WO2024046474A1 - 一种检测car拷贝数的方法 - Google Patents

一种检测car拷贝数的方法 Download PDF

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WO2024046474A1
WO2024046474A1 PCT/CN2023/116562 CN2023116562W WO2024046474A1 WO 2024046474 A1 WO2024046474 A1 WO 2024046474A1 CN 2023116562 W CN2023116562 W CN 2023116562W WO 2024046474 A1 WO2024046474 A1 WO 2024046474A1
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copy number
primer
car
seq
probe
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French (fr)
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刘进
曹乾升
陈庆庆
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南京传奇生物科技有限公司
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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

Definitions

  • the present disclosure belongs to the field of genetic analysis and detection technology, and specifically relates to a general method for quantitatively detecting CAR copy number in biological samples and its application.
  • Chimeric Antigen Receptor is a synthetic transmembrane receptor that can confer antigen-binding specificity to cells.
  • transgenic technology is used to express CAR on the surface of immune cells, and the CAR transgenic immune cells are expanded and injected into patients to specifically target and destroy lesions.
  • Significant clinical responses and high complete response rates have been observed in CAR-T cell therapy of B-cell malignancies.
  • CAR copy number detection which is a pharmacokinetic indicator of cell therapy drugs, is a crucial link in the development, preparation, in vivo and in vitro experiments of CAR-related products.
  • the present disclosure provides a method for detecting CAR copy number, as well as primer-probe combinations, kits and applications used in the method.
  • This disclosure attempts to perform accurate DNA input calibration by detecting double internal reference genes of humans and mice.
  • weight of the human genome is 6.6pg/diploid human cell (The American Journal of Human Genetics, 1999, 64(1):218-224), while the genomes of other species such as mice
  • This disclosure infers the weight of the genome of experimental mice, uses the internal reference gene copy numbers of both human and mouse species to correct the input amount, and proposes a corresponding calculation formula accordingly.
  • one of the technical solutions of the present disclosure relates to a method for detecting CAR copy number in biological samples, which includes the following steps:
  • the experimental animal is an animal injected with human cells expressing CAR, preferably an experimental mouse. After the experimental animal samples are injected with human cells expressing CAR, they become mixed samples. Three indicators of the samples are quantitatively detected, namely: the initial CAR copy number, the copy number of the human internal reference and the copy number of the experimental animal internal reference. The input of the internal reference to the sample is Calibrate the amount to obtain the corrected CAR copy number.
  • the methods described in this disclosure are applicable to all biological fluids and tissue samples.
  • the biological matrix sample is selected from blood, organ, or tumor homogenate.
  • the biological fluid sample is blood.
  • the visceral sample is liver or kidney.
  • the tissue sample is a tumor homogenate.
  • the biological sample is from experimental animal tissue or whole blood. Genomic DNA from tissue or whole blood is preferred. More preferred is whole blood genomic DNA.
  • a quantitative PCR method is used to detect the initial CAR copy number, the copy number of the human internal reference, and the copy number of the mouse internal control, preferably using fluorescence quantitative PCR, and more preferably ddPCR.
  • step (1) the initial CAR copy number of the sample is detected by detecting the universal vector sequence of the CAR.
  • the method is a universal method for quantitatively detecting CAR gene copy number.
  • primers and probes are designed for the LTR of the CAR vector, such as the region near the 5’ LTR.
  • the method is universal and there is no need to redesign primers and probes for different CAR-T products. Therefore, in some embodiments, the detection of the LTR uses the following primer probe combination: an upstream primer as shown in SEQ ID NO:1, a downstream primer as shown in SEQ ID NO:2, such as SEQ ID NO:3 Probe shown.
  • the non-homologous region is a non-homologous region of a human or mouse homologous gene or a non-homologous gene.
  • the non-homologous region is a non-homologous region of a human and mouse housekeeping gene.
  • the non-homologous region is derived from, but is not limited to, the non-homologous region of the following genes: ApoB, ACTB, ALB, tubulin, GAPDH, actin, RNase P, B2M, HPRT and TBP.
  • the double internal reference combination is: human ApoB gene exon 26, and mouse ApoB gene exon 26.
  • the double internal reference combination is: human ACTB gene and mouse ACTB gene, the first 1000 bp of which are of different origin.
  • the double internal reference combination is: exon 26 of the human ApoB gene, and the first 1000 bp of the mouse ACTB gene.
  • this formula is based on the industry consensus that the genome weight of each human cell is 6.6pg, and based on the ratio of the latest large version of the mouse and human genome sizes, the mouse genome weight is 5.6 pg (the genome size of the human GTCh38 version is 3,209,286,105bp, and the genome size of the mouse GRCm39 version is 2,728,222,451bp, so the calculated genome weight of the mouse is: 6.6 ⁇ 272822451/3209286105 ⁇ 5.6pg).
  • the CAR copy number is corrected by the following formula:
  • the CAR copy number on the left side of the equation is the corrected copy number;
  • the CAR copy number on the right side of the equation is the initial CAR copy number, that is, the CAR copy number directly obtained through detection means.
  • human genome copy number is corrected by the following formula:
  • the human genome copy number on the left side of the equation is the corrected copy number
  • the internal reference copy number on the right side of the equation is the initial copy number of the internal reference gene, that is, the copy number directly obtained by detection means.
  • the double internal reference combination is human ApoB gene and mouse ApoB gene.
  • the detection of the ApoB gene uses the following primer-probe combination:
  • Primer probe combination for detecting human ApoB gene including: an upstream primer as shown in SEQ ID NO:4, a downstream primer as shown in SEQ ID NO:5, as shown in SEQ ID NO:6 probe;
  • a primer-probe combination for detecting the ApoB gene of mice including: an upstream primer as shown in SEQ ID NO:7, a downstream primer as shown in SEQ ID NO:8, and a primer as shown in SEQ ID NO:9 probe shown.
  • the CAR copy number is corrected by the following formula:
  • step (1) it also includes (0) extracting genomic DNA from the biological sample.
  • the second technical solution of the present disclosure relates to a primer-probe combination for detecting CAR copy number.
  • the primer-probe combination includes: an upstream primer as shown in SEQ ID NO: 1, and an upstream primer as shown in SEQ ID NO: 2.
  • the downstream primer such as the probe shown in SEQ ID NO:3.
  • the third technical solution of the present disclosure relates to a primer-probe combination for detecting ApoB, which includes:
  • the upstream primer shown in SEQ ID NO:4 the downstream primer shown in SEQ ID NO:5, the probe shown in SEQ ID NO:6; and/or,
  • the upstream primer shown in SEQ ID NO:7 the downstream primer shown in SEQ ID NO:8, and the probe shown in SEQ ID NO:9.
  • the fourth technical solution of the present disclosure relates to a primer-probe set, which includes the primer-probe combination for detecting CAR copy number as described in the second technical solution and the primer-probe combination for detecting ApoB as described in the third technical solution.
  • Primer-probe combinations are included in the primer-probe set, which includes the primer-probe combination for detecting CAR copy number as described in the second technical solution and the primer-probe combination for detecting ApoB as described in the third technical solution.
  • the fifth technical solution of the present disclosure relates to a kit for detecting CAR copy number, which includes the primer-probe combination as described in the second technical solution of the present disclosure.
  • the kit further includes a primer-probe combination for detecting human and mouse ApoB genes, wherein,
  • the primer probe combination used to detect human ApoB gene includes: the upstream primer shown in SEQ ID NO:4, the downstream primer shown in SEQ ID NO:5, and the downstream primer shown in SEQ ID NO:6 probe;
  • the primer probe combination used to detect the mouse ApoB gene includes: the upstream primer shown in SEQ ID NO:7, the downstream primer shown in SEQ ID NO:8, as shown in SEQ ID NO:9 probe.
  • the kit includes one or more of a positive control plasmid and a negative control plasmid containing a part of the lentiviral vector sequence, a part of the hApoB sequence and a part of the mApoB sequence.
  • nucleotide sequence of a portion of the lentiviral vector sequence, a portion of the hApoB sequence, and a portion of the mApoB sequence is as shown in SEQ ID NO: 22.
  • the kit also includes instructions for use, which record:
  • the CAR copy number is corrected by the following formula:
  • the sixth technical solution of the present disclosure relates to a primer-probe combination as described in technical solution 2 or 3, a primer-probe set as described in technical solution 4, or a kit as described in technical solution 5 in detecting CAR copies. number or use in preparing diagnostic agents for detecting CAR copy number.
  • the seventh technical solution of the present disclosure relates to a system for detecting CAR copy number in biological samples.
  • the system includes:
  • (A) Input module Import the initial CAR copy number, the copy number of the human internal reference, the copy number of the experimental animal internal reference, the corresponding sample input amount of the human internal reference detection system and the corresponding sample input amount of the experimental animal internal reference detection system into the input module;
  • Experimental animals are, for example, mice;
  • (B) Calculation module The information obtained by the input module enters the calculation module to correct the CAR copy number and the human genome copy number; wherein, the CAR copy number is corrected by the following formula:
  • the human genome copy number is corrected by the following formula:
  • the information obtained by the input module in (A) comes from the method described in one of the technical solutions of the present disclosure.
  • system further includes (C) a printing module: printing the results obtained by the calculation module in (B).
  • the eighth technical solution of the present disclosure relates to an electronic device, which includes a memory and a processor; the memory includes a computer program stored therein that can be run on the processor; wherein,
  • the processor executes the computer program, it implements the functions of the system described in the seventh technical solution of the present disclosure.
  • the ninth technical solution of the present disclosure relates to a computer-readable storage medium on which a computer program is stored, wherein when the computer program is executed by a processor, the functions of the system as described in the seventh technical solution of the present disclosure can be realized. .
  • This disclosure uses double internal controls, which can effectively correct the sample loading, thereby ensuring the reliability of the data on the number of transgene copies per microgram of gDNA;
  • This disclosure uses double internal reference genes to also track the expansion of human cells in experimental animals
  • the theoretical input amount involved in this disclosure ranges from 1 to 100,000 copies, and the demand for sample loading is low, so the sampling amount of small animals, such as the amount of blood taken, can be saved;
