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WO2024245668A1 - Crth2 (ptgdr2) en tant que marqueur épigénétique pour l'identification de cellules immunitaires - Google Patents

Crth2 (ptgdr2) en tant que marqueur épigénétique pour l'identification de cellules immunitaires Download PDF

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WO2024245668A1
WO2024245668A1 PCT/EP2024/061805 EP2024061805W WO2024245668A1 WO 2024245668 A1 WO2024245668 A1 WO 2024245668A1 EP 2024061805 W EP2024061805 W EP 2024061805W WO 2024245668 A1 WO2024245668 A1 WO 2024245668A1
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cell
methylation
immune cells
cells
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Sven Olek
Udo Baron
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Precision for Medicine GmbH
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Precision for Medicine GmbH
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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
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • 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
    • C12Q2600/154—Methylation markers

Definitions

  • CRTH2 (PTGDR2) as epigenetic marker for the identification of immune cells
  • the present invention relates to a method, in particular an in vitro method, for identifying immune cells, comprising analyzing epigenetic modifications/properties of (including the methylation status) of at least one CpG position in the mammalian gene region for Prostaglandin D2 receptor 2 (PTGDR2), wherein a demethylation or lack of methylation of said at least one CpG position in the gene region to at least about 80% is indicative for eosinophils or basophils, when compared to a CRTH2- or CRTH+ leukocyte, CD8+ T cell, CD4+ T cell, CD 14+ monocyte, CD 19+ B cell, BDCA4+ pDC, CD56+ NK cell, and/or CD 15+ granulocyte.
  • PSGDR2 Prostaglandin D2 receptor 2
  • the analysis according to the invention can identify immune cells on an epigenetic level and distinguish them from all other cells in complex samples, such as, for example, other blood, non-blood or other immune cells.
  • the present invention furthermore provides an improved method for quantifying immune cells, in particular in complex samples.
  • the method can be performed without a step of purifying and/or enriching cells, preferably in whole blood and/or non-trypsinized tissue.
  • the present invention relates to a kit for performing the above methods as well as respective uses thereof. It is one aim of this invention to provide a novel, more robust means to quantitatively detect and measure immune cells of the blood within any solid organs, tissue or body fluid of a mammal, in particular whole blood.
  • Prostaglandin D2 receptor 2 (PTGDR2, DP2 or CRTH2) is a human protein encoded by the PTGDR2 gene. DP2 has also been designated as CD294 (cluster of differentiation 294). It is a member of the class of prostaglandin receptors which bind with and respond to various prostaglandins. DP2 along with Prostaglandin DPI receptor are receptors for prostaglandin D2 (PGD2). Activation of DP2 by PGD2 or other cognate receptor ligands has been associated with certain physiological and pathological responses, particularly those associated with allergy and inflammation, in animal models and certain human diseases. MisawaK, et al.
  • WO 2019/183121 Al discloses a method of characterizing a tumor, comprising quantifying or obtaining expression levels for a plurality of distinct genes, wherein the distinct genes are associated with respective distinct types of immune cells; determining over-expression or under-expression for each of the distinct genes relative to respective reference ranges, wherein the reference ranges are specific for a specific tumor type; and using the over-expression and/or under-expression of each of the distinct genes to infer activity and/or infiltration by the immune cells in the tumor.
  • One “distinct gene” as mentioned is PTGDR2.
  • JP 2020-178692A discloses methods for diagnosing and treating disorders associated with excessive number or activity of eosinophilic leucocytes including asthma, and methods for selecting or identifying patients for treating with TH2 pathway inhibitors.
  • the method comprises measuring the mRNA levels of at least one, at least two, or at least three markers of CSF1, MEIS2, LGALS12, IDO1, THBS4, OLIG2, ALOX15, SIGLEC8, CCL23, PYROXD2, HSD3B7, SORD, ASB2, CACNG6, GPR44, MGAT3, SLC47A1, SMPD3, CCR3, SLC47A1, SMPD3, CCR3, or CCR3, and the TH2 pathway inhibitor may target CRTH2.
