Lentiviral expression vector, and lentivirus and preparation method therefor and use thereof

Abstract

A lentivirus, comprising a BaEV envelope glycoprotein. The extracellular region of the BaEV envelope glycoprotein comprises at least one of the following sequences: a spacer sequence and a recognition sequence. The spacer sequence comprises a spacer unit, and the recognition sequence comprises BaEVSecSig-recognition protein-BaEVTM, wherein BaEVSecSig is a BaEV signal peptide, and BaEVTM is a BaEV transmembrane domain.

Description

Lentiviral expression vectors, lentiviruses, their preparation methods and applications

[0001] This application claims priority to Chinese patent application No. 202411799102.4, filed on December 6, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to the field of biomedical technology, and in particular to a lentiviral expression vector, lentivirus, its preparation method and application. Background Technology

[0003] Lentiviral vectors (LVs) are gene therapy vectors developed based on HIV-1 (human immunodeficiency virus type 1). Unlike typical retroviral vectors, they are capable of infecting both dividing and non-dividing cells.

[0004] This vector can effectively integrate exogenous genes into the host chromosome, thereby achieving persistent expression. In terms of infectivity, it can effectively infect various cell types, including neurons, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, and stem cells, thus achieving good gene therapy effects. Summary of the Invention

[0005] On one hand, a lentivirus is provided, comprising: BaEV envelope glycoprotein; wherein the extracellular region of the BaEV envelope glycoprotein comprises at least one of the following sequences: a spacer sequence and a recognition sequence, the spacer sequence comprising: a spacer unit; the recognition sequence comprising: BaEVSecSig-recognition protein-BaEVTM; wherein, BaEVSecSig is a BaEV signal peptide, and BaEVTM is a BaEV transmembrane domain.

[0006] In some embodiments, the spacer unit comprises a conserved amino acid.

[0007] In some embodiments, the spacing unit includes at least one of SAG, GAS, ASG, AGS, GSA, and SGA.

[0008] In some embodiments, the spacer sequence comprises 9 to 27 amino acids.

[0009] In some embodiments, the interval sequence includes at least one of: SAGSAGSAGSAGSAG, GASGASGASGASGAS, ASGASGASGASGASG, AGSAGSAGSAGSAGSAGS, GSAGSAGSAGSAGSAGSASA, and SGASGASGASGASGASGA.

[0010] In some embodiments, the insertion sites of the spacer sequence are positions 117 and 338 of the amino acid sequence of the BaEV envelope glycoprotein.

[0011] In some embodiments, the BaEV envelope glycoprotein comprising the spacer sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 1 to SEQ ID No. 6.

[0012] In some embodiments, the recognition protein includes: a receptor protein, an antibody, a single-chain recognition sequence modified from an antibody, a nanobody, a lectin, or a molecular chaperone protein.

[0013] In some embodiments, the recognition protein includes protein A or protein G.

[0014] In some embodiments, the recognition protein is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of either SEQ ID No. 7 or SEQ ID No. 8.

[0015] In some embodiments, the sequence of BaEVSecSig is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of SEQ ID No. 9; and / or, the sequence of BaEVTM is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of SEQ ID No. 10.

[0016] On the other hand, a lentiviral expression vector is provided, comprising: a nucleotide sequence expressing a lentivirus as described in any of the preceding embodiments.

[0017] In some embodiments, the nucleotide sequence expressing the spacer sequence of the BaEV envelope glycoprotein is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 11 to SEQ ID No. 16.

[0018] In some embodiments, the nucleotide sequence expressing the recognition sequence of the BaEV envelope glycoprotein is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of either SEQ ID No. 17 or SEQ ID No. 18.

[0019] On another front, a method for preparing lentivirus is provided, comprising: seeding packaging cells in a culture container and culturing them; adding a vector plasmid, a packaging plasmid, and a lentiviral expression vector to a cell culture medium to obtain a first solution; adding a transfection reagent to the cell culture medium and mixing to obtain a second solution; and when the confluence of the packaging cells reaches a preset value, mixing the first solution and the second solution and adding them to a culture container for transfection.

[0020] After transfection, the lentivirus was obtained;

[0021] The lentivirus includes: BaEV envelope glycoprotein; the extracellular region of the BaEV envelope glycoprotein includes at least one of the following sequences: a spacer sequence and a recognition sequence; the spacer sequence includes: a spacer unit; the recognition sequence includes: BaEVSecSig-recognition protein-BaEVTM; BaEVSecSig is a BaEV signal peptide, and BaEVTM is a BaEV transmembrane domain.

[0022] In some embodiments, the lentiviral expression vector comprises at least one of an amino acid sequence expressing a spacer sequence of the extracellular region of the BaEV envelope glycoprotein and an amino acid sequence expressing a recognition sequence of the extracellular region of the BaEV envelope glycoprotein.

[0023] In some embodiments, a lentiviral expression vector expressing a spacer sequence of BaEV envelope glycoprotein is referred to as a first vector; a lentiviral vector expressing a recognition sequence of BaEV envelope glycoprotein is referred to as a second vector; in the first solution, the lentiviral expression vector comprises: the first vector and the second vector, wherein the mass ratio of the first vector to the second vector is in the range of 5 to 10.

[0024] In some embodiments, the mass ratio of the vector plasmid, the packaging plasmid, and the lentiviral expression vector in the first solution is (3-5):3:(1-3).

[0025] In some embodiments, prior to seeding the packaged cells into the culture vessel for culture, the method further includes: modifying the culture vessel with a coagulated microgel.

[0026] In some embodiments, the condensate microgel comprises folic acid and a polycationic electrolyte.

[0027] In some embodiments, the polycationic electrolyte comprises at least one of protamine sulfate and polylysine.

[0028] In some embodiments, the condensate microgel modification of the culture vessel includes: mixing a folic acid solution with a polycationic electrolyte solution to obtain a third solution; coating the third solution onto the culture vessel; adding a phosphate solution to initiate gelation; and obtaining a culture vessel modified with condensate microgel.

[0029] On the other hand, we provide the application of lentivirus as described in any of the above embodiments in the transfection of human primary NK cells. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in this disclosure, the accompanying drawings used in some embodiments of this disclosure will be briefly described below. Obviously, the drawings described below are only drawings of some embodiments of this disclosure, and those skilled in the art can obtain other drawings based on these drawings. In addition, the drawings described below can be regarded as schematic diagrams and are not intended to limit the actual size of the product, the actual process of the method, etc. involved in the embodiments of this disclosure.