  • This disclosure designs a probe set based on the universal sequence of CAR, such as the LTR sequence. There is no need to redesign the primer probe set for different CAR products, and the method is universal;
  • This disclosure proposes for the first time the calculation formula of the double internal reference correction input amount for humans and mice.
  • FIG. 1 Schematic diagram of the design positions of CAR universal primers and probes.
  • Figure 5 System for detecting CAR copy number in samples.
  • Figure 6 is a schematic diagram of an electronic device of the present disclosure.
  • animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to a human at any stage of development.
  • Experimental animals generally refer to non-human animals whose functions, metabolism, structure and disease properties are similar to those of humans and are specially bred for experimental use. They mainly refer to scientific research, teaching, medical treatment and identification in medicine, pharmacy, biology, veterinary medicine, etc. Animals that need to be domesticated, bred, and raised for purposes such as diagnosis, manufacturing of biological products, etc. In some embodiments, experimental animals refer to non-human animals at any stage of development.
  • the non-human animal is a mammal, such as a mouse, rat, rabbit, dog, cat, sheep, cow, pig, horse, and/or a primate such as a monkey.
  • experimental animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms.
  • experimental animals may be transgenic animals, genetically engineered animals, and/or clones.
  • the experimental animals are animals injected with human CAR transgenic cells.
  • mice are cultivated from wild mice through long-term selection. They belong to the phylum Vertebrates, class Mammalia, order Rodentia, family Muridae, and genus Mus. Experimental mice are small, easy to feed and manage, and easy to control; they are produced and reproduced quickly. There are a large number of inbred strains, mutant strains and closed groups with different characteristics, and a variety of internationally recognized standard strains have been formed, which are widely used in Various experimental studies.
  • CAR Chimeric Antigen Receptor
  • a CAR includes an antigen-specific targeting domain, an extracellular domain, a transmembrane domain, optionally one or more costimulatory domains, and an intracellular signaling domain.
  • modified cells can be obtained from a patient or donor.
  • the modified cells are lymphocytes (eg, T cells), macrophages (eg, tumor-associated macrophages, TAMs).
  • cells are modified to express an exogenous construct, such as a CAR or T cell receptor (TCR), thereby obtaining CAR-T or CAR-M cells.
  • TCR T cell receptor
  • subject refers to a human or any non-human animal, such as a mouse, rat, rabbit, dog, cat, sheep, cow, pig, horse, and/or a primate (eg, a monkey).
  • a subject refers to a person who presents to a medical provider for diagnosis or treatment of a disease.
  • Subject is used interchangeably herein with “individual” or “patient.”
  • a subject may have, or be susceptible to, a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.
  • the subject is diagnosed with or exhibits symptoms or characteristics of one or more diseases, disorders and/or conditions.
  • the disease may include cancers such as lymphoma and leukemia.
  • sample is a part of the individuals observed or investigated, and the population is the entire research object.
  • Biological samples usually refer to plant flowers, leaves, stems, roots, seeds, etc., animal (including human) body fluids (such as urine, blood, saliva, bile, gastric juice, lymph fluid and other secretions of organisms, etc.), hair , muscles and some tissues and organs (such as thymus, pancreas, liver, lungs, brain, stomach, kidney, etc.) as well as various microorganisms.
  • a biological sample refers to a tissue sample from an experimental animal, or DNA extracted from the tissue.
  • tissue is the unit that constitutes the organs in the body.
  • Whole blood refers to the mixture formed by collecting blood from the human body into a blood collection container, which includes all components of blood cells and plasma.
  • treatment means the use to partially or completely alleviate, ameliorate, alleviate, inhibit, prevent, delay the onset of, or reduce the severity of one or more symptoms or characteristics of a particular disease, disorder, and/or condition. and/or any method to reduce its occurrence.
  • Subjects who show no signs of disease and/or who show only early signs of disease may be treated to reduce the risk of developing disease-related pathology.
  • PCR polymerase chain reaction
  • primer refers to a macromolecule having a specific oligonucleotide sequence that stimulates the initiation of synthesis when nucleotide polymerization is initiated.
  • Primers are usually two artificially synthesized oligonucleotide sequences.
  • One primer is complementary to a DNA template strand at one end of the target region (such as the 5' end) (called an upstream primer or forward primer), and the other primer is complementary to the other end of the target region.
  • the other DNA template strand (such as the 3' end) is complementary (called the downstream primer or reverse primer). Its function is to serve as the starting point for nucleotide polymerization.
  • Nucleic acid polymerase can start to synthesize a new nucleic acid strand from its 3' end. .
  • In vitro artificially designed primers are widely used in polymerase chain reaction, sequencing and probe synthesis.
  • fluorescent probe utilizes the principle of fluorescence resonance energy transfer (FRET), which usually includes an oligonucleotide sequence and a pair of energy donor and acceptor.
  • FRET fluorescence resonance energy transfer
  • the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor.
  • FRET fluorescence resonance energy transfer
  • the fluorescence energy generated by exciting the donor is absorbed by the nearby acceptor, causing the donor to
  • the fluorescence intensity emitted by the body is attenuated, and the fluorescence intensity of the acceptor fluorescent molecules is enhanced.
  • the spatial distance between the donor and the acceptor of the fluorescent probe is destroyed and the energy transfer is broken (for example, by the donor being cleaved from the fluorescent probe), so that the fluorescence emitted by the donor It cannot be absorbed by the receptor and detected by the fluorescence detection system.
  • a free fluorescent molecule reporter group
  • the detection of fluorescent signals can monitor the PCR process in real time. Accurately quantify PCR starting copy number.
  • primers are typically used in conjunction with fluorescent probes.
  • Real-time fluorescence quantitative PCR is a method that uses fluorescent chemicals to measure the total amount of product after each PCR cycle in a DNA amplification reaction.
  • Common qPCR include SYBRGreen and TaqMan probe methods.
  • ddPCR Droplet Digital Polymerase Chain Reaction
  • ddPCR Droplet Digital Polymerase Chain Reaction
  • Using microfluidic technology or other droplet generation methods dilute nucleic acid solutions are dispersed into microwells or droplets such that the ideal number of target nucleic acids per microwell or per droplet is less than or equal to one.
  • the reaction with the target molecule emits a positive fluorescence signal, while the reaction without the target molecule emits a negative fluorescence signal.
  • the nucleic acid concentration of the original nucleic acid solution can be obtained.
  • ddPCR can achieve absolute quantification of the starting DNA template through direct counting and Poisson correction analysis.
  • Gene refers to the sum of all genetic material of an organism. This genetic material includes DNA or RNA (viral RNA). For a haploid, the genome represents the total DNA or RNA of the organism. For diploid higher organisms, the sum of the DNA of their gametes is a set of subgenomes. Diploid has two homologous subgenomes. There are several subgenomes as there are several chromosome sets in eukaryotic cells. The DNA sequence that carries the genetic information for synthesizing proteins is the coding sequence, and the proportion of coding sequences in the genome varies greatly among different species. Non-coding sequences include introns, non-coding RNA sequences, regulatory DNA and repetitive DNA. Larger genomes do not necessarily contain more genes, and the proportion of non-repetitive DNA in complex eukaryotes decreases with increasing genome size, for example 98% of the human genome is non-coding sequence.
  • internal reference gene is an experimental reference introduced in quantitative genetic testing to control experimental errors caused by differences in experimental techniques or samples. It is a gene used to measure the accuracy of experimental results and correct the experimental results when necessary. .
  • several variables related to different steps of the experimental procedure can lead to considerable sample-to-sample variability and can lead to erroneous results: different amounts and qualities of starting material, RNA integrity, cDNA synthesis, and PCR amplification the efficiency of the increase and the overall transcriptional activity between tissues or cells.
  • the use of internal reference genes is generally considered the most reliable method to standardize qPCR and reduce possible errors in gene expression quantification. In this normalization strategy, the internal reference gene is used as an internal control, and the same experimental protocol as the GOI is used, and then the expression level of the target gene is normalized according to the value of the internal control. Housekeeping genes are usually used as internal reference genes.
  • House-keeping genes refer to a class of genes that are stably expressed in all cells of the same organism and are less affected by environmental factors. Their products are necessary to maintain the basic life activities of cells. Its expression is usually only affected by the promoter sequence or the interaction between the promoter and RNA polymerase, and is not regulated by other mechanisms, such as tubulin genes, glycolytic enzyme genes and ribosomal protein genes.
  • G3PDH glyceraldehyde-3-phosphate dehydrogenase
  • albumin actin
  • tubulin tubulin
  • B2M human ⁇ 2-microglobulin
  • TATA-binding protein TTP
  • cyclophilin hypoxanthine phosphoribosyltransferase (HRPT) and ribonuclease P (RNase P), etc.
  • HRPT hypoxanthine phosphoribosyltransferase
  • RNase P ribonuclease P
  • GAPDH is an important glycolytic pathway enzyme
  • the Albumin gene encodes the most abundant albumin in human blood (i.e., human serum albumin, ALB); actin includes ⁇ and ⁇ -actin, and tubulin includes ⁇ , ⁇ -tubulin, which is critical for the structure and dynamics of the cytoskeleton.
  • Ribonuclease P (RNase P) is composed of two subunits, protein and RNA. It is the enzyme responsible for the maturation of the 5' end of tRNA precursor.
  • ApoB is the gene encoding apolipoprotein B (apolipoprotein B) and is located at 2p24-p23 on chromosome 2.
  • the full length of the ApoB gene is 42.6kb, with a total of 29 exons.
  • the full length of the mRNA is 14.1nt, encoding a protein composed of 4,564 amino acid residues.
  • ApoB protein exists in two forms of isoforms in plasma, one is ApoB48 and the other is ApoB100. These two forms of ApoB protein are encoded by the same gene and the same pre-mRNA transcript with a length of more than 16kb. , share the same amino terminus.
  • Homologous genes are genes inherited in different species from a common ancestor. Although homologous genes are similar in sequence, similar sequences are not necessarily homologous. Homologous genes include orthologous genes, paralogous genes and heterologous genes. Orthologous genes refer to genes that evolved vertically from the same ancestor. Paralogous genes refer to homologous genes resulting from gene duplication, such as human ⁇ -globin gene and ⁇ -globin gene. Xenologous genes are produced by horizontal transfer of genes between different species. "Non-homologous genes” are genes inherited from different ancestors in the same species or in different species. For example, in this article, human ApoB and mouse ApoB are homologous genes; but human ApoB and human or mouse GAPDH are non-homologous genes.
