A baev-g envelope glycoprotein, its optimization method, and an envelope plasmid, a packaging method, and a kit involving the envelope glycoprotein

Abstract

The application provides a BaEV-G envelope glycoprotein, an optimization method thereof, and an envelope plasmid, a packaging method and a kit related to the envelope glyprotein, in particular, relates to an optimization method of a BaEV-G envelope glyprotein, a chimeric BaEV-G envelope glyprotein, a lentivirus envelope plasmid, a lentivirus pseudotyped packaging method and a kit. The BaEV-G envelope glyprotein is chimerized with the VSV-G envelope glyprotein to obtain the chimeric BaEV-G envelope glyprotein, so that the lentivirus packaging efficiency, biological activity and other performances can be effectively improved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a BaEV-G enveloped glycoprotein, its optimization method, and an envelope plasmid, packaging method, and kit relating to the enveloped glycoprotein. Background Technology

[0002] 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.

[0003] VSV-G (Vesicular Stomatitis Virus-Glycoprotein) is the envelope protein of herpes simplex virus. Due to its broad host range, high stability, and high efficiency, it is a commonly used envelope protein for lentiviral pseudotyped viruses. However, VSV-G also has drawbacks. Viruses packaged with VSV-G are inactivated by human serum complement, reducing their efficacy. Furthermore, in vivo applications suffer from cytotoxicity and insufficient targeting, limiting the application of this type of viral vector in certain gene therapy fields. BaEV-G (Baboon Endogenous Retrovirus-Glycoprotein) is derived from a baboon endogenous retrovirus. Although its pseudovirus infection range for different cell types is narrower than that of VSV-G, BaEV-G has lower toxicity and is less easily inactivated by the serum complement system compared to VSV-G. It also has a better bias towards blood cells, such as natural killer cells (NK cells) or hematopoietic stem cells (HSCs). It has received widespread attention in immunotherapy-related research and has therefore been used as a lentiviral pseudovirus vector to improve gene delivery efficiency and targeting for specific cell types.

[0004] Wild-type BaEV-G exhibits low packaging efficiency, thus researchers urgently seek a method to effectively improve its packaging efficiency. Patent CN104080917A (incorporated in its entirety herein) discloses a BaEV mutant, BaEV / TR, with higher packaging efficiency, referred to as BVTR in this invention. However, the improvement in its packaging efficiency remains limited. Therefore, how to further improve the packaging efficiency of BaEV-G while maintaining its low toxicity and bioactivity has become a pressing technical challenge in this field. Summary of the Invention

[0005] This invention provides an optimization method for BaEV-G enveloped glycoproteins. This method obtains a series of chimeric BaEV-G enveloped glycoproteins by rationally splicing BaEV-G and wild-type VSV-G enveloped glycoproteins. The packaging efficiency and activity are significantly higher than those of the currently known BaEV mutant BVTR enveloped glycoproteins.

[0006] On one hand, this invention provides an optimization method for BaEV-G envelope glycoprotein. The amino acid sequence of the BaEV-G envelope glycoprotein is selected from wild-type or mutant sequences. The amino acid sequence of the BaEV-G envelope glycoprotein is chimeric with the amino acid sequence of the VSV-G envelope glycoprotein to obtain a chimeric BaEV-G envelope glycoprotein. In some embodiments, the amino acid sequence of the BaEV-G envelope 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. In some embodiments, at least one region of the BaEV-G envelope glycoprotein is chimeric with the amino acid sequence of the wild-type VSV-G envelope glycoprotein. In some embodiments, two regions of the BaEV-G envelope glycoprotein are chimeric with the amino acid sequence of the VSV-G envelope glycoprotein. Specifically, the transmembrane region and the intracellular region of the BaEV-G envelope glycoprotein can be replaced with the corresponding regions of the wild-type VSV-G envelope glycoprotein to obtain a chimeric BaEV-G envelope glycoprotein. In some embodiments, three regions of the BaEV-G envelope glycoprotein are chimeric with the amino acid sequence of the VSV-G envelope glycoprotein. In some embodiments, the signal peptide region, transmembrane region, and intracellular region of the BaEV-G envelope glycoprotein are replaced with their corresponding regions in the wild-type VSV-G envelope glycoprotein to obtain a chimeric BaEV-G envelope glycoprotein. In some embodiments, the chimeric BaEV-G envelope glycoprotein retains the receptor-binding ability of BaEV-G while possessing some of the functional characteristics of VSV-G. By replacing different regions, the stability and membrane fusion ability of the chimeric protein can be modulated, thereby optimizing the transduction efficiency and host cell range of the viral vector. Furthermore, this chimeric envelope glycoprotein can be used to construct viral vector systems with broad host adaptability and higher safety, suitable for gene therapy and vaccine delivery.

