WO2017185086A1

WO2017185086A1 - Devices and methods for in vivo capture of biological samples and nucleic acids therein

Info

Publication number: WO2017185086A1
Application number: PCT/US2017/029117
Authority: WO
Inventors: Johan Karl Olov Skog
Original assignee: Exosome Diagnostics, Inc.
Priority date: 2016-04-22
Filing date: 2017-04-24
Publication date: 2017-10-26

Abstract

The invention provides devices and methods for isolating, detecting, and analyzing molecules, cells, and other biological samples or fractions thereof from a subject, and to methods of isolating, detecting, and analyzing nucleic acids from such biological samples or fractions thereof, including cell-free DNA and/or cell-free DNA and nucleic acids including at least RNA from microvesicles, and to methods for extracting nucleic acids from the microvesicles and/or from the biological samples and/or by using the device intracorporeally for collecting nucleic acids or cells or other target molecules by in-vivo collection. In one embodiment, a capturing agent for nucleic acids is bound to a polymer fibre. In another embodiment, the device contain an inner space with a functionalised surface for binding the relevant target structure and removing a portion of the body fluid.

Description

DEVICES AND METHODS FOR IN VIVO CAPTURE OF BIOLOGICAL SAMPLES AND NUCLEIC ACIDS THEREIN

RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Application No.

62/326,242, filed on April 22, 2016; the contents of which are hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention provides novel devices, compositions, methods, and kits for isolating, detecting, and analyzing molecules, cells, and other biological samples or fractions thereof from a subject, and to methods of isolating, detecting, and analyzing nucleic acids from such biological samples or fractions thereof, including cell-free DNA and/or cell-free DNA and nucleic acids including at least RNA from microvesicles, and to methods for extracting nucleic acids from the microvesicles and/or from the biological samples.

BACKGROUND

[0003] Current systems and methods for detecting nucleic acids in biological samples often fail to identify rare nucleic acids. For example, mutant nucleic acids in biological samples are often found in low copy numbers and cannot be readily and reliably identified in biological samples without the use of large sample volumes.

[0004] Accordingly, there is a need for devices, compositions, methods, and kits that readily and reliably identify low copy number nucleic acids in biological samples without the use of large sample volumes.

SUMMARY OF THE INVENTION

[0005] The invention provides novel devices, compositions, methods, and kits for isolating, detecting, and analyzing molecules, cells, and other biological samples or fractions thereof from a subject, and to methods of isolating, detecting, and analyzing nucleic acids from such biological samples or fractions thereof, including cell-free DNA and/or cell-free DNA and nucleic acids including at least RNA from microvesicles, and to methods for extracting nucleic acids from the microvesicles and/or from the biological samples.

[0006] The devices, compositions, methods, and kits of the disclosure are an improvement over current systems and methods for detecting nucleic acids such as cell-free DNA ("cfDNA") and/or circulating tumor cells ("CTC"). Mutant copies of nucleic acids, also referred to herein as "MT nucleic acids," in cfCDNA and/or CTCs are rare. In particular, cfDNA mutation copies are often limited, and even further limited in CTCs. (See e.g., Betegowda et al. Science Translational Medicine 2014, the contents of which are hereby incorporated by reference in their entirety). The devices, compositions, methods, and kits of the disclosure provide a solution to this problem of low copy number of mutant nucleic acids and thereby enable early screening. In contrast to the devices, compositions, methods, and kits of the disclosure, current systems and/or methods of detecting mutant nucleic acids in cfDNA ask for larger and larger blood volume draws to allow for the ability to detect rare copies of MT nucleic acids. The devices, compositions, methods, and kits of the disclosure avoid this problem by using in vivo isolation devices and methods to alleviate the need for large sample volumes of biofluids to ensure detection of MT nucleic acids.

[0007] In some embodiments, the devices, compositions, methods, and kits of the disclosure allow for the in vivo isolation or other capture of the microvesicle fraction from a biological sample.

[0008] In some embodiments, the devices, compositions, methods, and kits of the disclosure allow for the in vivo isolation or other capture of the microvesicle fraction and cfDNA from a biological sample.

[0009] In some embodiments, the devices, compositions, methods, and kits of the disclosure allow for the in vivo isolation or other capture of the microvesicle fraction, cfDNA, and CTCs from a biological sample.

[0010] In one aspect, a device of the disclosure is used to capture blood or a fraction thereof. In some embodiments, the device is inserted in a selected vein or artery depending on convenience and/or location of the disease. In some embodiments, the vein or artery is selected to increase sensitivity based on the disease location. Nucleic acids from the microvesicle fraction, e.g., biomarkers, and cfDNA are removed by the reticuloendothelial system and may reduce the analyte concentration after passage, for example, through lymph nodes, spleen and/or liver. [0011] In vivo capture of the nucleic acids from the microvesicle fraction and/or cfDNA, also referred to herein collectively as the biomarker analytes, is performed on a selective capture matrix. Any suitable selective capture matrix can be used. For example, a suitable selective capture matrix uses size selectivity (e.g., size exclusion and/or capture) and/or affinity selectivity, such as, by way of non-limiting examples, charge selectivity and/or use of a ligand as a means for selectivity. Suitable ligands include antibodies, e.g., antibodies that detect a marker such as, for example, EpCAM, a marker for epithelial cancer, EGFR, a marker for cancer, PSMA, a marker for prostate cancer, EGFRv3, a marker for glioblastoma, specific fusion antibodies, for example, ALK fusion antibodies like VENT ANA™™ anti-ALK antibody, and other antibody-based ligands; aptamers and other nucleic acid-based ligands; and ligand specific peptides and other polypeptide-based ligands.

[0012] Other suitable selective capture matrices include those described in PCT

Publication No. WO 2016/007755, the contents of which are hereby incorporated by reference in their entirety.

[0013] In some embodiments, the device includes medical stainless steel wire, for example, with a diameter of 0.5 mm, a rounded tip with a long layer of pure gold, e.g., a 2 cm long layer, which is a carrier of a three-dimensional polymer functionalized layer. In some embodiments, the device functionalizes through the use of a selective capture matrix or combination of selective captive matrices. In some embodiments, the device

functionalizes through the use of specific antibodies against cell surface antigens (e.g. anti- EpCAM antibodies).

