WO2024014439A1 - Method for quantifying target dna in living body - Google Patents

Abstract

Provided is a novel quantitative method capable of accurately evaluating the dynamics, etc. of a target gene in the body without being affected by the amount of genomic DNA (gDNA).　This problem was solved by the following method. A method for quantifying a target DNA that comprises: a step for preparing a liquid sample from a biological material of a measurement target; a step for quantifying the copy number of the target DNA contained in the liquid sample by digital PCR (dPCR); and a step for calculating the copy number of the target DNA per unit amount of the biological material with the use of the copy number of the target DNA in the liquid sample quantified above.

Description

In vivo target DNA quantification method

The present invention relates to the quantification of target DNA in vivo.

There is a need for a method to easily and accurately measure the distribution and behavior of target DNA in the living body of humans and animals.

For example, in vivo gene therapy using vectors such as recombinant adenovirus (rAd), recombinant adeno-associated virus (rAAV) or plasmid DNA (pDNA), or introducing specific genes into cells isolated from patients, There are various gene therapy methods such as ex vivo gene therapy such as CAR-T therapy, which returns these cells to the body, but the clinical efficacy and adverse effects of these existing and new gene therapy methods are unclear. When considering the relationship, it is essential to measure the pharmacokinetics of DNA introduced into the body for therapeutic purposes. Furthermore, regarding endogenous DNA, it may be necessary to measure the increase or decrease in DNA derived from blood cells before and after a specific treatment, or to compare the copy number of specific endogenous DNA in tissues.

Until now, when quantifying target DNA in a human or animal body, blood or tissue of the human or animal was collected, total DNA (genomic DNA) was extracted and measured, and then the amount of target DNA was determined. 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/μg genomic DNA (gDNA).

However, in order to calculate the target DNA concentration based on the amount of 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.

To avoid such risks, Yamamoto et al. have announced a quantitative PCR method that expresses the amount of target DNA in blood as copy number/μL by using a spike-in calibration curve with internal or external controls. (Non-patent document 1). Although this method allows the target DNA amount to be expressed in copy number/μL, it requires DNA extraction and an additional step of creating a spike-in standard curve using a control.

Furthermore, 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.

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.

In order to achieve the above object, the inventors discovered that the concentration of target DNA in a biological material can be expressed in units of copy number/μL or copy number/μg based on the volume or weight of the biomaterial, and that DNA extraction is not necessarily required. We have developed a method that allows the quantitative determination of target DNA in biological materials without the need for quantitation.

