WO2019014924A1 - 一种病毒转染增效剂和基于点击化学的病毒转染应用 - Google Patents

一种病毒转染增效剂和基于点击化学的病毒转染应用 Download PDF

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WO2019014924A1
WO2019014924A1 PCT/CN2017/093870 CN2017093870W WO2019014924A1 WO 2019014924 A1 WO2019014924 A1 WO 2019014924A1 CN 2017093870 W CN2017093870 W CN 2017093870W WO 2019014924 A1 WO2019014924 A1 WO 2019014924A1
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virus
dbco
pei
cells
transfection
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French (fr)
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蔡林涛
马轶凡
潘宏
金言
李萍
李文军
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中国科学院深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

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  • the invention belongs to the field of gene therapy, in particular, the invention relates to a virus transfection synergist - DBCO modified cationic polymer PEI (PEI-DBCO) and its application in virus transfection, especially based on click chemistry Viral transfection applications, as well as methods for the preparation of viral transfection synergists.
  • the invention also relates to a virus transfection vector system, which is a virus-PEI-DBCO complex formed by adsorbing PEI-DBCO on the surface of a virus particle, and having a DBCO group and a positive charge on the surface thereof, can effectively improve the virus transfection efficiency,
  • the invention also relates to the use of viral transfection vector systems.
  • the present invention also relates to a mammalian cell transfection method for efficient loading and transfection of mammalian cell viruses by bioorthogonal reactions, and for the application of azide modification of cells in mammalian cell transfection.
  • Gene therapy is not only an important means of treating gene-deficient diseases, but also an important strategy for treating tumors, infections, and neurological diseases in recent years.
  • Gene therapy includes two broad categories: gene replacement and gene editing.
  • Gene replacement is the replacement of defective genes with normal gene fragments to slow down or cure the disease.
  • Gene editing technology uses zinc finger protein nuclease (ZFN), transcription activator-like effector nuclease (TALENs), Crisper-Cas9 and other techniques to knock out, add, repair or remove diseased genes for specific DNA fragments. effect.
  • ZFN zinc finger protein nuclease
  • TALENs transcription activator-like effector nuclease
  • Crisper-Cas9 and other techniques to knock out, add, repair or remove diseased genes for specific DNA fragments. effect.
  • Traditional gene therapy involves injecting DNA or RNA sequences directly into a patient via a viral or non-viral vector.
  • the target cells are genetically engineered in vitro, and cultured and expanded back into the patient, which has become an important gene therapy.
  • a series of genetically engineered immune cells established using gene editing technology, Such as chimeric antigen receptor modified T cells (CAR-T), T cell receptor modified T cells (TCR-T), chimeric antigen receptor modified NK cells (CAR-NK), etc., in animal experiments and clinical trials Both show excellent tumor targeting, specificity, killing activity and persistence, which have become the hotspots of current tumor immunotherapy.
  • Gene vectors are the key to determining the success of gene therapy, including non-viral vectors and viral vectors.
  • Commonly used non-viral carriers are mainly cationic liposomes, cationic polymers, calcium phosphate, nanomaterials and the like.
  • these vectors are inefficient for gene transfection of suspension, primary cells, particularly immune cells, in a non-dividing state.
  • viral vectors have higher transfection efficiency, are capable of infecting both dividing cells and non-dividing cells, and can integrate the gene of interest into the host chromosome for stable and long-lasting expression.
  • suspended immune cells such as T cells and NK cells
  • viral vectors exhibit high transfection efficiency and are increasingly used in the field of genetically modified cell therapy technology.
  • Viral vectors for gene modification mainly include DNA viruses such as adenovirus (AdV), adeno-associated virus (AAV), Herpes simplex virus (HSV), and RNA viruses such as reverse transcription.
  • AdV adenovirus
  • AAV adeno-associated virus
  • HSV Herpes simplex virus
  • RNA viruses such as reverse transcription.
  • Virus Ribonucleic acid
  • MMV Murine leukemia viruses
  • lentivirus human immunodeficiency virus
  • polybrene is a kind of cationic polymer, which can neutralize the negative charge on the surface of the virus, promote the adsorption and infection of the virus on the target cells, and improve the transfection efficiency of the viral vector.
  • polybrene itself has strong cytotoxicity and affects the proliferation and activation of target cells.
  • Gene transfection techniques include viral vector-mediated and non-viral vector-mediated techniques.
  • Some traditional non-viral vectors and transfection reagents such as DEAE-dextran, phosphoric acid, liposomes, etc.
  • DEAE-dextran can only be transiently transfected and has certain toxicity to cells; calcium phosphate transfection is not suitable for primary cells, and the operation repeatability is poor; the cationic liposome method has wide applicability, but requires serum-free conditions. And the effect varies greatly with cell type.
  • Viral vector-mediated gene transfection technology is widely used for gene transfection of various types of cells.
  • cell types such as suspension cells and immune cells that are difficult to transfect
  • viral vectors also need to be transfected with synergists to increase transfection efficiency.
  • the widely used transfection synergist is polybrene, also known as hexadimethrine bromide, which is a cationic polymer that neutralizes the negative charge on the surface of the virus and promotes the virus against target cells. Adsorption and infection, thereby improving the transfection efficiency of the viral vector.
  • fibronectin is also used as a viral transfection enhancer because of its good intercellular fusion.
  • difficult-to-transfect suspension cells such as peripheral blood T cells
  • the effects of these transfection enhancers are still unsatisfactory.
  • the present invention discloses a novel viral transfection synergist and a viral vector transfection technique based on a "click-chemistry" reaction.
  • the virus transfection synergist is a cycloalkynyl (-DBCO) modified cationic polymer polyethyleneimine (PEI), PEI-DBCO, which can be encapsulated on the surface of the virus by electrostatic attraction and neutralizes the surface of the virus.
  • the negative charge causes the surface of the virus particles to carry DBCO groups.
  • the target cells are first incubated with a small molecule of choline/monosaccharide (Azide-Choline/Monosaccharide), and their azide group (-N 3 ) is labeled on the cell membrane by intracellular sugar metabolism/lipid metabolism pathway.
  • PEI-DBCO forms a polymer-virus complex with the virus, and the surface-DBCO and the -N 3 on the surface of the target cell membrane undergo a highly efficient and specific "click chemistry" reaction to form a covalent bond, thereby effectively assisting the virus to enter the target cell.
  • the positive charge carried on the surface of the virus-polymer complex further promotes the binding of the virus to the cell membrane, thereby synergistically improving the transfection efficiency of the viral vector.
  • the invention provides a viral transfection synergist which is modified by a DBCO group Cationic polymer PEI, ie PEI-DBCO.
  • the PEI is a dendritic PEI or a linear PEI.
  • the PEI has a molecular weight MW of from 600 to 25,000.
  • the invention provides a viral transfection vector system which is a virus transfection synergist-encapsulated virus, wherein the viral transfection synergist is PEI-DBCO.
  • the virus is a DNA virus or an RNA virus.
  • the DNA virus is selected from the group consisting of an adenovirus, an adeno-associated virus, and a herpes simplex virus
  • the RNA virus is selected from the group consisting of a retrovirus and a lentivirus.
  • the viral transfection vector is positively charged.
  • the invention provides a method of preparing the aforementioned viral transfection synergist comprising the steps of:
  • the molar ratio of PEI to DBCO modifying reagent molecules is from 1:1 to 1:10.
  • the stirring time is 2-4 hours.
  • the dialysis is dialyzed against ultrapure water for 3 days using a dialysis bag of MW1000.
  • the invention provides the use of PEI-DBCO in viral transfection.
  • the virus is a DNA virus or an RNA virus.
  • the DNA virus is selected from the group consisting of an adenovirus, an adeno-associated virus, and a herpes simplex virus
  • the RNA virus is selected from the group consisting of a retrovirus and a lentivirus.
  • the invention provides the use of the aforementioned viral transfection vector system for transfecting cells.
  • the cell is a suspended mammalian cell or an adherent mammalian cell.
  • the present invention provides the use of N 3 -DBCO click chemistry of the viral transfection.
  • the virus is a DNA virus or an RNA virus.
  • the DNA virus is selected from the group consisting of an adenovirus, an adeno-associated virus, and a herpes simplex virus
  • the RNA virus is selected from the group consisting of a retrovirus and a lentivirus.
  • the viral transfection is used to suspend mammalian cells or adherent mammalian cells.
  • the invention provides a virus transfection method comprising the steps of:
  • step (d) The cell virus mixture obtained by centrifugation in the step (c) is cultured in a CO 2 cell incubator for 4-6 hours, and then the culture medium is changed halfway, and the culture is continued.
  • the target cell is a suspended mammalian cell or an adherent mammalian cell.
  • step (a) the choline azide derivative or monosaccharide azide derivative is as shown in the following figure:
  • step (a) 1 x 10 6 of viable cells cultured in vitro and 5% of a choline azide derivative or a monosaccharide azide derivative at a concentration of 1-100 ⁇ M are at 37 ° C. Incubate for 48 hours in a CO 2 incubator.
  • the final concentration of PEI-DBOC is 0.01-10 ⁇ g/ml
  • incubation is carried out for 15 minutes at room temperature.
  • step (c) the virus-PEI-DBCO complex is incubated with the azide-modified target cell obtained in (a) at 37 ° C, and centrifuged under the conditions of 2500 rpm, 90 minutes, room temperature. .
  • the invention provides the use of azide modification of a cell in viral transfection.
  • the azide modification is the metabolic modification of a cell with a choline azide derivative or a monosaccharide azide derivative to carry an azide group on its surface.
  • the cell is a suspended mammalian cell or an adherent mammalian cell.
