Modified host cell as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a modified host cell as well as a preparation method and application thereof. All SLC1A5 homologous isomers of the transformed host cell are knocked out, and the transformed host cell is SLC1A4 negative. When the modified host cell provided by the invention is used as a packaging cell, the yield of the pseudotyped virus can be effectively increased.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a modified host cell and a preparation method and application thereof. Background Art

[0002] Virus pseudotyping refers to the modification of integrating envelope glycoproteins from other viruses into a target virus particle, so that the target virus particle retains its own genome while also having envelope proteins and related functions of other viruses. Through the pseudotyping of viruses, it is possible to adjust the cell types that virus particles can infect / transduce, improve the stability of virus particles, and change the immunogenicity of virus particles.

[0003] In the field of biomedicine, lentivirus and γ-retrovirus are usually pseudotyped with the envelope glycoprotein (VSV-G) derived from vesicular stomatitis virus (VSV) and are widely used important research tools. Generally speaking, pseudotyped lentivirus and γ-retrovirus are produced using specific packaging cell lines. By co-transfecting auxiliary plasmids (expressing viral structural proteins such as Gag, Pol and envelope proteins) and vector plasmids (carrying viral packaging element sequences and target genes), the process of viral genome transcription, viral particle assembly and budding can be initiated in packaging cells, so that viral particles carrying the target gene can be harvested in the culture supernatant.

[0004] Since the receptor of VSV-G is the low-density lipoprotein receptor (LDL-R) expressed on most cell membranes, VSV-G pseudotyped lentivirus and γ-retrovirus can transduce almost all types of mammalian cells, including dividing cells, non-dividing cells, primary cells, stable cell lines, stem cells, differentiated cells, adherent cells and suspended cells. In the field of immune cell therapy, transduction with high-titer VSV-G pseudotyped lentivirus or γ-retrovirus can efficiently deliver the target gene to immune-related cells (such as T cells), and stably integrate and express it.

[0005] However, the transduction efficiency of VSV-G pseudotyped viruses for some immune-related cells (such as hematopoietic stem cells, NK cells, B cells, etc.) is low. Some pseudotyped viruses can efficiently transduce the above immune-related cells, such as pseudotyped viruses related to the envelope glycoprotein GP70 of baboon endogenous retrovirus (Baboon endogenous virus, BaEV) (referred to as BaEV-GP70) and envelope glycoprotein GP70 of feline endogenous retrovirus RD114 (referred to as RD114-GP70). Therefore, optimizing the production process of such pseudotyped viruses and increasing their yield will help to make up for the shortcomings of VSV-G pseudotyped viruses in production and clinical applications. Summary of the invention

[0006] Based on this, one or more embodiments of the present application provide a genetically modified cell and a preparation method and application thereof, including the following technical solutions:

[0007] One or more embodiments of the present application provide a modified host cell, wherein relative to a wild-type host cell, none of the SLC1A5 isoforms in the modified host cell are expressed or are expressed at a low level.

[0008] In some embodiments of the present application, the SLC1A5 gene in the modified host cell is gene-edited.

[0009] In some embodiments of the present application, the common coding nucleic acid region of the SLC1A5 isoforms is mutated.

[0010] In some embodiments of the present application, the mutation includes one or a combination of two or more of an insertion mutation, a deletion mutation and a substitution mutation.

[0011] In some embodiments of the present application, the modified host cell is derived from any one of HEK293 cells and cell lines derived therefrom.

[0012] In some embodiments of the present application, the HEK293 cells and cell lines derived therefrom include HEK293, HEK293T, HEK293F and HEK293S.

[0013] Optionally, the nucleotide sequence of the common coding nucleic acid region of the SLC1A5 gene is shown in SEQ ID NO.9.

[0014] In some embodiments of the present application, the nucleotide sequence of the target editing region in the SLC1A5 gene common coding nucleic acid region is shown in SEQ ID NO.8.

[0015] Optionally, the target editing region is edited into a nucleic acid fragment having a nucleotide sequence as shown in SEQ ID NO.5 and SEQ ID NO.6, or is edited into a nucleic acid fragment having a nucleotide sequence as shown in SEQ ID NO.7.

[0016] In some embodiments of the present application, the modified host cells include adherent cells.

[0017] One or more embodiments of the present application provide a method for preparing the modified host cell, which comprises using gene editing technology to perform gene editing on the host cell to be modified so that it has the nucleic acid fragment.

[0018] In some embodiments of the present application, the gene editing technology includes CRISPR / Cas9 technology.

[0019] In some embodiments of the present application, the binding site of the sgRNA used in the preparation method is located in the common coding nucleic acid region of the SLC1A5 gene.

[0020] In some embodiments of the present application, the nucleotide sequence of the sgRNA is shown as SEQ ID NO.1 and SEQ ID NO.2.

[0021] One or more embodiments of the present application provide a method for producing a virus, wherein the method uses the modified host cell as a packaging cell to produce the virus;

[0022] The virus may or may not include a GOI in its genome.

[0023] In some embodiments of the present application, the virus includes one or more of a lentivirus and a retrovirus.

[0024] In some embodiments of the present application, the virus comprises a pseudotyped virus.

[0025] In some embodiments of the present application, the pseudotyped virus carries one or more of baboon endogenous retrovirus envelope glycoprotein GP70, cat endogenous retrovirus RD114 envelope glycoprotein GP70 and other proteins that bind to SLC1A5.

[0026] In some embodiments of the present application, the GOI includes one or more of a selectable marker gene and a target gene;

[0027] Optionally, the selection marker gene includes one or more of a luciferase gene, a fluorescent protein gene, a puromycin resistance gene, a blasticidin resistance gene, a geneticin resistance gene, a bleomycin resistance gene, a neomycin resistance gene, a hygromycin phosphotransferase gene, a dihydrofolate reductase gene, a thymidine kinase gene, a glutamine synthetase gene, an asparagine synthetase gene, a tryptophan synthetase gene, a histidinol dehydrogenase gene, an aminoglycoside phosphotransferase gene, and a tryptophan synthetase gene;

[0028] Optionally, the target gene encodes one or more of antibodies, recombinant proteins, polypeptides, enzymes, hormones, growth factors, receptors and non-coding RNAs.

[0029] One or more embodiments of the present application provide a method for producing modified immune cells, the production method comprising the following steps:

[0030] Producing the virus using the production method; and,

[0031] The virus is used to infect immune cells to be transformed to prepare transformed immune cells.

[0032] In some embodiments of the present application, the modified immune cells include one or more of T cells, TCR-T, CAR-T, TIL, DC-CIK and NK cells.

[0033] One or more embodiments of the present application provide a method for producing a drug, the method comprising the following steps:

[0034] Producing the modified immune cells using the production method; and,

[0035] The modified immune cells are prepared into medicines.

[0036] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The SLC1A5 gene, the coding regions of each SLC1A5 isoform, and the binding sites of sgRNA2 and sgRNA3;

[0038] Figure 2 For the detection of SLC1A5 expression in wild-type and SLC1A5 knockout 293T cells;

[0039] Figure 3Detection of GFP expression in wild-type and SLC1A5 knockout 293T cells after infection with reporter retrovirus pseudotyped with BaEV envelope protein;

[0040] Figure 4 The expression of GFP and SLC1A4 was detected in each SLC1A5 knockout 293T monoclonal cell after infection with reporter retrovirus pseudotyped with BaEV envelope protein;

[0041] Figure 5 For SLC1A5 genotype analysis of each 293T cell line;

[0042] Figure 6 The status and virus yield of each 293T cell line during retrovirus packaging;

[0043] Figure 7 The expression of the target gene mCherry in T cells after the viruses produced by 293T cells were transduced with equal MOI. DETAILED DESCRIPTION

[0044] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0045] According to a first aspect of the embodiments of the present application, a modified host cell is provided. Compared with a wild-type host cell, no SLC1A5 isoforms are expressed or are expressed at a low level in the modified host cell.

[0046] Using the modified host cells provided in the present application for virus packaging can rapidly increase virus production in a short period of time, for example, it can be increased by 3 times within 48 hours.

[0047] The term "host cell" refers to a cell that has been invaded or integrated by pathogens such as viruses, bacteria (such as the relationship between bacteriophages and bacterial hosts), fungi, parasites, or foreign genes (such as in genetic engineering). These pathogens or foreign genes rely on the mechanisms of host cells to carry out their own survival, replication, expression, etc. Host cells invaded by viruses to achieve the survival, replication, and expression of the virus itself can also be called virus packaging cells.

[0048] The term "expression" refers to the transcription and translation of a gene, polynucleotide, etc. into a polypeptide.

[0049] In order to make SLC1A5 isoforms not expressed or expressed at a low level, methods that can be used in the art include, but are not limited to: gene editing technology, RNA interference technology, small molecule inhibitors, antisense oligonucleotide technology, protein degradation targeted chimera technology, and changing culture conditions (including using special culture media).

[0050] The term "homologous isomers" refers to different protein forms that come from the same gene but are produced by mechanisms such as alternative splicing and post-translational modification after gene transcription. These different forms of proteins may differ in structure, function, intracellular localization or expression pattern. Taking SLC1A5 as an example, there are 3 isologous isomers.

