WO2019091429A1 - Empreintes génétiques uniques pour modèle tumoral murin et ses utilisations - Google Patents

Empreintes génétiques uniques pour modèle tumoral murin et ses utilisations Download PDF

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WO2019091429A1
WO2019091429A1 PCT/CN2018/114595 CN2018114595W WO2019091429A1 WO 2019091429 A1 WO2019091429 A1 WO 2019091429A1 CN 2018114595 W CN2018114595 W CN 2018114595W WO 2019091429 A1 WO2019091429 A1 WO 2019091429A1
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cell line
human tumor
determined
comprises seq
tumor cell
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PCT/CN2018/114595
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English (en)
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Jie Cai
Wubin QIAN
Xiaobo Chen
Bin Fan
Sheng Guo
Henry Li
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Crown Bioscience Inc. (Taicang)
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention generally relates to cancer diagnosis, prognosis and treatment.
  • the present invention relates to the identification of murine tumor models.
  • Tumor models are naturally existing or artificially induced systems that share features with human cancers.
  • Experimental systems for studying human cancer include cell lines and animals (e.g., mice) grafted with tumor cells line or tissues, which can be derived from human or non-human animals (e.g., mice) .
  • syngeneic and homograft murine tumor models become valuable experimental tools for preclinical efficacy evaluation of immune therapy and in combination with conventional or targeted cancer therapies.
  • murine tumor models were derived from limited number of laboratory strains of mice and do not have readily and sufficiently diversified common genotyping markers to differentiate individual tumor models. Hence, there is a need to develop markers and methods for identifying individual murine tumor models and for tracking the fidelity in these models.
  • the present disclosure provides a method for producing a non-human tumor model.
  • the method comprises the steps of transplanting a non-human tumor cell line or a primary tumor tissue to a non-human animal; raising the non-human animal in a condition that allows the non-human tumor cell line or the primary tumor tissue to develop into a tumor; detecting in the tumor the presence of a biomarker that is unique to the non-human tumor cell line or the primary tumor tissue; and determining the identification of the non-human tumor cell line or the primary tumor tissue.
  • the method comprises the steps of culturing a non-human tumor cell line in vitro; detecting in the non-human tumor cell line the presence of a biomarker that is unique to the non-human tumor cell line; determining the identification of the non-human tumor cell line; transplanting the non-human tumor cell line to a non-human animal; and raising the non-human animal in a condition that allows the non-human tumor cell line to develop into a tumor.
  • the non-human tumor model is a murine tumor model.
  • the non-human tumor cell line is selected from the group consisting of 4T1, A20, B16BL6, B16F0, B16F1, B16F10, C1498, Colon26, CT26, EG7_Ova, EL4, EMT6, H22, Hepa 1-6, J558, JC, KLN205, L1210, L5178-R, LL/2, MBT2, MPC-11, Neuro-2a, P388D1, P815, Renca, S91 and WEHI164.
  • the biomarker is a fusion gene.
  • the fusion gene is selected from the fusion genes in Table 1.
  • the fusion gene comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-75.
  • the biomarker is detected by an amplification assay, a hybridization assay, a sequencing assay or an array.
  • the biomarker is detected using a primer set capable of detecting a biomarker panel comprising SEQ ID NOs: 1-75.
  • the biomarker is detected using a microarray comprising probes capable of detecting a biomarker panel comprising SEQ ID NOs: 1-75.
  • the non-human tumor model is determined as cell line 4T1 when the biomarker comprises SEQ ID NO: 1.
  • the non-human tumor model is determined as cell line A20 when the biomarker comprises SEQ ID NO: 2 or 3.
  • the non-human tumor model is determined as cell line B16BL6 when the biomarker comprises SEQ ID NO: 4 or 5.
  • the non-human tumor model is determined as cell line B16F0 when the biomarker comprises SEQ ID NO: 6 or 7.
  • the non-human tumor model is determined as cell line B16F1 when the biomarker comprises SEQ ID NO: 8.