[0031] Figure 1 is a flowchart of a method for preparing lentivirus according to some embodiments of the present disclosure;

[0032] Figure 2 is a structural diagram of a lentivirus according to some embodiments of the present disclosure;

[0033] Figure 3 shows the percentage of GFP-positive cells according to some embodiments of this disclosure;

[0034] Figure 4 is another percentage diagram of GFP-positive cells according to some embodiments of the present disclosure;

[0035] Figure 5 shows another percentage of GFP-positive cells according to some embodiments of this disclosure;

[0036] Figure 6 is another percentage diagram of GFP-positive cells according to some embodiments of the present disclosure;

[0037] Figure 7 is a flow cytometry fluorescence diagram of antibody cell labeling according to Example 1 of this disclosure;

[0038] Figure 8 is a flow cytometry fluorescence diagram of antibody cell labeling according to Example 5 of this disclosure;

[0039] Figure 9 is a flow cytometry diagram of antibody cell labeling according to Example 6 of this disclosure. Detailed Implementation

[0040] The technical solutions in some embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments provided in this disclosure are within the scope of protection of this disclosure.

[0041] Unless the context otherwise requires, throughout the specification and claims, the term "comprise" and its other forms, such as the third-person singular "comprises" and the present participle "comprising," are interpreted as open-ended and encompassing, meaning "including, but not limited to." In the description of the specification, terms such as "one embodiment," "some embodiments," "exemplary embodiments," "example," "specific example," or "some examples," etc., are intended to indicate that a particular feature, structure, material, or characteristic associated with that embodiment or example is included in at least one embodiment or example of this disclosure. The illustrative representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics mentioned may be included in any suitable manner in any one or more embodiments or examples.

[0042] Hereinafter, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of embodiments of this disclosure, unless otherwise stated, "a plurality of" means two or more.

[0043] "At least one of A, B and C" has the same meaning as "at least one of A, B or C", both including the following combinations of A, B and C: only A, only B, only C, combinations of A and B, combinations of A and C, combinations of B and C, and combinations of A, B and C.

[0044] "A and / or B" includes the following three combinations: A only, B only, and a combination of A and B.

[0045] As used herein, “about” includes the value stated and the average value within an acceptable range of deviation from the given value, wherein the acceptable range of deviation is determined by a person skilled in the art taking into account the measurement under discussion and the error associated with the measurement of the given quantity (i.e., the limitations of the measurement system).

[0046] Cellular gene therapy (CGT) is an advanced technology combining gene therapy and cell therapy, aiming to treat and prevent diseases by modifying genes and cells. Gene therapy primarily involves introducing exogenous normal genes into target cells to correct or compensate for diseases caused by defective or abnormal genes. Common gene therapy techniques include gene augmentation and gene editing. The former uses vectors to introduce normal genes into the patient's body, while the latter directly edits gene sequences using technologies such as CRISPR (clustered regularly-interspaced short palindromic repeats). Cell therapy, on the other hand, involves using autologous or allogeneic cells, manipulated in vitro, and then introduced into the human body for disease treatment.

[0047] The most crucial technical step in cell gene therapy is the introduction of the target gene into target cells using gene delivery tools, and lentiviruses are one of the most important types of such tools. Lentiviral vectors (LVs) are retroviruses derived from human immunodeficiency virus (HIV-1). Their genome is a single-stranded positive-sense RNA, and the virulence genes have been removed and replaced by exogenous target genes, thus exhibiting high biosafety and stability.

[0048] Numerous reports have documented the application of lentiviral vectors in cell gene therapy, achieving promising results. For instance, vesicular stomatitis virus lentivirus (VSV-G-Lv) is widely used to encapsulate CRISPR-Cas genes and CAR (Chimeric antigen receptor) genes for targeted T-cell delivery. Furthermore, BaEV lentivirus (Baboonenvelope pseudotyped lentiviral vector, BaEV-LV) can effectively transduce various cell types, such as human B cells, T cells, and NK cells, particularly CD34+ stem cells, where its performance is even superior to VSV-G-LVs and RDTR-LVs.

[0049] BaEV lentivirus is a baboon-enveloped pseudolentiviral vector. NK cells (Natural killer cells) are also known as NK cells. RDTR is a mutant of the envelope glycoprotein of RD114 feline retrovirus.

[0050] Although lentiviral vectors have achieved great application prospects and commercial value, their production process is complex and costly. In particular, the production of high-titer vectors remains a major challenge for the industry, with the preparation of BaEV lentiviral vectors being especially difficult.

[0051] The envelope protein of BaEV lentivirus is the baboon endogenous retrovirus envelope glycoprotein (BaEV-G), which is expressed by the corresponding gene on the BaEV plasmid. The protein structure includes an extracellular domain, a transmembrane domain, and a tail domain, which is also the intracellular domain.

[0052] In some examples, optimization of BaEV lentiviruses is based on modifications to the intracellular domains, such as BaEV-Rless (no fusion-inhibiting R peptide) and BaEV / TR (replacing the tail domain with the tail domain of an MLV (Murine Leukemia Virus) envelope glycoprotein). However, expression of BaEV-Rless in 293T cells leads to the formation of numerous syncytia, resulting in significant cell death; while BaEV / TR exhibits significantly lower cytotoxicity and reduces syncytia formation, but its viral titer is lower than that of BaEV-Rless.

[0053] 293T cells are derived from 293 cells and are a human renal epithelial cell line expressing the SV40 large T antigen. 293T cells are widely used for transient transfection to overexpress various target proteins or for virus packaging. These cells are not demanding in terms of culture medium nutrients; they are adherent cells, but their adhesion is not strong, and slight disturbance can cause them to detach. When changing to fresh culture medium or rinsing with phosphate-buffered saline (PBS), fresh culture medium should be added carefully, ensuring that the liquid does not wash away adherent cells. The growth status of 293T cells directly affects the efficiency of virus packaging and transfection; cells with fewer passages and shorter total culture time should be selected whenever possible.

[0054] In other examples, constructing chimeric envelope glycoproteins by introducing the intracellular domain of VSV-G (Vesicularstomatitisvius) envelope glycoprotein improved the packaging efficiency of BaEV enveloped virus to some extent, but the improvement was still not significant, and the effect on infecting NK cells and T cells was poor.

[0055] Furthermore, due to the unstable structure of the envelope protein of BaEV lentivirus, it is easily degraded and inactivated, and it is easy to infect basal cells during packaging preparation, resulting in low lentivirus titers.

[0056] Therefore, how to systematically optimize the entire process of lentivirus preparation to obtain high-titer lentivirus vectors with better targeting and transfection efficiency is a core technical challenge that urgently needs to be solved in the industry.

[0057] The embodiments of this disclosure provide a method for preparing a lentivirus, as shown in Figures 1 and 2. The method for preparing a lentivirus includes steps R1 to R5.

[0058] R1. Seed the packaging cells into culture container 40 and culture them.

[0059] For example, 293T cells are seeded into culture container 40 (as shown in Figure 2). 293T cells are packaging cells, also known as basal cells.

[0060] R2. Add the vector plasmid, packaging plasmid, and lentiviral expression vector to the cell culture medium to obtain the first solution.

[0061] For example, the vector plasmid, packaging plasmid, and lentiviral expression vector are mixed in Opti-MEM serum-free medium to obtain a first solution.

