EP2310529A2 - Compositions et procédés à base de microvésicules - Google Patents

Compositions et procédés à base de microvésicules

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Publication number
EP2310529A2
EP2310529A2 EP09767814A EP09767814A EP2310529A2 EP 2310529 A2 EP2310529 A2 EP 2310529A2 EP 09767814 A EP09767814 A EP 09767814A EP 09767814 A EP09767814 A EP 09767814A EP 2310529 A2 EP2310529 A2 EP 2310529A2
Authority
EP
European Patent Office
Prior art keywords
microvesicles
rna
cancer
rna molecules
condition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09767814A
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German (de)
English (en)
Inventor
Zbigniew Pietrzkowski
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Proxy Life Science Holdings Inc
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Proxy Life Science Holdings Inc
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Filing date
Publication date
Application filed by Proxy Life Science Holdings Inc filed Critical Proxy Life Science Holdings Inc
Publication of EP2310529A2 publication Critical patent/EP2310529A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the field of the invention is detection and/or analysis of RNA in micro vesicles and their use in diagnosis, prognosis, and/or research of diseases and other conditions.
  • Microvesicles were historically regarded as cellular debris with no apparent function. However, a growing body of experimental data has suggested that microvesicles have numerous biological activities. For example, platelet-derived microvesicles were shown to stimulate selected cells via surface proteins on the microvesicles (e.g., Thromb. Haemost. (1999), 82:794, or J. Biol. Chem. (1999), 274:7545). In other examples, specific effects of bioactive lipids in platelet microvesicles on certain target cells were reported (e.g., J. Biol. Chem. (2001), 276: 19672; or Cardiovasc. Res. (2001), 49(5):88). In still further examples, platelet microvesicles increased adhesion of mobilized CD34+ endothelial cells by transfer of certain microvesicle surface components to the mobilized cells (e.g., Blood (2001), 89:3143).
  • platelet microvesicles increased adhesion of mobilized CD34+ endot
  • microvesicles have also been shown to comprise RNA that at least in part appeared to reflect the RNA content of the cell from which they originate. Microvesicles have also been shown to have significant biological effect on other cells, probably due to the RNA present in the microvesicles, and various examples and aspects for such microvesicles are described in our commonly owned International application (WO2005/121369), which expressly forms part of this application.
  • microvesicles were described as including non-coding miRNA (microRNA) that could potentially interfere or regulate gene expression in cells with which such microvesicles merge (PLoS November 2008, Vol. 3(11), e3694).
  • Other reports discuss in vitro cell-to-cell signaling via exosomal RNA (Cell Adh Migr 1 :3, 156-158; 2007; Cancer Immunol Immunother 2006 Jul;55(7):808-18; Blood. 2007 Oct 1;110(7):2440-8.).
  • the '634 patent elaborates on the chemically and structurally highly diverse nature of the RNA associated particles (presumably due to their diverse origin and manner of generation) and thus concludes that serum RNA is best isolated in an indiscriminate manner.
  • membrane associated or vesiculated RNA has more recently been reported, there is a large body of contradictory data and hypotheses with respect to the nature, quality, availability, and origin/manner of generation of microvesicles.
  • RNA-containing microvesicles has not been fully recognized in the art, and there is still a need for microvesicle-based diagnostic compositions and methods in which RNA from microvesicles is enriched and differentiated to so obtain a result that is indicative of the condition of tissue or organ from which the microvesicle originated.
  • microvesicles are employed in various diagnostic, prognostic, and/or analytic compositions and methods in which specific RNA content of microvesicles and optionally at least one more additional information bearing component of the microvesicle are used to obtain cell-, tissue-, organ-, and/or disease-specific information.
  • a biological sample e.g., plasma or serum
  • a living donor mammal preferably human
  • the microvesicles are then enriched and differentiated to produce a primary result based on one or more distinct RNA molecules, a secondary result based on at least two distinct RNA molecules, and/or a ternary result based on sub-segregated proxysomes and one or more distinct RNA molecules.
  • the RNA analysis in the differentiation may include analysis of a single RNA, of at least two distinct RNAs, and in some aspects even an entire RNA profile (typically obtained by analysis of an array of multiple distinct DNA or RNA).
  • the so obtained results are then correlated with a diagnosis (e.g. , cancer or a clinical stage of a cancer, including pre-cancerous stages) or prognosis/diagnosis of a condition of the mammal.
  • a diagnosis e.g. , cancer or a clinical stage of a cancer, including pre-cancerous stages
  • prognosis/diagnosis of a condition of the mammal.
  • the distinct RNA molecule(s) in the primary result is/are RNA that is overexpressed, underexpressed, and/or mutated, wherein the change in expression and/or the mutation is characteristic for the condition.
  • one of the two distinct RNA molecules in the secondary result is an RNA that are overexpressed, underexpressed, and/or mutated, wherein the change in expression and/or the mutation is characteristic for the condition
  • the other of the distinct RNA molecules in the secondary result is an RNA that is uniquely expressed in a specific tissue or organ of the mammal.
