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WO2024189064A1 - Arn circltbp2 circulaire en tant que biomarqueur et biocible dans des cholangiocarcinomes intrahépatiques - Google Patents

Arn circltbp2 circulaire en tant que biomarqueur et biocible dans des cholangiocarcinomes intrahépatiques Download PDF

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WO2024189064A1
WO2024189064A1 PCT/EP2024/056634 EP2024056634W WO2024189064A1 WO 2024189064 A1 WO2024189064 A1 WO 2024189064A1 EP 2024056634 W EP2024056634 W EP 2024056634W WO 2024189064 A1 WO2024189064 A1 WO 2024189064A1
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circltbp2
patient
level
circrna
mir
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Cédric COULOUARN
Corentin LOUIS
Julien EDELINE
Matthis DESOTEUX
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Institut National de la Santé et de la Recherche Médicale
Université De Rennes
Centre Eugène Marquis
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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
    • C12Q2600/118—Prognosis of disease development
    • 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
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • CCA Cholangiocarcinoma
  • CCA includes a heterogeneous group of tumors that can occur anywhere in the biliary system.
  • CCA is a rare cancer but both its incidence and mortality increased worldwide over the last decade. [1, 2] The silent presentation of CCA combined with its aggressive nature and resistance to chemotherapy, contributes to the high mortality rate of CCA, which represents 2% of all cancer-related deaths worldwide each year.
  • CircRNAs circular RNAs
  • miRNAs microRNAs
  • TGF ⁇ Transforming Growth Factor beta pathway
  • TGF ⁇ Transforming Growth Factor beta pathway
  • the present invention relates to the circular RNA circLTBP2 as a biomarker and biotarget in intrahepatic cholangiocarcinomas.
  • Intrahepatic cholangiocarcinoma is a deadly cancer worldwide with an increasing incidence and limited therapeutic options. Therefore, there is an urgent need to open the field to new concepts for identifying clinically relevant therapeutic targets and biomarkers.
  • CircLTBP2 (hsa-circ-0032603) was identified as a novel TGF ⁇ -induced circRNA in several CCA cell lines. CircLTBP2 promotes tumor cell proliferation, migration and resistance to gemcitabine-induced apoptosis in vitro and tumor growth in vivo.
  • CircLTBP2 acts as a competitive RNA regulating notably the activity of the tumor suppressor microRNA miR-338-3p, leading to the overexpression of its pro-metastatic targets.
  • the restoration of miR-338-3p levels in iCCA cells reversed the pro-tumorigenic effects driven by circLTBP2, including the resistance to gemcitabine-induced apoptosis.
  • circLTBP2 expression predicted a reduced survival, as detected in tumor tissues but also in serum exosomes isolated from patients with iCCA.
  • CircLTBP2 is a novel effector of the pro- tumorigenic arm of TGF ⁇ and a clinically relevant biomarker easily detected from liquid biopsies in iCCA.
  • the first object of the present invention relates to a method of diagnosing an intrahepatic cholangiocarcinoma in a subject comprising determining the level of circular RNA circLTBP2 in a sample obtained from the patient wherein said level indicates whether the patient suffers or not from an intrahepatic cholangiocarcinoma.
  • the term “patient” or “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate.
  • the patient is a human.
  • the patient is a human who is susceptible to have a intrahepatic cholangiocarcinoma as described above.
  • cholangiocarcinoma has its general meaning in the art and refers to a heterogeneous group of tumors that can occur anywhere in the biliary system.
  • intrahepatic cholangiocarcinoma or “iCCA” refers to a type of cholangiocarcinoma that occurs in the parts of the bile ducts within the liver.
  • diagnostic refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.
  • the method according to the invention allows to diagnose an intrahepatic cholangiocarcinoma.
  • high levels of circLTBP2 indicate that the patient suffers from an intrahepatic cholangiocarcinoma
  • low levels of circLTBP2 indicate that the patient does not suffer from an intrahepatic cholangiocarcinoma.
  • the term “high” refers to a measure that is greater than normal, greater than a standard such as a predetermined reference value or a subgroup measure or that is relatively greater than another subgroup measure.
  • high levels of circLTBP2 refers to a level of circLTBP2 that is greater than a normal circLTBP2 level.
  • a normal circLTBP2 level may be determined according to any method available to one skilled in the art.
  • High level of circLTBP2 may also refer to a level that is equal to or greater than a predetermined reference value, such as a predetermined cutoff.
  • High level of circLTBP2 may also refer to a level of circLTBP2 wherein a high circLTBP2 subgroup has relatively greater levels of circLTBP2 than another subgroup.
  • two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a subgroup whose measure is high (i.e., higher than the median) and another subgroup whose measure is low.
  • a “high” level may comprise a range of level that is very high and a range of level that is “moderately high” where moderately high is a level that is greater than normal, but less than “very high”.
  • the term “low” refers to a level that is less than normal, less than a standard such as a predetermined reference value or a subgroup measure that is relatively less than another subgroup level.
  • low level of circLTBP2 means a level of circLTBP2 that is less than a normal level of in a particular set of samples of patients.
  • a normal level of circLTBP2 measure may be determined according to any method available to one skilled in the art.
  • Low level of circLTBP2 may also mean a level that is less than a predetermined reference value, such as a predetermined cutoff.
  • Low level of circLTBP2 may also mean a level wherein a low level circLTBP2 subgroup is relatively lower than another subgroup.
  • two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a group whose measure is low (i.e., less than the median) with respect to another group whose measure is high (i.e., greater than the median).
  • the term “predetermined reference value” refers to a threshold value or a cut- off value.
  • a “threshold value”, “reference value” or “cut-off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art.
  • retrospective measurement of the level of the marker of the invention e.g. circLTBP2
  • the predetermined corresponding reference value is the median measured in the population of the patients for the marker of in the invention (circLTBP2 for example).
  • the threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).
  • the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • the level of the marker of the invention circLTBP2 for example
  • the full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests.
  • ROC curve is a comprehensive indicator the reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method.
  • a series of different cut-off values are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis.
  • AUC area under the curve
  • the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values.
  • the AUC value of the ROC curve is between 1.0 and 0.5.
  • a further aspect of the invention relates to a method for discriminating an intrahepatic cholangiocarcinoma from a hepatocellular carcinoma in a subject comprising i) determining in a sample obtained from the patient the level of circLTBP2; ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the patient has or is susceptible to have an intrahepatic cholangiocarcinoma when the level determined at step i) is higher than its predetermined reference value, or concluding that the patient has or is susceptible to have a hepatocellular carcinoma when the level determined at step i) is lower than its predetermined reference value.
  • a further object of the present invention relates to a method of predicting the survival time of a patient suffering from an intrahepatic cholangiocarcinoma comprising determining the level of circular RNA circLTBP2 in a sample obtained from the patient wherein said level indicates the survival time.
  • the method of the present invention is particularly suitable for predicting the duration of the overall survival (OS), progression-free survival (PFS), and/or the disease-free survival (DFS) of the cancer patient.
  • OS survival time is generally based on and expressed as the percentage of people who survive a certain type of cancer for a specific amount of time.
