WO2024014439A1 - Procédé de quantification d'adn cible dans un corps vivant - Google Patents

Procédé de quantification d'adn cible dans un corps vivant Download PDF

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WO2024014439A1
WO2024014439A1 PCT/JP2023/025502 JP2023025502W WO2024014439A1 WO 2024014439 A1 WO2024014439 A1 WO 2024014439A1 JP 2023025502 W JP2023025502 W JP 2023025502W WO 2024014439 A1 WO2024014439 A1 WO 2024014439A1
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dna
liquid sample
biological material
quantitative method
target dna
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PCT/JP2023/025502
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Japanese (ja)
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明宏 松本
陽介 山中
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アステラス製薬株式会社
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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
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to the quantification of target DNA in vivo.
  • rAd recombinant adenovirus
  • rAAV recombinant adeno-associated virus
  • pDNA plasmid DNA
  • target DNA DNA
  • genomic DNA genomic DNA
  • a method has been used in which the target DNA concentration is measured by PCR and expressed as the target DNA concentration per genomic DNA, ie, copy number/ ⁇ g genomic DNA (gDNA).
  • the amount of gDNA must be measured, which requires complicated operations. Furthermore, considering that the amount of gDNA itself changes, it is not necessarily appropriate to always use it for evaluation of target DNA. For example, the number of blood cells in the blood may change significantly due to the administration of chemotherapy drugs, steroids, etc. In such cases, using the copy number of target DNA relative to the amount of gDNA may lead to misinterpretation of the evaluation of the amount of target DNA. There is a big risk of doing so.
  • Non-patent document 1 a quantitative PCR method that expresses the amount of target DNA in blood as copy number/ ⁇ L by using a spike-in calibration curve with internal or external controls.
  • this method allows the target DNA amount to be expressed in copy number/ ⁇ L, it requires DNA extraction and an additional step of creating a spike-in standard curve using a control.
  • Non-Patent Document 2 Although there is an example of directly quantifying DNA of bacterial cells using droplet digital PCR (ddPCR) (Non-Patent Document 2), there is no known example of directly quantifying a gene of interest in animal tissue or blood.
  • ddPCR droplet digital PCR
  • An object of the present invention is to provide a new quantitative method that can easily and accurately evaluate the internal dynamics of target DNA.
  • the concentration of target DNA in a biological material can be expressed in units of copy number/ ⁇ L or copy number/ ⁇ g based on the volume or weight of the biomaterial, and that DNA extraction is not necessarily required.
  • the present invention provides the following [1] to [25].
  • [1] Preparing a liquid sample from the biological material to be measured; Quantifying the copy number of the target DNA contained in the liquid sample by digital PCR (dPCR), and A method for quantifying DNA of interest, comprising the step of calculating the number of copies of DNA of interest per unit amount of the biological material using the quantified copy number of DNA of interest in the liquid sample.
  • [2] The quantitative method according to [1] above, which does not include the step of extracting DNA from the biological material.
  • [3] The quantitative method according to [1] or [2] above, wherein the biomaterial is a liquid.
  • the step of quantifying the copy number of the target DNA by dPCR the step of setting a threshold for a negative signal based on a blank sample in which the dilution ratio of the biological material in the liquid sample is equal and does not contain the target DNA, and/ or any one of the above [1] to [12], for example, the above [1] or [2], further comprising the step of setting a threshold for a positive signal based on a spiked sample obtained by adding the target DNA to the blank sample.
  • Quantification method described in. [14] The quantitative method according to any one of [1] to [13] above, for example, [1] or [2] above, further comprising a step of performing an inactivation treatment on the liquid sample.
  • the introduced DNA in the biomaterial is derived from a vector, a cell, a nucleic acid drug, an oncolytic virus, or a phage.
  • the vector is derived from plasmid DNA (pDNA), recombinant adenovirus, recombinant adeno-associated virus (rAAV), recombinant lentivirus, recombinant Sendai virus, or recombinant retrovirus. Quantification method described in.
  • the target DNA is a recombinant adenovirus, recombinant adeno-associated virus (rAAV), recombinant lentivirus, recombinant Sendai virus, recombinant retrovirus, plasmid DNA (pDNA), CAR-T in the biological material.
  • FIG. 1 shows the results of quantifying EGFP cDNA in mouse blood by qPCR.
  • the vertical axis represents the Ct value, and the horizontal axis represents the logarithm of the copy number of EGFP cDNA per unit volume added.
