Nucleic acid extraction method for engineered cell

Abstract

Provided are a method for extracting nucleic acid in a cell, a reagent kit for extracting nucleic acid in a cell, and a use of a nuclease in the preparation of a pre-treatment reagent for extracting nucleic acid in a cell. The method comprises using a nuclease to treat an engineered cell.

Description

A method for extracting nucleic acid from engineered cells

[0001] This application claims priority to Chinese patent application No. 2023116567552, filed on December 6, 2023. This application incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0002] The present disclosure relates to methods of extracting nucleic acids from engineered cells. Background Art

[0003] Cell therapy has been used to treat diseases. It involves the introduction of exogenous nucleic acids into cells through genetic engineering and rigorous cell-based testing. However, the introduction of exogenous nucleic acids makes cells more susceptible to contamination, which can interfere with detection. Maintaining high detection efficiency while removing contamination remains a technical challenge.

[0004] Summary of the Invention

[0005] The present disclosure provides a method for extracting nucleic acids from engineered cells. The method includes a pretreatment step prior to the nucleic acid extraction step. The pretreatment step can remove exogenous nucleic acid contamination while maintaining the efficiency of nucleic acid extraction.

[0006] In one aspect, the method of the present disclosure comprises: a) a pretreatment step, comprising contacting the engineered cells with a nuclease; b) a step of extracting intracellular nucleic acids. In one aspect, the present disclosure also provides a method for pretreating engineered cells, comprising a pretreatment step, comprising contacting the engineered cells with a nuclease.

[0007] In one aspect, as described above, the nuclease is an endonuclease, an exonuclease or a combination thereof. In one aspect, the nuclease is a deoxyribonuclease, a ribonuclease or a universal nuclease. In one aspect, the nuclease is Benzonase or DnaseI.

[0008] In one aspect, the method as described above, wherein the contacting time is no more than 2 hours. In one aspect, the contacting time is 15 minutes to 60 minutes. In one aspect, the contacting time is about 30 minutes. In one aspect, the density of the engineered cells at the time of contacting is 1×10 4 / mL-1×10 8 In one aspect, the density of the engineered cells during contact is 5×10 5 / mL-1×10 7 In one aspect, the density of the engineered cells during contact is 5×10 5 / mL-7.5×10 6In one aspect, the concentration of the nuclease during contact is 50 to 2000 U / mL. In one aspect, the concentration of the nuclease during contact is 75-1500 U / mL. In one aspect, the concentration of the nuclease during contact is 100-1000 U / mL. In one aspect, the ratio of nuclease to cells during contact is 1×10 per 1000 U nuclease. 4 to 2×10 7 In one aspect, the ratio of nuclease to cells during contact is 5×10 per 1000 U nuclease. 5 to 1.5×10 7 In one aspect, the ratio of nuclease to cells during contact is 1×10 per 1000 U nuclease. 6 to 7.5×10 6 cells.

[0009] In one aspect, the method as described above, wherein the pretreatment step includes a step of freezing and thawing the engineered cells before the contacting step. In one aspect, the temperature of the frozen engineered cells does not exceed -20°C. In one aspect, the temperature of the frozen engineered cells does not exceed -80°C.

[0010] In one aspect, the method as described above comprises a) a pretreatment step, comprising contacting the engineered cells with the nuclease for 15 minutes to 60 minutes, wherein the density of the engineered cells during contact is 5×10 5 / mL-1×10 7 / m, the concentration of nuclease is 75-1500U / mL; b) the step of extracting intracellular nucleic acids. In one aspect, the pretreatment step includes the steps of freezing-thawing the engineered cells, and contacting the thawed engineered cells with the universal nuclease for about 30 minutes, at a density of 5×10 5 / mL-7.5×10 6 In one aspect, the pretreatment step comprises a freeze-thaw step of the engineered cells, and contacting the thawed cells with Benzonase for 30 minutes, at a density of 1×10 6 / mL-7.5×10 6 The concentration of nuclease was 1000 U / mL.

[0011] In one aspect, the method as described above comprises a) a pretreatment step, including a step of freezing and thawing the engineered cells, and a contacting step of contacting the engineered cells with a nuclease; and b) a step of extracting intracellular nucleic acids. In one aspect, the nuclease is Benzonase. In one aspect, the contacting time is 30 minutes to 60 minutes. In one aspect, the contacting time is 30 minutes. In one aspect, the density of the engineered cells at the time of contacting is 5×10 5 / mL-1×10 7 In one aspect, the density of the engineered cells during contact is 1×10 6 / mL-7.5×10 6 In one aspect, the ratio of nuclease to cells during contact is 1×10 per 1000 U nuclease. 6 to 1×10 7 In one aspect, the nuclease is Benzonase. In one aspect, the pretreatment step comprises the steps of freezing and thawing the engineered cells, and contacting the thawed cells with Benzonase for 30 minutes, at a density of 1×10 6 / mL-7.5×10 6 The ratio of nuclease to cells was 1 × 10 per 1000 U nuclease. 6 to 1×10 7 An engineered cell.