  • the expression “long survival time” indicates that the patient will have a survival time that will be higher than the median (or mean) observed in the general population of patients suffering from said cancer.
  • the patient will have a long survival time, it is meant that the patient will have a “good prognosis”.
  • high levels of circLTBP2 correlate with short survival time whereas low levels of circLTBP2 correlate with long survival time. More particularly, the higher the level of circLTBP2 is, the shorter will be the survival time of the patient whereas, the lower the level of circLTBP2 is, the longer will be the survival time of the patient.
  • the predetermined reference value may be determined by carrying out a method comprising the steps of a) providing a collection of samples; b) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding patient (i.e.
  • the level of circLTBP2 has been assessed for 100 samples of 100 patients.
  • the 100 samples are ranked according to the level of circLTBP2.
  • Sample 1 has the highest level and sample 100 has the lowest level.
  • a first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples.
  • the next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100.
  • Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated.
  • a further object of the present invention relates to a method of determining whether a patient suffering from an intrahepatic cholangiocarcinoma achieves a response to a therapy comprising determining the level of circular RNA circLTBP2 in a sample obtained from the patient wherein said level indicates whether the patient achieves or not a response to the therapy.
  • the predetermined level is the level determined in a sample obtained from the patient before the treatment.
  • the terms "achieve a response” or “respond” refer to the response to a therapy of the patient suffering from an intrahepatic cholangiocarcinoma.
  • Such therapy induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to intrahepatic cholangiocarcinoma.
  • the term “respond” refers to the ability of the therapy to an improvement of the pathological symptoms, thus, the patient presents a clinical improvement compared to the patient who does not receive the therapy.
  • the said patient is considered as a "responder” to the treatment.
  • the term “not respond” refers to a patient who does not present any clinical improvement to the treatment with the therapy.
  • This patient is considered as a "non-responder” to the therapy.
  • the patient as considered “non-responder” has a particular monitoring in the therapeutic regimen.
  • the response to a treatment is determined by Response evaluation criteria in solid tumors (RECIST) criteria.
  • This criteria refers to a set of published rules that define when the disease in the patients improve ("respond"), stay the same (“stabilize”), or worsen ("progress”) during treatment.
  • responder when the patient is identified as responder, it means that said patient improves overall and progression- free survival (OS/PFS).
  • OS/PFS progression- free survival
  • the method herein disclosed is particularly suitable for determining whether the achieves a response to chemotherapy.
  • chemotherapy has its general meaning in the art and is a cancer treatment that uses drugs to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing.
  • the said drug can be for example a small molecule: small molecules which can be conveniently used for the invention include in particular genotoxic drugs.
  • sample refers to any substance derived from a living organism.
  • a sample may be derived from blood as a urine sample, serum sample, a plasma sample, and or a whole blood sample.
  • a sample may be derived from a tissue collected, for example, by a biopsy. In a particular embodiment, the sample has been previously obtained from the subject.
  • the methods of the present invention are performed in vitro or ex vivo.
  • the biological is a tumor tissue sample.
  • tumor tissue sample means any tissue tumor sample derived from the patient. Said tissue sample is obtained for the purpose of the in vitro evaluation.
  • the tumor sample may result from the tumor resected from the patient.
  • the tumor sample may result from a biopsy performed in the primary tumour of the patient.
  • the tumor tissue sample can be subjected to a variety of well-known post-collection preparative and storage techniques (e.g., fixation, storage, freezing, etc.) prior to determining the level of circLTBP2 of interest.
  • TMA tissue microarrays
  • the sample is a blood sample.
  • blood sample means any blood sample derived from the subject. Collections of blood samples can be performed by methods well known to those skilled in the art.
  • the blood sample is a serum sample or a plasma sample.
  • the level of circLTBP2 is determined in the extracellular vesicles that are isolated from the blood sample of the patient.
  • extracellular vesicle or “EV” has its general meaning in the art and denotes a plasma membrane vesicle shed from an apoptotic or activated cell. Circulating extracellular vesicles can be isolated from the blood sample by coupling filtration and optionally contacting them with a set of binding partners directed against the specific surface markers of said extracellular vesicles. Typically, the extracellular vesicles are isolated as described in the EXAMPLE.
  • CircRNA refers to a non-coding RNA molecule harboring exons out of order from genomic context, a phenomenon termed “exon shuffling” or “non-colinear splicing”. CircRNA is thus a type of single-stranded RNA which, unlike linear RNA, forms a covalently closed continuous loop. In circular RNA, the 3' and 5' ends normally present in an RNA molecule have been joined together. Thus, a circRNA does not contain a free 3’-end or a free 5’ end, i.e. the entire nucleic acid is circularized.
  • the circRNA is preferably a circularized, single stranded RNA molecule.
  • the circRNA according to the present invention is a result of a head-to-tail splicing event that results in a discontinuous sequence with respect to the genomic sequence encoding the RNA.
  • a first sequence being present 5’-upstream of a second sequence in the genomic context, on the circRNA said first sequence at its 5’-end is linked to the 3’ end of said second sequence and thereby closing the circle.
  • the consequence of this arrangement is that at the junction where the 5’-end of said first sequence is linked to the 3’-end of said second sequence a unique sequence is build that is neither present in the genomic context nor in the normally transcribed RNA, e.g. mRNA.
  • junctions in all identified circRNAs, in the genomic context are flanked by the canonical splice sequence, the GT/AG splice signal known by the skilled person.
  • the skilled person will recognize that a usual mRNA transcript contains exon-exon junctions in a tail-to-head arrangement, i.e. the 3’ end (tail) of exon being upstream in the genomic context is linked to the 5’ end (head) of the exon being downstream in the genomic context.
  • the actual junction i.e. the point at which the one exon is linked to the other is also referred to herein as “breakpoint”. This feature confers numerous properties to circular RNA, many of which have only recently been identified.
  • circLTBP2 refers to the CircRNA available from the data base cirBase http://www.circbase.org/ under the accession number hsa_circ_0032603.
  • circLTBP2 is encoded by the nucleic acid sequences as set forth in SEQ ID NO:1.
  • SEQ ID NO:1 Homo sapiens latent transforming growth factor beta binding protein 2 (LTBP2), mRNA, NCBI Reference Sequence: NM_000428.3 1 ccaaaaataa aaccgtccgg gtcccttca gacggctgca ggcacaggga ggaggcgcga 61 aggtgcagca gccgtgcgag cccagctgga gtaggagcgc ggactcgagg ctcggggcgc 121 gcagccctcg ttccgcgag agccgggccc ccagtcggcccgggccctagact 181 cagagaagct ggccgcggg cggggccggg agaacagcccc agcgccccctagact 181 cagagaagct ggcc
  • the nucleic acid contained in the samples is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid- binding resins following the manufacturer's instructions.
