A chimeric envelope glycoprotein, a method for preparing the same, and an envelope plasmid, a packaging method and a kit relating to the same

Abstract

The application provides a chimeric envelope glycoprotein, a preparation method thereof, and an envelope plasmid, a packaging method and a kit related to the envelope glypoprotein, in particular, a preparation method of a chimeric envelope glycoprotein, a chimeric envelope glycoprotein, a lentivirus envelope plasmid, a lentivirus pseudotyped packaging method and a kit. The amino acid sequence of a wild type VSV-G envelope glycoprotein is chimerized with the amino acid sequence of other envelope glycoproteins, and the chimeric envelope glycoprotein obtained can effectively improve the lentivirus packaging efficiency, biological activity and other performances compared with the initial wild type envelope glycoprotein.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a chimeric envelope glycoprotein, a preparation method thereof, and an envelope plasmid, a packaging method and a kit involving the envelope glycoprotein. BACKGROUND

[0002] Lentiviral (LV) vector system is commonly used in cell gene therapy. It is usually obtained by co-transfecting host cells with packaging plasmid (providing viral structural proteins), envelope plasmid (determining host range), and transfer plasmid (containing target gene and necessary regulatory sequences) in a certain proportion, and the resulting virus-like particles (lentivirus) selectively infect receptor cells.

[0003] LV usually uses heterologous VSV-G as envelope glycoprotein for pseudovirus packaging. Using VSV-G instead of the gene encoding the viral envelope glycoprotein in the original virus can greatly increase the host cell range of the virus, giving the vector broad tropism and stability. VSV-G is derived from vesicular stomatitis virus and targets low-density lipoprotein receptors, as this receptor is ubiquitous in various cells, so LV packaged with VSV-G is considered a relatively broad-spectrum pseudovirus. However, VSV-G injection can easily trigger a host immune response, producing antibodies that neutralize the virus vector, reducing gene transduction efficiency, affecting treatment effectiveness, and limiting its persistence in gene therapy requiring long-term expression of target genes. VSV-G packaged viruses are also inactivated by human serum complement, which also reduces their efficacy, and have cytotoxicity when stably expressed in human cells, making them unsuitable for in vivo delivery and causing adverse complement-dependent immune responses against VSV-G in patients. These limitations make VSV-G-based LV less suitable for more cell gene therapy fields.

[0004] Currently, other envelope glycoprotein-packaged LVs have been developed in laboratories, such as Cocal vesiculovirus envelope glycoprotein (Cocal-G). Cocal vesiculovirus belongs to the same genus as vesicular stomatitis virus but is serologically different. Cocal-G envelope glycoprotein has 71.5% homology with VSV-G envelope glycoprotein at the amino acid level and also targets low-density lipoprotein, thus also having broad targeting. Moreover, Cocal-G-based LVs are more resistant to human serum complement inactivation and are less susceptible to human serum inactivation, have lower immunogenicity, thus improving transduction efficiency and gene expression persistence. Therefore, Cocal-G-based LVs can more effectively transduce human, non-human primate, and canine hematopoietic stem cells (HSCs) and CD34-positive T cells, and can be better applied to cell gene therapy, especially in the field of blood cells.

[0005] However, the packaging efficiency of Cocal-G based LV is relatively low compared to VSV-G based LV, and it is difficult to obtain high titer and high activity pseudovirus. In the published patent CN118085042A, a mutant version of Cocal-G (Cocal-VX24) was obtained through random evolution, but the improvement of its packaging titer is still limited. Other envelope proteins also have similar limitations, such as BaEV. Therefore, how to improve the high packaging titer of these envelope proteins and obtain high expression activity of envelope glycoprotein is still a problem to be studied.

[0006] The full text of CN118085042A is incorporated herein. SUMMARY

[0007] The present application provides a preparation method of a chimeric envelope glycoprotein, which combines and splices different domains (such as the membrane proximal region, the transmembrane region, the intracellular region, etc.) of wild-type envelope glycoprotein in a reasonable way, and a series of chimeric proteins are obtained, which have significantly higher packaging efficiency and activity than wild-type envelope glycoprotein.

[0008] In one aspect, the present application provides a preparation method of a chimeric envelope glycoprotein, in which the amino acid sequence of wild-type VSV-G envelope glycoprotein is chimerized with the amino acid sequence of wild-type Cocal-G envelope glycoprotein or the amino acid sequence of wild-type BaEV-G envelope glycoprotein to obtain a chimeric envelope glycoprotein. In some embodiments, the amino acid sequence of wild-type VSV-G envelope glycoprotein includes a signal peptide region, a non-membrane proximal domain of an extracellular region, a membrane proximal domain of an extracellular region, a transmembrane region, and an intracellular region, and at least one region of wild-type VSV-G envelope glycoprotein is chimerized with the amino acid sequence of wild-type Cocal-G envelope glycoprotein or the amino acid sequence of wild-type BaEV-G envelope glycoprotein.