[0051] In some embodiments of the present application, the SLC1A5 gene in the modified host cell is gene-edited.

[0052] The term "gene editing" is a genetic engineering technology that modifies specific target genes in the genome of an organism by deleting, inserting or replacing a fragment or specific base of the genome to cause specific changes in the genome, thereby achieving genetic modification of the DNA sequence.

[0053] In some embodiments of the present application, the common coding nucleic acid region of the SLC1A5 isoforms is mutated. The common coding nucleic acid region of the SLC1A5 isoforms described in the present application varies depending on the type of host cell. For example, when the host cell is a HEK293 cell or a cell line derived therefrom, the nucleotide sequence of the common coding nucleic acid region is as shown in SEQ ID NO.9.

[0054] In some embodiments of the present application, the mutation includes one or a combination of two or more of an insertion mutation, a deletion mutation and a substitution mutation.

[0055] In some embodiments of the present application, the modified host cell undergoes insertion mutation and deletion relative to the wild-type host cell. In some embodiments of the present application, the insertion mutation occurs in exon 5 of the SLC1A5 gene, accompanied by a large fragment deletion, such as the P1B7 cell in the example.

[0056] In some embodiments of the present application, relative to wild-type host cells, the modified host cells undergo a large fragment deletion, such as the P2G6 cells in the examples.

[0057] The term "insertion mutation" refers to the insertion of one or more additional nucleotides into a DNA sequence. These additional nucleotides can be single bases, short nucleotide sequences, or longer DNA fragments. The insertion location can be any region of the gene, such as the coding region, non-coding region, introns, or exons.

[0058] The term "deletion mutation" refers to the phenomenon that one or more nucleotides in a DNA sequence are missing. Deletions can range from a single base to large DNA regions, and similar to insertion mutations, deletions can occur anywhere in the gene.

[0059] The term "substitution mutation" refers to a situation in which a nucleotide in a DNA sequence is replaced by another nucleotide. This mutation involves only a change in a single base, so it is also called a point mutation.

[0060] In some embodiments of the present application, the modified host cell is derived from any one of HEK293 cells and cell lines derived therefrom. The HEK293 cells and cell lines derived therefrom include HEK293, HEK293T, HEK293F and HEK293S. In this case, the nucleotide sequence of the common coding nucleic acid region of the SLC1A5 gene is shown in SEQ ID NO.9.

[0061] In some embodiments of the present application, the SLC1A5 gene of the modified host cell is edited into a nucleic acid fragment having a nucleotide sequence such as SEQ ID NO.5 and SEQ ID NO.6 (for example, the P1B7 cell in the embodiment), or is edited into a nucleic acid fragment having a nucleotide sequence such as SEQ ID NO.7 (for example, the P2G6 cell in the embodiment).

[0062] In some embodiments of the present application, the modified host cells include adherent cells (eg, P1B7 cells and P2G6 cells in the examples).

[0063] According to a second aspect of an embodiment of the present application, a method for preparing the modified host cell is provided, the method comprising using gene editing technology to perform gene editing on the host cell to be modified so that the host cell has the nucleic acid fragment.

[0064] This application does not specifically limit the types of gene editing technology, including but not limited to: zinc finger nuclease technology (ZFN), transcription activator-like effector nuclease technology (TALEN), CRISPR / Cas system editing technology, single base editing technology, guide editing technology, and recombinase-mediated gene editing technology.

[0065] In some embodiments of the present application, the gene editing technology includes CRISPR / Cas9 technology.

[0066] In some embodiments of the present application, the binding site of the sgRNA used in the preparation method is located in the common coding nucleic acid region of the SLC1A5 gene.

[0067] In some embodiments of the present application, the nucleotide sequence of the sgRNA used in the preparation method is shown as SEQ ID NO.1 and SEQ ID NO.2.

[0068] In a third aspect of the embodiments of the present application, a method for producing a virus is provided, wherein the method uses the modified host cell as a packaging cell to produce the virus;

[0069] The virus may or may not include a GOI in its genome.

[0070] GOI is the abbreviation of Gene of Interest.

[0071] The present application does not specifically limit GOI, which can be used for different purposes. In some embodiments of the present application, the GOI includes one or more of a selection marker gene and a target gene.

[0072] In the present application, the selection marker gene includes but is not limited to: one or more of a luciferase gene, a fluorescent protein gene, a puromycin resistance gene, a blasticidin resistance gene, a geneticin resistance gene, a bleomycin resistance gene, a neomycin resistance gene, a hygromycin phosphotransferase gene, a dihydrofolate reductase gene, a thymidine kinase gene, a glutamine synthetase gene, an asparagine synthetase gene, a tryptophan synthetase gene, a histidinol dehydrogenase gene, an aminoglycoside phosphotransferase gene, and a tryptophan synthetase gene.

[0073] In the present application, the target protein encoded by the target gene includes but is not limited to antibodies, recombinant proteins, polypeptides, enzymes, hormones, growth factors, receptors and non-coding RNA.

[0074] In the present application, there is no particular limitation on the type of virus, including but not limited to lentivirus and retrovirus.

[0075] In some embodiments of the present application, the virus comprises a pseudotyped virus.

[0076] The term "pseudotyped virus" is a virus whose viral envelope protein is replaced by the envelope protein of other viruses after genetic engineering or natural recombination. Simply put, it has the genome of a virus, but the envelope comes from another virus, which is a form of "disguise" of the virus. The present application does not specifically limit the types of envelope proteins of other viruses, including but not limited to baboon endogenous retrovirus envelope glycoprotein GP70, cat endogenous retrovirus RD114 envelope glycoprotein GP70 and other proteins that bind to SLC1A5.

[0077] In a fourth aspect of the embodiments of the present application, a method for producing modified immune cells is provided, the production method comprising the following steps:

[0078] Producing the virus using the production method; and,

[0079] The virus is used to infect immune cells to be transformed to prepare transformed immune cells.

[0080] The term "immune cell" refers to cells involved in or associated with an immune response. They play a core role in the body's immune system and are responsible for identifying and removing foreign pathogens, abnormal cells of the body (such as tumor cells), and aging and damaged cells in the body, thereby maintaining the health of the body and the stability of the internal environment. This application does not specifically limit the types of immune cells to be modified, including but not limited to T cells, TCR-T, CAR-T, TIL, DC-CIK and NK cells.

[0081] In a fifth aspect of the embodiments of the present application, a method for producing a drug is provided, the method comprising the following steps:

[0082] Producing the modified immune cells using the production method; and,

[0083] The modified immune cells are prepared into medicines.

[0084] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0085] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0086] The present application examples involve the following experimental materials:

[0087] 4D-Nucleofector TM Electroporator: 4D-Nucleofector TM Core Unit+4D-Nucleofector TM XUnit Nucleocuvette TM 16-well electroporation strips.

[0088] 4D-Nucleofector for P3 Primary Cells TM X kit (Lonza, V4XP-3032).

[0089] DMEM culture medium (Gibco, 10569010).

[0090] FBS (LIFE, 10091148).

[0091] Trypsin-EDTA (0.25%), phenol red (Gibco, 25200072).

[0092] DPBS (without Ca2+ and Mg2+): (LIFE, 10099141).

[0093] MACS buffer (Miltenyi Biotec, 130-091-221).

[0094] Fixation and Permeabilization solution (BD, 554722).

[0095] Perm / Wash buffer (BD, 554723).

[0096] Cas9 protein: eSpCas9-N-NLS nuclease (GenScript, Z03470-300).

[0097] sgRNA: (GenScript synthesis).

[0098] AF647-labeled mouse anti-human SLC1A4 antibody (Santa Cruz Biotechnology, sc-393157).

[0099] Rabbit anti-human SLC1A5 antibody (Abcam, ab237704).

[0100] AF488-labeled goat anti-rabbit IgG antibody (Cell Signaling Technology, 4412).

[0101] AF647-labeled goat anti-rabbit IgG antibody (Cell Signaling Technology, 4414).

[0102] Example 1

[0103] 1. Construction of SLC1A5 knockout 293T cells

[0104] (1) Electroporation transfection of Cas9 / sgRNA complex

[0105] The SLC1A5 coding gene is approximately 13712 bp ( Figure 1 There are three different alternative splicing transcripts mRNA, encoding three different isoforms of SLC1A5 protein ( Figure 1 To completely knock out SLC1A5, the binding sites of the two sgRNAs targeting the SLC1A5 gene (SEQ ID NO.1 and SEQ ID NO.2) used in this experiment are located in the gene sequence shared by all isoforms of SLC1A5 ( Figure 1 ; SEQ ID NO.9), all SLC1A5 isoforms can be knocked out simultaneously.