  • homologous region refers to a highly conserved region in homologous genes, which usually occurs between pairs of chromosomes in the same subgenome or subgenomes from different ancestral sources.
  • non-homologous region refers to a region in homologous genes that is not highly conserved, or it can also refer to a region in which non-homologous genes are not conserved among themselves.
  • LTR long terminal repeat
  • Introducing CAR into T cells requires the use of a gene delivery vector carrying a CAR expression cassette.
  • retroviruses such as lentivirus are commonly used as CAR delivery vectors.
  • the CAR expression cassette between the two LTRs on the viral genome is randomly integrated into the host cell genome, thereby achieving long-term stable expression of CAR.
  • LTR is used as the target sequence to design a universal primer and/or probe pair for CAR copy number detection.
  • copy number refers to the number of a certain gene or a specific nucleotide sequence in the haploid genome of an organism. Determination of copy number is a commonly used technique in molecular biology research, such as cloning certain repetitive sequences, determining the copy number of foreign genes integrated in transfected cultured cells and transgenic animals and plants, etc., all of which require the target gene or sequence to be studied. The copy number was measured. Single copy means that there is only one gene or sequence in the haploid genome of the organism, while multi-copy means there are multiple copies. Copy number can usually be detected through technologies such as sequencing, southern blot, real-time fluorescence quantitative PCR or digital PCR.
  • the LTR primer probe set mentioned in this disclosure is used to detect the copy number and amplification status of CAR (actually designed near the vector universal sequence LTR) in the sample; the human ApoB (hApoB) primer probe set is used to detect hApoB in the sample The copy number and amplification status; the mouse ApoB (mApoB) primer probe set is used to detect the copy number and amplification status of mApoB in the sample.
  • a reaction system can be set up to detect up to two target genes, namely CAR.
  • the detection of hApoB and mApoB can be carried out in 2-3 reaction systems (including repeated detection of one of the genes).
  • the input amount of DNA sample is 10ng ⁇ 50 ⁇ 0.1ng, and the input amount of sample can be different for different reaction systems. Only one of these situations is listed below.
  • This disclosure provides upstream primers (F), downstream primers (R) and probes (P) for detecting CAR (actually designed near the vector universal sequence LTR, see Figure 1), hApoB and mApoB genes, their sequences and modification information As shown in Table 1, FAM and HEX are fluorescent modification groups, and MGB-NFQ and BHQ-1 are quenching groups:
  • the modifying group of the probe is not a limiting condition and can be replaced as needed.
  • the universal method for quantitatively detecting CAR gene copy number includes the following steps:
  • Negative control substance Use CAR-negative mouse gDNA as a negative control substance.
  • Two sets of PCR systems are prepared for each sample: one group detects CAR, the primer and probe mixture is the LTR primer and probe mixture described in Table 1, and the sample input amount is 50ng; the other group detects hApoB and mApoB at the same time , the primer-probe mixture is the hApoB and mApoB primer-probe mixture described in Table 1, and the sample input amount is 10ng.
  • 2 ⁇ digital PCR supermix was purchased from BioRad, product number 1863024.
  • the input amounts of the first group of systems and the second group of systems may be inconsistent, and the error value of the numerical range is ⁇ 0.1ng.
  • Threshold setting Manually set the thresholds of Channel 1 and Channel 2 to correctly distinguish negative and positive droplets.
  • the Channel 1 threshold is set to 6000; the Channel 2 threshold is set to 4000. If there are points that deviate significantly from the threshold, you can use the lasso tool to make fine adjustments.
  • the double internal reference is the non-homologous region of the human and mouse APOB gene, so the following formula is obtained:
  • the double internal reference is the non-homologous region of the human and mouse APOB gene, so the following formula is obtained:
  • the CAR-T sample involved in this disclosure is a sample self-made by Nanjing Legend Biotechnology Co., Ltd. (see WO2020043152A1, Example 3).
  • the CAR targets mesothelin, and its structure from the C terminus to the N terminus is CD8 ⁇ signal peptide, mesothelin binding domain, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, CD137 intracellular domain and CD3 ⁇ intracellular domain. costimulatory domain.
  • the DNA extracted therefrom can be replaced by an international standard (WHO 1st Reference Reagent for Lentiviral Vector Integration Site, NIBSC code: 18/144).
  • the positive plasmid (PCP) involved in this disclosure is provided by GenScript (Cat. No. SC1317), which is a linearized pcDNA3.1(+) plasmid containing part of the lentiviral vector sequence, part of the hApoB sequence and part of the mApoB sequence.
  • GenScript Cat. No. SC1317
  • the nucleotide sequence of a part of the lentiviral vector sequence, a part of the hApoB sequence and a part of the mApoB sequence contained in the positive plasmid is shown in SEQ ID NO: 22.
  • the negative plasmid (NCP) involved in this disclosure is the pcDNA3.1(+) plasmid.
  • the CAR primer probe set has no positive detection in the normal mouse and human genomes, hApoB is only positive in the normal human genome, and mApoB is only positive in the normal mouse genome. It shows that the method has good specificity.
  • the negative sample (mouse whole blood DNA) was added with 1 ⁇ 10 1 , 1 ⁇ 10 2 , 1 ⁇ 10 3 , 1 ⁇ 10 4 , and 4 ⁇ 10 4 copy numbers of PCP (positive plasmid quality control). Samples of the product) were tested three times, and all samples could be detected. The recovery rate of CAR copy number was in the range of 101.77% to 112.94%, and the CV did not exceed 30%. The copy number of CAR was proportional to the concentration of the test product. Linear relationship, and its R2 is greater than 0.999. The above results prove that using this method for CAR detection is Accuracy, repeatability and linearity are good.
  • Example 3 mApoB gene detection accuracy, repeatability and linearity verification
  • the negative sample human DNA
  • PCP positive plasmid quality control product
  • the negative sample (mouse whole blood DNA) was added with 1 ⁇ 10, 1 ⁇ 10 2 , 1 ⁇ 10 3 , 1 ⁇ 10 4 , and 4 ⁇ 10 4 copy numbers of PCP (positive plasmid quality control product). ) samples were tested three times, and all samples could be detected.
  • the recovery rate of hApoB copy number ranged from 85.63% to 111.70%, and the CV did not exceed 30%.
  • the copy number of hApoB was linear with the concentration of the test product. relationship, and its R2 is greater than 0.999. The above results show that this method has good accuracy, repeatability and linearity in detecting hApoB gene.
  • Example 7 System for detecting CAR copy number
  • This embodiment provides a system, as shown in Figure 5, including (1) an input module, (2) a calculation module; and optionally (3) a printing module.
  • Input module Import the CAR copy number, the copy number of the human internal reference, the copy number of the mouse internal reference, the corresponding sample input amount of the human internal reference detection system and the corresponding sample input amount of the mouse internal reference detection system obtained in the aforementioned embodiments into The input module;
  • the human genome copy number is corrected by the following formula:
  • the electronic device can be represented by a computing device (for example, it can be a server device), including a memory, a processor, and a computer program stored in the memory and runable on the processor, wherein the processing When the computer program is executed by the computer, the functions of the system in Embodiment 7 of the present disclosure can be realized.
  • a computing device for example, it can be a server device
  • FIG. 6 shows a schematic diagram of the hardware structure of this embodiment.
  • the electronic device 9 specifically includes:
  • At least one processor 91 at least one memory 92, and a bus 93 for connecting different system components (including the processor 91 and the memory 92), wherein:
  • Bus 93 includes a data bus, an address bus and a control bus.
  • Memory 92 includes volatile memory, such as random access memory (RAM) 921 and/or cache memory 922, and may further include read-only memory (ROM) 923.
  • RAM random access memory
  • ROM read-only memory
  • Memory 92 also includes a program/utility 925 having a set of (at least one) program modules 924, such program modules 924 including, but not limited to: an operating system, one or more application programs, other program modules, and program data, examples of which Each of these, or some combination thereof, may include the implementation of a network environment.
  • program modules 924 including, but not limited to: an operating system, one or more application programs, other program modules, and program data, examples of which Each of these, or some combination thereof, may include the implementation of a network environment.
  • the processor 91 executes computer programs stored in the memory 92 to execute various functional applications and data processing to implement the functions of the system in Embodiment 7 of the present disclosure.
  • Electronic device 9 further may communicate with one or more external devices 94 (eg, keyboard, pointing device, etc.). This communication may occur through an input/output (I/O) interface 95. Furthermore, the electronic device 9 may also communicate with one or more networks (eg, a local area network (LAN), a wide area network (WAN), and/or a public network, such as the Internet) through the network adapter 96. Network adapter 96 communicates with other modules of electronic device 9 via bus 93 . It should be understood that, although not shown in the figures, other hardware and/or software modules may be used in conjunction with the electronic device 9, including but not limited to: microcode, device drivers, redundant processors, external disk drive arrays, RAID (disk array) systems, tape drives, and data backup storage systems.
  • RAID disk array
  • Embodiment 9 Computer-readable storage medium
  • An embodiment of the present disclosure provides a computer-readable storage medium on which a computer program is stored.
  • the program is executed by a processor, the functions of the system described in Embodiment 7 of the present disclosure are implemented.
  • the readable storage medium that can be used may more specifically include but is not limited to: portable disk, hard disk, random access memory, read-only memory, erasable programmable read-only memory, optical storage device, magnetic storage device or any of the above. The right combination.
  • the present disclosure can also be implemented in the form of a program product, which includes program code.
  • program product which includes program code.
  • the program code is used to cause the terminal device to execute the implementation. Functions of the system described in Embodiment 7 of the present disclosure.
  • program code for executing the present disclosure can be written in any combination of one or more programming languages, and the program code can be completely executed on the user device, partially executed on the user device, as an independent
  • the software package executes partially on the user device, partially on the remote device, or entirely on the remote device.