  • FIG. 2 shows the results of quantifying EGFP cDNA in mouse blood by ddPCR method.
  • the vertical axis represents the signal intensity, and the horizontal axis represents the number of events.
  • FIG. 3 shows the results of examining the dilution ratio of mouse blood suitable for the ddPCR method.
  • the vertical axis represents the signal intensity, and the horizontal axis represents the number of events.
  • FIG. 4 shows the results of examining the dilution ratio of mouse blood suitable for the ddPCR method.
  • FIG. 5 shows the results of quantifying rAAV genomic DNA containing the target DNA in rAAV-administered mouse tissue samples (blood, liver, or brain) using the ddPCR method.
  • the vertical axis represents the number of copies of the target DNA per unit volume or unit weight, and the horizontal axis represents the number of days after rAAV administration.
  • FIG. 6 shows the results of quantifying rAAV genomic DNA containing the target DNA in rAAV-administered mouse tissue samples by qPCR.
  • the vertical axis represents the number of copies of target DNA per genomic DNA
  • the horizontal axis represents the number of days after rAAV administration.
  • the method for quantifying target DNA includes a preparation step of preparing a liquid sample from a biological material, and a quantitative step of quantifying the number of copies of the target DNA, and further includes determining the number of copies of the target DNA per unit volume or unit weight in the biological material.
  • the method may include a calculating step of calculating. Each step will be explained below.
  • liquid sample preparation step a liquid sample is prepared from the biological material to be measured.
  • the liquid sample is a target of a PCR reaction, and in this specification, the liquid sample is sometimes referred to as a PCR target sample.
  • a liquid sample can be prepared by directly diluting the biological material and further adding a PCR reaction reagent.
  • liquid samples can be prepared by first performing appropriate solubilization treatment, for example with protease, followed by dilution and addition of PCR reaction reagents.
  • a surfactant such as sodium dodecyl sulfate may be added to the solid biological material and the mixture may be stirred.
  • Biological materials are obtained from, for example, humans or experimental animals used for drug development (e.g., mice, rats, monkeys, dogs, rabbits, etc.), and include blood, cerebrospinal fluid, urine, saliva, aqueous humor, and tears. This includes liquids such as fluid, organs such as the liver, kidneys, spleen, gallbladder, heart, lungs, brain, testicles, and ovaries, and solid tissue pieces such as muscles, skin, and eyes.
  • the method for quantifying target DNA of the present invention can be applied to both liquid and solid biological materials, and it is possible to quantify target DNA in a wide range of measurement targets without any special restrictions.
  • the objective DNA quantification method of the present invention does not require the step of extracting DNA from biological materials, and allows efficient quantification with simple operations.
  • a liquid sample from a liquid biological material such as blood it can be prepared by diluting it directly with an appropriate dilution solvent such as a buffer, and then adding a PCR reaction reagent.
  • the reagent for PCR reaction may be any reagent that is compatible with the PCR used for measurement, and includes, for example, a commercially available PCR master mix, primer, and probe reagent.
  • the dilution ratio (volume ratio: e.g., volume of diluent (mL)/volume of biomaterial (mL)) of the liquid biomaterial in the liquid sample thus prepared, that is, the sample to be subjected to PCR, depends on the type of liquid sample.
  • the dilution ratio based on the final concentration of the liquid biomaterial is, for example, 20 to 1,000,000 times, 20 to 100,000 times, 25 to 10,000 times, 25 to 5,000 times, 30 to 2 ,000 times or 30 to 1,000 times, preferably 50 to 800 times, more preferably 70 to 600 times, even more preferably 100 to 600 times, particularly preferably 200 to 500 times.
  • the dilution ratio (volume ratio) based on the final concentration of the liquid biomaterial may be 200 times, 300 times, 400 times, 500 times, etc.
  • the dilution factor may be adjusted depending on whether the biomaterial is liquid or solid. It may be 100 to 1,000,000 times, 300 to 700,000 times, 500 to 500,000 times, 1,000 to 200,000 times, etc.
  • the dilution factor based on the final concentration of blood is, for example, 170 to 10,000,000 times, and below, a preferable range is defined as 200 to 1,000,000 times, etc. Yes, preferably 500 to 5,000,000 times, 1,000 to 1,000,000 times, and 5,000 to 500,000 times.