[0106] 293T cells were passaged 1-2 days before electroporation, and the cell confluence reached 60-80% on the day of electroporation. The sgRNA was dissolved in RNase / DNase-free and pyrogen-free ultrapure water to a final concentration of 100 μM. TM Electroporation solution (Nucleofector TM Solution) and supplement solution (Supplement) were mixed in a ratio of 4.5:1 (v:v) to prepare P3 electroporation buffer, and then balanced to room temperature. 5 μL P3 electroporation buffer, 1.2 μL (i.e. 120 pmol) of sgRNA (in the group of simultaneous transfection of sgRNA2 and sgRNA3, 0.6 μL of each of the two sgRNAs), and 1.6 μL (i.e. 40 pmol) of Cas9 protein were added to the centrifuge tube in sequence. After thorough mixing, incubate at room temperature for 10 minutes to form an RNP mixture. During the reagent incubation period, count and collect the required number of cells (each electroporation reaction requires 1×10 6 After centrifugation at 500 g for 5 min, the supernatant was discarded and the cells were carefully resuspended in 15 μL P3 electroporation buffer.

[0107] Take 15 μL of cell suspension and add it to the centrifuge tube containing Cas9 / sgRNA mixture and mix gently. Transfer the mixed solution to the Nucleocuvette TM In a 16-well electroporation plate, electroporation was performed using the CM-130 program. After electroporation, 80 μL of complete medium (DMEM medium + 10% FBS) preheated at 37°C was added to each well and incubated in an incubator (37°C, 5% CO 2 ) for 1 h. After incubation, the cells were transferred to a 6-well plate preheated at 37°C, supplemented with complete medium, and placed in an incubator (37°C, 5% CO 2 ) were cultured and expanded.

[0108] (2) Detection of SLC1A5 expression

[0109] After the cells to be tested have expanded to a sufficient number, trypsinize the cells. After washing the cells twice with MACS buffer, 1×10 6 The cells were resuspended in 100 μL of Fluxation and Permeabilization solution. After fixation and permeabilization at 4°C for 30 min, the cells were centrifuged at 500 g for 5 min, and the supernatant was discarded. The cells were washed twice with Perm / Wash buffer and resuspended in 100 μL of Perm / Wash buffer. Rabbit anti-human SLC1A5 antibody (1:400 dilution) was added. After incubation at 4°C for 1 h, the cells were centrifuged at 500 g for 5 min, and the supernatant was discarded. The cells were washed twice with Perm / Wash buffer and resuspended in 100 μL of Perm / Wash buffer. AF488-labeled goat anti-rabbit IgG antibody (1:400 dilution) was added. After incubation at 4°C for 1 h, the cells were centrifuged at 500 g for 5 min, and the supernatant was discarded. The cells were washed twice with Perm / Wash buffer, resuspended in 200 μL of Perm / Wash buffer, and the expression of SLC1A5 was detected using a BD FACSCelesta flow cytometer.

[0110] The results are as follows Figure 2 As shown in the figure, the expression rate of SLC1A5 in wild-type 293T cells was 14.2%, the expression rate of SLC1A5 in 293T cells transfected with sgRNA2 was 5.23%, the expression rate of SLC1A5 in 293T cells transfected with sgRNA3 was 4.64%, and the expression rate of SLC1A5 in 293T cells transfected with sgRNA2 and sgRNA3 was 3.18%, indicating that SLC1A5 can be effectively knocked out in 293T cells. The expression rate of SLC1A5 in 293T cells transfected with sgRNA2 and sgRNA3 was the lowest, so this cell line was selected for subsequent experiments.

[0111] (3) Detection of retrovirus receptivity of SLC1A5 knockout 293T cells

[0112] The wild-type and SLC1A5 knockout 293T cells were infected with BaEV envelope protein pseudotyped retrovirus carrying the GFP reporter gene at a multiplicity of infection (MOI) of 1TU / cell. 72 h after infection, the cells were trypsinized, washed twice with MACS buffer, and then 1×10 6 The cells were resuspended in 200 μL MACS buffer, and the expression of GFP was detected using a BD FACSCelesta flow cytometer.

[0113] The results are as follows Figure 3 As shown, the expression rate of GFP in wild-type 293T cells was 21.2%, and the expression rate of GFP in 293T cells transfected with sgRNA2 and sgRNA3 was 3.27%, indicating that knocking out SLC1A5 can significantly reduce the infection and transduction efficiency of retroviral vectors in 293T cells.

[0114] 2. Monoclonal Isolation and Genotyping

[0115] (1) Flow cytometry sorting of monoclonal cells

[0116] Among the 293T cells transfected with sgRNA2 and sgRNA3, 3.27% of the cells could still be infected by the retrovirus, indicating that these cells could be infected by the virus through SLC1A4.

[0117] The cells were trypsinized and washed twice with MACS buffer. 6 The cells were resuspended in 100 μL MACS buffer and AF647-labeled mouse anti-human SLC1A4 antibody was added. After incubation at 4°C for 1 hour, the cells were centrifuged at 500 g for 5 minutes and the supernatant was discarded. The cells were washed twice with MACS buffer and resuspended in 200 μL MACS buffer. The expression of SLC1A4 was detected using a BD FACSCelesta flow cytometer.

[0118] like Figure 4 As shown in Figure A, more than 6% of 293T cells are still SLC1A4 positive. Therefore, the monoclonal cells that are also negative for SLC1A4 were further sorted in the BD FACSAria III flow sorting instrument and inoculated into 96-well plates (a total of 192 monoclonal cells). After supplementing with complete medium, the cells were placed in an incubator (37°C, 5% CO 2 ) were cultured and expanded.

[0119] (2) Detection of monoclonal cell receptivity to retrovirus

[0120] The BaEV envelope protein-pseudotyped retrovirus carrying the GFP reporter gene was used to infect each 293T monoclonal cell line at an MOI of 1TU / cell. 72 h after infection, the cells were trypsinized, washed twice with MACS buffer, and then 1×10 6 The cells were resuspended in 100 μL MACS buffer and AF647-labeled mouse anti-human SLC1A4 antibody was added. After incubation at 4°C for 1 h, the cells were centrifuged at 500 g for 5 min and the supernatant was discarded. The cells were washed twice with MACS buffer and resuspended in 200 μL MACS buffer. The expression of GFP and SLC1A4 was detected using a BD FACSCelesta flow cytometer.

[0121] The results are as follows Figure 4 As shown in Figure B, among the 192 monoclonal cell lines, a total of 20 GFP-negative and SLC1A4-negative monoclonal cell lines were screened and obtained.

[0122] (3) Genotype identification

[0123] DNA of each monoclonal cell was extracted, and the gene region targeted by sgRNA was amplified by PCR using specific primers (SEQ ID NO.3 and SEQ ID NO.4), and then analyzed by 1% agarose gel electrophoresis. This example is demonstrated using three representative clones (P1B7, P2F7, and P2G6) as examples.

[0124] The results are as follows Figure 5 As shown, the size of the band amplified in the wild-type 293T cells was about 3900 bp, which was consistent with the predicted size; 500 bp and 3900 bp were detected in the P1B7 clone cells, indicating that a large deletion occurred in one of the SLC1A5 alleles; 500 bp, 700 bp and 3900 bp bands were detected in the P2F7 clone cells, indicating that the cell line may be impure and contain multiple clones; the size of the band amplified in the P2G6 clone cells was about 500 bp, and no wild-type-sized band was found, indicating that large deletions occurred in both SLC1A5 alleles in this clone.

[0125] To further confirm whether SLC1A5 was completely deleted in P1B7 and P2F7 clone cells, the bands obtained by PCR were subjected to Sanger sequencing.

[0126] The results showed that the SLC1A5 allele corresponding to the 3900bp fragment of P1B7 had a base deletion in intron 4 and a base insertion in exon 5 ( Figure 1As shown in Figure E, ▽ indicates a nucleotide deletion mutation, and ▼ indicates a nucleotide insertion mutation; SEQ ID NO.5), which ultimately leads to a frameshift mutation in the protein coding region; the sequencing results of the 500bp fragment of P1B7 showed that the SLC1A5 allele did have a large fragment deletion ( Figure 1 As shown in Figure F; SEQ ID NO.6). The Sanger sequencing results of P2F7 showed double peaks. The sequencing results of P2G6 showed that both SLC1A5 alleles had large fragment deletions ( Figure 1 As shown in Figure G; SEQ ID NO.7). Therefore, P1B7 and P2G6 clone cells are SLC1A5 completely deficient cell lines; P2F7 cells contain multiple SLC1A5 knockout clones.

[0127] 3. Comparison of packaging output

[0128] The lentiviral helper plasmid pRSV-Rev, pMDLg-pRRE, envelope protein expression plasmid pCMV-BaEV and lentiviral vector plasmid carrying mCherry reporter gene were mixed with Lipofectamin 3000 transfection reagent and co-transfected into P1B7, P2F7, P2G6 monoclonal and wild-type 293T cells.

[0129] After 48 hours of culture, the status of 293T cells in each group was observed under a microscope. It was found that a large number of wild-type 293T cells detached from the bottom of the culture flask, formed syncytia and apoptosis; on the contrary, the cloned cells still maintained a good adhesion state, and no obvious syncytia and apoptosis occurred ( Figure 6 The virus supernatants were collected separately, centrifuged at 4500 rpm for 15 min at 4°C, filtered through a 0.45 μm pore size filter membrane, and stored in separate devices at -80°C.