[0012] In one aspect, the method as described above, wherein the engineered cell is an engineered mammalian cell. In one aspect, the engineered cell is the engineered immune cell or engineered tumor cell. In one aspect, the engineered cell is an engineered T cell or an engineered NK cell. In one aspect, the engineered cell comprises an exogenous nucleic acid introduced via a viral vector, a non-viral vector or a physical method. In one aspect, the exogenous gene is introduced into the engineered cell via a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a cationic liposome, a polymer nanoparticle, a transposon, electroporation or microinjection. In one aspect, the exogenous gene is introduced into the engineered cell via a lentiviral vector.

[0013] In another aspect, the present disclosure further provides a kit for extracting nucleic acids from engineered cells, comprising a pretreatment reagent and a nucleic acid extraction reagent, wherein the pretreatment reagent comprises a nuclease. In one aspect, the nuclease is a deoxyribonuclease, a ribonuclease, or a universal nuclease; in one aspect, the nuclease is Benzonase or DNaseI.

[0014] In another aspect, the present disclosure further provides the use of a nuclease in preparing a pretreatment reagent for extracting nucleic acids from engineered cells. In one aspect, the nuclease is a deoxyribonuclease, a ribonuclease, or a universal nuclease; in one aspect, the nuclease is Benzonase or DnaseI. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Agarose electrophoresis was used to detect the effect of cell freezing on nuclease pretreatment

[0016] FIG2A to FIG2D show the DNA molecular weight distribution in lanes 1 to 4 of agarose electrophoresis, respectively. DETAILED DESCRIPTION

[0017] After reading this specification, it will become apparent to those skilled in the art how to implement the present invention in various alternative embodiments and alternative applications. However, the embodiments herein are presented by way of example only and not by way of limitation. As such, this detailed description should not be construed as limiting the scope or breadth of the present invention as set forth below.

[0018] I. Terminology

[0019] "A" or "an" may refer to one or more or one or more. "About" refers to a range in which the corresponding data value increases or decreases by up to 5%. "Including", "comprising" and "containing" should be understood as including any other feasible elements in addition to the elements included, as long as the addition of the elements does not make the technical solution containing the elements unworkable. "Composed of..." means that it only has the elements described by "...". "Comprising" covers the situation of "composed of..."

[0020] The term "engineered cell" refers to a cell that contains exogenous nucleic acid or genetic modifications.

[0021] The term "pretreatment" refers to any pretreatment that can improve the efficiency of nucleic acid extraction from cells, for example, in this context, pretreatment involves contacting cells with nucleases, surfactants, etc. The pretreatment does not or substantially does not result in cell lysis.

[0022] The term "nuclease" refers to an enzyme that is capable of catalyzing the hydrolysis of bonds between nucleic acids within a DNA or RNA molecule.

[0023] The term "contacting" refers to the interaction, reaction, or physical contact of two or more substances. For example, in this context, contacting includes the process of mixing engineered cells with nucleases. During contacting, the concentration or density of a substance is the concentration or density of the substance in the contact medium (e.g., a mixture).

[0024] The term "immune cell" refers to a cell that participates in an immune response intended to protect an organism from foreign substances, viruses, and cells. Immune cells can be derived from many organs and tissues, such as the thymus, spleen, lymph nodes, lymphoid tissue clusters (such as in the gastrointestinal tract and bone marrow). Such cells include T cells, B cells, natural killer cells, macrophages, neutrophils, tumor infiltrating lymphocytes, dendritic cells, mast cells, eosinophils and basophils, and the progenitor cells that develop into these cells. In this article, immune cells are transformed (such as introducing exogenous nucleic acids) into engineered cells before extracting nucleic acid.

[0025] II. Cell Treatment Methods

[0026] The present disclosure provides a method for extracting intracellular nucleic acids, which comprises a pretreatment step and an extraction step. The present disclosure also provides a pretreatment method for engineered cells, which comprises a pretreatment step. In one aspect, the intracellular nucleic acid is intracellular DNA. In one aspect, the intracellular nucleic acid is the genomic DNA of the cell. Surprisingly, the method of the present disclosure can greatly reduce the contamination of intracellular nucleic acids by impurities and retain a higher recovery rate compared to the intracellular nucleic acid extraction method that does not include a pretreatment step.

