Chimeric envelope glycoprotein, preparation method of chimeric envelope glycoprotein, envelope plasmid related to chimeric envelope glycoprotein, packaging method and kit

Abstract

The invention provides a chimeric envelope glycoprotein, a preparation method thereof and an envelope plasmid, a packaging method and a kit of the chimeric envelope glycoprotein, and particularly relates to a preparation method of the chimeric envelope glycoprotein, the chimeric envelope glycoprotein and a lentivirus envelope plasmid. The invention discloses a pseudotyped packaging method of lentivirus and a kit. The amino acid sequence of the wild type VSV-G envelope glycoprotein is chimeric with the amino acid sequences of other envelope glycoproteins, and compared with the initial wild type envelope glycoprotein, the obtained chimeric envelope glycoprotein can effectively improve the lentivirus packaging efficiency, biological activity and other properties.

Description

[0001] This application is a divisional application of Chinese patent application No. 2025115114075, filed on October 22, 2025, entitled "A chimeric enveloped glycoprotein, its preparation method, and an enveloped plasmid, packaging method and kit relating to the enveloped glycoprotein". Technical Field

[0002] This invention relates to the field of biomedicine, and in particular to a chimeric enveloped glycoprotein, its preparation method, and an envelope plasmid, packaging method, and kit relating to the enveloped glycoprotein. Background Technology

[0003] Lentiviral vector systems (LV) are commonly used in cell gene therapy. They are typically obtained by co-transfecting host cells with a packaging plasmid (which provides viral structural proteins), an envelope plasmid (which determines the host range), and a transfer plasmid (containing the target gene and essential regulatory sequences) in a certain ratio. The resulting virus-like particles (lentiviruses) selectively infect recipient cells.

[0004] Viral venovirus (LV) is typically packaged using heterologous VSV-G as the envelope glycoprotein in pseudoviruses. Replacing the gene encoding the viral envelope glycoprotein in the original virus with VSV-G significantly expands the virus's host cell range and endows the vector with broad targeting and stability. VSV-G originates from herpestostomosis virus and targets the low-density lipoprotein receptor (LDL-P). Because this receptor is ubiquitous in various cells, LV packaged with VSV-G is considered a relatively broad-spectrum pseudovirus. However, VSV-G injection easily triggers a host immune response, producing antibodies that neutralize the viral vector, reducing gene transduction efficiency, affecting therapeutic efficacy, and limiting its persistence in gene therapy requiring long-term expression of the target gene. The VSV-G-packaged virus is also inactivated by human serum complement, further reducing its efficacy. Furthermore, it exhibits cytotoxicity when stably expressed in human cells, making it unsuitable for in vivo delivery and causing unfavorable complement-dependent immune responses against VSV-G in patients. These limitations restrict the wider applicability of VSV-G-based LV to various cell gene therapy fields.

[0005] Laboratories have developed other envelope glycoprotein-packaged LVs, such as the Cocal vesicular virus envelope glycoprotein (Cocal-G). Cocal vesicular virus belongs to the same genus as herpestostomosis virus, but they differ serologically. The Cocal-G envelope glycoprotein shares 71.5% amino acid homology with the VSV-G envelope glycoprotein and also targets low-density lipoprotein, thus exhibiting broad targeting. Furthermore, Cocal-G-based LVs are more resistant to complement inactivation in human serum, are less easily inactivated by human serum, and have lower immunogenicity, thereby improving transduction efficiency and gene expression persistence. Therefore, Cocal-G-based LVs can more effectively transduce hematopoietic stem cells (HSCs) and CD34-positive T cells in humans, non-human primates, and dogs, and can be better applied to cell gene therapy, especially in the field of blood cells.

[0006] However, the packaging efficiency of Cocal-G-based LVs is relatively low compared to VSV-G-based LVs, making it difficult to obtain high-titer, highly active pseudoviruses. In the published patent CN118085042A, a Cocal-G mutant version (Cocal-VX24) was obtained through random evolution, but the improvement in its packaging titer remains limited. Other envelope proteins, such as BaEV, also have similar limitations. Therefore, how to improve the high packaging titer of these envelope proteins and obtain highly active envelope glycoproteins remains a problem that needs further investigation.

[0007] CN118085042A incorporates this invention in its entirety. Summary of the Invention

[0008] This invention provides a method for preparing chimeric enveloped glycoproteins. By rationally combining and splicing different structural domains (such as juxtamembrane region, transmembrane region, intracellular region, etc.) of wild-type enveloped glycoproteins, a series of chimeric proteins are obtained, whose packaging efficiency and activity are significantly higher than those of wild-type enveloped glycoproteins.

