Bioorthogonal-based viral modification technology and applications thereof

Abstract

The present application relates to a kind of virus modification technology based on biological ortho and its application, also provide a kind of surface modification has the virus carrier of orthogonal group fusion protein, the virus carrier is obtained after the virus packaging of modified orthogonal group fusion protein cell and virus packaging plasmid are mixed, carry out virus packaging;The virus packaging plasmid includes Packaging series plasmid, Envelope series plasmid and auxiliary system expression plasmid;And the coding sequence of the membrane fusion protein gene in the Envelope series plasmid is deleted;The coding sequence of the membrane fusion protein gene is as shown in SEQ ID NO:1.The present application provides a kind of novel specific virus transfection method based on biological ortho technology, realizes the efficient low-toxicity specific transfection of virus vector to a variety of host cells, improves the efficiency of target gene delivery, breaks the in-vivo gene delivery barrier.

Description

[0001] This application claims priority to Chinese Patent Application No. 202510516845.4, filed on April 23, 2025, entitled "A Virus Modification Technology Based on Bioorthogonality and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to a virus modification technology based on biological orthogonality and its application. Background Technology

[0003] Viral vectors are created by modifying viruses using genetic engineering techniques to deliver exogenous genes reliably and efficiently. These vectors carry therapeutic genes into target cells for long-term gene expression. As a biopharmaceutical delivery technology, engineered viral vectors offer advantages such as high transfection efficiency and high levels of exogenous gene expression, and are widely used in basic research and gene therapy. Currently, lentiviruses, adenoviruses, and adeno-associated viruses are among the more mature viral packaging technologies. Achieving efficient and specific in vivo gene delivery is a key objective of engineered viral vectors. However, in vivo gene delivery places high demands on the immunogenicity and safety of viral vectors. Furthermore, insufficient delivery efficiency and specificity can hinder the further development of vectors for live gene delivery.

[0004] Most current gene-editing clinical trials involve ex vivo editing, a method feasible for some important cell types such as hematopoietic stem cells (HSCs), but unsuitable for transplantation back into patients after ex vivo editing. In vivo gene editing, which involves directly delivering genes to edit cells within the body, offers a promising solution for treating genetic diseases. However, in vivo gene editing requires the efficient and safe delivery of editing elements directly to a sufficient number of relevant cells, a significant challenge. In vivo gene delivery vectors must effectively target the desired cells and deliver a sufficient quantity of editing factors to them. Other vectors have their own limitations in in vivo delivery: low transfection efficiency is a major problem for non-viral vectors such as liposomes, polymers, or nanoparticles. Because they lack the natural infection mechanism of viral vectors, they suffer from low cellular uptake or poor in vivo escape capabilities, leading to inefficiency. They also suffer from insufficient targeting, limited delivery capacity, and low stability. Viral vectors such as adenovirus and adeno-associated virus, due to their high immunogenicity, easily induce the production of neutralizing antibodies and T cell-mediated cytotoxicity, making it impossible to effectively target the desired cells. They lack specific targets, ultimately leading to insufficient in vivo delivery and off-target effects.

[0005] Bioorthogonal chemistry refers to a class of chemical reactions that can proceed rapidly in living systems. These reactions occur under complex physiological conditions within organisms, without reacting with other components or causing any interference or damage to the organism or target biomolecules. Bioorthogonal chemistry labeling strategies based on metabolic engineering are increasingly becoming an ideal in vivo and in vitro biomodification strategy due to their non-destructive, efficient, stable, and specific characteristics. It utilizes cellular metabolic engineering to non-destructively introduce specific chemical reporter groups into target molecules, and then forms stable covalent linkages with the target molecules carrying pairing groups through bioorthogonal reactions, achieving specific labeling and capture of the target molecules.

[0006] Given that the external structural components of viruses, such as the viral envelope and capsid, originate from the carbohydrates, proteins, and lipids of the host cell, during viral replication and assembly, metabolic engineering can be used to embed metabolic derivatives carrying reporter groups into the viral capsid and envelope, achieving non-destructive metabolic modification of the virus by chemical functional groups. Based on this, the chemical linkage reaction between target cells modified with specific paired groups and the groups carried by the virus can achieve highly efficient, mild, and specific functional enhancement of the virus. Different metabolic modification methods can be selected according to the differences in virus types. For example, enveloped viruses can use carbohydrate or lipid metabolism to modify phospholipids and glycoproteins in the viral envelope; non-enveloped viruses can use amino acid or carbohydrate metabolism to modify proteins and glycoprotein molecules on the capsid surface. Therefore, this non-destructive bioorthogonal modification method for viruses has good universality and application prospects.