  • the sub-segregated proxysome population is preferably obtained by isolating the proxysome population based on a surface molecule specific for origin of the proxysome population, and the distinct RNA molecule in the ternary result is RNA that is overexpressed, underexpressed, and/or mutated, wherein the change in expression and/or the mutation is characteristic for the condition.
  • the RNA encodes MMPl 1, BCARl, ERBB2, MKI67, PLAU, and/or TP53 where the condition is breast cancer; or encodes FGFRl, KRAS2, TGFBR2, MAP2K4, and/or CDKN2A where the condition is pancreatic cancer; or encodes KLK3, ERBB2, FGF8, PSCA, and/or CAVlwhere the condition is prostate cancer; or encodes BAX, SLC2A1, PTGS2, MUCl, and/or RUNX3 where the condition is gastric cancer; or encodes BCLlO, PAP, SPARC, CD44, and/or TP53 where the condition is liver cancer; or encodes a stem cell marker, and especially an adult stem cell marker (e.g., CD33, CD44, CXCR4, CXCR4+, Im-, CD45-, Oct-4, Nanog, SCAl,
  • an adult stem cell marker e.g., CD33, CD44, CXCR4, CXCR4+, Im-
  • the RNA encodes DCD, SCGB2A2, and/or ANKRD30A where the specific tissue or organ is breast tissue; or encodes UCN3, IPFl, and/or REGlB where the specific tissue or organ is a pancreas; or encodes UPK3 A, SEMG 1 , and/or PRAC where the specific tissue or organ is prostate tissue; or encodes GAST, GKNl, and/or TFF2 where the specific tissue or organ is gastric tissue; or encodes GYS2, F9, and/or HRG where the specific tissue or organ is a liver.
  • microvesicles are enriched via aggregation by interlinking the microvesicles with an interlinking composition (e.g., annexin V, fibrin, or an antibody or antibody fragment against a tetraspanin, ICAM-11, or CD86). Consequently, microvesicles especially contemplated herein have a membrane composition such that phosphatidylserine is on the outside of the membrane. It is further generally preferred that the primary or secondary result are obtained using a quantitative rtPCR for the distinct RNA molecule or via microarray technology.
  • an interlinking composition e.g., annexin V, fibrin, or an antibody or antibody fragment against a tetraspanin, ICAM-11, or CD86. Consequently, microvesicles especially contemplated herein have a membrane composition such that phosphatidylserine is on the outside of the membrane. It is further generally preferred that the primary or secondary result are obtained using a quantitative rtPCR for the distinct RNA
  • a method of staging a mammalian neoplasm (which also includes staging of precancerous lesions or growth) will include a step of obtaining a whole blood fraction (e.g., serum or plasma) that includes a plurality of microvesicles comprising a plurality of distinct RNA molecules.
  • a whole blood fraction e.g., serum or plasma
  • the microvesicles are enriched (e.g., via centrifugation and/or aggregation) and differentiated (e.g., using quantitative rtPCR) to produce a primary result based on at least one distinct RNA molecule, a secondary result based on at least two distinct RNA molecules, and/or a ternary result based on a sub-segregated proxysome population and at least one distinct RNA molecule.
  • the results are then correlated with a stage of the neoplasm in the mammal, typically via comparison with a reference result.
  • neoplasms especially contemplated neoplasms include acute lymphoblastic leukemia, bladder cancer, breast carcinoma, cervical cancer, colorectal cancer, lung cancer, ovarian cancer, and pancreatic adenocarcinoma. Therefore, one of the at least two distinct RNA molecules in the secondary result or one of the distinct RNA molecules in the primary or ternary result will be an RNA that encodes ERBB2.
  • Figure 1 is a graph depicting the results for in vitro expression of Her2-RNA in selected cell lines.
  • Figure 2A is a photograph of an agarose gel with Her2-RNA amplification products of RNA from microvesicles that were isolated from culture supernatants of the selected cell lines.
  • Figure 2B is a graph depicting the quantitative difference of the amplification products of Figure 2A.
  • Figure 3 A is a photograph of an agarose gel with Her2-RNA amplification products of RNA from micro vesicles that were isolated from murine serum of mice that developed tumors derived from one of the selected cell lines.
  • Figure 2B is a graph depicting the quantitative difference of the amplification products of Figure 3 A.
  • Figure 4A is a photograph of an agarose gel with Her2-RNA amplification products of RNA from micro vesicles that were isolated from human sera of patients with confirmed breast cancer diagnosis.
  • Figure 4B is a graph depicting the quantitative difference of the amplification products of Figure 3 A.
  • RNA and/or multiple non-RNA components e.g., soluble and/or membrane proteins, lipids, etc.
  • RNA content of the microvesicles is entirely or almost entirely representative of the cellular RNA of the cell from which the microvesicle originated, numerous uses are now envisioned.