[0130] The concentration of Jurkat cells was adjusted to 1 × 10 5 / 300μL, mix thoroughly and take 300μL of resuspended cells to each well of a 24-well plate. Take 70μL of lentiviral supernatant and dilute it 5 times with complete culture medium. Add the lentivirus of each dilution gradient to the above 24-well plate at a dosage of 200μL / well, so that the lentivirus infects Jurkat cells (only complete culture medium is added to the Jurkat cells in the negative control group), and culture them in a cell culture incubator (culture temperature is 37°C, carbon dioxide concentration is 5%). After 3 days of culture, the virus titer of each group was detected by qPCR. Specifically, after washing the cells twice with PBS, the DNA of each group of Jurkat cells was extracted, and specific primers were used to detect the copy number of the unique sequence WPRE in the lentiviral vector and the ALB gene in the Jurkat cells. The ALB copy number is recorded as A, the WPRE copy number is recorded as W, and the volume of the added lentivirus is recorded as V. The lentiviral titer at each dilution gradient is calculated by the following formula:

[0131] Lentivirus titer (IU / mL) = number of Jurkat cells per well × W × dilution factor / V × A

[0132] The titer of the lentivirus can be obtained by taking the average of the titers calculated under each dilution gradient.

[0133] The results are as follows Figure 6 As shown in Figure B, the titers of viral supernatants obtained by packaging with wild-type 293T cells, P1B7, P2G6, and P2F7 cells were 1.88×10 6 IU / mL, 3.70×10 6 IU / mL, 6.05×10 6 IU / mL, 3.97×10 6 IU / mL; compared with the lentivirus obtained in wild-type 293T cells, the titers of lentivirus obtained in P1B7, P2G6, and P2F7 cells increased by 1.9 times, 3.22 times, and 2.11 times, respectively (P<0.05).

[0134] 4. Comparison of viral transduction efficiency and target gene expression

[0135] PBMCs from healthy donors were revived in AIM V medium, and 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody and 300 IU / mL recombinant hIL-2 were added, and cultured in a cell culture incubator for 24 h (culture temperature was 37 ° C, carbon dioxide concentration was 5%). The obtained T cells were washed, and lentivirus was added at the same MOI (1 IU / cell) for transduction. At the same time, 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody and 300 IU / mL recombinant hIL-2 were supplemented and cultured in a cell culture incubator (culture temperature was 37 ° C, carbon dioxide concentration was 5%). After 24 h, the cell density was adjusted to 2×10 6 / mL, and supplemented with 300IU / mL of hIL-2. On the 4th day after transduction, the cells were washed to remove the residual lentiviral particles in the supernatant, and then cultured in a cell culture incubator for 5 days (culture temperature was 37°C, carbon dioxide concentration was 5%), during which the cell density was maintained at 1×10 6 / mL. The cells were collected on the 10th day after transduction. Flow cytometry was used to detect the expression of the target gene mCherry in each T cell group.

[0136] The results are as follows Figure 7 As shown in the figure, the positive rate of mCherry in T cells transduced by viruses obtained by wild-type 293T cells packaging was only 7.55%±2.14%; the positive rate of mCherry in T cells transduced by viruses obtained by P1B7 cells was 40.10%±3.10%; the positive rate of mCherry in T cells transduced by viruses obtained by P2G6 cells was 19.10%±6.89%; the positive rate of mCherry in T cells transduced by viruses obtained by P2F7 cells was 20.13%±4.80%. The expression rates of P1B7, P2G6, and P2F7 groups were significantly higher than those of the wild-type group (2.53 to 5.31 times, P<0.05). This result shows that the viruses packaged by P1B7, P2G6, and P2F7 cells are of higher quality and higher transduction efficiency, and less virus is required when transducing immune cells, which can effectively reduce the production cost of engineered immune cells. Existing theories cannot explain why SLC1A4 and / or SLC1A5-negative packaging cells can improve the transduction efficiency of lentivirus at a unit MOI, so this finding is unexpected.

[0137] In summary, the present application constructs SLC1A5 knockout packaging cells, which are also SLC1A4 negative. Based on the packaging cells, the present embodiment achieves a significant increase in the yield of BaEV-GP70 pseudotyped lentivirus per unit time, significantly shortens the virus production cycle, and improves the transduction efficiency of the virus under unit MOI. When producing the same amount of engineered immune cells, the required amount of virus can be significantly reduced, greatly reducing the production cost.

[0138] sgRNA2 (SEQ ID NO.1):

[0139] GGUCUCCUGGAUCAUGUGGU GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC.

[0140] sgRNA3 (SEQ ID NO.2):

[0141] CCACGAUCUUGCCAGCCACC GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC.

[0142] SLC1A5-F (SEQ ID NO. 3): CTGGAAAGAGGTCCCAAAGGCAG.

[0143] SLC1A5-R (SEQ ID NO. 4):CGATCTGTTCCTTGCCCACAG.

[0144] Nucleotide sequence 1 of the SLC1A5 gene cloned from P1B7 (SEQ ID NO.5):

[0145] ATGAACATCCTGGGCTTGGTAGTGTTTGCCATCGTCTTTGGTGTGGCGCTGCGGAAGCTGGGGCCTGAAGGGGAGCTGCTTATCCGCTTCTTCAA

[0146] CTCCTTCAATGAGGCCACCATGGTTCTGGTCTCCTGGATCATGTGTAGGTGGTGGGAGAGGAGGCTGGGGGTGGGTGGGGTTGGAGGGTCGGTC

[0147] TGCTGCCAGAGGCAGGTGCGGGGCATTCTGTTTGGGATGGGGCACCCTCTATTCACTCTTTGGAGCCAGATGACCTGCATTTAAATCCAGCATTGA

[0148] CCATTCCCTGTCTATGTGATTTTAGGCAAGTGATTTTCCTTCTCTGCTTCAGTTTCCTCATCTATAAAATGGGAACGGCACTTTGGGAGGAGGTGGG

[0149] TGGATCACTTGAGATCAGGAGTTCAAAACCAGCCTGGCCAACATGGTGAAACCCCATCTCTACTAAAAATACGAAAAAATTAGCCAGGCATGGG

[0150] GACACGCACCTGTAATCCCAGTTACTCGGGAACCTGAGGTATGAGAATCACTTAAGCCTGGGAGGCGGAAGTTGCAGTAAGCGGAGATCATGCC

[0151] ACTGCCTGGGCGACAGAGGGAAACTCTGTCTCAAGAAGAAAAGAAAAGAAAGTGAATACGGTACACGGAAGGTCTTAGGACAGTGCCTGGCTT

[0152] ATTGCGTGCCTACAGTAAATCCTAGTAGCTAATATTTATTGGGCTTTTTGCTTGTATAACTTCTGGATACTTCCCACTGGCCAGAACTTGGGTATCTA

[0153] GCCTCCCTTCTCTGCACAAGTGGCTGGGAAAATATGCCTTTCTTCTGTGACTTACAAGAAGGGACTGGGGAGATAACTAGTCAGTGATGATGGAT

[0154] GGGCGACCGATGATCGAGTAGTCTAGGGAAGTGATGTTGGGAGGCTTTTATTTTATTTTATTTTTTTGAGATGGAGTTTCGTTCTTGTTGCCCAGGC

[0155] TGGAGTGCAATGACGTGATCTCAGCTCACTGCAAACTCCGCCTCCCGGGTTCAAGAGATTCTCCTGCTTCAGTCTCCCGAGTAGCTGGGATTACA

[0156] GGCATGCGCCACCATGCCCGGCTAATTTTGTATTTTTAGTAGAGACGGGGTTTCTCCATGTTGGTCAGGCTGGTCTCAAACTCCCGACCTCAGGTG

[0157] ATCCGCCCACCTCGGCCTCCCGAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCGGACTTGGGAGTCCTCTTGGCTTGGAGCCATCCCTGG

[0158] GATCTTCACAGCAGGAGAGGGTGATAATAAACCCACCTGAAGTTTACAGATCACTTATTGTCTGTAAAAAGCTTTAGCTCCTTTCAACTGGCATCT

[0159] CATTGCATCCTCCCAGCATTCCCGTGAGGAATCTATTGGGTGTTGTGTAATCTGCAATCTGACTCATAGGACCTCCCTCCGGGGGTTGCTGGGAAG

[0160] ATTCAACATCATGTTCAAGGGCAGAGCACATCGCAAATGCTTCATACCTGGGAGTTGTTATTAAGACCCAGGCCATGGTCTTTCTAAAATTTGTATT

[0161] TATTTTATTTATTTATTTATTTAGAGACAGAGTCTTGCTCTGTCGCCCAGGCTGGAGGTGCAGTGGCACAGTCTCAGCTCACTGCAATGTCTGCCTC

[0162] CCATGTTCAAGTGATTCTCCTGCCTCAGCCTCCTGAGCAGCTGGGACCAAAGGCACGAACCACCATACCCAGCTAATTTTTGTATTTTTAGTAGAG

[0163] ACAGGGTTTTGCCATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAAGCGATCCGCCCTCTTCGGCCTCCCAAAGTGCTGGGATTACAGGTGT