[0007] Existing viral transfection techniques include: 1. Adding cationic polymers during viral transfection to improve transfection efficiency through electrostatic adsorption; 2. Adding viral transduction enhancers during viral transfection to alter host cell membrane permeability and transmembrane transport; 3. Concentrating and precipitating the virus using a flat-angle centrifuge to improve the efficiency of virus-cell contact. These methods all have drawbacks to varying degrees. 4. Current lentiviral vectors employ a four-plasmid packaging system, including envelope protein particles, such as those encoding vesicular stomatitis virus G protein (VSV-G), to broaden the target cell tropism range and stability of the vector. Method 1 is only applicable to adherent cells and neutralizes the charge on the cell surface and virus particle surface, affecting the normal physiological functions of both cells and viruses, exhibiting significant cytotoxicity. Method 2 is only applicable to specific viral vectors, affecting cell membrane permeability and transmembrane transport function, thus exhibiting significant toxicity to cells in complex environments, and has high reagent preparation costs and limited transfection-promoting ability. Method 3 is time-consuming, requires specialized equipment, and is ineffective on adherent cells. Method 4 exhibits low transfection efficiency for specific cell types and can negatively impact subsequent in vivo biodistribution and drug efficacy. Ultimately, while the aforementioned methods address the shortcomings of in vitro cell genetic engineering, they fail to provide a satisfactory solution to the critical issue of insufficient in vivo gene delivery.

[0008] Therefore, there is an urgent need to develop a novel specific viral transfection method based on bioorthogonal technology to achieve efficient and low-toxicity specific transfection of viral vectors into various host cells, improve the delivery efficiency of target genes, and overcome the barriers to gene delivery in vivo. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a virus modification technology based on bioorthogonalization and its applications. This invention overcomes the drawbacks of existing methods for promoting viral transfection of host cells, such as high cytotoxicity of transfection reagents, narrow applicability, susceptibility to environmental influences, low transfection efficiency, insufficient in vivo gene delivery, and low specificity. It provides a novel, specific virus transfection method based on bioorthogonalization technology, achieving highly efficient, low-toxicity, and specific transfection of viral vectors into various host cells, improving the efficiency of target gene delivery, and overcoming in vivo gene delivery barriers.

[0010] To achieve this objective, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides a viral vector of a membrane-free fusion protein with orthogonal groups modified on its surface, wherein the viral vector is obtained by mixing the viral vector packaging cells modified with orthogonal groups with a viral packaging plasmid and then packaging the virus.

[0012] The viral packaging plasmids include Packaging series plasmids, Envelope series plasmids, and helper expression plasmids; and the coding sequence of the membrane fusion protein gene in the Envelope series plasmids (viral envelope plasmids) has been deleted; the coding sequence of the membrane fusion protein gene is shown in SEQ ID NO:1;

[0013] The Packaging series plasmids include psPAX2 (12260, Didier Trono) or pCMV delta R8.2 (12263, Didier Trono).

[0014] The Envelope series of helper plasmids include PMD2.G (12259, Didier Trono), pCMV-VSV-G (8454, Bob Weinberg), pCAG-VSVG (35616, Arthur Nienhuis lab), or pLTR-G (17532, Jakob Reiser).

[0015] The helper expression plasmids include pZsGreenI (632187, Clontech) and / or p-Lvx-mCherry (VT1461, Youbio).

[0016] In this invention, information about the viral packaging plasmids mentioned above can be found on the following website (https: / / www.addgene.org / viral-vectors / lentivirus / ). Taking psPAX2 as an example, 12260 is the plasmid number, which comes from Didier Trono's laboratory; the source information for other plasmids is similar.

[0017] Currently, lentiviral vectors utilize viral fusion proteins that suffer from low specificity and high immunogenicity, hindering in vivo gene delivery. This invention knocks out viral fusion proteins in existing viral vectors (including but not limited to lentiviruses, adenoviruses, and adeno-associated viruses) and performs non-destructive labeling on the surface of these viral vectors through endogenous metabolic processes (such as oligosaccharide metabolic engineering, non-natural amino acid metabolic engineering, and choline lipid metabolism) or liposome membrane fusion technology. The modified derivatives, carrying orthogonal functional groups, are then implanted into the target cells and viral surfaces, enabling in vivo gene delivery, reducing in vivo immunogenicity, and enhancing immunogenicity against specific host cells.

