WO2018199136A1 - Procédé de mesure du niveau d'expression d'une mutation t315i d'abl1 - Google Patents

Procédé de mesure du niveau d'expression d'une mutation t315i d'abl1 Download PDF

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WO2018199136A1
WO2018199136A1 PCT/JP2018/016748 JP2018016748W WO2018199136A1 WO 2018199136 A1 WO2018199136 A1 WO 2018199136A1 JP 2018016748 W JP2018016748 W JP 2018016748W WO 2018199136 A1 WO2018199136 A1 WO 2018199136A1
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abl1
mutation
primer
mrna
nucleic acid
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PCT/JP2018/016748
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Japanese (ja)
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粛典 葛城
田中　秀明
隆太 伊藤
古賀　大輔
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大塚製薬株式会社
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Priority to SG11201909983Q priority Critical patent/SG11201909983QA/en
Priority to CN201880042856.1A priority patent/CN110997938A/zh
Priority to KR1020197034072A priority patent/KR20200002933A/ko
Priority to JP2019514562A priority patent/JPWO2018199136A1/ja
Publication of WO2018199136A1 publication Critical patent/WO2018199136A1/fr
Priority to PH12019502417A priority patent/PH12019502417A1/en

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the BCR-ABL1 fusion gene is a chromosomal abnormality found in chronic myelogenous leukemia (Chronic Myelogenous Leukemia: CML) and Philadelphia chromosome positive Acute Lymphocytic Leukemia: Ph positive ALL.
  • CML chronic myelogenous leukemia
  • Ph positive ALL Philadelphia chromosome positive Acute Lymphocytic Leukemia
  • the translocation t (9; 22) between chromosome 9 and chromosome 22 fused the ABL1 gene localized in the q34 band of chromosome 9 with the BCR gene localized in the q11 band of chromosome 22. It is formed.
  • the chimeric protein BCR-ABL1 encoded by the BCR-ABL1 fusion gene, has tyrosine kinase activity, constitutively stimulates cell proliferation signals, and suppresses apoptosis, thereby limiting the number of hematopoietic stem cells. Proliferate and cause abnormalities.
  • tyrosine kinase inhibitor tyrosine kinase inhibitor acting as a target molecule for BCR-ABL1
  • TKI tyrosine kinase inhibitor
  • a representative TKI, imatinib has demonstrated a breakthrough therapeutic effect on CML and Ph-positive ALL cases.
  • the effect was reduced and became a therapeutic issue.
  • Nilotinib and dasatinib were subsequently developed as effective TKIs against many of the BCR-ABL1 gene mutations that are resistant to imatinib.
  • T315I mutation isoleucine
  • Ponatinib was developed as a drug that exhibits a tyrosine kinase inhibitory action against BCR-ABL1 having this T315I mutation.
  • Ponatinib is useful for CML and Ph-positive ALL that are resistant or intolerant to existing TKIs, and shows a high effect particularly when it has a T315I mutation (Non-patent Documents 4 and 5).
  • the therapeutic effect can be monitored by measuring the expression level of BCR-ABL1 mRNA. Since the T315I mutation is resistant to many TKIs and has a poor prognosis, it is clinically useful to monitor the expression level of BCR-ABL1 mRNA having the T315I mutation in the same manner as the amount of BCR-ABL1 mRNA. Conceivable. It is also considered useful for evaluating the effects of ponatinib. However, the T315I mutation detection method developed so far lacks sufficient quantitativeness and is insufficient for these purposes.
  • the purpose of the present application is to provide a method for measuring the expression level of ABL1 T315I mutation in a subject, or a kit therefor.
  • the subject RNA sample is used with two reverse primers that bind upstream and downstream of the T315I mutation site of ABL1 mRNA. It was found that the expression level of the ABL1 T315I mutation can be measured by performing reverse transcription with and comparing the amount of reverse transcription product with the two primers.
  • the application is a method of measuring the expression level of an ABL1 T315I mutation in a subject, (1) In the presence of a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA, (a) a reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA; b) reverse transcription of the RNA sample of interest using a reverse primer that binds to the region upstream of the T315I mutation position of ABL1 mRNA in the same container; and (2) calculating the expression level of the ABL1 T315I mutation based on the ratio of the reverse transcription product of the primer of (a) to the reverse transcription product of the primer of (b),
  • a method comprising:
  • the application is a kit for measuring the expression level of an ABL1 T315I mutation in a subject comprising: (A) A reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA; (B) a reverse primer that binds to a region upstream of the T315I mutation position of ABL1 mRNA; and (C) a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA;
  • a kit is provided.
  • the expression level of ABL1 T315I mutation in a subject can be measured. This is expected to provide useful information for the treatment of patients with T315I mutation.
  • the base sequence of SEQ ID NO: 1 is shown.
  • the 947th cytosine base is shown in bold and underlined. This shows the base sequence of SEQ ID NO: 1 (continued).
  • the base sequence of SEQ ID NO: 3 is shown.
  • the 947th thymine base is shown in bold and underlined. This shows the base sequence of SEQ ID NO: 3 (continued).
  • FIG. 1 It is a schematic diagram of quantitative real-time PCR of T315I mutant ABL1 mRNA.
  • the amplification curve of amplification of cDNA derived from T315I reverse primer is shown.
  • the amplification curve of amplification of cDNA derived from ABL reverse primer is shown.
  • the ABL1 gene is a gene localized in the q34 band of chromosome 9.
  • BCR gene exon 1, exon 1-13 or 1-14 and ABL1 gene exon 2-4 are fused by translocation t (9; 22) between chromosome 9 and chromosome 22, chronic bone marrow BCR-ABL1 gene found in sexual leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia.