[0062] Vector plasmids contain target gene fragments and can introduce polynucleotide sequences, including the target gene sequence, into host cells to transform the host and promote the expression of the introduced sequence. Sequence expression includes, for example, transcription and translation. The vector plasmids are self-constructed, and the polynucleotide sequences within them can be chimeric antigen receptor coding sequences, immunoglobulin (Ig) gene coding sequences, cytokine gene coding sequences, etc., and can be used in gene and drug therapy fields.

[0063] For example, the packaging plasmid includes: pCMV-ΔR8.91 plasmid, which is a second-generation lentiviral packaging helper plasmid, containing viral functional genes such as gag, pol, rev and tat.

[0064] Lentiviral expression vectors, such as BaEV plasmids, encode the BaEV envelope glycoprotein (BaEV-G). This envelope glycoprotein typically comprises an extracellular region, a transmembrane domain, and a tail domain, sequentially from the amino terminus to the carboxyl terminus. In enveloped viruses, the transmembrane domain crosses the viral envelope, connecting the extracellular region located on the outer side of the viral envelope and the tail domain located on the inner side. The extracellular region corresponds to amino acid positions 1-488 (including the endpoints) of BaEV-G (NCBI sequence accession number YP_009109691.1). NCBI stands for National Center for Biotechnology Information, representing the National Center for Biotechnology Information in the United States.

[0065] Opti-MEM is a basic cell culture medium composed of the minimum concentration of nutrients necessary for the growth of the culture.

[0066] R3. Add the transfection reagent to the cell culture medium and mix to obtain the second solution.

[0067] For example, Lipofectamine 2000 is mixed with Opti-MEM medium to obtain a second solution.

[0068] Lipofectamine 2000 is a highly efficient transfection reagent suitable for a wide range of adherent and suspension cell lines. It exhibits excellent transfection efficiency, achieving very high protein expression levels.

[0069] R4. When the confluence of the cells to be packaged reaches the preset value, mix the first solution and the second solution and add them to the culture container for transfection.

[0070] For example, once the confluence of 293T cells reaches a preset value, the medium is replaced with serum-free DMEM medium.

[0071] For example, the preset range for the confluence of 293T cells is 70% to 90%.

[0072] For example, the first and second solutions are mixed and added dropwise to DMEM culture medium, and the mixture is gently shaken to cover all cells with the reagents.

[0073] For example, the culture container 40 includes: a T175 cell culture flask or a culture dish.

[0074] Cell confluence refers to the degree of adhesion and alignment among cells during proliferation in a culture vessel (40°C), usually expressed as a percentage. The concept of cell confluence is not merely about cell density; it comprehensively reflects the state of cell growth. Generally, when cells grow and confluence to form a monolayer, they become more crowded, their flattened shape decreases, and the surface area in contact with the culture medium also reduces. Simultaneously, some nutrients in the culture medium are gradually consumed, especially in areas immediately surrounding the cells. Cell division in this monolayer will cease. Therefore, assessing confluence provides a rough reference for selecting experimental timing and the passage time.

[0075] DMEM medium (Dulbecco's modified Eagle's medium) is a medium developed based on MEM medium, containing various amino acids and glucose. The characteristics of this medium are: (1) its amino acid content is twice that of Eagle's medium, and it contains non-essential amino acids such as glycine; (2) its vitamin content is four times that of Eagle's medium; (3) it contains pyruvate, an important substance in the glycolysis pathway; and (4) it contains trace amounts of iron ions.

[0076] R5. After transfection, lentivirus 100 is obtained.

[0077] For example, after culturing the culture vessel in a cell culture incubator, the culture medium is replaced with DMEM complete medium containing FBS, and the first supernatant is collected after transfection. After centrifuging the first supernatant, the second supernatant is collected, and the second supernatant is filtered to obtain lentivirus.

[0078] FBS (Fetal Bovine Serum) is a pale yellow, clear, non-hemolytic, foreign-free, slightly viscous liquid. The fetal bovine serum should be obtained from fetal bovines delivered by cesarean section. For example, the FBS content in the culture medium is 10% by mass. For example, cell pellets are removed by ultracentrifugation or PEG (polyethylene glycol) precipitation, and the resulting supernatant is collected and then filtered to obtain lentivirus.

[0079] As shown in Figure 2, BaEV Lentiviral 100 can be obtained from the above steps R1 to R5.

[0080] In BaEV lentivirus 100, the extracellular region of BaEV-G mediates the interaction between BaEV lentivirus 100 and target cell surface receptors, thereby affecting the lentivirus's ability to infect target cells.

[0081] In some examples, the extracellular amino acid sequence of BaEV-G is shown below.

[0082] SEQ ID No. 19:

[0083] The nucleotide sequence of the lentiviral expression vector corresponding to the amino acid sequence of SEQ ID No. 19 is as follows.

[0084] SEQ ID No. 20:

[0085] In some examples, the gene fragment corresponding to the extracellular region of BaEV-G in the lentiviral expression vector is modified, thereby altering the structure of BaEV-G to enhance the infectivity of BaEV lentivirus 100 on target cells.

[0086] The lentiviral expression vector is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 11 to SEQ ID No. 16.

[0087] SEQ ID No. 11:

[0088] SEQ ID No. 12:

[0089] SEQ ID No. 13:

[0090] SEQ ID No. 14:

[0091] SEQ ID No. 15:

[0092] SEQ ID No. 16:

[0093] For example, after any one of SEQ ID No. 11 to SEQ ID No. 16 is synthesized, its clone is replaced into a lentiviral expression vector by means of restriction endonuclease to obtain a modified lentiviral expression vector, referred to as the first vector.

[0094] Using the lentiviral expression vector in step R2, the first vector is used, which includes the sequence of spacer sequence 10 expressing the extracellular region of BaEV envelope glycoprotein. After the sequence of the first vector is expressed, the modified BaEV-G can be obtained.

[0095] As shown in Figure 2, the amino acid sequence of the extracellular region of the modified BaEV-G includes at least one spacer sequence 10, which includes a spacer unit.

[0096] For example, the spacer units include conserved amino acids.

[0097] For example, the spacer unit includes at least one of SAG, GAS, ASG, AGS, GSA, and SGA.

[0098] In some examples, the number of amino acids included in the spacer sequence 10 ranges from 9 to 27. For example, the number of amino acids included in the spacer sequence 10 may be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27, etc. There is no limit here.

[0099] For example, the interval sequence 10 includes at least one of the following sequences.

[0100] SEQ ID No. 21: SAGSAGSAGSAGSAG; SEQ ID No. 22: GASGASGASGASGAS; SEQ ID No. 23: ASGASGASGASGASG; SEQ ID No. 24: AGSAGSAGSAGSAGSAGS; SEQ ID No. 25: GSAGSAGSAGSAGSAGSA; SEQ ID No. 26: SGASGASGASGASGA.

[0101] For example, the amino acid sequence of the extracellular region of the modified BaEV-G is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 1 to SEQ ID No. 6.