[0018] Preferably, the orthogonal group is selected from any one of the following: Azide, Alkyne, BCN, Tz, DBCO, TCO, Cyclopropene, Norbornene, ketone, or hydroxylamine.

[0019] Preferably, the viral vector packaging cells are selected from any one or a combination of at least two of the following: HEK293T cells, Jurkat cells, RAW264.7 cells, HepG2 cells, MDA-MB-231 cells, NK92-MI cells, or Primary T cells.

[0020] Preferably, the method for modifying orthogonal groups includes: endogenous metabolic modification, chemical group modification, or liposome membrane fusion modification of viral vector packaging cells.

[0021] Preferably, the endogenous metabolic modification includes any one or a combination of at least two of the following: metabolic modification of modified sugars, metabolic modification of amino acid derivatives, or metabolic modification of choline derivatives.

[0022] Preferably, the modified sugar is selected from any one or a combination of at least two of Ac4ManNAz, Ac4GalNAz, Ac4GlcNAz, Ac4ManNBCN, Ac4GalNBCN, Ac4GlcNBCN, Ac4ManNAl, Ac4GalNAl, Ac4GlcNAl, Ac4ManNCycp, Ac4GalNCycp, or Ac4GlcNCycp.

[0023] Preferably, the amino acid derivative is selected from Aha and / or HPG.

[0024] Preferably, the choline derivative is AE-Cho.

[0025] This invention provides a method for viral engineering based on bioorthogonal technology. Specifically, it utilizes highly efficient and specific endogenous metabolic modifications (including but not limited to modified sugar metabolism, non-natural amino acid metabolism, and modified choline metabolism) or liposome membrane fusion to non-destructively label bioorthogonal groups on the cell surface. By introducing viral vector particles lacking viral envelope proteins (VSV-G protein), a viral vector with membrane fusion protein defects carrying bioorthogonal groups is packaged and generated. This rapid, specific, and targeted bioorthogonal reaction enables highly efficient, low-toxicity, and controllable transfection of the viral vector into host cells, improving the efficiency of target gene delivery and enabling in vivo gene delivery.

[0026] In a second aspect, the present invention provides a method for preparing a viral vector of a membrane-free fusion protein with surface-modified orthogonal groups as described in the first aspect, the method comprising:

[0027] (1) Viral vector packaging cells are modified by endogenous metabolism, chemical groups or liposome membrane fusion to obtain viral vector packaging cells modified with orthogonal groups;

[0028] (2) The viral vector packaging cells modified with orthogonal groups are mixed with viral packaging plasmids and viral packaging is performed to obtain a viral vector with a membrane-free fusion protein modified with orthogonal groups on the surface.

[0029] Thirdly, the present invention provides a virus modification technology based on biological orthogonality, the virus modification technology comprising:

[0030] (1) Viral vector packaging cells are modified by endogenous metabolism, chemical groups or liposome membrane fusion to obtain viral vector packaging cells modified with orthogonal groups;

[0031] (2) The viral vector packaging cells modified with orthogonal groups are mixed with viral packaging plasmids, and viral packaging is performed to obtain a viral vector with a membrane-free fusion protein modified with orthogonal groups on the surface; the viral packaging plasmids include Packaging series plasmids, Envelope series plasmids and helper series expression plasmids; and the coding sequence of the membrane fusion protein gene in the Envelope series plasmids is deleted; the coding sequence of the membrane fusion protein gene is shown in SEQ ID NO:1;

[0032] The Packaging series plasmids include psPAX2 or pCMV delta R8.2;

[0033] The Envelope series of helper plasmids include PMD2.G, pCMV-VSV-G, pCAG-VSVG, or pLTR-G;

[0034] The helper expression plasmids include pZsGreenI and / or p-Lvx-mCherry.

[0035] Preferably, the orthogonal group is selected from any one of: Azide, Alkyne, BCN, Tz, DBCO, TCO, Cyclopropene, Norbornene, ketone, or hydroxylamine.

[0036] Preferably, the viral vector packaging cells are selected from any one or a combination of at least two of the following: HEK293T cells, Jurkat cells, RAW264.7 cells, HepG2 cells, MDA-MB-231 cells, NK92-MI cells, or Primary T cells.

[0037] Preferably, the endogenous metabolic modification includes any one or a combination of at least two of the following: metabolic modification of modified sugars, metabolic modification of amino acid derivatives, or metabolic modification of choline derivatives.