[0009] On one hand, the present invention provides a method for preparing a chimeric enveloped glycoprotein, wherein the amino acid sequence of wild-type VSV-G enveloped glycoprotein is chimeric with the amino acid sequence of wild-type Cocal-G enveloped glycoprotein or wild-type BaEV-G enveloped glycoprotein to obtain a chimeric enveloped glycoprotein. In some embodiments, the amino acid sequence of wild-type VSV-G enveloped glycoprotein includes a signal peptide region, a non-proximal membrane domain of the extracellular region, a proximal membrane domain of the extracellular region, a transmembrane region, and an intracellular region, and at least one region of wild-type VSV-G enveloped glycoprotein is chimeric with the amino acid sequence of wild-type Cocal-G enveloped glycoprotein or wild-type BaEV-G enveloped glycoprotein.

[0010] In some embodiments, the wild-type VSV-G envelope glycoprotein is chimeric with the amino acid sequence of another wild-type envelope glycoprotein. In some embodiments, the wild-type VSV-G envelope glycoprotein is chimeric with the amino acid sequences of multiple wild-type envelope glycoproteins; specifically, it may be a chimeric combination of three amino acid sequences: wild-type VSV-G envelope glycoprotein, wild-type Cocal-G envelope glycoprotein, or wild-type VSV-G envelope glycoprotein.

[0011] In some embodiments, the amino acid sequence of the wild-type VSV-G envelope glycoprotein is chimeric 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 either the wild-type VSV-G envelope glycoprotein or the wild-type Cocal-G envelope glycoprotein; the non-proximal membrane domain of the extracellular region is selected from either the amino acid sequence of either the wild-type VSV-G envelope glycoprotein or the wild-type Cocal-G envelope glycoprotein; the proximal membrane domain of the extracellular region is selected from either the amino acid sequence of either the wild-type VSV-G envelope glycoprotein or the wild-type Cocal-G envelope glycoprotein; the transmembrane region is selected from either the amino acid sequence of either the wild-type VSV-G envelope glycoprotein or the wild-type Cocal-G envelope glycoprotein; and the intracellular region is selected from either the amino acid sequence of either the wild-type VSV-G envelope glycoprotein or the wild-type Cocal-G envelope glycoprotein.

[0012] In some embodiments, the amino acid sequence of the wild-type VSV-G envelope glycoprotein is chimeric 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 wild-type BaEV-G envelope glycoprotein; the non-proximal membrane domain of the extracellular region is selected from the amino acid sequence of the wild-type VSV-G envelope glycoprotein or the wild-type BaEV-G envelope glycoprotein; the proximal membrane domain of the extracellular region is selected from the amino acid sequence of the wild-type VSV-G envelope glycoprotein or 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 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 wild-type BaEV-G envelope glycoprotein.

[0013] Furthermore, the amino acid sequence of the signal peptide region of the wild-type VSV-G envelope glycoprotein is shown in SEQ ID NO: 1, the amino acid sequence of the non-proximal membrane domain of the extracellular region is shown in SEQ ID NO: 2, the amino acid sequence of the proximal membrane domain of the extracellular region is shown in SEQ ID NO: 3, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 4, and the amino acid sequence of the intracellular region is shown in SEQ ID NO: 5.

[0014] In some embodiments, the amino acid sequence of the wild-type VSV-G envelope glycoprotein includes a signal peptide region, a non-juxtamembrane domain of the extracellular region, a juxtamembrane domain of the extracellular region, a transmembrane region, and an intracellular region. The chimeric envelope glycoprotein is formed by replacing one to three regions of the wild-type VSV-G envelope glycoprotein with corresponding regions of the amino acid sequence of the wild-type Cocal-G envelope glycoprotein or the wild-type BaEV-G envelope glycoprotein. Further, the non-juxtamembrane domain of the extracellular region of the chimeric envelope glycoprotein is selected from the corresponding region of the wild-type Cocal-G envelope glycoprotein or the wild-type BaEV-G envelope glycoprotein.

[0015] Furthermore, the amino acid sequence of the signal peptide region of the wild-type Cocal-G envelope glycoprotein is shown in SEQ ID NO: 6, the amino acid sequence of the non-proximal membrane domain of the extracellular region is shown in SEQ ID NO: 7, the amino acid sequence of the proximal membrane domain of the extracellular region is shown in SEQ ID NO: 8, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 9, and the amino acid sequence of the intracellular region is shown in SEQ ID NO: 10.

[0016] In some embodiments, the amino acid sequence of the signal peptide region of the chimeric envelope glycoprotein is selected from SEQ ID NO: 1 or SEQ ID NO: 6, the amino acid sequence of the non-proximal membrane domain of the extracellular region is shown in SEQ ID NO: 7, the amino acid sequence of the proximal membrane domain of the extracellular region is selected from SEQ ID NO: 3 or SEQ ID NO: 8, the amino acid sequence of the transmembrane region is selected from SEQ ID NO: 4 or SEQ ID NO: 9, and the amino acid sequence of the intracellular region is selected from SEQ ID NO: 5 or SEQ ID NO: 10.