  • the diluting solvent for diluting the liquid biological material is not particularly limited, but includes, for example, Buffer AE manufactured by QIAGEN (consisting of 10 mM Tris-Cl and 0.5 mM EDTA, pH 9.0), ultrapure water. (MILLIQ water), phosphate buffered saline (PBS), physiological saline, etc., and preferably buffer AE is used.
  • Buffer AE manufactured by QIAGEN (consisting of 10 mM Tris-Cl and 0.5 mM EDTA, pH 9.0), ultrapure water. (MILLIQ water), phosphate buffered saline (PBS), physiological saline, etc., and preferably buffer AE is used.
  • the amount of liquid biomaterial used to prepare the liquid sample depends on the dilution factor and the number of measurements, but for example, 0.02 ⁇ L or more, 0.04 ⁇ L or more, 0.1 ⁇ L or more, 0.5 ⁇ L or more, 1
  • Solid Biomaterials When preparing a liquid sample from a solid biomaterial, such as a piece of tissue from an organ, the solid biomaterial is solubilized.
  • the solubilization method is not particularly limited as long as solubilization is possible, and protease may or may not be added to the solid biological material.
  • the comminuted biomaterial is suspended in a solvent such as a buffer containing the protease, ie, a protease treatment solvent such as a protease treatment buffer. If necessary, the biomaterial may be cut or pulverized in advance and then centrifuged.
  • An appropriate protease can be selected depending on the biomaterial used. For example, proteinase K may be mentioned.
  • the solvent for protease treatment may be selected according to the biological material, but examples include Buffer ATL manufactured by QIAGEN, ultrapure water (MILLIQ water), phosphate buffered saline (PBS), and physiological saline. etc., and preferably buffer ATL is used.
  • the above-mentioned solubilization treatment is performed, for example, by incubating a suspension of the biological material in a protease treatment solvent and performing the protease treatment.
  • the incubation temperature is selected depending on the type of protease and biomaterial used, and is, for example, 30 to 70°C, preferably 40 to 65°C, more preferably 50 to 60°C.
  • the incubation time is selected depending on the type of protease and biomaterial used, and is, for example, 10 to 24 hours, preferably 11 to 20 hours, more preferably 12 to 18 hours, most preferably 13 to 17 hours, and particularly preferably will be held for 16 hours.
  • the amount of the protease treatment solvent used in the solubilization treatment is appropriately adjusted depending on the amount of solid biomaterial, etc., but for example, it is 1 to 120 ⁇ L, preferably 2 to 50 ⁇ L, and more preferably 2 to 50 ⁇ L per 1 mg of biomaterial. About 5 to 30 ⁇ L is used.
  • the concentration of protease in the protease treatment solvent is, for example, 60 mAU/mL or more.
  • the suspension may be resuspended if necessary.
  • an inactivation treatment may be performed by heating the suspension containing the above-mentioned liquid sample by boiling treatment.
  • the boiling treatment includes boiling the suspension at, for example, 80 to 100°C, preferably 90 to 98°C, more preferably 95 to 96°C, for 2 to 30 minutes, preferably 5 to 20 minutes, or 5 to 25 minutes.
  • the inactivation treatment may be carried out by heating, more preferably for 8 to 12 minutes or 10 to 22 minutes, most preferably for 20 minutes, and in some cases for 10 minutes.
  • Such inactivation treatment does not affect the quantification results of the target DNA in the quantification step described below, but it also ensures safe subsequent handling even when using biological materials that may be biohazardous samples. make it possible.
  • the steps for the solid biomaterial described above can be employed, but preferably, addition of protease to the liquid biomaterial may be avoided.
  • the liquid biomaterial is not subjected to protease treatment, it may be inactivated by heating it by boiling.
  • the solution obtained by the protease treatment and/or inactivation treatment is sufficiently suspended, stirred or shaken as necessary, diluted as necessary with the above dilution solvent such as buffer AE, and then subjected to PCR.
  • a liquid sample is obtained by adding the reagents required for the reaction.
  • the reagent for PCR reaction may be any reagent that is compatible with the PCR used for measurement, and includes, for example, a commercially available PCR master mix, primer, and probe reagent.
  • the dilution factor of the solid biomaterial in the liquid sample thus prepared, that is, the PCR target sample is a value based on the final concentration (weight of biomaterial ( ⁇ g)/volume of diluent ( ⁇ L)), for example, 3.
  • the dilution factor based on the final concentration of the solid biomaterial is, for example, 15 to 250,000 times, 20 to 200,000 times, 25 to 10,000 times, 25 to 5,000 times, 3 to 1,000 times.