[0027] Preprocessing steps

[0028] In one aspect, the pretreatment step comprises a contacting step of contacting the engineered cells with a nuclease. In one aspect, the nuclease is an endonuclease, an exonuclease, or a combination thereof. In one aspect, the nuclease is a deoxyribonuclease, a ribonuclease, or a omnipotent nuclease. The omnipotent nuclease has both DNase and RNase activity. In one aspect, the nuclease has no or substantially no activity in degrading proteins or destroying cell membrane structure. In one aspect, the nuclease is Benzonase or DnaseI. In one aspect, the nuclease is Benzonase.

[0029] In one aspect, the contacting time is no more than 6 hours, such as no more than 1, 2, 3, 4, 5 or 6 hours. In one aspect, the contacting time is 1-60 minutes, such as 1, 5, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. In one aspect, the contacting time is 5 minutes to 60 minutes. In one aspect, the contacting time is 15 minutes to 60 minutes. In one aspect, the contacting time is 20 minutes to 40 minutes. In one aspect, the contacting time is about 30 minutes or 30 minutes. In one aspect, the contacting is carried out at 25 to 42°C. In one aspect, the contacting is carried out at 25, 37 or 42°C. In one aspect, the contacting is carried out at about 37°C or 37°C.

[0030] In one aspect, the density of the engineered cells during contacting is 1×10 4 / mL-1×10 8 In one aspect, the density of the engineered cells during contact is 1×10 4 / mL, 5×10 4 / mL, 1×10 5 / mL, 5×10 5 / mL, 1×10 6 / mL, 5×10 6 / mL, 7.5×10 6 / mL, 1×10 7 / mL, 5×10 7 / mL or 1×10 8 In one aspect, the density of the engineered cells during contact is 5×10 4 cells / mL to 1×10 7 In one aspect, the density of the engineered cells during contact is 1×10 5 cells / mL to 7.5×10 6 In one aspect, the density of the engineered cells during contact is 5×10 5 cells / mL to 7.5×10 6 In one aspect, the density of the engineered cells during contact is about 5×10 5 / mL, 1×10 6 / mL, 5×10 6 cells / mL or 7.5×10 6 pieces / mL.

[0031] In one aspect, the concentration of the nuclease during contact is 10 to 5000 U / mL. In one aspect, the concentration of the nuclease during contact is 50 to 2000 U / mL. In one aspect, the concentration of the nuclease during contact is 50 to 1500 U / mL. In one aspect, the concentration of the nuclease during contact is 100 to 1500 U / mL. In one aspect, the concentration of the nuclease during contact is 50 U / mL, 75 U / mL, 100 U / mL, 200 U / mL, 300 U / mL, 400 U / mL, 500 U / mL, 600 U / mL, 700 U / mL, 800 U / mL, 900 U / mL, 1000 U / mL, 1500 U / mL, 2000 U / mL, 3000 U / mL, 4000 U / mL, 5000 U / mL. In one aspect, the concentration of the nuclease during contact is 100-1000 U / mL. In one aspect, the concentration of the nuclease upon contacting is about 100, 500, or 1000 U / mL. In one aspect, the concentration of the nuclease upon contacting is about 1000 U / mL.

[0032] In one aspect, the ratio of nuclease to cells during contact is 1×10 4 to 1×10 8 In one aspect, the ratio of nuclease to cells during contact is 1×10 per 1000 U nuclease. 4 to 2×10 7 In one aspect, the ratio of nuclease to cells during contact is 1×10 per 1000 U nuclease. 4 , 5×10 4 1×10 5 5×10 5 1×10 6 5×10 6 7.5×10 6 1×10 7 2×10 7 5×10 7 or 1×10 8 In one aspect, the ratio of nuclease to cells during contact is 5×10 per 1000 U nuclease. 5 to 1.5×10 7 In one aspect, the ratio of nuclease to cells during contact is 5×10 per 1000 U nuclease. 5 to 1×10 7 In one aspect, the ratio of nuclease to cells during contact is 1×10 per 1000 U nuclease. 6 to 7.5×106 In one aspect, the nuclease concentration during contact is 100 U / mL and the cell density is 5×10 5 In one aspect, the nuclease concentration during contact is 500 U / mL and the cell density is 5×10 6 In one aspect, the nuclease concentration during contact is 1000 U / mL and the cell density is 1×10 6 In one aspect, the nuclease concentration during contact is 1000 U / mL and the cell density is 7.5×10 6 In one aspect, the nuclease concentration during contact is 1000 U / mL and the cell density is 1×10 7 indivual.