[0017] Specifically, the amino acid sequences or mutant sequences corresponding to the various chimeric Cocal-G envelope glycoproteins in this invention are each one of the following groups: a) VSV-G-wt: SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 4+ SEQ ID NO: 5; b)Cocal-G-wt: SEQ ID NO:6+SEQ ID NO:7+SEQ ID NO: 8+SEQ ID NO: 9+ SEQ ID NO: 10; c) CVGc: SEQ ID NO: 6 + SEQ ID NO: 7 + SEQ ID NO: 8 + SEQ ID NO: 9 + SEQ ID NO: 5; d) CVGmc: SEQ ID NO: 6 + SEQ ID NO: 7 + SEQ ID NO: 8 + SEQ ID NO: 4 + SEQ ID NO: 5; e) CVGpmc: SEQ ID NO: 6 + SEQ ID NO: 7 + SEQ ID NO: 3 + SEQ ID NO: 4 + SEQ ID NO: 5; f) GsCV: SEQ ID NO: 1 + SEQ ID NO: 7 + SEQ ID NO: 8 + SEQ ID NO: 9 + SEQ ID NO: 10; g) GsCVGc: SEQ ID NO: 1+SEQ ID NO: 7+SEQ ID NO: 8+SEQ ID NO: 9+ SEQ ID NO: 5; h) GsCVGmc: SEQ ID NO: 1+SEQ ID NO: 7+SEQ ID NO: 8+SEQ ID NO: 4+ SEQ ID NO: 5; i) GsCVGpmc: SEQ ID NO: 1 + SEQ ID NO: 7 + SEQ ID NO: 3 + SEQ ID NO: 4 + SEQ ID NO: 5.

[0018] In some embodiments, the amino acid sequence of the signal peptide region of the chimeric enveloped glycoprotein obtained by the optimized method of chimerizing wild-type VSV-G enveloped glycoprotein with wild-type Cocal-G enveloped glycoprotein is shown in SEQ ID NO: 1, the amino acid sequence of the non-proximal membrane domain of the extracellular region is shown in SEQ ID NO: 7, the amino acid sequence of the proximal membrane domain of the extracellular region is shown in SEQ ID NO: 8, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 9, and the amino acid sequence of the intracellular region is shown in SEQ ID NO: 5.

[0019] In some embodiments, wild-type VSV-G envelope glycoprotein is used in a chimera with wild-type BaEV-G envelope glycoprotein. The amino acid sequence of the signal peptide region of the wild-type BaEV-G envelope glycoprotein is shown in SEQ ID NO: 11, the amino acid sequence of the non-proximal membrane domain of the extracellular region is shown in SEQ ID NO: 12, the amino acid sequence of the proximal membrane domain of the extracellular region is shown in SEQ ID NO: 13, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 14, and the amino acid sequence of the intracellular region is shown in SEQ ID NO: 15.

[0020] Furthermore, the amino acid sequence of the signal peptide region of the chimeric envelope glycoprotein is selected from SEQ ID NO: 1 or SEQ ID NO: 11, the amino acid sequence of the non-proximal membrane domain of the extracellular region is shown in SEQ ID NO: 12, the amino acid sequence of the proximal membrane domain of the extracellular region is selected from SEQ ID NO: 3 or SEQ ID NO: 13, the amino acid sequence of the transmembrane region is selected from SEQ ID NO: 4 or SEQ ID NO: 14, and the amino acid sequence of the intracellular region is selected from SEQ ID NO: 5 or SEQ ID NO: 15.

[0021] Specifically, in this invention, wild-type VSV-G envelope glycoprotein is chimeric with wild-type BaEV-G envelope glycoprotein, and the amino acid sequences corresponding to the resulting chimeric envelope glycoproteins are any of the following groups: j) BVGmc: SEQ ID NO: 11 + SEQ ID NO: 12 + SEQ ID NO: 13 + SEQ ID NO: 4 + SEQ ID NO: 5; k) GsBVGmc: SEQ ID NO: 1 + SEQ ID NO: 12 + SEQ ID NO: 13 + SEQ ID NO: 4 + SEQ ID NO: 5;

[0022] In some embodiments, the amino acid sequence of the signal peptide region of the chimeric envelope glycoprotein of wild-type VSV-G envelope glycoprotein and wild-type BaEV-G envelope glycoprotein is selected from SEQ ID NO: 1 or SEQ ID NO: 11, the amino acid sequence of the non-proximal membrane domain of the extracellular region is shown in SEQ ID NO: 12, the amino acid sequence of the proximal membrane domain of the extracellular region is shown in SEQ ID NO: 13, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 4, and the amino acid sequence of the intracellular region is shown in SEQ ID NO: 5.