  • the non-human tumor model is determined as cell line B16F10 when the biomarker comprises SEQ ID NO: 9.
  • the non-human tumor model is determined as cell line C1498 when the biomarker comprises SEQ ID NO: 10, 11 or 12.
  • the non-human tumor model is determined as cell line Colon26 when the biomarker comprises SEQ ID NO: 13 or 14.
  • the non-human tumor model is determined as cell line CT26 when the biomarker comprises SEQ ID NO: 15 or 16.
  • the non-human tumor model is determined as cell line EG7_Ova when the biomarker comprises SEQ ID NO: 17.
  • the non-human tumor model is determined as cell line EL4 when the biomarker comprises SEQ ID NO: 18, 19 or 20.
  • the non-human tumor model is determined as cell line EMT6 when the biomarker comprises SEQ ID NO: 21, 22 or 23.
  • the non-human tumor model is determined as cell line H22 when the biomarker comprises SEQ ID NO: 24, 25, 26, or 27.
  • the non-human tumor model is determined as cell line Hepa 1-6 when the biomarker comprises SEQ ID NO: 28, 29 or 30.
  • the non-human tumor model is determined as cell line J558 when the biomarker comprises SEQ ID NO: 31 or 32.
  • the non-human tumor model is determined as cell line JC when the biomarker comprises SEQ ID NO: 33 or 34.
  • the non-human tumor model is determined as cell line KLN205 when the biomarker is detected as comprises SEQ ID NO: 35 or 36.
  • the non-human tumor model is determined as cell line L1210 when the biomarker comprises SEQ ID NO: 37, 28, 39 or 40.
  • the non-human tumor model is determined as cell line L5178-R when the biomarker comprises SEQ ID NO: 41, 42, 43, 44, 45, 46, 48, 49 or 50.
  • the non-human tumor model is determined as cell line LL/2 when the biomarker comprises SEQ ID NO: 51, 52 or 53.
  • the non-human tumor model is determined as cell line MBT-2 when the biomarker comprises SEQ ID NO: 54 or 55.
  • the non-human tumor model is determined as cell line MPC-11 when the biomarker comprises SEQ ID NO: 56 or 57.
  • the non-human tumor model is determined as cell line Neuro-2a when the biomarker comprises SEQ ID NO: 58, 59, 60 or 61.
  • the non-human tumor model is determined as cell line P388D1 when the biomarker comprises SEQ ID NO: 62, 63, 64 or 65.
  • the non-human tumor model is determined as cell line P815 when the biomarker comprises SEQ ID NO: 66, 67 or 68.
  • the non-human tumor model is determined as cell line Renca when the biomarker comprises SEQ ID NO: 69 or 70.
  • the non-human tumor model is determined as cell line S91 when the biomarker comprises SEQ ID NO: 71 or 72.
  • the non-human tumor model is determined as cell line WEHI164 when the biomarker comprises SEQ ID NO: 73, 74 or 75.
  • the non-human animal is a rodent. In certain embodiments, the non-human animal is a mouse.
  • the present disclosure provides a kit for detecting a fusion gene.
  • the fusion gene comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-75.
  • the present disclosure provides a hybridization probe for detecting a fusion gene selected from the group consisting of the fusion genes in Table 1.
  • the fusion gene comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-75.
  • the present disclosure provides a kit comprising primers for detecting a biomarker panel.
  • the biomarder panel comprises SEQ ID NOs: 1-75.
  • the present disclosure provides a microarray comprising probes for detecting a biomarker panel.
  • the biomarder panel comprises SEQ ID NOs: 1-75.
  • FIGs. 1A-1K illustrate the sequences of fusion genes that are unique to the murine tumor cell lines of the present invention.
  • the junction positions are marked with symbol “
  • cancer and “tumor” are interchangeable and refer to any diseases involving an abnormal cell growth and include all stages and all forms of the disease that affects any tissue, organ or cell in the body.
  • the term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and cancers of all stages and grades including pre-and post-metastatic cancers.