[0014] Devices of these embodiments and methods of use thereof overcome the restrictions of a limited blood sample. While most previous diagnostic approaches have focused on maximizing the efficient exploitation of a blood sample, this device is designed to collect the biomarker in vivo from the peripheral blood stream.

[0015] During the in vivo application of this device in a vessel, e.g., in cubital arm vein, e.g., a 30 minute in vivo application of the device, the device comes into contact with a large volume of the patient's total blood volume. This is a significantly higher quantity of blood than found in blood samples using in vitro methods. Even at low concentrations of CTCs in the blood, the approach of the device increases the chances of isolating the biomarker. [0016] In some embodiments, the device includes a capture structure that uses a plurality of carbon nanotubes or similar nanotubes that are appropriately spaced. In some embodiments, the nanotubes are functionalized with one or more target-specific ligands. Schematic representations of an exemplary device that uses carbon nanotubes is shown in Figures 1A, IB, and 2.

[0017] The spacing between nanotubes is optimized for the vesicle type intended to be isolated. For example, in some embodiments, the diameter and spacing between nanotubes should be modified to handle blood inside a vein and optimized for surface area to capture targets such as microvesicles and/or cfDNA.

[0018] In some embodiments, the spacing between nanotubes in the plurality of the nanotubes is in the range of about 10 nm to 2000 nm, or any range or value contained therein. In some embodiments, the spacing between nanotubes in the range of about 20 to 2000 nm. In some embodiments, the spacing between nanotubes is designed to allow for the isolation and capture of a majority of vesicles, while excluding platelets, such as for example, the spacing between nanotubes is in the range of 20 nm to 800 nm, which allows. In some embodiments, the spacing between nanotubes in the range of about 10 to 800 nm. In some embodiments, the spacing between nanotubes in the range of about 10 to 200 nm. In some embodiments, the spacing between nanotubes in the range of about 20 to 200 nm. In some embodiments, the spacing between nanotubes in the range of about 10 to 100 nm. In some embodiments, the spacing between nanotubes in the range of about 20 to 100 nm.

[0019] In some embodiments, the device allows targeting of different samples or fractions thereof, by controlling the functionalization strategy and/or the inter-carbon nanotube spacing (i.e., the forest porosity). Further flexibility is then provided by the possibility of tailoring the forest using standard photolithography, thus allowing for a variety of feature designs. Thus, the devices and methods of use thereof provided herein represent a versatile approach to multiscale bioparticle isolation applicable to a number of applications, from global health diagnostics and particle enrichment/depletion. Other suitable "tuning" of the carbon nanotubes includes any of a variety of art-recognized methods, such as, by way of non-limiting example, those described in Fachin, F. et al. "Integration of vertically-aligned carbon nanotube forests in microfluidic devices for multiscale isolation of bioparticles." 2010 IEEE SENSORS Journal, the contents of which are hereby incorporated by reference in their entirety. The methods described in Fachin et al, which use a microfluidic channel, are schematically represented in Figure 1, and these methods are modified in the devices and methods provided herein to fit on the devices of the disclosure.

[0020] The devices and the methods of use thereof are useful to capture

microvesicles and/or cfDNA. Without intending to be bound by theory, it is believed that most DNA is in vesicles (e.g., apoptotic bodies) or attached to the surface of the vesicles (e.g., exosomes, microvesicles, platelets and lymphocytes). Other cfDNA exist in nucleosome complexes or smaller free fragments. In the devices and methods of the disclosure, the vesicle DNA is captured by the microvesicle platform, and non-vesicle DNA is captured by multiple methods on the same or different region of the functionalized tip on the device. In some embodiments, the non-vesicle DNA is captured using a positive charge matrix, such as, for example, quaternary ammonium, an antibody or other ligand-based method, DNA, histones, and any other suitable capture method such as hybridization and/or intercalating.

[0021] In some embodiments, the device has optimized the surface area of the functionalized device to enable capture of large quantities of the target. The structure is optimized to maximize blood/surface interaction and enable antibody/aptamer engagement. Blood flow rate in the cubital veins are approximately 8 cm/sec. In some embodiments, the device has optimized compatibility to lysis buffers for nucleic acid (for example guanidinium thiocyanate). In some embodiments, the device has optimized safety for intravascular use and is non-reactive, for example, using any of a variety of art-recognized materials and methodologies used in the catheter space. Modem catheters are made of Teflon and polyvinyl chloride and polyethylene. In the United States, more than 25 million patients get a peripheral venous line each year.

[0022] In some embodiments, the device includes a flexible catheter. Flexible catheters can reduce endothelial injury which can lead to thrombosis, but they are more difficult to insert. The training and experience of the surgeon are vital factors in optimizing catheter implantation and ensuring successful outcomes.

[0023] Surface coatings modify catheter properties such as thrombogenicity, friction coefficient or antimicrobial properties, but in experimental surgery it must be remembered that coatings applied to implants may be biologically active and capable of influencing data. Pilot studies may be required to generate data characterizing changes caused by such materials and consideration should be given to suitable controls in experiments. [0024] The physical shape of a catheter tip can play a significant role in reducing endothelial trauma. Many commercially available catheters have a rounded tip which is considered to be less traumatic than square cut tubing or bevel ended tubing, although the latter is easiest to insert.

[0025] In some embodiments, the catheter in the device has a pore size that is sufficient large so as to minimize or otherwise avoid issues related to platelet reactivity to the material. In some embodiments, the catheter in the device has a pore size larger than 2 micrometer.

[0026] In some embodiments, the catheter used in a device of the disclosure exhibits one or more of these desirable biological properties: non-irritant - provokes minimal inflammatory response; non-carcinogenic - low tendency to cause neoplasia; non- thrombogenic - low tendency to cause blood clotting; non-toxic; resists microbial adhesion; and/or resists biofilm deposition.

[0027] In some embodiments, the catheter used in a device of the disclosure exhibits one or more of these desirable physical properties: high tensile strength; resists compression - maintains lumen patency; optimum flexibility; low friction coefficient; dimensional stability; tolerates physical sterilization methods (e.g. heat, steam, irradiation); ease of fabrication (e.g. heat forming or welding); non-permeable (water, gases, solvents); and/or radiopacity - ability to image catheter with X-rays.