[0033] In one aspect, the contacting is carried out in a liquid medium. In one aspect, the contacting is carried out in a liquid medium suitable for cell survival. In one aspect, the contacting is carried out in a buffer suitable for cell survival. In one aspect, the contacting is carried out in a phosphate buffer. In one aspect, the contacting is carried out in Dulbecco's phosphate buffered saline (DPBS). In one aspect, the buffer contains 1 to 10 mM Mg 2+ In one aspect, the buffer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mM Mg 2+ In one aspect, the buffer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mM Mg 2+ In one aspect, the buffer comprises 1 to 5 mM Mg 2+ In one aspect, the buffer comprises 1 to 3 mM Mg 2+ In one aspect, the buffer comprises about 2 mM or 2 mM Mg 2+ .

[0034] In one aspect, the method comprises a step of freezing and thawing the engineered cells prior to the contacting step. In one aspect, freezing is performed under conditions suitable for preserving the cells. In one aspect, the freezing temperature is no more than -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, or -100°C. In one aspect, the freezing temperature is between -20°C and -100°C. In one aspect, the freezing temperature is between -60°C and -80°C. In one aspect, the freezing temperature is approximately -80°C or -80°C. In one aspect, freezing is performed under liquid nitrogen. In one aspect, the freezing time is 1, 2, 3, 6, 12, 24, 48, or 72 hours, or 5, 7, or 14 days. In one aspect, the freezing time is between 6 hours and 7 days. In one aspect, the freezing time is between 24 hours and 3 days. In one aspect, thawing is performed at 0°C to 37°C. In one aspect, thawing is performed at room temperature, approximately 25°C, or 25°C. In one aspect, the engineered cells are present in a cryoprotectant. In one aspect, the cryoprotectant can be formulated in any manner known in the art. In one aspect, the cryoprotectant is DMEM. In one aspect, the cryoprotectant comprises 0-20% DMSO, 0-40% serum, and 50-100% DMEM. In one aspect, the cryoprotectant comprises 5-10% DMSO, 10-20% serum, and 70-85% DMEM. In one aspect, the cryoprotectant comprises 10% DMSO, 20% fetal bovine serum, and 70% DMEM.

[0035] In one aspect, the pretreatment step further comprises a washing step after the contacting step. In one aspect, the washing step comprises centrifuging and washing the cells with a buffer solution. In one aspect, the buffer solution is any buffer solution suitable for cell washing in the art. In one aspect, the buffer solution is PBS or DPBS. In one aspect, the volume of the buffer solution used for a single wash is 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mL. In one aspect, the cells are washed 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. In one aspect, the cells are centrifuged and washed 2 times with 1 mL of DPBS solution.

[0036] Extraction steps

[0037] In one aspect, the extraction step can use a variety of techniques known to those skilled in the art. An exemplary extraction method includes (i) destroying the cell membrane (i.e., lysing the cells), and (ii) extracting intracellular nucleic acids. Methods for lysing cells are well known to those skilled in the art, for example, a variety of mechanical shearing or ultrasonic techniques can be used to destroy the cell membrane. The cell lysis step also includes using detergents and surfactants to dissolve lipids on the cell membrane and nuclear membrane. In one aspect, the lysis step can further include using proteases to break down proteins, and / or using RNases to digest the RNA in the sample. Methods for nucleic acid extraction are also well known, for example, using an organic solvent (such as a mixture of phenol and chloroform) for extraction, followed by ethanol precipitation. Other suitable methods also include salting out DNA extraction, trimethylammonium bromide DNA extraction, and guanidine thiocyanate DNA extraction. A variety of kits are commercially available for extracting DNA from biological samples, for example, QIAamp from Qiagen (Germantown, MD), or QIAamp from Promega (Madison, WI). and ReliaPrep TM Series of kits.

[0038] III. Kit

[0039] In one aspect, the present disclosure provides a kit for intracellular nucleic acid extraction, which comprises a pretreatment reagent and an intracellular nucleic acid extraction reagent. In another aspect, the present disclosure provides the use of a nuclease in the preparation of a pretreatment reagent for intracellular nucleic acid extraction. In one aspect, the nuclease is a deoxyribonuclease, a ribonuclease, or a omnipotent nuclease. The omnipotent nuclease has DNase and RNase activity. In one aspect, the nuclease does not have or substantially does not have the activity of degrading proteins or destroying cell membrane structure.