[0164] GAGCCACCGCTCCTGGCCTTCAAATTTTTATTTTAAAAATGTGTGTTTTGGCTGGTTGTGGTACCTCACACCTGTAATCCCAGCACTTTGGGAGGC

[0165] TGAGGGGGGAGGATTGCTTGAGGCCAGGAGTTAGAGACTAGCCTGGTCAACATGGCGAAAAACCCTCTCTACAAAAAAATACAAATATTAGCTG

[0166] GGAATGGTGGTGTACACCTTTAATCCTAGCTACTACTCAGGAGGCTGAGGCATGAAAATCGCTTGAACCTGGGAGGCAAAGGTTGTAGTGAGCCA

[0167] AGATTGCAACAGTACACTCCAGCCTGGGCAACAGAACGAGACTGTCTAAATAATAATAATAATAGTAATTTAAAAAAACGTTTTTTGAACTAGGAT

[0168] CTCGCTCTGTTGCCTAGGTTGGAGTGCAGTGTGGCATGATCACAGCTCACTGCAGCCCGGACCTCCTGGGTTCAGGTGATCCTCCCACCTCAACC

[0169] TCCTAAGTGGCTGGGACTACAGACCCCAGCCACAAAGCCTAGCTCATTTTTTTTGTTGTTGTTGTTTAAGAAACTGGGTGGCCCAGTACGGAGGC

[0170] TCATGCCTATAATCCCAGCACTTTGGGAGGCTGAGGCAGGCAGATCACGAGGTCAGGAGATCGAGACCATCCTGGCTAACACAGAAAAACCCTG

[0171] TCTCTACTAAAAATACAAAAAATTAGCTGGGCGTGGTAGCACGCGCCTATAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTGAACC

[0172] TGGGAGGCGGAGGGTGCAGTGAGCCGAGATCATGCCACTGCACTCCAGCCTGGGCAACAGAGTGAGACTCCATCTCAAAAAAAAAAAAAGAG

[0173] AGACATGTTGCCCAGGCTGGTCTTGAACTCCTGGGCTCAAGCGATCTGTCTGCCTTCACCTCCCTGCATGCTGGGATTACAGGTATGAGCCACCGT

[0174] GCCTGGCCTATTTTTATTTTTTGTAGAGACTGGGTCTCGCTGTGTTGCCCAAGCTGGTCTCGAGAACTCCTGGCCTCAAGTAATCCTCCTGCTTTCA

[0175] CCTCCTAAAGTGCTGGGATTATAGGCATGAGCCACTGTGCCCAGCCTCTTTATCTTTCATCATCTGCAGAGAGCGTATTGGCTCAAGTCATGCTAGC

[0176] TGCCATAGCAAACCAACCCAGGAAGTGTGAATGGCGCACCTTGATGGACATTTCTTTCTGTCTCCCATGTGAAGTCCAAATCTGGTGACTGCTTC

[0177] ACATGGTGATGGAAGCACTGAGACTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAATGGCATGATCTT

[0178] GGCTCACAGCAACCTCTGCCTCCCTGATTCAAGCAATTCTCCTGCCTGAGCCTCCCAAGTAGCTGGGATTGCAGGCATGCGCCACCACGCCTGGT

[0179] TAATTTTGTATTTTTAGTAGAGACGGGGTTTCTCCATGTTGGTCAGGCTGGTCTTGAACTCCCGACCTCAGGTGATCCACCCACCTCAGCCTCCCA

[0180] AAGTGCTGGGATTACAGGCATGAGCCACCGTGCCCGGCCTGCACGGAGAGTTTTATCTCCGGGCTGCTCTACCTTTGAGGGGAAAGAGAGCATG

[0181] AGATTGGCATGGGGGCTTCAGGGGCCAGGCCTGGGAGTGGTGCCCATGCTGAAGCACACATTGCTGGGGCTTCTCCTGACCTCAGGAAGGCAGG

[0182] AAGATGTGGGTAGGGTGCTGAGCCTCACGCCTGGCCACTGGGCCCGTGGAAGAGGCAACAAGCTGTCTCCTGTGTTACTGATCAGGTACGCCCC

[0183] TGTGGGCATCATGTTCCTGGTGGGCTGGCAAGATCGTGGAGATGGAGGATGTGGGTTTACTCTTTGCCCGCCTTGGCAAGTACATTCTGTGCTGCC

[0184] TGCTGGGTCACGCCATCCATGGGCTCCTGGTACTGCCCCTCATCTACTTCCTCTTCACCCGCAAAAACCCCTACCGCTTCCTGTGGGGCATCGTGACGCCGCTGGCCACTGCCTTTGGGACCTCTTCCAG。

[0185] Nucleotide sequence 2 of SLC1A5 gene cloned by P1B7 (SEQ ID NO.6):

[0186] ATGAACATCCTGGGCTTGGTAGTGTTTGCCATCGTCTTTGGTGTGGCGCTGCGGAAGCTGGGGCCTGAAGGGGAGCTGCTTATCCGCTTCTTCAA

[0187] CTCCTTCAATGAGGCCACCATGGTTCTGGTCTCCTGGCTGGCAAGATCGTGGAGATGGAGGATGTGGGTTTACTCTTTGCCCGCCTTGGCAAGTA

[0188] CATTCTGTGCTGCCTGCTGGGTCACGCCATCCATGGGCTCCTGGTACTGCCCCTCATCTACTTCCTCTTCACCCGCAAAAACCCCTACCGCTTCCTGTGGGGCATCGTGACGCCGCTGGCCACTGCCTTTGGGACCTCTTCCAG。

[0189] Nucleotide sequence of the SLC1A5 gene cloned by P2G6 (SEQ ID NO.7):

[0190] ATGAACATCCTGGGCTTGGTAGTGTTTGCCATCGTCTTTGGTGTGGCGCTGCGGAAGCTGGGGCCTGAAGGGGAGCTGCTTATCCGCTTCTTCAA

[0191] CTCCTTCAATGAGGCCACCATGGTTCTGGTCTCCTGGATCATGTGGCTGGCAAGATCGTGGAGATGGAGGATGTGGGTTTACTCTTTGCCCGCCTT

[0192] GGCAAGTACATTCTGTGCTGCCTGCTGGGTCACGCCATCCATGGGCTCCTGGTACTGCCCCTCATCTACTTCCTCTTCACCCGCAAAAACCCCTACCGCTTCCTGTGGGGCATCGTGACGCCGCTGGCCACTGCCTTTGGGACCTCTTCCAG。

[0193] Nucleotide sequence of the target editing region (SEQ ID NO.8):

[0194] ATGAACATCCTGGGCTTGGTAGTGTTTGCCATCGTCTTTGGTGTGGCGCTGCGGAAGCTGGGGCCTGAAGGGGAGCTGCTTATCCGCTTCTTCAA

[0195] CTCCTTCAATGAGGCCACCATGGTTCTGGTCTCCTGGATCATGTGGTAGGTGGTGGGAGAGGAGGCTGGGGGTGGGTGGGGTTGGAGGGTCGGT

[0196] CTGCTGCCAGAGGCAGGTGCGGGGCATTCTGTTTGGGATGGGGCACCCTCTATTCACTCTTTGGAGCCAGATGACCTGCATTTAAATCCAGCATTG

[0197] ACCATTCCCTGTCTATGTGATTTTAGGCAAGTGATTTTCCTTCTCTGCTTCAGTTTCCTCATCTATAAAATGGGAACGGCACTTTGGGAGGAGGTGG

[0198] GTGGATCACTTGAGATCAGGAGTTCAAAACCAGCCTGGCCAACATGGTGAAACCCCATCTCTACTAAAAATACGAAAAAATTAGCCAGGCATGG

[0199] GGACACGCACCTGTAATCCCAGTTACTCGGGAACCTGAGGTATGAGAATCACTTAAGCCTGGGAGGCGGAAGTTGCAGTAAGCGGAGATCATGC

[0200] CACTGCCTGGGCGACAGAGGGAAACTCTGTCTCAAGAAGAAAAGAAAAGAAAGTGAATACGGTACACGGAAGGTCTTAGGACAGTGCCTGGCT

[0201] TATTGCGTGCCTACAGTAAATCCTAGTAGCTAATATTTATTGGGCTTTTTGCTTGTATAACTTCTGGATACTTCCCACTGGCCAGAACTTGGGTATCT

[0202] AGCCTCCCTTCTCTGCACAAGTGGCTGGGAAAATATGCCTTTCTTCTGTGACTTACAAGAAGGGACTGGGGAGATAACTAGTCAGTGATGATGGA

[0203] TGGGCGACCGATGATCGAGTAGTCTAGGGAAGTGATGTTGGGAGGCTTTTATTTTATTTTATTTTTTTGAGATGGAGTTTCGTTCTTGTTGCCCAGG

[0204] CTGGAGTGCAATGACGTGATCTCAGCTCACTGCAAACTCCGCCTCCCGGGTTCAAGAGATTCTCCTGCTTCAGTCTCCCGAGTAGCTGGGATTAC

[0205] AGGCATGCGCCACCATGCCCGGCTAATTTTGTATTTTTAGTAGAGACGGGGTTTCTCCATGTTGGTCAGGCTGGTCTCAAACTCCCGACCTCAGGT

[0206] GATCCGCCCACCTCGGCCTCCCGAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCGGACTTGGGAGTCCTCTTGGCTTGGAGCCATCCCTG