  • the primary target of mentioned DNA modifications is the two-nucleotide sequence Cytosine- Guanine (a 'CpG site'); within this context cytosine (C) can undergo a simple chemical modification to become formylated, methylated, hydroxymethylated, or carboxylated.
  • C Cytosine- Guanine
  • the CG sequence is much rarer than expected, except in certain relatively dense clusters called 'CpG islands'.
  • CpG islands are frequently associated with gene promoters, and it has been estimated that more than half of the human genes have CpG islands (Antequera and Bird, Proc Natl Acad Sci USA 90: 11995-9, 1993).
  • Methylation profiles have been recognized to be tumor specific (i.e., changes in the methylation pattern of particular genes or even individual CpGs are diagnostic of particular tumor types), and there is now an extensive collection of diagnostic markers for bladder, breast, colon, esophagus, stomach, liver, lung, and prostate cancers (summarized, for example, by Laird, Nature Reviews/Cancer 3:253-266, 2003).
  • WO 2012/162660 describes methods using DNA methylation arrays are provided for identifying a cell or mixture of cells and for quantification of alterations in distribution of cells in blood or in tissues, and for diagnosing, prognosing and treating disease conditions, particularly cancer.
  • the methods use fresh and archival samples.
  • the present invention solves the above object by providing a method for identifying immune cells in a sample, comprising analyzing the methylation status of at least one CpG position in the mammalian gene region for Prostaglandin D2 receptor 2 (PTGDR2) according to SEQ ID No. 1, wherein a demethylation or lack of methylation of said at least one CpG position in the gene region to at least about 80% is indicative for eosinophils or basophils, when compared to a CRTH2- or CRTH+ leukocyte, CD8+ T cell, CD4+ T cell, CD 14+ monocyte, CD19+ B cell, BDCA4+ pDC, CD56+ NK cell, and/or CD 15+ granulocyte.
  • PSGDR2 Prostaglandin D2 receptor 2
  • said at least one CpG position is selected from CpG position 4, 36, 42, 63, 68, 71, 75, 80, 99, 104, 110, 117, 122, 138, 148, 190, 219, 264, 267, 271, 273, 297, 321, 346, 366, 383, 386, 391, 401, 409, 414, and 417 according to SEQ ID No. 1, and preferably is selected from CpG position 271, 273, 297, 321, 346, 366, 383, and 386 according to SEQ ID No. 1.
  • a demethylation or lack of methylation of said at least one CpG position in the gene region to at least about 90% is indicative for eosinophils, wherein said at least one CpG position is selected from CpG position 297, 321, and 346 according to SEQ ID No. 1.
  • the present invention is based on the surprising identification of a region of the PTGDR2 gene according to SEQ ID NO: 1 by the inventors as specific epigenetic marker, allowing the identification of immune cells as well as the clinical routine application of said analysis.
  • the CpG positions are counted/numbered from the 5 ’-end of the sequence given in Figure 2, below. CpGs are underlined, and bold in the particularly preferred region of the assay according to the present invention. The positions as given correspond to the squares in the columns of Figure 1, counted starting from the left side of the Figure (see also legend).
  • the gene region shall comprise all of the genomic regions relating to and encoding for PTGDR2. Thus, included are enhancer regions, promoter region(s), introns, exons, and non-coding regions (5’- and/or 3 ’-regions) that belong to PTGDR2.
  • PTGDR2 Prostaglandin D2 receptor 2
  • ENSG00000183134 The sequence of homo sapiens Prostaglandin D2 receptor 2 (PTGDR2) can be taken from ENSG00000183134, the gene is located on Chromosome 11 : 60,850,933-60,855,950 reverse strand.
  • PTGDR2 and CRTH2 are used interchangeably, as they relate to the same gene and protein.
  • the genomic region for homo sapiens Prostaglandin D2 receptor 2 (PTGDR2), in particular according to SEQ ID No. 1 (Amp 3690), allows for the identification of eosinophils or basophils, when compared to a CD4+ T cell, CD8+ T cell, monocyte, B cell, neutrophil, pDC, or PTGDR2-negative cell, and preferably NK cells, and eosinophils, when compared to a CRTH2- or CRTH+ leukocyte, CD8+ T cell, CD4+ T cell, CD14+ monocyte, CD19+ B cell, BDCA4+ pDC, CD56+ NK cell, and/or CD15+ granulocyte, or more preferably the identification of eosinophils.