[0028] In some embodiments, the catheter used in a device of the disclosure exhibits one or more of these desirable chemical properties: absence of leachable additives (e.g. catalysts and plasticizers); stable during storage; stable on chemical sterilization; stable on implantation (non-biodegradable); permits adhesives in fabrication (possibility of bonding dissimilar materials); accepts surface coatings (e.g. hydrogel, antithrombotic, antibacterial); compatibility with chemical compounds and solvents (absence of absorption and chemical reaction); and/or MRI (Magnetic Resonance Imaging) compatibility.

[0029] The disclosure also provides devices and methods of use thereof that use extracorporeal filtration. In some embodiments, the device uses extracorporeal filtration to isolate a desired molecule, cell, and other target from a biological sample or a fraction thereof, and/or a nucleic acid contained therein. A schematic representation of an exemplary device that uses extracorporeal filtration is shown in Figure 3.

[0030] A device of the disclosure with extracorporeal removal of cfDNA, vesicles and CTCs (or combinations thereof) is similar to a dialysis machine, but the capture is targeted to these biomarkers. Capture is accomplished using any of a variety of selectivity criteria, including by way of non-limiting example, size selectivity, e.g., size exclusion and/or capture; affinity, charge, or ligand(s) directed to a specific target as a means for selectivity. Suitable ligands include antibodies, e.g., antibodies that detect a marker such as, for example, EpCAM, a marker for epithelial cancer, EGFR, a marker for cancer, PSMA, a marker for prostate cancer, EGFRv3, a marker for glioblastoma, specific fusion antibodies, for example, ALK fusion antibodies like VENT ANA™™ anti-ALK antibody, and other antibody-based ligands; aptamers and other nucleic acid-based ligands; and ligand specific peptides and other polypeptide-based ligands.

[0031] Other suitable selective capture matrices include those described in PCT

[0032] In some embodiments, the extracorporeal portion of the device includes a filter that is functionalized with one or more appropriate capture reagents. In some embodiments, the devices and methods of the disclosure are based on any of a variety of art- recognized devices and methods used in current dialysis technology that have been modified for use in the devices and methods provided herein.

[0033] In some embodiments, the devices and methods of use thereof that use extracorporeal filtration use plasmapheresis methodology. In some embodiments, the blood cells are returned to circulation, but a portion of the blood, e.g., the plasma and/or serum, is collected using a selective capture matrix that captures the intended target. For example, suitable selective capture matrices in these methods include those described in PCT

Publication No. WO 2016/007755, the contents of which are hereby incorporated by reference in their entirety. These devices and methods that use plasmapheresis follow current safety guidelines, such as those set forth by the FDA.

[0034] In any of the foregoing methods, the biological sample is a bodily fluid. The bodily fluids can be fluids isolated from anywhere in the body of the subject, preferably a peripheral location, including but not limited to, for example, blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid and combinations thereof. For example, the bodily fluid is blood or a compartment thereof, such as plasma and/or serum. [0035] In any of the foregoing methods, the nucleic acids are DNA and/or DNA and

RNA. Examples of RNA include messenger RNAs, transfer RNAs, ribosomal RNAs, small RNAs (non-protein-coding RNAs, non-messenger RNAs), microRNAs, piRNAs, exRNAs, snRNAs and snoRNAs.

[0036] In any of the foregoing methods, the nucleic acids are isolated from or otherwise derived from a sample, including RNA isolated from the microvesicle fraction of a sample.

[0037] In any of the foregoing methods, the nucleic acids are cell-free nucleic acids, also referred to herein as circulating nucleic acids. In some embodiments, the cell-free nucleic acids are DNA or RNA.

[0038] Various aspects and embodiments of the invention will now be described in detail. It will be appreciated that modification of the details may be made without departing from the scope of the invention. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0039] All patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representations as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

BRIEF DESCRIPTION OF THE FIGURES

[0040] Figure 1 A is a schematic representations demonstrating the use of carbon nanotubes on a microfluidic channel to isolate an intended target. The devices and methods of the disclosure that use carbon nanotubes modify the pore diameter and spacing between the nanotubes to fit on the device and to allow the device to process blood inside a vessel, e.g., artery or vein, and to optimize the surface area on the carbon nanotubes to capture the intended target, e.g., microvesicles and/or cfDNA.

[0041] Figure IB is a series of schematic representations of enlarged views of the functionalized tip of a device of the disclosure. [0042] Figure 2 is a schematic representation of embodiments that use carbon nanotubes.

[0043] Figure 3 is a schematic representation of an exemplary device that uses extracorporeal filtration.

DETAILED DESCRIPTION OF THE INVENTION

[0044] The invention provides novel devices, compositions, methods, and kits for isolating, detecting, and analyzing molecules, cells, and other biological samples or fractions thereof from a subject, and to methods of isolating, detecting, and analyzing nucleic acids from such biological samples or fractions thereof, including cell-free DNA and/or cell-free DNA and nucleic acids including at least RNA from microvesicles, and to methods for extracting nucleic acids from the microvesicles and/or from the biological samples.

[0045] The devices, compositions, methods, and kits of the disclosure are an improvement over current systems and methods for detecting nucleic acids such as cell-free DNA ("cfDNA") and/or circulating tumor cells ("CTC"). Mutant copies of nucleic acids, also referred to herein as "MT nucleic acids," in cfCDNA and/or CTCs are rare. In particular, cfDNA mutation copies are often limited, and even further limited in CTCs. (See e.g., Betegowda et al. Science Translational Medicine 2014, the contents of which are hereby incorporated by reference in their entirety). The devices, compositions, methods, and kits of the disclosure provide a solution to this problem of low copy number of mutant nucleic acids and thereby enable early screening. In contrast to the devices, compositions, methods, and kits of the disclosure, current systems and/or methods of detecting mutant nucleic acids in cfDNA ask for larger and larger blood volume draws to allow for the ability to detect rare copies of MT nucleic acids. The devices, compositions, methods, and kits of the disclosure avoid this problem by using in vivo isolation devices and methods to alleviate the need for large sample volumes of biofluids to ensure detection of MT nucleic acids.