  • the amount is preferably 5 to 700 times, more preferably 7 to 500 times, particularly preferably 10 to 300 times.
  • the dilution ratio of solid biomaterials is 12 times or more, 15 times or more, 20 times or more, 50 times or more, based on the final concentration (weight of biomaterial ( ⁇ g)/volume of diluent ( ⁇ L)). , 100 times or more, 150 times or more, etc.
  • the dilution factor may be adjusted depending on whether the biomaterial is solid or liquid, and the dilution factor based on the final concentration for solid biomaterials is, for example, 10 to 20,000,000 times, 10 to 10,000,000 times, 10 to 5,000,000 times, 10 to 2,000,000 times, 15 to 6,000,000 times, 15 to 3,000,000 It may be 15 times to 1,000,000 times, 20 to 10,000,000 times, 20 to 5,000,000 times, etc.
  • the dilution factor based on the final concentration of the solid biomaterial is preferably 20 to 200,000 times, 50 to 1,000,000 times, 50 to 100,000 times, 100 to 500,000 times, More preferably, it is 100 to 50,000 times, 200 to 50,000 times, 200 to 5,000 times, etc.
  • the dilution factor based on the final concentration of liver is, for example, 100 to 10,000,000 times, 100 to 1,000,000 times, 150 to 5,000,000 times, 150 to 500,000 times, etc., 200 to 1,000,000 times, 200 to 200,000 times, 500 to 100,000 times, 1,000 to 50,000 times, 2,200 to 4,600,000 times
  • the dilution factor based on the final concentration of quadriceps muscle is, for example, 5 to 10,000,000 times, 5 to 5,000,000 times, 5 to 1, 000,000 times, 10 to 1,000,000 times, 10 to 500,000 times, etc., 20 to 2,000,000 times, 20 to 200,000 times, 50 to 150,000 times, 100 to 100 ,000 times, 500 to 50,000 times, and preferably 460 to 4,600,000 times.
  • the dilution ratio when preparing a homogenate containing solid biomaterial and performing solubilization treatment is the value of the dilution ratio based on the amount of homogenate (weight of homogenate (mg) / volume of diluent ( ⁇ L)) ), and in that case, the numerical range of the dilution ratio based on the amount of homogenate is, for example, 1/50 to 1/5 of the value based on the final concentration described above.
  • the dilution rate based on the amount of homogenate is preferably within a range defined by a limit value of, for example, 1/50 times to 1/5 times the limit value (upper limit value and lower limit value) for the above-mentioned final concentration-based dilution rate. is a range defined by a limit value of 1/40 times to 1/7 times the limit value of the dilution ratio based on final concentration, and more preferably a limit value of 1/30 times to 1/10 times. This is the range in which
  • the dilution ratio of the solid biomaterial is preferably selected depending on the tissue of the living body, the type of diluent, etc., and a range different from the above-mentioned dilution ratio may be preferable.
  • the dilution ratio of a solid biomaterial is 10 to 500, based on the final concentration (weight of biomaterial ( ⁇ g)/volume of diluent ( ⁇ L)).
  • the final concentration is, for example, 3 times to 400 times, preferably 5 times. 300 times, more preferably 10 times to 250 times, most preferably 20 times to 200 times.
  • the dilution ratio is 10 times to 500 times, preferably 50 times to 400 times, more preferably 100 times to 300 times, based on the final concentration (weight of biomaterial ( ⁇ g)/volume of diluent ( ⁇ L)). , the most preferred specific example is 200 times.
  • the dilution ratio Examples are 10 times to 500 times, preferably 50 times to 400 times, more preferably 100 times to 300 times, based on the final concentration (weight of biomaterial ( ⁇ g)/volume of diluent ( ⁇ L)). The most preferred specific example is 200 times.
  • tissue pieces such as heart, lung, kidney, muscle, spleen, etc.
  • protease using the commercially available "Tissue Direct PCR Kit” and diluting the obtained solution by inactivation treatment if necessary.
  • the dilution ratio are 3 times to 200 times, preferably 5 times to 100 times, more preferably 5 times to 100 times, based on the final concentration (weight of biomaterial ( ⁇ g)/volume of diluent ( ⁇ L)).
  • the amount is 10 times to 50 times, and the most preferred example is 20 times.
