enzyme capable of degrading the second-phase polymer.

17. The kit of any one of claims 13 to 16 further comprising one or more reagents for isolating nucleic acids from the enriched EVs.

18. The kit of claim 17, wherein the nucleic acids are RNA.

19. The kit of claim 17, wherein the nucleic acids are DNA.

20. The kit of any one of claims 13 to 16 further comprising one or more reagents for antibody labelling.21 . The kit of any one of claims 13 to 20, wherein the first polymer and the second polymer are provided in solid form.

22. The kit of any one of claims 13 to 20, wherein the first polymer and the second polymer are provided in solution form.

Images