[0046] In some embodiments, the devices, compositions, methods, and kits of the disclosure allow for the in vivo isolation or other capture of the microvesicle fraction from a biological sample.

[0047] In some embodiments, the devices, compositions, methods, and kits of the disclosure allow for the in vivo isolation or other capture of the microvesicle fraction and cfDNA from a biological sample. [0048] In some embodiments, the devices, compositions, methods, and kits of the disclosure allow for the in vivo isolation or other capture of the microvesicle fraction, cfDNA, and CTCs from a biological sample.

[0049] In one aspect, a device of the disclosure is used to capture blood or a fraction thereof. In some embodiments, the device is inserted in a selected vein or artery depending on convenience and/or location of the disease. In some embodiments, the vein or artery is selected to increase sensitivity based on the disease location. Nucleic acids from the microvesicle fraction, e.g., biomarkers, and cfDNA are removed by the reticuloendothelial system and may reduce the analyte concentration after passage, for example, through lymph nodes, spleen and/or liver.

[0050] In vivo capture of the nucleic acids from the microvesicle fraction and/or cfDNA, also referred to herein collectively as the biomarker analytes, is performed on a selective capture matrix. Any suitable selective capture matrix can be used. For example, a suitable selective capture matrix uses size selectivity (e.g., size exclusion and/or capture) and/or affinity selectivity, such as, by way of non-limiting examples, charge selectivity and/or use of a ligand as a means for selectivity. Suitable ligands include antibodies, e.g., antibodies that detect a marker such as, for example, EpCAM, a marker for epithelial cancer, EGFR, a marker for cancer, PSMA, a marker for prostate cancer, EGFRv3, a marker for glioblastoma, specific fusion antibodies, for example, ALK fusion antibodies like VENT ANA™™ anti-ALK antibody, and other antibody-based ligands; aptamers and other nucleic acid-based ligands; and ligand specific peptides and other polypeptide-based ligands.

[0051] Other suitable selective capture matrices include those described in PCT

[0052] In some embodiments, the device includes medical stainless steel wire, for example, with a diameter of 0.5 mm, a rounded tip with a long layer of pure gold, e.g., a 2 cm long layer, which is a carrier of a three-dimensional polymer functionalized layer. In some embodiments, the device functionalizes through the use of a selective capture matrix or combination of selective captive matrices. In some embodiments, the device

functionalizes through the use of specific antibodies against cell surface antigens (e.g. anti- EpCAM antibodies). [0053] Devices of these embodiments and methods of use thereof overcome the restrictions of a limited blood sample. While most previous diagnostic approaches have focused on maximizing the efficient exploitation of a blood sample, this device is designed to collect the biomarker in vivo from the peripheral blood stream.

[0054] During the in vivo application of this device in a vessel, e.g., in cubital arm vein, e.g., a 30 minute in vivo application of the device, the device comes into contact with a large volume of the patient's total blood volume. This is a significantly higher quantity of blood than found in blood samples using in vitro methods. Even at low concentrations of CTCs in the blood, the approach of the device increases the chances of isolating the biomarker.

[0055] In some embodiments, the device includes a capture structure that uses a plurality of carbon nanotubes or similar nanotubes that are appropriately spaced. In some embodiments, the nanotubes are functionalized with one or more target-specific ligands. Schematic representations of an exemplary device that uses carbon nanotubes is shown in Figures 1A, IB, and 2.

[0056] The spacing between nanotubes is optimized for the vesicle type intended to be isolated. For example, in some embodiments, the diameter and spacing between nanotubes should be modified to handle blood inside a vein and optimized for surface area to capture targets such as microvesicles and/or cfDNA.

[0057] In some embodiments, the spacing between nanotubes in the plurality of the nanotubes is in the range of about 10 nm to 2000 nm, or any range or value contained therein. In some embodiments, the spacing between nanotubes in the range of about 20 to 2000 nm. In some embodiments, the spacing between nanotubes is designed to allow for the isolation and capture of a majority of vesicles, while excluding platelets, such as for example, the spacing between nanotubes is in the range of 20 nm to 800 nm, which allows. In some embodiments, the spacing between nanotubes in the range of about 10 to 800 nm. In some embodiments, the spacing between nanotubes in the range of about 10 to 200 nm. In some embodiments, the spacing between nanotubes in the range of about 20 to 200 nm. In some embodiments, the spacing between nanotubes in the range of about 10 to 100 nm. In some embodiments, the spacing between nanotubes in the range of about 20 to 100 nm.

[0058] In some embodiments, the device allows targeting of different samples or fractions thereof, by controlling the functionalization strategy and/or the inter-carbon nanotube spacing (i.e., the forest porosity). Further flexibility is then provided by the possibility of tailoring the forest using standard photolithography, thus allowing for a variety of feature designs. Thus, the devices and methods of use thereof provided herein represent a versatile approach to multiscale bioparticle isolation applicable to a number of applications, from global health diagnostics and particle enrichment/depletion. Other suitable "tuning" of the carbon nanotubes includes any of a variety of art-recognized methods, such as, by way of non-limiting example, those described in Fachin, F. et al. "Integration of vertically-aligned carbon nanotube forests in microfluidic devices for multiscale isolation of bioparticles." 2010 IEEE SENSORS Journal, the contents of which are hereby incorporated by reference in their entirety. The methods described in Fachin et al, which use a microfluidic channel, are schematically represented in Figure 1, and these methods are modified in the devices and methods provided herein to fit on the devices of the disclosure.

[0059] The devices and the methods of use thereof are useful to capture microvesicles and/or cfDNA. Without intending to be bound by theory, it is believed that most DNA is in vesicles (e.g., apoptotic bodies) or attached to the surface of the vesicles (e.g., exosomes, microvesicles, platelets and lymphocytes). Other cfDNA exist in nucleosome complexes or smaller free fragments. In the devices and methods of the disclosure, the vesicle DNA is captured by the microvesicle platform, and non-vesicle DNA is captured by multiple methods on the same or different region of the functionalized tip on the device. In some embodiments, the non-vesicle DNA is captured using a positive charge matrix, such as, for example, quaternary ammonium, an antibody or other ligand-based method, DNA, histones, and any other suitable capture method such as hybridization and/or intercalating.