  • the ABL1 gene typically has the base sequence of SEQ ID NO: 1, but many mutants are known.
  • the ABL1 gene includes a polynucleotide that hybridizes under stringent conditions to a sequence complementary to the polynucleotide having the sequence of SEQ ID NO: 1.
  • hybridize under stringent conditions the hybridization used here is in accordance with the usual method described in, for example, Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983) etc. It can be carried out.
  • “Stringent conditions” means, for example, 6 ⁇ SSC (a solution containing 1.5M NaCl, 0.15M trisodium citrate is 10 ⁇ SSC), 45 ° C.
  • the ABL1 gene has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity with a polynucleotide having the sequence of SEQ ID NO: 1.
  • a polynucleotide having Sequence identity means the degree of sequence similarity between two oligonucleotides. Two sequences that are aligned in an optimal state (a state that maximizes the sequence match) over the region of the base sequence to be compared. Determined by comparison. The sequence identity value (%) determines the same base present in both sequences to determine the number of matching sites, and then divides this number of matching sites by the total number of bases in the sequence region to be compared. It is calculated by multiplying the obtained numerical value by 100.
  • Algorithms for obtaining optimal alignment and sequence identity include various algorithms commonly available to those skilled in the art (eg, BLAST algorithm, FASTA algorithm, etc.).
  • sequence identity of the base sequence is determined using sequence analysis software such as BLAST and FASTA.
  • wild-type ABL1 gene the ABL1 gene having a nucleotide at the T315I mutation position having cytosine is referred to as “wild-type ABL1 gene”, and its mRNA is referred to as “wild-type ABL1 mRNA”.
  • Wild-type ABL1 mRNA typically has the nucleotide sequence of SEQ ID NO: 1, and the protein encoded thereby has the amino acid sequence of SEQ ID NO: 2.
  • “T315I mutation position” means the position corresponding to the 947th position of SEQ ID NO: 1 when the nucleotide sequence of a certain ABL1 gene is optimally aligned with the nucleotide sequence of SEQ ID NO: 1.
  • T315I mutation substitution in the ABL1 gene or ABL1 protein is referred to herein as the “T315I mutation”.
  • the ABL1 gene having a T315I mutation is referred to as “T315I mutant ABL1 gene”, and its mRNA is referred to as “T315I mutant ABL1BL mRNA”.
  • the T315I mutant ABL1 mRNA typically has the nucleotide sequence of SEQ ID NO: 3, and the protein encoded thereby has the amino acid sequence of SEQ ID NO: 4.
  • expression level of ABL1IT315I mutation refers to the expression level of all genes having a T315I mutation, ie, a translocated ABL1 gene having a T315I mutation, including a non-translocated ABL1 gene and a BCR-ABL1 gene. Means.
  • the subject is a human.
  • the subject may be a subject suffering from or suspected of having chronic myeloid leukemia (CML) or Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL).
  • CML chronic myeloid leukemia
  • Ph + ALL Philadelphia chromosome positive acute lymphoblastic leukemia
  • the subject may have a BCR-ABL1 gene.
  • an “RNA sample” is RNA extracted from a sample containing hematopoietic stem cells, leukocytes or leukemia cells derived from the subject, and can be, for example, RNA extracted from blood, bone marrow fluid, or lymph fluid. RNA extracted from isolated hematopoietic stem cells, blood cells, leukocytes or leukemia cells may be used. In certain embodiments, the RNA sample is RNA extracted from peripheral blood leukocytes or bone marrow nucleated cells. In certain embodiments, the RNA sample is RNA extracted from peripheral blood leukocytes.
  • RNA may be total RNA or purified mRNA.
  • a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA is used.
  • the modified nucleic acid is referred to as “T315I clamp”.
  • a modified nucleic acid contains one or more artificial nucleotides, forms a complementary strand more strongly than mRNA having a certain base sequence with respect to mRNA having a certain sequence, and is not degraded by the exonuclease activity of reverse transcriptase .
  • Reverse transcriptase stops the extension reaction at the binding site when the modified nucleic acid is bound to the template mRNA.
  • the T315I clamp binds to the wild-type ABL1 mRNA with a cytosine at the T315I mutation position, so that the nucleotide does not bind to the T315I variant ABL1 mRNA with thymine, and thus suppresses reverse transcription of the wild-type ABL1 mRNA.
  • the reverse transcription of T315I mutant ABL1 mRNA is designed not to be suppressed.
  • the T315I clamp is about 10-22, about 12-20, about 14-18, or about 15-17, for example about 16 comprising nucleotides at the T315I mutation position of wild-type ABL1 mRNA.
  • the T315I clamp comprises the nucleotide sequence of 5'-ATGAACTCAGTGATGA-3 '(SEQ ID NO: 5). In one embodiment, the T315I clamp consists of the base sequence of SEQ ID NO: 5.
  • the T315I clamp contains one or more artificial nucleotides.
  • the artificial nucleotide means one having a modified nucleoside (base moiety or sugar moiety) and having a structure different from that of a natural nucleotide.
  • an artificial nucleotide one that enhances nuclease resistance and binding affinity with a target sequence can be selected. For example, Deleavey, G. F., & Damha, M. J. (2012). Designing chemically modified oligonucleotides for targeted gene silencing. Chemistry & biology, 19 (8), 937 Artificial nucleotides described in -954 can be used.