  • the discriminatory pattern of bisulfite convertible and non-convertible cytosine is particularly and even exclusively limited to the genomic region of PTGDR2, in particular the amplicon according to SEQ ID
  • the inventors could demonstrate that in eosinophils the CpG motifs as disclosed are almost completely demethylated (i.e. to more than about 80%) in eosinophils or basophils, when compared to other immune cells, where the same motifs are nearly completely, and preferably completely, methylated.
  • the inventors could further demonstrate that in eosinophils the CpG motifs selected from CpG position 271, 273, 297, 321, 346, 366, 383, and 386 according to SEQ ID No. 1, and preferably selected from CpG position 297, 321, and 346 according to SEQ ID No. 1, are almost completely demethylated, i.e. to at least about 80%.
  • the region distinctive for eosinophils is and includes the positions as shown in Figure 1 according to SEQ ID No. 1 (see also Figure 2), and forms the basis of a preferred qPCR assay comprising positions 271, 273, 297, 321, 346, 366, 383, and 386, see examples and Figure 2, comprising the oligomers according to SEQ ID Nos. 4 to 6.
  • the differential methylation of the CpG motifs within the aforementioned regions is a valuable tool to identify the immune cell populations as indicated, such as will be required/or at least of some value for identifying and quantifying said cells in autoimmune diseases, transplant rejections, infection diseases, cancer, allergy, endometriosis, cardiovascular diseases, primary and secondary immunodeficiencies, such as, for example, HIV infections and AIDS, Graft versus Host (GvH), hematologic malignancies, rheumatoid arthritis, multiple sclerosis, or a cytotoxic T cell related immune status in any envisionable diagnostic context.
  • the assay allows measurement of immune cells without purification or any staining procedures.
  • Another preferred aspect of the method according to the present invention then further comprises a quantification of the relative amount of eosinophils based on comparing relative amounts of said methylation frequency in the genetic region for PTGDR2, preferably according to SEQ ID No. 1, more preferably selected from CpG position 297, 321, and 346 according to SEQ ID No. 1, as analyzed with relative amounts of the methylation frequency in a control gene, such as, for example, GAPDH.
  • a control gene such as, for example, GAPDH.
  • Said quantification is thus achieved based on the ratio of the bisulfite convertible DNA to nonconvertible DNA in the genetic regions for PTGDR2 (e.g. of SEQ ID No. 1) as described and analyzed herein.
  • a quantification of the relative amount of immune cells is based on an (preferably parallel or simultaneous) analysis of the relative amount of bisulfite convertible DNA of the cell-specific regions for PTGDR2 (in particular according to SEQ ID NO: 1), and of the relative amount of bisulfite convertible DNA of cell-unspecific genes (preferably designated “control genes” or “control regions”, such as, for example, the gene for GAPDH).
  • control genes control regions
  • control regions such as, for example, the gene for GAPDH
  • the region distinctive for eosinophils is and includes the positions as shown in Figure 1 according to SEQ ID No. 1 (see also Figure 2), and may form the basis of a preferred qPCR assay, see examples and Figure 2, comprising the oligomers according to SEQ ID Nos. 4 to 6.
  • said analysis of bisulfite convertibility comprises amplification with at least one primer of suitable primer pairs that can be suitably designed based on SEQ ID No. 1, preferably oligomers according to any of SEQ ID No. 2 to 6.
  • the measurement s) and analyses can be done independent of purification, storage - and to quite some extent - also to tissue quality.
  • the amplification involves a polymerase enzyme, a PCR or chemical amplification reaction, or other amplification methods as known to the person of skill as described below, e.g. in the context of MSP, HeavyMethyl, Scorpion, MS-SNUPE, MethylLight, bisulfite sequencing, methyl specific restriction assays and/or digital PCR (see, for example Kristensen and Hansen PCR-Based Methods for Detecting Single-Locus DNA Methylation Biomarkers in Cancer Diagnostics, Prognostics, and Response to Treatment Clinical Chemistry 55:8 1471— 1483 (2009)).