That is, 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.
[4] The quantitative method according to [3] above, wherein the liquid sample has a dilution factor of 20 to 5,000,000 times based on the final concentration of the biological material in the liquid.
[5] The quantitative method according to [4] above, wherein the liquid biological material is blood, and the dilution factor based on the final concentration of blood is 500 to 5,000,000 times.
[6] The quantitative method according to [3] above, wherein the liquid sample has a dilution factor of 30 to 2000 times based on the final concentration of the biological material in the liquid.
[7] The quantitative method according to [1] or [2] above, wherein the biomaterial is a solid.
[8] The quantitative method according to [7] above, wherein preparing the liquid sample includes solubilizing the solid biological material.
[9] The quantitative determination according to [7] or [8] above, for example, [7] above, wherein the dilution factor based on the final concentration of the solid biomaterial in the liquid sample is 20 to 5,000,000 times. Method.
[10] The quantitative method according to [7] or [8] above, for example, [7] above, wherein the dilution factor based on the final concentration of the solid biomaterial in the liquid sample is 3 to 1000 times.
[11] The solid biomaterial is liver, and the dilution factor based on the final concentration of the liver is 2,200 to 4,600,000 times, such as in [7] or [8] above, for example in [7] above. Quantification method as described.
[12] Any one of [7] to [9] above, wherein the solid biomaterial is quadriceps femoris muscle, and the dilution factor based on the final concentration of quadriceps femoris muscle is 460 to 4,600,000 times. Item 1, for example, the quantitative method described in [7] above.
[13] In 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.
[15] The quantitative method according to [14] above, wherein the step of inactivating the liquid sample is performed after the end of the PCR reaction and before measuring the signal.
[16] The quantitative method according to [14] or [15] above, for example, [15] above, wherein the inactivation treatment is a boiling treatment of the liquid sample.
[17] The quantitative method according to any one of [1] to [16] above, for example, [1] or [2] above, wherein the dPCR is droplet digital PCR (ddPCR).
[18] The quantitative method according to any one of [1] to [17] above, for example, [1] or [2] above, wherein the target DNA is introduced DNA or endogenous DNA in the biological material.
[19] The quantitative method according to [18] above, wherein the introduced DNA in the biomaterial is derived from a vector, a cell, a nucleic acid drug, an oncolytic virus, or a phage.
[20] The above-mentioned [19], wherein 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.
[21] The quantitative method according to [19] above, wherein the cells are derived from CAR-T cells, CAR-NK cells, or a cell therapy product derived from ES cells or iPS cells.
[22] The quantitative method according to any one of [18] to [21] above, for example, [18] above, wherein the endogenous DNA in the biomaterial is derived from blood cells.
[23] 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. Any one of the above [1] to [22], for example the above [ 1] or the quantitative method described in [2].
[24] Preparing a liquid sample from the biological material to be measured, and
A step of quantifying the number of copies of the target DNA contained in the liquid sample by digital PCR (dPCR),
A method for quantifying the number of copies of target DNA per unit amount of the biological material.
[25] A method for evaluating the biodistribution of a drug using any one of [1] to [24] above, for example, the quantitative method described in [1], [2], or [24] above.

Using the present invention, it becomes possible to accurately evaluate the pharmacokinetics of a target DNA using a simpler method, regardless of the amount of gDNA.

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. The vertical axis represents the signal intensity, and the horizontal axis represents the number of events. 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, and the horizontal axis represents the number of days after rAAV administration.

1. Method for quantifying target DNA According to the method for quantifying target DNA of the present invention, it is possible to quantify target DNA per unit volume or unit weight in a biological material.
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.

2. Liquid Sample Preparation Step In the 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.
When the biological material is a liquid, a liquid sample can be prepared by directly diluting the biological material and further adding a PCR reaction reagent. In the case of solid biomaterials, liquid samples can be prepared by first performing appropriate solubilization treatment, for example with protease, followed by dilution and addition of PCR reaction reagents. In addition, as a method of solubilization treatment, for example, 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. In this manner, 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.
In particular, 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.

2-1. Liquid Biological Materials When preparing 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. It is preferable to select a value based on the final concentration, for example, 10 to 20,000,000 times, 15 to 10,000,000 times, 20 to 5,000,000 times, etc. Further, 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. be. Further, 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.
Further, 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.
When the liquid biomaterial is blood, 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.
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 μL or more, 0.04 μL or more, 0.1 μL or more, 0.5 μL or more, 1 μL or more, 3 μL. The amount is 5 μL or more. As described above, in the quantification method of the present invention, DNA can be quantified with sufficiently high reliability using a small amount of liquid biological material.

2-2. 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. When adding a protease, 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.
Further, 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 μL, preferably 2 to 50 μL, and more preferably 2 to 50 μL per 1 mg of biomaterial. About 5 to 30 μL is used. The concentration of protease in the protease treatment solvent is, for example, 60 mAU/mL or more.

After protease treatment, the suspension may be resuspended if necessary. Further, after the protease treatment, 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.
Note that even when using a liquid biomaterial, the steps for the solid biomaterial described above can be employed, but preferably, addition of protease to the liquid biomaterial may be avoided. Furthermore, even if 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 (μg)/volume of diluent (μL)), for example, 3. It may be ~5,000,000 times, 5~1,000,000 times, 10~500,000 times, 15~200,000 times, etc. Further, 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. In addition, 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 (μg)/volume of diluent (μL)). , 100 times or more, 150 times or more, etc.