  • the virus is a DNA virus or an RNA virus.
  • the DNA virus is selected from the group consisting of an adenovirus, an adeno-associated virus, and a herpes simplex virus
  • the RNA virus is selected from the group consisting of a retrovirus and a lentivirus.
  • the N 3 -DBCO-based "click chemistry" reaction is highly efficient and specific, and is not interfered by other factors in the cell culture system. Therefore, the virus encapsulated by PEI-DBCO can rapidly form a -N 3 group on the surface of the target cell membrane. Covalent binding, thereby effectively assisting the virus into target cells.
  • PEI-DBCO forms a polymer-virus complex with the virus, and the positive charge carried on the surface further promotes the binding of the virus to the cell membrane, thereby synergistically improving the transfection efficiency of the viral vector.
  • the invention discloses a cationic polymer virus transfection synergist and a viral vector transfection technology based on a "click chemistry" reaction.
  • the technology uses a DBCO-modified cationic polymer polyethyleneimine (PEI-DBCO) as a viral transfection enhancer, which is coated on the surface of the viral carrier to carry a DBCO group on its surface.
  • PEI-DBCO DBCO-modified cationic polymer polyethyleneimine
  • the target cells are first incubated with a small molecule of the choline/monosaccharide azide derivative, such that the azide group (-N 3 ) is labeled on the cell membrane surface protein/phospholipid molecule by the intracellular sugar metabolism/lipid metabolism pathway.
  • the choline/monosaccharide azide derivative is co-cultured with the target cell, and the saccharide/lipid metabolic pathway derivative of the target cell is non-destructively modified and embedded in the cell membrane, wherein the azide group-N 3 is Exposed to the surface of the cell membrane;
  • the PEI-DBCO After the PEI-DBCO is incubated with the virus particles, the PEI-DBCO is encapsulated with the virus particles by electrostatic action to form a virus-cation complex complex;
  • the cationic polymer transfection synergist PEI-DBCO is characterized by a DBCO modified cationic polymer polyethyleneimine (PEI).
  • PEI DBCO modified cationic polymer polyethyleneimine
  • PEI-DBCO is formed on the PEI molecule.
  • the PEI-DBCO preparation method comprises the following steps: separately dissolving PEI and NHS-DBCO in PBS buffer, and slowly dropping the click chemical DBCO reagent solution into the PEI solution (the molar ratio of PEI to DBCO reagent molecules is 1:1-1-1). 10), the reaction was stirred for 2-4 hours, and then PEI-DBCO cationic polymer was obtained by dialysis in ultrapure water for 3 days using a MW1000 dialysis bag, and can be stored for a long time after vacuum freeze-drying (see Fig. 3).
  • the invention also provides a viral vector transfection technique based on a "click chemistry" reaction.
  • the technology combines the DBCO-modified polycationic viral vector with the azide group on the surface of the T cell membrane by azido molecule-mediated bioorthogonal ligation reaction to achieve efficient transfection of T cells. It is characterized in that the method comprises the following steps:
  • Azido (-N 3 ) modified target cells choline/monosaccharide azide derivatives (0.1-100 ⁇ M/10 6 cells) were added during target cell culture, and azide groups were cultured for 48-72 hours.
  • the pellet can be attached to the glycoprotein or phospholipid molecule of the target cell membrane by intracellular lipid metabolism/sugar metabolism.
  • the cells were washed twice with buffer at pH 7.2-7.6 and resuspended in fresh medium.
  • the target cells include all suspended and adherent mammalian cells.
  • the choline azide derivatives include AE-Cho, AP-Cho and other related lipid analogs, and the monosaccharide azide derivatives include Ac 4 GlcNAz, Ac 4 GalNAz, Ac 4 ManNAz and the like. .
  • the specific structure is shown in Figure 4.
  • PEI-DBCO polymer-encapsulated viral vector
  • the viral vector includes a DNA virus and an RNA virus.
  • Target cell transfection The virus-cation complex is added to the aforementioned azide (-N 3 ) modified target cell, and the mixture of the cell and the virus complex is centrifuged at room temperature (2500 rpm, 1.5 hours), and then placed. Continue to culture in a cell culture incubator. After 4-6 hours, the fresh medium was replaced by half a volume, and the culture was continued for 48-72 hours to obtain target cells for gene expression.
  • PEI Polyethyleneimine
  • PEI Polyethyleneimine
  • polyazide a water-soluble polymer.
  • Polyethyleneimine is divided into linear and branched, linear polyethylene. The imine contains all secondary amines, while the branched polyethyleneimine contains primary, secondary and tertiary amine groups.
  • Polyethylenimine condenses DNA into positively charged particles that bind to negatively charged cell surface residues and enter the cell by endocytosis.
  • Those skilled in the art can select a suitable PEI according to the actual situation, and these suitable PEIs are all within the protection scope of the present invention.
  • the cationic polymer of the present invention is not limited to PEI.
  • DBCO refers to a chemical group used in a click chemical reaction
  • DBCO modifier refers to a reagent used in a click chemical reaction, mainly for the modification of related targets.
  • Transfection synergist herein primarily refers to biochemical agents used to increase viral transfection efficiency, such as polybrane, fibnectin, and cationic targeting polymers protected by the present invention. Those skilled in the art can select suitable transfection synergists according to the actual situation, and these suitable transfection synergists are all within the scope of the present invention.
  • viral vector refers to a vector for carrying a virus, and those skilled in the art can select a suitable viral vector according to the actual situation, and these suitable viral vectors are all within the scope of the present invention.
  • DNA virus means that the nucleic acid of the virus is DNA, and in the present invention, including but not limited to adenovirus, adeno-associated virus, herpes simplex virus and the like. Those skilled in the art can select a suitable DNA virus according to the actual situation, and these suitable DNA viruses are all within the scope of the present invention.
  • RNA virus means that the viral nucleic acid is RNA, and in the present invention, including but not limited to lentivirus, Influenza virus, retrovirus, etc. Those skilled in the art can select a suitable RNA virus according to the actual situation, and these suitable RNA viruses are all within the scope of the present invention.
  • click chemistry also known as “link chemistry”
  • link chemistry aims to quickly and stably complete the chemical synthesis of various molecules through the connection reaction of small units (chemical groups). It has high-efficiency, specific, and stable characteristics, and the reaction is not interfered by complex biological systems, and it does not affect the biological system itself.
  • Hybrid mammalian cells mainly refers to mammalian cells that are grown in suspension, and include, but are not limited to, immune-related cells such as T cells, B cells, macrophages (THP-1, RAW cells), and NK cells in the present invention ( Including human and mouse sources).
  • immune-related cells such as T cells, B cells, macrophages (THP-1, RAW cells), and NK cells in the present invention ( Including human and mouse sources).
  • T cells T cells
  • B cells macrophages
  • RAW cells macrophages
  • NK cells Including human and mouse sources.
  • Adherent mammalian cells are primarily mammalian cells that are adherently grown and include, but are not limited to, HEK293T, 4T-1 cells, and the like (including human and murine sources) in the present invention. Those skilled in the art can select suitable adherent mammalian cells according to the actual situation, and these suitable adherent mammalian cells are all within the scope of the present invention.
  • Multiplicity of infection the ratio of the number of viruses to the number of cells at the time of infection, is used to determine the dose required for the virus to infect target cells, and the higher the MOI value, the greater the cytotoxicity.
  • Figure 1 shows that PEI-DBCO improves viral transfection efficiency through "click chemistry.”
  • Figure 2 shows the molecular structure of a click chemistry DBCO modification reagent.
  • Figure 3 shows the PEI-DBCO synthesis route.
  • Figure 4 is a structural formula of the azide derivative of choline/monosaccharide.
  • Figure 5 shows the modification of azide molecules in Jurkat cells by laser confocal, Western blot and flow cytometry.
  • Figure 6 shows the expression of GFP after transfection of Jurkat cells with PEI-DBCO.
  • Figure 7 shows the modification of surface azide molecules in human ⁇ T cells by laser confocal and flow cytometry.
  • Figure 8 shows the expression of GFP and the level of cell killing by PEI-DBCO carrying lentivirus transfected human ⁇ T cells.
  • Figure 9 shows the expression of GFP after transfection of human ⁇ T cells by PEI-DBCO with lentivirus.
  • Figure 10 shows the expression of GFP after transfection of Raji cells with PEI-DBCO.
  • FIG. 11 shows the expression of GFP after transfection of THP-1 cells with PEI-DBCO.
  • Figure 12 shows the expression of GFP after transfection of 293T cells with adenovirus carrying PEI-DBCO.
  • Figure 13 shows the expression of GFP after transfection of Raji cells by adenovirus carrying PEI-DBCO.
  • reagents, materials and equipment used in the examples are well known to those skilled in the art and are commercially available or readily available or available.
  • the various parameters and conditions therein, including quantity, ratio, molecular weight, temperature, time, etc., are not limited to the specific parameters and conditions in the embodiments, and those skilled in the art have the ability to adopt other parameters and conditions according to actual conditions, and Get the same or similar technical effects.
  • the cationic polymer PEI (1.8K, dendritic) (Sigma) was mixed with the bio-orthogonal reaction linker DBCO-PEG 4 -NHS (Click chemistry tools) at a molar ratio of 1:10, and incubated at room temperature for 3 hours, residual DBCO- The PEG 4 -NHS molecule was removed by 1 KDa dialysis bag dialysis (Merck Millipore).
  • the synthesized cationic targeting polymer (PEI-DBCO) was further characterized by mass spectrometry/nuclear magnetic analysis.
  • Jurkat cell culture Jurkat cells were cultured in RPM1640 complete medium containing ⁇ -mercaptoethanol. During the culture, cells were passaged or changed every other day.