  • microvesicles are shed by all cells in relatively high numbers, and as microvesicles are also present in equally high number in blood, the inventor concluded that, inter alia, gene expression, soluble/membrane proteins, and membrane composition of various cells, tissues, and organs can be easily determined by analyzing the corresponding microvesicles, where those microvesicles are obtained from a biological fluid, and especially blood.
  • microvesicle refers to a membranaceus particle having a diameter (or largest dimension where the particle is not spheroid) of between about 10 nm to about 5000 nm, more typically between 30 nm and 1000 nm, and most typically between about 50 nm and 750 nm, wherein at least part of the membrane of the microvesicle is directly obtained from a cell. Therefore, especially contemplated microvesicles include those that are shed from a donor cell, and will typically also include exosomes.
  • the microvesicles contemplated herein may exhibit different surface/lipid characteristics.
  • the microvesicles suitable for use in the present inventive subject matter may originate from cells membrane inversion, exocytosis, shedding, blebbing, or budding.
  • microvesicles suitable for use herein will be identifiable by having phosphatidylserine, a tetraspanin, ICAM-I, and/or CD86 on the outer surface.
  • a liposome made from isolated lipids will not include lipids or other components obtained from a cell-membrane, and is not considered a micro vesicle under the definition used herein. Therefore, most typically, contemplated microvesicles will have a lipid bilayer structure and are not multi-lamellar.
  • the microvesicles are generated from a differentiated cell, and most preferably from a terminally differentiated cell (i.e., a cell that has reached the end of its differentiation pathway). Therefore, microvesicles generated by blast cells, progenitor cells, and stem cells are excluded in at least some of the embodiments herein. However, and especially where the microvesicles are generated from a cancer cell, and where that cancer cell is derived from an adult stem cell (as opposed to an embryonic stem cell) less differentiated cells are also deemed suitable sources for the microvesicles.
  • the microvesicles are derived from cells that are diseased (e.g., neoplastic cell, infected cell, cell in an infected organ) or subject to an abnormal condition (e.g., metabolic abnormality, cell exposed to a drug, and more typically a drug that preferentially affects the cell relative to other cells, or dietary toxin, or damaged by free radicals).
  • the microvesicles are generated from a healthy, but ageing cell, i.e., a cell that has reached at least 50%, and more typically at least 70% of its ordinary number of cell divisions (Hayflick limit).
  • synaptosomes vesicular entities in the synaptic gap, but not present in the general circulation
  • microvesicles that are formed from platelets are expressly excluded.
  • RNA content of the microvesicles is thought to be characteristic of a particular condition (e.g., disease, age, stress, response to a chemical compound, senescence, inflammation, infection [e.g., viral, microbial, parasitic], immune status, regeneration, rejection, etc.).
  • Non-RNA information bearing components in microvesicles contemplated herein include various proteins (e.g., receptor, ligand, glycoprotein, etc.) that are associated with the microvesicle, which may be at least partially embedded in the membrane (or even be entirely enclosed), specific lipids or glycolipids that are associated with the micro vesicle, and/or a further nucleic acid (DNA and/or RNA) associated with the microvesicle.
  • non-RNA information bearing component is specific to a particular organ, tissue, and/or cell
  • analysis of the condition of the particular organ, tissue, and/or cell can be obtained in a highly simplified and even multiplexed manner. Consequently, and among other advantages, it should be especially appreciated that a serum or blood based test can be performed that provides (even multiple) organ specific results without the need for an otherwise required organ-specific biopsy. Still further, it should be noted that the methods contemplated herein will allow virtually unlimited and repeated real time access to an expression profile of the same tissue or organ.
  • the inventor contemplates a method of analyzing a biological sample of a mammal in which a plurality of microvesicles that include distinct RNA molecules is first obtained from a living donor mammal.
  • the microvesicles are then enriched and differentiated to produce a primary result based on one or more distinct RNA molecules (which are most typically a RNA that are uniquely expressed and/or mutated as a function of the condition of the cell from which it derived), a secondary result based on at least two distinct RNA molecules (with one of the molecules being RNA that is uniquely expressed in a specific tissue or organ, and with the other molecule being RNA that is uniquely expressed and/or mutated as a function of the condition of the cell from which it derived) and/or a ternary result based on a sub-segregated proxysome population (e.g., based on a specific surface marker of the cell from which the microvesicle was derived) and one or more distinct RNA molecules (most typically a RNA that is uniquely expressed and/or mutated as a function of the condition of the cell from which it derived).
  • the primary, secondary, and/or ternary results are then correlated with a diagnosis or prognosis
  • the microvesicles are obtained from whole blood, serum, plasma, or any other biological fluid, including urine, milk, tears, spinal fluid, amniotic fluid, etc., which are preferably obtained from a living mammal, and most preferably within the time limit acceptable for processing biological fluids for later clinical analysis.
  • microvesicles may also be obtained from stored materials (e.g., biological fluids, tissues, organs, etc.), wherein the time between obtaining the biological fluid, tissue, or organ, and enrichment of the microvesicles from the sample may be at least 12 hours, at least 24 hours, at least 2-5 days, or even at least one or more weeks.