[0007] In some embodiments, the amino acid sequence of the signal peptide region of the 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.

[0008] In some embodiments, the amino acid sequence of the signal peptide region of the BaEV-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.

[0009] In some embodiments, the amino acid sequence of the signal peptide region of the chimeric BaEV-G 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 selected from SEQ ID NO: 2 or 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.

[0010] In some embodiments, the amino acid sequence of the signal peptide region of the chimeric BaEV-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: 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: 4, and the amino acid sequence of the intracellular region is shown in SEQ ID NO: 5.

[0011] In some embodiments, the amino acid sequence of the signal peptide region of the chimeric BaEV-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: 4, and the amino acid sequence of the intracellular region is shown in SEQ ID NO: 5.

[0012] In some embodiments, the chimeric BaEV-G enveloped glycoprotein pseudolentiviral vector or its mutants include 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. In some embodiments, the chimeric BaEV-G enveloped glycoprotein pseudolentiviral vector or its mutants further include a fusion-inhibiting R peptide. Specifically, the corresponding amino acid sequences or their mutant sequences are as follows:

[0013] The amino acid sequence or mutant sequence of the signal peptide region is shown in SEQ ID NO:1 or SEQ ID NO:6;

[0014] The amino acid sequences or mutant sequences of the non-promembranous domains of the extracellular region are shown in SEQ ID NO:2 or SEQ ID NO:7;

[0015] The amino acid sequence or mutant sequence of the extracellular juxtamembrane domain is shown in SEQ ID NO:3 or SEQ ID NO:8;

[0016] The amino acid sequence or mutant sequence of the transmembrane region is shown in SEQ ID NO:4 or SEQ ID NO:9;

[0017] The intracellular region amino acid sequence or mutant sequence is shown in SEQ ID NO:5, SEQ ID NO:10 or SEQ ID NO:12;

[0018] The fusion inhibitory R peptide is shown in SEQ ID NO:11;

[0019] Preferably, the amino acid sequences or mutant sequences corresponding to each region of the chimeric BaEV envelope glycoprotein are any one of the following groups:

[0020] a) VSV-G-wt: SEQ ID NO:1+SEQ ID NO:2+SEQ ID NO: 3+SEQ ID NO: 4+ SEQ ID NO: 5;

[0021] b) BVTR: SEQ ID NO:6+SEQ ID NO:7+SEQ ID NO:8+SEQ ID NO:9+ SEQ ID NO:10+ SEQ ID NO: 11;

[0022] c) BVT: SEQ ID NO:6+SEQ ID NO:7+SEQ ID NO:8+SEQ ID NO:9+ SEQ ID NO:10;

[0023] d) BVRL: SEQ ID NO:6+SEQ ID NO:7+SEQ ID NO:8+SEQ ID NO:9+ SEQ ID NO:12;

[0024] e) BVGc: SEQ ID NO:6+SEQ ID NO:7+SEQ ID NO:8+SEQ ID NO:9+SEQ ID NO:5;

[0025] f) BVGmc: SEQ ID NO:6+SEQ ID NO:7+SEQ ID NO:8+SEQ ID NO: 4+ SEQ IDNO: 5;

[0026] g) BVGsmc: SEQ ID NO:6+SEQ ID NO:7+SEQ ID NO:3+SEQ ID NO:4+ SEQ IDNO: 5;

[0027] h) GsBVTR: SEQ ID NO:1+SEQ ID NO:7+SEQ ID NO: 8+SEQ ID NO: 9+ SEQ IDNO:10+ SEQ ID NO: 11;

[0028] i) GsBVT: SEQ ID NO:1+SEQ ID NO:7+SEQ ID NO: 8+SEQ ID NO: 9;

[0029] j) GsBVRL: SEQ ID NO:1+SEQ ID NO:7+SEQ ID NO: 8+SEQ ID NO: 9+SEQ IDNO: 12;

[0030] k) GsBVGc: SEQ ID NO:1+SEQ ID NO:7+SEQ ID NO: 8+SEQ ID NO: 9+SEQ IDNO: 5;

[0031] l) GsBVGmc: SEQ ID NO: 1 + SEQ ID NO: 7 + SEQ ID NO: 8 + SEQ ID NO: 4 + SEQ ID NO: 5.