[0009] In some embodiments, the amino acid sequence of wild-type VSV-G envelope glycoprotein is chimerized with the amino acid sequence of another wild-type envelope glycoprotein. In some embodiments, the amino acid sequence of wild-type VSV-G envelope glycoprotein is chimerized with the amino acid sequence of multiple wild-type envelope glycoproteins, specifically, the chimerization of the amino acid sequences of wild-type VSV-G envelope glycoprotein, wild-type Cocal-G envelope glycoprotein, and wild-type VSV-G envelope glycoprotein.

[0010] In some embodiments, the amino acid sequence of the wild-type VSV-G envelope glycoprotein is chimerized with the amino acid sequence of the wild-type Cocal-G envelope glycoprotein to obtain a chimeric envelope glycoprotein. Specifically, the signal peptide region is selected from the amino acid sequence of the wild-type VSV-G envelope glycoprotein or the amino acid sequence of the wild-type Cocal-G envelope glycoprotein; the non-membrane proximal domain of the extracellular region is selected from the amino acid sequence of the wild-type VSV-G envelope glycoprotein or the amino acid sequence of the wild-type Cocal-G envelope glycoprotein; the membrane proximal domain of the extracellular region is selected from the amino acid sequence of the wild-type VSV-G envelope glycoprotein or the amino acid sequence of the wild-type Cocal-G envelope glycoprotein; the transmembrane region is selected from the amino acid sequence of the wild-type VSV-G envelope glycoprotein or the amino acid sequence of the wild-type Cocal-G envelope glycoprotein; and the intracellular region is selected from the amino acid sequence of the wild-type VSV-G envelope glycoprotein or the amino acid sequence of the wild-type Cocal-G envelope glycoprotein.

[0011] In some embodiments, the amino acid sequence of the wild-type VSV-G envelope glycoprotein is chimerized with the amino acid sequence of the wild-type BaEV-G envelope glycoprotein to obtain a chimeric envelope glycoprotein. Specifically, the signal peptide region is selected from the amino acid sequence of the wild-type VSV-G envelope glycoprotein or the amino acid sequence of the wild-type BaEV-G envelope glycoprotein; the non-membrane proximal domain of the extracellular region is selected from the amino acid sequence of the wild-type VSV-G envelope glycoprotein or the amino acid sequence of the wild-type BaEV-G envelope glycoprotein; the membrane proximal domain of the extracellular region is selected from the amino acid sequence of the wild-type VSV-G envelope glycoprotein or the amino acid sequence of the wild-type BaEV-G envelope glycoprotein; the transmembrane region is selected from the amino acid sequence of the wild-type VSV-G envelope glycoprotein or the amino acid sequence of the wild-type BaEV-G envelope glycoprotein; and the intracellular region is selected from the amino acid sequence of the wild-type VSV-G envelope glycoprotein or the amino acid sequence of the wild-type BaEV-G envelope glycoprotein.

[0012] Further, the signal peptide region of the wild-type VSV-G envelope glycoprotein has the amino acid sequence shown in SEQ ID NO: 1, the non-membrane proximal domain of the extracellular region has the amino acid sequence shown in SEQ ID NO: 2, the membrane proximal domain of the extracellular region has the amino acid sequence shown in SEQ ID NO: 3, the transmembrane region has the amino acid sequence shown in SEQ ID NO: 4, and the intracellular region has the amino acid sequence shown in SEQ ID NO: 5.

[0013] In some embodiments, the amino acid sequence of the wild-type VSV-G envelope glycoprotein comprises a signal peptide region, a non-membrane proximal domain of extracellular region, a membrane proximal domain of extracellular region, a transmembrane region, and an intracellular region, the chimeric of the chimeric envelope glycoprotein is replacing 1 to 3 regions of the wild-type VSV-G envelope glycoprotein with the corresponding regions of the wild-type Cocal-G envelope glycoprotein amino acid sequence or the wild-type BaEV-G envelope glycoprotein amino acid sequence. Further, the non-membrane proximal domain of extracellular region of the chimeric envelope glycoprotein is selected from the corresponding regions of the wild-type Cocal-G envelope glycoprotein or the wild-type BaEV-G envelope glycoprotein.

[0014] Further, the signal peptide region amino acid sequence of the wild-type Cocal-G envelope glycoprotein is shown as SEQ ID NO: 6, the non-membrane proximal domain of extracellular region amino acid sequence is shown as SEQ ID NO: 7, the membrane proximal domain of extracellular region amino acid sequence is shown as SEQ ID NO: 8, the transmembrane region amino acid sequence is shown as SEQ ID NO: 9, and the intracellular region amino acid sequence is shown as SEQ ID NO: 10.

[0015] In some embodiments, the signal peptide region amino acid sequence of the chimeric envelope glycoprotein is selected from SEQ ID NO: 1 or SEQ ID NO: 6, the non-membrane proximal domain of extracellular region amino acid sequence is shown as SEQ ID NO: 7, the membrane proximal domain of extracellular region amino acid sequence is selected from SEQ ID NO: 3 or SEQ ID NO: 8, the transmembrane region amino acid sequence is selected from SEQ ID NO: 4 or SEQ ID NO: 9, and the intracellular region amino acid sequence is selected from SEQ ID NO: 5 or SEQ ID NO: 10.

