CN117343927B

CN117343927B - Nucleic acid extraction method of engineering cells

Info

Publication number: CN117343927B
Application number: CN202311656755.2A
Authority: CN
Inventors: 马超
Original assignee: Shanghai Yaoming Junuo Biotechnology Co ltd; Shanghai Yaoming Junuo Biomedical Research And Development Co ltd
Current assignee: Shanghai Yaoming Junuo Biotechnology Co ltd; Shanghai Yaoming Junuo Biomedical Research And Development Co ltd
Priority date: 2023-12-06
Filing date: 2023-12-06
Publication date: 2024-03-12
Anticipated expiration: 2043-12-06
Also published as: CN117343927A

Abstract

The present disclosure relates to methods for extracting intracellular nucleic acids, kits for intracellular nucleic acid extraction and the use of nucleases for the preparation of pretreatment reagents for intracellular nucleic acid extraction. The methods of the present disclosure include treating an engineered cell with a nuclease.

Description

Nucleic acid extraction method of engineering cells

Technical Field

The present disclosure relates to methods of extracting nucleic acids from engineered cells.

Background

Cell therapies have been used to treat diseases involving the introduction of exogenous nucleic acids into cells using genetically engineered methods and stringent cell detection procedures. However, the introduction of exogenous nucleic acid makes the cells more susceptible to contamination, thereby interfering with detection. How to remove the pollution while maintaining high detection efficiency is still a technical problem to be solved.

Disclosure of Invention

The present disclosure provides a method of extracting an engineered intracellular nucleic acid. The method comprises a pretreatment step prior to the nucleic acid extraction step. The pretreatment step can remove exogenous nucleic acid pollution and simultaneously maintain the efficiency of nucleic acid extraction.

In one aspect, a method of the present disclosure comprises: a) A pretreatment step comprising a contacting step of contacting the engineered cell with a nuclease; b) And extracting the nucleic acid in the cell. In one aspect, the present disclosure also provides a method of pretreatment of an engineered cell comprising a pretreatment step comprising a contacting step of contacting the engineered cell with a nuclease.

In one aspect, the nuclease is an endonuclease, an exonuclease, or a combination thereof, as previously described. In one aspect, the nuclease is a deoxyribonuclease, ribonuclease, or omnipotent nuclease. In one aspect, the nuclease is Benzonase or dnase i.

In one aspect, the method as described previously, wherein the contacting is for a time of no more than 2 hours. In one aspect, the contacting is for a period of 15 minutes to 60 minutes. In one aspect, the contacting is for about 30 minutes. In one aspect, the density of engineered cells upon contact is 1×10 ⁴ personal/mL-1X 10 ⁸ And each mL. In one aspect, the density of engineered cells upon contact is 5×10 ⁵ personal/mL-1X 10 ⁷ And each mL. In one aspect, the density of engineered cells upon contact is 5×10 ⁵ personal/mL-7.5X10 ⁶ And each mL. In one aspect, the concentration of nuclease at the time of the contacting is from 50 to 2000U/mL. In one aspect, the nuclease concentration upon contact is 75-1500U/mL. In one aspect, the nuclease concentration upon contact is 100-1000U/mL. In one aspect, the nuclease to cell ratio at contact is 1X 10 per 1000U nuclease ⁴ Up to 2X 10 ⁷ Individual cells. In one aspect, the nuclease to cell ratio at contact is 5X 10 per 1000U nuclease ⁵ Up to 1.5X10 ⁷ Individual cells. In one aspect, the nuclease to cell ratio at contact is 1X 10 per 1000U nuclease ⁶ From 7.5X10 ⁶ Individual cells.

In one aspect, the method as described previously, wherein the pre-treatment step comprises the step of freeze-thawing the engineered cells prior to the contacting step. In one aspect, the temperature of the freeze-engineered cells does not exceed-20 ℃. In one aspect, the temperature of the freeze-engineered cells does not exceed-80 ℃.