[0060] In some embodiments, the device has optimized the surface area of the functionalized device to enable capture of large quantities of the target. The structure is optimized to maximize blood/surface interaction and enable antibody/aptamer engagement. Blood flow rate in the cubital veins are approximately 8 cm/sec. In some embodiments, the device has optimized compatibility to lysis buffers for nucleic acid (for example guanidinium thiocyanate). In some embodiments, the device has optimized safety for intravascular use and is non-reactive, for example, using any of a variety of art-recognized materials and methodologies used in the catheter space. Modem catheters are made of Teflon and polyvinyl chloride and polyethylene. In the United States, more than 25 million patients get a peripheral venous line each year. [0061] In some embodiments, the device includes a flexible catheter. Flexible catheters can reduce endothelial injury which can lead to thrombosis, but they are more difficult to insert. The training and experience of the surgeon are vital factors in optimizing catheter implantation and ensuring successful outcomes.

[0062] Surface coatings modify catheter properties such as thrombogenicity, friction coefficient or antimicrobial properties, but in experimental surgery it must be remembered that coatings applied to implants may be biologically active and capable of influencing data. Pilot studies may be required to generate data characterizing changes caused by such materials and consideration should be given to suitable controls in experiments.

[0063] The physical shape of a catheter tip can play a significant role in reducing endothelial trauma. Many commercially available catheters have a rounded tip which is considered to be less traumatic than square cut tubing or bevel ended tubing, although the latter is easiest to insert.

[0064] In some embodiments, the catheter in the device has a pore size that is sufficient large so as to minimize or otherwise avoid issues related to platelet reactivity to the material. In some embodiments, the catheter in the device has a pore size larger than 2 micrometer.

[0065] In some embodiments, the catheter used in a device of the disclosure exhibits one or more of these desirable biological properties: non-irritant - provokes minimal inflammatory response; non-carcinogenic - low tendency to cause neoplasia; non- thrombogenic - low tendency to cause blood clotting; non-toxic; resists microbial adhesion; and/or resists biofilm deposition.

[0066] In some embodiments, the catheter used in a device of the disclosure exhibits one or more of these desirable physical properties: high tensile strength; resists compression - maintains lumen patency; optimum flexibility; low friction coefficient; dimensional stability; tolerates physical sterilization methods (e.g. heat, steam, irradiation); ease of fabrication (e.g. heat forming or welding); non-permeable (water, gases, solvents); and/or radiopacity - ability to image catheter with X-rays.

[0067] In some embodiments, the catheter used in a device of the disclosure exhibits one or more of these desirable chemical properties: absence of leachable additives (e.g. catalysts and plasticizers); stable during storage; stable on chemical sterilization; stable on implantation (non-biodegradable); permits adhesives in fabrication (possibility of bonding dissimilar materials); accepts surface coatings (e.g. hydrogel, antithrombotic, antibacterial); compatibility with chemical compounds and solvents (absence of absorption and chemical reaction); and/or MRI (Magnetic Resonance Imaging) compatibility.

[0068] In some embodiments, the device has a fluid removable section, which satisfies at least one of the following requirements: a. The fluid removal section comprises an opening through which the fluid passes into the inner space of the device; b. An inner space/interior of the fluid removal section communicates with the opening; c. The opening is formed at one end of the fluid removal section; d. Fluid The fluid removal section is formed from a biocompatible material; e. The fluid removal section is made of plastic, metal or glass, or a combination of these substances; f. The fluid removal section is designed as a cannula; g. The fluid removal section comprises an outer diameter of 0.25 to 3.5 mm; h. The opening comprises a diameter of 0.2 to 3.0 mm; i. The functionalized surface is located on an inner wall of the fluid removal section; and j. Fluid removal section comprises a material, which contains functional groups for covalent bonding of the detection molecules, and/or contains chemically or enzymatically fissile groups, to facilitate quantitative removal of bound target molecules, or target cells, and/or forms a matrix which prevents bonding of non-specific cells or interactions with cells or body fluids.

[0069] In some embodiments, the device has a pipe section, which satisfies at least one of the following requirements: a. An inner space of the pipe section communicates with an opening and/or an interior of a fluid removal section; b. The pipe section is connected to the fluid removal section; c. The pipe section is configured as a flexible hose; d. The pipe section has a larger internal diameter and/or outer diameter than the fluid removal section; e. The pipe section comprises an inner diameter of 0.25 to 3.5 mm; f. The pipe section comprises at least one branch pipe; g. The pipe section comprises at least an open branch pipe and/or at least one short-circuited branch pipe and/or at least one branching pipe; h. The pipe section comprises at least three branch pipes, which extend in different planes; i. The pipe section comprises at least one change in cross section; j. The functionalized surface is located on an inner wall of the pipe section; k. The pipe section is made of plastic; and 1. The pipe section comprises a material, which contains functional groups for covalent binding of the detection molecule, and/or chemically or enzymatically fissile groups to facilitate quantitative recovery of bound target molecules, or target cells, and/or forms a matrix which prevents binding of non-specific interactions with cells or body fluids.

[0070] In some embodiments, the device has a storage device which satisfies at least one of the following requirements: a. The storage device comprises a variable volume; b. The interior of the storage device communicates with the opening and/or with the interior of the fluid removal section and/or the interior of the pipe section; c. The storage device is connected to the pipe section and/or connected to the fluid removal section; d. The storage device is configured as a syringe; e. The functionalized surface is located on an inner wall of the storage device; and f. The memory storage device comprises a material, which contains functional groups for covalent binding of the detection molecule, and/or which contains chemically or enzymatically fissile groups, to facilitate quantitative recovery of bound target molecules, or target cells, and/or forms a matrix which prevents binding of non-specific interactions with cells or body fluids.