  • cancers can be categorized according to the tissue or organ from which the cancer is located or originated and morphology of cancerous tissues and cells.
  • cancer types include, without limitation, acute lymphoblastic leukemia (ALL) , acute myeloid leukemia, adrenocortical carcinoma, anal cancer, astrocytoma, childhood cerebellar or cerebral, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumor, brain cancer, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, Burkitt's lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, emphysema, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, retinoblastoma, gastric (stomach) cancer,
  • ALL acute lymph
  • complementarity refers to the ability of a nucleic acid to form hydrogen bond (s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%>, 70%>, 80%>, 90%, and 100%complementary) .
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100%over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
  • determining, ” “assessing, ” “assaying, ” “measuring” and “detecting” can be used interchangeably and refer to both quantitative and semi-quantitative determinations. Where either a quantitative and semi-quantitative determination is intended, the phrase “determining a level” of a polynucleotide or polypeptide of interest or “detecting” a polynucleotide or polypeptide of interest can be used.
  • hybridizing refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions.
  • stringent conditions refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences in a mixed population (e.g., a cell lysate or DNA preparation from a tissue biopsy) .
  • a “stringent hybridization” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization are sequence dependent, and are different under different environmental parameters.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on an array or on a filter in a Southern or northern blot is 42°C. using standard hybridization solutions (see, e.g., Sambrook and Russell Molecular Cloning: A Laboratory Manual (3rd ed. ) Vol. 1-3 (2001) Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY) .
  • An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2 ⁇ SSC wash at 65°C for 15 minutes. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is l ⁇ SSC at 45°C for 15 minutes.
  • An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4 ⁇ SSC to 6 ⁇ SSC at 40°C for 15 minutes.
  • nucleic acid and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA) , transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers.
  • the nucleic acid molecule may be linear or circular.
  • a “protein” is a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds) . Proteins may include moieties other than amino acids (e.g., may be glycoproteins) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence) , or can be a functional portion thereof. Those of ordinary skill will further appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
  • Tumor models refer to cells, tissues or animals used to study the development and progression of cancer, and to test treatments before they are given to human.
  • the tumor models provided herein are non-human animals grafted with tumor cell lines or primary tumor tissues.
  • the tumor cell lines used herein include, without limitation, 4T1, A20, B16BL6, B16F0, B16F1, B16F10, C1498, Colon26, CT26, EG7_Ova, EL4, EMT6, H22, Hepa 1-6, J558, JC, KLN205, L1210, L5178-R, LL/2, MBT2, MPC-11, Neuro-2a, P388D1, P815, Renca, S91 and WEHI164.
  • the 4T1 cell line is a mouse breast tumor cell line that is highly tumorigenic and invasive.
  • the 4T1 cell line can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain and bone.
  • 4T1 The following properties of 4T1 make it a suitable experimental animal model for human mammary cancer: (1) 4T1 cells are easily transplanted into the mammary gland so that the primary tumor grows in the anatomically correct site; (2) as in human breast cancer, 4T1 metastatic disease develops spontaneously from the primary tumor and the progressive spread of 4T1 metastases to the draining lymph nodes and other organs is very similar to that of human mammary cancer; (3) 4T1 is resistant to 6-thioguanine, hence enabling precise quantitation of metastatic cells, even when they are disseminated and at sub-microscopic levels in distant organ.
  • the A20 cell line is a BLAB/c B cell lymphoma line derived from a spontaneous reticulum cell neoplasm found in an old BALB/cAnN mouse.
  • the A20 cells express little surface immunoglobulin when grown in Click's medium; however, they express large amounts when grown in RPMI 1640 medium.
  • the A20 cells can present both alloantigens and protein antigens.
  • the B16BL6 cell line is a murine melanoma cell line derived from B16 cell line.
  • the B16BL6 cells display significantly stronger invasive activity than B16.
  • the B16F0 cell line is a murine melanoma cell line derived from B16 cell line. It is tumorigenic when being grafted in syngeneic mice but display less invasive activity than B16F10.