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Abstract

一种检测实验动物样本中CAR拷贝数的方法，其包括以下步骤：(1)获得所述样本中的CAR拷贝数；(2)获得所述样本中人内参的拷贝数及实验动物内参的拷贝数作为双内参组合以校正所述样本的投入量，由此获得校正后的CAR拷贝数。基于CAR通用序列设计检测方法，具有通用性；首次使用双内参，可以对上样量进行有效的校正，进而保证每微克gDNA中转基因拷贝数的数据的可靠性。

Description

一种检测CAR拷贝数的方法 技术领域
本公开属于基因分析检测技术领域，具体涉及定量检测生物样本中CAR拷贝数的通用型方法及其应用。
背景技术
嵌合抗原受体(Chimeric Antigen Receptor，CAR)是一种合成的跨膜受体，可赋予细胞抗原结合特异性。在细胞治疗领域中，使用转基因技术在免疫细胞表面表达CAR，将CAR转基因免疫细胞扩增并注入患者体内，用以特异性靶向攻击和摧毁病灶。在B细胞恶性肿瘤的CAR-T细胞治疗中，观察到了显著的临床反应和高完全缓解率。
整合到细胞基因组中的CAR拷贝数将直接决定这些基因工程细胞的临床效力(Journal of translational medicine,2020,18(1):1-7；Mol Ther.2007；15(3):445–56.)。因此在CAR相关产品开发、制备、体内体外实验过程中作为细胞治疗药物的药代动力学指标的CAR拷贝数检测是其中至关重要一个环节。
目前，通常有两种生物分析方法可用于检测和量化生物基质中的CAR-转基因细胞，即基于CAR基因拷贝数检测的定量聚合酶链式反应(qPCR)，及基于细胞表面CAR蛋白表达检测的流式细胞术。但是流式细胞检测要求新鲜有活力的细胞样本及特异性的CAR抗体，因此在一次包含大量样本的动物实验中的应用可行性极低。
现有的技术中主要存在以下几个缺陷：
(1)现有流式方法均为针对CAR不同结构区域的检测方法，检测方法仅针对特定的CAR产品，方法不具有通用性；其试验设计通常会包含数周内多个时间点的样本检测，每个时间点的样本必须现取现测，费时费力，且批次间的检测结果容易出现差异。(2)目前,在使用qPCR对CAR相关免疫治疗产品的药代动力学检测中缺乏对样本投入量进行准确校正的方法，进而也无法对CAR的拷贝数进行准确校正。
发明内容
为解决现有技术中的上述缺陷，本公开提供了一种检测CAR拷贝数的方法，以及该方法中使用的引物探针组合、试剂盒及其应用。
在CAR相关细胞治疗领域已发表的文章或者市面产品中，未发现使用人和小鼠双内参基因对两个物种的内参基因同时进行检测，并对投入量进行准确校正。本公开在实践 中发现，小鼠血液中的RNA含量非常高(可能有人类血液的10倍)，实际很难提取到很纯的DNA。因此，无论使用分光光度计(比如nanodrop)或者荧光计(比如Qubit)进行精确的DNA定量，其检测结果均严重偏离真实DNA浓度(过高的RNA含量会干扰DNA的定量)，进而影响最后的拷贝数报告值。另外，因为在实际试验中，人类细胞来源的CAR-T细胞在小鼠体内扩增后在小鼠血液中的比例会达到相当大的一个比例，比如～50％(Annals of Translational Medicine,2020,8(9):Figure 6)。因此，单独使用小鼠或人的内参基因进行投入量校正仍无法得到准确的DNA投入量。
本公开尝试通过检测人和小鼠的双内参基因进行准确的DNA投入量校准。然而，目前未在CAR相关细胞治疗领域已发表的文章或者市面产品中发现有明确使用多组引物探针组对两个物种的内参基因同时进行检测以对两个物种DNA投入量进行校正的计算公式。另外，人的基因组的重量有业界的共识，即为6.6pg/双倍体人细胞(The American Journal of Human Genetics,1999,64(1):218-224)，而小鼠等其他物种的基因组的重量并没有共识，本公开推断出实验小鼠的基因组的重量，将人、小鼠两个物种的内参基因拷贝数同时用于对投入量的校正，并据此提出了相应的计算公式。
由此，本公开的技术方案之一涉及一种检测生物样本中CAR拷贝数的方法，其包括以下步骤：
(1)获得所述生物样本中的初始CAR拷贝数；
(2)获得所述生物样本中人内参的拷贝数及实验动物内参的拷贝数作为双内参组合以校正所述样本的投入量，由此获得校正后的CAR拷贝数。
所述实验动物为注入表达CAR的人源细胞的动物，优选为实验小鼠。实验动物样本注入表达CAR的人源细胞后成为混合样本，通过定量检测样本的三个指标，即：初始CAR拷贝数，人内参的拷贝数和实验动物内参的拷贝数，通过内参对样本的投入量进行校正，获得校正后的CAR拷贝数。
在一些实施例中，本公开所述的方法可应用于所有生物流体和组织样本。在一些实施例中，生物基质样本选自血液、内脏或肿瘤匀浆。因此，在一些实施例中，生物流体样本是血液。在一些实施例中，内脏样本是肝脏或肾。在一些实施例中，组织样品是肿瘤匀浆。
在一些实施例中，所述生物样本来自实验动物组织或全血。优选为组织或全血的基因组DNA。更优选为全血基因组DNA。
在一些实施例中，使用定量PCR法检测所述初始CAR拷贝数、人内参的拷贝数及小鼠内参的拷贝数，优选使用荧光定量PCR更优选ddPCR。
在一些实施例中，步骤(1)中，通过检测CAR的通用载体序列来检测所述样本的初始CAR拷贝数。
所述方法是一种定量检测CAR基因拷贝数的通用型方法。如图1所示，引物及探针针对CAR载体的LTR例如5’LTR附近区域进行设计，方法具有通用性，无需对不同的CAR-T产品重新设计引物探针。因此，在一些实施例中，所述LTR的检测使用以下引物探针组合：如SEQ ID NO:1所示的上游引物，如SEQ ID NO:2所示的下游引物，如SEQ ID NO:3所示的探针。
双内参的引入是为了区分样本中人和小鼠的基因组，内参基因的选择具有较强的可替代性。在一些实施例中，步骤(2)中，所述非同源区为人和小鼠同源基因或非同源基因的非同源区。
在一些实施例中，所述非同源区为人和小鼠管家基因的非同源区。较佳地，所述非同源区来源但不限于以下基因的非同源区：ApoB、ACTB、ALB、tubulin、GAPDH、actin、RNase P、B2M、HPRT和TBP。
在一些具体的实施例中，所述双内参组合为：人的ApoB基因26号外显子，及小鼠的ApoB基因26号外显子。或，所述双内参组合为：人的ACTB基因及鼠的ACTB基因，其前1000bp是不同源的。或，所述双内参组合为：人的ApoB基因26号外显子，及鼠的ACTB基因前1000bp。
现有的在小鼠模型中检测CAR拷贝数的定量PCR仅以单内参校正样本投入量,但在小鼠模型中会注入表达CAR的人源细胞，且人和小鼠的基因组相差16％，根据单内参校正样本的投入量是不准确的。除此之外，在绝对定量PCR中，本领域其他检测方法大多是直接以分光光度计/荧光计的测量值来当作投入量的，而并不会引入内参来校正投入量。本公开开创性地在定量PCR中利用人和小鼠双内参校正样本投入量以获得更加准确的CAR在小鼠模型中的药代动力学结果。
以及使用双内参下的计算公式，该公式基于工业界共识的每个人细胞的基因组重量为6.6pg，以及根据小鼠及人的最新大版本的基因组大小的比例计算得到小鼠的基因组重量为5.6pg(人的GTCh38版本的基因组大小为3,209,286,105bp，小鼠的GRCm39版本的基因组大小为2,728,222,451bp，因此计算小鼠的基因组重量为：6.6×272822451/3209286105≈5.6pg)。
在一些实施例中，所述CAR拷贝数通过以下公式来校正：