[0038] Preferably, the modified sugar is selected from any one or a combination of at least two of Ac4ManNAz, Ac4GalNAz, Ac4GlcNAz, Ac4ManNBCN, Ac4GalNBCN, Ac4GlcNBCN, Ac4ManNAl, Ac4GalNAl, Ac4GlcNAl, Ac4ManNCycp, Ac4GalNCycp, or Ac4GlcNCycp.

[0039] Preferably, the amino acid derivative is selected from Aha and / or HPG.

[0040] Preferably, the choline derivative is AE-Cho.

[0041] Fourthly, the present invention provides the application of the viral vector of the membrane-free fusion protein with surface-modified orthogonal groups as described in the first aspect in the preparation of nucleic acid transfection products.

[0042] Fifthly, the present invention provides a viral vector of a membrane-free fusion protein with surface-modified orthogonal groups as described in the first aspect, or the application of the bioorthogonal-based viral modification technology as described in the third aspect in in vitro nucleic acid transfection or in vivo gene delivery.

[0043] The viral vector in this invention can achieve highly efficient and specific in vivo gene delivery using orthogonal biological reactions. Orthogonal biological reactions are introduced during the infection process of the viral vector with host cells and target cells (including but not limited to primary cells, immune cells, stem cells, and tumor cells). Modified host cells are co-incubated with an appropriate concentration of engineered viral vector. The adhesion and invasion of the virus to the cells are efficiently mediated by the orthogonal biological reaction. Based on target cell reinfusion technology and live virus injection technology, highly efficient and specific gene transfection of the viral vector into the aforementioned host cells is achieved in vivo.

[0044] In a sixth aspect, the present invention provides a nucleic acid transfection kit, the kit comprising a viral vector of a membrane-free fusion protein with orthogonal groups modified on its surface as described in the first aspect.

[0045] In a seventh aspect, the present invention provides a nucleic acid transfection method, the method comprising: linking a nucleic acid fragment to a viral vector containing a membrane-free fusion protein with orthogonal groups modified on its surface as described in the first aspect; transfecting a host cell with the viral vector, wherein the host cell surface is modified with orthogonal groups, the orthogonal groups comprising any one or a combination of at least two of the following: Azide, Alkyne, BCN, Tz, TCO, Cyclopropene, DBCO, Norbornene, ketone, or hydroxylamine.

[0046] Preferably, the method for modifying the host cell includes: endogenous metabolic modification or liposome membrane fusion modification.

[0047] Preferably, the host cell is selected from any one or a combination of at least two of the following: primary cells, immune cells, stem cells, or tumor cells.

[0048] The method of this invention is applicable to bioorthogonal metabolic labeling technology for immune cells, stem cells, and somatic cells. It achieves the implantation of bioorthogonal functional groups carried by modified derivatives (including but not limited to carbohydrate derivatives Ac4ManNAz, Ac4ManNBCN, Ac4ManNAl, Ac4GalNAz, Ac4GlcNAz or Ac4ManNCycp; amino acid derivatives Aha, HPG; choline derivatives AE-Cho) or liposome membrane fusion technology into cells through co-culturing. The bioorthogonal group labeling involved includes but is not limited to Azide, Alkyne, BCN, Tz, DBCO, TCO, Cyclopropene, Norbornene, ketone, or hydroxylamine.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] (1) This invention employs a novel bioorthogonal modification strategy to engineer viral vectors with knocked-out membrane fusion proteins, thereby providing the viral vectors with a novel invasion pathway for host cells while performing efficient labeling. This invention constructs stable, specific, and safe artificial targets on viral vectors with knocked-out membrane fusion proteins, achieving the "creation of targets from nothing." Furthermore, the knockout of membrane fusion proteins and the implantation of artificial targets alter the broad infectivity of existing viral vectors, achieving redirection between the viral vector and host cells, and significantly enhancing specificity. Targets based on bioorthogonal reactions facilitate rapid adsorption of viral vectors to the surface of host cells, with mild reaction conditions, high efficiency, strong specificity, and good stability, reducing the risk of off-target effects. The artificial targets constructed in this invention are universal and can be constructed between cells and viral vectors that originally lacked or had low targeting specificity, making them suitable for various types of viral vectors and host cells. Specifically, this invention is applicable to various types of viral vectors, including but not limited to lentiviruses, adenoviruses, and adeno-associated viruses; host cells include but are not limited to primary cells, immune cells, stem cells, and tumor cells of different species; and based on the principle of bioorthogonality, it can be applied to other organisms such as bacteria and fungi, and can be engineered for customization. The method of this invention does not compromise viral yield, engineered viral vectors can be mass-produced, and the small molecule chemical targets are low-cost and require small quantities, resulting in good economic benefits.