  • the B16F0 cell line is a murine melanoma cell line derived from B16 cell line. It is tumorigenic when being grafted in syngeneic mice but display less invasive activity than B16F10.
  • the B16F10 cell line is a murine melanoma cell line derived from B16 cell line.
  • the B16F10 cells display significantly stronger invasive activity than B16, B16F0 and B16F1.
  • the C1498 cell line is a murine AML cell line that arose spontaneously in a C57BL/6 mouse and grows aggressively in syngeneic mice.
  • the Colon26 cell line is a mouse colon adenocarcinoma derived from BALB/c.
  • the CT26 cell line is an N-nitroso-N-methylurethan-induced, undifferentiated colon carcinoma cell line.
  • the CT26 cell line has become a preclinical platform for evaluating the potential of drug combinations with immune checkpoint inhibitor antibodies.
  • the EG7_Ova cell line is a mouse thymoma EL4 cell line derived from C57BL/6.
  • the cell line is stably transfected with the cDNA of chicken ovalbumin (OVA) and thus express OVA epitope as a unique antigen.
  • OVA ovalbumin
  • the EL4 cell line is a 9, 10-dimethyl-1, 2-benzanthracene induced lymphoma cell line from a C57L mouse.
  • the EMT6 cell line was established from a transplantable murine mammary carcinoma that arose in a BALB/cCRGL mouse after implantation of a hyperplastic mammary alveolar nodule.
  • the resulting tumor line (named KHJJ) was propagated in BALB/cKa mice and adapted to tissue culture after the 25th animal passage, and the cell line was named EMT.
  • EMT6 is a clonal isolate of EMT isolated in 1971 at Stanford University.
  • the H22 cell line is a mouse hepatoma cell line.
  • the Hepa 1-6 cell line is derived from the BW7756 mouse hepatoma that arose in a C57/L mouse.
  • the J558 cell line is a mouse B myeloma cell line derived from a BALB/c mouse.
  • the JC cell line is an epithelial-like cell line derived from a spontaneous primary mammary adenocarcinoma along the milk line.
  • the KLN205 cell line is a murine lung carcinoma cell line, established form the Nettersheim lung carcinoma.
  • the L1210 cell line is a mouse lymphocytic leukemia cell line that is derived from the ascetic fluid of an 8-month-old female mouse.
  • the L5178-R cell line is derived from the L5178 thymic lymphoma induced by methylcholanthrene in a DBA/2 mouse.
  • the LL/2 cell line is a Lewis lung carcinoma cell line established from the lung of a C57B mouse bearing a tumor resulting from an implantation of primary Lewis lung carcinoma.
  • the MBT2 cell line is a murine bladder carcinoma cell line.
  • the MPC11 cell line is a murine myeloma cell line.
  • the Neuro-2a cell line is a mouse neuroblastoma cell line. It displays a neuronal and amoeboid stem cell morphology.
  • the P388D1 cell line is a murine macrophage cell line.
  • the P815 cell line is a mouse mastocytomacell line derived from DBA/2 mice.
  • the Renca cell line is an adenocarcinoma cell line derived from a tumor that arose spontaneously as a renal cortical adenocarcinoma in Balk/cCr mice.
  • the S91 cell line is mouse melanoma cell line.
  • the WEHI164 cell line is a mouse fibrosarcoma cell line induced by subcutaneous injection of 3-methylcholanthrene.
  • the present disclosure provides novel fusion genes in murine tumor models.
  • Fusion gene and “gene fusion” are used interchangeably herein and are intended to encompass both DNA and RNA, including but not limited to fusion of two or more separate genes at DNA level (such as genomic DNA or cDNA) , RNA level (such as mRNA) .
  • the two genes are fused at genomic DNA level due to chromosome rearrangement, such as a translocation, interstitial deletion, or chromosomal inversion. Fusion gene may be transcribed and/or translated to its gene product, which can be RNA or protein.