  • Azide modification of Jurkat cells The medium of the above cells was replaced with fresh medium, and the monosaccharide azide derivative Ac 4 GalNAz at a final concentration of 50 ⁇ M was co-cultured with the cells for 48 hours, and then washed away with PBS buffer. Excess modifiers, cells were stained with DBCO-Fluor 488. The cells were harvested and analyzed by laser confocal microscopy, Western blotting and cell flow analysis on the cell surface. As shown in Figure 5, the azide molecule has been expressed on the cell surface and is dose dependent.
  • Cationic targeting polymer-encapsulated lentivirus The cationic targeting polymer (PEI-DBCO) was diluted into serum-free RPMI-1640 medium (final concentration 0.02 ⁇ g/ml) and the virus was infected at a multiplicity of 10 The dose was diluted to the above medium and incubated for 15 minutes at room temperature to obtain a lentiviral-cation targeting polymer.
  • PEI-DBCO cationic targeting polymer
  • Lentivirus-cation targeting polymer was transfected into Jurkat cells: The above modified cells were washed twice with PBS to remove residual Ac 4 GalNAz molecules. The above lentivirus-cation targeting polymer was added to the Jurkat cell culture solution modified in the above step, centrifuged at 37 ° C (2500 rpm, 1.5 hours), placed in an incubator for 5 hours, and then replaced with fresh medium for 48 hours. The residue was washed away with PBS buffer, and the virus-transfected cells were harvested for use.
  • the lentivirus used in this experiment carries the green fluorescent protein (GFP) reporter gene
  • GFP expression of Jurkat cells transfected with lentivirus can be observed by fluorescence microscopy.
  • the lentiviral-cation targeting polymer treated Jurkat cells had significant fluorescent signals compared to the control groups.
  • the number of positive cells infected and the fluorescence intensity were determined by flow cytometry.
  • the results are shown in Figure 6b. It can be seen that the cells infected with the lentivirus-cation targeting polymer have a high GFP + cell number and a strong fluorescence intensity, indicating that the cationic targeting polymer viral vector can efficiently transfect Jurkat cells.
  • Example 3 Lentivirus-mediated transfection of human primary ⁇ T lymphocytes
  • PBMC Peripheral blood mononuclear cells
  • Azide modification of human primary ⁇ T lymphocytes After culture for 3 days, the monosaccharide azide derivatives Ac 4 GalNAz, Ac 4 GlcNAz and Ac 4 ManNAz with a final concentration of 25-100 ⁇ M were co-cultured with ⁇ T cells, respectively. After an hour, the excess modification reagent was washed away with PBS buffer, and the cells were harvested and subjected to DBCO-Fluor 488 fluorescence staining, and the expression of the azide molecule on the cell surface was analyzed by laser confocal microscopy and cell flow analysis. As shown in Figure 7, after the metabolically modified cells of Ac 4 GalNAz and Ac 4 GlcNAz, the azide molecule has been expressed on the cell surface and has a significant dose-dependent nature.
  • the cationic targeting polymer encapsulates the lentivirus: the cationic targeting polymer viral vector is diluted into serum-free AIM-V medium (0.02 ⁇ g/ml), and the virus is diluted to the above medium at a dose of 10 in a multiplicity of infection. Incubate for 15 minutes at room temperature.
  • Lentivirus-cation targeting polymer was transfected into human primary T lymphocytes: The above modified T cells were washed twice with PBS to remove residual saccharide derivatives. The lentivirus-cation targeting polymer was added to the human primary T lymphocyte culture solution modified in the above step, centrifuged at 28 ° C (2500 rpm, 1.5 hours), and placed in an incubator for 5 hours, and then replaced with fresh culture solution. After 96 hours of culture, the residue was washed away with PBS buffer, and the virus-transfected cells were harvested for use.
  • the transfection efficiency of lentivirus to ⁇ T cells can be evaluated by detecting the expression level of GFP and the killing ability (CTL) of effector T cells.
  • CTL killing ability
  • lentiviral (GFP)-cation targeting polymer treated cells had significant green fluorescent protein signal compared to each control group.
  • the number of positive cells infected and the fluorescence intensity were determined by flow cytometry. The results are shown in Figure 8b.
  • the results showed that the cells infected with the lentivirus (GFP)-cation targeting polymer had a high GFP + cell number (70-80%) and strong fluorescence intensity; at the same time, the effector T cell killing results indicated that the lentivirus (CD19- CAR)-Cell-targeted polymer-infected cells have strong tumor cell killing ability (see Figure 8c). These results indicate that the cationic targeting polymer viral vector can efficiently transfect human primary ⁇ T lymphocytes.
  • PBMC Peripheral blood mononuclear cells
  • Azide modification of ⁇ T lymphocytes After culture for 3 days, the monosaccharide azide derivatives Ac 4 GalNAz and Ac 4 GlcNAz at a final concentration of 25-100 M were co-cultured with cells for 48 hours, respectively, and then washed with PBS buffer. The excess modification reagent was removed, and the cells were harvested and subjected to DBCO-Fluor488 fluorescent staining, and the expression of the azide molecule on the cell surface was analyzed by flow cytometry. As shown in Figure 9a, after the Ac 4 GalNAz was metabolically modified, the azide molecule was successfully modified on the cell surface.
  • Cationic targeting polymer-encapsulated lentivirus The cationic targeting polymer viral vector was diluted into serum-free RPM1640 medium (0.01 ⁇ g/ml), and the virus was diluted to the above medium at a dose of 10, and incubated at room temperature. 15 minutes.
  • Lentivirus-cation targeting polymer was transfected into human primary ⁇ T lymphocytes: The above modified cells were washed twice with PBS to remove residual saccharide derivatives. The lentiviral-cation targeting polymer was added to the cell culture solution obtained in the above step, centrifuged at 28 ° C (2500 rpm, 1.5 hours), placed in an incubator for 5 hours, and then replaced with fresh medium for 96 hours, using PBS. The buffer was washed away and the virus-transfected cells were harvested for use.
  • the transfection efficiency of lentivirus to ⁇ T cells can be evaluated by detecting the expression level of GFP.
  • the lentiviral-cation targeting polymer infected cells had a high GFP + cell number (60%) and a strong fluorescence intensity compared to the control cells.
  • Raji cell culture Raji cells were cultured in RPM1640 complete medium containing 10% FBS. Cell passage was performed every other day during the cultivation.
  • Azidation modification of Raji cells The above cells were replaced with fresh medium, and a monosaccharide azide derivative Ac 4 GlcNAz with a final concentration of 50 M was co-cultured with the cells for 48 hours, and the excess modifier was washed away with PBS buffer. The cells were stained with DBCO-Fluor 488. Cells were harvested and flow cytometric analysis of the expression of azide molecules on the cell surface. As shown in Figure 10a, the azide molecule is highly expressed on the surface of Raji cells.
  • Cationic targeting polymer encapsulation of lentivirus The cationic targeting polymer viral vector was diluted into serum-free RPM1640 medium (final concentration 0.05 ⁇ g/ml) and the virus was diluted to a dose of 5 at the multiplicity of infection to the above culture. Base, incubate for 15 minutes at room temperature.
  • the lentivirus used in this experiment carries the green fluorescent protein (GFP) reporter gene, and the expression of GFP in Raji cells transfected with lentivirus can be observed by laser confocal microscopy.
  • GFP green fluorescent protein
  • the lentiviral-cation targeting polymer treated Raji cells had a significant green fluorescent protein signal compared to each control group.
  • flow cytometric analysis confirmed the positive cells of the infection and the fluorescence intensity, and the results are shown in Fig. 10c. It can be seen that the cells infected with the lentivirus-cation targeting polymer have a high GFP + cell number and a strong fluorescence intensity, indicating that the cationic targeting polymer viral vector can efficiently transfect Raji cells.
  • THP-1 cell culture THP-1 cells were cultured in RPM1640 complete medium containing 10% FBS. Cell passage was performed every other day during the cultivation.
  • Azide modification of THP-1 cells The above cells were replaced with fresh medium, and then the monosaccharide azide derivative Ac 4 GalNAz at a final concentration of 50 ⁇ M was co-cultured with the cells for 48 hours, and then washed away with PBS buffer. Excess modifiers, cells were stained with DBCO-Fluor 488. Cells were harvested and flow cytometry was used to analyze the expression of azide on the cell surface. As shown in Figure 11a, the azide molecule is highly expressed on the surface of THP-1 cells.
  • Cationic targeting polymer-encapsulated lentivirus The cationic targeting polymer viral vector was diluted into serum-free RPM1640 medium (final concentration 0.1 ⁇ g/ml), and the virus was diluted to a dose of 10 at the multiplicity of infection to the above culture. Base, incubate for 15 minutes at room temperature.
  • Lentiviral-cationic targeting polymer was transfected into THP-1 cells: The above modified cells were washed twice with PBS to remove residual saccharide derivatives. The lentivirus-cation targeting polymer is added to the THP-1 cell culture solution modified in the above step, centrifuged at room temperature (2500 rpm, 1.5 hours), and placed in an incubator for 4-6 hours to replace the fresh culture solution. After 96 hours of culture, the residue was washed away with PBS buffer, and the virus-transfected cells were harvested for use.
  • the lentivirus used in this experiment carries the green fluorescent protein (GFP) reporter gene
  • GFP expression in Lentiviral-transfected THP-1 cells can be observed by laser confocal microscopy.
  • the lentiviral-cation targeting polymer treated THP-1 cells had a significant green fluorescent protein signal compared to each control group.