  • an amplicon of the PTGDR2 gene region is produced that is a particularly preferred “tool” for performing the method(s) according to the present invention. Consequently, oligomers according to any of SEQ ID No. 2 to 6 or an amplicon as amplified by a primer pair based on SEQ ID No. 2 and 3 or 4 and 5 as mentioned herein constitute preferred embodiments of the present invention.
  • the sequence of SEQ ID No. 1 (and, if needed, the complementary sequences thereto) can be used to design primers for amplifications, i.e. serve as “beacons” in the sequence as relevant.
  • additional primers and probes can be designed based on the amplicon according to SEQ ID No. 1. Amplification can take place either in the genomic and/or bisulfite (i.e. “converted”) DNA sequence.
  • the present invention furthermore relates to a method for producing an (isolated) amplicon sequence in the mammalian (e.g. human) gene region for PTGDR2, preferably according to SEQ ID No. 1 of eosinophils or basophils, comprising a bisulfite treatment of the genomic DNA derived from a mammalian (e.g. human) sample comprising immune cells, and amplification of said gene region or a part thereof (e.g. located according to the sequence of SEQ ID No.
  • a method for producing an (isolated) amplicon sequence in the mammalian (e.g. human) gene region for PTGDR2, preferably according to SEQ ID No. 1 of eosinophils or basophils comprising a bisulfite treatment of the genomic DNA derived from a mammalian (e.g. human) sample comprising immune cells, and amplification of said gene region or a part thereof (e.g. located according to the sequence of SEQ ID No.
  • said amplicon sequence exhibits a demethylation or lack of methylation of at least one CpG position in the amplicon to at least about 80%, when compared to a CRTH2- or CRTH+ leukocyte, CD8+ T cell, CD4+ T cell, CD14+ monocyte, CD19+ B cell, BDCA4+ pDC, CD56+ NK cell, and/or CD 15+ granulocyte.
  • the amplicon sequence exhibits a demethylation or lack of methylation of at least one CpG position in the amplicon to at least about 90%, preferably at least about 95%, in eosinophils, wherein said at least one CpG position is selected from CpG position 297, 321, and 346 according to SEQ ID No. 1, when compared to immune cells other than eosinophils.
  • qPCR quantitative PCR
  • the person of skill will furthermore be able to select specific subsets of CpG positions in order to minimize the amount of sites to be analyzed, for example at least one of CpG position selected from a CpG position in an amplicon according to SEQ ID No. 1, and is preferably selected from the CpG 4, 36, 42, 63, 68, 71, 75, 80, 99, 104, 110, 117, 122, 138, 148, 190, 219, 264, 267, 271, 273, 297, 321, 346, 366, 383, 386, 391, 401, 409, 414, and 417 in the amplicon AMP 3690 according to SEQ ID No. 1.
  • CpG positions 271, 273, 297, 321, 346, 366, 383, and 386 for distinguishing eosinophils from other blood (immune) cells, more preferred are CpG positions 297, 321, and 346 according to SEQ ID No. 1.
  • the analysis of the methylation status comprises a method selected from methylation specific enzymatic digests, bisulphite sequencing, analysis selected from promoter methylation, CpG island methylation, MSP (methylation-specific PCR), HeavyMethyl, MethyLight, Ms-SNuPE or other methods relying on a detection of amplified DNA.
  • said method is suitable for routine application, for example on a DNA- such as an oligonucleotide-based chip. Based on the above information and the respective literature, the person of skill will be able to adjust the method as above to such settings.
  • said method is performed without a step of purifying and/or enriching said cells to be identified, preferably using whole blood and/or non-trypsinized tissue.
  • the identification comprises a distinction of said immune cells as above from all major peripheral blood cell types and/or non-blood cells, or cord blood cells, and/or non-blood cells such as from at least one of the cell type selected from endothelial cells, smooth muscle cells (aortic or intestine), and dermal fibroblasts.