[0051] (2) The viral vector constructed in this invention can be used for in vivo gene delivery. The knockout of membrane fusion proteins and the targeting effect based on novel biological orthogonal reactions greatly reduce immunogenicity, reduce serum complement inactivation, and lower the body's immune level against transgenic products, thus reducing off-target problems in in vivo gene delivery.

[0052] (3) In relatively complex biological systems such as blood and human tissues, the bioorthogonal reaction of this gene delivery enhancement strategy can occur under complex physiological conditions in the body. It will not react with other components in the body, nor will it cause any interference or damage to the organism or the target biomolecule.

[0053] (4) This gene delivery enhancement strategy is simple and easy to implement, effectively improves infection efficiency, and has low toxicity to host cells.

[0054] (5) The engineered viral vector of the present invention has a wide range of applications, including cell therapy, gene therapy, disease model construction (including but not limited to tumors, inflammation, aging, metabolic diseases or degenerative diseases), anti-infection or liposome membrane fusion technology, etc. Attached Figure Description

[0055] Figure 1 This is a schematic diagram of the bioorthogonal group metabolic labeling of host cells and viral vectors.

[0056] Figure 2 This is a data graph showing the successful construction of a viral vector that does not express the membrane fusion protein VSV-G and is modified with bioorthogonal groups.

[0057] Figure 3 This involves laser confocal imaging analysis of the metabolic expression of the Az group in HEK 293T cells.

[0058] Figure 4 It uses flow cytometry to analyze the surface modifications of different cells by lipid insertion.

[0059] Figure 5 This is a schematic diagram illustrating how biological orthogonal groups mediate viral vector infection of target cells.

[0060] Figure 6 It is the expression of GFP in HEK 293T, RAW264.7, and Jurkat cells after viral infection modified by bioorthogonal groups.

[0061] Figure 7 The study measured the efficiency of viral vector infiltration into tumors and the CAR positivity rate in blood and spleen by in vitro transfection of viruses without biological orthogonal groups or in vivo transfection of viruses with biological orthogonal groups. Detailed Implementation

[0062] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0063] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0064] Example 1

[0065] Modification of bioorthogonal groups on the surface of viral vectors

[0066] Figure 1 This is a schematic diagram of the metabolic labeling of viral vector packaging cells and viral vector bioorthogonal groups. Figure 2 This is a data graph showing the successful construction of a viral vector that does not express the membrane fusion protein VSV-G and is modified with bioorthogonal groups.

[0067] The viral vector-packaged cells were seeded into cell culture flasks. After the cells adhered, endogenous metabolic sugar derivatives (such as Ac4manNAz) were added and the cells were cultured until the next day when the cell aggregate was about 70%.

[0068] Viral packaging plasmids (such as lentiviral packaging plasmids pZsGreenI, psPAX2, PMD2.G) were added to pure cell culture medium in appropriate proportions. After thorough mixing, liposome transfection reagent was added, and the mixture was allowed to stand at room temperature. The mixture was then gently mixed to prepare the transfection complex. The complex was then slowly added dropwise to the packaging cell culture medium and incubated for transfection under cell culture conditions. The culture medium was then replaced with a complete culture medium containing sugar derivatives, and the cells were cultured for another 36-48 hours before the virus was isolated and purified.

[0069] The harvested cell culture supernatant containing the virus was centrifuged at 4°C to remove a small number of cells, and then filtered through a 0.45 μm sterile filter membrane to remove cell debris. The collected virus solution was added to a virus concentration reagent, gently inverted to mix, and allowed to stand at 4°C. After standing, it was centrifuged at high speed, and the supernatant was removed to obtain a white precipitate, which was the virus. The white precipitate was resuspended in PBS to obtain concentrated virus solution, which was aliquoted and stored at -80°C to avoid repeated freeze-thaw cycles.

[0070] The viral packaging plasmids include Packaging series plasmids, Envelope series plasmids, and helper expression plasmids; and the coding sequence of the membrane fusion protein gene in the Envelope series plasmids has been deleted;

[0071] The Packaging series plasmids include psPAX2 or pCMV delta R8.2;

[0072] The Envelope series of helper plasmids include PMD2.G, pCMV-VSV-G, pCAG-VSVG, or pLTR-G;

[0073] The helper expression plasmids include pZsGreenI and / or p-Lvx-mCherry.