[0102] SEQ ID No. 1:

[0103] SEQ ID No. 2:

[0104] SEQ ID No. 3:

[0105] SEQ ID No.4:

[0106] SEQ ID No. 5:

[0107] SEQ ID No. 6:

[0108] The insertion site of spacer sequence 10 is a site where the amino acid sequence has a very low prediction score in antigenic determinant prediction (antigenic determinant prediction (v1), detaibio.com). After inserting spacer sequence 10, the prediction is re-performed, and the prediction score of this site is higher than zero, thereby generating more antigenic determinants.

[0109] For example, the insertion sites of spacer sequence 10 are positions 117 and 338 of the BaEV envelope glycoprotein amino acid sequence.

[0110] Therefore, the modified BaEV lentivirus 100 increases the number of spacer sequences 10 to improve the recognition range between the recognition region and the target cell surface receptor protein, thereby improving the lentivirus infection efficiency and having a relatively high infectivity for target cells.

[0111] The inventors discovered that BaEV lentivirus 100 transfects target cells through the interaction between the extracellular region of BaEV-G and receptors on the target cell surface. However, for human T cells and NK cells, the expression of the corresponding receptors on their surface is often low, resulting in generally low infection efficiency of BaEV lentivirus 100. Therefore, by leveraging the interaction between antibodies and antigens to enhance the types and strength of interactions between BaEV lentivirus 100 and target cells, the transfection efficiency of BaEV lentivirus 100 can be improved.

[0112] Since the recognition protein can recognize and capture most common proteins, introducing the recognition protein into the lentiviral expression vector can improve the transfection efficiency of BaEV lentivirus 100.

[0113] For example, recognition proteins include: receptor proteins, antibodies, single-chain recognition sequences modified from antibodies, nanobodies, lectins, or molecular chaperone proteins.

[0114] For example, receptor proteins include hormones, neurotransmitters, or growth factors; lectins include concanavalin A; and molecular chaperone proteins include heat shock protein 70 (Hsp70).

[0115] Protein A and protein G are two of the most commonly used antibody molecules.

[0116] For example, the NCBI sequence accession number for protein A is ACH54083.1. Protein A comes from Staphylococcus pseudintermedius ED99, which is a Gram-positive bacterium belonging to the genus Staphylococcus.

[0117] For example, the gene sequence of protein G (GenBank) is CAA37410.1. Protein G comes from strptococcus, which is a bacterium of the genus Streptococcus.

[0118] Therefore, as shown in Figure 2, embodiments of this disclosure introduce protein A or protein G into the lentiviral expression vector backbone. When packaged into BaEV lentivirus 100, recognition motif 20, including protein A and / or protein G, is introduced on the surface of the lentiviral capsid protein. This recognition motif 20 is targeted and can mediate the recognition of the lentivirus on the target cell, thereby improving the targeting of the lentivirus and enhancing the transfection efficiency of BaEV lentivirus 100.

[0119] For example, a lentiviral expression vector that introduces protein A and / or protein G into BaEV lentivirus 100 may be represented as a second vector. The second vector includes a sequence expressing a recognition sequence of a BaEV envelope glycoprotein.

[0120] In some examples, BaEV lentivirus 100, after introducing protein A and / or protein G onto the surface of the lentiviral capsid protein, includes a recognition sequence, which is recognition motif 20; the recognition sequence includes: BaEVSecSig-recognition protein-BaEVTM. BaEVSecSig is the BaEV signal peptide, and BaEVTM is the BaEV transmembrane domain.

[0121] Signal peptides are short peptide chains (5 to 30 amino acids in length) that guide the transfer of newly synthesized proteins into the secretory pathway.

[0122] For example, the recognition protein is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of either SEQ ID No. 7 or SEQ ID No. 8.

[0123] SEQ ID No. 7:

[0124] SEQ ID No. 8:

[0125] The sequence of BaEVSecSig is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of SEQ ID No. 9, SEQ ID No. 9: MGFTTKIIFLYNLVLVYA. The sequence of BaEVTM is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of SEQ ID No. 10, SEQ ID No. 10: YLLPFLGPLLTLLLLLTIGPCIF.

[0126] BaEV lentivirus 100 includes a recognition sequence, and a recognition motif 20 of protein A or protein G is introduced on the surface of the lentiviral capsid protein. This recognition motif 20 is targeted and mediates the recognition of the lentivirus on the target cell, thereby improving the targeting of the lentivirus and thus enhancing the transfection efficiency of BaEV lentivirus 100.

[0127] The nucleotide sequence of protein A is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of SEQ ID No. 27.

[0128] SEQ ID No. 27:

[0129] The nucleotide sequence of protein G is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of SEQ ID No. 28.

[0130] SEQ ID No. 28:

[0131] Based on the nucleotide sequences of proteins A and G mentioned above, the nucleotide sequence of the second vector includes either the nucleotide sequence of protein A or protein G.

[0132] For example, the second carrier comprises at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% derived from either SEQ ID No. 17 or SEQ ID No. 18.

[0133] SEQ ID No. 17:

[0134] SEQ ID No. 18:

[0135] As shown in Figure 2, an embodiment of this disclosure also provides a lentivirus, which is BaEV lentivirus 100. The extracellular region of the envelope glycoprotein of BaEV lentivirus 100 includes at least one of the following sequences: spacer sequence 10 and recognition sequence.

[0136] For example, the extracellular region of the envelope glycoprotein of BaEV lentivirus 100 may include an amino acid sequence and a nucleic acid sequence.

[0137] For example, the spacer sequence 10 includes: a spacer unit; the recognition sequence includes: BaEVSecSig-recognition protein-BaEVTM; wherein, BaEVSecSig is a BaEV signal peptide and BaEVTM is a BaEV transmembrane domain.

[0138] For example, the extracellular region of the envelope glycoprotein of BaEV lentivirus 100 includes the amino acid sequence expressed by the first vector, that is, the extracellular region of BaEV-G has at least one spacer sequence 10, the description of which is as described above and will not be repeated here.

[0139] For example, the amino acid sequence of the extracellular region of BaEV-G is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 1 to SEQ ID No. 6. The descriptions of SEQ ID No. 1 to SEQ ID No. 6 are as described above and will not be repeated here.

[0140] By using at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the envelope glycoprotein of BaEV lentivirus 100 derived from any one of SEQ ID No. 1 to SEQ ID No. 6, the recognition range between the recognition region and the target cell surface receptor protein can be increased, thereby improving the lentivirus infection efficiency and exhibiting a relatively high infectivity to target cells.

[0141] For example, the extracellular region of the envelope glycoprotein of BaEV lentivirus 100 includes the amino acid sequence expressed by the second vector. This sequence is represented as: BaEVSecSig-recognition protein-BaEVTM. For an introduction to BaEVSecSig-recognition protein-BaEVTM, please refer to the above content, and it will not be repeated here.

[0142] By including the amino acid sequence expressed by the second vector in the extracellular region of the envelope glycoprotein of BaEV lentivirus 100, the targeting of lentivirus can be improved, thereby enhancing the transfection efficiency of BaEV lentivirus 100.

[0143] The embodiments of this disclosure also provide the application of lentivirus as described in any of the foregoing embodiments in the transfection of human primary NK cells.