  • flow cytometric analysis confirmed the positive cells of the infection and the fluorescence intensity, and the results are shown in Fig. 11c. It can be seen that the cells infected with the lentivirus-cation targeting polymer have a high GFP + cell number and a strong fluorescence intensity, indicating that the cationic targeting polymer viral vector can efficiently transfect THP-1 cells.
  • Example 7 Adenovirus (AdV) mediates transfection of human embryonic kidney cells (HEK 293T)
  • HEK 293T cell culture HEK293T cells were cultured in DMEM complete medium containing 10% FBS. Cell passage was performed every other day during the cultivation.
  • Azide modification of HEK 293T cells The above cells were replaced with fresh medium, and the monosaccharide azide derivative Ac 4 GalNAz with a final concentration of 25 ⁇ M was co-cultured with the cells for 48 hours, and the excess modification was washed away with PBS buffer. The cells were stained with DBCO-Fluor 488. Cells were harvested and flow cytometric analysis of the expression of azide molecules on the cell surface.
  • Cationic targeting polymer-encapsulated adenovirus Dilute the cationic targeting polymer viral vector into serum-free DMEM medium (final concentration 0.5 ⁇ g/ml) and dilute the virus to the above culture at a dose of 5 Base, incubate for 15 minutes at room temperature.
  • Adenovirus-cation targeting polymer was transfected into HEK293T cells: The treated cells were washed twice with PBS to remove residual saccharide derivatives. The lentivirus-cation targeting polymer is added to the HEK 293T cell culture solution modified in the above step, and centrifuged at 2500 rpm for 1.5 hours at room temperature, and then placed in an incubator for 5 hours, and then the fresh culture solution is replaced; the culture is continued. After 48 hours, the residue was washed away with PBS buffer, and the virus-transfected cells were harvested for use.
  • the adenovirus used in this experiment carries the green fluorescent protein (GFP) reporter gene, and the expression of GFP in HEK 293T cells transfected with adenovirus can be observed by laser confocal microscopy.
  • GFP green fluorescent protein
  • adenovirus-cation targeting polymer treated HEK293T cells had significant green fluorescent protein signal compared to each control group.
  • flow cytometric analysis confirmed the positive cells of the infection and the fluorescence intensity, and the results are shown in Figure 12b.
  • Example 8 Adenovirus (AdV) mediates transfection of human B cells
  • Raji cell culture Raji cells were cultured in RPM1640 complete medium containing 10% FBS. Cell passage was performed every other day during the cultivation.
  • Azide modification of Raji cells The above cells were replaced with fresh medium, and a monosaccharide azide derivative Ac 4 GlcNAz with a final concentration of 50 ⁇ M was co-cultured with the cells for 48 hours, and the excess modifier was washed away with PBS buffer. The cells were stained with DBCO-Fluor 488. Cells were harvested and flow cytometric analysis of the expression of azide molecules on the cell surface. As shown in Figure 10a, the azide molecule is highly expressed on the surface of Raji cells.
  • Cationic targeting polymer-encapsulated adenovirus Dilute the cationic targeting polymer viral vector into serum-free RPM1640 medium (final concentration 0.5 ⁇ g/ml) and dilute the virus to the above culture at a dose of 2 Base, incubate for 15 minutes at room temperature.
  • Transfection of Raji cells with adenovirus-cation targeting polymer Wash the above treated cells with PBS Twice twice to remove residual sugar derivatives.
  • the above adenovirus-cation targeting polymer is added to the Raji cell culture solution obtained by the above step, and centrifuged at 2500 rpm for 1.5 hours at room temperature, and then placed in an incubator for 5 hours, and then replaced with fresh culture solution; After hours, the residue was washed away with PBS buffer, and the virus-transfected cells were harvested for use.
  • the adenovirus used in this experiment carries a green fluorescent protein (GFP) reporter gene, and GFP expression in Raji cells transfected with adenovirus can be observed by laser confocal microscopy.