  • the fusion genes provided herein comprise a first encoding sequence for a first gene ( “upstream gene” or “up gene” ) covalently linked to a second encoding sequence for a second gene ( “downstream gene” or “down gene” ) .
  • the upstream gene and the downstream gene as disclosed herein can refer the gene (e.g. the DNA sequence) , the gene transcript (e.g. mRNA) , or the protein product (i.e., the amino acid sequence) , and people skilled in the art can understand the meaning from the context.
  • “Encoding sequence” as used herein refers to the polynucleotide sequence which encodes at least a fragment of a protein product.
  • the encoding sequence can be a DNA sequence such as genomic DNA or cDNA, and can also be a RNA sequence such as mRNA.
  • the fusion of the two encoding sequences can be in frame, such that after being translated into its protein product, a protein fragment of the upstream gene is fused to a protein fragment of the downstream gene.
  • the upstream gene and the downstream gene can be found in Table 1.
  • the first encoding sequence for the upstream gene and the second encoding sequence for the downstream gene can be found in Table 1.
  • fusion junction The site where the upstream gene sequence fuses to the downstream gene sequence is referred to as fusion junction.
  • the fusion genes provided herein comprise a fusion junction as disclosed in Table 1.
  • the fusion gene detected in a murine tumor model is unique to the murine tumor model when the fusion gene is not detected in other murine tumor models.
  • the method comprises: obtaining a nucleic acid containing sample from the murine tumor model, contacting the sample with a detecting agent which specifically detects a target polynucleotide comprising a fusion of a first encoding sequence for a upstream gene and a second encoding sequence for a downstream gene, and detecting the presence of the target polynucleotide.
  • the nucleic acid-containing sample can be derived from a cell or a tissue of the murine tumor model.
  • “Nucleic acid” as used herein can be a polymer of RNA or a polymer of DNA.
  • the sample may contain isolated nucleic acid such as isolated RNA or cDNA.
  • the sample may contain nucleic acid in its natural or unpurified or unamplified state, for example, the sample may be an isolated cell or tissue, optionally pretreated to release the nucleic acid contained therein.
  • the nucleic acid in the sample may be amplified, e.g. by PCR reaction or PCR following reverse transcription.
  • the sample is derived from a non-human animal transplanted with the murine tumor model.
  • the sample can be any suitable biological material collected from the non-human animal, such as body fluid (e.g. blood) and a biopsy sample (e.g. cells or tissues from a disease affected area) .
  • the sample can be treated to extract the nucleic acid.
  • the sample is contacted with an oligonucleotide which specifically detects a target polynucleotide comprising the fusion gene.
  • the target polynucleotide can be cDNA, DNA, or mRNA, depending on the type of nucleic acid contained in the sample.
  • the target polynucleotide can comprise any of the fusion genes as provided herein.
  • the target polynucleotide can be detected based on any suitable methods known in the art, for example but not limited to, hybridization-based methods and amplification-based methods.
  • Hybridization-based methods usually involve using a probe to hybridize and detect the target sequence. Examples of hybridization-based methods include, Northern blot, DNA microarray, whole genome sequencing, RNA sequencing (RNA-seq) , quantitative real time PCR (qRT-PCR) , digital multiplexed gene expression analysis method (see, e.g., Kulkarni MM, Curr Protoc Mol Biol. 2011 Apr, Chapter 25: Unit25B. 10.
  • FISH method Fluorescence In Situ Hybridization
  • CISH Chromogenic In Situ Hybridization
  • SISH silver in situ hybridization
  • Amplification-based methods usually involve using primers, polymerase and mixture of nucleotide monomers to synthesize nascent polynucleotide chain based on the base sequence of the target template polynucleotide.
  • amplification-based methods include, PCR (polymerase chain reaction) , LCR (Ligase chain reaction) , SDA (Strand displacement amplification) , isothermal and chimeric primer-initiated amplification of nucleic acids) , loop-mediated isothermal amplification, transcription-mediated amplification and the like.