  • Conventional methods and reagents for isolating RNA from a sample comprise High Pure miRNA Isolation Kit (Roche), Trizol (Invitrogen), Guanidinium thiocyanate-phenol-chloroform extraction, PureLinkTM miRNA isolation kit (Invitrogen), PureLink Micro-to- Midi Total RNA Purification System (invitrogen), RNeasy kit (Qiagen), miRNeasy kit (Qiagen), Oligotex kit (Qiagen), phenol extraction, phenol-chloroform extraction, TCA/acetone precipitation, ethanol precipitation, Column purification, Silica gel membrane purification, PureYieldTM RNA Midiprep (Promega), PolyATtract System 1000 (Promega), Maxwell® 16 System (Promega), SV Total RNA Isolation (Pro
  • rRNA ribosomal RNAs
  • the content of rRNA in the sample should be depleted to less than 20%, preferably less than 10%, more preferably less than 2% with respect to the total RNA content.
  • the rRNA depletion may be performed as known in the art, e.g. it may be facilitated by commercially available kits (e.g. Ribominus, Themo Scientific) or enzymatic methods (Xian Adiconis et al.
  • the level of circLTBP2 in the sample may be determined by any suitable method. Any reliable method for measuring the level or amount of circRNA in a sample may be used.
  • circRNA can be detected and quantified from a sample (including fractions thereof), such as samples of isolated RNA by various methods known for mRNA, including, for example, amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.), hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.), and sequencing-based methods (e.g., next- generation sequencing methods, for example, using the Illumina or IonTorrent platforms).
  • amplification-based methods e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.
  • hybridization-based methods e.g.
  • exemplary techniques include ribonuclease protection assay (RPA) and mass spectroscopy.
  • the circRNA according to the present invention may be detected using different techniques. As outlined herein, the exon-exon junction in a head-to-tail arrangement is unique to the circRNAs. Hence, the detection of these is preferred. Nucleic acid detection methods are commonly known to the skilled person and include probe hybridization based methods, nucleic acid amplification based methods, and nucleic acid sequencing, or combinations thereof. Hence, in a some embodiments circRNA is detected using a method selected from the group consisting of probe hybridization based methods, nucleic acid amplification based methods, and nucleic acid sequencing.
  • Probe hybridization based method employ the feature of nucleic acids to specifically hybridize to a complementary strand.
  • nucleic acid probes may be employed that specifically hybridize to the exon-exon junction in a head-to-tail arrangement of the circRNA, i.e. to a sequence spanning the exon-exon junction, preferably to the region extending from 10 nt upstream to 10 nt downstream of the exon-exon junction, preferably to the region from 20 nt upstream to 20 nt downstream of the exon-exon junction, or even a greater region spanning the exon-exon junction.
  • hybridization probes specifically hybridizing to the respective sequence of the circRNA may be used, as well as hybridization probes specifically hybridizing to the reverse complement sequence thereof, e.g. in case the circRNA is previously reverse transcribed to cDNA and/or amplified.
  • Hybridization can also be used as a measure of homology between two nucleic acid sequences.
  • a nucleic acid sequence hybridizing specifically to an exon-exon junction in a head-to-tail arrangement according to the present invention may be used as a hybridization probe according to standard hybridization techniques.
  • hybridization of the probe to DNA or RNA from a test source is an indication of the presence of the relevant circRNA in the test source.
  • a test source e.g., the bodily fluid, like whole blood, or amplified nucleic acids from the sample of the bodily fluid
  • Hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1991.
  • specific hybridization refers to hybridization under stringent conditions.
  • “Stringent conditions” are defined as equivalent to hybridization in 6 ⁇ sodium chloride/sodium citrate (SSC) at 45° C., followed by a wash in 0.2 ⁇ SSC, 0.1% SDS at 65° C.; or as equivalent to hybridization in commercially available hybridization buffers (e.g. ULTRAHyb, ThermoScientific) for blotting techniques and 5 ⁇ SSC 0.5% SDS (750 mM NaCl, 75 mM sodium citrate, 0.5% sodiumdodecylsulfate, pH 7.0) for array based detection methods at 65° C.
  • the means and methods of the present invention preferably comprise the use of nucleic acid probes.
  • a nucleic acid “probe” is an oligonucleotide, nucleic acid or a fragment thereof, which is substantially complementary to a specific nucleic acid sequence. “substantially complementary” refers to the ability to hybridize to the specific nucleic acid sequence under stringent conditions.
  • the skilled person knows means and methods to determine the levels of nucleic acids in a sample and compare them to control levels. Such methods may employ labeled nucleic acid probes according to the invention.
  • “Labels” include fluorescent or enzymatic active labels as further defined herein below. Such methods include real-time PCR methods and microarray methods, like Affimetrix®, nanostring and the like. The determination of the circRNAs or their level may also be detected using sequencing techniques.
  • Sequencing techniques include but are not limited to Maxam-Gilbert Sequencing, Sanger sequencing (chain-termination method using ddNTPs), and next generation sequencing methods, like massively parallel signature sequencing (MPSS), polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, or ion torrent semiconductor sequencing or single molecule, real-time technology sequencing (SMRT).
  • MPSS massively parallel signature sequencing
  • polony sequencing 454 pyrosequencing
  • Illumina (Solexa) sequencing SOLiD sequencing
  • SMRT real-time technology sequencing
  • the level of a circRNA is determined by detection of an exon-exon-junction in a head-to-tail arrangement.
  • Circular RNAs are a large class of animal RNAs with regulatory potency. Nature. 2013; 495(7441):333-338, which is incorporated herein by reference in particular as relates to the detection and annotation of circRNAs.
  • the biogenesis of many mammalian circRNAs depends on complementary sequences within flanking introns Hence, in some embodiments, the two introns upstream and downstream of and direct adjacent in the genomic context to the exons of the exon-exon junction (i.e. forming the exon-exon junction) in a head to tail arrangement often contain complementary sequences, e.g.
  • RNA of a sample is sequenced after reverse transcription and library preparation. Afterwards, the sequences are analyzed for the presence of exon-exon junctions in a head-to-tail arrangement. Further, in some embodiments, RNA sequences which map continuously to the genome by aligning without any trimming (end-to-end mode) are neglected. Reads not mapping continuously to the genome are preferably used for circRNA candidate detection.
  • the terminal sequences (anchors) from the sequences e.g.20 nt or more, may be extracted and re-aligned independently to the genome.
  • sequences may be extended until the full circRNA sequence is covered, i.e. aligned.
  • Consecutively aligning anchors indicate linear splicing events whereas alignment in reverse orientation indicates head-to-tail splicing as observed in circRNAs.
  • the so identified resulting splicing events are filtered using the following criteria 1) GT/AG signal flanking the splice sites in the genomic context; 2) the breakpoint, i.e. the exon-exon-junction can be unambiguously detected; and 3) no more than 100 kilobases distance between the two splice sites in the genomic context.
  • further optional criteria may be used, depending on the method chosen; e.g.
  • the detection/determination of the circRNAs and the respective level may also employ nucleic acid amplification method alone or in combination with the sequencing and/or hybridization method. Nucleic acid amplification may be used to amplify the sequence of interest prior to detection. It may however also be used for quantifying a nucleic acid, e.g. by real-time PCR methods. Such methods are commonly known to the skilled person.