In one aspect, a method as described previously comprises a) a pretreatment step comprising contacting an engineered cell with a nuclease for 15 minutes to 60 minutes at a density of 5 x 10 of the engineered cell ⁵ personal/mL-1X 10 ⁷ The concentration of nuclease per m is 75-1500 U/mL; b) And extracting the nucleic acid in the cell. In one aspect, the pretreatment step comprises the steps of freezing-thawing the engineered cells, and contacting the thawed engineered cells with a totipotent nuclease for about 30 minutes at a density of 5X 10 of the engineered cells ⁵ personal/mL-7.5X10 ⁶ The concentration of nuclease is 100-1000U/mL. In one aspect, the pretreatment step comprises the steps of freezing-thawing the engineered cells, and contacting the thawed cells with Benzonase for 30 minutes at a density of 1X 10 of the engineered cells ⁶ personal/mL-7.5X10 ⁶ The concentration of nuclease was 1000U/mL.

In one aspect, a method as previously described comprises a) a pretreatment step comprising the steps of freeze-thawing the engineered cells, and a contacting step of contacting the engineered cells with a nuclease; b) And extracting the nucleic acid in the cell. In one aspect, the nuclease is Benzonase. In one aspect, the contacting is for a time of 30 minutes to 60 minutes. In one aspect, the time of contact is 30 minutes. In one aspect, the density of engineered cells upon contact is 5×10 ⁵ personal/mL-1X 10 ⁷ And each mL. In one aspect, the density of engineered cells upon contact is 1×10 ⁶ personal/mL-7.5X10 ⁶ And each mL. In one aspect, the nuclease to cell ratio at contact is 1X 10 per 1000U nuclease ⁶ Up to 1X 10 ⁷ And engineering the cells. In one aspect, the nuclease is Benzonase. In one aspect, the pretreatment step comprises the steps of freezing-thawing the engineered cells, and contacting the thawed cells with Benzonase for 30 minutes at a density of 1X 10 of the engineered cells ⁶ personal/mL-7.5X10 ⁶ The nuclease to cell ratio was 1X 10 per 1000U nuclease per mL ⁶ Up to 1X 10 ⁷ And engineering the cells.

In one aspect, the method as described previously, 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 lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, cationic liposomes, polymeric nanoparticles, transposons, electroporation, or microinjection. In one aspect, the exogenous gene is introduced into the engineered cell via a lentiviral vector.

In another aspect, the present disclosure also provides a kit for extracting nucleic acid of an engineered cell comprising a pretreatment reagent comprising a nuclease and a nucleic acid extraction reagent. In one aspect, the nuclease is a deoxyribonuclease, ribonuclease, or omnipotent nuclease; in one aspect, the nuclease is Benzonase or dnase i.

In another aspect, the present disclosure also provides the use of a nuclease in the preparation of a pretreatment reagent for the extraction of nucleic acids in an engineered cell. In one aspect, the nuclease is a deoxyribonuclease, ribonuclease, or omnipotent nuclease; in one aspect, the nuclease is Benzonase or dnase i.

Drawings

FIG. 1 shows the effect of cell cryopreservation on nuclease pretreatment using agarose electrophoresis;

FIGS. 2A to 2D show the molecular weight distribution of DNA in lanes 1-4 of agarose electrophoresis, respectively.

Detailed Description

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

I. Terminology

"a" or "an" may mean one or more or one or more. "about" refers to a range in which its corresponding data value increases or decreases by up to 5%. It should be understood that "comprising," "including," and "containing" are intended to include any viable elements other than the elements so long as the addition of such elements does not render the solution containing such elements impractical. "consisting of … …" means having only the elements described by "… …". "comprising" encompasses the situation "consisting of … …".

The term "engineered cell" refers to a cell comprising an exogenous nucleic acid or genetic modification.