  • GFP green fluorescent protein
  • the adenovirus-cation targeting polymer treated Raji cells had significant green fluorescent protein signal compared to each control group.
  • flow cytometric analysis confirmed the positive cells of the infection and the fluorescence intensity, and the results are shown in Figure 13b. It can be seen that cells infected with adenovirus-cation targeting polymer have high GFP+ cell number and strong fluorescence intensity, indicating that the cationic targeting polymer (adenovirus) vector can efficiently transfect Raji cells.

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Abstract

涉及一种病毒转染增效剂——DBCO修饰的阳离子聚合物PEI(PEI-DBCO)和其在病毒转染中的应用，特别是基于点击化学的病毒转染应用，以及病毒转染增效剂的制备方法。还涉及病毒转染载体系统，其为PEI-DBCO吸附在病毒颗粒表面而形成的病毒-PEI-DBCO复合物，其表面具有DBCO基团和正电荷，能够有效地提高病毒转染效率，以及病毒转染载体系统的应用。最后，涉及一种哺乳动物细胞转染方法，通过生物正交反应实现哺乳动物细胞病毒的高效装载及细胞转染，以及细胞的叠氮修饰在哺乳动物细胞转染中的应用。

Description

一种病毒转染增效剂和基于点击化学的病毒转染应用 技术领域
本发明属于基因治疗领域，具体地，本发明涉及一种病毒转染增效剂——DBCO修饰的阳离子聚合物PEI(PEI-DBCO)和其在病毒转染中的应用，特别是基于点击化学的病毒转染应用，以及涉及病毒转染增效剂的制备方法。本发明还涉及病毒转染载体系统，其为PEI-DBCO吸附在病毒颗粒表面，形成的病毒-PEI-DBCO复合物，其表面具有DBCO基团和正电荷，能够有效地提高病毒转染效率，本发明也涉及病毒转染载体系统的应用。最后，本发明还涉及一种哺乳动物细胞转染方法，通过生物正交反应实现哺乳动物细胞病毒的高效装载及细胞转染，以及涉及细胞的叠氮修饰在哺乳动物细胞转染中的应用。
背景技术
基因治疗不但是治疗基因缺陷疾病的重要手段，近年来也成为治疗肿瘤、感染、神经系统性疾病的重要策略。基因治疗包括两大类：基因替代和基因编辑。基因替代是用正常的基因片段代替缺陷基因，以实现减缓或者治愈疾病目。基因编辑技术则是用锌指蛋白核酸酶(ZFN)、转录激活因子样效应物核酸酶(TALENs)、Crisper-Cas9等技术对特定DNA片段敲除、加入、修复或去除病变基因，从而达到治疗效果。传统的基因治疗是将DNA或RNA序列通过病毒载体或非病毒载体直接注入患者体内。此外，将靶细胞在体外进行基因改造后，培养扩增回输至患者体内，也成为一种重要的基因治疗方式。近年来，利用基因编辑技术建立的系列基因改造免疫细胞， 如嵌合抗原受体修饰T细胞(CAR-T)、T细胞受体修饰T细胞(TCR-T)、嵌合抗原受体修饰NK细胞(CAR-NK)等，在动物实验和临床试验中均显示出优良的肿瘤靶向性、特异性、杀伤活性和持久性，成为当前肿瘤免疫治疗的热点领域。
基因载体是决定基因治疗成功与否的关键环节，主要包括非病毒载体和病毒载体两大类。常用的非病毒载体主要有阳离子脂质体、阳离子聚合物、磷酸钙，纳米材料等。然而，这些载体对处于非分裂状态的悬浮、原代细胞，特别是免疫细胞进行基因转染效率低下。与非病毒载体相比，病毒载体则具有更高的转染效率，对分裂细胞和非分裂细胞均具有感染能力，并且可将目的基因整合到宿主染色体上获得稳定持久表达。尤其是对于T细胞和NK细胞等悬浮的免疫细胞，病毒载体展现出较高的转染效率，在基因修饰细胞治疗技术领域应用越来越广泛。用于基因修饰的病毒载体主要有DNA病毒，如腺病毒(adenovirus，AdV)、腺相关病毒(adeno-associated virus，AAV)、单纯疱疹病毒(Herpes simplex virus，HSV)；RNA病毒，如逆转录病毒(Retrovirus，Murine leukemia viruses(MLV))和慢病毒(lentivirus，Human immunodeficiency virus HIV)。
在对难转染的细胞进行病毒转染时，通常需要添加病毒转染增效剂以促进病毒对目的细胞的吸附，从而提高基因转染效率。聚凝胺作为一种常用的转染增效剂是一类阳离子聚合物，可以中和病毒表面的负电荷，促进病毒对目的细胞的吸附和侵染，从而提高病毒载体的转染效率。然而，聚凝胺自身具有较强的细胞毒性，对目的细胞的增殖活化等均有影响。
因此，亟需开发一种高效安全的病毒转染增效剂和病毒转染技术。
发明内容
本发明要解决的技术问题：
基因转染技术包括病毒载体介导和非病毒载体介导两大类技术。一些传统的非病毒载体和转染试剂，如DEAE-右旋糖苷、磷酸、脂质体等，均 存在各自的优缺点。DEAE-右旋糖苷法只能进行瞬时转染，而且对细胞有一定毒性；磷酸钙转染不适合原代细胞，操作重复性较差；阳离子脂质体法适用性广，但是需要无血清条件而且效果随细胞类型变化大。对于难转染的细胞，通过电穿孔法虽然可实现高效的目的基因转染，但是核酸用量多，且转染时需要高压脉冲刺激，会降低细胞的存活率，这给该方法的应用推广带来困难。
病毒载体介导基因转染技术广泛用于各类细胞的基因转染。但是对难转染的悬浮细胞和免疫细胞等细胞类型，病毒载体还需要借助转染增效剂，从而提高转染效率。目前广泛使用的转染增效剂是聚凝胺，又名为溴化己二甲铵(hexadimethrine bromide)，是一种阳离子聚合物，可以中和病毒表面的负电荷，促进病毒对靶细胞的吸附和侵染，从而提高病毒载体的转染效率。此外，纤粘连蛋白(Fibronectin)由于具有良好的促细胞间融合的作用，也被作为一种病毒转染增效剂。然而，在难转染的悬浮细胞中(如外周血T细胞)，这些转染增效剂的效果仍不理想。
本发明公开了一种新型的病毒转染增效剂和一种基于“点击化学(click-chemistry)”反应的病毒载体转染技术。这种病毒转染增效剂是环炔基(-DBCO)修饰的阳离子聚合物聚乙烯亚胺(PEI)，即PEI-DBCO，可通过静电吸引包裹在病毒表面，一方面可以中和病毒表面的负电荷，另一方面使得病毒颗粒表面携带DBCO基团。靶细胞则先与胆碱/单糖的叠氮衍生物(Azide-Choline/Monosaccharide)小分子共孵育，使其叠氮基团(-N3)通过细胞内糖代谢/脂代谢途径标记在细胞膜表面蛋白/磷脂分子上。PEI-DBCO与病毒形成聚合物-病毒复合物，其表面的-DBCO与靶细胞膜表面的-N3发生高效、特异的“点击化学”反应，形成共价结合，从而有效地协助病毒进入靶细胞。此外，病毒-聚合物复合物表面携带的正电荷也进一步促进病毒与细胞膜结合，进而协同提高病毒载体的转染效率。
本发明提供的具体方案如下：
一方面，本发明提供了一种病毒转染增效剂，其为DBCO基团修饰的 阳离子聚合物PEI，即PEI-DBCO。
在一些实施方式中，所述PEI为枝状PEI或直链PEI。
优选地，PEI的分子量MW为600-25000。
优选地，用于制备所述PEI-DBCO的DBCO修饰试剂选自DBCO-NHS酯；DBCO-硫代-NHS酯；DBCO-PEGn-NHS酯，其中n＝1-20；DBCO-C6-NHS酯；DBCO-酸；DBCO-C6-酸；DBCO-PEGn-酸，其中n＝1-20。
另一方面，本发明提供了一种病毒转染载体系统，其为病毒转染增效剂包裹的病毒，其中所述病毒转染增效剂为PEI-DBCO。
在一些实施方式中，所述病毒为DNA病毒或RNA病毒。
优选地，所述DNA病毒选自腺病毒、腺相关病毒、单纯疱疹病毒，所述RNA病毒选自逆转录病毒和慢病毒。
优选地，所述病毒转染载体带正电荷。
另一方面，本发明提供了一种制备前述病毒转染增效剂的方法，其包括如下步骤：
(a)分别溶解PEI与DBCO修饰试剂；
(b)将所述DBCO修饰试剂溶液滴加入所述PEI溶液中，并搅拌；
(c)透析，得到DBCO基团修饰的阳离子聚合物PEI，即PEI-DBCO。
优选地，所述DBCO修饰试剂选自DBCO-NHS酯；DBCO-硫代-NHS酯；DBCO-PEGn-NHS酯，其中n＝1-20；DBCO-C6-NHS酯；DBCO-酸；DBCO-C6-酸；DBCO-PEGn-酸，其中n＝1-20。
优选地，在步骤(b)中，PEI与DBCO修饰试剂分子的摩尔比为1∶1-1∶10。
优选地，在步骤(b)中，搅拌时间为2-4小时。
优选地，在步骤(c)中，所述透析为采用MW1000的透析袋在超纯水中透析3天。
另一方面，本发明提供了PEI-DBCO在病毒转染中的应用。
在一些实施方式中，所述病毒为DNA病毒或RNA病毒。
优选地，所述DNA病毒选自腺病毒、腺相关病毒、单纯疱疹病毒，所述RNA病毒选自逆转录病毒和慢病毒。
另一方面，本发明提供前述病毒转染载体系统用于转染细胞的应用。
在一些实施方式中，其中所述细胞为悬浮哺乳动物细胞或贴壁哺乳动物细胞。
另一方面，本发明提供了N3-DBCO的点击化学在病毒转染中的应用。
在一些实施方式中，所述病毒为DNA病毒或RNA病毒。