[0023] This invention also provides a lentiviral envelope plasmid capable of transcribing any of the aforementioned chimeric envelope glycoproteins. The envelope plasmid comprises a promoter, a coding sequence for the chimeric BaEV-G envelope glycoprotein, and a transcription termination signal, wherein the promoter is located upstream of the coding sequence and the transcription termination signal is located downstream of the coding sequence, ensuring efficient expression and proper processing of the chimeric envelope glycoprotein in host cells. This lentiviral envelope plasmid is applicable to various viral vector systems, significantly improving viral titers and infection efficiency, providing a reliable tool for gene therapy and vaccine development.

[0024] This invention also provides a method for packaging lentiviruses, using any of the aforementioned chimeric envelope glycoproteins or envelope plasmids. The method involves co-transfecting a chimeric envelope glycoprotein or its encoding plasmid with a viral structural protein expression plasmid into host cells, achieving efficient packaging of lentiviral particles through transient or stable transfection. The resulting viral vector exhibits higher infection efficiency and a wider host range in target cells, making it particularly suitable for primary cells and stem cells that are difficult to transfect. Furthermore, this packaging method is simple to operate, highly reproducible, and its stability and reliability have been verified in multiple rounds of experiments, providing strong technical support for gene function research and clinical gene therapy applications.

[0025] This invention also provides a kit comprising the aforementioned chimeric envelope glycoprotein or envelope plasmid. Optionally, the kit includes helper plasmids required for viral packaging, transfection reagents, and instructions for use, suitable for research and preclinical studies. The components are optimized in proportion to ensure efficient viral packaging and stable production. The chimeric envelope glycoprotein in the kit mediates viral targeting of specific cell types, significantly improving transduction efficiency while reducing the risk of immunogenicity.

[0026] This invention also provides a pseudotyped lentivirus comprising the chimeric envelope glycoprotein defined above. It also relates to a drug comprising the pseudotyped lentivirus as defined above as an active ingredient. This pseudotyped lentivirus achieves efficient recognition and infection of specific cell types through the chimeric BaEV-G envelope glycoprotein displayed on its surface, significantly improving the transduction efficiency and bioavailability of the viral vector in vivo and in vitro. The therapeutic gene it carries can be stably integrated into the host genome, achieving long-term expression, and is suitable for the treatment of various diseases, including genetic diseases, tumors, and viral infections.

[0027] The invention also relates to pharmaceutical compositions comprising a pseudotyped lentivirus as defined above and a pharmaceutically acceptable carrier. These pharmaceutical compositions can be administered via various routes, including but not limited to intravenous injection, local injection, or oral administration, and can be formulated and dose-optimized according to disease type and treatment needs to ensure effective drug concentration and safety at the target site. The invention further relates to a method for treating a subject in need of the invention, comprising administering a therapeutically effective amount of a pseudotyped lentivirus as defined above to the subject in need of the invention.

[0028] The present invention also relates to a method for treating a subject in need of the virus, comprising administering to the subject in need a therapeutically effective amount of pseudotyped lentivirus as defined above.

[0029] In the context of this invention, "subject" refers to a human or non-human mammal, such as rodents (rats, mice, rabbits), primates (chimpanzees), felines (cats), and canines (dogs). Preferably, the subject is a human.

[0030] Furthermore, this invention provides a highly efficient viral packaging system comprising a plasmid encoding the aforementioned chimeric BaEV-G envelope glycoprotein, a viral structural protein expression plasmid, and a host cell line. This system, through a synergistic expression mechanism, effectively improves the assembly efficiency and titer of viral particles, making it suitable for large-scale production of viral vectors.

[0031] The method for preparing chimeric envelope glycoproteins provided by this invention involves chimerizing the amino acid sequences of wild-type VSV-G envelope glycoprotein with those of wild-type Cocal-G envelope glycoprotein or wild-type BaEV-G envelope glycoprotein. The resulting chimeric protein, compared to the initial wild-type envelope glycoprotein, effectively improves lentiviral packaging efficiency and bioactivity. By rationally designing and recombining functional domains from envelope glycoprotein sequences from different sources, the resulting chimeric protein, while maintaining its original receptor recognition ability, further enhances the stability of viral particles and transmembrane fusion efficiency. Attached Figure Description

[0032] To better understand the present invention and more clearly demonstrate how to implement it, features of embodiments according to the present invention are now illustrated by way of example and with reference to the accompanying drawings, wherein:

[0033] Figure 1 : Schematic diagram of the sequence regions of various enveloped glycoproteins used in this invention.

[0034] Figure 2 Image of fluorescent protein expression in 293TH cells 48 hours after infection with wild-type VSV-G and wild-type Cocal-G chimeric envelope glycoprotein pseudovirus.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] As used herein, the term "Cocal-G envelope glycoprotein" refers to the wild-type form of the Cocal-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 Cocal-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.