  • the amount of solid biomaterial used in the method of the present invention depends on the type of biomaterial, dilution factor, number of measurements, etc., but is, for example, 1 to 100 mg, preferably 3 to 50 mg, and more preferably The amount is 5 to 20 mg, 8 to 20 mg, 5 to 10 mg, or 5 to 15 mg, and more preferably 8 to 10 mg or 8 to 12 mg.
  • PCR kits such as the Tissue Direct PCR Kit manufactured by Omega BIO-TEK, can also be used.
  • the step of preparing a liquid sample does not require the step of extracting DNA from a biological material, and the method for quantifying target DNA of the present invention allows simple and rapid quantification. Furthermore, there is no need for a spike-in calibration curve for internal or external controls during quantitation, and there is no need to add DNA that is not derived from the biological material to the liquid sample prepared in the preparation step.
  • the copy number of the target DNA contained in the liquid sample can be quantified by dPCR.
  • dPCR absolute quantification of target DNA is possible by performing PCR on a liquid sample distributed in minute wells and measuring the reaction rate in negative wells.
  • ddPCR droplet digital PCR
  • chip-based digital PCR etc.
  • ddPCR is preferably used.
  • a known ddPCR device and system can be used, such as those manufactured by Bio-Rad or BioTNS.
  • ddPCR it is also possible to add oil to the liquid sample obtained in the liquid sample preparation step to prepare a measurement sample, if necessary, and quantify the target DNA in the measurement sample.
  • the threshold for a negative signal is determined based on a blank sample that has the same concentration (dilution ratio) of biological materials such as blood in the liquid sample obtained in the liquid sample preparation step and does not contain DNA. It is preferable to set a threshold value for a positive signal based on a spiked sample obtained by adding target DNA to the blank sample. It is more preferable to set both a negative signal threshold and a positive signal threshold. Furthermore, in dPCR, it is preferable to appropriately adjust the concentrations of primers and probe reagents.
  • the liquid sample is treated with a drug (e.g., glutaraldehyde, sodium hypochlorite, formalin, methanol, or acetonitrile, etc.), autoclaved, or boiled before measuring the signal after the PCR reaction is completed.
  • a drug e.g., glutaraldehyde, sodium hypochlorite, formalin, methanol, or acetonitrile, etc.
  • Inactivation treatment may be further performed by treatment or the like.
  • the liquid sample after measuring the signal, may be subjected to the inactivation treatment described above.
  • the boiling treatment may be performed, for example, by heating under the conditions described in column 2-2 above.
  • the inactivation treatment is preferably performed by boiling the liquid sample before measuring the signal after the PCR reaction is completed.
  • the copy number of the target DNA in the liquid sample can be determined by a simple method.
  • the copy number of the target DNA in the liquid sample obtained in this way is useful data for calculating the number of DNA copies per unit amount of biological material, as will be described in detail later.
  • the number of copies of the target DNA per unit amount of the biomaterial ( copy number/ ⁇ L or copy number/ ⁇ g) can be calculated. That is, the number of copies of the DNA of interest per unit volume or weight of biomaterial is determined by dividing the number of copies of the DNA of interest in the quantified liquid sample by the amount of biomaterial used to prepare the liquid sample. can be calculated.
  • the target DNA to be quantified includes introduced DNA and endogenous DNA present in the biological material.
  • the introduced DNA is not particularly limited as long as it is foreign DNA, and includes, for example, DNA derived from regenerative/cell therapy, in vivo gene therapy, ex vivo gene therapy using cells into which genes have been introduced, and nucleic acid medicine. is included.
  • the introduced DNA includes those derived from vectors such as plasmid vectors or viral vectors, those derived from cells used for cell therapy or ex vivo gene therapy, nucleic acid medicines, oncolytic viruses, and Includes those derived from phages (bacteriophages).
  • the introduced DNA includes vector-derived DNA, such as plasmid DNA (pDNA), recombinant adenovirus (rAd), recombinant adeno-associated virus (rAAV), recombinant lentivirus, recombinant Includes those derived from recombinant Sendai virus, recombinant retrovirus, etc.
  • vector-derived DNA such as plasmid DNA (pDNA), recombinant adenovirus (rAd), recombinant adeno-associated virus (rAAV), recombinant lentivirus, recombinant Includes those derived from recombinant Sendai virus, recombinant retrovirus, etc.
  • rAAV examples include rAAV serotype 1 (rAAV1), rAAV serotype 2 (rAAV2), rAAV serotype 3 (rAAV3), rAAV serotype 4 (rAAV4), rAAV serotype 5 (rAAV5), rAAV serotype 6 (rAAV6), and rAAV serotype 7.