其中，等式左边的CAR拷贝数为校正后的拷贝数；等式右边的CAR拷贝数为初始CAR拷贝数，即通过检测手段直接获得的CAR拷贝数。
在一些实施例中，人基因组拷贝数通过以下公式来校正：
其中，等式左边的人基因组拷贝数为校正后的拷贝数；等式右边的内参拷贝数为内参基因的初始拷贝数，即通过检测手段直接获得的拷贝数。
在一些实施例中，所述双内参组合为人ApoB基因和小鼠ApoB基因。
较佳地，所述ApoB基因的检测使用以下引物探针组合：
(a)用于检测人的ApoB基因的引物探针组合，包括：如SEQ ID NO:4所示的上游引物，如SEQ ID NO:5所示的下游引物，如SEQ ID NO:6所示的探针；
(b)用于检测小鼠的ApoB基因的引物探针组合，包括：如SEQ ID NO:7所示的上游引物，如SEQ ID NO:8所示的下游引物，如SEQ ID NO:9所示的探针。
当所述双内参组合为人ApoB基因和小鼠ApoB基因时，所述CAR拷贝数通过以下公式来校正：
人基因组拷贝数通过以下公式来校正：
更佳地，步骤(1)前，还包括(0)从生物样品中提取基因组DNA。
本公开的技术方案之二涉及一种用于检测CAR拷贝数的引物探针组合，所述引物探针组合包括：如SEQ ID NO:1所示的上游引物，如SEQ ID NO:2所示的下游引物，如SEQ ID NO:3所示的探针。
本公开的技术方案之三涉及一种用于检测ApoB的引物探针组合，其包括：
如SEQ ID NO:4所示的上游引物，如SEQ ID NO:5所示的下游引物，如SEQ ID NO:6所示的探针；和/或，
如SEQ ID NO:7所示的上游引物，如SEQ ID NO:8所示的下游引物，如SEQ ID NO:9所示的探针。
本公开的技术方案之四涉及一种引物探针组，其包括如技术方案之二所述的用于检测CAR拷贝数的引物探针组合和如技术方案之三所述的用于检测ApoB的引物探针组合。
本公开的技术方案之五涉及一种用于检测CAR拷贝数的试剂盒，其包括如本公开的技术方案之二所述的引物探针组合。
在一些具体实施例中，所述试剂盒还包括用于检测人和小鼠的ApoB基因的引物探针组合，其中，
用于检测人的ApoB基因(hApoB)的引物探针组合包括：如SEQ ID NO:4所示的上游引物，如SEQ ID NO:5所示的下游引物，如SEQ ID NO:6所示的探针；
用于检测小鼠的ApoB基因(mApoB)的引物探针组合包括：如SEQ ID NO:7所示的上游引物，如SEQ ID NO:8所示的下游引物，如SEQ ID NO:9所示的探针。
在一些实施例中，所述的试剂盒包括包含一部分慢病毒载体序列、一部分hApoB序列和一部分mApoB序列的阳性对照质粒和阴性对照质粒中的一种或多种。
在一些具体的实施例中，一部分慢病毒载体序列、一部分hApoB序列、一部分mApoB序列的核苷酸序列如SEQ ID NO:22所示。
在一些具体的实施例中，所述的试剂盒还包括使用说明书，所述使用说明书中记载：
所述CAR拷贝数通过以下公式来校正：
和/或，人基因组拷贝数通过以下公式来校正：
本公开的技术方案之六涉及一种如技术方案之二或三所述的引物探针组合、技术方案之四所述的引物探针组或技术方案之五所述的试剂盒在检测CAR拷贝数或制备检测CAR拷贝数的诊断剂中的用途。
本公开的技术方案之七涉及一种检测生物样本中CAR拷贝数的系统，所述系统包括：
(A)输入模块：将初始CAR拷贝数、人内参的拷贝数、实验动物内参的拷贝数、对应人内参检测体系样本投入量和对应实验动物内参检测体系样本投入量导入到所述输入模块；实验动物例如为小鼠；
(B)计算模块：所述输入模块获得的信息进入所述计算模块，以校正CAR拷贝数和人基因组拷贝数；其中，所述CAR拷贝数通过以下公式来校正：
和/或，所述人基因组拷贝数通过以下公式来校正：
在一些具体的实施例中，(A)中所述输入模块获得的信息来自如本公开的技术方案之一所述的方法。
在一些优选的实施例中，所述系统还包括(C)打印模块：打印(B)中所述计算模块获得的结果。
本公开的技术方案之八涉及一种电子设备，其包括存储器和处理器；所述存储器包括存储在其中的可在处理器上运行的计算机程序；其中，
所述处理器执行所述计算机程序时实现如本公开的技术方案之七所述的系统的功能。
本公开的技术方案之九涉及一种计算机可读存储介质，其上存储有计算机程序，其中，所述计算机程序被处理器执行时可实现如本公开的技术方案之七所述的系统的功能。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本公开各较佳实例。
如未特殊说明，本公开所用试剂和原料均市售可得。
本公开的积极进步效果在于：
1.本公开使用双内参，可以对上样量进行有效的校正，进而保证每微克gDNA中转基因拷贝数的数据的可靠性；
2.本公开使用双内参基因还可以跟踪人源细胞在实验动物体内的扩增情况；
3.本公开涉及的理论投入量范围1～100000个拷贝，对样本的上样量需求较低，因此可以节省小动物的采样量例如取血量；
4.本公开基于CAR的通用序列，例如LTR序列设计探针组，无需针对CAR产品的不同而重新设计引物探针组，方法具有通用性；
5.本公开首次提出人和小鼠双内参校正投入量的计算公式。
附图说明
图1 CAR通用引物探针设计位置示意图。
图2 CAR检测线性拟合结果。
图3 mApoB检测线性拟合结果。
图4 hApoB检测线性拟合结果。
图5检测样本中CAR拷贝数的系统。
图6本公开的电子设备示意图。
具体实施方式
下面通过实施例的方式进一步说明本公开，但并不因此将本公开限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
定义
术语“动物”是指动物界的任何成员。在一些实施方案中，“动物”是指处于任何发育阶段的人。“实验动物”一般为功能、代谢、结构及疾病性质与人相似的、专门培育供实验用的非人动物，主要指作为医学、药学、生物学、兽医学等的科研、教学、医疗、鉴定、诊断、生物制品制造等需要为目的而驯养、繁殖、育成的动物。在一些实施方案中，实验动物是指处于任何发育阶段的非人动物。在一些实施方案中，非人动物是哺乳动物，例如小鼠、大鼠、兔子、狗、猫、羊、牛、猪、马和/或灵长类动物例如猴子。在一些实施例中，实验动物包括但不限于哺乳动物、鸟类、爬行动物、两栖动物、鱼类、昆虫和/或蠕虫。在一些实施方案中，实验动物可以是转基因动物、基因工程动物和/或克隆。在本文的一些实施方案中，实验动物为注入人源CAR转基因细胞的动物。
“实验小鼠”由野生小鼠经过长期选择培育而成，属于脊椎动物门，哺乳纲、啮齿目(Rodentia)、鼠科(Muridae)、鼠属(Mus)动物。实验小鼠体小，饲养管理方便，易于控制；生产繁殖快，已拥有大量的具有不同特点的近交系、突变系和封闭群，并已形成多种国际公认的标准品系，被广泛应用于各种实验研究中。
“嵌合抗原受体”(Chimeric Antigen Receptor，CAR)是一种合成的跨膜受体，可赋予细胞(例如NK细胞、T细胞如幼稚T细胞、中枢记忆T细胞、效应记忆T细胞或其组合)抗原特异性。在一些实施方案中，CAR包括抗原特异性靶向结构域、胞外结构域、跨膜结构域、任选的一个或多个共刺激结构域和胞内信号结构域。
术语“工程化的”(engineered)是指修饰细胞基因组的方法，包括但不限于删除编码或非编码区或其一部分，或插入编码区或其一部分。在一些实施方案中，修饰的细胞可以从患者或供体获得。在一些实施方案中，修饰的细胞为淋巴细胞(例如T细胞)、巨噬细胞(例如肿瘤相关巨噬细胞，TAM)。在一些实施方案中，修饰细胞以表达外源构建体，例如CAR或T细胞受体(TCR)，由此获得CAR-T或CAR-M细胞。
术语“受试者”指人或任何非人动物，例如小鼠、大鼠、兔子、狗、猫、羊、牛、猪、马和/或灵长类动物(例如猴子)。在一些实施例中，受试者指的是到医疗提供处进行疾病的诊断或治疗的人。受试者在本文中可与“个体”或“患者”互换使用。受试者可能患有或易患疾病或紊乱，但可能表现出或不表现出疾病或紊乱的症状。在一些实施例中，受试者被诊断出患有或表现出一种或多种疾病、紊乱和/或病症的症状或特征。该疾病可能包括癌症，例如淋巴瘤和白血病。
术语“样本”(specimen，sample)是观测或调查的一部分个体，总体是研究对象的全部。生物样本通常是指植物的花、叶、茎、根、种子等，动物(包括人)的体液(如尿、血、唾液、胆汁、胃液、淋巴液及生物体的其他分泌液等)、毛发、肌肉和一些组织器官(如胸腺、胰腺、肝、肺、脑、胃、肾等)以及各种微生物。在本文的一些实施方案中，生物样本指来自实验动物的组织样本，或从组织中提取的DNA。“组织”为机体中构成器官的单位，是由许多形态和功能相同的细胞按一定的方式结合而成的。人和动物具有上皮组织、结缔组织、肌肉组织和神经组织。“全血”指将人体内血液采集到采血容器内所形成的混合物，即包括血细胞和血浆的所有成分。
如本文所用，术语“治疗”是指用于部分或完全缓解、改善、减轻、抑制、预防、延迟特定疾病、紊乱和/或病症的一种或多种症状或特征的发作、降低其严重性和/或降低其发生率的任何方法。可对未表现出疾病体征和/或仅表现出疾病早期体征的受试者给予治疗，以降低发生与疾病相关的病理的风险。
术语“聚合酶链式反应”(PCR)是一种用于放大扩增特定的DNA片段的分子生物学技术，利用DNA在体外高温时变性会变成单链，低温时引物与单链按碱基互补配对的原则结合，再调温度至DNA聚合酶最适反应温度，DNA聚合酶沿着磷酸到五碳糖(5'-3')的方向合成互补链。
术语“引物”指在核苷酸聚合作用起始时刺激合成启动的一种具有特定寡核苷酸序列的大分子。引物通常是人工合成的两段寡核苷酸序列，一个引物与靶区域一端(例如5'端)的一条DNA模板链互补(称上游引物或正向引物)，另一个引物与靶区域另一端(例如3'端)的另一条DNA模板链互补(称下游引物或反向引物)，其功能是作为核苷酸聚合作用的起始点，核酸聚合酶可由其3'端开始合成新的核酸链。体外人工设计的引物被广泛用于聚合酶链反应、测序和探针合成等。
术语“荧光探针”利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)的原理，其通常包括一条寡核苷酸序列和一对能量供体和受体。其中供体的发射光谱与受体的吸收光谱重叠，当它们在空间上相互接近到一定距离(例如1～10nm)时，激发供体而产生的荧光能量正好被附近的受体吸收，使得供体发射的荧光强度衰减，受体荧光分子的荧光强度增强。在目标核酸扩增过程中，荧光探针的供体与受体的空间距离被破坏后打破能量的传递(例如通过供体被从荧光探针上切割下来而实现)，从而供体发射的荧光不能被受体吸收而被荧光检测系统探测到。通常每扩增一条DNA链，就对应有一个游离的荧光分子(报告基团)形成，保证了荧光信号的累积与PCR产物形成完全同步，因此对荧光信号进行检测就可以实时监控PCR的过程，准确定量PCR的起始拷贝数。本公开中，引物通常与荧光探针配合使用。
实时荧光定量PCR(Quantitative PCR，qPCR)是一种在DNA扩增反应中，以荧光化学物质测每次PCR循环后产物总量的方法。通过内参或者外参法对待测样品中的特定DNA序列进行定量分析的方法。常见的qPCR包括SYBRGreen和TaqMan探针法等。
如本文所用，“ddPCR”即液滴数字聚合酶链式反应(Droplet Digital Polymerase Chain Reaction)，是一种允许基于单分子PCR进行绝对核酸定量的方法。利用微流控技术或其他液滴生成方法，将稀释的核酸溶液分散到微孔或液滴中，使每个微孔或每个液滴的靶核酸的理想数量小于或等于一个。扩增后，带有靶分子的反应发出阳性荧光信号，而没有靶分子的反应发出阴性荧光信号，根据相对比率和反应的体积，可以得出原始核酸溶液的核酸浓度。与传统的定量PCR不同，ddPCR可以通过直接计数和泊松修正分析实现起始DNA模板的绝对定量。
“基因组”(genome)是指生物体所有遗传物质的总和。这些遗传物质包括DNA或 RNA(病毒RNA)。对单倍体而言，基因组表示这种生物的总DNA或RNA。对于二倍体的高等生物，其配子的DNA总和即为一组亚基因组。二倍体有两份同源的亚基因组，真核生物细胞中有几个染色体组，就有几个亚基因组。携带合成蛋白质遗传信息的DNA序列是编码序列，不同物种中编码序列占基因组的比例差异很大。非编码序列包括内含子，非编码RNA的序列，调控DNA和重复DNA。较大的基因组不一定含有更多的基因，并且复杂真核生物中非重复DNA的比例随着基因组大小的增加而减少，例如人类基因组的98％属于非编码序列。
术语“内参基因”是在基因定量检测中，为控制由于实验技术或样本差异带来的实验误差而引入的实验参照，用于衡量实验结果的准确性并在必要时对实验结果进行校正的基因。例如，在qPCR中，实验程序的不同步骤相关的几个变量可能导致相当大的样品间差异，并可能导致错误的结果：起始材料的不同数量和质量、RNA完整性、cDNA合成和PCR扩增的效率以及组织或细胞之间总体转录活性的差异。内参基因的使用通常被认为是标准化qPCR和减少基因表达定量中可能产生的误差的最可靠方法。在这种标准化策略中，内参基因被用作内部对照，并采用与GOI相同的实验方案，然后根据内部对照的值对目标基因的表达水平进行标准化。内参基因通常选用管家基因。
“管家基因”(house-keeping genes)是指在同一生物体的所有细胞中均稳定表达的一类基因，受环境因素影响较小，其产物是对维持细胞基本生命活动所必需的。它的表达通常只受启动序列或启动子与RNA聚合酶相互作用的影响，而不受其他机制调节，如微管蛋白基因、糖酵解酶系基因与核糖体蛋白基因等。
常见的管家基因还包括甘油醛-3-磷酸脱氢酶(G3PDH，又称GAPDH)、白蛋白、肌动蛋白(actin)、微管蛋白(tubulin)、人β2-微球蛋白(B2M)、TATA结合蛋白(TATA-binding protein，TBP)、亲环素、次黄嘌呤磷酸核糖转移酶(HRPT)和核糖核酸酶P(RNase P)等。例如，GAPDH为重要的糖酵解途径酶；Albumin基因编码了人类血液中最丰富的白蛋白(即人血清白蛋白，ALB)；肌动蛋白包括β、γ-肌动蛋白，微管蛋白包括α、β-微管蛋白，它们对细胞骨架的结构和动力学至关重要。核糖核酸酶P(RNase P)由蛋白质和RNA两种亚单位组成，是负责tRNA前体5'端成熟的酶。
ApoB为编码apolipoprotein B(载脂蛋白B)的基因，位于第二号染色体2p24-p23位置。ApoB基因全长42.6kb，共有29个外显子，mRNA全长14.1nt，编码4,564个氨基酸残基组成的蛋白。ApoB蛋白在血浆中以两种形式的亚型存在，一种为ApoB48，另一种为ApoB100，这两种形式的ApoB蛋白都是由同一个基因同一个长度超过16kb的mRNA前转录子进行编码的，共用相同的氨基末端。
“同源基因”是由一个共同祖先在不同物种中遗传的基因。虽然同源基因在序列上是相似的，但相似的序列不一定同源。同源基因包括直系同源基因、旁系同源基因和异源同源基因。直系同源基因(orthologous gene)是指从同一祖先垂直进化而来的基因。旁系同源基因(paralogous gene)是指由于基因复制而产生的同源基因，例如人γ-珠蛋白基因和β-珠蛋白基因。异源同源基因(xenologous gene)是由于基因在不同物种间的横向转移(horizontal transfer)而产生的。“非同源基因”即同一物种或不同物种中的由不同祖先遗传的基因。例如，在本文中，人的ApoB和小鼠的ApoB属于同源基因；但人的ApoB和人或小鼠的GAPDH属于非同源基因。
术语“同源区”指同源基因中具有高度保守性的区域，通常出现在同个亚基因组或不同祖先来源的亚基因组的成对染色体间。术语“非同源区”指同源基因中不具有高度保守性的区域，也可以指非同源基因相互之间不具有保守性的区域。
术语“LTR”(long terminal repeat)即长末端重复序列，包括5’LTR和3’LTR。向T细胞引入CAR需要使用携带CAR表达盒的基因递送载体，目前通常使用逆转录病毒例如慢病毒作为CAR递送载体。逆转录病毒转导T细胞后，病毒基因组上的两个LTR之间的CAR表达盒随机整合至宿主细胞基因组，从而实现CAR的长期稳定表达。本公开的一些实施例中，以LTR为目标序列来设计通用型的CAR的拷贝数检测用的引物和/或探针对。
术语“拷贝数”，是指某基因或某一段特定的核苷酸序列在某一生物的单倍体基因组中的个数。拷贝数的测定是分子生物学研究常用的技术，例如克隆某些重复序列、测定外源基因在转染培养细胞中和转基因动植物中整合的拷贝数等，都需要对所研究目的基因或序列的拷贝数进行测定。单拷贝即基因或序列在该生物的单倍体基因组中只有一个，多拷贝则指有多个。通常可以通过测序、southern blot、实时荧光定量PCR或数字PCR等技术进行拷贝数的检测。
在本申请中，“或”的使用表示“和/或”，除非另有说明。如本文所用，单数形式“一个”、“一种”和“所述”包括单数和复数指代物，除非上下文另有明确说明。
实验方法与试剂
本公开提及的LTR引物探针组用于检测样本中CAR(实际设计在载体通用序列LTR附近)的拷贝数及扩增情况；人ApoB(hApoB)的引物探针组用于检测样本中hApoB的拷贝数及扩增情况；小鼠ApoB(mApoB)的引物探针组用于检测样本中mApoB的拷贝数及扩增情况。
为保证结果的准确性，一个反应体系最多可以设置两个靶基因的检测，即CAR， hApoB及mApoB的检测可放在2-3个反应体系中进行(包括其中某个基因的重复检测)。DNA样本的投入量为10ng～50±0.1ng，不同反应体系的样本投入量可以不同。下述仅列举其中的一种情况。
本公开提供用于检测CAR(实际设计在载体通用序列LTR附近，见图1)，hApoB以及mApoB基因的上游引物(F)，下游引物(R)和探针(P)，其序列及修饰信息如表1所示，其中，FAM及HEX为荧光修饰基团，MGB-NFQ及BHQ-1为淬灭基团：
表1.本公开所涉及的引物/探针序列及其修饰信息
本公开中，探针的修饰基团并不作为限制条件，可依需更换。
所述定量检测CAR基因拷贝数通用型方法包括如下步骤：
1.引物探针准备
表2引物探针工作浓度