[0044] As used herein, the term "BaEV-G envelope glycoprotein" refers to the wild-type form of BaEV-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 BaEV-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.

[0045] As used herein, "chimeric enveloped glycoprotein" refers to a chimeric protein composed of VSV-G enveloped glycoprotein and either Cocal-G or BaEV-G enveloped glycoprotein. The term "chimeric" in this article refers to... Figure 1 The signal peptide region, non-proximal domain of the extracellular region, proximal domain of the extracellular region, transmembrane region, and intracellular region of the envelope glycoprotein shown are composed of the corresponding regions of wild-type VSV-G envelope glycoprotein and wild-type Cocal-G envelope glycoprotein or wild-type BaEV-G envelope glycoprotein.

[0046] As used in this article, "pseudovirus" or "pseudotyped lentivirus" are interchangeable terms. The process of introducing a heterologous envelope glycoprotein into the core of a lentiviral vector is called "pseudotypening." Pseudotyped lentiviruses are typically produced by co-transfecting a plasmid encoding a heterologous envelope glycoprotein into packaging cells along with a lentiviral vector system, resulting in recombinant lentiviral particles carrying the heterologous envelope glycoprotein on their surface. These particles have a core structure similar to the original lentivirus, but their surface envelope glycoprotein determines the virus's targeting and invasion efficiency in infecting host cells. Through pseudotypening technology, envelope glycoproteins from different sources 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.

[0047] Unless otherwise specified, the experimental methods described below are standard methods, and the experimental materials used can be easily obtained from commercial companies unless otherwise specified.

[0048] Examples: The present invention will be more readily understood by referring to the following examples, which are used to illustrate the invention and should not be construed as limiting the scope of the invention in any way.

[0049] Unless otherwise defined or the context clearly requires, 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 invention pertains. It should be understood that any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention.

[0050] Although the invention has been described in detail with reference to embodiments thereof, these embodiments are provided for illustration and not limitation. Other embodiments that can be obtained according to the principles of the invention fall within the scope defined by the claims of the invention.

[0051] Experimental methods not specifically described in this invention are performed according to the methods described in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (4th Edition) or according to the relevant product instructions. When used herein, unless otherwise stated, all terms in this invention should be understood in their ordinary meaning as known in the art. Unless otherwise specified, all biological reagents used in this invention are commercially available.

[0052] Example 1: Construction of expression plasmids for VSV-G and various chimeric Cocal-G envelope glycoproteins

[0053] Ten groups of envelope glycoproteins were designed, and plasmids of wild-type VSV-G and various chimeric Cocal-G envelope glycoproteins were synthesized. Group 1, denoted as VSV-G-wt, is the wild-type VSV-G; Group 2, denoted as Cocal-G-wt, is the wild-type Cocal-G; Groups 3 through 9, denoted as CVGc, CVGmc, CVGpmc, GsCV, GsCVGc, GsCVGmc, and GsCVGpmc, are chimeric VSV-G and Cocal-G, respectively; Group 10, Cocal-VX24, was prepared according to CN118085042A and served as a positive control, while NC served as a negative control. The nucleic acid sequences corresponding to the above 10 protein sequences were constructed into pCMV vectors through gene synthesis. After obtaining the correct plasmids, large-scale extraction was performed for lentiviral packaging experiments. The amino acid sequences of each envelope glycoprotein in groups 1-9 are shown in Table 1.

[0054] Table 1. Amino acid sequences of various envelope glycoproteins

[0055] Example 2: Packaging lentiviruses using HEK293TH suspension cells

[0056] HEK293TH seeding: In T125mL cell culture flasks, at a concentration of 1-1.5 × 10⁻⁶ cells / mL. 6 Cells were seeded at 25 mL of BalanCD HEK293 (FUJIFILM) + 4 mM L-Glutamine + 1% penicillin / streptomycin culture system and cultured at 37°C, 5% CO2 incubator, and 180 rpm for 18-20 h.

[0057] Virus Packaging: A four-plasmid virus packaging system was used. The shuttle plasmid (expressing the NeonGreen-teLuc reporter gene), helper plasmid 1 (expressing Gag-pol protein), helper plasmid 2 (expressing Rev protein), and envelope glycoprotein plasmids (expressing proteins 1-10 from Example 1) were added sequentially at a mass ratio of 50 μg total plasmids of 10:5:2:3. After mixing, 300 μl of BalanCD HEK293 medium was added and thoroughly mixed to prepare a DNA solution. 300 μL of BalanCD HEK293 medium and 100 μL of PEI pro (polyplus transfection) were added to prepare a PEI solution. The PEI solution was added dropwise to the DNA solution, vortexed thoroughly, and incubated for 15-20 minutes. The mixture was then added dropwise to cultured cells for transfection. HEK293TH culture flasks were incubated at 37°C and 180 rpm in a 5% CO2 incubator.