  • the introduced DNA includes those derived from cells, such as those derived from cell therapy products derived from CAR-T cells, CAR-NK cells, ES cells, or iPS cells.
  • the introduced DNA includes those derived from nucleic acid medicines, such as siRNA, miRNA, mRNA, antisense, aptamer, decoy, ribozyme, CpG oligo, and the like.
  • nucleic acid medicines such as siRNA, miRNA, mRNA, antisense, aptamer, decoy, ribozyme, CpG oligo, and the like.
  • Specific examples of endogenous DNA include DNA derived from blood cells contained in human or animal blood, DNA derived from specific tissues, and the like.
  • Biodistribution Test The biodistribution of a drug can be evaluated using the method for quantifying target DNA of the present invention. Using the biodistribution evaluation results further enables pharmacokinetic analysis and systems pharmacology analysis.
  • Targeted drugs include drugs containing the introduced DNA described in column 5 above, such as vectors such as plasmid vectors or viral vectors, cells used for cell therapy or ex vivo gene therapy, nucleic acid drugs, oncolytic viruses, and phage.
  • the master mix includes TaqMan Gene Expression Assays (Thermo Fisher Scientific, 4331182, Assay ID Mr04097229_Mr), TaqPath qPCR Mast er Mix, CG (Thermo Fisher Scientific, A16245) was used. 4 ⁇ L of master mix was added to a 384-well plate, 1 ⁇ L of each sample was added, and the plate was sealed to serve as a measurement plate. Measurements and analyzes were performed with QuantStudio 12K Flex (Applied Biosystems). Amplification was performed under the following conditions. 1 cycle of 50°C, 2 minutes 1 cycle of 95°C, 10 minutes 40 cycles of 95°C, 15 seconds and 60°C, 1 minute Hold at 4°C *Temperature increase/decrease rate is 1.6°C/sec
  • Target DNA containing the EGFP gene sequence (hereinafter simply referred to as target DNA) was quantified.
  • concentration of the target DNA relative to the blood volume used for measurement was expressed as copy number/ ⁇ L.
  • a more specific method for quantifying EGFP cDNA by ddPCR will be described based on Examples.
  • Example 1 Quantification of EGFP cDNA in mouse blood by ddPCR method using diluted blood EGFP cDNA was added to diluted mouse blood (dilution solvent: Buffer AE; dilution ratio: 50 to 500,000 times) or added to Buffer AE.
  • the copy number of EGFP cDNA in the obtained solution was measured by a ddPCR method, which is a type of dPCR method. The specific steps are as follows. A master mix of 9 times the amount of the measurement sample was added to prepare a ddPCR reaction solution (the measurement sample in the ddPCR reaction solution was diluted 10 times).
  • the master mix included TaqMan Gene Expression Assays (Thermo Fisher Scientific, 4331182, Assay ID Mr04097229_Mr), ddPCR Supermix for Probe (no dU TP), X2 (Bio-Rad, 1863024), RT-PCR Grade Water (Thermo Fisher Scientific, AM9935 )It was used.
  • the QX200 system Bio-Rad was used for sample measurement by ddPCR method. Droplets were created using a Droplet Generator using 20 ⁇ L of the ddPCR reaction solution according to the instruction manual, and transferred to a 96-well plate (Bio-Rad, 12001925).
  • Tables 1 and 2 show the results of (A) one cycle of 98°C for 10 minutes, and Table 3 shows the results of (B) one cycle of 98°C for 20 minutes.
  • Example 2 Quantification of EGFP cDNA in human blood by ddPCR using diluted blood A ddPCR method using human blood as a matrix was investigated. Human blood was purchased from KAC Corporation. EGFP cDNA (the amount added was under the three conditions of addition conditions 1 to 3 in the table below) was added to human blood diluted 50 times or to buffer AE. The EGFP cDNA copy number in the solution was measured by the ddPCR method in the same manner as in Example 1 ((B) 98° C., 20 minutes for one cycle).
  • Example 3 Examination of inactivation treatment (boiling) conditions Mouse blood (dilution solvent: buffer AE; dilution ratio: 50 times) in which EGFP cDNA (addition amount is the two conditions of addition conditions 1 and 2 in Table 5 below) was diluted. added to. The obtained solution was boiled (95°C, 20 minutes) or left at 4°C for 20 minutes before the amplification reaction, and then the EGFP cDNA copy number in the solution was determined using the same master mix as in Example 1. Amplification was performed under the following conditions (condition b and condition a in Table 5, respectively). The sample after amplification was detected with Droplet Reader and analyzed with QuantaSoft.