  • Nucleic acid amplification methods for example include rolling circle amplification (such as in Liu, et al., “Rolling circle DNA synthesis: Small circular oligonucleotides as efficient templates for DNA polymerases,” J. Am. Chem. Soc.118:1587-1594 (1996).), isothermal amplification (such as in Walker, et al., “Strand displacement amplification—an isothermal, in vitro DNA amplification technique,” Nucleic Acids Res.
  • rolling circle amplification such as in Liu, et al., “Rolling circle DNA synthesis: Small circular oligonucleotides as efficient templates for DNA polymerases,” J. Am. Chem. Soc.118:1587-1594 (1996).
  • isothermal amplification such as in Walker, et al., “Strand displacement amplification—an isothermal, in vitro DNA amplification technique”
  • ligase chain reaction such as in Landegren, et al., “A Ligase-Mediated Gene Detection Technique,” Science 241:1077-1080, 1988, or, in Wiedmann, et al., “Ligase Chain Reaction (LCR)—Overview and Applications,” PCR Methods and Applications (Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, N Y, 1994) pp. S51-S64.)).
  • Nucleic-acid amplification can be accomplished by any of the various nucleic-acid amplification methods known in the art, including but not limited to the polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), nucleic acid sequence based amplification (NASBA), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustaining sequence replication (3SR) and Q ⁇ amplification.
  • PCR polymerase chain reaction
  • LCR transcription-based amplification system
  • TAS transcription-based amplification system
  • NASBA nucleic acid sequence based amplification
  • RCA rolling circle amplification
  • TMA transcription-mediated amplification
  • Q ⁇ amplification Q ⁇ amplification.
  • the amplification of the circRNA may start with a reverse transcription of the RNA into complementary DNA (cDNA), optionally followed by amplification of the so produced cDNA.
  • the methods of the present invention further comprises determining the level of miR-338-3p in the sample obtained from the subject wherein the combined measures of circLTBP2 and miR-338-3p indicate whether: - the subject suffers or not from an intrahepatic cholangiocarcinoma, or - the subject will have a long or short survival time, or - the subject achieves or not a response to the therapy.
  • high levels of circLTBP2 and low levels of miR-338-3p indicate that: - the subject suffers from an intrahepatic cholangiocarcinoma, or - the subject will have a short survival time - the subject does not achieve a response to the therapy.
  • miRNA refers to an RNA molecule that is approximately 21-23 nucleotides (nt) in length. miRNAs can range between 18-26 nucleotides in length. Typically, miRNAs are single-stranded. However, in some embodiments, miRNAs may be at least partially double-stranded.
  • miRNAs may comprise an RNA duplex (referred to herein as a “duplex region”) and may optionally further comprises one or two single-stranded overhangs.
  • the miRNA comprises a duplex region ranging from 15 to 29 by in length and optionally further comprising one or two single- stranded overhangs.
  • a miRNA may be formed from two RNA molecules that hybridize together or may alternatively be generated from a single RNA molecule that includes a self-hybridizing portion. In general, free 5’ends of miRNA molecules have phosphate groups, and free 3’ ends have hydroxyl groups.
  • the duplex portion of a miRNA usually, but does not necessarily, comprise one or more bulges consisting of one or more unpaired nucleotides.
  • One strand of a miRNA includes a portion that hybridizes with a target RNA.
  • one strand of the miRNA is not precisely complementary with a region of the target RNA, meaning that the miRNA hybridizes to the target RNA with one or more mismatches.
  • one strand of the miRNA is precisely complementary with a region of the target RNA, meaning that the miRNA hybridizes to the target RNA with no mismatches.
  • miRNAs are thought to mediate inhibition of gene expression by inhibiting translation of target transcripts.
  • miRNAs may mediate inhibition of gene expression by causing degradation of target transcripts.
  • miR-338-3p has its general meaning in the art and refers to the miRNA available from the data base http://mirbase.org under the miRBase accession number MIMAT0000763 (hsa-miR-338-3p).
  • the nucleic acid sequence of miR-338-3p is represented by SEQ ID NO:2.
  • the expression level of miR-338-3p in the sample may be determined by any suitable method.
  • miRNA can be detected and quantified from a sample (including fractions thereof), such as samples of isolated RNA by various methods known for mRNA, including, for example, amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.), hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.), and sequencing-based methods (e.g., next- generation sequencing methods, for example, using the Illumina or IonTorrent platforms).
  • amplification-based methods e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.
  • hybridization-based methods e.g.,
  • Methods of therapy Further aspect of the present invention relates to methods of treating intrahepatic cholangiocarcinoma.
  • the methods of therapy as herein disclosed a carried out once the patient has been diagnosed according to the methods of diagnosis according to the present invention.
  • the term "treatment” or “treat” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase "induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • loading regimen may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • the phrase "maintenance regimen” or “maintenance period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • circRNA inhibitors In particular, a further object of the present invention relates to a method of treating an intrahepatic cholangiocarcinoma in a patient in need thereof comprising administering to the patient an circRNA inhibitor of circLTBP2.
  • the term “circRNA inhibitor” refers to any compound that blocks circRNA expression, processing and/or function.
  • the circRNA inhibitor of the present invention is a compound that inhibits or reduces the activity or expression of circLTBP2.
  • the term “inhibiting circLTBP2 expression” means that the production of circLTBP2 in target cells after treatment is less than the amount produced prior to treatment or neutralize the activity of existent amount.
  • One skilled in the art can readily determine whether circLTBP2 expression has been inhibited in the target cells, using for example the techniques for determining miRNA transcript level.
  • the circRNA inhibitor of the present invention is a compound such as nucleic acid that hybridizes with circLTBP2 or having sequence complementarity to that of circLTBP2.
  • circRNA inhibitor of the present invention is a compound such as nucleic acid having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 9899 or 100% sequence complementarity to that of circLTBP2.
  • Suitable circRNA inhibitors include double-stranded RNA (such as short- or small-interfering RNA or "siRNA"), antisense nucleic acids, and enzymatic RNA molecules such as ribozymes. Each of these compounds can be targeted to a given circRNA and destroy or induce the destruction of the target circRNA.
  • Suitable circRNA inhibitors include double-stranded RNA (such as short- or small-interfering RNA or "siRNA”), antagomirs, antisense nucleic acids, and enzymatic RNA molecules such as ribozymes.
  • siRNA short- or small-interfering RNA
  • antagomirs antisense nucleic acids
  • enzymatic RNA molecules such as ribozymes.
  • Each of these compounds can be targeted to a given miRNA and destroy or induce the destruction of the target miRNA.
  • expression of a given miRNA can be inhibited by inducing RNA interference of the miRNA with an isolated double-stranded RNA ("dsRNA") molecule which has at least 90%, for example 90%; 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence homology with at least a portion of the miRNA.
  • dsRNA isolated double-stranded RNA
  • the dsRNA molecule is a "short or small interfering RNA” or "siRNA".
  • siRNA useful in the present methods comprise short double-stranded RNA from about 17 nucleotides to about 29 nucleotides in length, preferably from about 19 to about 25 nucleotides in length.