[0058] Twenty-four hours after cell transfection, BalanCD HEK293 Viral Feed (FUJIFILM) was added at 12% of the system volume.

[0059] Forty-eight hours after cell transfection, the viral supernatant was collected, centrifuged at 300g for 5 min to collect the supernatant culture medium, and then centrifuged at 25,000 rpm for 1.5 hours using a Beckman ultracentrifuge with the sucrose pad method. After discarding the supernatant, an appropriate amount of viral preservation solution was added to dissolve the viral particles for at least 2 hours, centrifuged at 300g for 1 min at 4°C, and the solution was collected at the bottom of the tube. 10% DMSO was added and mixed well. The mixture was then aliquoted and stored at -80°C.

[0060] Example 3 Lentiviral infection of HEK293TH cells

[0061] 4×10 5HEK293TH cells were resuspended in 1 mL BalanCD HEK293 medium + 4 mM L-Glutamine + 1% penicillin / streptomycin medium and seeded in 24-well plates. 16 μL of concentrated virus solutions with different viral envelope structures prepared in Example 2 were added and mixed thoroughly, resulting in a final infection volume of 1 mL. After incubation at 37°C for 48 hours, the proportion of cells expressing green fluorescent protein was detected by fluorescence microscopy, luciferase activity assay, and flow cytometry to compare the lentivirus infection efficiency. The results of fluorescent protein expression are shown below. Figure 2 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, with CVGpmc, GsCV, GsCVGc and GsCVGmc being particularly significant.

[0062] Luciferase assay results are as follows Figure 3 The results showed that, compared with the luciferase activity of pseudolentiviruses packaged with Cocal-G-wt, the luciferase activity of pseudolentiviruses packaged with several chimeric Cocal-G envelope glycoproteins was increased to varying degrees after infection of cells. These included CVGc, CVGmc, CVGpmc, GsCV, GsCVGc, GsCVGmc, and GsCVGpmc. Among them, the pseudolentiviruses of chimeric envelope glycoproteins CVGc, CVGpmc, GsCV, GsCVGc, and GsCVGmc were significantly better than those of pseudolentiviruses packaged with VSV-G-wt.

[0063] The flow cytometry results are shown in Table 2. Compared with the NeonGreen positivity rate of cells infected with pseudolentiviruses packaged in Cocal-G-wt, the NeonGreen positivity rate of cells infected with several chimeric Cocal-G envelope glycoproteins showed varying degrees of improvement, including CVGc, CVGmc, CVGpmc, GsCV, GsCVGc, and GsCVGmc. Among them, the NeonGreen positivity rate of pseudolentiviruses packaged in chimeric Cocal-G envelope glycoproteins such as CVGc, CVGpmc, GsCV, GsCVGc, and GsCVGmc was also significantly better than that of pseudolentiviruses packaged in VSV-G-wt.

[0064] Table 2. Flow cytometry results of HEK293TH cells after infection

[0065] Example 4 Lentiviral infection of Jurkat cells

[0066] 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.

[0067] The calculation formula is: Titer = (Number of cells at infection (number) * Positive cells%) / Virus volume (mL).

[0068] 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.

[0069] 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.

[0070] 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.

[0071] Example 5: Construction of expression plasmids for VSV-G and BaEV-G chimeric envelope glycoproteins

[0072] Referring to Example 1, two groups of chimeric envelope glycoproteins were designed, and plasmids expressing high levels of VSV-G and BaEV-G chimeric envelope glycoproteins were synthesized. The first group was labeled BVGmc, and the amino acid sequences of the signal peptide region of the envelope glycoprotein are shown in SEQ ID NO: 11, the amino acid sequences of the non-juxtamembrane domains of the extracellular region are shown in SEQ ID NO: 12, the amino acid sequences of the juxtamembrane domains of the extracellular region are shown in SEQ ID NO: 13, the amino acid sequences of the transmembrane region are shown in SEQ ID NO: 4, and the amino acid sequences of the intracellular region are shown in SEQ ID NO: 5. The second group was labeled GsBVGmc, and the amino acid sequences of the signal peptide region of the envelope glycoprotein are shown in SEQ ID NO: 1, the amino acid sequences of the non-juxtamembrane domains of the extracellular region are shown in SEQ ID NO: 12, the amino acid sequences of the juxtamembrane domains of the extracellular region are shown in SEQ ID NO: 13, the amino acid sequences of the transmembrane region are shown in SEQ ID NO: 4, and the amino acid sequences of the intracellular region are shown in SEQ ID NO: 5. Then, referring to Example 2, the two obtained plasmids were packaged into lentiviruses using HEK293TH suspension cells. Finally, the obtained lentiviruses were used to infect NK cells and the results were analyzed.