[0071] In some embodiments, the device has a detection device which satisfies at least one of the following requirements: a. The detection device is configured as a functionalized chip; b. The detection device provides optical, detection and/or identification of target molecules, or target cells; c. The detection device provides computer-aided detection and/or identification of the target molecules, or target cells; d. The detection device is integrated into the fluid removal section and/or in the pipe section and/or the storage device; e. The inner space of the detection device communicates with the opening and/or with the interior of the fluid removal section and/or the interior of the pipe section and/or with the interior of the storage device; f. The detection device is connected to the fluid removal section and/or to the pipe section and/or to the storage device; g. The functionalized surface is located on an inner wall of the detection device; and h. The detection device comprises a material, which contains functional groups for covalent binding of the detection molecule, and/or that contains chemically or enzymatically fissile groups, to facilitate quantitative recovery of bound target molecules, or target cells, and/or forms a matrix which prevents binding of non-specific interactions with cells or body fluids.

[0072] In some embodiments, the device has a secondary layer which satisfies at least one of the following requirements: a. The secondary layer is designed in the form of as a polymer layer; b. The secondary layer contains functional groups for covalent bonding of the detection molecules; c. The secondary layer comprises chemically or enzymatically fissile groups, to facilitate quantitative removal of bound target molecules, or target cells; d. The secondary layer forms a matrix, which prevents bonding of non-specific cells or interactions with body fluids; and e. At the secondary layer detection molecules are covalently bonded. [0073] In some embodiments, the functionalized surface has a patterning, with projections and/or recesses, which are at least one of cylindrical, spherical segment, conical or frustoconical, pyramidal or truncated pyramidal shape, or ridges or furrows.

[0074] The disclosure also provides devices and methods of use thereof that use extracorporeal filtration. In some embodiments, the device uses extracorporeal filtration to isolate a desired molecule, cell, and other target from a biological sample or a fraction thereof, and/or a nucleic acid contained therein. A schematic representation of an exemplary device that uses extracorporeal filtration is shown in Figure 3.

[0075] A device of the disclosure with extracorporeal removal of cfDNA, vesicles and CTCs (or combinations thereof) is similar to a dialysis machine, but the capture is targeted to these biomarkers. Capture is accomplished using any of a variety of selectivity criteria, including by way of non-limiting example, size selectivity, e.g., size exclusion and/or capture; affinity, charge, or ligand(s) directed to a specific target as a means for selectivity. Suitable ligands include antibodies, e.g., antibodies that detect a marker such as, for example, EpCAM, a marker for epithelial cancer, EGFR, a marker for cancer, PSMA, a marker for prostate cancer, EGFRv3, a marker for glioblastoma, specific fusion antibodies, for example, ALK fusion antibodies like VENT ANA™™ anti-ALK antibody, and other antibody-based ligands; aptamers and other nucleic acid-based ligands; and ligand specific peptides and other polypeptide-based ligands.

[0076] Other suitable selective capture matrices include those described in PCT

[0077] In some embodiments, the extracorporeal portion of the device includes a filter that is functionalized with one or more appropriate capture reagents. In some embodiments, the devices and methods of the disclosure are based on any of a variety of art- recognized devices and methods used in current dialysis technology that have been modified for use in the devices and methods provided herein.

[0078] In some embodiments, the devices and methods of use thereof that use extracorporeal filtration use plasmapheresis methodology. In some embodiments, the blood cells are returned to circulation, but a portion of the blood, e.g., the plasma and/or serum, is collected using a selective capture matrix that captures the intended target. For example, suitable selective capture matrices in these methods include those described in PCT

[0079] In some embodiments, the devices and methods use a selective capture reagent to remove a desired target from the blood or plasma of a subject by obtaining blood or plasma from the individual, passing the blood or plasma through a porous hollow fiber membrane wherein the selective capture reagent or combination of selective capture reagents is/are immobilized within the porous exterior portion of the membrane, collecting pass-through blood or plasma.

[0080] Passage of the blood through the hollow fibers having the immobilized selective capture reagent(s) causes the molecules, cells, or fractions thereof, e.g., microvesicle fractions, that contain the desired target to bind the selective capture reagent, thereby removing the target from the effluent.

[0081] The disclosure also provides devices comprising porous hollow fibers, wherein the exterior surface of the fibers is in close proximity with immobilized selective capture reagent(s).

[0082] The devices and methods provided herein can be used to isolate target nucleic acids. The isolated nucleic acids, e.g., DNA and/or DNA and RNA, can then be subject to further analysis using any of a variety of downstream assays. In some embodiments, the combined detection of DNA and RNA is used to increase the sensitivity for actionable mutations. There are multiple potential sources of detectable mutations in circulating nucleic acids. For example, living tumor cells are a potential source for RNA and DNA isolated from the microvesicle fraction of a sample, and dying tumor cells are potential sources for cell-free DNA sources such as, for example, apoptotic vesicle DNA and cell-free DNA from necrotic tumor cells. As mutated nucleic acids are relatively infrequent in circulation, the maximization of detection sensitivity becomes very important. Combined isolation of DNA and RNA delivers comprehensive clinical information to assess progression of disease and patient response to therapy. However, in contrast to the methods and kits provided herein, commercially available kits for detecting circulating nucleic acids are only able to isolate cfDNA from plasma, i.e., from dying cells.

[0083] As used herein, the term "nucleic acids" refer to DNA and RNA. The nucleic acids can be single stranded or double stranded. In some instances, the nucleic acid is DNA. In some instances, the nucleic acid is RNA. RNA includes, but is not limited to, messenger RNA, transfer RNA, ribosomal RNA, non-coding RNAs, microRNAs, and HERV elements. [0084] As used herein, the term "biological sample" refers to a sample that contains biological materials such as DNA, RNA and protein.

[0085] In some embodiments, the biological sample may suitably comprise a bodily fluid from a subject. The bodily fluids can be fluids isolated from anywhere in the body of the subject, such as, for example, a peripheral location, including but not limited to, for example, blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid and cell culture supernatant, and combinations thereof. Biological samples can also include fecal or cecal samples, or supernatants isolated therefrom.

[0086] The devices, methods, and kits of the disclosure are suitable for use with samples derived from a human subject. The devices, methods, and kits of the disclosure are suitable for use with samples derived from a human subject. In addition, the devices, methods, and kits of the disclosure are also suitable for use with samples derived from a human subject. The m devices, methods, and kits of the disclosure are suitable for use with samples derived from a non-human subject such as, for example, a rodent, a non-human primate, a companion animal (e.g., cat, dog, horse), and/or a farm animal (e.g., chicken).