  • the detecting step involves an amplification step.
  • the detecting agent comprises at least a pair of primers which can hybridize to the target polynucleotide and amplify a target region encompassing the fusion junction in the presence of a polymerase.
  • the detecting agent comprises a first primer directed to the first encoding sequence for the upstream gene, and a second primer directed to the second encoding sequence for the downstream gene.
  • a primer or a probe “directed to” a sequence means that the primer or the probe has sufficient identity with or complementarity to at least a portion of the sequence such that the primer or the probe can specifically hybridize to the sequence or to its complementary strand.
  • “Specifically hybridize” as used herein means the primer or probe can hybridize to the intended sequence under stringent conditions.
  • Stringent condition refers to hybridizing at 42 °C in a solution consisting of 5 ⁇ SSPE, 5 ⁇ Denhardt’s solution, 0.5%SDS, and 100 ug/mL denatured salmon sperm DNA, and then washing at 42 °C with a solution comprising 0.5 ⁇ SSC and 0.1%SDS.
  • the detecting agent comprises a junction primer directed to a fragment containing the fusion junction, and a non-junction primer directed to the first or the second encoding sequence.
  • the junction primer would specifically hybridize to the fusion junction, thereby specifically enabling the amplification when the target polynucleotide is present. Otherwise, if the nucleic acid in the sample does not contain the target polynucleotide, the junction primer would not specifically hybridize to its target sequence, and cannot effectuate a meaningful amplification.
  • the amplification product After amplification by a suitable nucleic acid amplification method such as PCR, the amplification product is detected.
  • the amplification product has a length of 100bp-1500bp (e.g. 100bp-1000bp, 100bp-900bp, 100bp-800bp, 100bp-700bp, 100bp-600bp, 100bp-500bp, 100bp-400bp, 100bp-350bp, 100bp-300bp, 200bp-1000bp, 200bp-900bp, 200bp-800bp, 200bp-700bp, 200bp-600bp, 200bp-500bp, 200bp-400bp, 200bp-350bp, 200bp-300bp, etc.
  • 100bp-1500bp e.g. 100bp-1000bp, 100bp-900bp, 100bp-800bp, 100bp-700bp, 100bp-
  • the presence of the amplification product would be indicative of the presence of the target polynucleotide.
  • the molecular weight or size or sequence of the amplification product is further detected, and a desired size or sequence of the amplification product indicates presence of the target polynucleotide.
  • the amplification step may optionally further comprises a reverse transcription step to produce cDNA of the RNA in the sample.
  • the cDNA is then amplified using the primers to allow detection of presence of the fusion junction.
  • the first primer or the second primer is directed to a region at least 80bp upstream or downstream of the fusion junction of the fusion gene.
  • the fusion gene comprises a fusion junction as disclosed in Table 1.
  • the first primer and the second primer are useful of amplifying an amplicon having a length of about 200bp to 400bp.
  • the non-junction primer can be designed based on the desired length of the amplification product, once the junction primer is determined. For example, when it is desired to have a 300bp amplification product, then the non-junction primer can be designed to be complementary to the target polynucleotide about 300bp 5’ upstream the fusion junction or 3’ downstream of the fusion junction.
  • the detecting step involves a hybridization step.
  • Probes can be designed to specifically hybridize to the target polynucleotide, thereby allowing its detection.
  • Probes provided herein can have a suitable length, for example, about 20-200bp (e.g. 20-190bp, 20-150bp, 20-120bp, 20-100bp, 20-90bp, 20-80bp, 20-70bp, 20-60bp, 20-50bp, 20-40bp, and etc. ) .
  • the detecting agent comprises a first probe directed to the first encoding sequence for the upstream gene, and a second probe directed to the second encoding sequence for the downstream gene.
  • one of the first and the second probes can be a capture probe which further comprises an immobilizing moiety capable of associating with a substrate through a covalent or a non-covalent bond
  • the other probe can be a detecting probe which further comprises a detectable label.