Furthermore, as mentioned above, 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. Further, 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.
When the solid biomaterial is liver, 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 It is preferable that
When the solid biomaterial is quadriceps muscle, 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.
In addition, 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 (μL)) ), 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. For example, when using liver or brain tissue pieces, the dilution ratio of a solid biomaterial is 10 to 500, based on the final concentration (weight of biomaterial (μg)/volume of diluent (μL)). times, preferably 50 times to 400 times, more preferably 100 times to 300 times, most preferably 200 times, and when using tissue pieces such as heart, kidney, spleen, etc., for example, 3 times to 200 times, preferably is 5 times to 100 times, more preferably 10 times to 50 times, and most preferably 20 times. In addition, when using tissue pieces such as lungs and muscles, the final concentration (weight of biomaterial (μg)/volume of diluent (μL)) 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.

For example, when a tissue piece such as liver, brain, lung, muscle, etc. is treated with protease using the buffer ATL and proteinase K mentioned above, and the obtained solution is diluted by inactivation treatment as necessary, the dilution ratio is An example of this 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 (μg)/volume of diluent (μL)). , the most preferred specific example is 200 times.

For example, when treating a piece of tissue such as liver or brain with protease using the commercially available "Tissue Direct PCR Kit" described below, and diluting the resulting solution by performing inactivation treatment if necessary, 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 (μg)/volume of diluent (μL)). The most preferred specific example is 200 times.

In addition, when treating tissue pieces such as heart, lung, kidney, muscle, spleen, etc. with protease using the commercially available "Tissue Direct PCR Kit" and diluting the obtained solution by inactivation treatment if necessary. Examples of 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 (μg)/volume of diluent (μL)). 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.

In the liquid sample preparation step using solid biomaterials, existing PCR kits, such as the Tissue Direct PCR Kit manufactured by Omega BIO-TEK, can also be used.

As described above, 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.

3. DNA Copy Number Quantification Step In the quantification step, the copy number of the target DNA contained in the liquid sample can be quantified by dPCR. According to 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.
There are no particular restrictions on the dPCR method used in the quantitative step, and for example, droplet digital PCR (ddPCR), chip-based digital PCR, etc. may be used, and ddPCR is preferably used.

In the quantitative step, a known ddPCR device and system can be used, such as those manufactured by Bio-Rad or BioTNS. In 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.

In dPCR, it is usually required to set appropriate thresholds for positive and negative signals. Therefore, in the quantification step that employs dPCR, 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.

In the quantitative step, 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. Inactivation treatment may be further performed by treatment or the like. In another embodiment, after measuring the signal, the liquid sample 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.

According to the method for quantifying the copy number of the target DNA, which includes the step of preparing a liquid sample and the step of quantifying the DNA copy number described above, 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.

4. Calculating the number of DNA copies per unit amount of biomaterial In the calculation step, the number of copies of the target DNA per unit amount of the biomaterial ( copy number/μL or copy number/μg) 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.

5. purpose DNA
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.
In one aspect of the present invention, 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). In one aspect of the present invention, 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. Examples of rAAV 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. (rAAV7), rAAV serotype 8 (rAAV8), rAAV serotype 9 (rAAV9), rAAV serotype 10 (rAAV10), rAAV serotype 11 (rAAV11), or artificially modified serotypes derived from these AAVs. .
In one aspect of the present invention, 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.
In one aspect of the present invention, the introduced DNA includes those derived from 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.

6. 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.

Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples are not intended to limit the present invention.