2.质控品准备
(1)提取CAR-T细胞及小鼠全血基因组DNA(gDNA)，随后使用ddPCR绝对定量，并根据定量的结果制备阳性对照品。
(2)阴性对照品：以CAR阴性小鼠gDNA作为阴性对照品。
(3)阳性对照品：以1×low TE[IDTE(1X TE Solution)，购自IDT公司]作为稀释液，利用CAR-T细胞gDNA及CAR阴性小鼠gDNA制备为约1％CAR阳性的参考品。
(4)制备的质控品(阴阳性对照品)，分装储存在≤-15℃
3.反应体系制备
每个样本进行检测时配制两组PCR体系：一组检测CAR，引物探针混合液为表1中所述的LTR引物探针混合液，样本投入量为50ng；另一组同时检测hApoB及mApoB，引物探针混合液为表1中所述的hApoB及mApoB引物探针混合液，样本投入量为10ng。其中2×digital PCR supermix购自BioRad，货号1863024。
表3PCR体系
*第一组体系及第二组体系的投入量可以不一致，且所述数值范围误差值≤0.1ng。
4.微滴生成
(1)将微滴发生卡放入配套的底座中
(2)将配制好的20μL PCR体系加入到芯片SAMPLE孔，如样本不足8孔以水补齐，注意在加样过程中避免产生气泡，如有气泡，以枪头扎破
(3)在OIL孔中各加入70μL微滴生成油
(4)盖上密封垫(gasket)，将芯片及底座放置于微滴生成仪中，开始生成微滴。
(5)微滴生成过程结束后，将DROPLETS孔中的样本转移40μl至96孔板中
(6)微滴转入96孔板内后，将热封膜覆盖于96孔板上。然后放入预热好的PCR热 封仪中进行封膜，运行程序为：180℃，10s。
5.PCR反应
将热封后的96孔板加入PCR仪，并按照以下程序进行PCR扩增
表4 PCR运行程序
6.微滴读取和分析
(1)将PCR反应结束后的96孔板放入微滴读取仪
(2)参数设置：打开QuantaSoft软件，双击任意样本孔在新弹窗内进行设置：
依次设置Name(实验名称)，Experiment(ABS)，Supermix(ddPCRTMSupermix for Probes(No dUTP))，Target Name(LTR/mApoB/hApoB)，Target Type(Ch1Unknown/Ch2Unknown)
完成设置后点击“Apply”应用。完成所有编辑后点击“Run”运行。
(3)阈值设置：手动设置Channel 1及Channel 2的阈值，使阴阳性微滴正确区分开。Channel 1阈值设置为6000；Channel 2阈值设置为4000。若有明显偏离阈值的点，可通过套索工具进行微调
(5)结果分析
根据以下公式计算每μg基因组DNA中CAR的拷贝数：
此时双内参为人和小鼠APOB基因的非同源区，因此得如下公式：
根据以下公式计算每μg基因组DNA中人单倍体基因组的拷贝数：
此时双内参为人和小鼠APOB基因的非同源区，因此得如下公式：
实施例1 特异性验证
本公开涉及的CAR-T样本为南京传奇生物科技有限公司自制的样本(见WO2020043152A1，Example 3)。所述CAR靶向间皮素(mesothelin)，其从C端到N端的结构依次为CD8α信号肽，间皮素结合结构域，CD8α铰链区，CD8α跨膜区，CD137胞内域以及CD3ζ胞内共刺激域。在本公开的案例中，由其提取的DNA可由国际标准品(WHO 1st Reference Reagent for Lentiviral Vector Integration Site，NIBSC code:18/144)替代。
本公开涉及到的阳性质粒(PCP)为金斯瑞提供(货号SC1317)，其为包含一部分慢病毒载体序列、一部分hApoB序列和一部分mApoB序列的线性化pcDNA3.1(+)质粒。阳性质粒包含的一部分慢病毒载体序列、一部分hApoB序列和一部分mApoB序列的核苷酸序列如SEQ ID NO:22所示。本公开涉及到的阴性质粒(NCP)为pcDNA3.1(+)质粒。
SEQ ID NO:22