  • artificial nucleotides include, for example, abasic nucleosides; arabino nucleosides, 2′-deoxyuridines, ⁇ -deoxyribonucleosides, ⁇ -L-deoxyribonucleosides, nucleosides with other sugar modifications; peptide nucleic acids (PNA) ), A peptide nucleic acid to which a phosphate group is bound (PHONA), a cross-linked artificial nucleic acid (LNA), a 2′-O, 4′-C-ethylene cross-linked nucleic acid (ENA), a constrained ethyl (cEt), a morpholino nucleic acid, etc. .
  • PNA peptide nucleic acids
  • Nucleosides having sugar modifications include substituted pentose monosaccharides such as 2′-O-methyl ribose, 2′-O-methoxyethyl ribose, 2′-deoxy-2′-fluororibose, 3′-O-methyl ribose; Included are nucleosides having 1 ', 2'-deoxyribose; arabinose; substituted arabinose sugars; hexose and alpha-anomeric sugar modifications.
  • modified base examples include pyrimidines such as 5-hydroxycytosine, 5-methylcytosine, 5-fluorouracil and 4-thiouracil; purines such as 6-methyladenine and 6-thioguanosine; and other heterocyclic bases Is mentioned.
  • the artificial nucleotides in the T315I clamp may all be of the same type, or two or more different artificial nucleotides may be present.
  • the T315I clamp includes one or more PNAs as artificial nucleotides.
  • PNA generally has a structure in which a phosphate bond of DNA is replaced by a peptide bond by N- (2-aminoethyl) glycine binding to a peptide (Nielsen et al. 1991 Science 254, 1457-1500).
  • PNA is resistant to various nucleolytic enzymes and forms complementary strands with DNA or RNA by molecular recognition similar to DNA or RNA.
  • the affinity between PNA and DNA is higher than the affinity between DNA and DNA or between DNA and RNA.
  • the nucleotides in the T315I clamp are all PNA.
  • the addition amount of the T315I clamp may be an amount that can suppress reverse transcription of wild-type ABL1 mRNA, and is, for example, about 0.1 ⁇ M to 10 ⁇ M or about 0.5 ⁇ M to 5 ⁇ M at the final volume molar concentration of the reverse transcription reaction solution. For example, it can be about 2 ⁇ M.
  • step (1) for reverse transcription, two reverse primers, (a) a reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA, and (b) a T315I mutation position of ABL1 mRNA.
  • a reverse primer that binds to the upstream region The reverse primer in (a) reverse transcribes a portion including a region to which the T315I clamp of ABL1 mRNA binds. In the present specification, the reverse primer is referred to as “T315I reverse primer”.
  • the reverse primer in (b) reversely transcribes the part upstream of the region to which the T315I clamp binds in ABL1 mRNA. In this specification, the reverse primer of (b) is referred to as “ABL reverse primer”. Methods for designing primers suitable for reverse transcription are well known to those skilled in the art.
  • step (1) reverse transcription using a T315I reverse primer and an ABL reverse primer is performed in one step, that is, in the same container, simultaneously or successively.
  • reverse transcription with T315I reverse primer and ABL reverse primer is performed simultaneously in the same container.
  • Schematic diagrams of reverse transcription reactions of wild-type ABL1 mRNA and T315I mutant ABL1 mRNA using these reverse transcription primers and T315I clamp are shown in Figs. 3 and 4, respectively.
  • reagents such as reverse transcriptase, dNTP, and buffer
  • conditions such as amounts of various reagents, reaction time, and temperature are described in the instructions attached to the reverse transcriptase. It can be appropriately determined by a known method such as a description or a commonly used protocol.
  • the reverse transcriptase is any known reverse transcriptase that can be used in molecular biology experiments, such as Tth DNA polymerase, rTth DNA polymerase, AMV reverse transcriptase, MMLV reverse transcriptase, HIV reverse transcriptase, etc. Derivatives can be used.
  • step (2) the expression level of the ABL1 T315I mutation is calculated based on the ratio of the reverse transcription product produced by the T315I reverse primer of (a) to the reverse transcription product produced by the ABL reverse primer of (b).
  • the value obtained by quantifying the reverse transcription product by the T315I reverse primer in (a) divided by the value quantifying the reverse transcription product by the ABL reverse primer in (b) is a measured value of the expression level of the ABL1 T315I mutation.
  • Quantification of reverse transcripts may be performed by any method known in the art.
  • step (2) of the above method comprises quantifying the reverse transcription product by the T315I reverse primer of step (2-1) (a) by quantitative PCR, and step (2-2) (b)
  • the method further comprises the step of quantitatively quantifying the reverse transcription product of the ABL reverse primer of said product by quantitative PCR.
  • Quantitative PCR may be quantitative real-time PCR, for example.
  • Various fluorescent PCR techniques can be used for quantitative real-time PCR.
  • Examples of the fluorescent PCR technique include an intercalator method using a fluorescent nucleic acid labeling agent such as SYBR GREEN I (for example, LightCycler (registered trademark) (Roche), ABI Prizm 7700 Sequence Detection System (registered trademark) (Perkin Elmer, Applied Biosystems), TaqMan probe method for monitoring amplification in real time using 5 ′ exonuclease activity of DNA polymerase, RNase activity of RNase H enzyme and cycling probe using dedicated chimeric RNA probe Law, etc., but is not limited to this.
  • SYBR GREEN I for example, LightCycler (registered trademark) (Roche), ABI Prizm 7700 Sequence Detection System (registered trademark) (Perkin Elmer, Applied Biosystems), TaqMan probe method for monitoring amplification in real time using 5 ′ exonuclease activity of DNA polymerase, RNase activity of RNase H enzyme and cycling probe using dedicated chimeric RNA
  • quantitative real-time PCR is performed by the TaqMan probe method.