[0207] GGATCTTCACAGCAGGAGAGGGTGATAATAAACCCACCTGAAGTTTACAGATCACTTATTGTCTGTAAAAAGCTTTAGCTCCTTTCAACTGGCATC

[0208] TCATTGCATCCTCCCAGCATTCCCGTGAGGAATCTATTGGGTGTTGTGTAATCTGCAATCTGACTCATAGGACCTCCCTCCGGGGGTTGCTGGGAA

[0209] GATTCAACATCATGTTCAAGGGCAGAGCACATCGCAAATGCTTCATACCTGGGAGTTGTTATTAAGACCCAGGCCATGGTCTTTCTAAAATTTGTAT

[0210] TTATTTTATTTATTTATTTATTTAGAGACAGAGTCTTGCTCTGTCGCCCAGGCTGGAGGTGCAGTGGCACAGTCTCAGCTCACTGCAATGTCTGCCT

[0211] CCCATGTTCAAGTGATTCTCCTGCCTCAGCCTCCTGAGCAGCTGGGACCAAAGGCACGAACCACCATACCCAGCTAATTTTTGTATTTTTAGTAGA

[0212] GACAGGGTTTTGCCATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAAGCGATCCGCCCTCTTCGGCCTCCCAAAGTGCTGGGATTACAGGTG

[0213] TGAGCCACCGCTCCTGGCCTTCAAATTTTTATTTTAAAAATGTGTGTTTTGGCTGGTTGTGGTACCTCACACCTGTAATCCCAGCACTTTGGGAGG

[0214] CTGAGGGGGGAGGATTGCTTGAGGCCAGGAGTTAGAGACTAGCCTGGTCAACATGGCGAAAAACCCTCTCTACAAAAAAATACAAATATTAGCT

[0215] GGGAATGGTGGTGTACACCTTTAATCCTAGCTACTACTCAGGAGGCTGAGGCATGAAAATCGCTTGAACCTGGGAGGCAAAGGTTGTAGTGAGCC

[0216] AAGATTGCAACAGTACACTCCAGCCTGGGCAACAGAACGAGACTGTCTAAATAATAATAATAATAGTAATTTAAAAAAACGTTTTTTGAACTAGGA

[0217] TCTCGCTCTGTTGCCTAGGTTGGAGTGCAGTGTGGCATGATCACAGCTCACTGCAGCCCGGACCTCCTGGGTTCAGGTGATCCTCCCACCTCAAC

[0218] CTCCTAAGTGGCTGGGACTACAGACCCCAGCCACAAAGCCTAGCTCATTTTTTTTGTTGTTGTTGTTTAAGAAACTGGGTGGCCCAGTACGGAGG

[0219] CTCATGCCTATAATCCCAGCACTTTGGGAGGCTGAGGCAGGCAGATCACGAGGTCAGGAGATCGAGACCATCCTGGCTAACACAGAAAAACCCT

[0220] GTCTCTACTAAAAATACAAAAAATTAGCTGGGCGTGGTAGCACGCGCCTATAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTGAAC

[0221] CTGGGAGGCGGAGGGTGCAGTGAGCCGAGATCATGCCACTGCACTCCAGCCTGGGCAACAGAGTGAGACTCCATCTCAAAAAAAAAAAAAGA

[0222] GAGACATGTTGCCCAGGCTGGTCTTGAACTCCTGGGCTCAAGCGATCTGTCTGCCTTCACCTCCCTGCATGCTGGGATTACAGGTATGAGCCACC

[0223] GTGCCTGGCCTATTTTTATTTTTTGTAGAGACTGGGTCTCGCTGTGTTGCCCAAGCTGGTCTCGAGAACTCCTGGCCTCAAGTAATCCTCCTGCTTT

[0224] CACCTCCTAAAGTGCTGGGATTATAGGCATGAGCCACTGTGCCCAGCCTCTTTATCTTTCATCATCTGCAGAGAGCGTATTGGCTCAAGTCATGCTA

[0225] GCTGCCATAGCAAACCAACCCAGGAAGTGTGAATGGCGCACCTTGATGGACATTTCTTTCTGTCTCCCATGTGAAGTCCAAATCTGGTGACTGCT

[0226] TCACATGGTGATGGAAGCACTGAGACTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAATGGCATGATC

[0227] TTGGCTCACAGCAACCTCTGCCTCCCTGATTCAAGCAATTCTCCTGCCTGAGCCTCCCAAGTAGCTGGGATTGCAGGCATGCGCCACCACGCCTG

[0228] GTTAATTTTGTATTTTTAGTAGAGACGGGGTTTCTCCATGTTGGTCAGGCTGGTCTTGAACTCCCGACCTCAGGTGATCCACCCACCTCAGCCTCC

[0229] CAAAGTGCTGGGATTACAGGCATGAGCCACCGTGCCCGGCCTGCACGGAGAGTTTTATCTCCGGGCTGCTCTACCTTTGAGGGGAAAGAGAGCA

[0230] TGAGATTGGCATGGGGGCTTCAGGGGCCAGGCCTGGGAGTGGTGCCCATGCTGAAGCACACATTGCTGGGGCTTCTCCTGACCTCAGGAAGGCA

[0231] GGAAGATGTGGGTAGGGTGCTGAGCCTCACGCCTGGCCACTGGGCCCGTGGAAGAGGCAACAAGCTGTCTCCTGTGTTACTGATCAGGTACGCC

[0232] CCTGTGGGCATCATGTTCCTGGTGGCTGGCAAGATCGTGGAGATGGAGGATGTGGGTTTACTCTTTGCCCGCCTTGGCAAGTACATTCTGTGCTGC

[0233] CTGCTGGGTCACGCCATCCATGGGCTCCTGGTACTGCCCCTCATCTACTTCCTCTTCACCCGCAAAAACCCCTACCGCTTCCTGTGGGGCATCGTGACGCCGCTGGCCACTGCCTTTGGGACCTCTTCCAG。

[0234] Consensus nucleotide sequence of each SLC1A5 isoform (SEQ ID NO.9):

[0235] ATGAACATCCTGGGCTTGGTAGTGTTTGCCATCGTCTTTGGTGTGGCGCTGCGGAAGCTGGGGCCTGAAGGGGAGCTGCTTATCCGCTTCTTCAA

[0236] CTCCTTCAATGAGGCCACCATGGTTCTGGTCTCCTGGATCATGTGGTAGGTGGTGGGAGAGGAGGCTGGGGGTGGGTGGGGTTGGAGGGTCGGT

[0237] CTGCTGCCAGAGGCAGGTGCGGGGCATTCTGTTTGGGATGGGGCACCCTCTATTCACTCTTTGGAGCCAGATGACCTGCATTTAAATCCAGCATTG

[0238] ACCATTCCCTGTCTATGTGATTTTAGGCAAGTGATTTTCCTTCTCTGCTTCAGTTTCCTCATCTATAAAATGGGAACGGCACTTTGGGAGGAGGTGG

[0239] GTGGATCACTTGAGATCAGGAGTTCAAAACCAGCCTGGCCAACATGGTGAAACCCCATCTCTACTAAAAATACGAAAAAATTAGCCAGGCATGG

[0240] GGACACGCACCTGTAATCCCAGTTACTCGGGAACCTGAGGTATGAGAATCACTTAAGCCTGGGAGGCGGAAGTTGCAGTAAGCGGAGATCATGC

[0241] CACTGCCTGGGCGACAGAGGGAAACTCTGTCTCAAGAAGAAAAGAAAAGAAAGTGAATACGGTACACGGAAGGTCTTAGGACAGTGCCTGGCT

[0242] TATTGCGTGCCTACAGTAAATCCTAGTAGCTAATATTTATTGGGCTTTTTGCTTGTATAACTTCTGGATACTTCCCACTGGCCAGAACTTGGGTATCT

[0243] AGCCTCCCTTCTCTGCACAAGTGGCTGGGAAAATATGCCTTTCTTCTGTGACTTACAAGAAGGGACTGGGGAGATAACTAGTCAGTGATGATGGA

[0244] TGGGCGACCGATGATCGAGTAGTCTAGGGAAGTGATGTTGGGAGGCTTTTATTTTATTTTATTTTTTTGAGATGGAGTTTCGTTCTTGTTGCCCAGG

[0245] CTGGAGTGCAATGACGTGATCTCAGCTCACTGCAAACTCCGCCTCCCGGGTTCAAGAGATTCTCCTGCTTCAGTCTCCCGAGTAGCTGGGATTAC

[0246] AGGCATGCGCCACCATGCCCGGCTAATTTTGTATTTTTAGTAGAGACGGGGTTTCTCCATGTTGGTCAGGCTGGTCTCAAACTCCCGACCTCAGGT

[0247] GATCCGCCCACCTCGGCCTCCCGAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCGGACTTGGGAGTCCTCTTGGCTTGGAGCCATCCCTG