[0016] Specifically, the respective amino acid sequence or mutant sequence of each of the plurality of chimeric Cocal-G envelope glycoproteins in the present application is any one of the following groups:

[0017] a) VSV-G-wt: SEQ ID NO: 1 + SEQ ID NO: 2 + SEQ ID NO: 3 + SEQ ID NO: 4 + SEQ ID NO: 5;

[0018] b) Cocal-G-wt: SEQ ID NO: 6 + SEQ ID NO: 7 + SEQ ID NO: 8 + SEQ ID NO: 9 + SEQ ID NO: 10;

[0019] c) CVGc: SEQ ID NO: 6 + SEQ ID NO: 7 + SEQ ID NO: 8 + SEQ ID NO: 9 + SEQ ID NO: 5;

[0020] d) CVGmc: SEQ ID NO: 6 + SEQ ID NO: 7 + SEQ ID NO: 8 + SEQ ID NO: 4 + SEQ ID NO: 5;

[0021] e) CVGpmc: SEQ ID NO: 6 + SEQ ID NO: 7 + SEQ ID NO: 3 + SEQ ID NO: 4 + SEQ ID NO: 5;

[0022] f) GsCV: SEQ ID NO: 1 + SEQ ID NO: 7 + SEQ ID NO: 8 + SEQ ID NO: 9 + SEQ ID NO: 10;

[0023] g) GsCVGc: SEQ ID NO: 1 + SEQ ID NO: 7 + SEQ ID NO: 8 + SEQ ID NO: 9 + SEQ ID NO: 5;

[0024] h) GsCVGmc: SEQ ID NO: 1 + SEQ ID NO: 7 + SEQ ID NO: 8 + SEQ ID NO: 4 + SEQ ID NO: 5;

[0025] i) GsCVGpmc: SEQ ID NO: 1 + SEQ ID NO: 7 + SEQ ID NO: 3 + SEQ ID NO: 4 + SEQ ID NO: 5.

[0026] In some embodiments, the signal peptide region amino acid sequence of the chimeric envelope glycoprotein obtained using the optimized method of chimerization of wild-type VSV-G envelope glycoprotein with wild-type Cocal-G envelope glycoprotein is as set forth in SEQ ID NO: 1, the non-membrane proximal domain amino acid sequence of the extracellular region is as set forth in SEQ ID NO: 7, the membrane proximal domain amino acid sequence of the extracellular region is as set forth in SEQ ID NO: 8, the transmembrane region amino acid sequence is as set forth in SEQ ID NO: 9, and the intracellular region amino acid sequence is as set forth in SEQ ID NO: 5.

[0027] In some embodiments, the signal peptide region amino acid sequence of the chimeric envelope glycoprotein is selected from SEQ ID NO: 1 or SEQ ID NO: 11, the non-membrane proximal domain amino acid sequence of the extracellular region is as set forth in SEQ ID NO: 12, the membrane proximal domain amino acid sequence of the extracellular region is selected from SEQ ID NO: 3 or SEQ ID NO: 13, the transmembrane region amino acid sequence is selected from SEQ ID NO: 4 or SEQ ID NO: 14, and the intracellular region amino acid sequence is selected from SEQ ID NO: 5 or SEQ ID NO: 15.

[0028] Further, the signal peptide region amino acid sequence of the chimeric envelope glycoprotein is selected from SEQ ID NO: 1 or SEQ ID NO: 11, the non-membrane proximal domain amino acid sequence of the extracellular region is as set forth in SEQ ID NO: 12, the membrane proximal domain amino acid sequence of the extracellular region is selected from SEQ ID NO: 3 or SEQ ID NO: 13, the transmembrane region amino acid sequence is selected from SEQ ID NO: 4 or SEQ ID NO: 14, and the intracellular region amino acid sequence is selected from SEQ ID NO: 5 or SEQ ID NO: 15.

[0029] In particular, the wild type VSV-G envelope glycoprotein and the wild type BaEV-G envelope glycoprotein are chimerized in the present application, and the amino acid sequences of the respective chimeric envelope glycoproteins obtained are any one of the following groups:

[0030] j) BVGmc: SEQ ID NO: 11 + SEQ ID NO: 12 + SEQ ID NO: 13 + SEQ ID NO: 4 + SEQ ID NO: 5;

[0031] k) GsBVGmc: SEQ ID NO: 1 + SEQ ID NO: 12 + SEQ ID NO: 13 + SEQ ID NO: 4 + SEQ ID NO: 5;

[0032] In some embodiments, the signal peptide region amino acid sequence of the chimeric envelope glycoprotein is selected from SEQ ID NO: 1 or SEQ ID NO: 11, the non-membrane proximal domain amino acid sequence of the extracellular region is as set forth in SEQ ID NO: 12, the membrane proximal domain amino acid sequence of the extracellular region is selected from SEQ ID NO: 3 or SEQ ID NO: 13, the transmembrane region amino acid sequence is selected from SEQ ID NO: 4 or SEQ ID NO: 14, and the intracellular region amino acid sequence is selected from SEQ ID NO: 5 or SEQ ID NO: 15.