  • an oligonucleotide in which the 5 'end is modified with a fluorescent substance and the 3' end with a quencher substance is added to the PCR reaction system.
  • the TaqMan probe specifically hybridizes to the template DNA at the annealing stage. However, since a quencher is present on the probe, the generation of fluorescence is suppressed even when irradiated with excitation light.
  • TaqMan probe hybridized to the template is decomposed by the 5 'exonuclease activity of Taq polymerase in the extension reaction step, the fluorescent dye is separated from the quencher and emits fluorescence.
  • TaqMan probes can bind anywhere in the PCR product and can be designed by methods well known in the art. Also, any combination of fluorescent material and quencher material can be used.
  • a standard solution containing a known concentration of cDNA and an unknown concentration of cDNA are amplified simultaneously, and the horizontal axis represents the number of amplification cycles, and the vertical axis represents the fluorescence intensity (logarithmic conversion value) of the reporter dye.
  • An amplification curve may be created. A line parallel to the horizontal axis is drawn near the central value of the fluorescence intensity where the amplification curve becomes linear, and the number of amplification cycles when the line and each amplification curve intersect can be obtained.
  • the sample amplification cycle number is applied to this standard curve.
  • the concentration of can be calculated.
  • primer pairs used in steps (2-1) and (2-2) are designed so that the reverse transcription product of step (1) can be amplified.
  • Methods for designing primers suitable for PCR are well known to those skilled in the art.
  • Primer pairs are designed so that the amplified nucleic acid has a length suitable for quantification, eg, about 10-1000, about 20-500, about 50-300, about 100-200 nucleotides, eg, about 150 nucleotides in length. obtain.
  • the primer pair used in step (2-1) is designed so that a region including the T315I mutation position of the reverse transcription product can be amplified. That is, a forward primer having a base sequence of a region containing the T315I mutation position of the ABL gene or a region upstream thereof, and a base sequence complementary to the base sequence of the region containing the T315I mutation position of the ABL1 gene or a region downstream thereof Use a reverse primer with The forward primer used in step (2-1) has a base sequence in the region containing the T315I mutation position of the ABL gene, and the nucleotide corresponding to the nucleotide at the T315I mutation position is replaced with uracil ribonucleotide. Also good.
  • the same T315I reverse primer as in step (1) (a) is used in step (2-1).
  • step (2-2) the same ABL reverse primer as in step (1) (b) is used.
  • the reverse transcription product by the T315I reverse primer may be quantified by RNaseH-dependent quantitative PCR specific for the T315I mutation.
  • RNaseH-dependent PCR is a sequence-specific PCR method using RNaseH (Boucard AA, et. Al. J Biol Chem, 289 (1): 387-402; Dobosy JR, et al., BMC Biotechnol, 11 ( 80): 1-18).
  • RNase H recognizes an RNA / DNA heteroduplex and cleaves a phosphodiester bond with the 5'-end DNA of RNA.
  • At least one base of the primer is RNA to form a template DNA and an RNA / DNA heteroduplex, and a region (blocking region) where DNA polymerase cannot bind is provided on the 3 ′ side of RNA.
  • the RNA in the primer must be complementary to the template DNA and not mismatched.
  • step (2-1) the nucleotide sequence of the region containing the T315I mutation position of the ABL1 gene has been substituted, and the nucleotide corresponding to the nucleotide at the T315I mutation position has been replaced with uracil ribonucleotide.
  • a forward primer having a blocking region at the 3 ′ end is used.
  • the forward primer is referred to as “T315I forward primer”.
  • the uracil ribonucleotide in the T315I forward primer causes a mismatch with the nucleotide of the template in PCR of wild type ABL cDNA, so that the blocking region is not cleaved and PCR does not proceed.
  • T315I mutant ABL cDNA can be specifically amplified by RNaseH-dependent PCR using T315I forward primer.
  • the T315I forward primer includes a sequence in a region upstream of the T315I mutation position of the ABL1 gene, and is designed so that the remaining oligonucleotide functions as a forward primer for the PCR reaction after the RNA and the blocking region are separated.
  • the T315I forward primer includes, for example, a sequence of about 8-60, 10-30, 15-25 or 19-23 nucleotides upstream of the T315I mutation position of ABL1 mRNA, eg, about 20, 21 or 22 nucleotides.
  • the nucleotide corresponding to the nucleotide at the T315I mutation position in the T315I forward primer is a uracil ribonucleotide.
  • the blocking region on the 3 'side includes mismatching DNA and blocking groups such as C3 spacer.
  • the blocking region consists of 4 deoxyribonucleotides having the same bases as the 945th to 948th nucleotides of the ABL1 gene, one deoxyribonucleotide having a different base from the 949th nucleotide, and one blocking group.
  • the blocking region is one deoxyribonucleotide having the same base as the 945th nucleotide of the ABL1 gene, two blocking groups, one deoxyribonucleotide having the same base as the 946th nucleotide, 947th It consists of one deoxyribonucleotide having a base different from that of the nucleotide.
  • the T315I forward primer is 5'-GAGCCCCCGTTCTATACATCATArUT / iSpC3 // iSpC3 / GC-3 '(SEQ ID NO: 6, where rU is uracil RNA and iSpC3 is spacer C3).
  • Steps (2-1) and (2-2) may be performed in the presence of a T315I clamp. Therefore, the reaction can be carried out by adding reagents necessary for the step (2-1) or (2-2) to all or a part of the product of the step (1).
  • the steps (1) and (2-1) or the steps (1) and (2-2) can be performed in one step, that is, in the same container, simultaneously or successively.