[0073] NK cell transduction efficiency test

[0074] ① NK cell infection experiment

[0075] Based on the NK cell viable cell count of 4 × 10⁶ per well 5 The cells were seeded into 24-well cell culture plates, and 16 μL of each of the two pseudotyped lentiviruses obtained by BVGmc and GsBVGmc were added to transduce NK cells, with a final volume of 1 mL in each well. Polybrene was added to the wells to a final concentration of 6 μg / mL. After mixing thoroughly, the cells were incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0076] ② Results of NK cell transduction efficiency

[0077] Luciferase activity assay requires the cell samples to be tested to be prepared at a ratio of 1 × 10⁻⁶ viable cells per well. 4 The cells were seeded into 96 cell culture plates, with a final volume of 100 μl / well. At least three parallel samples were prepared for each cell type. 10 μl of cell lysis buffer (10% Triton X-100) was added to each well, and lysis was performed at room temperature for 5-10 min. Using an ELISA reader (Feyond-A300), an equal volume of Diphenylterazine (2X) solution was added to the 96 cell culture plate. Luciferase detection was performed using LUM measurement mode and endpoint assay. Results are as follows: Figure 6As shown, the virus particles prepared by the optimized chimeric GsBVGmc and BVGmc of this invention, after being transduced into NK cells for 48 hours, showed better luciferase readings than wild-type VSV-G-wt.

[0078] Although the invention has been described in detail with reference to embodiments thereof, these embodiments are provided for illustration and not limitation. Other embodiments that can be obtained according to the principles of the invention fall within the scope defined by the claims of the invention.

[0079] The present invention relates to a partial amino acid sequence SEQ ID NO: 1: Amino acid sequence of wild-type VSV-G signal peptide region MKCLLYLAFLFIGVNC SEQ ID NO: 2: Amino acid sequence of the non-juxtamembrane domain of the extracellular region of wild-type VSV-G KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQG TWLNPGFPPQSCGYATVTDAEAAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGF RSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMGHGMLDSDLHLSSKAQVFEHPHIQ SEQ ID NO: 3: Amino acid sequence of the juxtamembrane domain of the extracellular region of wild-type VSV-G DAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWK SEQ ID NO: 4: Amino acid sequence of the transmembrane region of wild-type VSV-G SSIASFFFIIGLIIGLFLVLRV SEQ ID NO: 5: Intracellular amino acid sequence of wild-type VSV-G GIHLCIKLKHTKKRQIYTDIEMNRLGK SEQ ID NO: 6: Amino acid sequence of the wild-type Cocal-G signal peptide region MNFLLLTFIVLPLCSHA SEQ ID NO: 7: The amino acid sequence of the non-membrane domain of the extracellular region of wild-type Cocal-G.

[0080] KFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQG TWMSPGFPPQNCGYATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGY RSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVDQDVYAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPK NPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGPNGILKTPTGYKFPLFMIGHGMLDSDLHKTSQAEVFEHPHLA SEQ ID NO: 8: Amino acid sequence of the juxtamembrane domain of the extracellular region of wild-type Cocal-G EAPKQLPEEEETLFFGDTGISKNPVELIEGWFSSWK SEQ ID NO:9: Amino acid sequence of the transmembrane region of wild-type Cocal-G STVVTFFFAIGVFILLYVVARIV SEQ ID NO:10: Intracellular amino acid sequence of wild-type Cocal-G IAVRYRYQGSNNKRIYNDIEMSRFRK SEQ ID NO:11: Amino acid sequence of wild-type BaEV-G signal peptide region MGFTTKIIFLYNLVLVYA SEQ ID NO:12: Amino acid sequence of the non-proximal membrane domain of the extracellular region of wild-type BaEV-G GFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPKDTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTGGTS DVQVLGSTNKLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQRKLEEIHKALYPELQYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSNTSLVDDCWLCLKL GPPTPLAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVAFSNCTSITNVTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLATLLPDID IIPGDEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISDVQILSSTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFY SEQ ID NO:13: Amino acid sequence of the juxtamembrane domain of the extracellular region of wild-type BaEV-G VNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLP SEQ ID NO:14: Amino acid sequence of the transmembrane region of wild-type BaEV-G YLLPFLGPLLTLLLLLTIGPCIF SEQ ID NO:15: Intracellular amino acid sequence of wild-type BaEV-G NRLTAFINDKLNIIHAM.