  • Example 4 Homogenate preparation of mouse organs Organs were collected from mice and crushed as follows.
  • the liver and quadriceps muscle were each placed in a 3 mL cryo-fracture tube for Multi-Bead Shocker (Yasui Kikai Co., Ltd., ST-0320PCF), and a metal cone for Multi-Shocker (Yasui Kikai Co., Ltd., MC-0316s) was placed over it. I put one in.
  • the tube was set in a multi-bead shocker (Yasui Kikai Co., Ltd., MB701PU(s)) and crushed at 2500 rpm for 20 seconds.
  • the brain, lung, spleen, and kidney were each placed in a tube, and two 5 mm Stainless steel beads (Qiagen) were placed on top of the tube.
  • the tube was set in Shake Master BMS-A20TP (Biomedical Science) and crushed by repeating 1 minute at 1100 rpm three times. The homogenate was stored at -80°C until immediately before use.
  • Example 5 Solubilization of mouse organ homogenate using Tissue Direct PCR Kit Solubilization of mouse homogenate was investigated using Tissue Direct PCR Kit (Omega BIO-TEK, TQ2310). 8 to 53 mg (see Table 6) of the liver and quadriceps muscle homogenate was weighed into a tube, and 100 ⁇ L of L1 buffer and 20 ⁇ L of L2 buffer included in the kit were added. The mixture was set in an Eppendorf Thermomixer R T3317 (Eppendorf) and incubated at 56° C. for 12 hours or more while stirring at approximately 500 rpm. It was then incubated at 95° C.
  • sample was returned to room temperature, 100 ⁇ L of the Tissue direct PCR Neutralization buffer attached to the kit was added, and the sample was sufficiently stirred with a vortex to prepare a test sample. Samples were stored frozen (below -20°C) until immediately before use.
  • Example 7 Examination of solubilization conditions for mouse organ homogenate The samples obtained in Example 5 and Example 6 were visually confirmed for solubilization and evaluated by ddPCR method.
  • the ddPCR method was performed as follows. Nine times the amount of master mix was added to the suitably diluted sample to prepare a dPCR reaction solution.
  • the master mix included TaqMan Gene Expression Assays (Thermo Fisher Scientific, 4331182, Assay ID Mr04097229_Mr), ddPCR Supermix for Probe (no dU TP), X2 (Bio-Rad, 1863024), Direct PCR buffer (10X) (Tissue Direct PCR, Omega BIO-TEK, TQ2310) and RT-PCR Grade Water (Thermo Fisher Scientific, AM9935) were used.
  • the PCR reaction by the ddPCR method and the subsequent steps were carried out in the same manner as in Example 1 ((B) conditions of 98° C. and 20 minutes for one cycle).
  • Example 8 Examination of dilution ratio of solubilized homogenate of each mouse organ. Homogenate of brain, lung, liver, kidney, quadriceps muscle, and spleen solubilized by the method of Example 5 or 6 was diluted with buffer AE at 1 to 10%. ,000 times diluted. EGFP cDNA was diluted in a dilution series using Buffer AE as a solvent at a final concentration of 5 x 10 1 to 5 x 10 4 copies/ ⁇ L. The diluted solution of the solubilized homogenate of Example 5 and each concentration of EGFP cDNA were mixed at a ratio of 1:1.
  • Example 7 the diluted solution of the solubilized homogenate of Example 6 and EGFP cDNA at a final concentration of 5 ⁇ 10 2 copies/ ⁇ L were mixed at a ratio of 1:1.
  • the EGFP cDNA copy number in the sample was evaluated by ddPCR method. Measurement of the sample by ddPCR method was carried out in the same manner as in Example 7.
  • Table 7 shows the dilution ratio at which the sample could be measured by the ddPCR method.
  • the dilution ratio (20 to 200,000 times) based on the amount of homogenate suitable for the ddPCR measurement method; 460 to 4,600,000 times)).
  • the brain, lung, and liver solubilized by the method of Example 6 only samples at a dilution rate of 200 times based on the homogenate amount (the dilution rate in terms of final concentration is 2,200 times) were measured.
  • Example 9 Biodistribution test of recombinant AAV (rAAV)
  • target DNA concentration of target DNA containing the EGFP gene sequence (hereinafter simply referred to as target DNA) in rAAV-administered mouse tissues was determined by the previously described ddPCR method.