[0144] The following section further optimizes the conditions for the preparation method of lentiviruses.

[0145] In some examples, the lentiviral expression vector in step R2 includes a first vector and a second vector, wherein the mass ratio of the first vector to the second vector ranges from 5 to 10.

[0146] For example, the ratio of the mass of the first carrier to the mass of the second carrier can be 5, 6, 7, 8, 9 or 10, etc., and there is no limit here.

[0147] Setting the ratio of the mass of the first vector to the mass of the second vector to be within the range of 5 to 10 can improve the transfection capability of BaEV lentivirus 100. For details of the experimental data, please refer to the following content, which will not be described in detail here.

[0148] In some examples, in step R2, the mass ratio of the vector plasmid, packaging plasmid, and lentiviral expression vector in the first solution is (3-5):3:(1-3).

[0149] The mass fraction of the lentiviral expression vector can be the sum of the mass of the first vector and the mass of the second vector. Alternatively, the lentiviral expression vector is an unmodified BaEV plasmid derived from the nucleotide sequence of SEQ ID No. 20.

[0150] For example, the mass ratio of the vector plasmid, packaging plasmid, and lentiviral expression vector can be 3:3:1, 3:3:1.5, 3:3:2, 3:3:2, 3.5:3:1, 3.5:3:1.5, 3.5:3:2, 3.5:3:3, 4:3:1, 4:3:1.5, 4:3:2, 5:3:1, 5:3:2, or 5:3:3, etc., and there are no limitations here.

[0151] By setting the mass ratio of vector plasmid, packaging plasmid, and lentiviral expression vector to (3-4):3:(1-2), the yield of BaEV lentivirus 100 can be increased. Specific experimental data will be provided later and will not be elaborated here.

[0152] In some embodiments, before step R1 involves seeding the packaged cells into the culture container for culture, the process further includes: modifying the culture container 40 with a condensate microgel 30.

[0153] Microgels are colloidal dispersion systems of gel particles at the micrometer or submicrometer scale, possessing intramolecular cross-linked structures. Agglomerate microgels 30 are special microgel systems formed during preparation or application by causing microgel particles to aggregate or condense through some means (physical or chemical action).

[0154] By modifying the culture container 40 with the aggregate microgel 30, the adhesion of basal cells to the culture substrate can be enhanced, preventing them from detaching during toxin production. The microgel encapsulates culture factors, enabling their slow release through external stimulation, thereby increasing the activity and toxin production time of basal cells.

[0155] For example, the polymer microgel includes folic acid and a polycationic electrolyte, such as protamine sulfate and polylysine.

[0156] In some examples, the modification of the culture vessel 40 with the aggregate microgel 30 includes the following steps: mixing a folic acid solution with a polycationic electrolyte solution to obtain a third solution; applying the third solution to the culture vessel 40; adding a phosphate solution to initiate gelation, thereby obtaining the culture vessel 40 modified with the aggregate microgel 30.

[0157] For example, a folic acid solution with a concentration ranging from 11 mmol / mL to 45 mmol / mL is mixed with a polycationic electrolyte solution with a concentration ranging from 15 mmol / mL to 88 mmol / mL to obtain a third solution with a pH range of 8.5 to 10.5; wherein the molar ratio of folic acid to polycationic electrolyte is in the range of 1:1.5 to 1:6. The third solution is coated onto a culture vessel 40, and a phosphate solution with a pH range of 6.5 to 8.0 is added to initiate gelation, resulting in a culture vessel 40 modified with aggregate microgel 30; wherein the volume ratio of phosphate solution to folic acid solution is in the range of 1:5 to 1:20.

[0158] For example, the concentration of folic acid solution can be 11 mmol / mL, 15 mmol / mL, 18 mmol / mL, 20 mmol / mL, 24 mmol / mL, 28 mmol / mL, 32 mmol / mL, 36 mmol / mL or 45 mmol / mL, etc., and there is no limit here.

[0159] For example, the concentration of the polycationic electrolyte solution is 15 mmol / mL, 20 mmol / mL, 30 mmol / mL, 35 mmol / mL, 40 mmol / mL, 55 mmol / mL, 60 mmol / mL, 80 mmol / mL or 88 mmol / mL, etc., and there is no limit here.

[0160] For example, the pH of the third solution may be 8.5, 9, 9.5 or 10, etc., and there is no limitation here.

[0161] For example, the ratio of the number of moles of folic acid to the number of moles of polycationic electrolyte is 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5 or 1:6, etc., and there is no limitation here.

[0162] For example, the phosphate solution is 20 mmol / L sodium dihydrogen phosphate, and the pH of the phosphate solution is 6.5, 7, 7.5 or 8, etc., without limitation.

[0163] For example, the volume ratio of phosphate solution to folic acid solution can be 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:15, 1:16, 1:18, 1:19 or 1:20, etc., and there is no limit here.

[0164] In the culture vessel 40 modified with the coagulated microgel 30, there are cations + and anions - formed by polyelectrolytes, and cations + are more abundant than anions -. Since basal cells contain more negative charges, the cations + can adsorb basal cells onto the culture medium substrate, enhancing the adhesion of basal cells to the substrate and preventing them from detaching during toxin production.

[0165] Therefore, the above embodiments modify the 293T cell growth substrate to improve its adhesion, thereby effectively preventing the BaEV lentivirus 100 produced from inducing the formation of syncytia, reducing the probability of BaEV lentivirus 100 shedding and death, and increasing the yield of BaEV lentivirus 100.

[0166] Based on the above-described method for preparing lentiviruses, the following examples are provided.

[0167] Example 1

[0168] 1. Virus packaging.

[0169] (1) 20-24 hours before transfection, 3×10 5 Cells (cells) / 20mL (DMEM medium containing 10% FBS): 293T cells were seeded into T175 cell culture flasks (or 15cm culture dishes, without any modification or treatment).

[0170] (2) When the 293T cells reach 70%–90% confluence during transfection, replace the medium with serum-free DMEM (0.6 x 10⁻⁶). 5 cells / cm 2 In 15 mL of medium, there are 0.6 x 10 per square centimeter. 5 The cells were cultured in a dish containing 15 ml of culture medium.

[0171] (3) Take a 15mL centrifuge tube and prepare the solution according to the scheme in the table below. Gently invert the tube to mix.

[0172] pCMV-ΔR8.91 is the packaging plasmid.

[0173] Refer to the table below for specific preparation methods.

[0174] (4) Add the liquid (plasmid solution) in tube 1 drop by drop (or slowly add by inserting the pipette tip into the liquid and rotating) to tube 2 (Lipofectamine mixture), gently invert and mix for 10 to 15 seconds, and let stand at room temperature for 5 minutes.

[0175] (5) Add the reagent obtained in (4) dropwise to the cell culture dish and gently shake to cover all cells. After culturing in a cell culture incubator for 4 hours, replace the culture medium with preheated DMEM complete medium containing 10% FBS and collect the supernatant (approximately 20 mL) from 48 to 72 hours after transfection.