Reference Example 1 Quantification of EGFP cDNA in mouse blood by qPCR method using diluted blood First, DNA containing the artificially synthesized Enhanced Green Fluorescent Protein (EGFP) gene sequence was added to diluted mouse blood. Hereinafter referred to as EGFP cDNA) was added. At that time, the mouse blood was diluted 250 times in terms of final concentration. As a control group, the same amount of EGFP cDNA was added to buffer AE (Qiagen). The EGFP cDNA copy number in the obtained solution was measured by qPCR method.
In the qPCR method, 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 μL of master mix was added to a 384-well plate, 1 μL 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℃, 2 minutes 1 cycle of 95℃, 10 minutes 40 cycles of 95℃, 15 seconds and 60℃, 1 minute Hold at 4℃ *Temperature increase/decrease rate is 1.6℃/sec

The results are shown in Figure 1. When the Threshold Cycle (Ct) values of EGFP cDNA in mouse blood and Buffer AE were compared, the Ct value in mouse blood containing EGFP cDNA was higher than the Ct value in Buffer AE as a control. It was also high, about 5. Since a matrix effect due to blood components was observed, it was suggested that it is difficult to directly quantify the target DNA in blood using the qPCR method.

[Quantification of EGFP cDNA in mouse blood by ddPCR (Example 1)]
Next, we first attempted to quantify EGFP cDNA in mouse blood by ddPCR using the method outlined below.
[Method]
First, artificially synthesized DNA containing the Enhanced Green Fluorescent Protein (EGFP) gene sequence (hereinafter referred to as EGFP cDNA) was added to diluted mouse blood, and the resulting solution was used to perform ddPCR. We considered the conditions.
On the other hand, as a control, EGFP cDNA was added to the buffer solution and measured by ddPCR under the same conditions. When the measured amounts of EGFP cDNA in the blood and buffer solution were expressed as copy number/μL and compared, the concentration in the blood to which EGFP cDNA had been added was similar to that in the buffer solution used as a control. . Therefore, this measurement method was found to be suitable.

Next, for example, by collecting the blood of the mouse after a single intravenous administration of rAAV carrying the EGFP gene to the mouse (Example 9 described later), ddPCR was used based on the conditions optimized above. Target DNA containing the EGFP gene sequence (hereinafter simply referred to as target DNA) was quantified. The concentration of the target DNA relative to the blood volume used for measurement was expressed as copy number/μL. The result of the concentration of the target DNA per unit volume of blood (copy number/μL) obtained in this way and the target DNA concentration per gDNA (copy number/μL) measured using the same sample using the conventional quantitative PCR method. gDNA).
Hereinafter, 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 (addition amount is under three conditions of addition conditions 1 to 3 in Table 3 below) 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 μL of the ddPCR reaction solution according to the instruction manual, and transferred to a 96-well plate (Bio-Rad, 12001925). After sealing the plate with a foil heat seal (Bio-Rad, 1814040) and a PX1 PCR plate sealer (Bio-Rad), amplification was performed in a C1000 Touch Thermal Cycler (Bio-Rad) under the following conditions. The sample after amplification was detected with Droplet Reader and analyzed with QuantaSoft.
1 cycle of 95℃, 10 minutes 40 cycles of 94℃, 30 seconds and 60℃, 1 minute (A) 1 cycle of 98℃, 10 minutes or (B) 1 cycle of 98℃, 20 minutes Hold at 4℃ * Heating rate is 2℃/sec

The results are shown in Figures 2 to 4 and Tables 1 to 3. 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.

A comparison of the amount of EGFP cDNA in the mouse blood and Buffer AE obtained by measurement, expressed as copy number/μL, showed that in the mouse blood sample diluted 500 times in terms of final concentration, the amount of EGFP cDNA in Buffer AE, which was used as a control, was compared. The concentrations were similar (Figure 2 and Table 1). Furthermore, the concentration of a mouse blood sample diluted 5,000,000 times in terms of final concentration was similar to that in buffer AE, which was used as a control (FIG. 4 and Table 3). Therefore, it was found that this measurement method is suitable for blood diluted in the range of 500-5,000,000 times in terms of final concentration.