[0032] On the other hand, the present invention also provides a chimeric BaEV-G envelope glycoprotein, which is optimized from any of the aforementioned VSV-G envelope glycoproteins using an optimization method. This chimeric BaEV-G envelope glycoprotein, by fusing an optimized sequence with the VSV-G domain, significantly improves the stability and targeted transduction efficiency of viral vectors, especially exhibiting higher fusion activity and lower immunogenicity in mammalian cells, making it suitable for various gene delivery scenarios.

[0033] 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.

[0034] This invention also provides a method for packaging lentiviruses, using any of the aforementioned chimeric envelope glycoproteins or envelope plasmids to package lentiviruses. The method involves co-transfecting the 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, 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.

[0035] This invention also provides a kit comprising the aforementioned chimeric envelope glycoprotein or envelope plasmid. The kit further includes viral structural protein expression plasmids, transfection reagents, and antibodies for detecting envelope glycoprotein expression levels. All components undergo rigorous quality control to ensure the accuracy and reproducibility of experimental results. The kit is suitable for research laboratories and biopharmaceutical companies, and has broad application prospects in gene editing, functional genomics research, and personalized medicine.

[0036] 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.

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

[0038] 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.

[0039] 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.

[0040] The present invention provides an optimization method for BaEV-G envelope glycoprotein, which involves chimerizing the amino acid sequence of wild-type or mutant BaEV-G envelope glycoprotein with a portion of the amino acid sequence of VSV-G envelope glycoprotein. The resulting chimeric BaEV-G protein can effectively improve lentiviral packaging efficiency, bioactivity, and other properties. By rationally combining specific functional domains of BaEV-G and VSV-G, the optimized chimeric protein significantly enhances viral particle stability and infection efficiency while retaining the targeting specificity of BaEV-G. These advantages make this chimeric BaEV-G envelope glycoprotein a promising candidate for applications in gene therapy, vaccine delivery, and biological research. Attached Figure Description

[0041] 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:

[0042] Figure 1 Schematic diagram of the sequence regions of various enveloped glycoproteins.

[0043] Figure 2 Statistical graph of live cell density and cell viability 48 hours after HEK293TH suspension cells packaged with various envelope glycoprotein pseudotypes of lentivirus.

[0044] Figure 3 Images of fluorescent protein expression in NK cells 48 hours after infection with various enveloped glycoprotein pseudotypes of lentiviruses.

[0045] Figure 4 Results of luciferase activity and cell viability of NK cells 48 hours after infection with various enveloped glycoprotein pseudotypes of lentiviruses.

[0046] Figure 5 The percentage of NeonGreen-positive cells 48 hours after infection of NK cells with various enveloped glycoprotein pseudotypes of lentiviruses. Detailed Implementation

[0047] 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.

[0048] 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.

[0049] 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.

[0050] As used herein, the term "VSV envelope glycoprotein" or "VSV-G" refers to the wild-type form of the VSV-G envelope glycoprotein or a mutant of the 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.

[0051] As used herein, the term “BaEV envelope glycoprotein” or “BaEV-G” refers to the wild-type form of the BaEV envelope glycoprotein or a mutant of the 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 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.

[0052] As used herein, the term "mutant" refers to a polypeptide having one or more amino acid substitutions, deletions, or insertions compared to a wild-type sequence, wherein the one or more substitutions are preferably conserved amino acid substitutions.

[0053] As used herein, the term "enveloping glycoprotein" refers to glycosylated proteins located on the outer membrane of viral particles that mediate the specific binding of the virus to receptors on the surface of host cells and the subsequent membrane fusion process, thereby determining the host range and tissue tropism of viral infection. Envelope glycoproteins comprise a signal peptide region, an extracellular domain, a transmembrane domain, and an intracellular domain, among which the signal peptide region...

[0054] The extracellular domains are responsible for recognizing and binding to specific receptors on the host cell surface, while the transmembrane domains anchor proteins to the viral membrane and participate in membrane fusion. The synergistic effect of these domains determines the efficiency and specificity of viral infection.