  • microvesicles may also be obtained from an in vitro source, and most typically from cell or tissue culture, or even organ culture. In yet further contemplated aspects, it should be noted that deceased donors are also deemed suitable as a source for the microvesicles. Most commonly, however, microvesicles will be obtained from blood, which may be immediately or within a few hours (less than 12 hours) processed to form serum or plasma, which is then either stored, shipped, and/or further processed o enrich the microvesicles.
  • isolation or enrichment of microvesicles it is contemplated that all known manners of isolation of microvesicles are deemed suitable for use herein.
  • isolation or “isolating” in conjunction with microvesicles are interchangeably used with the terms “enrichment” or “enriching”, and refer to one or more process steps that result in an increase of the fraction of microvesicles in a sample as compared to the fraction of microvesicles in the obtained biological sample.
  • microvesicles may be purified to homogeneity, purified to at least 90% (with respect to non- microvesicle particulate matter), less preferably at least 80%, even less preferably at least 50%, and least preferably at least 20% (or even less).
  • physical properties of microvesicles/proxysomes may be employed to separate them from a medium or other source material, and especially preferred physical methods include separation on the basis of electrical charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieving, etc), density (e.g., regular or gradient centrifugation), Svedberg constant (e.g., sedimentation with or without external force, etc).
  • isolation may be based on one or more biological properties, and especially suitable isolation methods may employ surface markers (e.g., for precipitation, reversible binding to solid phase, FACS separation, specific ligand binding, non-specific ligand binding such as annexin V, etc.).
  • the microvesicles may also be fused using chemical and/or physical methods, including PEG-induced fusion and/or ultrasonic fusion.
  • enriching can be done in a general and nonselective manner (typically including serial centrifugation), and may be performed by aggregation where the microvesicles are interlinked with an interlinking composition (e.g., annexin V, fibrin, or an antibody or fragment thereof against at least one of a tetraspanin, ICAM-I, and CD86).
  • an interlinking composition e.g., annexin V, fibrin, or an antibody or fragment thereof against at least one of a tetraspanin, ICAM-I, and CD86.
  • enriching can be done in a more specific and selective manner (e.g., using tissue or cell specific surface markers).
  • tissue or cell specific surface markers may be used in immunoprecipitation, FACS sorting, bead-bound ligands for magnetic separation etc.
  • RNA of a specific type of micro vesicle e.g., microvesicle generated by hepatocyte
  • proxy RNA RNA of a specific type of micro vesicle
  • proxysomes microvesicles that are specific to a single type of origin
  • proxysomes will consequently have an RNA content and membrane composition (especially in terms of surface markers, but also to at least some degree in terms of lipid composition) that is consistent with the RNA content of a cell from which the proxysome is produced.
  • the microvesicles/proxysomes are isolated from a biological source (e.g., whole blood or serum) or mixed cell or tissue culture, it should be noted that the isolation may produce a heterogeneous population of microvesicles/proxysomes with respect to the celWtissue-, and/or organ-type from which the microvesicles/proxysomes were produced.
  • a biological source e.g., whole blood or serum
  • mixed cell or tissue culture e.g., whole blood or serum
  • the isolated population may already be homogenous with respect to the cell-/tissue-, and/or organ-type from which the microvesicles/proxysomes were produced.
  • the mechanism of release of the vesicles from the cell may be used to further differentiate, even among proxysomes.
  • vesicular architectures e.g., inside-out, etc.
  • a population of microvesicles obtained from a biological fluid will typically represent a plurality of cells, tissues, and organs, differentiation of the heterogeneous population is often desirable to obtain a cell, tissue, or organ specific result.
  • Differentiation e.g., cancer, infection, senescence, inflammation, etc.
  • a meaningful result may be based on the expression profile and/or specific sequence of one or more distinct RNA molecules without further normalization of the signal.
  • ERBB2 ERBB2
  • CML CML
  • Bcr/Abl fusion mutant
  • a condition e.g., cancer, infection, senescence, inflammation, etc.
  • a condition e.g., cancer, infection, senescence, inflammation, etc.
  • a condition of a cell, tissue, or organ may be associated with a unique and distinguishable cell, tissue, or organ type and may therefore be specifically characterized by normalizing an expression profile and/or specific sequence of one or more distinct RNA molecules against the expression profile and/or specific sequence of one or more further distinct RNA molecules that are uniquely present or expressed in the specific cell, tissue, or organ.
  • a secondary result can be obtained based on the expression profile and/or specific sequence of at least two distinct RNA molecules without physical sub-segregation of the micro vesicles.
  • a condition e.g., cancer, infection, senescence, inflammation, etc.
  • a condition e.g., cancer, infection, senescence, inflammation, etc.
  • a condition e.g., cancer, infection, senescence, inflammation, etc.
  • a condition e.g., cancer, infection, senescence, inflammation, etc.
  • a condition e.g., cancer, infection, senescence, inflammation, etc.