Furthermore, as shown in FIG. 3 and Table 2, it was found that the background signal intensity and the signal intensity of positive droplets changed in the sample containing diluted blood compared to the buffer AE sample used as a control. Whether droplets obtained by ddPCR are positive or negative is determined by a threshold based on the signal intensity they emit. In order to accurately quantify the target DNA, it was considered effective to set a threshold value based on positive and negative controls having the same matrix composition as the measurement target.

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).

The results are shown in Table 4.

When the amount of EGFP cDNA in human blood and buffer AE obtained by measurement was compared expressed as copy number/μL, the concentration in human blood sample diluted 500 times in terms of final concentration was higher than that in the control buffer. The concentration was similar to that in AE. Therefore, based on the results of Example 1, it was found that this measurement method is applicable not only to mouse blood but also to human blood.

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.
1 cycle of 95℃, 10 minutes 40 cycles of 94℃, 30 seconds and 60℃, 1 minute 1 cycle of 98℃, 10 minutes Hold at 4℃ *Heating rate is 2℃/second Next, 4℃, 20 minutes For the sample that had been left to stand, the EGFP cDNA copy number was measured by ddPCR using the same master mix and amplification conditions as in Example 1 ((B) 98°C, 20 minutes for 1 cycle). c).

The results are shown in Table 5.

When comparing the amount of EGFP cDNA in the mouse blood obtained by measurement expressed as copy number/μL, it was found that the mouse blood sample that was boiled before the amplification reaction (condition b) was compared with the sample that was not boiled (condition b). The concentrations were not similar to those in condition a). On the other hand, in the mouse blood sample that was boiled after the amplification reaction and before signal measurement (condition c), the concentration was similar to that in the sample that was not boiled (condition a). From the above, it was considered that performing a boiling operation after the amplification reaction (ddPCR reaction) and before signal measurement is effective as an inactivation treatment for a sample that may be a biohazard.

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.

Reference Example 2 Solubilization of Mouse Organ Homogenate by Vortex Operation Approximately 25 mg of liver and quadriceps muscle homogenate was weighed into a tube, and buffer AE was added so that the mass concentration was 2%. A vortex operation was performed to examine whether the homogenate was uniformly dispersed in the solution. As a result, vortexing was not effective in uniformly dispersing the homogenate in the solution. It was found that it was necessary to consider the conditions for solubilizing the homogenate in order to prepare a homogeneous sample. Therefore, solubilization using protease was investigated as detailed in Examples 5-7.

[Study of solubilization conditions for mouse organ homogenate (Examples 5 to 7)]
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 μL of L1 buffer and 20 μL 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. for 10 minutes while stirring at approximately 500 rpm. The sample was returned to room temperature, 100 μL 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 6 Solubilization of mouse organ homogenate using proteinase K Solubilization of mouse homogenate using proteinase K was investigated. Liver and quadriceps homogenates were weighed out at 10-52 mg (see Table 6) and 90 μL of buffer ATL (Qiagen, 19076) and 10 μL of proteinase K (Qiagen, 19133) 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. for 10 minutes while stirring at approximately 500 rpm. The sample was returned to room temperature and used as a test sample. The 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).

The results are shown in Table 6.

We were able to find homogenate solubilization conditions suitable for ddPCR measurements. That is, it was found that the samples obtained in Examples 5 and 6 both needed to be treated with about 10 mg of homogenized organs, for example, about 5 mg to 20 mg or 8 mg to 15 mg.

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/μL. The diluted solution of the solubilized homogenate of Example 5 and each concentration of EGFP cDNA were mixed at a ratio of 1:1. Further, the diluted solution of the solubilized homogenate of Example 6 and EGFP cDNA at a final concentration of 5×10 ² copies/μL 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.

For each solubilized homogenate, Table 7 shows the dilution ratio at which the sample could be measured by the ddPCR method.

For each solubilized homogenate, 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)). However, for 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. Ta.

Example 9 Biodistribution test of recombinant AAV (rAAV) Next, the 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. Alternatively, as a comparative test, 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.