The term "pretreatment" refers to any pretreatment that increases the acid extraction efficiency within a cell, e.g., as used herein, the pretreatment brings a nuclease, surfactant, etc. into contact with the cell. Pretreatment does not or substantially not result in cell lysis.

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

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 an engineered cell with a nuclease. The concentration or density of a substance during contact is that of the substance in the contact medium (e.g., a mixed liquor).

The term "immune cell" refers to a cell that is involved in an immune response intended to protect an organism from foreign substances, viruses and cells. Immune cells may be derived from a number of organs and tissues, such as thymus, spleen, lymph nodes, lymphoid tissue clusters (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 progenitor cells that develop into these cells. In this context, immune cells are engineered (e.g., introduced with exogenous nucleic acid) into engineered cells prior to nucleic acid extraction.

II cell treatment method

The present disclosure provides methods of extracting intracellular nucleic acids comprising a pretreatment step and an extraction step. The present disclosure also provides a method of pretreatment of an engineered cell comprising a pretreatment step. In one aspect, the intracellular nucleic acid is intracellular DNA. In one aspect, the intracellular nucleic acid is genomic DNA of a cell. Surprisingly, the methods of the present disclosure are capable of greatly reducing contamination of intracellular nucleic acids with impurities and retaining higher recovery rates than intracellular nucleic acid extraction methods that do not include a pretreatment step.

2.1. Pretreatment step

In one aspect, the pretreatment step comprises a contacting step of contacting the engineered cell 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, ribonuclease, or omnipotent nuclease. The omnipotent nuclease has DNase and RNase activities. In one aspect, the nuclease has no or substantially no activity to degrade proteins or disrupt cell membrane structures. In one aspect, the nuclease is Benzonase or dnase i. In one aspect, the nuclease is Benzonase.

In one aspect, wherein the time of contacting is no more than 6 hours, such as no more than 1,2, 3,4, 5, or 6 hours. In one aspect, wherein the contacting is for a time of 1 to 60 minutes, such as 1, 5, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In one aspect, the contacting is for a time of from 5 minutes to 60 minutes. In one aspect, the contacting is for a period of 15 minutes to 60 minutes. In one aspect, the contacting is for a time of 20 minutes to 40 minutes. In one aspect, the time of contact is about 30 minutes or 30 minutes. In one aspect, the contacting is performed at 25 to 42 ℃. In one aspect, the contacting is performed at 25, 37, or 42 ℃. In one aspect, the contacting is performed at about 37 ℃ or 37 ℃.

In one aspect, the density of engineered cells upon contact is 1×10 ⁴ personal/mL-1X 10 ⁸ And each mL. In one aspect, the density of engineered cells upon contact is 1×10 ⁴ Per mL, 5X 10 ⁴ Per mL, 1X 10 ⁵ Per mL, 5X 10 ⁵ Per mL, 1X 10 ⁶ Per mL, 5X 10 ⁶ personal/mL, 7.5X10 ⁶ Per mL, 1X 10 ⁷ Per mL, 5X 10 ⁷ individual/mL or 1X 10 ⁸ And each mL. In one aspect, the density of engineered cells upon contact is 5×10 ⁴ From 1 to 10 per mL ⁷ And each mL. In one aspect, the density of engineered cells upon contact is 1×10 ⁵ From 7.5X10 g/mL ⁶ And each mL. In one aspect, the density of engineered cells upon contact is 5×10 ⁵ From 7.5X10 g/mL ⁶ And each mL. In one aspect, the density of engineered cells upon contact is about 5 x 10 ⁵ Per mL, 1X 10 ⁶ Per mL, 5X 10 ⁶ individual/mL or 7.5X10 ⁶ And each mL.