Claims

1. A method for preparing a chimeric encapsulated glycoprotein, characterized in that, The amino acid sequences of a first wild-type envelope glycoprotein and a second wild-type envelope glycoprotein are intercalated, wherein the first wild-type envelope glycoprotein is selected from wild-type VSV-G envelope glycoprotein, and the second wild-type envelope glycoprotein is selected from one or both of wild-type Cocal-G envelope glycoprotein and wild-type BaEV-G envelope glycoprotein. The amino acid sequence of the envelope glycoprotein includes a signal peptide region, a non-juxtamembrane domain in the extracellular region, a juxtamembrane domain in the extracellular region, a transmembrane region, and an intracellular region. The chimerism involves replacing the non-juxtamembrane domain of the extracellular region of the wild-type VSV-G envelope glycoprotein with the corresponding region of the wild-type Cocal-G envelope glycoprotein or the wild-type BaEV-G envelope glycoprotein. One to three regions of the wild-type VSV-G envelope glycoprotein—the signal peptide region, the juxtamembrane domain, the transmembrane region, and the intracellular region—are replaced with the corresponding regions of the amino acid sequence of the wild-type Cocal-G envelope glycoprotein or the wild-type BaEV-G envelope glycoprotein. The chimeric enveloped glycoprotein is not a protein composed of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:

5.

2. The method for preparing the chimeric encapsulated glycoprotein according to claim 1, characterized in that, The amino acid sequence of the signal peptide region of the wild-type VSV-G envelope glycoprotein is shown in SEQ ID NO: 1, the amino acid sequence of the non-proximal membrane domain of the extracellular region is shown in SEQ ID NO: 2, the amino acid sequence of the proximal membrane domain of the extracellular region is shown in SEQ ID NO: 3, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 4, and the amino acid sequence of the intracellular region is shown in SEQ ID NO:

5.

3. The method for preparing the chimeric encapsulated glycoprotein according to claim 2, characterized in that, The amino acid sequence of the signal peptide region of the wild-type Cocal-G envelope glycoprotein is shown in SEQ ID NO: 6, the amino acid sequence of the non-proximal membrane domain of the extracellular region is shown in SEQ ID NO: 7, the amino acid sequence of the proximal membrane domain of the extracellular region is shown in SEQ ID NO: 8, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 9, and the amino acid sequence of the intracellular region is shown in SEQ ID NO:

10.

4. The method for preparing the chimeric encapsulated glycoprotein according to claim 2, characterized in that, The amino acid sequence of the signal peptide region of the wild-type BaEV-G envelope glycoprotein is shown in SEQ ID NO: 11, the amino acid sequence of the non-proximal membrane domain of the extracellular region is shown in SEQ ID NO: 12, the amino acid sequence of the proximal membrane domain of the extracellular region is shown in SEQ ID NO: 13, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 14, and the amino acid sequence of the intracellular region is shown in SEQ ID NO:

15.

5. The method for preparing the chimeric encapsulated glycoprotein according to claim 3, characterized in that, The amino acid sequence of the signal peptide region of the chimeric envelope glycoprotein is selected from SEQ ID NO: 1 or SEQ ID NO: 6; the amino acid sequence of the non-proximal membrane domain of the extracellular region is shown in SEQ ID NO: 7; the amino acid sequence of the proximal membrane domain of the extracellular region is selected from SEQ ID NO: 3 or SEQ ID NO: 8; the amino acid sequence of the transmembrane region is selected from SEQ ID NO: 4 or SEQ ID NO: 9; and the amino acid sequence of the intracellular region is shown in SEQ ID NO:

5. The chimeric enveloped glycoprotein is not a protein formed by linking the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:

5.

6. The method for preparing the chimeric encapsulated glycoprotein according to claim 4, characterized in that, The amino acid sequence of the signal peptide region of the chimeric envelope glycoprotein is selected from SEQ ID NO: 1 or SEQ ID NO: 11; the amino acid sequence of the non-proximal membrane domain of the extracellular region is shown in SEQ ID NO: 12; the amino acid sequence of the proximal membrane domain of the extracellular region is selected from SEQ ID NO: 3 or SEQ ID NO: 13; the amino acid sequence of the transmembrane region is selected from SEQ ID NO: 4 or SEQ ID NO: 14; and the amino acid sequence of the intracellular region is shown in SEQ ID NO:

5.

7. A chimeric enveloped glycoprotein, characterized in that, The chimeric encapsulated glycoprotein is prepared using the method for preparing chimeric encapsulated glycoproteins according to any one of claims 1 to 6; The chimeric enveloped glycoprotein is not a protein formed by linking the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:

5.

8. A lentiviral envelope plasmid, characterized in that, The envelope plasmid is capable of transcribing the chimeric envelope glycoprotein as described in claim 7.

9. A method for pseudo-packaging lentiviruses, characterized in that, The lentivirus is pseudo-packaged using the chimeric envelope glycoprotein of claim 7 or the lentivirus envelope plasmid of claim 8.

10. A reagent kit, characterized in that, The kit contains the chimeric envelope glycoprotein as described in claim 7 or the lentiviral envelope plasmid as described in claim 8.

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