[0074] The coding sequence of the membrane fusion protein gene includes that shown in SEQ ID NO:1:

[0075]

[0076] Example 2

[0077] Cellular endogenous metabolic markers bioorthogonal groups

[0078] HEK 293T cells were cultured in medium supplemented with a carbohydrate derivative (Ac4ManNAz) to a final concentration of 30 μM for 48 h. The cells were then incubated with the fluorescent probe DBCO-AZDye 488 at room temperature for 20 min, washed with PBS, and then incubated with Hoechst 33342 nuclear staining agent for nuclear staining. After washing with PBS, images were captured using a laser confocal microscope. Figure 3 As shown, strong fluorescence was detected on the cell membrane surface of the experimental group by confocal microscopy, while only weak background fluorescence was detected in the control group, proving that at the experimental concentration, the bioorthogonal group can be efficiently integrated into the cell membrane surface of HEK 293T cells via metabolic pathways.

[0079] Example 3

[0080] Multiple cellular lipids inserted into biological orthogonal groups

[0081] Add orthogonalized phospholipid PEG polymer (DSPE-PEG-DBCO) to the culture medium of different cell lines (HEK293T, Jurkat, RAW264.7, HepG2, MDA-MB-231, NK92-MI, PrimaryT) to a final concentration of 100 μM, gently mix, and incubate at 37°C in a shaker for 40 minutes. Add the fluorescent probe N3-AZDye 488 and incubate at room temperature for 20 minutes. After washing with PBS, analyze the fluorescence signal using flow cytometry. Figure 4 As shown, the flow cytometry results indicate that over 90% of the cells carry fluorescent signals, the cell membrane surface is implanted with DBCO groups, and the cells maintain high viability. The inserted groups also exhibit stability.

[0082] Example 4

[0083] Bio-orthogonal groups enhance the virus's ability to infect host cells. Figure 5 This is a schematic diagram illustrating how biological orthogonal groups mediate viral vector infection of target cells.

[0084] Approximately 10 RAW264.7 metabolically modified mononuclear macrophage cells were used. 4 ~10 6 Each virus was inoculated into a well plate, and an appropriate amount of concentrated viral vector particles modified with bioorthogonal groups was added at a multiplicity of infection of 20. After adding the virus, the plate was centrifuged at 2000 rpm for 40 min in a flat-angle centrifuge, and then incubated at 37°C.

[0085] Six to 12 hours after infection, the virus-containing culture medium was replaced with complete culture medium, and the culture was continued for 24 to 36 hours. The expression of green fluorescence was observed using an inverted fluorescence microscope, and the fluorescence was quantitatively detected by flow cytometry. Figure 5 As shown, the proportion of GFP gene-positive cells in the experimental treatment group was significantly higher than that in the control group. This experimental result proves that introducing bioorthogonal reaction groups during viral infection can effectively improve viral infection efficiency, and this effect is applicable to common difficult-to-transfect cells, such as RAW264.7 mononuclear macrophage tumor cells and Jurkat T lymphoma cells.

[0086] Example 5

[0087] In vivo delivery and transfection to generate CAT-T cells

[0088] T cells were stained with the live-cell tracer CMFDA (green). A culture medium containing a phospholipid derivative with a bio-orthogonal group (DSPE-PEG-DBCO, concentration 50-100 μM) was prepared. Fluorescently labeled T cells were cultured in this medium at 37°C in a shaker for 40 min to allow the surface to acquire the bio-orthogonal group DBCO. The cells were then resuspended in PBS and intravenously injected with 10 μL of PBS. 5 ~10 7 The above cells were introduced into mice, and 10 mg was injected intravenously 2-4 hours later. 6 The viral vector N3-LV (CAR•EGFRvIII), modified with the orthogonal group (N3-), was used to transfect host cells in vivo. CAR-T cells transfected with LV (mCAR•EGFRvIII) in vitro served as a control group. T cells were then used at 10... 5 Seeds were inoculated into culture plates, lentiviral solution (volume calculated by MOI) and polybrene (final concentration 5-8 μg / mL) were added, and the cells were centrifuged for infection. After incubation at 37°C for 24 hours, the culture medium was replaced with fresh medium to obtain in vitro transfected CAR-T cells. 10 μg / mL of the culture medium was injected intravenously. 5 ~10 7 CAR-T cells transduced in vitro were introduced into mice. Seven days after in vivo transfection, mouse blood and spleen were collected, ground, filtered, and prepared into single-cell suspensions for flow cytometry analysis. Fluorescence signals were detected using a flow cytometer; cells were first sorted using CMFDA green fluorescence, and then the corresponding antibody fluorescence signals were measured.