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

In one aspect, the nuclease to cell ratio at contact is 1X 10 per 1000U nuclease ⁴ Up to 1X 10 ⁸ And engineering the cells. In one aspect, the nuclease to cell ratio at contact is 1X 10 per 1000U nuclease ⁴ Up to 2X 10 ⁷ And engineering the cells. In one aspect, the nuclease to cell ratio at contact is 1X 10 per 1000U nuclease ⁴ 、5×10 ⁴ Respectively, 1×10 ⁵ Personal, 5×10 ⁵ Respectively, 1×10 ⁶ Personal, 5×10 ⁶ Personal, 7.5X10) ⁶ Respectively, 1×10 ⁷ 2X 10 ⁷ Personal, 5×10 ⁷ Or 1X 10 ⁸ And engineering the cells. In one aspect, the nuclease to cell ratio at contact is 5X 10 per 1000U nuclease ⁵ Up to 1.5X10 ⁷ And engineering the cells. In one aspect, the nuclease to cell ratio at contact is 5X 10 per 1000U nuclease ⁵ Up to 1X 10 ⁷ And engineering the cells. In one aspect, the nuclease to cell ratio at contact is 1X 10 per 1000U nuclease ⁶ From 7.5X10 ⁶ And engineering the cells. In one aspect, the nuclease concentration at contact is 100U/mL and the cell density is 5X 10 ⁵ And each. In one aspect, the nuclease concentration at contact is 500U/mL and the cell density is 5X 10 ⁶ And each. In one aspect, the nuclease concentration at contact is 1000U/mL and the cell density is 1X 10 ⁶ And each. In one aspect, the nuclease concentration at contact is 1000U/mL and the cell density is 7.5X10 ⁶ And each. In one aspect, the nuclease concentration at contact is 1000U/mL and the cell density is 1X 10 ⁷ And each.

In one aspect, the contacting is performed in a liquid medium. In one aspect, the contacting is performed in a liquid medium suitable for cell survival. In one aspect, the contacting is performed in a buffer suitable for cell survival. In one aspect, the contacting is performed in phosphate buffer. In one aspect, the contacting is performed in Du's Phosphate Buffer (DPBS). In one aspect, the buffer comprises 1 to 10mM Mg ²⁺ . In one aspect, the buffer comprises 1,2, 3,4, 5, 6, 7, 8, 9, or 10mM Mg ²⁺ . In one aspect, the buffer comprises 1,2, 3,4, 5, 6, 7, 8, 9, or 10mM Mg ²⁺ . In one aspect, the buffer comprises 1 to 5mM Mg ²⁺ . In one aspect, the buffer comprises 1 to 3mM Mg ²⁺ . In one aspect, the buffer comprises about 2mM or 2mM Mg ²⁺ 。

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

In one aspect, the pretreatment step further comprises a washing step after the contacting step. In one aspect, the washing step comprises washing the cells with a buffer centrifuge. In one aspect, the buffer is any buffer suitable in the art for cell washing. In one aspect, the buffer is PBS or DPBS. In one aspect, the volume of buffer 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, cells are washed 2 times with DPBS solution of 1 mL.

2.2. Extraction step

In one aspect, the extraction step may use a variety of techniques known to those skilled in the art. A first partExemplary extraction methods include (i) disrupting cell membranes (i.e., lysing cells), and (ii) extracting intracellular nucleic acids. Methods of lysing cells are well known to those skilled in the art, and for example, various mechanical shearing or ultrasonic techniques can be used to disrupt the cell membrane. The cell lysis step also includes the use of detergents and surfactants to solubilize lipids on the cell membrane and nuclear membrane. In one aspect, the cleaving step may further comprise using a protease to cleave a protein, and/or using an rnase to digest RNA in the sample. Methods of nucleic acid extraction are also well known, for example, using extraction with an organic solvent (e.g., a mixture of phenol and chloroform) followed by precipitation with ethanol. Other suitable methods also include salting out DNA extraction, trimethyl ammonium bromide salt DNA extraction, and guanidine thiocyanate DNA extraction. A variety of kits are commercially available for extracting DNA from biological samples, e.g., QIAamp from Qiagen (Germanown, MD) or Maxwell from Promega (Madison, wis.), among others ^® And the reliaPrep ™ series of kits.