[0033] The present application also provides a lentivirus envelope plasmid, which can transcribe any of the above chimeric envelope glycoproteins. The envelope plasmid comprises a promoter, a coding sequence of the chimeric BaEV-G envelope glycoprotein, and a transcription termination signal, wherein the promoter is upstream of the coding sequence and the transcription termination signal is downstream of the coding sequence, ensuring efficient expression and correct processing of the chimeric envelope glycoprotein in host cells. The lentivirus envelope plasmid is suitable for various viral vector systems and can significantly improve the titer and infection efficiency of viral particles, providing a reliable tool for gene therapy and vaccine development.

[0034] The present application also provides a packaging method for lentivirus, which uses any of the above chimeric envelope glycoproteins or envelope plasmids to package lentivirus. The method comprises co-transfecting the chimeric envelope glycoprotein or its coding plasmid with a viral structural protein expression plasmid into host cells to achieve efficient packaging of lentivirus particles through transient transfection or stable transfection. The packaged viral vectors exhibit higher infection efficiency and wider host range in target cells, and are particularly suitable for primary cells and stem cells that are difficult to transfect. In addition, the packaging method is simple and reproducible, and its stability and reliability have been verified in multiple experiments, providing strong technical support for gene function research and clinical gene therapy applications.

[0035] The present application also provides a kit comprising the above chimeric envelope glycoprotein or envelope plasmid. The kit optionally comprises auxiliary plasmids, transfection reagents, and instructions for use required for viral packaging, and is suitable for scientific research and preclinical research. The components are optimally matched to ensure efficient packaging and stable production of viruses. The chimeric envelope glycoprotein in the kit can mediate viral targeting to specific cell types, significantly improving transduction efficiency while reducing immunogenicity risk.

[0036] The present application also provides a pseudotyped lentivirus comprising the above defined chimeric envelope glycoprotein. It also relates to a medicament comprising the pseudotyped lentivirus as defined above as an active ingredient. The pseudotyped lentivirus achieves efficient recognition and infection of specific cell types through surface-displayed chimeric BaEV-G envelope glycoproteins, significantly improving the transduction efficiency and bioavailability of viral vectors in vitro and in vivo. The therapeutic genes carried by it can be stably integrated into the host genome, achieving long-term expression, and are suitable for the treatment of various diseases such as genetic diseases, tumors, and viral infections.

[0037] Also contemplated is a pharmaceutical composition comprising the pseudotyped lentivirus as defined above and a pharmaceutically acceptable carrier. The pharmaceutical composition can be administered via various routes, including but not limited to intravenous injection, local injection or oral administration, and the dosage form and dosage can be optimized according to the type of disease and the treatment requirement to ensure the effective concentration and safety of the drug at the target site. The present application also relates to a method for treating a subject in need thereof, which comprises administering to the subject in need thereof a therapeutically effective amount of the pseudotyped lentivirus as defined above.

[0038] The present application also relates to a method for treating a subject in need thereof, which comprises administering to the subject in need thereof a therapeutically effective amount of the pseudotyped lentivirus as defined above.

[0039] In the context of the present application, "subject" refers to a human or a non-human mammal, such as a rodent (rat, mouse, rabbit), a primate (chimpanzee), a feline (cat), a canine (dog). Preferably, the subject is a human.

[0040] In addition, the present application also provides an efficient virus packaging system, which comprises a plasmid encoding the above-mentioned chimeric BaEV-G envelope glycoprotein, a virus structural protein expression plasmid and a host cell line. The system effectively improves the assembly efficiency and titer of virus particles through a synergistic expression mechanism, and is suitable for large-scale production of virus vectors.

[0041] The present application provides a preparation method of the chimeric envelope glycoprotein, which adopts the amino acid sequence of the wild-type VSV-G envelope glycoprotein and the amino acid sequence of the wild-type Cocal-G envelope glycoprotein or the amino acid sequence of the wild-type BaEV-G envelope glycoprotein for chimerization, so that the chimeric protein can effectively improve the lentivirus packaging efficiency, biological activity and other properties compared with the initial wild-type envelope glycoprotein. By rationally designing and functionally recombining the envelope glycoprotein sequences from different sources, the chimeric protein further enhances the stability and transmembrane fusion efficiency of the virus particles on the basis of maintaining the original receptor recognition ability. BRIEF DESCRIPTION OF DRAWINGS

[0042] In order to better understand the present application and more clearly show how to implement the present application, the features of the embodiments according to the present application are described by way of example and with reference to the accompanying drawings, in which:

[0043] Figure 1 : Sequence region schematic diagram of various envelope glycoproteins used in the present application.

[0044] Figure 2 : Expression image of fluorescent protein after wild-type VSV-G and wild-type Cocal-G chimeric envelope glycoprotein pseudotyped lentivirus infected 293TH cells for 48 hours.

[0045] Figure 3 Results of luciferase activity and percentage of NeonGreen flow cytometry-positive cells 48 hours after infection of 293TH cells with wild-type VSV-G and wild-type Cocal-G chimeric envelope glycoprotein pseudotype lentivirus.

[0046] Figure 4 Image of fluorescent protein expression in Jurkat cells 48 hours after infection with wild-type VSV-G and wild-type Cocal-G chimeric envelope glycoprotein pseudovirus.