  • the capture probe can be first contacted with the sample to allow hybridization with the nucleic acid, then the complex is immobilized on a substrate via the immobilizing moiety on the capture probe, and the unbound molecules are removed.
  • the detecting probe is then added to the immobilized complexes to allow hybridization to occur. After washing away the excess probe, the detectable label immobilized on the substrate is detected.
  • the immobilizing moiety include, but are not limited to, biotin, streptavidin, antigen, antibody, protein A, protein G, oligonucleotide, etc.
  • the detectable label on the detecting probe can be, for example, fluorescent dye, radioisotope, antibody, enzyme, and oligonucleotide (e.g. an oligonucleotide barcode) .
  • one of the first and the second probes further comprises a fluorescent dye and the other probe comprises a quencher.
  • fluorescent dye include, but are not limited to fluorescein isothiocyanate (FITC) , Alexa 488, Alexa 532, cy3, cy5, 6-joe, EDANS; rhodamine 6G (P6G) and its derivatives (tetramethyirhodamine (TMR) , tetramethylrhodamine isothiocyanate (TMRITC) , x-rhodamine, Texas red, "BODJPY FL" (trade name, product of Molecular Probes, Inc.
  • FITC fluorescein isothiocyanate
  • TMR rhodamine 6G
  • TMR tetramethyirhodamine
  • TRITC tetramethylrhodamine isothiocyanate
  • x-rhodamine Texas red
  • BODJPY FL trade name, product of Molecular Probes, Inc.
  • quencher examples include, but are not limited to, Dabcyl, "QSY7” (Molecular Probes) , “QSY33” (Molecular Probes) , Ferrocene and its derivatives, methyl viologen, and N, N'-dimethyl-2, 9-diazopyrenium and the like.
  • the detecting agent comprises a junction probe directed to a fragment containing the fusion junction.
  • the junction probe may further comprise a detectable label.
  • the nucleic acid in the sample may be immobilized on a substrate, and then contacted with the probe which recognizes the fusion junction. After washing away the unreacted probes, the substrate can be detected for presence of the probe, which can indicate the presence of the fusion junction of the fusion gene.
  • the junction probe can comprise both a fluorescent dye and a quencher, such that the quencher quenches the fluorescence of the dye when the probe is intact.
  • the probe can be used in an amplification method in which a target region encompassing the fusion junction is to be amplified using a polymerase having 5’ -3’ exonuclease activity (such as Taq polymerase) .
  • a polymerase having 5’ -3’ exonuclease activity such as Taq polymerase
  • the probe which hybridizes to the fusion junction can be degraded by the polymerase as it proceeds along the target polynucleotide, thereby separating the fluorescent dye and the quencher on the probe, and allow the fluorescent dye to emit its signal to be detected.
  • the present disclosure further provides methods of detecting the fusion gene provided herein in a protein-containing sample, comprising contacting the sample with a detecting agent which specifically detects a fusion protein encoded by the fusion gene, and detecting the presence of the fusion protein.
  • the presence and level of the fusion protein encoded by the fusion gene can be detected.
  • the sample may be contacted with an antibody specific for the fusion protein, and formation of a complex between the antibody and the fusion protein can be detected using methods known in the art, such as, for example, an immunoassay, Western blot method, ELISA, ELIFA, fluorescence immunoassay method, radioimmunoassay method, enzymatic immunoassay method, double antibodies sandwich method, and etc.
  • primer set or probe sets or junction probe as provided herein are useful in detecting the novel fusion genes in the murine tumor models disclosed herein. Therefore, another aspect of the present disclosure relates to kits comprising the primer sets, or the probe sets, or the junction probe described herein.
  • kits comprise a first primer directed to a first encoding sequence for a first gene, and a second primer directed to a second encoding sequence for a second gene.
  • the kits comprise a junction primer directed to a fragment containing the fusion junction of the first gene and the second gene, and a non-junction primer directed to the first encoding sequence for the first gene or the second encoding sequence for the second gene.