III kit

In one aspect, the present disclosure provides a kit for intracellular nucleic acid extraction comprising 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, ribonuclease, or omnipotent nuclease. The omnipotent nuclease has DNase and RNase activities. In one aspect, the nuclease has no or substantially no activity to degrade proteins or disrupt cell membrane structures.

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

In one aspect, the kit comprises an intracellular nucleic acid extraction reagent. Such nucleic acid extraction reagents are well known to those skilled in the art, for example, cell-cleavable detergents, cell membrane and nuclear membrane-soluble lipids, proteases, rnases, and organic solvents (e.g., mixtures of phenol and chloroform), salts, trimethylammonium bromide salts, guanidine thiocyanate, and the like for extracting nucleic acids. In one aspect, the kit comprises commercially available reagents, e.g., QIAamp from Qiagen, or Maxwell from Promega ^® And the reliaPrep ™ series of reagents. In one aspect, the pretreatment kit intracellular nucleic acid extraction reagents are packaged in combination or separately. It is also within the scope of the present disclosure that the kit contains only pretreatment reagents and that the intracellular nucleic acid extraction reagents are described in the specification.

IV cells

In one aspect, the cells involved in the methods of the present disclosure are engineered to contain an exogenous gene. In one aspect, the cell is a mammalian cell. In one aspect, the cell is a human cell. In one aspect, the cell is an immune cell or an enriched immune cell. In one aspect, the cell is a tumor cell. In one aspect, the cell is a T cell or an enriched T cell. In one aspect, the cell is CD4 ⁺ T cells or enriched CD4 ⁺ T cells. In one aspect, the cell is CD8 ⁺ T cells or enriched CD8 ⁺ T cells. In one aspect, the cell is CD4 ⁺ T cells and CD8 ⁺ T cells. In one aspect, the cell is enriched for CD4 ⁺ T cells and enriched CD8 ⁺ T cells. In one aspect, the cells comprise engineered cells or an enriched population of engineered cells. In one aspect, theThe cells comprise genetically engineered T cells or an enriched population of genetically engineered T cells. In one aspect, the cell comprises a Chimeric Antigen Receptor (CAR) expressing T cell or an enriched CAR expressing T cell. In one aspect, the cells undergo a freeze-thaw step.

In one aspect, the method of making the engineered cells comprises cell isolation, selection, activation, transduction, incubation, expansion, washing, suspension, dilution, concentration, and/or formulation. In one aspect, the method comprises isolating cells, and preparing, processing, culturing the 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 acid 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 acid into the cells, and culturing the transduced cells. In one aspect, the isolating comprises the step of selecting cells. In one aspect, the transduction is performed after stimulation of the isolated cells with a stimulating agent.

In one aspect, the method of preparation includes 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 leukocyte apheresis product); (b) Isolating (e.g., selecting) desired cells (e.g., CD 4) from the sample ⁺ 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 cell, such as by incubating the isolated (e.g., selected) cell with a viral vector encoding the recombinant receptor; and (d) culturing or expanding the cells. In one aspect, the method may further comprise the step of activating the cells by exposing the cells to a stimulating condition, which may be performed before, during and/or after incubating the cells with the viral vector, e.g., atBetween step (b) and step (c). In one aspect, the viral vector is a lentiviral vector. In one aspect, a washing and/or suspending step may also be performed before or after any of the above steps. In one aspect, one, more 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.