  • the sample is selected from a mammalian body fluid, including human blood samples, human samples comprising immune cells, cord blood sample, or a tissue, organ or a sample of lymphocytes or a purified or separated fraction of such tissue, organ or lymphocytes or a cell type sample.
  • a mammal is a mouse, goat, dog, pig, cat, cow rat, monkey or human.
  • the samples can be suitably pooled, if required.
  • said cells are human cells, such as immune cell preparations.
  • Another preferred aspect of the method according to the present invention then further comprises the step of concluding on the immune and/or disease status of said mammal based on said immune cells as identified.
  • the immune cells as identified can be quantified and be used as a benchmark to relatively quantify further detailed subpopulations, or it can be used as a predictive and/or screening and/or diagnostic and/or prognostic and/or adverse events detecting factor, or it can be used to finally detect this population to determine the overall immune or disease activity status.
  • the mammal suffers from or is likely to suffer from autoimmune diseases, transplant rejections, infection diseases, cancer, and/or allergy as but not limited to Trypanosoma cruzi-miQ(P on, malaria and HIV infection; hematologic malignancies as but not limited to chronic myelogenous leukemia, multiple myeloma, Non Hodgkin's lymphoma, Hodgkin's Disease, chronic lymphocytic leukemia, graft versus host and host versus graft disease, mycosis fungoides, extranodal T cell lymphoma, cutaneous T cell lymphomas, anaplastic large cell lymphoma, angioimmunoblastic T cell lymphoma and other T-cell, B-cell and NK cell neoplasms, endometriosis, cardiovascular diseases, T cell deficiencies such as but not limited to lymphocytopenia, severe combined immunodeficiency (SCID), Omenn
  • Another preferred aspect of the method according to the present invention relates to a method as above, further comprising measuring and/or monitoring the amount of the immune cells as identified in response to chemical and/or biological substances that are provided to said mammal, i.e. in response to a treatment of said patient.
  • Said method comprises the steps as above, and comparing said relative amount of said cells as identified to a sample taken earlier or in parallel from the same mammal, and/or to a control sample. Based on the results as provided by the method(s) of the invention, the attending physician will be able to conclude on the immune status of the patient, and adjust a treatment of the underlying disease accordingly.
  • said method is performed without a step of purifying and/or enriching cells, preferably in whole blood and/or non-trypsinized tissue, or any other biological sample potentially containing said immune cells as e.g. a sample for cell transfer into a patient.
  • Another preferred aspect of the method according to the present invention then relates to a method as above, further comprising formulating said immune cells as identified for transplantation into a patient.
  • Pharmaceutical preparations for these purposes and methods for their production are performed according to methods known in the art of transplantation medicine.
  • Another preferred aspect of the method according to the present invention then further then relates to a method for treating a condition or disease in a mammal, in particular in a human, comprising a method according to the invention as above, and the step of transplanting the immune cells as identified, i.e. basophils and eosinophils, preferably eosinophils, and/or as isolated/multiplied in cell culture into a patient.
  • Pharmaceutical preparations for these purposes and methods for their production are performed according to methods known in the art of transplantation medicine.
  • the transplant can be autologous or allogenic.
  • Another preferred aspect of the method according to the present invention then further then relates to a method for treating and/or preventing a condition or disease in a mammal, in particular in a human, comprising a method according to the invention as above including a suitable treatment for said condition or disease comprising providing chemical and/or biological substances as above, and adjusting said treatment of the underlying disease or condition based on the results as provided by the method(s) of the invention.
  • This may comprise the step of concluding on the immune or disease status of said mammal based on said immune cells as identified.
  • the immune cells can be quantified and be used as a benchmark to relatively quantify further detailed subpopulations, or it can be used as a predictive and/or screening and/or diagnostic and/or prognostic and/or adverse events detecting factor, or it can be used to finally detect this population to determine the overall immune activity status.
  • This basis allows for adjusting said treatment, if necessary.
  • Such adjustments may comprise the step of transplanting the immune cells as identified and isolated/multiplied in cell culture into a patient as above, and/or providing additional chemical and/or biological substances for adjusting said treatment and/or prevention.