[0087] The term "subject" is intended to include all animals shown to or expected to have nucleic acid-containing particles. In particular embodiments, the subject is a mammal, a human or nonhuman primate, a dog, a cat, a horse, a cow, other farm animals, or a rodent (e.g. mice, rats, guinea pig. etc.). A human subject may be a normal human being without observable abnormalities, e.g., a disease. A human subject may be a human being with observable abnormalities, e.g., a disease. The observable abnormalities may be observed by the human being himself, or by a medical professional. The term "subject," "patient," and "individual" are used interchangeably herein.

[0088] In embodiments where the capture surface is a membrane, the device for isolating the molecules, cells and/or fraction thereof, e.g., microvesicle fraction from a biological sample contains at least one membrane. In some embodiments, the device comprises one, two, three, four, five or six membranes. In some embodiments, the device comprises three membranes. In embodiments where the device comprises more than one membrane, the membranes are all directly adjacent to one another at one end of the column. In embodiments where the device comprises more than one membrane, the membranes are all identical to each other, i.e. , are of the same charge and/or have the same functional group.

[0089] It should be noted that capture by filtering through a pore size smaller than the microvesicles is not the primary mechanism of capture by the methods provided herein. However, filter pore size is nevertheless very important, e.g. because mRNA gets stuck on a 20nm filter and cannot be recovered, whereas microRNAs can easily be eluted off, and e.g. because the filter pore size is an important parameter in available surface capture area.

[0090] The methods provided herein use any of a variety of capture surfaces. In some embodiments, the capture surface is a membrane, also referred to herein as a filter or a membrane filter. In some embodiments, the capture surface is a commercially available membrane. In some embodiments, the capture surface is a charged commercially available membrane. In some embodiments, the capture surface is neutral. In some embodiments, the capture surface is selected from Mustang® Ion Exchange Membrane from PALL

Corporation; Vivapure ® Q membrane from Sartorius AG; Sartobind Q, or Vivapure® Q Maxi H; Sartobind ® D from Sartorius AG, Sartobind (S) from Sartorius AG, Sartobind ® Q from Sartorius AG, Sartobind ® IDA from Sartorius AG, Sartobind® Aldehyde from Sartorius AG, Whatman® DE81 from Sigma, Fast Trap Virus Purification column from EMD Millipore; Thermo Scientific* Pierce Strong Cation and Anion Exchange Spin Columns.

[0091] In embodiments where the capture surface is charged, the capture surface can be a charged filter selected from the group consisting of 0.65um positively charged Q PES vacuum filtration (Millipore), 3-5um positively charged Q RC spin column filtration (Sartorius), 0.8um positively charged Q PES homemade spin column filtration (Pall), 0.8um positively charged Q PES syringe filtration (Pall), 0.8um negatively charged S PES homemade spin column filtration (Pall), 0.8um negatively charged S PES syringe filtration (Pall), and 50nm negatively charged nylon syringe filtration (Sterlitech). Preferably, the charged filter is not housed in a syringe filtration apparatus, as Qiazol/RNA is harder to get out of the filter in these embodiments. Preferably, the charged filter is housed at one end of a column.

[0092] In embodiments where the capture surface is a membrane, the membrane can be made from a variety of suitable materials. In some embodiments, the membrane is polyethersulfone (PES) (e.g., from Millipore or PALL Corp.). In some embodiments, the membrane is regenerated cellulose (RC) (e.g., from Sartorius or Pierce). [0093] In some embodiments, the capture surface is a positively charged membrane.

In some embodiments, the capture surface is a Q membrane, which is a positively charged membrane and is an anion exchanger with quaternary amines. For example, the Q membrane is functionalized with quaternary ammonium, R-CH2-N⁺(CH3)3. In some embodiments, the capture surface is a negatively charged membrane. In some embodiments, the capture surface is an S membrane, which is a negatively charged membrane and is a cation exchanger with sulfonic acid groups. For example, the S membrane is functionalized with sulfonic acid, R-CH2-SO3^". In some embodiments, the capture surface is a D membrane, which is a weak basic anion exchanger with diethylamine groups, R-CH2- NH⁺(C2H5)2. In some embodiments, the capture surface is a metal chelate membrane. For example, the membrane is an IDA membrane, functionalized with minodiacetic acid - N(CH2COOH^")2. In some embodiments, the capture surface is a microporous membrane, functionalized with aldehyde groups, -CHO. In other embodiments, the membrane is a weak basic anion exchanger, with diethylaminoethyl (DEAE) cellulose. Not all charged membranes are suitable for use in the methods provided herein, e.g., RNA isolated using Sartorius Vivapure S membrane spin column showed RT-qPCR inhibition and, thus, unsuitable for PCR related downstream assay.

[0094] In embodiments where the capture surface is charged, microvesicles can be isolated with a positively charged filter.

[0095] In embodiments where the capture surface is charged, the pH during microvesicle capture is a pH <7. In some embodiments, the pH is greater than 4 and less than or equal to 8.

[0096] In embodiments where the capture surface is a positively charged Q filter, the buffer system includes a wash buffer comprising 250mM Bis Tris Propane, pH6.5-7.0. In embodiments where the capture surface is a positively charged Q filter, the lysis buffer is Qiazol. In embodiments where the capture surface is a positively charged Q filter, the lysis buffer is present at one volume. In embodiments where the capture surface is a positively charged Q filter, the lysis buffer is present at more than one volume.

[0097] Depending on the membrane material, the pore sizes of the membrane range from 3 μπι to 20 nm.

[0098] The surface charge of the capture surface can be positive, negative or neutral.