In one aspect, the cell culture method comprises a step of genetic engineering. In one aspect, this step introduces exogenous nucleic acid into the cell. In one aspect, the genetic engineering step comprises introducing the 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 viral particles, such as vectors of adenovirus, adeno-associated virus (AAV) and Human Immunodeficiency Virus (HIV), as well as recombinant lentiviral vectors or retroviral vectors (e.g., gamma retroviral vectors). In one aspect, the non-viral system includes a transposon system, such as a PiggyBac or a Sleeping Beauty gene transfer system. In one aspect, this step is performed by electroporation. In one aspect, the step is performed by transduction, transposon, electroporation, or a combination thereof. Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection, tungsten particle-promoted microprojectile bombardment, and strontium phosphate DNA co-precipitation.

V. exogenous nucleic acid

In one aspect, the cells involved in the methods of the present disclosure comprise an exogenous nucleic acid encoding a recombinant protein. 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.

In one aspect, the recombinant protein is a Chimeric Antigen Receptor (CAR). In one aspect, the antigen is selectively expressed or over-expressed on cells of a disease or disorder (e.g., tumor or pathogenic cells) as compared to normal or non-targeted cells. In one aspect, the diseases and conditions include proliferative, neoplastic and malignant diseases and disorders, including cancers and tumors, including hematological cancers, cancers of the immune system, such as lymphomas, leukemias and/or myelomas, such as B-leukemia, T-leukemia and myelogenous leukemia, lymphomas and multiple myelomas. In one aspect, the CAR contains an extracellular antigen recognition domain that specifically binds to an antigen. Thus, the extracellular antigen-recognition domain that specifically binds an antigen includes one or more antigen-binding molecules, such as one or more antigen-binding fragments, domains, or portions, 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 an scFv. In one aspect, the CAR comprises a spacer, which may be or comprise 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 comprises a transmembrane domain. In one aspect, the transmembrane domain is derived from a natural source or from a synthetic source. Where 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 α, β or ζ chains of T cell receptors, CD28, CD3 epsilon, 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 comprises predominantly hydrophobic amino acid residues, such as leucine and valine. In one aspect, the CAR contains an intracellular signaling domain comprising a cytoplasmic signaling domain, e.g., an intracellular domain capable of inducing a primary activation signal in a T cell, e.g., the zeta chain of the CD3 zeta chain; and/or the intracellular signaling domain comprises an immune receptor tyrosine based activation motif (ITAM). In one aspect, the CAR contains a co-stimulatory domain. In one aspect, the co-stimulatory domain is the signaling region and/or transmembrane portion of CD28, 4-1BB, OX40, DAP10 or ICOS. In some aspects, the same CAR includes both the primary activation signaling region and the co-stimulatory component. In one aspect, the same CAR includes both a primary activation signaling domain and a co-stimulatory domain.

VI. Examples

In the examples below, unless otherwise indicated, the frozen stock solution contained 10% DMSO, 20% fetal bovine serum, and 70% DMEM medium. The DNA extraction process comprises the following steps: lysing the cells, and proteinase K treatment to remove proteins adsorbed on the DNA; adsorbing free DNA by using a silica gel column or magnetic beads; washing the DNA by using an ethanol solution to remove impurities; the DNA adsorbed on the silica gel column or the magnetic beads is eluted using a low-salt solution. The copy number of the gene is detected using techniques well known in the art or commercially available kits.

Example 1

This example describes the effect of different treatments on DNA extraction efficiency and cell detection. The cells of this example are engineered T cells. Prior to detection, exogenous nucleic acid is introduced into the cell using a lentiviral vector. The cells were then cultured at 1X 10 ⁷ Up to 2X 10 ⁷ The cell density of each/mL is dispersed in the frozen stock solution, and the frozen stock solution is placed at-80 ℃ or frozen in liquid nitrogen for at least 12 hours, thawed at room temperature and centrifugally collected. Preparation of a solution containing 100U/mL nuclease Benzonase and 2mM Mg ²⁺ The DPBS solution of (2) is used as a nuclease treatment solution. Cells were treated as described in groups 1-6 below.