  • a condition of a cell, tissue, or organ may be associated with a unique and distinguishable cell, tissue, or organ type and may therefore be specifically characterized by physical sub-segregation (infra) of the micro vesicles prior to analysis of the expression profile and/or specific sequence of one or more distinct RNA molecules in the sub-segregated microvesicles.
  • differentiation is preferably done using one or more additional information bearing components in and/or on the microvesicle to correlate the result with the additional information to so obtain normalized cell-, tissue-, organ-, and/or disease-specific information.
  • differentiation may therefore include a sub-segregation of a subset of proxysomes from the micro vesicles, and/or a determination of a reference marker that is specific to a subset of proxysomes of the isolated population.
  • the differentiation may also include detection or use of a component (or portion thereof) on a microvesicle or proxysome that is otherwise not accessible on the cell. Such is especially true where the microvesicle or proxysome has an inside-out architecture that exposes on the outside of the microvesicle or proxysome the component that has an orientation normally oriented towards the interior of the cell from which the vesicle is produced.
  • differentiation may be based on the unique sequence, expression profile, mutation, or other character of a particular RNA and thus will typically not require physical subsegregation and/or normalization against other RNA and/or protein markers.
  • presence, abundance, and/or sequence of one or more RNA molecules in the microvesicle population can be directly attributed to a specific disease or condition of a cell, tissue, or organ.
  • RNA is normally not present in measurable quantities in micro vesicles.
  • a relevant oncogene e.g., BRCA, Her2/neu, etc.
  • differentiation may be based on normalization of the RNA under investigation against one or more other constituent parts of the micro vesicles/proxysomes. Therefore, it should be appreciated that differentiation can be performed on a non-segregated pool of microvesicles, and even on the source material where the microvesicles are present. In such case, it is generally preferred that the additional information bearing component is specific to a particular subset of proxysomes.
  • a gene may be identified that is uniquely (greater or equal 90%) or predominantly (greater or equal 75%) expressed in the mammary gland tissue.
  • the proxysome RNA content is an at least partial, and in many cases a complete representation of the RNA content of the mammary gland cell from which the proxysome is derived, the mammary gland proxysome is expected also to comprise the uniquely or predominantly expressed gene.
  • dermcidin is >95% selectively expressed in mammary gland tissue and therefore also expected to be uniquely present in mammary gland proxysomes.
  • non-nucleic acid components can be used to differentiate and may include disease specific markers ⁇ e.g., tumor specific markers such as CEA), organ specific markers (e.g., PSA), and/or cell-specific markers ⁇ e.g., CD4 for T-helper cells), etc.
  • disease specific markers e.g., tumor specific markers such as CEA
  • organ specific markers e.g., PSA
  • cell-specific markers e.g., CD4 for T-helper cells
  • additional information bearing components may then be used for physical subsegregation in a manner as described above.
  • RNA information infra
  • molecular analysis for differentiation can be done with high specificity to a particular cell type, tissue type, and/or organ type.
  • Physical subsegregation of a subset of proxysomes may be performed using antibodies or other binding agents that binds selectively to a component that is specific to particular cell, tissue, or organ.
  • Such components are typically cell-, tissue-, or organ-specific marker, receptors, structural components, glycoproteins, CDs, etc., all of which may be further derivatized.
  • Suitable organ, tissue, and cell specific surface markers are well known in the art and can be found in numerous publications (e.g., for natural receptors see e.g., Cell Surface Receptors: A Short Course on Theory and Methods by Lee E. Limbird Springer; 2nd ed.
  • subsegregation can be performed specific to a cell type, tissue type, and/or organ type.
  • a solid phase to which a compound is bound that (preferably releasably) binds to one or more of the component that is specific to particular cell, tissue, or organ.
  • a compound preferably releasably binds to one or more of the component that is specific to particular cell, tissue, or organ.
  • the solid phase is a multi-well plate
  • proxysomes may be separated into separate wells.
  • the solid phase is a magnetic or color coded bead
  • the proxysomes may be isolated using a magnet or light activated sorter.
  • the solid phase may be a dipstick or membrane and separation will (preferably releasably) bind the proxysomes on the solid phase.
  • subsegregation may also be performed on an array in which antibodies are coupled to the surface of the array in predetermined positions to so specifically bind distinct proxysomes in distinct and predetermined positions on the array. Detection of the bound proxysomes may then be performed in a non-specific manner (e.g., using a labeled form of annexin V) or a specific manner (e.g., using a labeled antibody against another surface marker of the proxysome).
  • a non-specific manner e.g., using a labeled form of annexin V
  • a specific manner e.g., using a labeled antibody against another surface marker of the proxysome.
  • subsegregation may be done after isolation of a population of micro vesicles, but may also be directly performed on the source material of the micro vesicles/proxysomes. In such case, the steps of isolation and subsegregation are combined. Most typically, and depending on the surface marker used, the subsegregation will produce a subpopulation of proxysomes that is substantially homogeneous (i.e., at least 70%, more typically at least 80%, most typically at least 90%) with respect to the tissue from which they originate (e.g., microvesicle from liver, prostate, mammary gland, etc.). It should further be especially appreciated that the subsegregation may also be performed (e.g., without RNA analysis) to determine numerical ratios and/or absolute numbers of microvesicles/proxysomes.