  • One particular example is a method for treating and/or preventing a condition or disease in a mammal, in particular in a human, wherein first a medicament is provided to said mammal.
  • Respective medication strategies are known, optionally with suitable carriers and adjuvants. Therefore, the method then comprises measuring and/or monitoring the amount of immune cells in response to said medication that is/are provided to said mammal.
  • said treatment here: medication
  • said treatment is adjusted, i.e. more drug is given.
  • the method may be repeated until sufficient desired cells (i.e. a substantial population of immune cells) can be detected.
  • Treatment and/or prevention shall herein relate to the curing, prevention or alleviation of a disorder or malfunction of the body, i.e. bringing a body back to its healthy state.
  • compositions for these purposes and methods for their production are performed according to methods known in the art of a treatment using chemical and/or biological substances or transplantation medicine. Again, the transplant can be autologous or allogenic.
  • Another preferred aspect of the method according to the present invention relates to an oligomer according to any of SEQ ID No. 2 to 6, or an amplicon according to SEQ ID No. 1.
  • kits for identifying, quantifying, and/or monitoring immune cells selected from basophils and eosinophils, in particular eosinophils, in a mammal based on the analysis of the bisulfite accessibility of CpG positions in the genetic region for PTGDR2, in particular according to SEQ ID NO: 1, comprising components for performing a method according to invention as described herein, in particular a kit comprising a) a bisulfite reagent, and b) materials for the analysis of the methylation status of CpG positions selected from the CpG positions in the region according to SEQ ID NO: 1, such as an oligomer selected from the sequences according to SEQ ID No. 2 to 6, or an amplicon as amplified by a primer pair based on SEQ ID No. 2 and 3 or 4 and 5, respectively.
  • the present invention also encompasses the use of oligomers or amplicon or a kit according to the present invention for identifying and/or for monitoring immune cells in a mammal as described herein, in particular eosinophils.
  • non-bisulfite convertible cytosine encompasses 5-methylcytosine (mC) and 5- hydroxymethylcytosine (hmC), and (ii) bisulfite convertible (i.e.
  • cytosine encompasses 5-formylcytosine (fC), 5-carboxycytosine (cC), as well as non-modified cytosine. Additionally, past inventions are based on (i) the ratio of bisulfite convertible cytosine to whole amount of chromatin (cell-type independent, 100% bisulfite convertible DNA locus) or (ii) on the ratio of bisulfite convertible cytosine (fC, cC, non-modified cytosine) to non-bisulfite convertible cytosine (hmC and mC). These ratios characterize cell type, cell differentiation, cell stage as well as pathological cell stages. Therefore, new techniques will result in novel, more specific ratios and might supplement current cell specific, cell state specific as well as pathological patterns of epigenetic modifications and therefore, define potential novel biomarkers. Novel ratios to be discovered as biomarkers can be defined as:
  • epigenetic modifications in the DNA sequence is referred to by the terminology of (i) bisulfite convertible cytosine (5- formylcytosine, (fC) and/or 5-carboxy cytosine (cC)) and (ii) non-bisulfite convertible cytosine ((including 5-methylcytosine (mC), 5-hydroxymethylcytosine, (hmC)).
  • fC, cC bisulfite convertible cytosine
  • non-bisulfite convertible cytosine (including 5-methylcytosine (mC), 5-hydroxymethylcytosine, (hmC)).
  • mC 5-methylcytosine
  • hmC 5-hydroxymethylcytosine
  • methylated DNA encompasses mC as well as hmC.
  • non-methylated DNA encompasses fC, cC, and non-modified DNA. It is expected that novel variants of DNA modifications will be discovered in future. Each type of modification will be either bisulfite convertible or not. However, since the present method reliably distinguishes between the two groups, these novel modifications will also be usable as markers.
  • histones undergo posttranslational modifications that alter their interaction with DNA and nuclear proteins. Modifications include methylation, acetylation, phosphorylation, ubiquitination, sumoylation, citrullination, and ADP-ribosylation.