[0248] GGATCTTCACAGCAGGAGAGGGTGATAATAAACCCACCTGAAGTTTACAGATCACTTATTGTCTGTAAAAAGCTTTAGCTCCTTTCAACTGGCATC

[0249] TCATTGCATCCTCCCAGCATTCCCGTGAGGAATCTATTGGGTGTTGTGTAATCTGCAATCTGACTCATAGGACCTCCCTCCGGGGGTTGCTGGGAA

[0250] GATTCAACATCATGTTCAAGGGCAGAGCACATCGCAAATGCTTCATACCTGGGAGTTGTTATTAAGACCCAGGCCATGGTCTTTCTAAAATTTGTAT

[0251] TTATTTTATTTATTTATTTATTTAGAGACAGAGTCTTGCTCTGTCGCCCAGGCTGGAGGTGCAGTGGCACAGTCTCAGCTCACTGCAATGTCTGCCT

[0252] CCCATGTTCAAGTGATTCTCCTGCCTCAGCCTCCTGAGCAGCTGGGACCAAAGGCACGAACCACCATACCCAGCTAATTTTTGTATTTTTAGTAGA

[0253] GACAGGGTTTTGCCATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAAGCGATCCGCCCTCTTCGGCCTCCCAAAGTGCTGGGATTACAGGTG