[0047] Figure 5 Results of luciferase activity and percentage of NeonGreen flow cytometry-positive cells 48 hours after infection of Jurkat cells with wild-type VSV-G and wild-type Cocal-G chimeric envelope glycoprotein pseudovirus.

[0048] Figure 6 Results of luciferase activity in NK cells 48 hours after infection with wild-type VSV-G and wild-type BaEV-G chimeric envelope glycoprotein pseudovirus. Detailed Implementation

[0049] Definitions: To provide a clear and consistent understanding of the terminology used in this specification, some definitions are provided below. Furthermore, unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0050] When used in conjunction with the term "comprising" in the claims and / or specification, the word "a" can mean "one," but it is also known to mean "one or more," "at least one," and "one or more." Similarly, the word "another" can mean at least a second or more.

[0051] As used in this specification and claims, the words “comprising” (and any form of inclusion, such as “comprising” and “including”), “having” (and any form of having, “having,” “including,” and “containing”) are inclusive and open-ended and do not exclude additional unlisted elements or processing steps.

[0052] As used herein, the term "VSV-G envelope glycoprotein" refers to the wild-type form of the VSV-G envelope glycoprotein or a mutant of the wild-type BaEV-G envelope glycoprotein that is at least 80%, preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% identical to the wild-type VSV-G envelope glycoprotein, provided that the mutant glycoprotein retains the ability of the wild-type glycoprotein to bind to and fuse with the hematopoietic cell membrane.

[0053] As used herein, the term "Cocal-G envelope glycoprotein" refers to a wild-type form of a Cocal-G envelope glycoprotein or a mutant of said wild-type Cocal-G envelope glycoprotein which is at least 80%, preferably at least 85%, still preferably at least 90%, more preferably at least 95%, still more preferably at least 99% identical to said wild-type BaEV-G envelope glycoprotein, provided that said mutant glycoprotein retains the ability of the wild-type glycoprotein to bind to hematopoietic cell membranes and to fuse with hematopoietic cell membranes.

[0054] As used herein, the term "BaEV-G envelope glycoprotein" refers to a wild-type form of a BaEV-G envelope glycoprotein or a mutant of said wild-type BaEV-G envelope glycoprotein which is at least 80%, preferably at least 85%, still preferably at least 90%, more preferably at least 95%, still more preferably at least 99% identical to said wild-type BaEV-G envelope glycoprotein, provided that said mutant glycoprotein retains the ability of the wild-type glycoprotein to bind to hematopoietic cell membranes and to fuse with hematopoietic cell membranes.

[0055] As used herein, "chimeric envelope glycoprotein" refers to a chimeric protein of a VSV-G envelope glycoprotein and a Cocal-G envelope glycoprotein or a BaEV-G envelope glycoprotein. "Chimeric" herein means that the signal peptide region, the non-membrane proximal domain of the extracellular region, the membrane proximal domain of the extracellular region, the transmembrane region, and the intracellular region of the envelope glycoprotein as shown are spliced together from the corresponding regions of a wild-type VSV-G envelope glycoprotein and a wild-type Cocal-G envelope glycoprotein or a wild-type BaEV-G envelope glycoprotein. Figure 1

[0056] As used herein, "pseudotype" or "pseudotyped lentivirus" generically, the process of introducing a heterologous envelope glycoprotein onto the core of a lentiviral vector is termed "pseudotyping". Pseudotyped lentivirus is usually achieved by co-transfecting a plasmid encoding a heterologous envelope glycoprotein into packaging cells with a lentiviral vector system, resulting in recombinant lentivirus particles that carry the heterologous envelope glycoprotein on their surface. These particles have a similar core structure as the original lentivirus, but their surface envelope glycoprotein determines the targeting and invasion efficiency of the virus to infect host cells. Through the pseudotyping technique, different sources of envelope glycoproteins can be flexibly replaced to regulate the tissue specificity or cell tropism of the viral vector, thereby expanding its application potential in gene therapy, vaccine delivery, and functional genomics research.

[0057] The following experimental methods are all conventional methods unless otherwise specified, and the experimental materials used are all readily available from commercial companies unless otherwise specified.

[0058] ​Embodiments: The present application will be more readily understood by reference to the following examples, which are intended to illustrate the present application and are not to be construed as limiting the scope of the present application.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It will be apparent to those skilled in the art that any methods and materials equivalent to those described herein can be used in practice or testing of the present application.

[0060] While the present application has been described in detail with reference to embodiments thereof, it is to be understood that the embodiments are for illustration only and that other embodiments can be obtained from the teachings of the present application without departing from the scope of the present application.

[0061] Experimental methods not specifically described in the present application are carried out according to the specific methods in the book of Molecular Cloning: A Laboratory Manual (Fourth Edition) by J. Sambrook, or according to the instructions of the relevant products. When used in the present application, all terms should be understood in accordance with their ordinary meanings known in the art, unless otherwise specified. Biological reagents used in the present application, unless otherwise specified, can be obtained from commercial channels.