[0055] As used herein, the term "chimeric VSV-G envelope glycoprotein" refers to a chimeric protein consisting of BaEV envelope glycoprotein and VSV-G envelope glycoprotein. In this context, "chimeric" refers to the combination and splicing of one or more of the signal peptide region, the non-juxtamembrane domain of the extracellular region, the juxtamembrane domain of the extracellular region, the transmembrane region, and the intracellular region of the BaEV envelope glycoprotein by replacing them with corresponding regions of the VSV-G envelope glycoprotein.

[0056] As used herein, the term "fusion-inhibiting R-peptide" refers to the C-terminal portion of the cytoplasmic tail domain of an envelope glycoprotein, which carries the tyrosine endocytosis signal -YXXL and is cleaved by viral proteases during viral particle maturation, thereby enhancing membrane fusion of envelope glycoproteins.

[0057] As used herein, the terms "pseudovirus" or "pseudotyped lentivirus" are interchangeable. 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 into host cells. Pseudotypening technology allows for the flexible replacement of envelope glycoproteins from different sources to regulate the tissue specificity or cell tropism of viral vectors, thereby expanding their potential applications in gene therapy, vaccine delivery, and functional genomics research.

[0058] 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.

[0059] 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.

[0060] Unless otherwise defined or the context clearly specifies otherwise, 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.

[0061] 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.

[0062] 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.

[0063] Example 1: Construction of a chimeric BaEV-G pseudotyped lentiviral vector

[0064] Twelve groups of envelope glycoproteins were designed, and plasmids for the BaEV-G envelope glycoprotein were synthesized. Group 1, labeled VSV-G-wt, represents the wild-type VSV-G; Group 2, a positive control labeled BVTR, represents a beneficial mutant of BaEV-G; and Groups 3 through 12, labeled BVT, BVRL, BVGc, BVGmc, BVGsmc, GsBVTR, GsBVT, GsBVRL, GsBVGc, and GsBVGmc, respectively, represent mutants or chimeric forms of BaEV-G with VSV-G. The corresponding nucleic acid sequences of these 12 proteins 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 1-12 are shown below. Figure 1 As shown.

[0065] Example 2: Chimeric BaEV-G enveloped glycoprotein pseudotyped lentiviral vector lentiviral packaging

[0066] ① HEK293TH seeding: based on a live cell count of 1×10⁻⁶ 6Cells were seeded at a rate of 1 / mL into 125 mL triangular cell culture flasks, and cultured in a 25 mL BalanCD HEK293 (FUJIFILM) + 4 mM L-Glutamine + 1% penicillin / streptomycin culture system. The flasks were then placed in a shaking incubator at 37°C, 180 rpm, and 5% CO2 in the dark. Virus packaging was performed 18-20 hours after cell culture. A four-plasmid virus packaging system was used, with the following components added sequentially at a mass ratio of 10:5:2:3: 50 μg total plasmid. The transfer plasmid (expressing the NeonGreen-teLuc reporter gene), packaging plasmid (expressing Gag-pol protein), packaging plasmid (expressing Rev protein), and vector expressing the envelope glycoprotein (groups 1-12 in Example 1) were added. These were thoroughly mixed with the 2.4% BalanCD HEK293 medium in the culture system, and then PEI pro (polyplustransfection) at twice the volume of the plasmid was added. The mixture was incubated at room temperature for 15-20 minutes, and then added dropwise to the cells for transfection. HEK293TH triangular cell culture flasks were placed in a shaking incubator at 37°C, 180 rpm, and 5% CO2, protected from light.

[0067] ② 24 hours after cell transfection, add 12% volume of BalanCD HEK293 Viral Feed (FUJIFILM) medium.

[0068] ③ Forty-eight hours after cell transfection, collect the viral supernatant, centrifuge at 300g for 5 min to collect the supernatant culture medium, and centrifuge at 25,000 rpm for 1.5 hours using a Beckman ultracentrifuge with the sucrose pad method. After discarding the supernatant, add an appropriate amount of viral preservation solution to dissolve the viral particles for at least 2 hours, centrifuge at 300g for 1 min at 4℃, collect the solution at the bottom of the tube, add 10% DMSO and mix well, aliquot and store at -80℃.

[0069] ④ Results of live cell density and cell viability after 48 hours of HEK293TH cell lentivirus packaging are as follows: Figure 2 As shown in Table 1, the cell survival rates of pseudo-lentiviral vectors containing novel chimeric BaEV-G envelope glycoproteins were all above 80%, with some reaching over 90%, which is basically consistent with the cell survival rates of wild-type VSVG-G-wt and mutant BVTR.