[0176] (6) After collecting about 20 mL of supernatant, remove the cell precipitate by ultracentrifugation or PEG precipitation, collect the supernatant, and then filter it with a 0.45 μm filter to obtain the virus.

[0177] 2. Virus concentration.

[0178] (1) Slowly add 5 mL of 5XPEG-3000NaCl stock solution to every 20 mL of filtered virus solution and place at 4°C.

[0179] PEG-3000 refers to polyethylene glycol with a molecular weight of 3000. 5XPEG-3000NaCl stock solution refers to a solution formed by dissolving 8.766g of NaCl and 50g of PEG-3000 in 200mL of pure water.

[0180] (2) Mix every 20 to 30 minutes, repeat 3 to 5 times; place at 4°C overnight.

[0181] (3) At 4℃, centrifuge at 4000 x g for 20 min to make the lentivirus particles gather at the bottom of the centrifuge tube, and obvious precipitation can be seen.

[0182] (4) Discard the supernatant and centrifuge again at 4000 x g for 5 min (or let the tube stand for 1 min to 2 min) at 4℃ to remove the residual liquid.

[0183] (5) Add an appropriate amount of HBSS / PBS / sterile double-distilled water to each tube. In this case, add 200μL of PBS to each tube corresponding to 25mL of virus stock solution to dissolve the lentivirus precipitate and store at 4℃ for 4 hours.

[0184] Among them, HBSS (Hank's Balanced Salt Solution) is Hank's balanced salt solution, which is generally used to prepare culture media or wash cells during cell culture.

[0185] (6) Collect the precipitated virus particles, aliquot them into 10μL to 50μL tubes, store them at -80℃, and use them as needed.

[0186] 3. Virus titration.

[0187] (1) 20-24 hours before transfection, 3×10 4Spread 100 μL of cells per well (DMEM medium containing 10% FBS) into a 96-well cell culture vessel.

[0188] (2) Perform 8 viral gradients: 5-fold dilution, with an initial viral load of 3 μL / well.

[0189] ①The PEG-concentrated virus stock solution was serially diluted 5-fold using DMEM medium containing 2% FBS and 2 μg / mL polybrene according to the limiting dilution method. The dilutions were repeated for 8 consecutive times, and the culture was carried out at 37°C and 5% CO2.

[0190] Polybrene, also known as hexadimethrine bromide, is a cationic polymer.

[0191] ② The dilution method is as follows: Prepare eight 1.5 mL centrifuge tubes, and add 40 μL of complete culture medium to each tube; add 10 μL of virus stock solution to the first tube, mix well, and then add 10 μL to the second tube and mix well. Continue in this manner. Then mix the diluted virus solution (15 μL / well) with the cells (50 μL / well) and add the mixture to the corresponding wells of the plate. Centrifuge at 5000 rpm for 3 hours.

[0192] ③ After culturing at 37℃ for 6-8 hours, add 200 μL of DMEM medium containing 10% FBS to promote cell growth, and incubate overnight at 37℃ and 5% CO2.

[0193] (3) Wash once with RPMI 1640 medium, resuspend in 200 μL of medium, and incubate in 96-well plates for 2-3 days.

[0194] RPMI is an abbreviation for Roswell Park Memorial Institute, referring to the Roswell Park Memorial Institute. RPMI is a type of cell culture medium developed by the institute, and 1640 is the code for the culture medium.

[0195] (4) The proportion of GFP was detected by flow cytometry, and the viral titer was calculated from samples with a proportion of 10% to 30%.

[0196] GFP stands for Green Fluorescent Protein.

[0197] The formula for calculating viral titer is: Viral titer = Cell number × Fluorescence percentage × 10 3 / volume of original virus solution (μL).

[0198] Example 2

[0199] Based on Example 1, the plasmid mixing ratio in step (3) of the virus packaging was adjusted as shown in the table below.

[0200] Refer to the table below for specific preparation methods.

[0201] In Example 1, the mass ratio of the vector plasmid, the packaging plasmid, and the BaEV plasmid was 1:1:1. In Example 2, the mass ratio of the vector plasmid, the packaging plasmid, and the BaEV plasmid was 4:3:2.

[0202] Example 3

[0203] Based on Example 1, the plasmid mixing ratio in step (3) of the virus packaging was adjusted as shown in the table below.

[0204] Refer to the table below for specific preparation methods.

[0205] In Example 3, the mass ratio of the vector plasmid, the packaging plasmid, and the BaEV plasmid was 3:3:1.

[0206] Example 4: Culture container 40 modified with aggregate microgel 30.

[0207] (1) Preparation of condensate microdroplets.

[0208] Prot / FA condensate microdroplets were prepared by mixing 20 μL of folic acid solution (FA, 11.08 mg / ml, 25 mM, pH = 10.0) and 100 μL of protamine sulfate solution (Prot, 4.25 mg / ml, average amino acid molecular weight 170 Da, monomer concentration 25 mM, pH = 10.0).

[0209] (2) Culture container 40 substrate modification.

[0210] The above-mentioned coagulated microdroplet solution was spread on T175 cell culture flasks (or 15cm culture dishes), and then 2μL of phosphate solution (20mM sodium dihydrogen phosphate, pH=7.0) was added to initiate gelation; then it was incubated at 37°C for 1 hour.

[0211] (3) The plasmid mixing ratio is as shown in Example 2, and the other packaging preparation operations and steps remain unchanged.

[0212] Example 5: Modification of the extracellular region of BaEV-G.

[0213] (1) The original amino acid sequence of the extracellular region of the BaEV envelope glycoprotein is derived from SEQ ID No. 19.

[0214] (2) The nucleotide sequence corresponding to the original amino acid sequence of the extracellular region of BaEV envelope glycoprotein is derived from SEQ ID No. 20.

[0215] (3) The amino acid sequence of the modified extracellular region of BaEV envelope glycoprotein is derived from SEQ ID No. 1.

[0216] (4) The nucleotide sequence corresponding to the modified amino acid sequence of the extracellular region of BaEV envelope glycoprotein is derived from SEQ ID No. 11.

[0217] After synthesis of SEQ ID No. 11, it was cloned into the extracellular region of the BaEV envelope glycoprotein using restriction endonuclease to replace it; the plasmid mixing ratio was as shown in Example 2, and the remaining packaging preparation operations and steps remained unchanged.

[0218] Example 6: Add identification element 20.

[0219] (1) Plasmid construction.

[0220] Synthesize the following gene sequences: BaEVSecSig-protein A-BaEVTM and / or BaEVSecSig-protein G-BaEVTM.

[0221] BaEVSecSig refers to the BaEV signal peptide; BaEVTM refers to the BaEV transmembrane domain.

[0222] The specific sequence is as follows:

[0223] BaEVSecSig:MGFTTKIIFLYNLVLVYA.

[0224] BaEVTM:YLLPFLGPLLTLLLLLTIGPCIF.

[0225] The nucleotide sequence corresponding to the gene is derived from SEQ ID No. 17.