[0062] Example 1: Construction of expression plasmids of VSV-G and various chimeric Cocal-G envelope glycoproteins

[0063] Ten groups of envelope glycoproteins were designed, and plasmids of wild-type VSV-G and various chimeric Cocal-G envelope glycoproteins were synthesized, wherein the first group is denoted as VSV-G-wt, which is the wild type of VSV-G; the second group is denoted as Cocal-G-wt, which is the wild type of Cocal-G; the third to ninth groups are denoted as CVGc, CVGmc, CVGpmc, GsCV, GsCVGc, GsCVGmc, GsCVGpmc, respectively, which are chimeric types of VSV-G and Cocal-G; the tenth group Cocal-VX24 is prepared according to CN118085042A and is used as a positive control; and NC is a negative control. The nucleic acid sequences corresponding to the above-mentioned ten protein sequences are respectively constructed on the pCMV vector by gene synthesis, and after obtaining the correct plasmid, a large amount of extraction is carried out for the slow virus packaging experiment. The amino acid sequences of the envelope glycoproteins of the first to ninth groups are shown in Table 1.

[0064] Table 1 Amino acid sequences of various envelope glycoproteins

[0065]

[0066] Example 2: Packaging of slow virus using suspension cells HEK293TH

[0067] HEK293TH inoculation: In a T125 mL cell culture flask, inoculate cells at 1-1.5 x 10 6 cell / mL, follow the 25 mL BalanCD HEK293 (FUJIFILM) + 4 mM L-Glutamine + 1% Penicillin / Streptomycin culture system, 37°C, 5% CO2 incubator, 180 rpm culture for 18-20 h;

[0068] Virus packaging: Use a four-plasmid virus packaging system, follow the total plasmid 50 μg, mass ratio 10:5:2:3, add the shuttle plasmid (expressing NeonGreen-teLuc reporter gene), helper plasmid 1 (expressing Gag-pol protein), helper plasmid 2 (expressing Rev protein) and envelope glycoprotein plasmid (expressing proteins 1-10 in Example 1, respectively) in turn, mix well after adding 300 μl BalanCD HEK293 culture medium to prepare a DNA solution, add 300 μL BalanCD HEK293 culture medium and 100 μL of PEI pro (polyplus transfection) to prepare a PEI solution, add the PEI solution dropwise to the DNA solution, vortex well to mix, incubate for 15-20 min, then add dropwise to the cultured cells for cell transfection. Place the HEK293TH culture flask in a 37°C, 5% CO2 incubator at 180 rpm.

[0069] After 24 hours of cell transfection, add BalanCD HEK293 Viral Feed (FUJIFILM) according to the system 12% by volume.

[0070] After 48 hours of cell transfection, collect the virus supernatant, centrifuge at 300g for 5 min to collect the supernatant culture medium, use a Beckman ultracentrifuge to centrifuge at 25000 rpm for 1.5 hours with a sucrose pad. Discard the supernatant and add an appropriate amount of virus preservation solution to dissolve the virus particles for more than 2 hours at 4°C, 300g centrifugation for 1 min, concentrate the solution at the bottom of the tube, mix well with 10% DMSO, and store at -80°C after aliquoting.

[0071] Example 3 Lentivirus infection of HEK293TH cells

[0072] Inoculate 4 x 10 5HEK293TH cells were resuspended in 1 mL of BalanCD HEK293 medium + 4 mM L- glutamine + 1% penicillin / streptomycin medium and seeded in a 24-well plate. Different virus concentration of virus envelope structure prepared in Example 2 were added respectively 16 μL and mixed evenly, and the final volume of the infection system was 1 mL. After 48 hours of culture at 37°C, the infection efficiency of lentivirus was compared by taking pictures under fluorescence microscope, luciferase activity determination, and flow cytometry detection of the proportion of green fluorescent protein expressing cells. The results of green fluorescent protein expression are shown in Figure 3. Figure 2 It is shown that compared with Cocal-G-wt and VSV-G-wt packaged pseudotyped lentivirus, several chimeric Cocal-G envelope glycoprotein packaged pseudotyped lentivirus show stronger infection activity, among which CVGpmc, GsCV, GsCVGc and GsCVGmc are particularly significant.

[0073] The results of luciferase determination are shown in Figure 4. Figure 3 It is shown that compared with the luciferase activity of Cocal-G-wt packaged pseudotyped lentivirus after infecting cells, the luciferase activity of several chimeric Cocal-G envelope glycoprotein packaged pseudotyped lentivirus after infecting cells shows different degrees of improvement, including CVGc, CVGmc, CVGpmc, GsCV, GsCVGc, GsCVGmc and GsCVGpmc, among which the chimeric envelope glycoprotein pseudotyped lentivirus of CVGc, CVGpmc, GsCV, GsCVGc and GsCVGmc is also significantly better than VSV-G-wt packaged pseudotyped lentivirus.

[0074] The flow detection results are shown in Table 2. Compared with the NeonGreen positive rate of Cocal-G-wt packaged pseudotyped lentivirus after infecting cells, the NeonGreen positive rate of several chimeric Cocal-G envelope glycoprotein after infecting cells shows different degrees of improvement, including CVGc, CVGmc, CVGpmc, GsCV, GsCVGc and GsCVGmc, among which the NeonGreen positive rate of chimeric Cocal-G envelope glycoprotein packaged pseudotyped lentivirus of CVGc, CVGpmc, GsCV, GsCVGc and GsCVGmc after infecting cells is also significantly better than VSV-G-wt packaged pseudotyped lentivirus.