  • step (2-1) When RNaseH-dependent PCR is performed in step (2-1) and steps (1) and (2-1) are performed in a single step, part of the T315I clamp and part of the T315I forward primer are complementarily bound. You may be able to.
  • the ability of an oligonucleotide to discriminate a single base mutation is believed to be maximized when the mutation site is in the middle of the oligonucleotide, but to reduce this complementary binding, the T315I clamp is It may be designed such that the nucleotide corresponding to the mutation position is located 3 ′ from the center of the T315I clamp.
  • the T315I clamp can be designed so that the complementary region of the T315I clamp and the T315I forward primer is less than 50% of the total length of the T315I clamp.
  • the T315I clamp consisting of the nucleotide sequence of SEQ ID NO: 5 has been optimized so that it has less complementary binding to the forward primer used for RNase H-dependent PCR and has the ability to identify mutations.
  • Such adjustment of the modified nucleic acid sequence is applicable not only to the detection of T315I mutation but also to the detection of other mutations.
  • a portion of less than 50% of the total length of the modified nucleic acid is a forward primer for RNase H-dependent PCR. It is better to adjust it so that it is complementary.
  • reagents such as DNA polymerase, RNaseH, dNTP, and buffer
  • the conditions such as the amount of each reagent, reaction time, temperature, etc. are described in the instructions attached to the enzyme. And can be appropriately determined by a known method such as a commonly used protocol.
  • DNA polymerase any known DNA polymerase that can be used for molecular biology experiments and the like, for example, rTth DNA polymerase, Taq polymerase, and derivatives thereof can be used.
  • RNaseH may be any known RNaseH that can be used for molecular biology experiments, for example, RNaseH2.
  • the primers, clamps and probes listed in the table below are used. All of these may be used in combination, or at least one may be used.
  • kits for performing the above methods is at least (A) A reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA; (B) a reverse primer that binds to a region upstream of the T315I mutation position of ABL1 mRNA; and (C) a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA; including.
  • the kit may further include at least one of a forward primer for amplifying the reverse transcription product of ABL1 mRNA by the reverse primer of (a) and (b) and a further reverse primer for PCR.
  • the primer may be for RNase H dependent PCR.
  • the kit may further include a probe for quantitative PCR.
  • the kit can further comprise a standard, eg, at least one of a known amount of wild-type ABL1 mRNA and T315I mutant ABL1 mRNA.
  • the kit may further contain reagents necessary for carrying out the above method, for example, reverse transcriptase, DNA polymerase, RNaseH, dNTP, buffer, etc. for reverse transcription, PCR, quantitative PCR or RNaseH-dependent PCR.
  • the kit may further include other components desirable from a commercial and user standpoint, such as a package insert containing instructions for use (eg, a written or storage medium).
  • kits Each component included in the kit is either separately or, if possible, mixed, dissolved in water or a suitable buffer, or lyophilized and placed in a suitable container.
  • Suitable containers include bottles, vials, test tubes, tubes, plates, multiwell plates and the like.
  • the container may be formed from at least one material such as glass, plastic, metal, and the like.
  • the container may have a label.
  • the expression level of T315I mutant ABL1 mRNA in which the 944th base of ABL1 mRNA in RNA extracted from peripheral blood leukocytes or bone marrow nucleated cells is mutated from cytosine to thymine is measured.
  • the measurement principle is a two-step quantitative RT-PCR method consisting of two steps: reverse transcription reaction (RT) that synthesizes complementary DNA (cDNA) using RNA as a template, and real-time PCR that quantifies cDNA using a fluorescently labeled probe. It is.
  • a region including the T315I mutation position of T315I mutant ABL1 mRNA and a region not including the T315I mutation position of all ABL1 mRNA are amplified, and a ratio obtained by dividing the former quantitative value by the latter quantitative value is used as a report value.
  • the outline of the reverse transcription reaction is shown in FIG.
  • the ABL1 mRNA whose 944th base is cytosine is defined as wild-type ABL1 mRNA
  • the ABL1 mRNA whose thymine is defined as T315I mutant ABL1 mRNA.
  • Two primers, ABL reverse primer and T315I reverse primer are used to reverse transcribe the region for quantifying all ABL1 mRNA and the region for quantifying T315I mutant ABL1 mRNA, respectively.
  • the ABL reverse primer and the T315I reverse primer bind to complementary sequences contained in the measurement sample (FIGS. 5 (1a) and (1b)).
  • the first strand cDNA is synthesized by the reverse transcription activity of Tth DNA polymerase contained in the reaction solution (FIG. 5 (2a) or (2b)).
  • the reaction solution contains a T315I clamp having a sequence complementary to the wild-type ABL1 mRNA.
  • the T315I clamp specifically binds, Since the reverse transcription reaction is inhibited (FIG.
  • ABL1 mRNA-derived cDNA synthesized by reverse transcription reaction is amplified by real-time PCR (FIG. 6).
  • the fluorescently labeled probe (ABL probe) bound to one side of the double-stranded cDNA is decomposed by the 5′-3 ′ exonuclease activity of Tth DNA polymerase in the reaction solution, and the reporter dye is released.
  • the increase in the amount of cDNA derived from ABL1 mRNA is measured in real time by measuring the fluorescence intensity of the released dye for each cycle (FIG. 6 (6)).
  • rhPCR RNase H-dependent PCR
  • RNase H2 a nucleolytic enzyme used for rhPCR, has the property of recognizing RNA / DNA heteroduplexes and cleaving the phosphodiester bond with the 5 ′ terminal DNA of RNA.