[0040] In one aspect, the concentration of the nuclease in the pretreatment reagent is 10 to 5000 U / mL. In one aspect, the concentration of the nuclease in the pretreatment reagent is 50 to 2000 U / mL. In one aspect, the concentration of the nuclease in the pretreatment reagent is 50 to 1500 U / mL. In one aspect, the concentration of the nuclease in the pretreatment reagent is 100 to 1500 U / mL. In one aspect, the concentration of the nuclease in the pretreatment reagent is 50 U / mL, 75 U / mL, 100 U / mL, 200 U / mL, 300 U / mL, 400 U / mL, 500 U / mL, 600 U / mL, 700 U / mL, 800 U / mL, 900 U / mL, 1000 U / mL, 1500 U / mL, 2000 U / mL, 3000 U / mL, 4000 U / mL, 5000 U / mL. In one aspect, the concentration of the nuclease in the pretreatment reagent is 100-1000 U / mL. In one aspect, the concentration of the nuclease is about 100, 500 or 1000 U / mL. In one aspect, the concentration of the nuclease is about 1000 U / mL.

[0041] In one aspect, the kit comprises a reagent for extracting nucleic acids from cells. Such nucleic acid extraction reagents are well known to those skilled in the art, for example, detergents that can lyse cells, surfactants that can dissolve lipids on cell membranes and nuclear membranes, proteases, RNA enzymes, and organic solvents (such as a mixture of phenol and chloroform), salts, trimethylammonium bromide salts, guanidine thiocyanate, etc. for extracting nucleic acids. In one aspect, the kit comprises commercially available reagents, for example, Qiagen's QIAamp, or Promega's and ReliaPrep TM In one aspect, the pretreatment kit and the intracellular nucleic acid extraction reagent are packaged in combination or separately. Kits containing only pretreatment reagents and describing intracellular nucleic acid extraction reagents in the instructions are also within the scope of protection of the present disclosure.

[0042] IV cells

[0043] In one aspect, the cells involved in the methods of the present disclosure are engineered and contain exogenous genes. In one aspect, the cells are mammalian cells. In one aspect, the cells are human cells. In one aspect, the cells are immune cells or enriched immune cells. In one aspect, the cells are tumor cells. In one aspect, the cells are T cells or enriched T cells. In one aspect, the cells are CD4 + T cells or enriched CD4 + In one aspect, the cells are CD8 + T cells or enriched CD8 +In one aspect, the cells are CD4 + T cells and CD8 + In one aspect, the cells are enriched for CD4 + T cells and enriched CD8 + T cells. In one aspect, the cells comprise engineered cells or enriched engineered cell colonies. In one aspect, the cells comprise genetically engineered T cells or enriched genetically engineered T cell colonies. In one aspect, the cells comprise chimeric antigen receptor (CAR) expressing T cells or enriched CAR expressing T cells. In one aspect, the cells have undergone a freeze-thaw step.

[0044] In one aspect, the method for preparing the engineered cells comprises cell separation, selection, activation, transduction, cultivation, amplification, washing, suspension, dilution, concentration and / or formulation. In one aspect, the method comprises isolating cells, and preparing, processing, and culturing cells under stimulating conditions. In one aspect, the method comprises isolating cells from a biological sample, incubating the isolated cells with a viral vector to transduce exogenous nucleic acids into the cells. In one aspect, the method comprises isolating cells from a biological sample, incubating the isolated cells with a viral vector to transduce exogenous nucleic acids into the cells, and culturing the transduced cells. In one aspect, the separation comprises the step of selecting cells. In one aspect, the transduction is performed after stimulating the isolated cells with a stimulating reagent.

[0045] In one aspect, the preparation method comprises one or more of the following: (a) pre-washing a biological sample containing cells (e.g., a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukapheresis product); (b) isolating (e.g., selecting) desired cells (e.g., CD4 + and / or CD8 +T cells), for example, by incubating the cells with an immunoaffinity reagent; (c) introducing a vector encoding an exogenous nucleic acid (e.g., a recombinant receptor) into the isolated or selected cells, such as by incubating the isolated (e.g., selected) cells with a viral vector encoding the recombinant receptor; and (d) culturing or expanding the cells. In one aspect, the method may further include a step of activating the cells by exposing them to stimulating conditions, which may be performed before, during, and / or after incubating the cells with the viral vector, for example, between steps (b) and (c). In one aspect, the viral vector is a lentiviral vector. In one aspect, washing and / or suspension steps may also be performed before or after any of the above steps. In one aspect, one, more than one, or all of the steps of the cell culture method are performed under sterile conditions. In some embodiments of this method, cell isolation, transduction, washing, optional activation or stimulation, and formulation are all performed within a closed system.