  • the siRNA comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions (hereinafter "base-paired").
  • the sense strand comprises a nucleic acid sequence which is substantially identical to a nucleic acid sequence contained within the target miRNA.
  • a nucleic acid sequence in a siRNA which is "substantially identical" to a target sequence contained within the target mRNA is a nucleic acid sequence that is identical to the target sequence, or that differs from the target sequence by one or two nucleotides.
  • the sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules, or can comprise a single molecule in which two complementary portions are base- paired and are covalently linked by a single-stranded "hairpin" area.
  • the siRNA can also be altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non- nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, or modifications that make the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA with deoxyribonucleotides.
  • One or both strands of the siRNA can also comprise a 3 overhang.
  • a "3' overhang" refers to at least one unpaired nucleotide extending from the 3'-end of a duplexed RNA strand.
  • the siRNA comprises at least one 3' overhang of 1 to about 6 nucleotides (which includes ribonucleotides or deoxyribonucleotides) in length, preferably from 1 to about 5 nucleotides in length, more preferably from 1 to about 4 nucleotides in length, and particularly preferably from about 2 to about 4 nucleotides in length.
  • the 3' overhang is present on both strands of the siRNA, and is 2 nucleotides in length.
  • each strand of the siRNA can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu").
  • the siRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described above. Exemplary methods for producing and testing dsRNA or siRNA molecules are described in U.S. published patent application 2002/0173478 to Gewirtz and in U.S. published patent application 2004/0018176 to Reich et al., the entire disclosures of which are herein incorporated by reference.
  • the circRNA of the present invention is an antisense nucleic acid.
  • an "antisense nucleic acid” refers to a nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-peptide nucleic acid interactions, which alters the activity of the target RNA.
  • Antisense nucleic acids suitable for use in the present methods are single-stranded nucleic acids (e.g., RNA, DNA, RNA-DNA chimeras, PNA) that generally comprise a nucleic acid sequence complementary to a contiguous nucleic acid sequence in a miRNA.
  • the antisense nucleic acid comprises a nucleic acid sequence that is 50-100% complementary, more preferably 75-100% complementary, and most preferably 95-100% complementary to a contiguous nucleic acid sequence in the circRNA.
  • the antisense nucleic acid activates RNase H or some other cellular nuclease that digests the circRNA/antisense nucleic acid duplex and thus inhibits the expression of the targeted circRNA.
  • the antisense binds to the miR-338-3p target site in the circLTBP2 nucleic acid sequence.
  • binding of the nucleic acid antisense to the miR-338-3p mRNA target site does not induce gene silencing (e.g. by circRNA degradation or translational repression) of said target circRNA. Binding of the antisense to a miR-338-3p mRNA target site may occur via complementary base pairing, as described above.
  • binding between the antisense and the miR-338-3p mRNA target site occurs via complementary base pairing between at least one nucleotide present in the antisense and a corresponding nucleotide present in the miR-338-3p mRNA target site, such that at least a portion of the antisense and the miR-338-3p mRNA target site together define a base-paired nucleic acid duplex.
  • Said complementary base pairing can occur over a region of two or more contiguous nucleotides of the miR-338-3p mRNA target site (e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides).
  • a base- paired nucleic acid duplex formed when the antisense binds to the miR-338-3p mRNA target may comprise one or more mismatch pairings.
  • two or more regions of complementary base-paired nucleic acid duplex e.g.3, 4, 5 or 6) are formed, wherein each region is separated from the next by one or more mismatch pairings.
  • Antisense nucleic acids can also contain modifications of the nucleic acid backbone or of the sugar and base moieties (or their equivalent) to enhance target specificity, nuclease resistance, delivery or other properties related to efficacy of the molecule.
  • circRNA inhibitor of the present invention is an endonuclease.
  • the endonuclease is CRISPR-cas.
  • the endonuclease is CRISPR-cas13 (“cas13”).
  • Cas13 uses a guide RNA (CRISPR-RNA, aka crRNA) to identify its substrate, which is RNA rather than DNA.
  • CRISPR-RNA aka crRNA
  • the crRNA used by Cas13 forms a short hairpin structure next to a short spacer sequence (28–30 nucleotides) that is specific to the target transcript. Since CRISPR/Cas13 mediates RNA degradation, it holds the promise to replace or complement RNA interference (RNAi) approaches or other systems that interfere with transcript levels, such as CRISPRi.
  • RNAi RNA interference
  • the circRNA inhibitor of the present invention can be obtained using a number of standard techniques.
  • the circRNA inhibitor of the present invention can be chemically synthesized or recombinantly produced using methods known in the art.
  • the circRNA inhibitors of the present invention are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • Commercial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, 111., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK).
  • the circRNA inhibitor of the present invention is resistant to degradation by nucleases.
  • nucleic acids which are nuclease resistant, for example by incorporating one or more ribonucleotides that are modified at the 2'- position into the miRNAs.
  • Suitable 2'-modified ribonucleotides include those modified at the 2'-position with fluoro, amino, alkyl, alkoxy, and O-allyl.
  • the circRNA inhibitor of the present invention can be expressed from recombinant linear or circular DNA plasmids using any suitable promoter.
  • Suitable promoters for expressing RNA from a plasmid include, e.g., the U6 promoter sequence, or the cytomegalovirus promoters.
  • the recombinant plasmids of the present invention can also comprise inducible or regulatable promoters for expression of the circRNA inhibitor of the present invention in target cells.
  • the circRNA inhibitor of the present invention that is expressed from recombinant plasmids can be isolated from cultured cell expression systems by standard techniques.
  • the circRNA inhibitor of the present invention which is expressed from recombinant plasmids can also be delivered to, and expressed directly in, target cells. The use of recombinant plasmids to deliver the circRNA inhibitor of the present invention to target cells is discussed in more detail below.
  • the circRNA inhibitor of the present invention can be expressed from a separate recombinant plasmid, or can be expressed from a unique recombinant plasmid.
  • the circRNA inhibitor of the present invention is expressed as the nucleic acid precursor molecules from a single plasmid, and the precursor molecules are processed into the functional circRNA inhibitor by a suitable processing system, including processing systems extant within target cells.
  • suitable processing systems include, e.g., the in vitro Drosophila cell lysate system as described in U.S. published application 2002/0086356 to Tuschl et al. and the E. coli RNAse III system described in U.S.
  • a plasmid expressing the circRNA inhibitor of the present invention comprises a sequence encoding a circRNA inhibitor precursor under the control of the CMV intermediate early promoter.
  • a promoter under the control of a promoter means that the nucleic acid sequences are located 3' of the promoter, so that the promoter can initiate transcription of the circRNA inhibitor coding sequences.
  • the circRNA inhibitor of the present invention can also be expressed from recombinant viral vectors. It is contemplated that the circRNA inhibitor of the present invention can be expressed from separate recombinant viral vectors, or from a unique viral vector. The circRNA inhibitor expressed from the recombinant viral vectors either can be isolated from cultured cell expression systems by standard techniques or can be expressed directly in target cells. The use of recombinant viral vectors to deliver the circRNA inhibitor to target cells is discussed in more detail below.