In some embodiments, the capture surface is positively charged. [0099] In some embodiments, the extracted nucleic acid comprises DNA and/or

DNA and RNA. In embodiments where the extracted nucleic acid comprises DNA and RNA, the RNA is preferably reverse-transcribed into complementary DNA (cDNA) before further amplification. Such reverse transcription may be performed alone or in combination with an amplification step. One example of a method combining reverse transcription and amplification steps is reverse transcription polymerase chain reaction (RT-PCR), which may be further modified to be quantitative, e.g., quantitative RT-PCR as described in US Patent No. 5,639,606, which is incorporated herein by reference for this teaching. Another example of the method comprises two separate steps: a first of reverse transcription to convert RNA into cDNA and a second step of quantifying the amount of cDNA using quantitative PCR. As demonstrated in the examples that follow, the RNAs extracted from nucleic acid- containing particles using the methods disclosed herein include many species of transcripts including, but not limited to, ribosomal 18S and 28S rRNA, microRNAs, transfer RNAs, transcripts that are associated with diseases or medical conditions, and biomarkers that are important for diagnosis, prognosis and monitoring of medical conditions.

[00100] For example, RT-PCR analysis determines a Ct (cycle threshold) value for each reaction. In RT-PCR, a positive reaction is detected by accumulation of a fluorescence signal. The Ct value is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e., exceeds background level). Ct levels are inversely proportional to the amount of target nucleic acid, or control nucleic acid, in the sample (i.e., the lower the Ct level, the greater the amount of control nucleic acid in the sample).

[00101] In another embodiment, the copy number of the control nucleic acid can be measured using any of a variety of art-recognized techniques, including, but not limited to, RT-PCR. Copy number of the control nucleic acid can be determined using methods known in the art, such as by generating and utilizing a calibration, or standard curve.

[00102] In some embodiments, one or more biomarkers can be one or a collection of genetic aberrations, which is used herein to refer to the nucleic acid amounts as well as nucleic acid variants within the nucleic acid-containing particles. Specifically, genetic aberrations include, without limitation, over-expression of a gene (e.g., an oncogene) or a panel of genes, under-expression of a gene (e.g., a tumor suppressor gene such as p53 or RB) or a panel of genes, alternative production of splice variants of a gene or a panel of genes, gene copy number variants (CNV) (e.g., DNA double minutes) (Hahn, 1993), nucleic acid modifications (e.g., methylation, acetylation and phosphorylations), single nucleotide polymorphisms (SNPs), chromosomal rearrangements (e.g., inversions, deletions and duplications), and mutations (insertions, deletions, duplications, missense, nonsense, synonymous or any other nucleotide changes) of a gene or a panel of genes, which mutations, in many cases, ultimately affect the activity and function of the gene products, lead to alternative transcriptional splice variants and/or changes of gene expression level, or combinations of any of the foregoing.

[00103] The analysis of nucleic acids present in the isolated particles is quantitative and/or qualitative. For quantitative analysis, the amounts (expression levels), either relative or absolute, of specific nucleic acids of interest within the isolated particles are measured with methods known in the art (described below). For qualitative analysis, the species of specific nucleic acids of interest within the isolated microvesicles, whether wild type or variants, are identified with methods known in the art.

[00104] The present invention also includes various uses of the new methods of isolating molecules, cells, and/or fractions thereof, e.g., microvesicles, from a biological sample for high quality nucleic acid extraction from a for (i) aiding in the diagnosis of a subject, (ii) monitoring the progress or reoccurrence of a disease or other medical condition in a subject, or (iii) aiding in the evaluation of treatment efficacy for a subject undergoing or contemplating treatment for a disease or other medical condition; wherein the presence or absence of one or more biomarkers in the nucleic acid extraction obtained from the method is determined, and the one or more biomarkers are associated with the diagnosis, progress or reoccurrence, or treatment efficacy, respectively, of a disease or other medical condition.

Other Embodiments

[00105] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following.

Claims

What is claimed is:

1. A device for detecting one or more nucleic acids in a biological sample, the device comprising a polymer fiber and one or more capturing reagents, wherein the one or more capturing reagents bind to the nucleic acid(s).

2. The device of claim 1, wherein the polymer fiber is flexible.

3. The device of claim 1 or claim 2, wherein the polymer fiber has a shape of a cylinder or a tube.

4. The device of claim 1 or claim 2, wherein the fiber is round-shaped or bow-shaped at one or both ends of the fiber.

5. The device of any one of claims 1 to 4, wherein the fiber comprises a material selected from the group consisting of nylon, polyimide, Teflon, polyurethane, polystyrene, polyethylene, epoxy, polycarbonate and/or composite materials hereof.

6. The device of any one of claims 1 to 5, wherein the capturing reagent is selected from the group consisting of a capture surface, antibodies, antigens, receptors,

polynucleotides, polypeptides, DNA probes, RNA probes, proteins and/or cells.

7. The device of any one of claims 1 to 6, wherein the capturing reagents are connected to the polymeric fiber.

8. The device of any one of claims 1 to 7, wherein the space between the capturing molecules is between about 20 to 800 nm.

9. A method for detecting one or more nucleic acids using the device of any preceding claim, the method comprising: (i) introduction of a biological sample into the device, (ii) binding of the nucleic acid(s) to the capturing reagent(s), (iii) separation of the nucleic acid(s) and/or capturing reagent(s), and (iv) analysis of the nucleic acid(s).

10. The method of claim 9 further comprising introduction of the device into an organism as the biological sample.

1 1. A device comprising a functionalized surface for isolation of molecules or cells or a fraction thereof from a human body, wherein the device is configured to remove a fluid from the human body, and to absorb it into an inner space of the device, wherein the functionalized surface is oriented to the inner space of the device.

12. The device according to claim 1 1, wherein the functionalized surface is at least partially occupied with detection molecules.

13. The device according to claim 12, wherein the detection molecule includes capture surfaces, antibodies, nucleic acid structures, carbohydrate structures, and synthetic structures.

14. The device according to claim 1 1, wherein the detection molecules are connected and oriented to the functionalized surface.

15. The device according to claim 1 1, wherein the detection molecules are covalently attached to the functionalized surface.

16. A method for detecting one or more nucleic acids using the device of any of claims 1 1 to 15, the method comprising: (i) introduction of a biological sample into the device, (ii) binding of the nucleic acid(s) to the functionalized surface, (iii) separation of the nucleic acid(s) and/or functionalized surface, and (iv) analysis of the nucleic acid(s).

17. The method of claim 16 further comprising introduction of the device into an organism as the biological sample.