[0070] Table 1 HEK293TH cell lentivirus packaging 48 hours

[0071] .

[0072] Example 3: NK cell transduction efficiency test

[0073] ①NK cell infection experiment

[0074] Based on the NK cell viable cell count of 4 × 10⁶ per well 5 NK cells were seeded into 24-well cell culture plates, and 16 μL of each of the various pseudotyped lentiviruses obtained in step 3 of Example 2 were added to each well to transduce NK cells, with a final volume of 1 mL. Polybrene was added to a final concentration of 6 μg / mL, and the mixture was incubated at 37°C in a 5% CO2 incubator for 48 hours. The intensity of cell fluorescence expression was observed under a microscope, luciferase activity was detected by enzyme-linked immunosorbent assay (ELISA), and the proportion of cells expressing NeonGreen+ was detected by flow cytometry. The optimal chimeric BaEV-G envelope glycoprotein pseudotyped lentiviral vector was screened by comparing with the control group (BVTR, VSVG-G-wt).

[0075] ②Results of NK cell transduction efficiency

[0076] The results of fluorescence expression intensity observation under a microscope are as follows: Figure 3 As shown, the viral particles prepared from the optimized chimeric GsBVGmc and BVGmc envelope glycoprotein pseudotyped lentiviral vectors of this invention, after being transduced into NK cells for 48 hours, showed enhanced fluorescence intensity compared to the control group BVTR, and even stronger than wild-type VSV-G-wt.

[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 per 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 4 As shown in Table 2, after 48 hours of transduction of NK cells with the optimized chimeric GsBVGmc and BVGmc, the luciferase reading value of the virus particles was significantly higher than that of the control group mutant BVTR, and significantly better than that of wild-type VSV-G-wt.

[0078] Table 2 Virus particle transduction of NK cells 48 hours

[0079] .

[0080] The unit for viral titer is TU / mL, which represents the number of transduction-functional viral particles per milliliter of viral fluid.

[0081] The calculation formula is: Titer = (Number of cells at infection (cells) * Positive cells %) / Virus volume (mL)

[0082] The viral volume and cell count at infection are known; flow cytometry is needed to confirm the percentage of positive cells. Therefore, to detect the packaging efficiency of the lentivirus, flow cytometry (BD Biosciences) was used. The cell samples were washed with DPBS to avoid the culture medium components affecting the accuracy of the results. Since the viral particles prepared in this invention contain the NeonGreen-teLuc reporter gene, after transduction into cells and expression for 48 hours, green fluorescence can be detected under excitation light, allowing for flow cytometry detection without antibody incubation. Flow cytometry positive detection results are as follows: Figure 5 As shown, after 48 hours of transduction of NK cells with chimeric BaEV-G viral particles, compared with the control group mutant BVTR, GsBVGmc and BVGmc exhibited consistently high transduction and packaging efficiencies in different cell subtypes. Particularly in refractory NK cells, the proportion of NeonGreen+ cells was nearly doubled compared to BVTR, indicating that the optimized envelope glycoprotein has wider applicability and stronger penetration ability. This characteristic provides important support for its future application in personalized immunotherapy.

[0083] In summary, this invention significantly improves the efficiency of cell transduction using pseudoformed lentiviral vectors by optimizing the structure of the chimeric BaEV-G envelope glycoprotein. Experimental data show that chimeric BaEV-G outperforms existing technologies such as BVTR or VSV-G-wt in terms of fluorescence intensity, post-infection cell viability, and the proportion of transduced NeonGreen+ cells, demonstrating higher transduction efficiency and application potential. This improvement provides a more efficient and stable tool for lentiviral vector-based immunocellular therapy research and lays a solid foundation for the optimization of subsequent gene therapy strategies.

[0084] 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.