  • the recombinant viral vectors of the present invention comprise sequences encoding the circRNA inhibitor compound of the present invention and any suitable promoter for expressing the circRNA inhibitor sequences.
  • suitable promoters include, for example, the U6 or HI RNA pol III promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art.
  • the recombinant viral vectors of the present invention can also comprise inducible or regulatable promoters for expression of the circRNA inhibitor in target cells.
  • Any viral vector capable of accepting the coding sequences for The circRNA inhibitor of the present invention can be used; for example, vectors derived from adenovirus (AV); adenoassociated virus (t); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.
  • AV adenovirus
  • t adenoassociated virus
  • retroviruses e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus
  • herpes virus and the like.
  • the tropism of the viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
  • lentiviral vectors of the present invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like.
  • AAV vectors of the present invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes.
  • an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2.
  • This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector.
  • the circRNA inhibitor can be administered to a patient by any means suitable for delivering these compounds to target cells.
  • the circRNA inhibitor can be administered by methods suitable to transfect cells of the patient with these compounds, or with nucleic acids comprising sequences encoding these compounds.
  • the cells are transfected with a plasmid or viral vector comprising sequences encoding at least one circRNA inhibitor.
  • the circRNA inhibitor can be administered to a patient by any suitable enteral or parenteral administration route.
  • Suitable enteral administration routes for the present methods include, e.g., oral, rectal, or intranasal delivery.
  • Suitable parenteral administration routes include, e.g., intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra- arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., intra-retinal injection, or subretinal injection); subcutaneous injection or deposition, including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest, for example by a catheter or other placement device (e.g., an implant comprising a porous, non-porous, or gelatinous material); and inhalation.
  • intravascular administration e.g., intravenous bolus injection, intravenous infusion, intra-
  • Preferred administration routes are injection, infusion and direct injection into the tumor tissue.
  • a therapeutically effective amount of the circRNA inhibitor to be administered to a given patient by taking into account factors such as the size and weight of the patient; the extent of disease penetration; the age, health and sex of the patient; the route of administration; and whether the administration is regional or systemic.
  • An effective amount of said compound can be based on the approximate or estimated body weight of a patient to be treated.
  • such effective amounts are administered parenterally or enterally, as described herein.
  • an effective amount of the compound administered to a patient can range from about 5-10000 micrograms/kg of body weight and is preferably between about 5-3000 micrograms/kg of body weight, and is preferably between about 700- 1000 micrograms/kg of body weight, and is more preferably greater than about 1000 micrograms/kg of body weight.
  • the compound can be administered to the patient once (e.g., as a single injection or deposition).
  • the circRNA inhibitor of the present invention is administered to the patient in combination with one or more TGF-beta inhibitor(s).
  • TGF- beta has its general meaning in the art and refers to one or more members of the transforming growth factor-beta family of proteins, e.g., TGF-beta 1 , TGF- beta 2, and TGF- beta 3.
  • TGF-beta inhibitor refers to an agent having the ability to directly or indirectly inhibit a biological function of TGF-beta.
  • TGF-beta inhibitors include, but are not limited to, inhibitors (e.g., blocking (neutralizing) antibodies) specific for TGF-beta, soluble TGF-beta receptors (which would competitively inhibit TGF-beta), membrane-bound TGF- beta receptors, protease inhibitors that inactivate a protease responsible for activating a precursor TGF-beta into mature TGF-beta, inhibitors (e.g., antibodies or small molecules) specific to TGF-beta receptors (Types I, II or III) that prevent TGF-beta binding to the receptor, siRNA or antisense RNA that block expression of TGF-beta or combinations of the foregoing.
  • inhibitors e.g., blocking (neutralizing) antibodies
  • soluble TGF-beta receptors which would competitively inhibit TGF-beta
  • membrane-bound TGF- beta receptors membrane-bound TGF- beta receptors
  • protease inhibitors that inactiv
  • the TGF-beta inhibitor is fresolimumab.
  • the circRNA inhibitors of the present invention are preferably formulated as pharmaceutical compositions, prior to administering to a patient, according to techniques known in the art.
  • Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free.
  • pharmaceutical formulations include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the present invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
  • the present pharmaceutical formulations comprise circRNA inhibitor (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a pharmaceutically-acceptable carrier.
  • the pharmaceutical formulations of the present invention can also comprise circRNA inhibitor which are encapsulated by liposomes and a pharmaceutically-acceptable carrier.
  • Preferred pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
  • Pharmaceutical compositions of the present invention can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
  • Suitable additives include, e.g., physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (such as, for example, calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • physiologically biocompatible buffers e.g., tromethamine hydrochloride
  • additions of chelants such as, for example, DTPA or DTPA-bisamide
  • calcium chelate complexes such as, for example, calcium DTPA, CaNaDTPA-bisamide
  • calcium or sodium salts for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate.
  • Pharmaceutical compositions of the present invention can be packaged for use in liquid form or can be lyophilized.
  • a further object of the present invention relates to a method of treating an intrahepatic cholangiocarcinoma in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a miR-338-3p mimic.
  • miRNA mimic refers to synthetic small non-coding RNAs capable of entering the RNAi pathway and regulating gene expression.
  • synthetic miRNA refers to any type of miRNA sequence, other than an endogenous miRNA.
  • miRNA mimics imitate the function of endogenous miRNAs and can be designed as mature, double stranded (duplex) molecules or mimic precursors e.g., pri-miRNAs or pre-miRNAs.
  • the miRNA mimic may comprise an effector sequence which is substantially identical to the effector sequence of the corresponding endogenous miRNA.
  • miRNA mimics can be comprised of modified and/or unmodified RNA, DNA, RNA-DNA hybrids or alternative nucleic acid chemistries.
  • CircLTBP2 a TGF ⁇ -induced circRNA, predicts a poor prognosis in iCCA patients.
  • CircLTBP2 (hsa_circ_0032603) relative expression in 20 freshly frozen iCCA tumors from a French national cohort (separated in two groups: circLTBP2 low and circLTBP2 high , according to their circLTBP2 median expression).
  • B Kaplan-Meier plots and log-rank statistics analysis revealed a significant decreased overall survival (OS) and relapse free survival (RFS) for iCCA patients with a high expression of circLTBP2.
  • OS overall survival
  • RFS relapse free survival
  • D Kaplan-Meier plots and log-rank statistics analysis endorsed the association of circLTBP2 expression with OS in the independent cohort of CCA while no correlation was found in HCC. Data are presented as means ⁇ SD. (* P-value ⁇ 0.05 ** P- value ⁇ 0.01, *** P-value ⁇ 0.001, n ⁇ 3).
  • circLTBP2 Overexpression of circLTBP2 promotes iCCA cell proliferation, migration, and resistance to gemcitabine-induced apoptosis in vitro and tumor growth in vivo.
  • HuCC-T1 cells resistance to gemcitabine-induced apoptosis (10 ng/mL) was evaluated every 4 hours using a red fluorescent Annexin V labelling, in both the circLTBP2 overexpressing and control groups.