优选地，所述DNA病毒选自腺病毒、腺相关病毒、单纯疱疹病毒，所述RNA病毒选自逆转录病毒和慢病毒。
在一些实施方案中，所述病毒转染用于悬浮哺乳动物细胞或贴壁哺乳动物细胞。
另一方面，本发明提供了一种病毒转染方法，包括以下步骤：
(a)将靶细胞与胆碱叠氮衍生物或单糖叠氮衍生物共孵育，得到叠氮修饰的靶细；
(b)将PEI-DBCO与病毒混合并孵育，获得病毒-PEI-DBCO复合物；
(c)将步骤(b)中得到的病毒-PEI-DBCO复合物与(a)中得到的叠氮修饰的靶细胞孵育后，离心；
(d)将步骤(c)中离心得到的细胞病毒混合液在CO2细胞培养箱中培养4-6小时后，半量更换培养液后继续培养。
在一些实施方式中，所述靶细胞为悬浮哺乳动物细胞或贴壁哺乳动物细胞。
在一些实施方式中，在步骤(a)中，所述的胆碱叠氮衍生物或单糖叠氮衍生物如下图所示：
胆碱-叠氮类似物：
Figure PCTCN2017093870-appb-000001
单糖-叠氮类似物：
Figure PCTCN2017093870-appb-000002
在一些实施方式中，在步骤(a)中，将在体外培养的活细胞1×106与浓度为1-100μM的胆碱叠氮衍生物或单糖叠氮衍生物在37℃的5％CO2培养箱中孵育48小时。
在一些实施方式中，在步骤(b)中，PEI-DBOC终浓度为0.01-10μg/ml，病毒感染复数MOI＝0.1-200，室温孵育15分钟。
在一些实施方式中，在步骤(c)中，将病毒-PEI-DBCO复合物与(a)中得到的叠氮修饰的靶细胞在37℃孵育后，离心，条件是2500rpm，90分钟，室温。
在再一个方面，本发明提供了细胞的叠氮修饰在病毒转染中的应用。
在一些实施方式中，所述的叠氮修饰是利用胆碱叠氮衍生物或单糖叠氮衍生物对细胞进行代谢修饰，使其表面携带叠氮基团。
在一些实施方式中，所述细胞为悬浮哺乳动物细胞或贴壁哺乳动物细胞。
在一些实施方式中，所述病毒为DNA病毒或RNA病毒。
优选地，所述DNA病毒选自腺病毒、腺相关病毒、单纯疱疹病毒，所述RNA病毒选自逆转录病毒和慢病毒。
本发明的主要技术效果：
(1)基于N3-DBCO的“点击化学”反应高效特异，且不受细胞培养体 系中其他因素的干扰，因此利用PEI-DBCO包裹的病毒可以迅速与靶细胞膜表面的-N3基团形成共价结合，从而有效地协助病毒进入靶细胞。
(2)PEI-DBCO与病毒形成聚合物-病毒复合物，其表面携带的正电荷也进一步促进病毒与细胞膜结合，进而协同提高病毒载体的转染效率。
(3)通过代谢标记技术对靶细胞进行叠氮化修饰，高效低毒，避免了化学偶联对细胞表面活性分子的影响。
发明详述：
本发明公开了一种阳离子聚合物病毒转染增效剂和一种基于“点击化学”反应的病毒载体转染技术。该技术采用DBCO基团修饰的阳离子聚合物聚乙烯亚胺(PEI-DBCO)作为病毒转染增效剂，包裹在病毒载体表面，使其表面携带DBCO基团。靶细胞则先与胆碱/单糖的叠氮衍生物小分子共孵育，使叠氮基团(-N3)通过细胞内糖代谢/脂代谢途径标记在细胞膜表面蛋白/磷脂分子上。PEI-DBCO包裹的病毒-阳离子聚合物复合体，其表面的-DBCO与靶细胞膜表面的-N3发生高效、特异的“点击化学”反应，形成共价结合，从而有效地协助病毒进入靶细胞。(见图1)。对图1的解释如下：
(1)将胆碱/单糖的叠氮衍生物与靶细胞共培养，通过靶细胞的糖类/脂类代谢途径衍生物被无损的修饰嵌入细胞膜中，其中叠氮基团-N3被裸露在细胞膜表面；
(2)PEI-DBCO与病毒颗粒孵育后，通过静电作用PEI-DBCO与病毒颗粒包裹，形成病毒-阳离子复合物复合体；
(3)其表面基团-DBCO与靶细胞膜表面-N3发生“点击化学”反应形成共价结合，同时复合物表面的正电荷也可协助自身与细胞结合，从而高效地将病毒导入靶细胞内。
所述的阳离子聚合物转染增效剂PEI-DBCO，其特征是一种DBCO修饰的阳离子聚合物聚乙烯亚胺(PEI)。采用枝状/直链PEI分子(MW＝600-25000)与点击化学DBCO基团及其衍生物反应，通过-NHS或-COOH与PEI分子的伯氨基缩合形成酰胺键，从而将DBCO基团偶联在 PEI分子上，形成PEI-DBCO。点击化学DBCO试剂包括，DBCO-NHS酯、DBCO-硫代-NHS酯、DBCO-PEGn-NHS酯(其中n＝1-20)、DBCO-C6-NHS酯、DBCO-Acid、DBCO-C6-Acid、DBCO-PEGn-Acid(其中n＝1-20)等，具体结构详见图2。
PEI-DBCO制备方法包括以下步骤：将PEI与NHS-DBCO分别溶解在PBS缓冲液中，将点击化学DBCO试剂溶液缓慢滴加入PEI溶液中(PEI与DBCO试剂分子的摩尔比为1∶1-1∶10)，搅拌反应2-4小时，随后采用MW1000的透析袋在超纯水中透析3天即可得到PEI-DBCO阳离子聚合物，真空冷冻干燥后可长期保存(见图3)。
本发明还提供了一种基于“点击化学”反应的病毒载体转染技术。该技术通过叠氮分子介导的生物正交连接反应，将DBCO修饰的多聚阳离子的病毒载体与T细胞膜表面的叠氮基团吸附结合，以实现对T细胞的高效转染。其特征在于所述方法包括以下步骤：
(1)叠氮(-N3)修饰靶细胞：在靶细胞培养过程中添加胆碱/单糖叠氮衍生物(0.1-100μM/106细胞)，培养48-72小时后，叠氮基团可通过细胞内脂代谢/糖代谢连接到靶细胞膜的糖蛋白或磷脂分子上。用pH在7.2-7.6的缓冲液洗涤细胞2次，重悬于新鲜培养基中。
所述的靶细胞包括所有悬浮和贴壁哺乳动物细胞。所述的胆碱叠氮衍生物包括AE-Cho、AP-Cho等相关的脂类类似物，单糖叠氮衍生物包括Ac4GlcNAz、Ac4GalNAz、Ac4ManNAz等相关的糖类类似物。具体结构见图4。
(2)PEI-DBCO聚合物包裹病毒载体：将PEI-DBCO与病毒颗粒混合(PEI-DBCO终浓度为0.01-10μg/ml，病毒载体的剂量(感染复数，multiplicity of infection，MOI)为0.1-200)室温孵育15分钟，获得病毒-阳离子复合物。所述病毒载体包括DNA病毒和RNA病毒。
(3)靶细胞转染：将病毒-阳离子复合物加入前述叠氮(-N3)修饰靶细胞，将细胞与病毒复合物的混合液在室温下低速离心(2500rpm，1.5小时)，再置于细胞培养箱继续培养。4-6小时后半量更换新鲜培养液，继续 培养48-72小时，即获得基因表达的靶细胞。
术语定义：
“PEI”，是指聚乙烯亚胺(Polyethyleneimine，PEI)又称聚氮杂环丙烷，是一种水溶性高分子聚合物，聚乙烯亚胺分为线性和分枝状两种，线性聚乙烯亚胺包含的全是仲胺，而分枝状聚乙烯亚胺中有伯胺、仲胺和叔胺基。聚乙烯亚胺能将DNA缩合成带正电荷的微粒，这些微粒可以黏合到带有负电荷的细胞表面残基，并通过胞吞作用进入细胞。本领域技术人员根据实际情况，能够选用合适的PEI，这些合适的PEI都在本发明的保护范围之内。此外，本发明的阳离子聚合物并不限于PEI。
“DBCO”，是指一种用于点击化学反应的化学基团，“DBCO修饰剂”是指用于点击化学反应的试剂，主要用于相关目标物的修饰。DBCO修饰剂包括但不限于如下：DBCO-NHS酯、DBCO-硫代-NHS酯、DBCO-PEGn-NHS酯(其中n＝1-20)、DBCO-C6-NHS酯、DBCO-Acid、DBCO-C6-Acid、DBCO-PEGn-Acid(其中n＝1-20)。本领域技术人员根据实际情况，能够选用合适的DBCO修饰剂，这些合适的DBCO修饰剂都在本发明的保护范围之内。
“转染增效剂”，这里主要是指用于提高病毒转染效率的生化试剂，如聚凝胺(polybrane)、纤连蛋白(fibnectin)及本发明所保护的阳离子靶向聚合物。本领域技术人员根据实际情况，能够选用合适的转染增效剂，这些合适的转染增效剂都在本发明的保护范围之内。
“病毒载体”，本发明中的病毒载体是指用于负载病毒的载体，本领域技术人员根据实际情况，能够选用合适的病毒载体，这些合适的病毒载体都在本发明的保护范围之内。
“DNA病毒”，指病毒的核酸是DNA，在本发明中，包括但不限于腺病毒、腺相关病毒、单纯疱疹病毒等。本领域技术人员根据实际情况，能够选用合适的DNA病毒，这些合适的DNA病毒都在本发明的保护范围之内。
“RNA病毒”，指病毒核酸是RNA，在本发明中，包括但不限于慢病毒、 流感病毒、逆转录病毒等。本领域技术人员根据实际情况，能够选用合适的RNA病毒，这些合适的RNA病毒都在本发明的保护范围之内。
“胆碱叠氮衍生物”，叠氮修饰过的胆碱衍生物，包括但不限于本发明列出的种类。本领域技术人员根据实际情况，能够选用合适的胆碱叠氮衍生物，这些合适的胆碱叠氮衍生物都在本发明的保护范围之内。
“单糖叠氮衍生物”，叠氮修饰过的单糖衍生物，包括但不限于本发明列出的种类。本领域技术人员根据实际情况，能够选用合适的单糖叠氮衍生物，这些合适的单糖叠氮衍生物都在本发明的保护范围之内。
“点击化学(click chemistry)”，又称为“链接化学”，主旨是通过小单元(化学基团)的连接反应，来快速稳定地完成各种分子的化学合成。它具有高效、特异、稳定的优良特征，反应既不被复杂生物体系所干扰，其自身也不对生物体系产生影响。
“悬浮哺乳动物细胞”，主要是指悬浮生长的哺乳动物细胞，在本发明中包括但不限于T细胞、B细胞、巨噬细胞(THP-1，RAW细胞)、NK细胞等免疫相关细胞(包括人源与鼠源)。本领域技术人员根据实际情况，能够选用合适的悬浮哺乳动物细胞，这些合适的悬浮哺乳动物细胞都在本发明的保护范围之内。
“贴壁哺乳动物细胞”，主要是指贴壁生长的哺乳动物细胞，在本发明中包括但不限于HEK293T、4T-1细胞等(包括人源与鼠源)。本领域技术人员根据实际情况，能够选用合适的贴壁哺乳动物细胞，这些合适的贴壁哺乳动物细胞都在本发明的保护范围之内。