(a) Administration of rAAV to mice, sampling, and crushing and solubilization of organs The 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.

(b) Quantification of rAAV genomic DNA containing target DNA in rAAV-administered mouse tissue samples by ddPCR method A portion of each homogenate prepared in Example 9(a) was weighed and solubilized by the method described in Example 6. did. Thereafter, rAAV genomic DNA containing the target DNA in each sample was quantified using the same method as in Examples 1 and 7 for the solubilized homogenate and blood.

The results are shown in Figure 5.

(c) DNA Extraction from Mouse Organs Next, in order to quantify rAAV genomic DNA by qPCR, a portion of each homogenate prepared in Example 9(a) was weighed and DNA was extracted. DNeasy Blood & Tissue Kit (Qiagen, 69581) was used for DNA extraction. The specific steps are as follows. 30 to 40 mg of the organ crushed as described above was weighed, and 180 μL of buffer ATL and 20 μL of proteinase K were added. The mixture was set in Eppendorf Thermomixer R T3317 (Eppendorf) and incubated at 56° C. for 17 hours or more while stirring at 500 rpm. Once the homogenate was solubilized, 4 μL of RNase (Qiagen, 19101) was added, vortexed well, and incubated for 5 minutes at room temperature. Add 410 μL of Buffer AL included with the kit and add the amount of ethanol listed on the container, stir thoroughly by vortexing, then transfer the sample to each well of the DNeasy 96 plate, seal with AirPore Tape Sheet, and centrifuge. did. 500 μL of buffer AW1 included in the kit was added, sealed with AirPore Tape Sheet, and centrifuged. Subsequently, 500 μL of buffer AW2 included in the kit was added, sealed with AirPore Tape Sheet, and centrifuged. A DNeasy 96 plate was set on a new rack of Elution Microtube RS, 50 μL 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.

DNeasy Blood & Tissue Kit (Qiagen, 69504) was used to extract DNA from blood. 100 μL of blood was weighed out, and 100 μL of PBS and 20 μL of proteinase K were added. 4 μL of RNase (Qiagen, 19101) was added, mixed well by vortexing, and incubated at room temperature for 5 minutes. After adding 200 μL 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. After adding 200 μL of 100% ethanol and stirring well with a vortex, 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 μL of Buffer AW1 was added, and centrifuged at 20,000×g and 4° C. for 2 minutes. 500 μL 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 μL 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.

(d) Quantification of rAAV genomic DNA containing target DNA in rAAV-administered mouse tissue samples by qPCR method A standard curve was prepared by diluting 13.3 pg/μL EGFP cDNA (FASMAC) with L-gDNA. The master mix includes TaqMan Gene Expression Assays (Thermo Fisher Scientific, 4331182, Assay ID Mr04097229_Mr), TaqPath qPCR Master Mix, CG (Thermo Fisher Scientific, A16245), TaqMan Copy Number Reference Assay, mouse, Tfrc (Thermo Fisher Scientific, 4458366 , which was diluted 20 times with ultrapure water [MilliQ water]) was used. Each DNA sample was diluted with buffer AE to a concentration of 100 ng/μL or less. 4 μL of the master mix was added to a 384-well plate, 1 μL 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℃, 2 minutes 1 cycle of 95℃, 10 minutes 40 cycles of 95℃, 15 seconds and 60℃, 1 minute Hold at 4℃ *Temperature increase/decrease rate is 1.6℃/sec

The results are shown in Figure 6. From the results of Example 9(b) (Fig. 5) and Fig. 6, it can be seen that the time course of the target DNA concentration in mouse tissues after a single intravenous administration of rAAV is the copy number/μL or copy number by this method. The target DNA concentration expressed in number/mg was similar to that evaluated in units of copy number/μg gDNA by the conventional qPCR method. Therefore, with this method, the biodistribution of rAAV can be quantitatively evaluated using a simpler method.

According to 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.