  • kits comprise a first probe directed to a first encoding sequence for the first gene, and a second probe directed to a second encoding sequence for the second gene.
  • the kits comprise a junction probe directed to a fragment containing the fusion junction.
  • kits provided herein may further comprise one or more components useful for the detection, for example, polymerase, a buffer useful for amplification, and/or a buffer useful for probe hybridization.
  • This example shows the identification of unique gene signature (genetic fingerprints) of individual murine tumor homografts derived from murine cell lines or murine primary tumors.
  • RNAseq Whole transcriptome sequencing
  • other forms of NGS e.g. WGS or WES
  • the unique gene features with respect to a given syngeneic cell line or tumor sample were obtained through bioinformatics analysis of the NGS data sets. Once identified by bioinformatics analysis, the unique gene features (e.g. gene fusions or mutations) were validated by RT-PCR or PCR followed by Sanger sequencing. Once validated, the unique gene features of a given cell line or murine tumor can be used as a genetic fingerprint to track the passage of murine cell lines or murine tumors of the same lineage.
  • a predetermined number of cells suspended in 0.1ml PBS were inoculated within the right flank of immunocompetent mice (C57BL/6, BALB/c, or C3H) .
  • immunocompetent mice C57BL/6, BALB/c, or C3H
  • spontaneous tumors from GEMM models were surgically removed from GEMM model and subsequently implanted within the right flank of immunocompetent mice of the same genetic background. Tumor bearing mice were monitored twice a week and tumors were collected when the mean tumor size reached 250 ⁇ 350mm 3 .

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Abstract

La présente invention concerne un procédé de production d'un modèle tumoral murin. Dans un mode de réalisation, le procédé comprend la transplantation d'une lignée cellulaire tumorale de souris vers une souris ; l'élevage de la souris dans un état qui permet à la lignée cellulaire tumorale de se développer en une tumeur ; la détection dans la tumeur de la présence d'un marqueur biologique qui est unique à la lignée cellulaire tumorale de souris ; et la détermination de l'identification de la lignée cellulaire tumorale de souris.
PCT/CN2018/114595 2017-11-08 2018-11-08 Empreintes génétiques uniques pour modèle tumoral murin et ses utilisations WO2019091429A1 (fr)

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KR20200125061A (ko) * 2019-04-25 2020-11-04 주식회사 대웅제약 만성 골수성 백혈병의 진단용 바이오마커

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US7259290B1 (en) * 2002-11-06 2007-08-21 Central Institute For Experimental Animals Animal model of human hematopoietic tumor
CN101380478A (zh) * 2007-09-06 2009-03-11 复旦大学附属中山医院 荧光可视高转移人肝癌裸鼠模型的建立方法
WO2009122443A2 (fr) * 2008-03-31 2009-10-08 Council Of Scientific & Industrial Research Système de modèle de tumeur utile pour étudier un cancer à multiples stades
CN102939934A (zh) * 2012-11-08 2013-02-27 同济大学 整体可视化人肺腺癌h1650裸鼠模型及其建立与应用

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Publication number Priority date Publication date Assignee Title
US7259290B1 (en) * 2002-11-06 2007-08-21 Central Institute For Experimental Animals Animal model of human hematopoietic tumor
CN101380478A (zh) * 2007-09-06 2009-03-11 复旦大学附属中山医院 荧光可视高转移人肝癌裸鼠模型的建立方法
WO2009122443A2 (fr) * 2008-03-31 2009-10-08 Council Of Scientific & Industrial Research Système de modèle de tumeur utile pour étudier un cancer à multiples stades
CN102939934A (zh) * 2012-11-08 2013-02-27 同济大学 整体可视化人肺腺癌h1650裸鼠模型及其建立与应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200125061A (ko) * 2019-04-25 2020-11-04 주식회사 대웅제약 만성 골수성 백혈병의 진단용 바이오마커
KR102351603B1 (ko) 2019-04-25 2022-01-18 주식회사 대웅제약 만성 골수성 백혈병의 진단용 바이오마커

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