“感染复数(Multiplicity of infection，MOI)”，感染时病毒和细胞数量的比值，用于确定病毒感染靶细胞所需剂量，同时MOI的值越高，对细胞毒性越大。
附图说明
图1为PEI-DBCO通过“点击化学”提高病毒转染效率。
图2为点击化学DBCO修饰试剂分子结构。
图3为PEI-DBCO合成路线。
图4为胆碱/单糖的叠氮衍生物结构式。
图5为激光共聚焦，蛋白质印迹及流式细胞分析Jurkat细胞叠氮分子修饰情况。
图6为PEI-DBCO携带慢病毒转染Jurkat细胞后GFP的表达情况。
图7为激光共聚焦与流式细胞分析人源αβT细胞表面叠氮分子修饰情况。
图8为PEI-DBCO携带慢病毒转染人源αβT细胞后GFP的表达与细胞杀伤水平。
图9为PEI-DBCO携带慢病毒转染人源γδT细胞后GFP的表达情况。
图10为PEI-DBCO携带慢病毒转染Raji细胞后GFP的表达情况。
图11为PEI-DBCO携带慢病毒转染THP-1细胞后GFP的表达情况。
图12为PEI-DBCO携带腺病毒转染293T细胞后GFP的表达情况。
图13为PEI-DBCO携带腺病毒转染Raji细胞后GFP的表达情况。
具体实施方式
下面以实例对本技术发明做进一步的说明，但是不限制本发明的内容。
实施例中所用试剂、原料和设备均为本领域技术人员熟知，且均为市场上能够购买到或容易获得或制得。其中的各种参数和条件，包括数量、比例、分子量、温度、时间等等，并不限于实施例中的具体参数和条件，本领域技术人员根据实际情况有能力采用其他的参数和条件，并取得相同或相似的技术效果。
实施例1：PEI-DBCO制备
将阳离子聚合物PEI(1.8K，枝状)(Sigma)与生物正交反应连接剂DBCO-PEG4-NHS(Click chemistry tools)按1∶10摩尔比混合，室温孵育3 小时，残留的DBCO-PEG4-NHS分子通过1KDa透析袋透析(Merck Millipore)去除。合成的阳离子靶向聚合物(PEI-DBCO)进一步通过质谱/核磁分析、鉴定。
实施例2：慢病毒介导的Jurkat细胞转染
Jurkat细胞培养：Jurkat细胞培养在含有β-巯基乙醇的RPM1640完全培养基中。在培养过程中，每隔一天进行细胞传代或换液。
Jurkat细胞的叠氮化修饰：将上述细胞的培养基换成新鲜的培养基，将终浓度为50μM的单糖叠氮衍生物Ac4GalNAz与细胞共培养48小时，然后用PBS缓冲液洗去多余的修饰试剂，细胞进行DBCO-Fluor 488染色。收获细胞并运用激光共聚焦显微镜，蛋白免疫印迹与细胞流式分析叠氮分子在细胞表面的表达情况。如图5所示，叠氮分子已经表达在细胞表面，并且具有剂量依赖的特点。
阳离子靶向聚合物包裹慢病毒：将阳离子靶向聚合物(PEI-DBCO)稀释到无血清的RPMI-1640培养基中(终浓度为0.02μg/ml)，并将病毒按照感染复数为10的剂量稀释到上述培养基，室温孵育15分钟，得到慢病毒-阳离子靶向聚合物。
慢病毒-阳离子靶向聚合物转染Jurkat细胞：将上述修饰过的细胞用PBS洗涤两次，去除残留的Ac4GalNAz分子。将上述慢病毒-阳离子靶向聚合物加入以上步骤修饰得到的Jurkat细胞培养液中，在37℃下离心(2500rpm，1.5小时)，置于培养箱培养5小时后更换新鲜的培养液培养48小时，用PBS缓冲液洗去残留物，收获被病毒转染的细胞备用。
由于该实验采用的慢病毒携带绿色荧光蛋白(GFP)报告基因，因此通过荧光显微镜可观察到被慢病毒转染Jurkat细胞的GFP表达情况。如图6a所示，与各对照组相比，慢病毒-阳离子靶向聚合物处理的Jurkat细胞具有显著的荧光信号。同时流式细胞分析确定感染的阳性细胞数与荧光强度，结果见图6b。可以看到慢病毒-阳离子靶向聚合物感染的细胞具有很高的GFP+细胞数和较强的荧光强度，说明阳离子靶向聚合物病毒载体能够高效 转染Jurkat细胞。
实施例3：慢病毒介导的人原代αβT淋巴细胞的转染
人原代αβT淋巴细胞培养：从健康志愿者外周血中分离得到外周血单核细胞(PBMC)，并将其培养在含有Anti-CD3/CD28抗体包被的磁珠及IL-2(300U/mL)的AIM-V(2％FBS)培养基中。在培养过程中，每隔一天进行细胞换液。其中上述方法获得的CD3+T细胞可达98％。
人原代αβT淋巴细胞的叠氮化修饰：细胞在培养3d后，将终浓度为25-100μM的单糖叠氮衍生物Ac4GalNAz、Ac4GlcNAz及Ac4ManNAz分别与αβT细胞共培养48小时，然后用PBS缓冲液洗去多余的修饰试剂，收获细胞并对其进行DBCO-Fluor 488荧光染色，运用激光共聚焦显微镜与细胞流式分析叠氮分子在细胞表面的表达情况。如图7所示，Ac4GalNAz与Ac4GlcNAz经代谢修饰细胞后，叠氮分子已经表达在细胞表面，并且具有显著的剂量依赖的特点。
阳离子靶向聚合物包裹慢病毒：将阳离子靶向聚合物病毒载体稀释到无血清的AIM-V培养基中(0.02μg/ml)，并将病毒按照感染复数为10剂量稀释到上述培养基，室温孵育15分钟。
慢病毒-阳离子靶向聚合物转染人原代T淋巴细胞：将上述修饰过的T细胞用PBS洗涤两次，去除残留的糖类衍生物。将慢病毒-阳离子靶向聚合物加入以上步骤修饰得到的人原代T淋巴细胞培养液中，在28℃下离心(2500rpm，1.5小时)，置于培养箱培养5小时后更换新鲜的培养液培养96小时，用PBS缓冲液洗去残留物，收获被病毒转染的细胞备用。
由于该实验采用的慢病毒携带绿色荧光蛋白(GFP)或CD19-CAR基因，因此慢病毒对αβT细胞的转染效率可以通过检测GFP的表达水平与效应T细胞的杀伤能力(CTL)来评价。如图8a所示，与各对照组相比，慢病毒(GFP)-阳离子靶向聚合物处理的细胞具有显著的绿色荧光蛋白信号。同时流式细胞分析确定感染的阳性细胞数与荧光强度，结果见图8b。结果显示慢病毒(GFP)-阳离子靶向聚合物感染的细胞具有很高的GFP+细胞数 (70-80％)与较强的荧光强度；同时，效应T细胞杀伤结果表明慢病毒(CD19-CAR)-阳离子靶向聚合物感染的细胞具有很强的肿瘤细胞杀伤能力(见图8c)。这些结果表明阳离子靶向聚合物病毒载体能高效转染人原代αβT淋巴细胞。
实施例4：慢病毒介导人原代γδT淋巴细胞的转染
人原代γδT淋巴细胞培养：从健康志愿者外周血中分离得到外周血单核细胞(PBMC)，并将其培养在含有Zoledronicacidhydrate(1μmol/L)与IL-2(100U/mL)的RPM1640培养基中。在培养过程中，每隔一天进行细胞换液。该方法获得的γδT细胞为30％。
γδT淋巴细胞的叠氮化修饰：细胞在培养3d后，将终浓度为25-100M的单糖叠氮衍生物Ac4GalNAz与Ac4GlcNAz分别与细胞共培养48小时，然后用PBS缓冲液洗去多余的修饰试剂，收获细胞并对其进行DBCO-Fluor488荧光染色，运用细胞流式分析叠氮分子在细胞表面的表达情况。如图9a所示，Ac4GalNAz经代谢修饰细胞后，叠氮分子成功修饰在细胞表面。
阳离子靶向聚合物包裹慢病毒：将阳离子靶向聚合物病毒载体稀释到无血清的RPM1640培养基中(0.01μg/ml)，并将病毒按照感染复数为10剂量稀释到上述培养基，室温孵育15分钟。
慢病毒-阳离子靶向聚合物转染人原代γδT淋巴细胞：将上述修饰过的细胞用PBS洗涤两次，去除残留的糖类衍生物。将慢病毒-阳离子靶向聚合物加入以上步骤所得的细胞培养液中，在28℃下离心(2500rpm，1.5小时)，置于培养箱培养5小时后更换新鲜的培养液培养96小时，用PBS缓冲液洗去残留物，收获被病毒转染的细胞备用。
由于该实验采用的慢病毒携带绿色荧光蛋白(GFP)报告基因，因此慢病毒对γδT细胞的转染效率可以通过检测GFP的表达水平来评价。如9b、c所示，与对照组细胞相比，慢病毒-阳离子靶向聚合物感染的细胞具有很高的GFP+细胞数(60％)与较强的荧光强度。这些结果表明阳离子靶向聚合物病毒载体能高效转染人原代γδT淋巴细胞。
实施例5：慢病毒介导人源B细胞的转染
Raji细胞培养：Raji细胞培养在含有10％FBS的RPM1640完全培养基中。在培养过程中，每隔一天进行细胞传代。
Raji细胞的叠氮化修饰：将上述细胞换成新鲜的培养基，将终浓度为50M的单糖叠氮衍生物Ac4GlcNAz与细胞共培养48小时，用PBS缓冲液洗去多余的修饰剂，细胞进行DBCO-Fluor 488染色。收获细胞并用细胞流式分析叠氮分子在细胞表面的表达情况。如图10a所示，叠氮分子高效表达在Raji细胞表面。
阳离子靶向聚合物包裹慢病毒：将阳离子靶向聚合物病毒载体稀释到无血清的RPM1640培养基中(终浓度为0.05μg/ml)，并将病毒按照感染复数为5的剂量稀释到上述培养基，室温孵育15分钟。
慢病毒-阳离子靶向聚合物转染Raji细胞：将上述处理的细胞用PBS洗涤两次，去除残留的糖类衍生物。将上述慢病毒-阳离子靶向聚合物加入以上步骤修饰得到的Raji细胞培养液中，在室温条件下，2500rpm，离心1.5小时，置于培养箱培养5小时后更换新鲜的培养液；继续培养96小时，用PBS缓冲液洗去残留物，收获被病毒转染的细胞备用。
该实验采用的慢病毒携带绿色荧光蛋白(GFP)报告基因，可通过激光共聚焦显微镜可观察到被慢病毒转染的Raji细胞中GFP表达情况。如图10b所示，与各对照组相比，慢病毒-阳离子靶向聚合物处理的Raji细胞具有显著的绿色荧光蛋白信号。同时流式细胞分析确定感染的阳性细胞与荧光强度，结果见图10c。可以看到慢病毒-阳离子靶向聚合物感染的细胞具有很高的GFP+细胞数和较强的荧光强度，说明阳离子靶向聚合物病毒载体能够高效转染Raji细胞。
实施例6：慢病毒介导人源巨噬细胞THP-1的转染
THP-1细胞培养：THP-1细胞培养在含有10％FBS的RPM1640完全培养基中。在培养过程中，每隔一天进行细胞传代。