[0046] In one aspect, the cell culture method includes a genetic engineering step. In one aspect, this step introduces exogenous nucleic acid into the cell. In one aspect, the genetic engineering step includes introducing recombinant protein into the cell via a vector. Such vectors include viral and non-viral systems. In one aspect, the viral system includes recombinant infectious virus particles, such as vectors of adenovirus, adeno-associated virus (AAV) and human immunodeficiency virus (HIV), and recombinant lentiviral vectors or retroviral vectors (such as gamma retroviral vectors). In one aspect, the non-viral system includes a transposon system, such as the gene transfer system of PiggyBac or Sleeping Beauty. In one aspect, this step is performed by electroporation. In one aspect, this step is performed by transduction, transposon, electroporation or a combination thereof. Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection, tungsten particle-promoted microparticle bombardment and strontium phosphate DNA co-precipitation.

[0047] V. Exogenous Nucleic Acids

[0048] In one aspect, the cells involved in the methods of the present disclosure comprise exogenous nucleic acids encoding recombinant proteins. In one aspect, the recombinant protein is a chimeric receptor, a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric antibody-T cell construct (caTCR), a chimeric signaling receptor (CSR), or a combination thereof.

[0049] In one aspect, the recombinant protein is a chimeric antigen receptor (CAR). In one aspect, the antigen is selectively expressed or overexpressed on the cells (for example, tumors or pathogenic cells) of a disease or disorder compared to normal or non-targeted cells. In one aspect, the disease and disorder include proliferative, neoplastic and malignant diseases and disorders, including cancer and tumors, including blood cancers, immune system cancers, such as lymphomas, leukemias and / or myeloma, such as type B leukemia, T-type leukemia and myeloid leukemia, lymphoma and multiple myeloma. In one aspect, the CAR contains an extracellular antigen recognition domain specifically bound to an antigen. Therefore, the extracellular antigen recognition domain specifically bound to an antigen includes one or more antigen binding molecules, such as one or more antigen binding fragments, domains or parts, or one or more antibody variable domains, and / or antibodies. In one aspect, the antigen binding molecule is a full-length antibody, such as a single-domain antibody, a monospecific antibody or a multispecific antibody; or an antibody fragment, such as scFv. In one aspect, the CAR includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or a variant or modified form thereof, such as a hinge region and / or CH1 / CL and / or Fc region. In one aspect, the spacer is located between the extracellular antigen recognition domain and the transmembrane domain. In one aspect, the CAR includes a transmembrane domain. In one aspect, the transmembrane domain is derived from a natural source or from a synthetic source. When the source is natural, the transmembrane domain is derived from any membrane-bound protein or transmembrane protein. The transmembrane domain includes one or more of the following: α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In one aspect, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain mainly comprises hydrophobic amino acid residues, such as leucine and valine. In one aspect, the CAR contains an intracellular signaling domain, and the intracellular signaling domain includes a cytoplasmic signaling domain, for example, an intracellular domain capable of inducing a primary activation signal in a T cell, for example, a ζ chain of a CD3ζ chain; and / or the intracellular signaling domain includes an activation motif (ITAM) based on immunoreceptor tyrosine. In one aspect, the CAR contains a costimulatory domain. In one aspect, the costimulatory domain is a signaling region and / or a transmembrane portion of CD28, 4-1BB, OX40, DAP10 or ICOS. In some aspects, the same CAR includes both primary activation signaling region and costimulatory components. In one aspect, the same CAR includes both primary activation signaling domain and costimulatory domain.

[0050] VI. Examples

[0051] After reading this specification, it will become apparent to those skilled in the art how to implement the present invention in various alternative embodiments and alternative applications. However, the embodiments herein are presented by way of example only and not by way of limitation. As such, this detailed description should not be construed as limiting the scope or breadth of the present invention as set forth below.

[0052] In the following examples, unless otherwise specified, the freezing medium contained 10% DMSO, 20% fetal bovine serum, and 70% DMEM medium. The DNA extraction process involves: cell lysis, proteinase K treatment to remove proteins adsorbed to the DNA; adsorption of free DNA using a silica gel column or magnetic beads; washing the DNA with an ethanol solution to remove impurities; and elution of DNA adsorbed to the silica gel column or magnetic beads using a low-salt solution. Gene copy number was determined using techniques known in the art or commercially available kits.

[0053] Example 1

[0054] This example describes the effects of different treatments on DNA extraction efficiency and cell detection. The cells in this example are engineered T cells. Before detection, exogenous nucleic acid was introduced into the cells using a lentiviral vector. The cells were cultured at 1×10 7 to 2×10 7 Disperse the cells at a density of 100 U / mL in freezing medium and freeze at -80℃ or in liquid nitrogen for at least 12 hours. Thaw at room temperature and collect the cells by centrifugation. 2+ The cells were treated with the following method described for Groups 1-6.