  • the specific RNA of the proxysome of interest can most typically be analyzed using all known manners of RNA analysis, and particularly preferred manners include rtPCR, quantitative PCR (rt or otherwise), primer extension analysis (with or without prior amplification), and all solid-phase based methods (e.g., using arrays, magnetic or color coded beads, etc.). While not limiting to the inventive subject matter, it is generally preferred that the specific RNA is (1) native to the cell, tissue or organ, i.e., not introduced (directly or indirectly) by a pathogen, and/or (2) specific or indicative to a predisposition, condition, disease, response to a stimulus, and/or pathogen.
  • suitable specific RNAs include genes that are specifically and exclusively expressed or overexpressed in cells, tissues, or organs affected with a neoplastic disease, a metabolic disease, inflammation, senescence, hypoxia, an infection, and/or inheritable disease.
  • RNA specific for breast cancer includes RNA that encodes MMPl 1, BCARl, ERBB2, MKI67, PLAU, and/or TP53
  • RNA specific for pancreatic cancer includes RNA that encodes FGFRl, KRAS2, TGFBR2, MAP2K4, and/or CDKN2A
  • RNA specific for prostate cancer includes RNA that encodes KLK3, ERBB2, FGF8, PSCA, and/or CAVl
  • RNA specific for gastric cancer includes RNA that encodes BAX, SLC2A1, PTGS2, MUCl, and/or RUNX3
  • RNA specific for liver cancer includes RNA that encodes BCLlO, PAP, SPARC, CD44, and/or TP53
  • RNA found for various cancers includes RNA
  • RNA can be found in various publicly or commercially available database (e.g. , http://wvyw.caMeTM incorporated by reference herein).
  • more than one specific RNA is measured to so improve a diagnostic finding.
  • ERBB2 and VEGFR may be measured in a population of proxysomes from mammary gland tissue.
  • RNA of interest need not be limited to an RNA that is associated with a particular disease, but that suitable information may be generated from analysis of the expression pattern on of or more distinct RNAs. For example, it is contemplated that total RNA from a non-segregated microvesicle population is analyzed on a chip of other multiplexed platform to so arrive at a genome wide expression profile. Such expression profile may then be used as a basis for diagnosis of a condition or disease where an expression profile of a healthy individual is known. Consequently, it should be appreciated that for the first time, one or more systemic expression profiles may be obtained from the same individual (or group of individuals) in a manner that does not require multiple biopsies.
  • tissue specific expression profiles may be obtained from the same individual (or group of individuals) in a manner that does not require biopsies.
  • RNA is an RNA that is coding and can give rise to a protein via ribosomal translation. Therefore, most typically RNA will include a 3'-polyA tail and may further include a 5'-cap structure (typically 7- methylated guanine nucleotide bound to mRNA via 5'-5' triphosphate group, but less common structures also deemed suitable and include methylation of the 2' hydroxy-groups of the first 3 ribose sugars of the 5' end of the mRNA). Furthermore, it should also be noted that while mRNA is deemed especially useful in conjunction with the teachings presented herein, other forms of RNA are also expressly contemplated herein.
  • RNA for analysis include single stranded and double stranded RNA, heterogenous RNA (hnRNA), small interfering RNA (siRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), mitochondrial RNA (mtRNA), capped RNA and uncapped RNA, polyadenylated and non- polyadenylated RNA, etc.
  • hnRNA heterogenous RNA
  • siRNA small interfering RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • mtRNA mitochondrial RNA
  • capped RNA and uncapped RNA uncapped RNA
  • polyadenylated and non- polyadenylated RNA etc.
  • RNA specific for a disease/condition at least one RNA specific for a disease/condition and at least one additional representative component of the cell from which that proxysome was produced (e.g., RNA of a tissue-specific expressed gene, a cytoplasmic component, a membrane composition, membrane associated component (typically comprising a protein).
  • additional representative component of the cell from which that proxysome was produced e.g., RNA of a tissue-specific expressed gene, a cytoplasmic component, a membrane composition, membrane associated component (typically comprising a protein).
  • the specific RNA and the additional component (which may be another RNA that is cell- or tissue specific, a protein, a lipid, or any combination thereof) are then employed to analyze a condition (e.g., disease or predisposition, age, reaction to physical or chemical stimulus [e.g., drug, food, radiation, etc.]) in a highly cell-, tissue-, and/or organ-specific manner without the need to directly sample the cell-, tissue-, and/or organ.
  • Analysis may be typically be specific with respect to a set of markers. However, analysis may also be analyzed as a function of time to see fluctuations in total microvesicles and/or proxysomes or proxysome populations. Analysis may be focused on time course, quantity, and/or association with further markers.