[0089] like Figure 6As shown, compared with the CAR-T treatment group transfected with viral vectors without bioorthogonal groups in vitro, the bioorthogonal group modified CCAR-T group had a significantly higher proportion of CAR+ cells in both blood and spleen (blood: 28.5±4.8%, spleen: 6.4±1.3%), and could achieve better intratumoral CAR+ cell infiltration. This indicates that the engineered virus modified with bioorthogonal groups can effectively improve viral infectivity, significantly enhance its T cell transfection efficiency in vivo, and promote the killing effect of T cells on tumors.

[0090] Comparative Example 1

[0091] As described above, equal amounts of lentiviral vectors LV-G (without knockout membrane fusion protein), LV-ΔG (with knockout membrane fusion protein), and LV-AzΔG (with knockout membrane fusion protein and modified with bioorthogonal groups) were prepared. These vectors were then incubated at 37°C for 1 hour in culture media with / without fresh serum. After removing the serum and isolating and purifying the virus, the physical titer of the virus was determined. Simultaneously, the three viral vectors were added to 293T cell culture media with / without fresh serum for cell transfection. After centrifugation and washing, the cells were recovered, and the GFP-positive cell recovery rate was obtained by measuring the GFP-positive signal of the cells, thus detecting the gene delivery capability of the viral vectors. Figure 7 As shown, the titer recovery rate of the lentiviral vector LV-G without knockout of the membrane fusion protein in serum-containing medium was significantly lower than that in serum-containing medium, indicating that the unmodified viral vector can bind to serum without specificity. The titer recovery rate of the viral vector after knockout of the membrane fusion protein was significantly increased, indicating that the probability of its interaction and binding with serum was greatly reduced. Meanwhile, the recovery rate of GFP-positive cells in the experimental group was significantly higher than that in the control group, indicating that the modified viral vector had a significantly enhanced gene delivery capacity and possessed specificity.

[0092] In summary, this invention provides an improvement to viral vectors based on bioorthogonal technology by knocking out viral fusion proteins in existing viral vectors (including but not limited to lentiviruses, adenoviruses, and adeno-associated viruses) and non-destructively labeling the surface of these viral vectors through endogenous metabolic processes (such as oligosaccharide metabolic engineering, non-natural amino acid metabolic engineering, and choline lipid metabolism) or liposome membrane fusion technology. The bioorthogonal functional groups carried by the modified derivatives are then implanted into the surface of target cells and viruses, enabling gene delivery in vivo, reducing their immunogenicity in vivo, and enhancing their immunogenicity against specific host cells.

[0093] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A viral vector containing a membrane-free fusion protein with surface-modified orthogonal groups, characterized in that, The viral vector is obtained by mixing viral vector packaging cells modified with orthogonal groups with viral packaging plasmids and then packaging the virus. The viral packaging plasmids include Packaging series plasmids, Envelope series plasmids, and helper expression plasmids; and the coding sequence of the membrane fusion protein gene in the Envelope series plasmids has been deleted; the coding sequence of the membrane fusion protein gene is shown in SEQ ID NO:1; The Packaging series plasmids include psPAX2 or pCMV delta R8.2; The Envelope series of helper plasmids include PMD2.G, pCMV-VSV-G, pCAG-VSVG, or pLTR-G; The helper expression plasmids include pZsGreenI and / or p-Lvx-mCherry.

2. The viral vector of a membrane-free fusion protein with surface-modified orthogonal groups according to claim 1, characterized in that, The orthogonal group is selected from any one of: Azide, Alkyne, BCN, Tz, DBCO, TCO, Cyclopropene, Norbornene, ketone, or hydroxylamine.

3. The viral vector of a membrane-free fusion protein with surface-modified orthogonal groups according to claim 1 or 2, characterized in that, The viral vector packaging cells are selected from any one or a combination of at least two of the following: HEK293T cells, Jurkat cells, RAW264.7 cells, HepG2 cells, MDA-MB-231 cells, NK92-MI cells, or Primary T cells.