  • the core of the histones H2A, H2B, and H3 can also be modified. Histone modifications act in diverse biological processes such as gene regulation, DNA repair, chromosome condensation (mitosis) and spermatogenesis (meiosis).
  • a specific pattern of modification is specific for different cell types, cell stages, differentiation status and such a pattern can be analyzed for bisulfite convertibility or similar methods in order to identify certain cells and cell stages.
  • the present invention also encompasses a use of these modifications.
  • the inventors using the PTGDR2 genetic region and in particular the amplicon according to SEQ ID NO: 1 as described herein as a marker, more particularly the region of the qPCR assay comprising the CpG position 271, 273, 297, 321, 346, 366, 383, and 386, preferably CpG position 297, 321, and 346 as described herein (Example and Figure 1), the inventors very specifically identified, quantified and in particular differentiated immune cells, and in their relation to other cell types in a sample, for example to other blood cells.
  • Figure 1 shows the analysis of CpG sites on amplicon AMP3690 (SEQ ID No. 1) according to the invention.
  • the rows in the table correspond to the cell types as analyzed and the columns correspond to the CpG positions in the amplicon as analyzed (e.g. CpG 1, 2, etc.) with the positions indicated (AMP3690:297 corresponding to CpG at position 297 of Amplicon 3690 according to SEQ ID No. 1, ...etc.).
  • the CRTH2+ leukocytes were enriched to 80% purity (upper preparation), and 45% purity (lower preparation), respectively.
  • the Th2 cells were gated for CD3+/CD4+/CD45RA-/CCR6-/CXCR3- expression, and not for CRTH2 expression.
  • the Thl cells were gated for CD3+/CD4+/CD45RA-/CCR6-/CXCR3+.
  • Figure 2 shows the genomic sequence of the amplicon (AMP3690) according to the present invention (SEQ ID No. 1).
  • the positions of the primers according to SEQ ID Nos: 4 and 5 are double underlined, the probe according to SEQ ID No: 6 is indicated in italic, the CpG positions inside the preferred qPCR assay region are indicated in bold.
  • SEQ ID No. 1 shows the genomic sequence of amplicon No. 3690.
  • SEQ ID Nos. 2 to 6 show the sequences of specific oligomers (primers and probes) according to the present invention.
  • Oligonucleotides of qPCR Assay (TpG Variant i.e., demethylation-specific; 5' - 3')

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Abstract

La présente invention concerne un procédé, plus particulièrement un procédé in vitro, pour identifier les cellules immunitaires, comprenant l'analyse des modifications/propriétés épigénétiques (y compris l'état de méthylation) d'au moins une position CpG dans la région du gène mammifère pour le récepteur 2 de la prostaglandine D2 (PTGDR2), dans lequel une déméthylation ou une absence de méthylation d'au moins une position CpG dans la région du gène à au moins 80 % indique la présence d'éosinophiles ou de basophiles, par comparaison avec un leucocyte CRTH2 ou CRTH+, un lymphocyte T CD8+, un lymphocyte T CD4+, un monocyte CD14+, un lymphocyte B CD19+, un pDC BDCA4+, une cellule NK CD56+, et/ou un granulocyte CD15+. L'analyse selon l'invention permet d'identifier les cellules immunitaires au niveau épigénétique et de les distinguer de toutes les autres cellules dans des échantillons complexes, tels que, par exemple, d'autres cellules sanguines, non sanguines ou d'autres cellules immunitaires. La présente invention concerne en outre un procédé amélioré de quantification des cellules immunitaires, plus particulièrement dans des échantillons complexes. Le procédé peut être réalisé sans étape de purification et/ou d'enrichissement des cellules, de préférence dans du sang total et/ou des tissus non trypsinisés.
PCT/EP2024/061805 2023-05-26 2024-04-29 Crth2 (ptgdr2) en tant que marqueur épigénétique pour l'identification de cellules immunitaires Pending WO2024245668A1 (fr)

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DE102023114008.6A DE102023114008B3 (de) 2023-05-26 2023-05-26 CRTH2 (PTGDR2) als epigenetischer Marker zur Identifizierung von Immunzellen
DE102023114008.6 2023-05-26

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