  • analytic results will typically include variants, quantities, and/or or time course expression of the specific RNA, which will most typically be correlated with one or more results obtained from the differentiation (which may be a quantitative measure of mRNA, a peptide, a membrane component, etc.).
  • the additional information bearing component comprises a peptide
  • especially preferred manners include use of antibodies (or fragments thereof), ligands (synthetic or natural), etc., all of which may be labeled or otherwise modified for qualitative or quantitative analysis.
  • the additional information bearing component is a polysaccharide
  • lectins may be used
  • specific ligands e.g., annexin V
  • the additional information bearing component may be used to sub-segregate a population of micro vesicle into a population that is enriched in one specific type of microvesicle (enrichment preferably >80%).
  • RNA and manner of differentiation may vary to at least some degree.
  • correlation may advantageously be done by comparison of the result(s) with result(s) obtained from a microvesicle population obtained from a reference patient (which may be healthy, or representative to a specific disease stage or state).
  • Similar correlation can be performed for tests in which the RNA is normalized against another RNA or non-RNA compound from a microvesicle, or is normalized meg of total RNA/mL of serum, total RNA per mg of MVs proteins isolated from 1 mL of serum, or other preferably fixed parameter.
  • correlation is typically performed against a known standard that is indicative for a particular disease or condition.
  • the comparison of the results may be performed one a single RNA basis, on the basis of two or more RNA results, or in at least some cases, on a genome or organ- specific basis. In such case, the correlation will include comparison of multiple RNA- specific results with multiple reference results, and all possible cross-relations thereof.
  • the inventor especially contemplates a method of confirming and/or staging a mammalian neoplasm in which in one step a whole blood fraction is obtained that includes a plurality of micro vesicles comprising a plurality of distinct RNA molecules.
  • the microvesicles are enriched and differentiated to so produce a primary result based on one or more distinct RNA molecules, a secondary result based on at least two distinct RNA molecules, and/or a ternary result based on a sub- segregated proxysome population and at least one of the plurality of distinct RNA molecules.
  • the primary, secondary, and/or ternary results are correlated with a stage of the neoplasm in the mammal.
  • RNA coding for ERRB2 overexpression can be quantified in numerous manners, however, it is generally preferred that the step of differentiating comprises quantitative rtPCR and/or reverse transcription and solid phase (e.g., microarray) hybridization.
  • the step of correlating comprises comparison of the primary, secondary, and/or ternary results with one or more reference results that are characteristic of a healthy cell and/or a diseased cell, tissue or organ in a specific diseased, compromised, or aged state.
  • contemplated uses of diagnostic tests and compositions include diagnosis, predisposition, and prognosis of the metabolic state of a cell, tissue, and/or organ, analysis of the response of a cell, tissue, and/or organ to a physical and more typically chemical stimulus, and/or diagnosis or determination of predisposition for a disease or disorder in patient harboring a cell, tissue, and/or organ from which the proxysomes were analyzed.
  • the methods according to the inventive subject matter are employed in the diagnosis of cancer, a pre-cancerous condition or predisposition, and/or a clinical stage of a cancer.
  • contemplated methods are also deemed suitable for prenatal diagnosis as fetal microvesicles have been identified in (Kidney International, 2007; 72(9):1095-102) amniotic fluid, placenta, and possibly also blood of the pregnant mother.
  • microvesicle/proxysome compositions and methods according to the inventive subject matter can be used in bioinformatic analysis in which 'normal' proxysome quantity and composition is acquired and then compared to proxysome quantity and composition from cells treated with one or more agents or conditions.
  • Wile contemplated compositions and methods typically reply on already known specific RNAs and additional markers and information bearing components, it should be appreciated that new specific RNAs and new information bearing component can be relatively easy obtained.
  • a cell culture of diseased cells or cells from a disease model can be propagated in vitro. The supernatant is then collected and microvesicles are isolated from the supernatant.
  • protein is extracted from the microvesicles and antibodies generated.
  • the antibody collection is then subtracted against normal tissue to obtain disease specific antibodies.
  • corresponding experiments can be performed in silico based on expression profiling, or subtractive nucleic acid libraries of diseased and corresponding healthy cells can be generated.
  • the microvesicle diagnostic methods presented herein can be advantageously employed in drug discovery where multiple cell cultures are separately exposed to multiple drugs.
  • Genomics and proteomics analyses can be performed as known in the art.
  • proxysomes can be collected from the individual cultures and analyzed as described above. The so obtained results can then be followed in vivo to ascertain that in vitro findings correlate with prior in vitro results.
  • drug discovery using proxysomes dramatically increases throughput in drug discovery.
  • RNA(a) RNA(a) and additional information bearing components
  • the microvesicle/proxysome components can be analyzed in relative and absolute proportions to obtain a measure for a particular clinical parameter.
  • the methods and compositions according to the inventive subject matter will may also be advantageously employed in personalized medicine and personalized nutrition in which the effect of administration of nutraceuticals (and medications) on microvesicles and proxysomes can be monitored in a highly simplified manner.