对各种空白对照、阴阳性对照品应用本公开的方法进行多个孔的检测，下表每个数字代表一个孔的检测结果：
表5 特异性验证结果

如上表，以5拷贝为cutoff值，CAR引物探针组在正常的小鼠及人基因组中均无阳性检出，hApoB只在正常人基因组有阳性，mApoB也只在正常小鼠基因组有阳性,说明该方法特异性很好。
实施例2 CAR基因检测准确性、重复性及线性验证
将不同拷贝数的阳性质粒混合到小鼠全血DNA中，应用本公开进行CAR拷贝数检测：
表6 CAR检测准确性、重复性及线性验证结果
如表5及图2，阴性样本(小鼠全血DNA)加入1×101、1×102、1×103、1×104、4×104拷贝数的PCP(阳性质粒质控品)的样本进行3次重复检测，所有样本均可检出，CAR拷贝数的其回收率在101.77％～112.94％范围内，CV均不超过30％，CAR的拷贝数与供试品浓度成线性关系，且其R2均大于0.999。上述结果证明，使用该方法对CAR检测的 准确性、重复性及线性较好。
实施例3 mApoB基因检测准确性、重复性及线性验证
将不同拷贝数的阳性质粒分别混合到人DNA中，应用本公开进行mApoB检测：
表7 mApoB检测准确性、重复性及线性验证结果
如表6及图3，阴性样本(人DNA)加入1×101、1×102、1×103、1×104、4×104拷贝数的PCP(阳性质粒质控品)的样本进行3次重复检测，所有样本均可检出，mApoB拷贝数的其回收率在104.72％～110.15％范围内，CV均不超过30％，mApoB的拷贝数与供试品浓度成线性关系，且其R2均大于0.999。上述结果证明，使用该方法对mApoB检测的准确性、重复性及线性较好。
实施例4 hApoB基因检测准确性、重复性及线性验证
将不同拷贝数的阳性质粒混合到小鼠全血DNA中，应用本公开进行hApoB拷贝数检测：
表8 hApoB检测准确性、重复性及线性验证结果

如表7及图4，阴性样本(小鼠全血DNA)加入1×10、1×102、1×103、1×104、4×104拷贝数的PCP(阳性质粒质控品)的样本进行3次重复检测，所有样本均可检出，hApoB拷贝数的其回收率在85.63％～111.70％范围内，CV均不超过30％，hApoB的拷贝数与供试品浓度成线性关系，且其R2均大于0.999。上述结果说明，本方法对hApoB基因检测的准确性、重复性及线性较好。
实施例5 公开方法精密度验证
将CAR-T细胞DNA与小鼠全血DNA混合在一起，配制成CAR-T细胞与小鼠全血细胞比例理论0.4％(CAR-T与小鼠全血gDNA的质量比)的样本，对这个样本不同时间用不同人员进行测试：
表9 精密度验证结果

数据如上表，组间CV均小于30％，说明本公开方法精密度很好。
按照平均值根据以下公式计算每μg基因组DNA中CAR的拷贝数：
即为：(48.67÷50)÷(69.28×3.3÷10+602.72×2.8÷10)×1000000＝5079.7拷贝/μg gDNA。
按照平均值根据以下公式计算每μg基因组DNA中人单倍体基因组的拷贝数：
即为：(69.28÷10)÷(69.28×3.3÷10+602.72×2.8÷10)×1000000＝36154.1拷贝/μg gDNA。
实施例6 CAR通用引物探针组合的优化
本公开对CAR通用引物探针组合进行了优化，以下(表10)为部分引物探针组合的例子，经试验发现引物探针组合5的效果最佳，因此选择用于本公开上述实施例中。实验方法同实施例1前具体实施方式部分。
表10 部分引物探针组合

实施例7 检测CAR拷贝数的系统
本实施例提供了一种系统，其如图5所示，包括(1)输入模块、(2)计算模块；可选地还包括(3)打印模块。
(1)输入模块：将前述的实施例获得的CAR拷贝数、人内参的拷贝数、小鼠内参的拷贝数、对应人内参检测体系样本投入量和对应小鼠内参检测体系样本投入量导入到所述输入模块；
(2)计算模块：所述输入模块获得的信息进入所述计算模块，以校正CAR拷贝数和人基因组拷贝数；其中，所述CAR拷贝数通过以下公式来校正：
和/或，所述人基因组拷贝数通过以下公式来校正：
实施例8 电子设备
本实施例提供了一种电子设备，电子设备可以通过计算设备的形式表现(例如可以为服务器设备)，包括存储器、处理器及存储在存储器上并可在处理器上运行的计算机程序，其中处理器执行计算机程序时可以实现本公开实施例7的系统的功能。
图6示出了本实施例的硬件结构示意图，电子设备9具体包括：
至少一个处理器91、至少一个存储器92以及用于连接不同系统组件(包括处理器91和存储器92)的总线93，其中：
总线93包括数据总线、地址总线和控制总线。
存储器92包括易失性存储器，例如随机存取存储器(RAM)921和/或高速缓存存储器922，还可以进一步包括只读存储器(ROM)923。
存储器92还包括具有一组(至少一个)程序模块924的程序/实用工具925，这样的程序模块924包括但不限于：操作系统、一个或者多个应用程序、其它程序模块以及程序数据，这些示例中的每一个或某种组合中可能包括网络环境的实现。
处理器91通过运行存储在存储器92中的计算机程序，从而执行各种功能应用以及数据处理以实现本公开实施例7中的系统的功能。
电子设备9进一步可以与一个或多个外部设备94(例如键盘、指向设备等)通信。这种通信可以通过输入/输出(I/O)接口95进行。并且，电子设备9还可以通过网络适配器96与一个或者多个网络(例如局域网(LAN)，广域网(WAN)和/或公共网络，例如因特网)通信。网络适配器96通过总线93与电子设备9的其它模块通信。应当明白，尽管图中未示出，可以结合电子设备9使用其它硬件和/或软件模块，包括但不限于：微代码、设备驱动器、冗余处理器、外部磁盘驱动阵列、RAID(磁盘阵列)系统、磁带驱动器以及数据备份存储系统等。
应当注意，尽管在上文详细描述中提及了电子设备的若干单元/模块或子单元/模块，但是这种划分仅仅是示例性的并非强制性的。实际上，根据本申请的实施方式，上文描述的两个或更多单元/模块的特征和功能可以在一个单元/模块中具体化。反之，上文描述的一个单元/模块的特征和功能可以进一步划分为由多个单元/模块来具体化。
实施例9 计算机可读存储介质
本公开实施例提供了一种计算机可读存储介质，其上存储有计算机程序，程序被处理器执行时实现本公开实施例7中所述系统的功能。
其中，可读存储介质可以采用的更具体可以包括但不限于：便携式盘、硬盘、随机存取存储器、只读存储器、可擦拭可编程只读存储器、光存储器件、磁存储器件或上述的任意合适的组合。
在可能的实施方式中，本公开还可以实现为一种程序产品的形式，其包括程序代码，当所述程序产品在终端设备上运行时，所述程序代码用于使所述终端设备执行实现本公开本公开实施例7中所述系统的功能。
其中，可以以一种或多种程序设计语言的任意组合来编写用于执行本公开的程序代码，所述程序代码可以完全地在用户设备上执行、部分地在用户设备上执行、作为一个独立的软件包执行、部分在用户设备上部分在远程设备上执行或完全在远程设备上执行。
虽然以上描述了本公开的具体实施方式，但是本领域的技术人员应当理解，这仅是举例说明，本公开的保护范围是由所附权利要求书限定的。本领域的技术人员在不背离本公开的原理和实质的前提下，可以对这些实施方式做出多种变更或修改，但这些变更和修改均落入本公开的保护范围。