  • target DNA concentration of target DNA containing the EGFP gene sequence
  • the applicability of the above-mentioned ddPCR method was investigated by evaluating the method by extracting DNA and quantifying it by qPCR. Details are below.
  • rAAV8 and rAAV9 to be administered contain the EGFP expression gene, as well as the CMV enhancer, CMV promoter, WPRE sequence, and bGH poly(A) signal. Contains encoding nucleic acid.
  • Each rAAV was diluted to 2.0 ⁇ 10 12 vg/mL with DPBS (Life Technologies). A single dose of 5 mL of rAAV diluted solution per kg of body weight was administered into the tail vein of mice, and blood, liver, and brain were collected and frozen at 4, 24, and 48 hours and 1, 2, and 4 weeks after administration. saved. The liver and brain were crushed using the method described in Example 4.
  • a DNeasy 96 plate was set on a new rack of Elution Microtube RS, 50 ⁇ L of Buffer AE was added, the plate was sealed with AirPore Tape Sheet, and the plate was left at room temperature for 1 minute, then centrifuged to collect DNA. DNA was extracted in the same manner from mouse livers to which rAAV had not been administered, and a liver genomic DNA solution (referred to as L-gDNA) was obtained.
  • L-gDNA liver genomic DNA solution
  • DNeasy Blood & Tissue Kit (Qiagen, 69504) was used to extract DNA from blood. 100 ⁇ L of blood was weighed out, and 100 ⁇ L of PBS and 20 ⁇ L of proteinase K were added. 4 ⁇ L of RNase (Qiagen, 19101) was added, mixed well by vortexing, and incubated at room temperature for 5 minutes. After adding 200 ⁇ L of Buffer AL and thoroughly stirring with a vortex, it was set in Eppendorf Thermomixer R T3317 (Eppendorf) and incubated at 56° C. for 10 minutes while stirring at 500 rpm.
  • the mixture was transferred to a DNeasy Mini Spin Column and centrifuged at 20,000 ⁇ g and 4° C. for 1 minute.
  • the filter was transferred to a new collection tube, 500 ⁇ L of Buffer AW1 was added, and centrifuged at 20,000 ⁇ g and 4° C. for 2 minutes.
  • 500 ⁇ L of Buffer AW2 was added, and the mixture was further centrifuged at 20,000 ⁇ g and 4° C. for 3 minutes.
  • a filter was set in a new tube, 50 ⁇ L of Buffer AE was added, and the tube was left to stand at room temperature for 1 minute, then centrifuged at 6000 ⁇ g and 4° C. for 2 minutes to collect DNA.
  • Each DNA sample was diluted with buffer AE to a concentration of 100 ng/ ⁇ L or less. 4 ⁇ L of the master mix was added to a 384-well plate, 1 ⁇ L of each sample was added, and the plate was sealed to serve as a measurement plate. Measurements and analyzes were performed with QuantStudio 12K Flex (Applied Biosystems). Amplification was performed under the following conditions. 1 cycle of 50°C, 2 minutes 1 cycle of 95°C, 10 minutes 40 cycles of 95°C, 15 seconds and 60°C, 1 minute Hold at 4°C *Temperature increase/decrease rate is 1.6°C/sec
  • the present invention it is possible to quantify the target gene in the body based on the volume or weight. As a result, new considerations regarding the quantitative relationship between pharmacological and toxicological effects will become possible, which may greatly benefit patients undergoing gene therapy, cell therapy, etc.

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Abstract

La présente invention concerne un nouveau procédé quantitatif pouvant évaluer avec précision, entre autres, la dynamique d'un gène cible dans l'organisme sans être affecté par la quantité d'ADN génomique (ADNg).　La solution selon la présente invention consiste en l'application du procédé suivant. Ce procédé de quantification d'un ADN cible comprend les étapes suivantes : préparation d'un échantillon liquide à partir d'un matériau biologique d'une cible de mesure ; quantification du nombre de copies de l'ADN cible contenu dans l'échantillon liquide par PCR numérique (dPCR) ; et calcul du nombre de copies de l'ADN cible par unité de matériau biologique à l'aide du nombre de copies de l'ADN cible dans l'échantillon liquide quantifié ci-dessus.
PCT/JP2023/025502 2022-07-11 2023-07-10 Procédé de quantification d'adn cible dans un corps vivant WO2024014439A1 (fr)

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