4. The viral vector of a membrane-free fusion protein with surface-modified orthogonal groups according to any one of claims 1-3, characterized in that, The methods for modifying orthogonal groups include: endogenous metabolic modification, chemical group modification, or liposome membrane fusion modification of viral vector packaging cells; Preferably, the endogenous metabolic modification includes any one or a combination of at least two of the following: metabolic modification of modified sugars, metabolic modification of amino acid derivatives, or metabolic modification of choline derivatives. Preferably, the modified sugar is selected from any one or a combination of at least two of the following: Ac4ManNAz, Ac4GalNAz, Ac4GlcNAz, Ac4ManNBCN, Ac4GalNBCN, Ac4GlcNBCN, Ac4ManNAl, Ac4GalNAl, Ac4GlcNAl, Ac4ManNCycp, Ac4GalNCycp, or Ac4GlcNCycp; Preferably, the amino acid derivative is selected from Aha and / or HPG; Preferably, the choline derivative is AE-Cho.

5. A method for preparing a viral vector of a non-membrane fusion protein with surface-modified orthogonal groups as described in any one of claims 1-4, characterized in that, The preparation method includes: (1) Viral vector packaging cells are modified by endogenous metabolism, chemical groups or liposome membrane fusion to obtain viral vector packaging cells modified with orthogonal groups; (2) The viral vector packaging cells modified with orthogonal groups are mixed with viral packaging plasmids and viral packaging is performed to obtain a viral vector with a membrane-free fusion protein modified with orthogonal groups on the surface.

6. A virus modification technology based on bioorthogonality, characterized in that, The viral modification technology includes: (1) Viral vector packaging cells are modified by endogenous metabolism, chemical groups or liposome membrane fusion to obtain viral vector packaging cells modified with orthogonal groups; (2) The viral vector packaging cells modified with orthogonal groups are mixed with viral packaging plasmids, and viral packaging is performed to obtain a viral vector with a membrane-free fusion protein modified with orthogonal groups on the surface; the viral packaging plasmids include Packaging series plasmids, Envelope series plasmids and helper series expression plasmids; and the coding sequence of the membrane fusion protein gene in the Envelope series plasmids is deleted; the coding sequence of the membrane fusion protein gene is shown in SEQ ID NO:1; The Packaging series plasmids include psPAX2 or pCMV delta R8.2; The Envelope series of helper plasmids include PMD2.G, pCMV-VSV-G, pCAG-VSVG, or pLTR-G; The helper expression plasmids include pZsGreenI and / or p-Lvx-mCherry; Preferably, the orthogonal group is selected from any one of: Azide, Alkyne, BCN, Tz, DBCO, TCO, Cyclopropene, Norbornene, ketone, or hydroxylamine; Preferably, the viral vector packaging cells are selected from any one or a combination of at least two of the following: HEK293T cells, Jurkat cells, RAW264.7 cells, HepG2 cells, MDA-MB-231 cells, NK92-MI cells, or Primary T cells; Preferably, the endogenous metabolic modification includes any one or a combination of at least two of the following: metabolic modification of modified sugars, metabolic modification of amino acid derivatives, or metabolic modification of choline derivatives. Preferably, the modified sugar is selected from any one or a combination of at least two of the following: Ac4ManNAz, Ac4GalNAz, Ac4GlcNAz, Ac4ManNBCN, Ac4GalNBCN, Ac4GlcNBCN, Ac4ManNAl, Ac4GalNAl, Ac4GlcNAl, Ac4ManNCycp, Ac4GalNCycp, or Ac4GlcNCycp; Preferably, the amino acid derivative is selected from Aha and / or HPG; Preferably, the choline derivative is AE-Cho.

7. The use of the viral vector of the non-membrane fusion protein with surface-modified orthogonal groups as described in any one of claims 1-4 in the preparation of nucleic acid transfection products.

8. The viral vector of the non-membrane fusion protein with surface-modified orthogonal groups as described in any one of claims 1-4, or the application of the bioorthogonal-based viral modification technology as described in claim 6, in in vitro nucleic acid transfection or in vivo gene delivery.

9. A nucleic acid transfection kit, characterized in that, The kit includes a viral vector of a membrane-free fusion protein with surface-modified orthogonal groups as described in any one of claims 1-4.

10. A method for nucleic acid transfection, characterized in that, The method comprises: linking a nucleic acid fragment to a viral vector of a membrane-free fusion protein with an orthogonal group modified on the surface according to any one of claims 1-5; transfecting a host cell with the viral vector, wherein the host cell surface is modified with an orthogonal group, the orthogonal group comprising any one or a combination of at least two of Azide, Alkyne, BCN, Tz, TCO, Cyclopropene, DBCO, Norbornene, ketone, or hydroxylamine; Preferably, the method for modifying the host cells includes: endogenous metabolic modification or liposome membrane fusion modification; Preferably, the host cell is selected from any one or a combination of at least two of the following: primary cells, immune cells, stem cells, or tumor cells.

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