[0085] Some amino acid sequences involved in this article:

[0086] SEQ ID NO: 1: Amino acid sequence of wild-type VSV-G signal peptide region

[0087] MKCLLYLAFLFIGVNC

[0088] SEQ ID NO: 2: Amino acid sequence of the non-promembranous domain of the extracellular region of wild-type VSV-G

[0089] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQG TWLNPGFPPQSCGYATVTDAEAAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGF RSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMGHGMLDSDLHLSSKAQVFEHPHIQ

[0090] SEQ ID NO: 3: Amino acid sequence of the juxtamembrane domain of the extracellular region of wild-type VSV-G

[0091] DAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWK

[0092] SEQ ID NO: 4: Amino acid sequence of the transmembrane region of wild-type VSV-G

[0093] SSIASFFFIIGLIIGLFLVLRV

[0094] SEQ ID NO: 5: Intracellular amino acid sequence of wild-type VSV-G

[0095] GIHLCIKLKHTKKRQIYTDIEMNRLGK

[0096] SEQ ID NO: 6: Amino acid sequence of wild-type BaEV-G signal peptide region

[0097] MGFTTKIIFLYNLVLVYA

[0098] SEQ ID NO: 7: Amino acid sequence of the non-juxtamembrane domain of the extracellular region of wild-type BaEV-G

[0099] KFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMGFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPKDTSPSGPL QECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTGGTSDVQVLGSTNKLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQRKLEEIHKALYPELQYHPLAIPKVRDNLMVDAQ TLNILNATYNLLLMSNTSLVDDCWLCLKLGPPTPLAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVAFSNCTSITNVTGPICAVNGSVFLCGNNMAYTYLPTNW TGLCVLATLLPDIDIIPGDEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISDVQILSSTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFY

[0100] SEQ ID NO: 8: Amino acid sequence of the juxtamembrane domain of the extracellular region of wild-type BaEV-G

[0101] VNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLP

[0102] SEQ ID NO:9: Amino acid sequence of the transmembrane region of wild-type BaEV-G

[0103] YLLPFLGPLLTLLLLLTIGPCIF

[0104] SEQ ID NO:10: Amino acid sequence of the mutated intracellular region of BaEV-G

[0105] NRLVQFVKDRISVVQAL

[0106] SEQ ID NO:11: Amino acid sequence of fusion inhibitory R peptide

[0107] VLTQQYHQLKPLEYEP

[0108] SEQ ID NO:12: Intracellular amino acid sequence of wild-type BaEV-G

[0109] NRLTAFINDKLNIIHAM

Claims

1. A method for optimizing BaEV-G enveloped glycoprotein, characterized in that, The transmembrane and intracellular regions of the BaEV-G envelope glycoprotein are replaced with the corresponding regions of the VSV-G envelope glycoprotein to obtain a chimeric BaEV-G envelope glycoprotein; or, the signal peptide region, transmembrane region, and intracellular region of the BaEV-G envelope glycoprotein are replaced with the corresponding regions of the VSV-G envelope glycoprotein to obtain a chimeric BaEV-G envelope glycoprotein. The amino acid sequence of the BaEV-G envelope glycoprotein consists of a signal peptide region, a non-proximal membrane domain in the extracellular region, a proximal membrane domain in the extracellular region, a transmembrane region, and an intracellular region; the amino acid sequence of the VSV-G envelope glycoprotein consists of a signal peptide region, a non-proximal membrane domain in the extracellular region, a proximal membrane domain in the extracellular region, a transmembrane region, and an intracellular region. The amino acid sequence of the signal peptide region of the 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. The amino acid sequence of the signal peptide region of the BaEV-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 or SEQ ID NO:

12.

2. The method for optimizing BaEV-G enveloped glycoprotein according to claim 1, characterized in that, The amino acid sequence of the signal peptide region of the chimeric BaEV-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: 4, and the amino acid sequence of the intracellular region is shown in SEQ ID NO:

5.

3. The method for optimizing BaEV-G enveloped glycoprotein according to claim 1, characterized in that, The amino acid sequence of the signal peptide region of the chimeric BaEV-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: 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: 4, and the amino acid sequence of the intracellular region is shown in SEQ ID NO:

5.

4. A chimeric BaEV-G enveloped glycoprotein, characterized in that, The chimeric BaEV-G enveloped glycoprotein is optimized by the optimization method of the BaEV-G enveloped glycoprotein according to any one of claims 1 to 3.

5. A lentiviral envelope plasmid, characterized in that, The envelope plasmid is capable of transcribing the chimeric BaEV-G envelope glycoprotein of claim 4.

6. A method for pseudo-packaging lentiviruses, characterized in that, The lentivirus is pseudo-packaged using the chimeric BaEV-G envelope glycoprotein of claim 4 or the lentivirus envelope plasmid of claim 5.

7. A reagent kit, characterized in that, The kit contains the chimeric BaEV-G envelope glycoprotein as described in claim 4 or the lentiviral envelope plasmid as described in claim 5.

Images