[0226] SEQ ID No. 17 was inserted into the first vector to replace the SAG region of the BaEV plasmid, resulting in the second vector.

[0227] (2) The first carrier and the second carrier are mixed at a mass ratio of 9:1 and then added as a coating plasmid to form the first solution.

[0228] (3) The plasmid mixing ratio is as shown in Example 2, and the other packaging preparation operations and steps remain unchanged.

[0229] Example 7

[0230] The lentiviruses prepared in Examples 5 and 6 were used to transfect human primary NK cells to test their transfection efficiency.

[0231] The specific operating steps are as follows:

[0232] (1) After NK cells were revived and cultured for 48 hours, the lentivirus was thawed on ice and mixed by pipetting.

[0233] (2) Calculate the volume of lentivirus solution required for transfection: number of cells exposed to lentivirus min =700 / 0.1×300=2×10 6 MOI = 10, where MOI is an abbreviation for multiplicity of infection, indicating the plural of infection.

[0234] (3) Resuscitate Vectofusin-1 (infectious virus enhancer) at room temperature and vortex mix.

[0235] (4) The final concentration of Vectofusin-1 in the final culture medium was 2.5 μg / mL.

[0236] (5) Dilute the lentivirus solution and Vectofusin-1 separately with culture medium, and ensure that their final volumes are the same.

[0237] (6) Mix the two solutions and blow them together.

[0238] (7) Add the above mixture (<10 min) to the cell suspension and mix by pipetting.

[0239] (8) Centrifuge at 32℃ and 400 x g for 2 hours.

[0240] (9) Culture at 37℃ with 5% to 10% CO2, and wash with culture medium every 24 hours after transfection.

[0241] Monitor the culture progress daily and divide the culture into smaller flasks as needed. On day 4 after transfection, remove the supernatant as much as possible without touching the cells. Replenish the culture medium to the original volume with pre-warmed medium containing 5% AB, IL-2, and IL-15. Replace the medium every 2-3 days, or when cell confluence is nearly 80% and the medium is depleted. Culture for 12 days. Note that IL-2 and IL-15 are cytokines, and 5% AB refers to 5% human AB serum. The percentage of GFP-positive cells was then detected by flow cytometry.

[0242] The following describes the viral titers in the viral stock solutions obtained by the lentivirus preparation methods provided in Examples 1 to 6. Here, titer is the reciprocal of dilution. Viral titer is the concentration of the viral suspension, also known as viral potency. The unit is transfection units / mL, i.e., TU / mL.

[0243] The calculation formula used is: Viral titer = Cell number × Fluorescence percentage × 10 3 / The viral stock solution volume (μL) is used to calculate the titer of the lentiviruses prepared in Examples 1 to 7. The cell number is the cell base used in the examples of this disclosure, with a value of 3 x 10. 5 The volume of the original virus solution is 100 μL.

[0244] Figure 3 shows the percentage of GFP-positive cells. The horizontal axis represents fluorescence intensity in au (arbitrary units), and the vertical axis represents the number of cells. The blue area represents the blank control data, and the pink area represents the fluorescence intensity data of NK cells transfected with the lentivirus prepared in Example 1. As can be seen from Figure 3, the fluorescence percentage of NK cells transfected with the lentivirus prepared in Example 1 is 2.3%. Therefore, according to the formula: Virus titer = Number of cells × Fluorescence percentage × 10... 3 Based on the volume of the original viral fluid, the viral titer is calculated to be 3 x 10. 5 x2.3% x10 3 / 100 = 6.9 x 10 4 TU / mL, which is the viral titer of the lentivirus prepared in Example 1, is 6.9 x 10. 4 TU / mL.

[0245] Figure 4 shows another percentage of GFP-positive cells. The blue area represents the blank control data, and the pink area represents the fluorescence intensity data of NK cells transfected with the lentivirus prepared in Example 2. As can be seen from Figure 4, the fluorescence percentage of NK cells transfected with the lentivirus prepared in Example 2 is 3.4%. Therefore, according to the formula: Virus titer = Cell number × Fluorescence percentage × 10... 3 Based on the volume of the original viral fluid, the viral titer is calculated to be 3 x 10. 5 x3.4% x10 3 / 100 = 1.02 x 10 5 TU / mL, which is the viral titer of the lentivirus prepared in Example 2, is 1.02 x 10⁻¹⁰. 5 TU / mL. Compared to Example 1, Example 2, by setting the mass fractions of the vector plasmid to 4 parts, the mass fraction of the packaging plasmid to 3 parts, and the mass fraction of the BaEV plasmid to 2 parts, can increase the yield of BaEV lentivirus 100 by approximately 2 times.

[0246] Figure 5 shows another percentage of GFP-positive cells. The blue area represents the blank control data, and the pink area represents the fluorescence intensity data of NK cells transfected with the lentivirus prepared in Example 3. As can be seen from Figure 5, the fluorescence percentage of NK cells transfected with the lentivirus prepared in Example 3 is 4.1%. Therefore, according to the formula: Virus titer = Cell number × Fluorescence percentage × 10...3 Based on the volume of the original viral fluid, the viral titer is calculated to be 3 x 10. 5 x4.1% x10 3 / 100 = 1.23 x 10 5 TU / mL, which is the viral titer of the lentivirus prepared in Example 3, is 1.23 x 10⁻¹⁰. 5 TU / mL. Compared to Example 1, Example 3, by setting the mass fractions of the vector plasmid to 3 parts, the mass fraction of the packaging plasmid to 3 parts, and the mass fraction of the BaEV plasmid to 1 part, can increase the yield of BaEV lentivirus 100 by more than 2 times.

[0247] Figure 6 shows another percentage of GFP-positive cells. The blue area represents the blank control data, and the pink area represents the fluorescence intensity data of NK cells transfected with the lentivirus prepared in Example 4. As can be seen from Figure 6, the fluorescence percentage of NK cells transfected with the lentivirus prepared in Example 4 is 58.2%. Therefore, according to the formula: Virus titer = Cell number × Fluorescence percentage × 10... 3 Based on the volume of the original viral fluid, the viral titer is calculated to be 3 x 10. 5 x58.2% x10 3 / 100 = 1.7 x 10 6 TU / mL, which means the viral titer of the lentivirus prepared in Example 4 is 1.7 x 10⁻¹⁰. 6 TU / mL. Compared to Example 1, Example 4, by modifying the culture vessel 40 with aggregate microgel 30, can increase the yield of BaEV lentivirus 100 by one order of magnitude.

[0248] Therefore, as can be seen from Figures 7 to 9, adjusting the mass mixing ratio of the vector plasmid, packaging plasmid, and BaEV plasmid can improve the lentivirus packaging effect and increase the yield of lentivirus.