[0075] Table 2 Flow detection results after HEK293TH cell infection

[0076] .

[0077] Example 4 Lentivirus infection of Jurkat cells

[0078] Resuscitate Jurkat cells. Incubate Jurkat cells at a rate of 4 × 10⁶ cells / year. 5 Each well was inoculated with RPMI 1640 culture medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Subsequently, 16 μL of each of the different viral envelope structures prepared in step 1 of Example 3 was added, along with polybrene transfection aid to a final concentration of 6 μg / mL, resulting in a final infection volume of 1 mL. After thorough mixing, the mixture was incubated at 37°C for 48 hours. The cells were photographed using a fluorescence microscope, and the proportion of cells expressing green fluorescent protein was determined by luciferase activity assay to compare the lentiviral infection efficiency. The unit of viral titer is TU / mL, representing the number of transduction-functional viral particles per milliliter of viral solution.

[0079] The calculation formula is: Titer = (Number of cells at infection (cells) * Positive cells %) / Virus volume (mL).

[0080] Since the viral volume and cell count at infection are known, flow cytometry is needed to confirm the percentage of positive cells. Therefore, flow cytometry is used to detect lentiviral packaging efficiency.

[0081] Fluorescent protein expression results as follows Figure 4 The results showed that, compared with pseudolentiviruses packaged with Cocal-G-wt and VSV-G-wt, several chimeric pseudolentiviruses packaged with Cocal-G envelope glycoproteins exhibited stronger infectivity, including CVGpmc, GsCVGc, and GsCVGmc.

[0082] Luciferase assay results are as follows Figure 5 The results showed that, compared with the luciferase activity of pseudolentiviruses packaged with Cocal-G-wt and VSV-G-wt after cell infection, the luciferase activity of several chimeric Cocal-G envelope glycoprotein-packaged pseudolentiviruses after cell infection was significantly higher than that of the positive control VX24. CVGpmc, GsCVGc, and GsCVGmc were even significantly better than wild-type Cocal-G-wt and VSV-G-wt.

[0083] The flow cytometry results are shown in Table 3. Compared with the NeonGreen positivity rate of cells infected with pseudo-lentiviruses packaged with Cocal-G-wt and VSV-G-wt, the NeonGreen positivity rates of cells infected with several chimeric Cocal-G envelope glycoproteins were all better than the positive control VX24. CVGpmc, GsCV, GsCVGc, and GsCVGmc were even significantly better than wild-type Cocal-G-wt and VSV-G-wt.

[0084] Table 3. Flow cytometry results after Jurkat cell infection.

[0085] .

[0086] Example 6 Construction of expression plasmids of VSV-G and BaEV-G chimeric envelope glycoprotein

[0087] Referring to Example 1, two groups of chimeric envelope glycoproteins were designed, and plasmids for high expression of VSV-G and BaEV-G chimeric envelope glycoproteins were synthesized, respectively. The first group was labeled as BVGmc, the signal peptide region of the envelope glycoprotein had an amino acid sequence as shown in SEQ ID NO: 11, the non-membrane proximal domain of the extracellular region had an amino acid sequence as shown in SEQ ID NO: 12, the membrane proximal domain of the extracellular region had an amino acid sequence as shown in SEQ ID NO: 13, the transmembrane region had an amino acid sequence as shown in SEQ ID NO: 4, and the intracellular region had an amino acid sequence as shown in SEQ ID NO: 5. The second group was labeled as GsBVGmc, the signal peptide region of the envelope glycoprotein had an amino acid sequence as shown in SEQ ID NO: 1, the non-membrane proximal domain of the extracellular region had an amino acid sequence as shown in SEQ ID NO: 12, the membrane proximal domain of the extracellular region had an amino acid sequence as shown in SEQ ID NO: 13, the transmembrane region had an amino acid sequence as shown in SEQ ID NO: 4, and the intracellular region had an amino acid sequence as shown in SEQ ID NO: 5. Then, referring to Example 2, the two obtained plasmids were used to package lentivirus by using suspension cells HEK293TH, and finally the obtained lentivirus was used to infect NK cells and detection was performed.

[0088] Test of transduction efficiency of NK cells

[0089] ① NK cell infection experiment

[0090] According to the number of viable cells of NK cells per well, 4x10 5 Inoculation into a 24-well cell culture plate, 16 μL of two pseudotyped lentiviruses obtained by implementing BVGmc and GsBVGmc were added to transduce NK cells, the final volume in the well was 1 mL, and polybrene (polybrene) transfection aid was added to a final concentration of 6 μg / mL, and then mixed uniformly and placed in a 37°C, 5% CO2 incubator for 48 hours.