[0055] Table 1. Cell treatment methods

[0056] DNA was extracted and the DNA concentration was quantified using a UV spectrophotometer. The relative recovery rate of each treatment method was calculated based on the DNA concentration of Group 1. VSV-G (vesicular stomatitis virus glycoprotein G) can be adsorbed on the cell membrane surface during lentiviral transduction. The ACTB (human β-actin) gene is present in the cell genome. 20 ng / μL of DNA was taken, and fluorescence quantitative PCR was used to detect the effects of different treatment methods on the detection of intranuclear DNA and extracellular adsorbed DNA. The relative impurity removal rate of each treatment method was calculated based on the VSV-G content of Group 1. The results are shown in Table 2.

[0057] Table 2. Cell treatment efficiency

[0058] The results showed that nuclease treatment was more effective in removing surface-adsorbed impurities than conventional washing with surfactants and buffer. Surprisingly, 30 minutes of nuclease treatment not only effectively removed impurities but also maintained a comparable recovery rate to the untreated group. Furthermore, none of the treatments resulted in a change in the Ct value for the ACTB gene, indicating that the genomic DNA in each cell group was not degraded.

[0059] Example 2

[0060] This example evaluates the effects of different assay conditions on cell detection. The cells in this example are human T cells. Before the assay, exogenous nucleic acid was introduced into the cells using a lentiviral vector. The cells were cultured at 1×10 7 to 2×10 7 Disperse the cells at a density of 100 cells / mL in freezing medium and freeze at -80℃ or in liquid nitrogen for at least 12 hours. Thaw at room temperature and collect the cells by centrifugation. 2+ 1mL DPBS solution was used as a blank treatment solution (Group 1), and Benzonase was added to the blank treatment solution to obtain a treatment solution containing nuclease (Groups 2-5). Cells were added to 1mL of treatment solution and incubated for 30 minutes. The cells were washed twice by centrifugation with 1mL of DPBS solution, and DNA was extracted. The results showed that the treatment solution of the present invention is suitable for samples with different cell densities. At the same cell number and enzyme ratio, reducing the cell density can improve the plasmid residual clearance rate.

[0061] Table 3. Effects of nuclease treatment on gene stability

[0062] Example 3

[0063] This example evaluates the effect of cell freezing on cell detection. The cells in this example are human T cells. Before detection, exogenous nucleic acid was introduced into the cells using a lentiviral vector. The transduced cells were divided into two parts. One part of the cells was 1×10 5 ~1×10 7 The cells were dispersed in the culture medium and centrifuged. The other part of the cells was dispersed in the culture medium at a density of 1×10 5 ~1×10 7 Disperse the cells at a density of 1000 U / mL in freezing medium and store at -80℃ for at least 12 hours. Thaw the frozen cells at room temperature and centrifuge. Prepare a solution containing 1000 U / mL nuclease, 2 mM Mg 2+ Use 1 mL of DPBS as the nuclease treatment solution. Add 1 mL of nuclease treatment solution to unfrozen cells or frozen cell pellets and incubate at 37°C for 30 minutes. Wash the cells twice with 1 mL of DPBS by centrifugation and extract DNA.

[0064] ALB (albumin, human albumin) was quantitatively detected using qPCR. The results are shown in Table 4. The frozen cells showed enhanced resistance to nucleases, and their genomic DNA stability was higher than that of the unfrozen group.

[0065] Table 4. Effect of cryopreservation on gene stability after nuclease treatment

[0066] Example 4

[0067] This example evaluates the effect of cell freezing on cell detection. The cells in this example are HT1080. Before detection, exogenous nucleic acid was introduced into the cells using a lentiviral vector. The transduced cells were divided into two parts. One part of the cells was 1×10 5 ~1×10 7 The cells were dispersed in the culture medium and centrifuged. The other part of the cells was dispersed in the culture medium at a density of 1×10 5 ~1×10 7 Disperse the cells at a density of 1000 U / mL in freezing medium and store at -80℃ for at least 12 hours. Thaw the frozen cells at room temperature and centrifuge. Prepare a solution containing 1000 U / mL nuclease, 2 mM Mg 2+ DPBS solution is used as nuclease treatment solution. Add 1 mL of nuclease treatment solution to unfrozen cells or frozen cell pellets and incubate at 37°C for 30 minutes. Use 1 mL of DPBS solution to centrifuge and wash the cells twice and then extract DNA. Detect the extracted DNA by agarose gel electrophoresis. Use 1×TEA solution to prepare 0.8% agarose gel. Add Gelred stain to the sample and marker. After staining, spot a DNA marker (molecular weight 250b~10000b) in the leftmost lane, add 100 ng of unfrozen extracted DNA to the first lane, 200 ng of unfrozen extracted DNA to the second lane, 100 ng of frozen extracted DNA to the third lane, and 200 ng of DNA to the fourth lane. Electrophoresis is performed at 140V for 30 minutes. Use Gel Doc TM Agarose gel electrophoresis imaging was performed using the EZ Gel Imager.