Claims (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 claim 1, which does not include the step of extracting DNA from the biological material.
3. The quantitative method according to claim 1 or 2, wherein the biological material is a liquid.
4. The quantitative method according to claim 3, wherein the liquid sample has a dilution factor of 20 to 5,000,000 times based on the final concentration of the biological material in the liquid.
5. The quantitative method according to claim 4, wherein the liquid biological material is blood, and the dilution factor based on the final concentration of blood is 500 to 5,000,000 times.
6. The quantitative method according to claim 3, wherein the liquid sample has a dilution factor of 30 to 2000 times based on the final concentration of the biological material in the liquid.
7. The quantitative method according to claim 1 or 2, wherein the biomaterial is a solid.
8. The quantitative method according to claim 7, wherein preparing the liquid sample includes solubilizing the solid biological material.
9. The quantitative method according to claim 7, wherein the dilution factor based on the final concentration of the solid biomaterial in the liquid sample is 20 to 5,000,000 times.
10. The quantitative method according to claim 7, wherein the dilution factor based on the final concentration of the solid biomaterial in the liquid sample is 3 to 1000 times.
11. The quantitative method according to claim 7, wherein the solid biomaterial is liver, and the dilution factor based on the final concentration of liver is 2,200 to 4,600,000 times.
12. The quantitative method according to claim 7, wherein the solid biomaterial is quadriceps femoris muscle, and the dilution factor based on the final concentration of quadriceps femoris muscle is 460 to 4,600,000 times.
13. In the step of quantifying the copy number of the target DNA by dPCR, a step of setting a threshold for a negative signal based on a blank sample in which the biological material is diluted at an equal dilution rate in the liquid sample and does not contain the target DNA, and/or the blank 3. The quantification method according to claim 1, further comprising the step of setting a threshold for a positive signal based on a spiked sample obtained by adding target DNA to the sample.
14. The quantitative method according to claim 1 or 2, further comprising the step of performing an inactivation treatment on the liquid sample.
15. 15. The quantitative method according to claim 14, wherein the step of inactivating the liquid sample is performed after the PCR reaction is completed and before the signal is measured.
16. The quantitative method according to claim 15, wherein the inactivation treatment is a boiling treatment of the liquid sample.
17. The quantitative method according to claim 1 or 2, wherein the dPCR is droplet digital PCR (ddPCR).
18. The quantitative method according to claim 1 or 2, wherein the target DNA is introduced DNA or endogenous DNA in the biological material.
19. The quantitative method according to claim 18, wherein the introduced DNA in the biological material is derived from a vector, a cell, a nucleic acid drug, an oncolytic virus, or a phage.
20. 20. Quantification according to claim 19, wherein the vector is derived from plasmid DNA (pDNA), recombinant adenovirus, recombinant adeno-associated virus (rAAV), recombinant lentivirus, recombinant Sendai virus, or recombinant retrovirus. Method.
21. The quantification method according to claim 19, wherein the cells are derived from CAR-T cells, CAR-NK cells, or a cell therapy product derived from ES cells or iPS cells.
22. The quantitative method according to claim 18, wherein the endogenous DNA in the biomaterial is derived from blood cells.
23. The target DNA is a recombinant adenovirus, recombinant adeno-associated virus (rAAV), recombinant lentivirus, recombinant Sendai virus, recombinant retrovirus, plasmid DNA (pDNA), CAR-T cell, CAR in the biological material. - The quantitative method according to claim 1 or 2, which is derived from a cell therapy product derived from NK cells, ES cells, or iPS cells, blood cells, oncolytic viruses, phages, or nucleic acid medicines.
24. preparing a liquid sample from the biological material to be measured; and
    A step of quantifying the number of copies of the target DNA contained in the liquid sample by digital PCR (dPCR),
    A method for quantifying the number of copies of target DNA per unit amount of the biological material.
25. A method for evaluating the biodistribution of a drug using the quantitative method according to claim 1, 2, or 24.