  • RNA and DNA heteroduplex are formed by changing one deoxyribonucleotide of a primer to a ribonucleotide, and a region (blocking region) where DNA polymerase cannot bind is provided on the 3 ′ side of RNA.
  • PCR proceeds only when RNase H2 recognizes and cleaves an RNA / DNA heteroduplex.
  • RNase H2 recognizes and cleaves an RNA / DNA heteroduplex.
  • cDNA derived from T315I mutant ABL1 mRNA in which the 944th base of ABL1 mRNA is thymine is amplified.
  • An outline of quantitative real-time PCR of T315I mutant ABL1 mRNA is shown in FIG.
  • the portion corresponding to the 944th base of the T315I mutant ABL1 mRNA is substituted from DNA (thymine) to RNA (uracil) (FIG. 7 (1 a)). Since the RNA-substituted base of the T315I forward primer does not form an RNA / DNA heteroduplex with cDNA derived from wild-type ABL1 mRNA, the cleavage of the blocking region by RNase H2 does not occur, and the PCR reaction does not occur (FIG. 7 ( 2a)).
  • the RNA-substituted base of the T315I forward primer forms an RNA / DNA heteroduplex with respect to the cDNA derived from T315I mutant ABL1 mRNA
  • the blocking region is cleaved by RNase H2
  • the cDNA derived from T315I mutant ABL1 mRNA Specific amplification proceeds (FIGS. 7 (1b) and (2b)).
  • Specific amplification of cDNA derived from T315I mutant ABL1 mRNA is repeated by PCR (FIGS. 7 (3) to (9)).
  • the fluorescently labeled probe (ABLT315I probe) bound to one side of the double-stranded cDNA is decomposed by the 5'-3 'exonuclease activity of Tth DNA polymerase in the reaction solution, and the reporter dye is released.
  • the increase in the amount of cDNA derived from T315I mutant ABL1 mRNA is measured in real time (FIG. 7 (6)).
  • the present application is a method for detecting an ABL1 T315I mutation in a subject, comprising: (1) In the presence of a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA, using a reverse primer that binds to the region downstream of the T315I mutation position of ABL1 mRNA, Reverse transcription of an RNA sample; (2) Using a forward primer having a base sequence of the region containing the T315I mutation position of ABL1 mRNA, the nucleotide corresponding to the nucleotide at the T315I mutation position is uracil ribonucleotide, and having a blocking region at the 3 ′ end ( Amplifying the reverse transcription product of 1) by RNase H-dependent PCR; (3) a step of determining that the subject expresses the ABL1 T315I mutation when the nucleic acid is amplified in step (2);
  • a method comprising:
  • the present application is a kit for performing the above method, comprising: (A) A forward primer having a base sequence of a region containing the T315I mutation position of ABL1 mRNA, a nucleotide corresponding to the nucleotide at the T315I mutation position being replaced with uracil ribonucleotide, and a blocking region at the 3 ′ end; (B) a reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA; and (C) a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA; A kit is provided.
  • a method for measuring the expression level of an ABL1 T315I mutation in a subject (1) In the presence of a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA, (a) a reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA; b) reverse transcription of the RNA sample of interest using a reverse primer that binds to the region upstream of the T315I mutation position of ABL1 mRNA in the same container; and (2) calculating the expression level of the ABL1 T315I mutation based on the ratio of the reverse transcription product of the primer of (a) to the reverse transcription product of the primer of (b), Including methods.
  • Step (2) Quantifying the reverse transcription product by the primer of step (2-1) (a) by quantitative PCR; and Quantifying the reverse transcript by the primer of step (2-2) (b) by quantitative PCR;
  • the method of claim 1 further comprising: [3] Quantitative PCR in step (2-1) (X) the nucleotide sequence of the region containing the T315I mutation position of the ABL gene, wherein the nucleotide corresponding to the nucleotide at the T315I mutation position is replaced with uracil ribonucleotide, or (Y) having a base sequence in a region upstream of the T315I mutation position of ABL mRNA, Item 3.
  • a part of the modified nucleic acid and a part of the forward primer of (X) are complementary, and the complementary region is less than 50% of the total length of the modified nucleic acid, The method according to any one.
  • the method according to any one of items 1 to 9 wherein the nucleotide corresponding to the T315I mutation position in the modified nucleic acid is located 3 ′ from the center of the modified nucleic acid.
  • the method according to any one of items 1 to 10 wherein the modified nucleic acid comprises the base sequence of SEQ ID NO: 5.
  • step (a) comprises the sequence of SEQ ID NO: 7.
  • step (b) comprises the sequence of SEQ ID NO: 10.
  • step (2-1) is performed in the presence of the modified nucleic acid used in step (1).
  • a kit for measuring the expression level of ABL1 T315I mutation in a subject (A) A reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA; (B) a reverse primer that binds to a region upstream of the T315I mutation position of ABL1 mRNA; and (C) a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA; Including kit. [25] The kit according to item 24, further comprising a forward primer for amplifying the reverse transcription product of ABL mRNA by the reverse primer of (a) and (b) by PCR.
  • the forward primer is (X) the nucleotide sequence of the region containing the T315I mutation position of ABL1 mRNA, and the nucleotide corresponding to the nucleotide of the T315I mutation is replaced with uracil ribonucleotide, or (Y) having a base sequence in a region upstream from the T315I mutation position of ABL1 mRNA, 26.
  • the kit according to item 25 which is a forward primer.
  • the kit according to item 26 wherein the forward primer is the forward primer of (X).
  • the forward primer has a blocking region at the 3 ′ end.
  • the kit according to any one of items 25 to 28, wherein the forward primer comprises the sequence of SEQ ID NO: 6.