[0091] ② NK cell transduction efficiency results

[0092] For luciferase activity detection, the cell sample to be detected was prepared according to the number of viable cells per well, 1x10 4Inoculate into 96 cell culture plates, the final volume in the hole is 100 μl / hole, the parallel sample amount of each cell to be detected is not less than 3, add 10 μl cell lysis solution (10% Triton X-100) to the hole, and lyse at room temperature for 5-10 min, use enzyme-linked immunoassay instrument (Feyond-A300) to add equal volume of Diphenylterazine (2X) solution into 96 cell culture plates, use LUM measurement mode, end point method measurement type to measure luciferase. The results are shown in Figure 6 As shown in the table, the luciferase reading of the NK cells transduced by the virus particles prepared from the optimized chimeric GsBV Gmc and BV Gmc in the application is better than that of the wild type VSV-G-wt after 48 hours.

[0093] Although the present application is described in detail with reference to the embodiments thereof, the embodiments are provided for illustration purposes and are not intended to limit the present application. Other embodiments obtained according to the principles of the present application all fall within the scope defined by the claims of the present application.

[0094] Part of the amino acid sequences involved in the present application

[0095] SEQ ID NO: 1: Amino acid sequence of wild type VSV-G signal peptide region

[0096] MKCLLYLAFLFIGVNC

[0097] SEQ ID NO: 2: Amino acid sequence of non-membrane proximal domain of wild type VSV-G extracellular region

[0098] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQ

[0099] SEQ ID NO: 3: Amino acid sequence of the membrane proximal domain of the wild type VSV-G ectodomain

[0100] DAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWK

[0101] SEQ ID NO: 4: Amino acid sequence of the transmembrane region of the wild type VSV-G

[0102] SSIASFFFIIGLIIGLFLVLRV

[0103] SEQ ID NO: 5: Amino acid sequence of the wild type VSV-G endodomain

[0104] GIHLCIKLKHTKKRQIYTDIEMNRLGK

[0105] SEQ ID NO: 6: Amino acid sequence of the signal peptide region of the wild type Cocal-G

[0106] MNFLLLTFIVLPLCSHA

[0107] SEQ ID NO: 7: Amino acid sequence of the non-membrane proximal domain of the wild type Cocal-G ectodomain.

[0108] KFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQNCGYATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVDQDVYAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGPNGILKTPTGYKFPLFMIGHGMLDSDLHKTSQAEVFEHPHLA

[0109] SEQ ID NO: 8: Amino acid sequence of the membrane proximal domain of the wild type Cocal-G ectodomain ​

[0110] EAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWK

[0111] SEQ ID NO:9: Amino acid sequence of the transmembrane region of wild-type Cocal-G

[0112] STVVTFFFAIGVFILLYVVARIV

[0113] SEQ ID NO:10: Intracellular amino acid sequence of wild-type Cocal-G

[0114] IAVRYRYQGSNNKRIYNDIEMSRFRK

[0115] SEQ ID NO:11: Amino acid sequence of wild-type BaEV-G signal peptide region

[0116] MGFTTKIIFLYNLVLVYA

[0117] SEQ ID NO:12: Amino acid sequence of the non-proximal membrane domain of the extracellular region of wild-type BaEV-G

[0118] GFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPKDTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTGGTS DVQVLGSTNKLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQRKLEEIHKALYPELQYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSNTSLVDDCWLCLKL GPPTPLAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVAFSNCTSITNVTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLATLLPDID IIPGDEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISDVQILSSTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFY

[0119] SEQ ID NO:13: Amino acid sequence of the juxtamembrane domain of the extracellular region of wild-type BaEV-G

[0120] VNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLP

[0121] SEQ ID NO: 14: Wild-type BaEV-G transmembrane region amino acid sequence

[0122] YLLPFLGPLLTLLLLLTIGPCIF

[0123] SEQ ID NO: 15: Wild-type BaEV-G intracellular region amino acid sequence

[0124] NRLTAFINDKLNIIHAM.

Claims

1. A method for producing a chimeric envelope glycoprotein, characterized by, The amino acid sequence of the chimeric envelope glycoprotein is sequentially connected from N terminus a signal peptide region shown as SEQ ID NO: 1, a non-membrane proximal domain of extracellular region shown as SEQ ID NO: 7, a membrane proximal domain of extracellular region shown as SEQ ID NO: 8, a transmembrane region shown as SEQ ID NO: 9, and an intracellular region shown as SEQ ID NO:

5.

2. A chimeric envelope glycoprotein, characterized by, The amino acid sequence of the chimeric envelope glycoprotein is sequentially connected from N terminus a signal peptide region shown as SEQ ID NO: 1, a non-membrane proximal domain of extracellular region shown as SEQ ID NO: 7, a membrane proximal domain of extracellular region shown as SEQ ID NO: 8, a transmembrane region shown as SEQ ID NO: 9, and an intracellular region shown as SEQ ID NO:

5.

3. A lentiviral envelope plasmid, characterized in that, The envelope plasmid is capable of translating the chimeric envelope glycoprotein of claim 2.

4. A method for pseudotyping a lentivirus, characterized in that, The lentivirus is pseudotyped packaged with the chimeric envelope glycoprotein of claim 2 or with the lentivirus envelope plasmid of claim 3.

5. A kit characterized in that, The kit comprises the chimeric envelope glycoprotein of claim 2 or the lentivirus envelope plasmid of claim 3.

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