  • Mouse subcutaneous tumor model was used to evaluate the impact of circLTBP2 overexpression on tumor growth (D) and volume (E).5 mice were included in the control group and 6 mice were included in the circLTBP2 overexpression group.
  • Statistical analyses for (E) were performed by a Mann–Whitney test.
  • OS overall survival
  • RFS relapse free survival
  • Circulating vesicles containing circLTBP2 in serum are associated with poor OS in iCCA patients.
  • (B) Positive correlation of circLTBP2 expression in matched tissues and serum samples (patients for which both samples are available, n 17)
  • pLV-circLTBP2 (circLTBP2 overexpression vector), pLV- circRNA (empty vector).
  • RNA CircRNA microarray analysis
  • Total RNA was purified with an miRNeasy kit (Qiagen, 217004).
  • Arraystar Human circRNA Microarray v2.0 (Arraystar, Rockville, MD, USA) were used to screen for novel TGF ⁇ -regulated circRNAs in human CCA cell lines.
  • q-RT-PCR, western blot and immunoprecipitation (IP) Quantitative reverse-transcription PCR (q-RT-PCR) and western blot were performed as previously described.
  • Primers and antibodies are listed in Table S1.
  • MiRNA IP was carried out according to the manufacturer’s instructions (miRNA Target IP kit #25500, Active Motif, Inc).
  • miR-338-3p mimics (miRCURY LNA miRNA Mimic, Qiagen, 5nM) were transfected into HuCC-T1 cells for 24 hours.
  • the IP was performed using a pan-Ago antibody to identify miRNA/mRNA and miRNA/circRNA complexes.
  • circRNA pulldown assay A circLTBP2 pulldown assay was carried out by Creative Biogene (NY, USA). Briefly, a labeled circRNA expression vector was generated and co-transfected into HuCC-T1 cells with a capture protein expression vector.
  • a pulldown assay was done by using a labeled antibody that binds to the induced capture protein, which allows for the pulldown of circLTBP2-miRNA complexes. Eluted miRNA were identified by miRNA-sequencing.
  • Tumor xenograft model HuCC-T1 cells were modified to overexpress either an empty vector (pLV-circRNA-GFP) or circLTBP2 (pLV-circLTBP2-GFP) and were infected with GL261-Luc (CMV-Firefly luciferase lentivirus (Neo), PLV-10064-50, Cellomics Technology, USA).4,000,000 cells were implanted on the flanks of 8-week-old female NSG mice (Charles River, USA). All animal procedures followed the European Community Directive guidelines (Agreement B35- 639238- 40, Biosit Rennes, France; DIR #7163) and were approved by the local ethics committee. Tumor growth was evaluated by measuring the size of the tumor with caliper.
  • mice 90 days after implantation, mice were sacrificed and the tumors, liver, and lung tissues were isolated and analyzed at the molecular level. The presence of metastases in the liver and in the lungs was evaluated by bioluminescence.
  • Statistical analyses All statistical analyses were conducted using Prism 8 software (from GraphPad Software) and the results were presented as mean values ⁇ standard deviation or median values. Comparison between variables was made using either Student’s t-test or Mann-Whitney U test. For multiple sets of multivariate comparisons, two-way analysis of variance was applied. Survival curves were analyzed through Kaplan-Meier method and log-rank test. A P value ⁇ 0.05 was considered statistically significant.
  • HuCC-T1 is a TGF ⁇ -responsive iCCA cell line CCA is a rare cancer with limited characterized in vitro models [14]. Therefore, we first characterized 3 iCCA (HuCC-T1, HuH-28, SG231) and 3 eCCA (Mz-ChA-1, TFK-1, SK-ChA- 1) cell lines. Mz-ChA1 and TFK-1 displayed an epithelial phenotype (high expression of E- Cadherin, CDH1 and no expression of Vimentin, VIM) while HuH-28 displayed a mesenchymal phenotype (high VIM / no CDH1).
  • SK-ChA-1, SG231 and HuCC-T1 exhibited an intermediate phenotype with expression of both CDH1 and VIM (data not shown).
  • TGF ⁇ target genes SERPINE1, SMAD7, BIRC3
  • HuCC- T1 and HuH-28 cell lines were fully responsive to TGF ⁇ , with a significant SMAD3 phosphorylation, up-regulation of SERPINE1, SMAD7 and down-regulation of BIRC3.
  • these effects were reversed in presence of galunisertib (LY2157299), a selective inhibitor of TGFBR1 (data not shown).
  • CircLTBP2 is a TGF ⁇ -induced circRNA predictive of poor prognosis in iCCA Gene expression profiling using Arraystar microarrays identified 119 circRNAs significantly (P ⁇ 0.001 and FC>2) deregulated by TGF ⁇ (1 ng/mL, 16 hours) in HuCC-T1, including 85 up- and 34 down-regulated circRNAs (data not shown). Interestingly, more than 50% of circRNAs and their linear counterparts were co-regulated in response to TGF ⁇ (data not shown). Out of the 119 TGF ⁇ -regulated circRNAs, 43 were predicted to act as miRNA sponges data not shown).
  • CircLTBP2 is a large exonic circRNA (2165 bases) composed of exons 2 to 16 of LTBP2 mRNA. Its circular structure was confirmed by Sanger sequencing (data not shown).
  • circLTBP2 overexpression promoted resistance to gemcitabine-induced apoptosis (Fig. 2C).
  • the pro-tumorigenic potential of circLTBP2 was further evaluated in vivo by injecting genetically engineered HuCC-T1 cells into NSG mice (data not shown). Tumors developed in mice injected with cells overexpressing circLTBP2 were significantly bigger in size (856 ⁇ 336 vs 1478 ⁇ 292.07 mm 3 , P ⁇ 0.001) and weight (0.6 ⁇ 021 vs 1.12 ⁇ 0.41 g, P ⁇ 0.05) (Fig. 2D-E). CircLTBP2 overexpression in the resected tumors was confirmed by q-RT-PCR (data not shown).
  • CircLTBP2 acts as a sponge for miR-338-3p, a tumor suppressor miRNA
  • circLTBP2 pull-down assay followed by miRNA sequencing. Due to the large size of circLTBP2 and the diverse functional impact of circLTBP2, we hypothesized that circLTBP2 may act as a sponge for tumor suppressor miRNAs. A decision-making pattern was established to select the most promising miRNA candidates (data not shown). By circRNA pull-down, we identified 94 miRNAs possibly interacting with circLTBP2, among them 69 were recorded in databases.
  • miRNAs also relied on a strong dependency to circLTBP2, as determined by the number of predicted binding sites (>2) and the enrichment score (>1.3) of the predicted miRNA targets in the gene expression profile of HuCC-T1 cells stimulated by TGF ⁇ .
  • 8 miRNAs met the selection criteria (miR-338-3p, miR-3064-5p, miR1270, miR-1294, miR-34c-5p, miR324-3p, miR-1914-3p, miR-34a-5p) (data not shown).
  • EVs (mainly exosomes) were isolated by ultracentrifugation and qualified by qNano, transmission electron microscopy and expression of specific surface markers (data not shown).