Claims (16)

  1. 一种检测生物样本中CAR拷贝数的方法，其特征在于，其包括以下步骤：
    (1)获得所述生物样本的初始CAR拷贝数；
    (2)获得所述生物样本中人内参的拷贝数及实验动物内参的拷贝数作为双内参组合以校正所述样本的投入量，由此获得校正后的CAR拷贝数；
    所述实验动物优选为实验小鼠。
  2. 如权利要求1所述的方法，其特征在于，
    所述生物样本来自实验动物组织或全血；优选为组织或全血的基因组DNA，更优选为全血基因组DNA。
  3. 如权利要求2所述的方法，其特征在于，使用定量PCR法检测所述初始CAR拷贝数、人内参的拷贝数及小鼠内参的拷贝数，优选使用荧光定量PCR更优选ddPCR。
  4. 如权利要求3所述的方法，其特征在于，步骤(1)中，通过检测CAR的通用载体序列来检测所述样本的初始CAR拷贝数；优选所述通用载体序列为LTR；
    较佳地，所述LTR的检测使用以下引物探针组合：如SEQ ID NO:1所示的上游引物，如SEQ ID NO:2所示的下游引物，如SEQ ID NO:3所示的探针。
  5. 如权利要求1～4任一项所述的方法，其特征在于，步骤(2)中，所述内参为人和实验动物的同源基因或非同源基因的非同源区，优选为人和小鼠同源基因或非同源基因的非同源区；
    较佳地，所述内参为人和小鼠管家基因的非同源区；
    更佳地，所述内参来源但不限于以下基因的非同源区：ApoB、ALB、tubulin、GAPDH、actin、RNase P、B2M、HPRT和TBP。
  6. 如权利要求1～5任一项所述的方法，其特征在于，所述初始CAR拷贝数通过以下公式来校正：
    和/或，人基因组拷贝数通过以下公式来校正：

  7. 如权利要求1～6任一项所述的方法，其特征在于，所述双内参组合为人ApoB基因(hApoB)和小鼠ApoB基因(mApoB)；
    较佳地，所述ApoB基因的检测使用以下引物探针组合：
    (a)用于检测人的ApoB基因的引物探针组合，包括：如SEQ ID NO:4所示的上游引物，如SEQ ID NO:5所示的下游引物，如SEQ ID NO:6所示的探针；
    (b)用于检测小鼠的ApoB基因的引物探针组合，包括：如SEQ ID NO:7所示的上游引物，如SEQ ID NO:8所示的下游引物，如SEQ ID NO:9所示的探针；
    更佳地，步骤(1)前，还包括(0)从生物样品中提取基因组DNA。
  8. 一种用于检测CAR拷贝数的引物探针组合，其特征在于，所述引物探针组合包括：如SEQ ID NO:1所示的上游引物，如SEQ ID NO:2所示的下游引物，如SEQ ID NO:3所示的探针。
  9. 一种用于检测ApoB的引物探针组合，其特征在于，所述引物探针组合包括：
    如SEQ ID NO:4所示的上游引物，如SEQ ID NO:5所示的下游引物，如SEQ ID NO:6所示的探针；和/或，
    如SEQ ID NO:7所示的上游引物，如SEQ ID NO:8所示的下游引物，如SEQ ID NO:9所示的探针。
  10. 一种引物探针组，其特征在于，其包括如权利要求8所述的用于检测CAR拷贝数的引物探针组合和如权利要求9所述的用于检测ApoB的引物探针组合。
  11. 一种用于检测CAR拷贝数的试剂盒，其特征在于，其包括如权利要求8所述的引物探针组合；
    较佳地，所述试剂盒还包括如权利要求9所述的引物探针组合。
  12. 如权利要求11所述的试剂盒，其特征在于，所述试剂盒还包括包含一部分慢病毒载体序列、一部分hApoB序列和一部分mApoB序列的阳性对照质粒和阴性对照质粒中的一种或多种；
    较佳地，所述试剂盒还包括使用说明，所述使用说明中记载：
    所述CAR拷贝数通过以下公式来校正：

    和/或，人基因组拷贝数通过以下公式来校正：
  13. 如权利要求8或9所述的引物探针组合、权利要求10所述的引物探针组或权利要求11或12所述的试剂盒在检测CAR拷贝数或制备检测CAR拷贝数的诊断剂中的用途。
  14. 一种检测小鼠样本中CAR拷贝数的系统，其特征在于，所述系统包括：
    (A)输入模块：将初始CAR拷贝数、人内参的拷贝数、小鼠内参的拷贝数、对应人内参检测体系样本投入量和对应小鼠内参检测体系样本投入量导入到所述输入模块；
    (B)计算模块：所述输入模块获得的信息进入所述计算模块，以校正CAR拷贝数和人基因组拷贝数；其中，所述CAR拷贝数通过以下公式来校正：
    和/或，所述人基因组拷贝数通过以下公式来校正：
    较佳地，(A)中所述输入模块获得的信息来自如权利要求1～5所述的方法；
    更佳地，所述系统还包括(C)打印模块：打印(B)中所述计算模块获得的结果。
  15. 一种电子设备，其包括存储器和处理器；所述存储器包括存储在其中的可在处理器上运行的计算机程序；其特征在于，
    所述处理器执行所述计算机程序时实现如权利要求14所述的系统的功能。
  16. 一种计算机可读存储介质，其上存储有计算机程序，其特征在于，所述计算机程序被处理器执行时可实现如权利要求14所述的系统的功能。
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109576799A (zh) * 2018-11-30 2019-04-05 安吉康尔（深圳）科技有限公司 Fh测序文库的构建方法和引物组及试剂盒
CN111334565A (zh) * 2020-03-31 2020-06-26 时力生物科技（北京）有限公司 一种荧光定量pcr检测t淋巴细胞中car基因拷贝数的方法
CN111500690A (zh) * 2020-04-15 2020-08-07 深圳科诺医学检验实验室 一种检测car-t细胞基因组中病毒载体拷贝数的方法
CN112481361A (zh) * 2020-11-30 2021-03-12 北京鼎成肽源生物技术有限公司 检测平均单个car-t细胞中car基因拷贝数的引物组、荧光探针组、试剂盒及方法
US20210332448A1 (en) * 2020-03-09 2021-10-28 Janssen Pharmaceutica Nv Compositions and methods for quantifying integration of recombinant vector nucleic acid
WO2022079489A1 (en) * 2020-10-12 2022-04-21 Takeda Pharmaceutical Company Limited Determination of cellular kinetics and biodistribution for car-t cells
CN114686576A (zh) * 2022-04-18 2022-07-01 武汉科技大学 一种用于检测单个car-t细胞的慢病毒载体拷贝数的方法及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109576799A (zh) * 2018-11-30 2019-04-05 安吉康尔（深圳）科技有限公司 Fh测序文库的构建方法和引物组及试剂盒
US20210332448A1 (en) * 2020-03-09 2021-10-28 Janssen Pharmaceutica Nv Compositions and methods for quantifying integration of recombinant vector nucleic acid
CN111334565A (zh) * 2020-03-31 2020-06-26 时力生物科技（北京）有限公司 一种荧光定量pcr检测t淋巴细胞中car基因拷贝数的方法
CN111500690A (zh) * 2020-04-15 2020-08-07 深圳科诺医学检验实验室 一种检测car-t细胞基因组中病毒载体拷贝数的方法
WO2022079489A1 (en) * 2020-10-12 2022-04-21 Takeda Pharmaceutical Company Limited Determination of cellular kinetics and biodistribution for car-t cells
CN112481361A (zh) * 2020-11-30 2021-03-12 北京鼎成肽源生物技术有限公司 检测平均单个car-t细胞中car基因拷贝数的引物组、荧光探针组、试剂盒及方法
CN114686576A (zh) * 2022-04-18 2022-07-01 武汉科技大学 一种用于检测单个car-t细胞的慢病毒载体拷贝数的方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
THORNE LEIGH B., CIVALIER CHRIS, BOOKER JESSICA, FAN HONGXIN, GULLEY MARGARET L.: "Analytic Validation of a Quantitative Real-time PCR Assay to Measure CMV Viral Load in Whole Blood", DIAGNOSTIC MOLECULAR PATHOLOGY, LIPPINCOTT WILLIAMS AND WILKINS, US, vol. 16, no. 2, 30 June 2007 (2007-06-30), US , pages 73 - 80, XP009553274, ISSN: 1052-9551, DOI: 10.1097/PDM.0b013e318033ab9e *

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