DNA was extracted and DNA concentration was quantified using an ultraviolet spectrophotometer. The relative recovery rate of each treatment mode was calculated based on the DNA concentration of group 1. VSV-G (vesicular stomatitis virus glycoprotein G) can adsorb to the cell membrane surface during lentivirus transduction. The ACTB (human beta actin) gene is present in the cell genome. The effect of different treatment modes on detection of DNA in the nucleus and DNA adsorbed outside the cell is detected by taking 20 ng/mu L of DNA and adopting fluorescent quantitative PCR. The relative impurity removal rates for each treatment regime were calculated based on the VSV-G content of group 1. The results are shown in Table 2.

The results show that the surface adsorbed impurities can be removed more effectively by nuclease treatment than by conventional surfactant and buffer washes. Surprisingly, not only was the nuclease treatment for 30 minutes effective in removing impurities, but recovery was maintained comparable to the nuclease-untreated group. Furthermore, no change in Ct value of ACTB gene was caused by each treatment, indicating that genomic DNA of each group of cells was not degraded.

Example 2

This example evaluates the effect of different detection conditions on cell detection. The cells of this example are human T cells. Prior to detection, exogenous nucleic acid is introduced into the cell using a lentiviral vector. The cells were then cultured at 1X 10 ⁷ Up to 2X 10 ⁷ The cell density of each/mL is dispersed in the frozen stock solution, and the frozen stock solution is placed at-80 ℃ or frozen in liquid nitrogen for at least 12 hours, thawed at room temperature and centrifugally collected. According to Table 3, a solution containing 2mM Mg was used ²⁺ The DPBS solution of (1) was used as a blank treatment solution, and Benzonase was added to the blank treatment solution to obtain a nuclease-containing treatment solution (groups 2 to 5). Cells were added to 1mL of the treatment solution and incubated for 30 minutes. Cells were washed 2 times with 1mL DPBS solution and DNA was extracted. The results show that the treatment fluid is suitable for samples with different cell densities, and the plasmid residue clearance can be improved by reducing the cell density under the condition of the same cell number and enzyme proportion.

Example 3

This example evaluates the effect of cell cryopreservation on cell detection. The cells of this example are human T cells. Prior to detection, exogenous nucleic acid is introduced into the cell using a lentiviral vector. The transduced cells were split into two parts. A fraction of cells were grown in 1X 10 cells ⁵ ~1×10 ⁷ Is dispersed in a culture medium solution and centrifuged. Another part of the cells was used as 1X 10 ⁵ ~1×10 ⁷ The cell density of each mL is dispersed in the frozen stock solution and is frozen at-80 ℃ for no less than 12 hours. Cryopreserved cells were thawed at room temperature and centrifuged. Preparation of a kit containing 1000U/mL nuclease and 2mM Mg ²⁺ The DPBS solution of (2) is used as a nuclease treatment solution. The unfrozen cells or frozen cell pellet were added to 1mL nuclease treatment solution and incubated at 37 ℃ for 30min. The cells were washed 2 times with 1mL DPBS solution and the DNA was extracted.

ALB (Albumin, human Albumin) was quantitatively detected by qPCR. The results are shown in Table 4. The frozen cells showed a stronger resistance to nucleases and their genomic DNA stability was higher than the non-frozen groups.