  • circLTBP2 was expressed in EVs secreted by HuCC-T1 cells and its expression in EVs was greater in cells pre-treated by TGF ⁇ (data not shown).
  • LTBP2 associates with ECM and it has been suggested that it may indirectly regulate the activation of TGF ⁇ by releasing LTBP-1 from microfibrils.
  • Increased expression of LTBP2 has been reported in several cancers and associated with tumor progression, notably in HCC.
  • This miRNA plays a crucial role in tumor progression and its activity is tightly regulated in cancer. Many pathways depend on its activity, including WNT, MAPK, and PI3K/AKT, all of which are involved in the progression of iCCA.
  • miR-338-3p controls numerous hallmarks of cancer cells, such as inhibiting cell proliferation signals, inducing cell death, or decreasing angiogenesis.
  • MiR-338-3p has also a strong prognostic value for biliary tract cancers. Low levels of miR-338-3p have been found in several advanced stage (TNM3) biliary tract tumors associated with significant lymph node infiltration. MiR-338-3p has also been identified in a signature of 3 miRNAs that predict survival in patients with iCCA. Patients with low levels of miR-338-3p had the worse OS and RFS.
  • miR-338-3p possibly targets SOX4, a key gene involved in epithelial-to-mesenchymal transition (EMT), modulating master regulators TWIST1, SNAI1 and ZEB1.
  • EMT epithelial-to-mesenchymal transition
  • TWIST1 master regulators
  • ZEB1 ZEB1.
  • the regulation of SOX4 by miR-338-3p has already been described in several cancers (e.g. breast cancer, kidney cancer) and associated with cell proliferation and migration.
  • miR-338-3p In lung cancer, miR-338-3p has been shown to suppress metastasis by targeting SOX4.
  • Only few studies have reported in depth functional studies of circRNAs driving iCCA oncogenesis so far.
  • circACTN4 acts as a signaling nexus allowing for the coordinated activation of Hippo and Wnt pathways in iCCA.
  • circACTN4 is able to sponge miR-424-5p in the cytoplasm allowing for the expression of Yes-associated protein 1 (YAP1) and to recruit YBX1 at the promoter of FZD7 in the nucleus.
  • YAP1 Yes-associated protein 1
  • CircRNA MBOAT2 promotes iCCA progression and lipid metabolism reprogramming by stabilizing PTBP1 to facilitate FASN mRNA cytoplasmic export.
  • some circRNAs could be translated.
  • IL6-induced circGGNBP2 was shown to promote CCA cell growth and metastasis, notably through a positive regulatory loop modulating IL-6/STAT3 signaling.
  • Secreted circRNAs could also play a key role in CCA carcinogenesis, as illustrated by circ_0020256 in exosomes from tumor-associated M2 macrophages which has been shown to promote CCA cell proliferation, migration, and invasion by modulating a miR-432-5p/E2F3 axis.
  • the current first-line reference treatment is a combination of gemcitabine and cisplatin.
  • the benefit of this treatment is modest, and iCCA remains a poor prognosis cancer.
  • Our data further suggest that inhibiting circLTBP2 (e.g. by using antisense oligonucleotides) could be relevant to sensitize iCCA cells to gemcitabine. Translational studies supporting this point will be required.
  • our study demonstrates that circRNAs modulate iCCA carcinogenesis and may serve as clinically relevant predictive biomarker for a better management of patients. Additionally, the study provides robust evidence for the regulatory role of the TGF ⁇ /circLTBP2/miR-338-3p axis in iCCA.
  • Circular RNAs in cholangiocarcinoma Cancer Lett 2023;553:215980.
  • Louis C, Leclerc D Coulouarn C. Emerging roles of circular RNAs in liver cancer. JHEP Rep 2022;4:100413.
  • Chen Q Wang H, Li Z, Li F, Liang L, Zou Y, et al.
  • Circular RNA ACTN4 promotes intrahepatic cholangiocarcinoma progression by recruiting YBX1 to initiate FZD7 transcription. J Hepatol 2021.
  • Li H, Lan T Liu H, Liu C, Dai J, Xu L, et al.
  • IL-6-induced cGGNBP2 encodes a protein to promote cell growth and metastasis in intrahepatic cholangiocarcinoma. Hepatology 2021.
  • Louis C Coulouarn C. One stone, two birds: circACTN4, a nexus for a coordinated activation of Hippo and Wnt/beta-catenin pathways in cholangiocarcinoma. J Hepatol 2022;76:8-10.
  • Bevant K Desoteux M, Angenard G, Pineau R, Caruso S, Louis C, et al.
  • TGFbeta- induced FOXS1 controls epithelial-mesenchymal transition and predicts a poor prognosis in liver cancer.
  • CircRNA MBOAT2 promotes intrahepatic cholangiocarcinoma progression and lipid metabolism reprogramming by stabilizing PTBP1 to facilitate FASN mRNA cytoplasmic export.
  • IL-6-induced cGGNBP2 encodes a protein to promote cell growth and metastasis in intrahepatic cholangiocarcinoma. Hepatology 2022;75:1402-1419.

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Abstract

Le cholangiocarcinome intrahépatique (iCCA) est un cancer mortel dans le monde entier, dont l'incidence est en augmentation et les options thérapeutiques limitées. Il est donc urgent d'ouvrir le domaine à de nouveaux concepts permettant d'identifier des cibles thérapeutiques et des biomarqueurs cliniquement pertinents. Ici, les inventeurs ont étudié le rôle et la pertinence clinique de l'ARN circulaire circLTBP2 dans l'iCCA. Plus particulièrement, le CircLTBP2 (hsa_circ_0032603) a été identifié comme un nouveau circARN induit par le TGFβ dans plusieurs lignées cellulaires de CCA. Le CircLTBP2 favorise la prolifération des cellules tumorales, la migration et la résistance à l'apoptose induite par la gemcitabine in vitro et la croissance tumorale in vivo. Mécaniquement, le circLTBP2 agit comme un ARN concurrent régulant notamment l'activité du microARN suppresseur du tumeur miR-338-3p, conduisant à la surexpression de ses cibles pro-métastatiques. La restauration des niveaux de miR-338-3p dans les cellules iCCA a inversé les effets pro-tumorigènes induits par circLTBP2, y compris la résistance à l'apoptose induite par la gemcitabine. En outre, l'expression de circLTBP2 prédit une réduction de la survie, comme cela a été détecté dans les tissus tumoraux mais aussi dans les exosomes sériques isolés chez les patients atteints d'iCCA. En conclusion, le CircLTBP2 constitue un nouvel effecteur du bras pro-tumorigène du TGFβ et un biomarqueur cliniquement pertinent, facilement détecté à partir de biopsies liquides dans l'iCCA. La présente invention concerne donc l'ARN circulant circLTBP2 en tant que biomarqueur et biotarget dans les cholangiocarcinomes intrahépatiques.
PCT/EP2024/056634 2023-03-14 2024-03-13 Arn circltbp2 circulaire en tant que biomarqueur et biocible dans des cholangiocarcinomes intrahépatiques WO2024189064A1 (fr)

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