[0254] TGAGCCACCGCTCCTGGCCTTCAAATTTTTATTTTAAAAATGTGTGTTTTGGCTGGTTGTGGTACCTCACACCTGTAATCCCAGCACTTTGGGAGG

[0255] CTGAGGGGGGAGGATTGCTTGAGGCCAGGAGTTAGAGACTAGCCTGGTCAACATGGCGAAAAACCCTCTCTACAAAAAAATACAAATATTAGCT

[0256] GGGAATGGTGGTGTACACCTTTAATCCTAGCTACTACTCAGGAGGCTGAGGCATGAAAATCGCTTGAACCTGGGAGGCAAAGGTTGTAGTGAGCC

[0257] AAGATTGCAACAGTACACTCCAGCCTGGGCAACAGAACGAGACTGTCTAAATAATAATAATAATAGTAATTTAAAAAAACGTTTTTTGAACTAGGA

[0258] TCTCGCTCTGTTGCCTAGGTTGGAGTGCAGTGTGGCATGATCACAGCTCACTGCAGCCCGGACCTCCTGGGTTCAGGTGATCCTCCCACCTCAAC

[0259] CTCCTAAGTGGCTGGGACTACAGACCCCAGCCACAAAGCCTAGCTCATTTTTTTTGTTGTTGTTGTTTAAGAAACTGGGTGGCCCAGTACGGAGG

[0260] CTCATGCCTATAATCCCAGCACTTTGGGAGGCTGAGGCAGGCAGATCACGAGGTCAGGAGATCGAGACCATCCTGGCTAACACAGAAAAACCCT

[0261] GTCTCTACTAAAAATACAAAAAATTAGCTGGGCGTGGTAGCACGCGCCTATAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTGAAC

[0262] CTGGGAGGCGGAGGGTGCAGTGAGCCGAGATCATGCCACTGCACTCCAGCCTGGGCAACAGAGTGAGACTCCATCTCAAAAAAAAAAAAAGA

[0263] GAGACATGTTGCCCAGGCTGGTCTTGAACTCCTGGGCTCAAGCGATCTGTCTGCCTTCACCTCCCTGCATGCTGGGATTACAGGTATGAGCCACC

[0264] GTGCCTGGCCTATTTTTATTTTTTGTAGAGACTGGGTCTCGCTGTGTTGCCCAAGCTGGTCTCGAGAACTCCTGGCCTCAAGTAATCCTCCTGCTTT

[0265] CACCTCCTAAAGTGCTGGGATTATAGGCATGAGCCACTGTGCCCAGCCTCTTTATCTTTCATCATCTGCAGAGAGCGTATTGGCTCAAGTCATGCTA

[0266] GCTGCCATAGCAAACCAACCCAGGAAGTGTGAATGGCGCACCTTGATGGACATTTCTTTCTGTCTCCCATGTGAAGTCCAAATCTGGTGACTGCT

[0267] TCACATGGTGATGGAAGCACTGAGACTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAATGGCATGATC

[0268] TTGGCTCACAGCAACCTCTGCCTCCCTGATTCAAGCAATTCTCCTGCCTGAGCCTCCCAAGTAGCTGGGATTGCAGGCATGCGCCACCACGCCTG

[0269] GTTAATTTTGTATTTTTAGTAGAGACGGGGTTTCTCCATGTTGGTCAGGCTGGTCTTGAACTCCCGACCTCAGGTGATCCACCCACCTCAGCCTCC

[0270] CAAAGTGCTGGGATTACAGGCATGAGCCACCGTGCCCGGCCTGCACGGAGAGTTTTATCTCCGGGCTGCTCTACCTTTGAGGGGAAAGAGAGCA

[0271] TGAGATTGGCATGGGGGCTTCAGGGGCCAGGCCTGGGAGTGGTGCCCATGCTGAAGCACACATTGCTGGGGCTTCTCCTGACCTCAGGAAGGCA

[0272] GGAAGATGTGGGTAGGGTGCTGAGCCTCACGCCTGGCCACTGGGCCCGTGGAAGAGGCAACAAGCTGTCTCCTGTGTTACTGATCAGGTACGCC

[0273] CCTGTGGGCATCATGTTCCTGGTGGCTGGCAAGATCGTGGAGATGGAGGATGTGGGTTTACTCTTTGCCCGCCTTGGCAAGTACATTCTGTGCTGC

[0274] CTGCTGGGTCACGCCATCCATGGGCTCCTGGTACTGCCCCTCATCTACTTCCTCTTCACCCGCAAAAACCCCTACCGCTTCCTGTGGGGCATCGTG

[0275] ACGCCGCTGGCCACTGCCTTTGGGACCTCTTCCAGGTACGGCCTTGGGGTGGGCTAGGGTGGGATGTTTAATCCGCTAAGCGGCATGGAGCGGAT

[0276] GAAATTTTCTCTGCCCGGGACATTGTATCTGGGTACAGGGGCCATACTTCAGGAGATTCTCATAAGGTCCTGGATTTATCCCCACCCTTAAAAAGAT

[0277] AGGAAATGATTGCCCTAGAGATTTATTTCCCTCTTTCTTACACTGCCTCCCCGTGGCCCTGGAGGCACTCTTTTTTTTCTTTTGTTTTAAACAATGG

[0278] CGTCTCGCTCTGTTGCCCAGGCTGGAGTGTAGTGGTGTGATCTCAGCTCACTGCAACCTCTGCCTCCTGGGTTCAAGTGATTCTCTGCCTCAGCCT

[0279] CCCGAGTAGCTGGGACTGCAAGTGTCCCCCACCATACCCAGCTAATTTTTGTGTTTTTAGTAGAGACGGGGTTTCGCCATGTTGGCCAGGCTGGTC

[0280] TCAAACTCCTGGCCTCAAGTGATCCGCCCACCTTGGCCTCCAAAAGTGCTGGGATTATAGGTGTGAGCTGCCATGCCCCTGGGGGGCTCTTAAGT

[0281] TCTGCTCTGATTCAACCTCAGAACTCCTTCCACCGTCTCTCCTGGGCATTTCATTCATCCCTGCTGGCCCTGTCCCCTCCCAAGACAATAGTAATTC

[0282] TTAGTGTATTATTGGTGAGAGTCAGCAGGACCCAGACAGTAGAATTTAGGTGTGAATTGTGTGGGTATTTTGGGAGGTAGTAGTCTATACTCTCCC

[0283] AGGGGTTCCAGGACTCATGGATTTTGTAATCCTGGTATTGAGTTCCCTAGTGCTGCCTGCTTGTGGGAAAAGAGCTTAGATATAGCAAGTGAGGGC

[0284] TGGGGGTGGAGTCAGGACATGGTAGGAGGGAAGGGGGGTCTAGGTTAGATGTGGGGGCCTTGGGCCAAGGGACAGACTGGGGACGTGGGTGG

[0285] GACTTCGGCTGGGCAGGTGGTATGGGCAGGATCTGGTGGGCGGGGCTTTGGCTGGGCAAGTGGTATGGGCGGGATCTGGGTGGGCGGGGCTTTG

[0286] GCTGGGCAGGTGGTGTGGGTGGGCTCTGGGTGGGCGGGGCTTCAACTGGGTGGGTGGTGCAGGTCAGACCTGGAGGGTGGTTGGGTGACAGGG

[0287] CTTTGCTTAGGCAGGCTCTGGTGGGGATCCACTGGGTGGGGTGGGTGGGGCTTTGGCTGGTTGGGGTGGGGTAGGCGAGGCCTCACCTGGGCTG

[0288] GACCTGGAGGGTGGGCGGAGCCCCGGTCAGCAGGTTAACCTAACTCAGCTCCCCCATCCCTTGCAGTTCCGCCACGCTGCCGCTGATGATGAAG

[0289] TGCGTGGAGGAGAATAATGGCGTGGCCAAGCACATCAGCCGTTTCATCCTGCCCATCGGCGCCACCGTCAACATGGACGGTGCCGCGCTCTTCCA

[0290] GTGCGTGGCCGCAGTGTTCATTGCACAGCTCAGCCAGCAGTCCTTGGACTTCGTAAAGATCATCACCATCCTGTGAGTGTCAGGGGGCTGCGGAT

[0291] GGGACCCCCGGACCCCTGTTGACCCCCATCTTCTCCCACAAGACAGGCATCTGCCTAACCTCCCACCTTCTCCCTCAGGGTCACGGCCACAGCGT

[0292] CCAGCGTGGGGGCAGCGGGCATCCCTGCTGGAGGTGTCCTCACTCTGGCCATCATCCTCGAAGCAGTCAACCTCCCGGTCGACCATATCTCCTTG

[0293] ATCCTGGCTGTGGACTGGCTAGTGTGAGTGTGGGTCTGGAGACTGGGGCAGGGCCAGGGGTACCCCTGGGAAAGAGGATGAGTGAGTGAGAGT

[0294] CCTTAGAGCCTCTGGACAGAAGAGGAAGAGAAGAAGGAAGGAATTCTGGAGGAAATGGGGGGAACCTTGAGCATTCCTGAGGGCAAGGAATG

[0295] GCATCTTTGTGGCTCTAAGAGGGATGTCAGAAAAGTAGAGCGTCTGTGTGGTGAATACAGCAGACTCTTCAGCCAGACTGCTCAGGTTCACATCC

[0296] CAGCTCCAACATTCACTGTTGTGACCTCGGGAAGTGCTTAATCTTCCTGCGTCTCAGTTTCTCCATCTGTAAAACGGGGAGAAGAACAGTCTCTC

[0297] CCTAAGGATTAGATAACAGACACACCTCCCTTAGAGCAGCCCCCAGTAAGCAGTAATTGCTACATAATTATTTGCTATCACGATTAAGCTATTAAGA

[0298] GTATAAAGATTGGAAAGCAGCCAGGCACGGTGGCTCACACCTGTAATCCCAACATTTTGGGAGGCCCAGGCAGGTGGATTATTTGAGGTTAGGAG

[0299] TTTGAGACAAGCCTGACCAACATGGTGAAACCCCATCTCCACTAAAAATACAAAAAAATTAGCCGGGTATGGTGGTGTACGCCTGTCCTGTGATC

[0300] TCAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTTGAACTTGGGAGGTGGAGGTTGCAGTGAGCCGAGATTGTGCCACTGCACTCCAGCCTG

[0301] GGCAACAGAGTGAGATCCTGTCTCAAAAAAAAAAAAAAAAAAAAATTCTGGAATTAATTATTGGCAATGGTTGCACAAGTCTGAATATACTAAAA

[0302] AATACCAAGATGGACACTTTTAAAAGGTTAATTTTATGGTAGGTGAATTATAATTTTTTTTCGAGACACAGTCTGGCTCTGTCACCCAGGCTGGAGT

[0303] GTAGTGATGTGATCATGGTTGTGATCACTGCAGCCTCCACCTCCCAGGCTCCAGCGATCCTCCTGCCCCAGCCTCCTGAGTAGCTGGGACTATAGG

[0304] TGTGCACTACCATGCCTGGCTAATTTTTTTTTTTTTTAAATATATATAAAGATGGGATCTCACTATGTTGCCCAGGCTGACTCCAACTCTTAATTTTTA

[0305] CTTTTTAGATTCCAGGGGGAGAGAGGCAGGAGGCTTAGGCCTTGGCCCTTTCCTTCCCTCTCACCCTCTCCCCTCCCGCTTACTTTGCTGTCATTT

[0306] CTACCCCTTTAGCGACCGGTCCTGTACCGTCCTCAATGTAGAAGGTGACGCTCTGGGGGCAGGACTCCTCCAAAATTACGTGGACCGTACGGAGT

[0307] CGAGAAGCACAGAGCCTGAGTTGATACAAGTGAAGAGTGAGCTGCCCCTGGATCCGCTGCCAGTCCCCACTGAGGAAGGAAACCCCCTCCTCA

[0308] AACACTATCGGGGGCCCGCAGGGGATGCCACGGTCGCCTCTGAGAAGGAATCAGTCATGTAAACCCCGGGAGGGACCTTCCCTGCCCTGCTGGG

[0309] GGTGCTCTTTGGACACTGGATTATGAGGAATGGATAAATGGATGAGCTAGGGCTCTGGGGGTCTGCCTGCACACTCTGGGGAGCCAGGGGCCCC

[0310] AGCACCCTCCAGGACAGGAGATCTGGGATGCCTGGCTGCTGGAGTACATGTGTTCACAAGGGTTACTCCTCAAAACCCCCAGTTCTCACTCATGT

[0311] CCCCAACTCAAGGCTAGAAAACAGCAAGATGGAGAAATAATGTTCTGCTGCGTCCCCACCGTGACCTGCCTGGCCTCCCCTGTCTCAGGGAGCA

[0312] GGTCACAGGTCACCATGGGGAATTCTAGCCCCCACTGGGGGGATGTTACAACACCATGCTGGTTATTTTGGCGGCTGTAGTTGTGGGGGGATGTG

[0313] TGTGTGCACGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTCTGTGACCTCCTGTCCCCATGGTACGTCCCACCCTGTCCCCAGATCCC

[0314] CTATTCCCTCCACAATAACAGAAACACTCCCAGGGACTCTGGGGAGAGGCTGAGGACAAATACCTGCTGTCACTCCAGAGGACATTTTTTTTAGCAATAAAATTGAGTGTCAACTATTTAA。

[0315] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0316] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A modified host cell, characterized in that Compared with wild-type host cells, the homologous isoforms of SLC1A5 in the modified host cells are not expressed or are expressed at a low level; Optionally, the SLC1A5 gene in the modified host cell is gene-edited; Optionally, the common coding nucleic acid region of the SLC1A5 isoforms is mutated; Optionally, the mutation includes one or a combination of two or more of an insertion mutation, a deletion mutation and a substitution mutation.

2. The modified host cell according to claim 1, characterized in that The modified host cell satisfies one or more of the conditions shown in (1) and (2): (1) The modified host cell is derived from any one of HEK293 cells and cell lines derived therefrom; Optionally, the HEK293 cells and cell lines derived therefrom include HEK293, HEK293T, HEK293F and HEK293S; Optionally, the nucleotide sequence of the common coding nucleic acid region of the SLC1A5 gene is shown in SEQ ID NO.9; Optionally, the nucleotide sequence of the target editing region in the common coding nucleic acid region of the SLC1A5 gene is shown in SEQ ID NO.8; Optionally, the target editing region is edited into a nucleic acid fragment with a nucleotide sequence as shown in SEQ ID NO.5 and SEQ ID NO.6, or is edited into a nucleic acid fragment with a nucleotide sequence as shown in SEQ ID NO.7; and, (2) The modified host cells include adherent cells.

3. A method for preparing a modified host cell according to any one of claims 1 to 2, characterized in that: The preparation method comprises using gene editing technology to perform gene editing on a host cell to be transformed so that the host cell has the nucleic acid fragment.

4. The method for preparing the modified host cell according to claim 3, characterized in that: The gene editing technology includes CRISPR / Cas9 technology; Optionally, the binding site of the sgRNA used in the preparation method is located in the common coding nucleic acid region of the SLC1A5 gene; Optionally, the nucleotide sequence of the sgRNA is shown in SEQ ID NO.1 and SEQ ID NO.

2.

5. A method for producing a virus, characterized in that: The production method uses the modified host cell according to any one of claims 1 to 2 as a packaging cell to produce the virus; The virus may or may not include a GOI in its genome.

6. The method for producing a virus according to claim 5, characterized in that: The virus includes one or more of a lentivirus and a retrovirus.

7. The method for producing a virus according to claim 5, characterized in that: The viruses include pseudotyped viruses; Optionally, the pseudotyped virus carries one or more of baboon endogenous retrovirus envelope glycoprotein GP70, feline endogenous retrovirus RD114 envelope glycoprotein GP70 and other proteins that bind to SLC1A5.

8. The method for producing a virus according to any one of claims 5 to 7, characterized in that The GOI includes one or more of a selection marker gene and a target gene; Optionally, the selection marker gene includes one or more of a luciferase gene, a fluorescent protein gene, a puromycin resistance gene, a blasticidin resistance gene, a geneticin resistance gene, a bleomycin resistance gene, a neomycin resistance gene, a hygromycin phosphotransferase gene, a dihydrofolate reductase gene, a thymidine kinase gene, a glutamine synthetase gene, an asparagine synthetase gene, a tryptophan synthetase gene, a histidinol dehydrogenase gene, an aminoglycoside phosphotransferase gene, and a tryptophan synthetase gene; Optionally, the target gene encodes one or more of antibodies, recombinant proteins, polypeptides, enzymes, hormones, growth factors, receptors and non-coding RNAs.

9. A method for producing modified immune cells, characterized in that: The production method comprises the following steps: The virus is produced by the production method according to any one of claims 5 to 8; and Infecting the immune cells to be transformed with the virus to prepare the transformed immune cells; Optionally, the modified immune cells include one or more of T cells, TCR-T, CAR-T, TIL, DC-CIK and NK cells.

10. A method for producing a drug, characterized in that: The production method comprises the following steps: Producing the modified immune cells using the production method of claim 9; and, The modified immune cells are prepared into medicines.

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