[0068] The agarose gel electrophoresis results are shown in Figure 1. The unfrozen sample (lanes 1 and 2) shows three bands, located above 10 kb, 500-1000 bp, and 200-500 bp, respectively. The frozen sample (lanes 3 and 4) shows only one band located above 10 kb. Gel imaging software was used to process the images, and the results for lanes 1-4 are shown in Figures 2A-2D, respectively. Figures 2A and 2B show that 80% of the DNA is larger than 10 kb, and approximately 20% is located in the small fragment region <1000 bp. Figures 2C and 2D show that 100% of the DNA is larger than 10 kb. Nuclease treatment of unfrozen cells can easily lead to genomic DNA degradation and affect the test results.

Claims

1. A method for extracting nucleic acid from engineered cells, comprising: a) a pretreatment step, including a step of freezing-thawing the engineered cells, and a contacting step of contacting the engineered cells with a nuclease; b) The step of extracting intracellular nucleic acid.

2. The method of claim 1, wherein the nuclease is an endonuclease, an exonuclease, or a combination thereof; Preferably, the nuclease is a deoxyribonuclease, a ribonuclease or a universal nuclease; More preferably, the nuclease is Benzonase or DNaseI.

3. The method according to claim 1 or 2, wherein the contacting time is no more than 2 hours; Preferably, the contacting time is 15 minutes to 60 minutes; More preferably, the contacting time is about 30 minutes.

4. The method according to any one of claims 1 to 3, wherein the density of the engineered cells during contacting is 1×10 4 / mL-1×10 8 Pieces / mL; Preferably, the density of the engineered cells during contact is 5×10 5 / mL-1×10 7 Pieces / mL; More preferably, the density of the engineered cells during contacting is 5×10 5 / mL-7.5×10 6 Pieces / mL.

5. The method according to any one of claims 1 to 4, wherein the concentration of the nuclease during the contacting is 50 to 2000 U / mL; Preferably, the concentration of the nuclease during the contacting is 75-1500 U / mL; More preferably, the concentration of the nuclease during the contacting is 100-1000 U / mL.

6. The method according to any one of claims 1 to 5, wherein the ratio of nuclease to cells during contact is 1×10 4 to 2×10 7 cells; Preferably, the ratio of nuclease to cells during contact is 5×10 5 to 1.5×10 7 cells; More preferably, the ratio of nuclease to cells during contact is 1×10 6 to 7.5×10 6 cells.

7. The method according to any one of claims 1 to 6, wherein the temperature of the frozen engineered cells does not exceed -20°C; Preferably, the temperature of the frozen engineered cells is about -80°C.

8. A method for extracting nucleic acid from engineered cells, comprising a) a pretreatment step, comprising contacting the engineered cells with the nuclease for 15 to 60 minutes, at a density of 5×10 5 / mL-1×10 7 / m, the concentration of nuclease is 75-1500U / mL; b) a step of extracting nucleic acid from cells; Preferably, the method comprises a) a pretreatment step, comprising a step of freezing and thawing the engineered cells, and contacting the thawed engineered cells with a universal nuclease for about 30 minutes, wherein the density of the engineered cells during the contacting is 5×10 5 / mL-7.5×10 6 / mL, the concentration of nuclease is 100-1000U / mL; b) a step of extracting intracellular nucleic acid; More preferably, the method comprises a) a pretreatment step, including a freeze-thaw step of the engineered cells, and contacting the thawed cells with Benzonase for 30 minutes, at a density of 1×10 6 / mL-7.5×10 6 / mL, the concentration of nuclease was 1000U / mL; b) The step of extracting intracellular nucleic acid.

9. The method of any one of claims 1 to 8, wherein the engineered cell is an engineered mammalian cell; Preferably, the engineered cells are the engineered immune cells or engineered tumor cells; More preferably, the engineered cells are engineered T cells or engineered NK cells.

10. The method of any one of claims 1 to 9, wherein the engineered cells comprise exogenous nucleic acids introduced via viral vectors, non-viral vectors or physical methods; Preferably, the exogenous gene is introduced into the engineered cell via a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a cationic liposome, a polymer nanoparticle, a transposon, electroporation or microinjection; More preferably, the exogenous gene is introduced into the engineered cells via a lentiviral vector.

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