Example 4

This example evaluates the effect of cell cryopreservation on cell detection. The cell of this example is HT1080. Prior to detection, exogenous nucleic acid is introduced into the cell using a lentiviral vector. The transduced cells were split into two parts. A fraction of cells were grown in 1X 10 cells ⁵ ~1×10 ⁷ Is dispersed in a culture medium solution and centrifuged. Another part of the cells was used as 1X 10 ⁵ ~1×10 ⁷ The cell density of each mL is dispersed in the frozen stock solution and is frozen at-80 ℃ for no less than 12 hours. Cryopreserved cells were thawed at room temperature and centrifuged. Preparation of a kit containing 1000U/mL nuclease and 2mM Mg ²⁺ The DPBS solution of (2) is used as a nuclease treatment solution. Adding unfrozen cells or frozen cell pellet into 1mL nuclease treatment solution, and adding into the solutionIncubate at 37 ℃ for 30min. The cells were washed 2 times with 1mL DPBS solution and the DNA was extracted. The extracted DNA was detected by agarose gel electrophoresis experiments. A0.8% agarose gel was prepared using a 1 XTEA solution. Gelred staining was added to the samples and markers. After the dyeing treatment, a DNA marker (molecular weight 250 b-10000 b) is spotted on the leftmost lane, 100ng of DNA which is not subjected to freeze-preservation extraction is added in lane 1, 200ng of DNA which is not subjected to freeze-preservation extraction is added in lane 2, 100ng of DNA which is subjected to freeze-preservation extraction is added in lane 3, and 200ng of DNA which is added in lane 4. Electrophoresis was performed at 140V for 30min. Agarose electrophoresis imaging was performed using a Gel Doc ™ EZ Gel imager.

As shown in FIG. 1, the agarose electrophoresis results show that the samples (lanes 1 and 2) which are not subjected to cell cryopreservation have three bands which are respectively more than 10kb, 500-1000bp and 200-500 bp; whereas samples subjected to cell cryopreservation (lanes 3, 4) showed only one band located over 10kb. Image processing was performed using gel imager software and the processing results for lanes 1-4 are shown in FIGS. 2A-2D, respectively. FIGS. 2A and 2B show that 80% of the DNA has a molecular weight greater than 10kb, and about 20% of the DNA is located in a small fragment region of <1000 bp; FIGS. 2C and 2D show that 100% of the DNA has a molecular weight of more than 10kb. The nuclease is adopted to treat the cells which are not frozen, which is easy to degrade the genome DNA and affects the detection result.

Claims

1. A method of extracting an engineered intracellular nucleic acid comprising

a) A pretreatment step comprising the step of freezing-thawing said engineered cells, said frozen temperature not exceeding-80 ℃, said engineered cells being present in a cryoprotectant; and a contacting step of contacting the thawed engineered cells with a nuclease;

b) A step of extracting intracellular nucleic acid;

wherein the nuclease is Benzonase and the contacting is for 30 minutes to 60 minutes, and the ratio of nuclease to cell at the time of contacting is 5X 10 per 1000U of nuclease ⁵ Up to 1X 10 ⁷ An engineered cell;

the engineered cell is an engineered mammalian cell.

2. The method of claim 1, wherein the time of contacting is 30 minutes.

3. The method of claim 1, wherein the density of engineered cells upon contact is 5 x 10 ⁵ personal/mL-1X 10 ⁷ And each mL.

4. The method of claim 3, wherein the engineered cells have a density of 1 x 10 at contact ⁶ personal/mL-7.5X10 ⁶ And each mL.

5. The method of claim 1, wherein the nuclease to cell ratio at the time of contact is 1X 10 per 1000U of nuclease ⁶ Up to 1X 10 ⁷ And engineering the cells.

6. A method of extracting an engineered intracellular nucleic acid comprising

a) A pretreatment step comprising the step of freezing-thawing the engineered cells, said frozen temperature not exceeding-80 ℃, said engineered cells being present in a cryoprotectant; and contacting the thawed cells with Benzonase for 30 minutes at a density of 1X 10 engineered cells ⁶ personal/mL-7.5X10 ⁶ The nuclease to cell ratio was 1X 10 per 1000U nuclease per mL ⁶ Up to 1X 10 ⁷ An engineered cell;

b) A step of extracting intracellular nucleic acid;

the engineered cell is an engineered mammalian cell.

7. The method of any one of claims 1 to 6, wherein the engineered cell is an engineered T cell.

8. The method of any one of claims 1 to 6, wherein the engineered cell comprises an exogenous nucleic acid introduced via a lentiviral vector.