  • the RNA signal is normalized against a second signal, typically derived from the additional information bearing components (e.g., total signal from proxysome membrane [e.g., via labeled annexin V]).
  • a range of normal can be established from a clinically healthy population, again using a signal from an additional information bearing component.
  • a known microvesicle ELISA which measures amount of PS, phosphatidylserine, which is highly correlated with microvesicles.
  • RNA Her2 could be calculated per total microvesicles, measured PS, or HER2 RNA signal per (phosphatidylserine - Platelet microvesicles phosphatidylserine). If using realtime PCR, one would compare the number of cycles per phosphatidylserine. In case off an arras, the array signal is correlated to per phosphatidylserine.
  • Real time PCR reaction was performed with using Real Time PCR System ABI 7500 (Applied Biosystems), using following settings: 50 0 C 2 min, 95°C 5 min, 95°C 15 sec, 60 0 C 60 sec; Last two cycles were repeated 40 times.
  • Relative quantitation of Her-2 mRNA expression was calculated with the comparative Ct method.
  • Regular PCR Using RNA isolated as described above, a regular PCR was performed. Briefly, RNA was reverse transcribed to cDNA with using Applied Biosystem reverse transcription kit following the manufacturer's protocol, and the so prepared cDNA was used for PCR studies. Regular PCR reaction used the following Her-2 primers (5'- to 3'- end):
  • Isolation of Microvesicles The tumor derived microvesicles were isolated from culture conditioned media. Briefly, the supernatant was spun at about 850 g for 10 minutes at 4 0 C. The supernatant was collected and the pellets were discarded. The supernatants were again spun at 24,000 g for 2 hr at 4 0 C and the supernatant was discarded. PBS supplemented with HEPES (5mM) was added to pellet that now contained microvesicles and the resuspended microvesicles were transferred to Eppendorf tubes (1.6 ml) in which they were spun at maximum speed for 60 min at 4 0 C.
  • PCR Analysis Using the methods described for cells above, total RNA was isolated from the microvesicles, and real time PCR and regular PCR were performed. When using real time PCR, Her-2 was detected only in microvesicles from the SKOV cell culture but not from T47D and MCF cell culture. However, when using regular PCR, Her-2 was detectable on the mRNA level from all three cell cultures by agarose gel electrophoresis as can be seen in Figure 2A. Not surprisingly, a quantitative analysis of the agarose gel electrophoresis showed that microvesicles from the SKOV cell culture provided the strongest her-2 signal, which was followed by the T47D cell culture, which was in turn followed by the MCF cell culture. A quantitative graph illustrating these results is shown in Figure 2B. It should therefore be appreciated that cellular expression levels of Her-2 are paralleled by microvesicular Her-2 RNA quantities detected in the culture supernatants of the respective cell lines.
  • mice with SKOV-derived Tumors Athymic nude mide were injected with 3x10 6 SKOV3 cells using standard protocol to establish solid tumors in these mice. After three weeks, presence of palpable/measurable tumors was confirmed, and serum was collected from the animals as well as from control mice without tumors (no SKOV injection).
  • microvesicles were isolated from the serum following the general protocol as outlined above.
  • the total RNA obtained from the microvesicles was evaluated for the presence of Her-2 RNA using the PCR protocols as provided above.
  • PCR products were quantified and checked via agarose gel electrophoresis and exemplary results are shown in Figure 3A (to ensure analysis of equal amounts, control PCR was performed using beta-2- microglobulin as standard).
  • a graphic representation of the Her-2 quantification of corresponding rtPCR is shown in Figure 3B.
  • SKOV-tumor derived microvesicles that were generated in vivo were not only positive for Her-2 RNA, but that such RNA can also be detected from a relatively small blood sample with a high degree of sensitivity and specificity.
  • Microvesicles were isolated from actual patient and control sera following the general protocol as outlined above, and total RNA obtained from the microvesicles was evaluated for the presence of Her-2 RNA using the PCR protocols as provided above.
  • the PCR products were quantified and checked via agarose gel electrophoresis and the results for these experiments are shown in Figure 4A (to ensure analysis of equal amounts control PCR was again performed using beta-2-microglobulin as standard).
  • none of the control sera from healthy volunteers showed any detectable Her-2 RNA as tested by rtPCR and normal PCR, but all three of the patients with confirmed breast cancer exhibited a significant and strong signal.

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Abstract

La présente invention concerne des procédés et des compositions pour le diagnostic et/ou l’analyse d’une pathologie chez un mammifère dans lesquels de l’ARN de microvésicules est enrichi et différencié de manière à obtenir un résultat qui est indicatif de la pathologie du tissu ou de l’organe duquel les microvésicules proviennent. Dans des modes de réalisation particulièrement préférés, la pathologie est une maladie néoplasique d’un humain et peut être identifiée et stadifiée par analyse différentielle d’un ou plusieurs ARN distincts, éventuellement avec l’identification et l’analyse d’un composant non-ARN de la microvésicule.
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