THP-1细胞的叠氮化修饰：将上述细胞换成新鲜的培养基，然后将终浓度为50μM的单糖叠氮衍生物Ac4GalNAz与细胞共培养48小时后，用PBS缓冲液洗去多余的修饰试剂，细胞进行DBCO-Fluor 488染色。收获细胞并运用流式分析叠氮在细胞表面的表达情况。如图11a所示，叠氮分子高效表达在THP-1细胞表面。
阳离子靶向聚合物包裹慢病毒：将阳离子靶向聚合物病毒载体稀释到无血清的RPM1640培养基中(终浓度为0.1μg/ml)，并将病毒按照感染复数为10的剂量稀释到上述培养基，室温孵育15分钟。
慢病毒-阳离子靶向聚合物转染THP-1细胞：将上述修饰过的细胞用PBS洗涤两次，去除残留的糖类衍生物。将上述慢病毒-阳离子靶向聚合物加入以上步骤修饰得到的THP-1细胞培养液中，在室温下离心(2500rpm，1.5小时)，置于培养箱培养4-6小时后更换新鲜的培养液培养96小时，用PBS缓冲液洗去残留物，收获被病毒转染的细胞备用。
由于该实验采用的慢病毒携带绿色荧光蛋白(GFP)报告基因，因此通过激光共聚焦显微镜可观察到被慢病毒转染的THP-1细胞中GFP表达情况。如图11b所示，与各对照组相比，慢病毒-阳离子靶向聚合物处理THP-1细胞具有显著的绿色荧光蛋白信号。同时流式细胞分析确定感染的阳性细胞与荧光强度，结果见图11c。可以看到慢病毒-阳离子靶向聚合物感染的细胞具有很高的GFP+细胞数和较强的荧光强度，说明阳离子靶向聚合物病毒载体能够高效转染THP-1细胞。
实施例7：腺病毒(AdV)介导人胚肾细胞(HEK 293T)的转染
HEK 293T细胞培养：HEK293T细胞培养在含有10％FBS的DMEM完全培养基中。在培养过程中，每隔一天进行细胞传代。
HEK 293T细胞的叠氮化修饰：将上述细胞换成新鲜的培养基，将终浓度为25μM的单糖叠氮衍生物Ac4GalNAz与细胞共培养48小时，用PBS缓冲液洗去多余的修饰剂，细胞进行DBCO-Fluor 488染色。收获细胞并用细胞流式分析叠氮分子在细胞表面的表达情况。
阳离子靶向聚合物包裹腺病毒：将阳离子靶向聚合物病毒载体稀释到无血清的DMEM培养基中(终浓度为0.5μg/ml)，并将病毒按照感染复数为5的剂量稀释到上述培养基，室温孵育15分钟。
腺病毒-阳离子靶向聚合物转染HEK293T细胞：将上述处理的细胞用PBS洗涤两次，去除残留的糖类衍生物。将上述慢病毒-阳离子靶向聚合物加入以上步骤修饰得到的HEK 293T细胞培养液中，在室温条件下，2500rpm，离心1.5小时，置于培养箱培养5小时后更换新鲜的培养液；继续培养48小时，用PBS缓冲液洗去残留物，收获被病毒转染的细胞备用。
该实验采用的腺病毒携带绿色荧光蛋白(GFP)报告基因，可通过激光共聚焦显微镜可观察到被腺病毒转染的HEK 293T细胞中GFP表达情况。如图12a所示，与各对照组相比，腺病毒-阳离子靶向聚合物处理的HEK293T细胞具有显著的绿色荧光蛋白信号。同时流式细胞分析确定感染的阳性细胞与荧光强度，结果见图12b。可以看到腺病毒-阳离子靶向聚合物感染的细胞具有非常高的GFP+细胞数和较强的荧光强度，说明阳离子靶向聚合物(腺病毒)载体能够高效转染HEK 293T细胞。
实施例8：腺病毒(AdV)介导人源B细胞的转染
Raji细胞培养：Raji细胞培养在含有10％FBS的RPM1640完全培养基中。在培养过程中，每隔一天进行细胞传代。
Raji细胞的叠氮化修饰：将上述细胞换成新鲜的培养基，将终浓度为50μM的单糖叠氮衍生物Ac4GlcNAz与细胞共培养48小时，用PBS缓冲液洗去多余的修饰剂，细胞进行DBCO-Fluor 488染色。收获细胞并用细胞流式分析叠氮分子在细胞表面的表达情况。如图10a所示，叠氮分子高效表达在Raji细胞表面。
阳离子靶向聚合物包裹腺病毒：将阳离子靶向聚合物病毒载体稀释到无血清的RPM1640培养基中(终浓度为0.5μg/ml)，并将病毒按照感染复数为2的剂量稀释到上述培养基，室温孵育15分钟。
腺病毒-阳离子靶向聚合物转染Raji细胞：将上述处理的细胞用PBS洗 涤两次，去除残留的糖类衍生物。将上述腺病毒-阳离子靶向聚合物加入以上步骤修饰得到的Raji细胞培养液中，在室温条件下，2500rpm，离心1.5小时，置于培养箱培养5小时后更换新鲜的培养液；继续培养96小时，用PBS缓冲液洗去残留物，收获被病毒转染的细胞备用。
该实验采用的腺病毒携带绿色荧光蛋白(GFP)报告基因，可通过激光共聚焦显微镜可观察到被腺病毒转染的Raji细胞中GFP表达情况。如图13a所示，与各对照组相比，腺病毒-阳离子靶向聚合物处理的Raji细胞具有显著的绿色荧光蛋白信号。同时流式细胞分析确定感染的阳性细胞与荧光强度，结果见图13b。可以看到腺病毒-阳离子靶向聚合物感染的细胞具有很高的GFP+细胞数和较强的荧光强度，说明阳离子靶向聚合物(腺病毒)载体能够高效转染Raji细胞。
以上实施例所用的具体的阳离子聚合物(PEI)、生物正交修饰剂(DBCO)、病毒载体、靶细胞、叠氮衍生物等，并不构成对本发明保护范围的限定。任何根据本发明的技术构思所做出的各种其他相应的改变与变形，均应包含在本发明权利要求的保护范围内。
以上所述仅是本发明的较佳实施例而已，并非对本发明做任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本专业的技术人员，在不脱离本发明技术方案的范围内，当可利用上述揭示的技术内容作出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。

Claims (28)

  1. 一种病毒转染增效剂，其为DBCO基团修饰的阳离子聚合物PEI，PEI-DBCO。
  2. 如权利要求1所述的病毒转染增效剂，其中，所述PEI为枝状PEI或直链PEI。
  3. 如权利要求1所述的病毒转染增效剂，其中，PEI的分子量MW为600-25000。
  4. 如权利要求1-3任一项所述的病毒转染增效剂，其中，用于制备所述PEI-DBCO的DBCO修饰试剂选自DBCO-NHS酯；DBCO-硫代-NHS酯；DBCO-PEGn-NHS酯，其中n＝1-20；DBCO-C6-NHS酯；DBCO-酸；DBCO-C6-酸；DBCO-PEGn-酸，其中n＝1-20。
  5. 一种病毒转染载体系统，其为病毒转染增效剂包裹的病毒，其中所述病毒转染增效剂为PEI-DBCO。
  6. 如权利要求5所述的病毒转染载体系统，其中，所述病毒为DNA病毒或RNA病毒。
  7. 如权利要求6所述的病毒转染载体系统，其中，所述DNA病毒选自腺病毒、腺相关病毒、单纯疱疹病毒，所述RNA病毒选自逆转录病毒和慢病毒。
  8. 如权利要求5所述的病毒转染载体系统，其中，所述病毒转染载体带正电荷。
  9. 一种制备权利要求1-4任一项所述的病毒转染增效剂的方法，其包括如下步骤：
    (a)分别溶解PEI与DBCO修饰试剂；
    (b)将所述DBCO修饰试剂溶液滴加入所述PEI溶液中，并搅拌；
    (c)透析，得到DBCO基团修饰的阳离子聚合物PEI，PEI-DBCO。
  10. 如权利要求9所述的方法，其中，所述DBCO修饰试剂选自DBCO-NHS酯；DBCO-硫代-NHS酯；DBCO-PEGn-NHS酯，其中n＝1-20；DBCO-C6-NHS酯；DBCO-酸；DBCO-C6-酸；DBCO-PEGn-酸，其中n＝1-20。
  11. 如权利要求9所述的方法，其中，在步骤(b)中，PEI与DBCO修饰试剂分子的摩尔比为1∶1-1∶10。
  12. PEI-DBCO在病毒转染中的应用。
  13. 如权利要求12所述的应用，其中，所述病毒为DNA病毒或RNA病毒。
  14. 如权利要求13所述的应用，其中，所述DNA病毒选自腺病毒、腺相关病毒、单纯疱疹病毒，所述RNA病毒选自逆转录病毒和慢病毒。
  15. 权利要求5-8任一项所述的病毒转染载体系统用于转染细胞的应用。
  16. 如权利要求15所述的应用，其中所述细胞为悬浮哺乳动物细 胞或贴壁哺乳动物细胞。
  17. N3-DBCO的点击化学在病毒转染中的应用。
  18. 如权利要求17所述的应用，其中，所述病毒为DNA病毒或RNA病毒。
  19. 如权利要求18所述的应用，其中，所述DNA病毒选自腺病毒、腺相关病毒、单纯疱疹病毒，所述RNA病毒选自逆转录病毒和慢病毒。
  20. 如权利要求17所述的应用，其中，所述病毒转染用于悬浮哺乳动物细胞或贴壁哺乳动物细胞。
  21. 一种病毒转染方法，包括以下步骤：
    (a)将靶细胞与胆碱叠氮衍生物或单糖叠氮衍生物共孵育，得到叠氮修饰的靶细胞；
    (b)将PEI-DBCO与病毒混合并孵育，获得病毒-PEI-DBCO复合物；
    (c)将步骤(b)中得到的病毒-PEI-DBCO复合物与(a)中得到的叠氮修饰的靶细胞孵育后，离心；
    (d)将步骤(c)中离心得到的细胞病毒混合液在CO2细胞培养箱中培养4-6小时后，半量更换培养液后继续培养。
  22. 如权利要求21所述的方法，其中，所述靶细胞为悬浮哺乳动物细胞或贴壁哺乳动物细胞。
  23. 如权利要求21所述的方法，在步骤(a)中，所述的胆碱叠氮衍生 物或单糖叠氮衍生物如下图所示：
    Figure PCTCN2017093870-appb-100001
  24. 细胞的叠氮修饰在病毒转染中的应用。
  25. 如权利要求24所述的应用，其中，所述的叠氮修饰是利用胆碱叠氮衍生物或单糖叠氮衍生物对细胞进行代谢修饰，使其表面携带叠氮基团。
  26. 如权利要求24所述的应用，其中，所述细胞为悬浮哺乳动物细胞或贴壁哺乳动物细胞。
  27. 如权利要求24所述的应用，其中，所述病毒为DNA病毒或RNA病毒。
  28. 如权利要求27所述的应用，其中，所述DNA病毒选自腺病毒、腺相关病毒、单纯疱疹病毒，所述RNA病毒选自逆转录病毒和慢病毒。
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