  • the forward primer consists of the sequence of SEQ ID NO: 6.
  • a part of the modified nucleic acid is complementary to a part of the forward primer of (X), and the complementary region is less than 50% of the total length of the modified nucleic acid.
  • the kit according to any one. [32] The kit according to any one of items 24 to 31, wherein the nucleotide corresponding to the T315I mutation position in the modified nucleic acid is located 3 ′ from the center of the modified nucleic acid. [33] The kit according to any one of items 24 to 32, wherein the modified nucleic acid comprises the sequence of SEQ ID NO: 5. [34] The kit according to any one of items 24 to 33, wherein the modified nucleic acid comprises the sequence of SEQ ID NO: 5.
  • the kit according to any one of items 24 to 34, wherein the modified nucleic acid comprises PNA.
  • the reverse primer of (a) comprises the sequence of SEQ ID NO: 7.
  • the reverse primer of (b) comprises the sequence of SEQ ID NO: 10.
  • the kit according to any one of items 24 to 36, wherein the reverse primer of (a) comprises the sequence of SEQ ID NO: 7.
  • the reverse primer of (b) comprises the sequence of SEQ ID NO: 10.
  • kits according to any one of items 24 to 39 further comprising at least one of wild-type ABL1 mRNA and ABL1 mRNA having a T315I mutation.
  • the kit according to any one of items 24 to 40 which comprises at least one of the primer, clamp and probe described in Table 1.
  • the kit according to any one of items 24 to 41 comprising the primer, clamp and probe described in Table 1.
  • Primer and probe design The following primer sets and probes were designed and synthesized.
  • the fluorescently labeled probe was labeled with HEX (6-carboxyfluorescein) at the 5 ′ end of the probe and labeled with Iowa Black FQ (Integrated DNA technologies) as the quenching dye at the 3 ′ end of the probe. Details of primers and probes in this example are shown in Table 2.
  • [Test 1] Preparation of standard product (ABL1 T315I mutant RNA)
  • synthetic RNA containing the sequence of ABL1 mRNA having T315I mutation was used.
  • a plasmid vector containing a PCR amplification region using the above-described primers in wild-type ABL1 mRNA and a T7 promoter sequence serving as a starting point for RNA synthesis was prepared.
  • a single base mutation was introduced into the portion corresponding to the T315I mutation of the ABL1 mRNA-derived sequence contained in this plasmid vector by site-directed mutagenesis.
  • the plasmid vector introduced with this mutation was transformed into E. coli.
  • Escherichia coli was cultured to prepare a large amount of plasmid vector, which was cleaved at one site with a restriction enzyme to prepare a linear DNA fragment having a sequence encoding ABL1 mRNA having a T315I mutation.
  • RNA containing the sequence of ABL1 mRNA having the T315I mutation was synthesized using the DNA fragment as a template.
  • the synthesized RNA was diluted with TE buffer containing 100 ng / ⁇ L of E. coli transfer RNA to prepare an RNA standard.
  • the ABL1 T315I mutant RNA standard thus prepared was adjusted to a concentration of 1 ⁇ 10 2 , 1 ⁇ 10 3 , 1 ⁇ 10 4 , 1 ⁇ 10 5 , 1.0 ⁇ 10 6 , 1 ⁇ 10 7 copies / test. .
  • As a negative control TE buffer containing 100 ng / ⁇ L of E. coli transfer RNA was used.
  • Reverse transcription reaction (2-1) Preparation of reaction solution
  • the volume of the reaction solution is 50 ⁇ L
  • T315I reverse primer and ABL reverse primer (Integrated DNA technologies) are 0.2 ⁇ M final concentration and dNTPs (Toyobo), respectively.
  • Final concentration 0.1 mM
  • MnOAc (Toyobo) 2.4 mM
  • PNA clamp (Panagene) 2 ⁇ M
  • rTth DNA polymerase (Toyobo) 2.5 U per reaction
  • test RNA per reaction It prepared so that it might become 1 microgram.
  • One sample standard for each concentration was used in the reaction.
  • the PNA clamp had a base sequence of ATGAACTCAGTGATGA (SEQ ID NO: 5), and all nucleotides were peptide-bonded.
  • (2-2) Reaction conditions The reverse transcription reaction was performed at 60 ° C. for 60 minutes.
  • (3-2) Measurement of cDNA derived from ABL reverse primer (a) Preparation of reaction solution The volume of the reaction solution was 30 ⁇ L, and ABL forward primer and ABL reverse primer (Integrated DNA technologies) were each at a final concentration of 0.3 ⁇ M and ABL fluorescence. Labeled probe (Integrated DNA technologies) at a final concentration of 0.2 ⁇ M, dNTPs (Toyobo) at a final concentration of 0.4 mM, MnOAc (Toyobo) at a final concentration of 2.0 mM, Tth DNA polymerase at 1.125 U per reaction, The reverse transcription reaction product was prepared to be 5 ⁇ L per reaction. A single measurement of the reverse transcription reaction product at each concentration was performed.
  • T315I mutant RNAs used for the standard product of Test 1 were used for the RNA extracted from human leukemia-derived cell HL60 in order to obtain the detection lower limit of the ABL1 T315I mutation measurement system.
  • T315I reverse primer-derived cDNA and ABL reverse primer-derived cDNA were measured using a sample added at a concentration of 1, and the ratio obtained by dividing the former measured value by the latter measured value was determined.