[0249] Figure 7 shows the antibody-cell labeling flow cytometry fluorescence image of Example 1. Figure 8 shows the antibody-cell labeling flow cytometry fluorescence image of Example 5, and Figure 9 shows the antibody-cell labeling flow cytometry fluorescence image of Example 6. The horizontal axis, CD56-APC, represents the relative intensity of the CD56 labeling signal. Antibody CD56 is a glycoprotein distributed on the cell surface, commonly found on the surface of human nerve cells and astrocytes, and also present in small amounts on the surface of lymphocytes. Antibody CD56 is a specific marker for NK cells. The vertical axis, CAR-GFP, represents the relative intensity of the expressed protein signal. The antibody-cell labeling flow cytometry fluorescence image is divided into four regions. The upper right region is where both antibody CD56 and CAR are expressed; the higher the value in this region, the stronger the lentivirus's infectivity on primary NK cells.

[0250] The value in the upper right region of Figure 7 is 18.7%, the value in the upper right region of Figure 8 is 24.1%, and the value in the upper right region of Figure 9 is 31.1%. It can be seen that, compared with Example 1, the lentivirus has enhanced infection ability on primary NK cells after BaEV-G extracellular region modification in Example 5; after BaEV-G extracellular region modification in Example 6, recognition motif 20 is further added to the surface of the lentivirus capsid protein, which further enhances the infection ability of lentivirus on primary NK cells.

[0251] Therefore, as shown in Figures 7-9, the modification of the BaEV-G extracellular region increased the recognition range between the recognition region and the target cell surface receptor protein, thereby improving the lentiviral infection efficiency and giving the lentiviral virus a relatively high infectivity to target cells. Introducing a targeting recognition motif 20 onto the surface of the lentiviral capsid protein mediates the lentiviral recognition of target cells, thereby improving the lentiviral targeting and further enhancing its infectivity to target cells.

[0252] The above description is merely a specific embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any variations or substitutions conceived by those skilled in the art within the scope of the technology disclosed in this disclosure should be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.

Claims

1. A lentivirus comprising: BaEV membrane glycoprotein; The extracellular region of the BaEV envelope glycoprotein includes at least one of the following sequences: Interval sequence, including: interval units; The recognition sequence includes: BaEVSecSig-recognition protein-BaEVTM; wherein, BaEVSecSig is a BaEV signal peptide, and BaEVTM is a BaEV transmembrane domain.

2. The lentivirus according to claim 1, wherein, The spacer unit comprises conserved amino acids.

3. The lentivirus of claim 2, wherein, The spacer unit includes at least one of SAG, GAS, ASG, AGS, GSA, and SGA.

4. The lentivirus of claim 3, wherein, The number of amino acids included in the spacer sequence ranges from 9 to 27.

5. The lentivirus of claim 4, wherein, The interval sequence includes at least one of: SAGSAGSAGSAGSAG, GASGASGASGASGAS, ASGASGASGASGASG, AGSAGSAGSAGSAGSAGS, GSAGSAGSAGSAGSAGSA, and SGASGASGASGASGASGA.

6. The lentivirus according to any one of claims 1 to 5, wherein, The insertion sites of the spacer sequence are positions 117 and 338 of the amino acid sequence of the BaEV envelope glycoprotein.

7. The lentivirus according to any one of claims 1 to 6, wherein, The BaEV envelope glycoprotein containing the spacer sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 1 to SEQ ID No.

6.

8. The lentivirus according to any one of claims 1 to 7, wherein, The recognition proteins include: receptor proteins, antibodies, single-chain recognition sequences modified from antibodies, nanobodies, lectins, or molecular chaperone proteins.

9. The lentivirus according to any one of claims 1 to 8, wherein, The recognition proteins include protein A or protein G.

10. The lentivirus according to any one of claims 1 to 9, wherein, The recognition protein is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of either SEQ ID No. 7 or SEQ ID No.

8.

11. The lentivirus according to any one of claims 1 to 10, wherein, The sequence of the BaEVSecSig is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of SEQ ID No. 9; and / or, the sequence of the BaEVTM is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of SEQ ID No.

10.

12. A lentiviral expression vector comprising: Expresses the nucleotide sequence of the lentivirus as described in any one of claims 1 to 11.

13. The lentiviral expression vector of claim 12, wherein, The nucleotide sequence expressing the spacer sequence of the BaEV envelope glycoprotein is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 11 to SEQ ID No.

16.

14. The lentiviral expression vector of claim 12 or 13, wherein, The nucleotide sequence expressing the recognition sequence of the BaEV envelope glycoprotein is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of either SEQ ID No. 17 or SEQ ID No.

18.

15. A method for preparing a lentivirus, comprising: The packaging cells were seeded into culture containers and cultured. The vector plasmid, packaging plasmid, and lentiviral expression vector were added to the cell culture medium to obtain the first solution; The transfection reagent was added to the cell culture medium and mixed to obtain the second solution; When the confluence of the packaged cells reaches the preset value, the first solution and the second solution are mixed and added to the culture container for transfection; After transfection, the lentivirus was obtained; The lentivirus includes: BaEV envelope glycoprotein; the extracellular region of the BaEV envelope glycoprotein includes at least one of the following sequences: a spacer sequence and a recognition sequence; the spacer sequence includes: a spacer unit; the recognition sequence includes: BaEVSecSig-recognition protein-BaEVTM; BaEVSecSig is a BaEV signal peptide, and BaEVTM is a BaEV transmembrane domain.

16. The method of preparing a lentivirus according to claim 15, wherein, The lentiviral expression vector comprises at least one of the amino acid sequences of a spacer sequence expressing BaEV envelope glycoprotein and an amino acid sequence of a recognition sequence expressing BaEV envelope glycoprotein.

17. The method of producing a lentivirus according to claim 16, wherein, Lentiviral expression vectors expressing the spacer sequence of BaEV envelope glycoproteins are designated as the first vector; lentiviral vectors expressing the recognition sequence of BaEV envelope glycoproteins are designated as the second vector. In the first solution, the lentiviral expression vector comprises: the first vector and the second vector, wherein the mass ratio of the first vector to the second vector is in the range of 5 to 10.

18. The method of producing a lentivirus according to any one of claims 15 to 17, wherein, In the first solution, the mass ratio of the vector plasmid, the packaging plasmid, and the lentiviral expression vector is (3-5):3:(1-3).

19. The method of producing a lentivirus according to any one of claims 15 to 18, wherein, Before the packaged cells are seeded into the culture container for culture, the method further includes: modifying the culture container with a condensate microgel.

20. The method of producing a lentivirus according to claim 19, wherein, The condensate microgel comprises folic acid and polycationic electrolytes.

21. The method of producing a lentivirus according to claim 19 or 20, wherein, The polycationic electrolyte includes at least one of protamine sulfate and polylysine.

22. The method for preparing a lentivirus according to any one of claims 19 to 21, wherein, The microgel modification of the culture vessel includes: The folic acid solution was mixed with the polycationic electrolyte solution to obtain the third solution; The third solution is coated onto the culture container, and a phosphate solution is added to initiate gelation, resulting in a culture container modified with aggregate microgels.

23. The use of a lentivirus as described in any one of claims 1 to 11 in the transfection of human primary NK cells.

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