  • Test sample a sample obtained by adding ABL1 T315I mutant RNA used as a standard product at various concentrations to RNA extracted from human leukemia cell line HL60 which is negative for BCR-ABL1 was used. Specifically, ABL1 T315I mutant RNA used as a standard product was added to Total RNA extracted from HL60 at a concentration of 25, 50, 100 copies / test, and the final RNA concentration was 100 ng / ⁇ L using TE buffer. Prepared. As a control product, total RNA extracted from HL60 and adjusted to 100 ng / ⁇ L using TE buffer was used.
  • Results A ratio obtained by dividing the measured value of cDNA derived from T315I reverse primer by Table 4, the measured value of cDNA derived from ABL reverse primer by Table 5, and the measured value of cDNA derived from T315I reverse primer divided by the measured value of cDNA derived from reverse primer (ABL1 Table 6 shows T315I mutation / ABL1 ratio).
  • the measured values of cDNA derived from T315I reverse primer in samples added with 100, 50, and 25 copies / test ABL1 T315I mutant RNA were 109.4, 47.4, and 27.3 on average, which were consistent with the theoretical values. .
  • no fluorescence amplification was detected in the measurement of cDNA derived from T315I reverse primer of HL60 (control) not containing ABL1 T315I mutant RNA.
  • the measured value of cDNA derived from ABL reverse primer was 2.08 ⁇ 10 5 on average because HL60 used for dilution expressed a certain amount of ABL1 mRNA, and almost the same value was shown in all measured samples. Thus, even when RNA extracted from HL60 cells expressing 10 5 or more copies of wild-type ABL was measured, cDNA derived from T315I reverse primer of HL60 not containing ABL1 T315I mutant RNA was not detected. This measurement system is considered to have very high specificity for ABL1 T315I mutant RNA.
  • the ABL1 T315I mutant / ABL1 ratio of the sample added with 100, 50 and 25 copies / test ABL1 T315I mutant RNA was 0.052%, 0.023%, and 0.013%, respectively, on average. From this, the mutation detection rate of this measurement system was very high sensitivity of about 0.01%.
  • Test 3 Reaction inhibitory effect by PNA clamp The purpose of this test was to confirm the effect of PNA clamp in reverse transcription reaction.
  • PCR PCR was performed in the same manner as in Test 2.
  • Results A ratio obtained by dividing the measured value of cDNA derived from T315I reverse primer by Table 7, the measured value of cDNA derived from ABL reverse primer by Table 8, and the measured value of cDNA derived from T315I reverse primer divided by the measured value of cDNA derived from reverse primer (ABL1 T315I mutation / ABL1 ratio) is shown in Table 9.
  • the measured value of T315I reverse primer-derived cDNA in the sample to which 100, 50 and 25 copies / test of ABL1 T315I mutant RNA was added was 333.7, 235.3, and 202.3 copies / test on average.
  • the measured value of T315I reverse primer-derived cDNA of RNA extracted from HL60 (control) was 186.9 copies / test.
  • the measured value of cDNA derived from ABL reverse primer was 2.29 ⁇ 10 5 on average because HL60 used for dilution expressed a certain amount of ABL1 mRNA, and almost the same value was shown in all measured samples.
  • the ABL1 T315I mutation / ABL1 ratio of the sample to which 100, 50, and 25 copies / test ABL1 T315I mutant RNA was added was 0.147%, 0.107%, and 0.090%, respectively, on average.
  • the ABL1 T315I mutation / ABL1 ratio of HL60 (control) containing no ABL1 T315I mutant RNA was 0.077% on average.
  • the T315I reverse primer-derived cDNA of the sample not added with ABL1 T315I mutant RNA was measured at 186.9 copies / test, and the difference from the sample added with ABL1 T315I mutant RNA I could not get enough. Therefore, the use of a PNA clamp in the reverse transcription reaction is considered important for detecting the ABL13T315I mutation with high sensitivity.
  • the present application can provide a highly sensitive ABL1AT315I mutation detection or quantification method compared to conventional methods. That is, by using the above method or kit, the expression level of the ABL1BLT315I mutation can be quantified with high sensitivity.
  • the expression level of the ABL1 T315I mutation thus quantified is expected to be a useful index in the diagnosis of onset and recurrence of leukemia, determination of prognosis, and timing of bone marrow transplantation.

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Abstract

La présente invention concerne un procédé qui comprend (1) une étape de transcription inverse d'un échantillon d'ARN cible en présence d'un acide nucléique modifié ayant une séquence de base complémentaire d'une région qui contient la position de mutation T315I de l'ARNm ABL1 de type sauvage, en utilisant, dans le même récipient, (a) une amorce inverse qui se lie à une région en aval de la position de mutation T315I de l'ARNm ABL1 et (b) une amorce inverse qui se lie à une région en amont de la position de mutation T315I de l'ARNm ABL1, et (2) une étape de détermination du niveau d'expression de la mutation T315I d'ABL1 sur la base du rapport du produit de transcription inverse dû à l'amorce (a) par rapport au produit de transcription inverse dû à l'amorce (b). L'invention concerne également un kit pour ce procédé.
PCT/JP2018/016748 2017-04-26 2018-04-25 Procédé de mesure du niveau d'expression d'une mutation t315i d'abl1 WO2018199136A1 (fr)

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CN201880042856.1A CN110997938A (zh) 2017-04-26 2018-04-25 Abl1 t315i突变的表达水平的测定方法
KR1020197034072A KR20200002933A (ko) 2017-04-26 2018-04-25 Abl1 t315i 변이의 발현 레벨의 측정 방법
JP2019514562A JPWO2018199136A1 (ja) 2017-04-26 2018-04-25 Ａｂｌ１ ｔ３１５ｉ変異の発現レベルの測定方法
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