Packaging system, lentivirus obtained by packaging system and application of lentivirus

Abstract

The invention discloses a packaging system which comprises a transfer plasmid, a packaging plasmid and an envelope plasmid, and the envelope plasmid comprises a nucleotide sequence of a gene for coding vesicular stomatitis virus glycoprotein and a nucleotide sequence of a gene for coding a baboon endogenous virus envelope. The envelope plasmid of the packaging system comprises a nucleotide sequence for coding VSVG (Vesicular Stomatitis Virus Glycoprotein) and a nucleotide sequence for coding BaEV, the technical problem of low transduction rate of CAR-NK cells in the prior art is solved, the transduction rate is improved, and the treatment effect of the CAR-NK cells is further improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to a lentivirus packaging system, a virus prepared by the system, a cell transduced by the virus and the application of the cell. BACKGROUND

[0002] NK cells have the following advantages, first, because CAR-NK does not require donor HLA compatibility, it can be mass-produced to reduce costs, unlike CAR-T which must provide its own T cells for each patient, so CAR-NK has a cost advantage. Second, NK itself has killing power. NK cells themselves express PRRs (Pattern Recognition Receptors), which can recognize and non-specifically kill foreign bacteria, viruses and other microorganisms or tumor cells that are harmful to the body. Third, if NK cells are modified and added with CAR to specifically recognize the target of tumor cells, it becomes an important direction in the field of immune cell therapy. When using viruses to deliver CAR, the membrane protein on the surface of the virus plays a crucial role. The reason is that when the virus membrane protein receptor expressed on the NK cell is matched with the virus membrane protein, the transduction effect will be significantly increased. Because NK itself expresses PRR, it has very high resistance to virus transduction, which also leads to low transduction rate or sometimes high transduction rate but very poor repeatability.

[0003] Currently, the main application in the field is to use VSVG single membrane protein or other single membrane proteins such as BaEV, MV-LV, RD114 to package lentivirus for transduction of NK or NK cell lines. (WO2013045639 LENTIVIRAL VECTORSPSEUDOTYPED WITH MUTANT BaEV GLYCOPROTEINS) discloses the membrane protein obtained by using BaEV and HLV fusion.

[0004] BaEV and VSV-G fusion membrane proteins can transfect human T, B cells (Baboon Envelope Pseudotyped“Nanoblades”Carrying Cas9 / gRNA Complexes Allow Efficient Genome Editing in Human T, B, and CD34+ Cells and Knock-in of AAV6-Encoded Donor DNA in CD34+ Cells, Front. Genome Ed., 09 February 2021).

[0005] This dual-membrane protein is used for CAR-NK cells, and the transduction rate is higher than that of single use (Simultaneous engineering of natural killer cells for CAR transgenesis and CRISPR-Cas9 knockout using retroviral particles, Molecular Therapy Methods & Clinical Development, Volume 29, 8 June 2023, Pages 173-184).

[0006] Although the BaEv-TR mentioned in the paper works better with VSVG, the transduction rate is still low. A packaging system that can improve the transduction rate of CAR-NK cells is needed. SUMMARY

[0007] In order to achieve the above invention purpose, the present application provides a new packaging system, the envelope plasmid of the packaging system includes a nucleotide sequence encoding VSVG (vesicular stomatitis virus glycoprotein) and a nucleotide sequence encoding BaEV, which solves the technical problem of low transduction rate of CAR-NK cells in the prior art, improves the transduction rate, and further increases the treatment effect of CAR-NK cells.

[0008] The first invention purpose of the present application is a packaging system, comprising:

[0009] transfer plasmid, packaging plasmid and envelope plasmid,

[0010] The envelope plasmid contains a nucleotide sequence of a gene encoding vesicular stomatitis virus glycoprotein and a nucleotide sequence of a gene encoding baboon endogenous virus envelope.

[0011] Further, the envelope plasmid includes at least one plasmid.

[0012] Further, the nucleotide sequence of the gene encoding vesicular stomatitis virus glycoprotein and the nucleotide sequence of the gene encoding baboon endogenous virus envelope are on the same envelope plasmid.

[0013] Further, the nucleotide sequence of the gene encoding vesicular stomatitis virus glycoprotein and the nucleotide sequence of the gene encoding baboon endogenous virus envelope are on the same envelope plasmid.

[0014] Further, the packaging system is a four-plasmid system.

[0015] Further, the packaging system is a five-plasmid system.

[0016] Further, the nucleotide sequence of the gene encoding the baboon endogenous virus envelope is BaEvRless.

[0017] The packaging system of the present application comprises a transfer plasmid carrying the gene of interest and elements, a packaging plasmid, and an envelope plasmid. The envelope plasmid carries a nucleotide sequence encoding VSVG (vesicular stomatitis virus glycoprotein) and a nucleotide sequence encoding BaEv (baboon endogenous virus envelope). Further, the nucleotide sequence of BaEv (baboon endogenous virus envelope) used in the present application is BaEvRless. BaEvRless refers to the cytoplasmic tail of the BaEV envelope glycoprotein having no R peptide (Rless) or a R peptide having a length of 3 consecutive amino-terminal amino acids or less relative to the length of the R peptide of the wild-type BaEV envelope glycoprotein.

[0018] The nucleotide sequence encoding VSVG (vesicular stomatitis virus glycoprotein) of the present application is shown in Seq NO. 1, and the nucleotide sequence encoding BaEv (baboon endogenous virus envelope) is shown in Seq NO. 2.

[0019] The nucleotide sequence of VSVG (vesicular stomatitis virus glycoprotein) and the nucleotide sequence of BaEv (baboon endogenous virus envelope) of the present application are located on two different envelope plasmids, or on the same envelope plasmid. Most preferably, they are located on the same envelope plasmid.

[0020] The packaging plasmid in the packaging system is at least one, and the packaging plasmid can be a four-plasmid system or a five-plasmid system. Most preferably, it is a four-plasmid system. In the four-plasmid system, the expression ratio of the two envelopes VSVG:BaEV is 1:0.2-5, further 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5.

[0021] The transfer plasmid of the present application carries the gene of interest and elements, and the elements on the transfer plasmid include an anti-kana mycin protein sequence, an EF-1a promoter, a CD8SP, a FMC63, a CD8Hinge, a CD8TM, a CD28, a 4-1BB, an OX40, a DAP10, a DAP12, a CD3Z, a WPRE, etc., and further uses a transfer plasmid from Suzhou BoTeng Biological

[0022] The packaging plasmid of the present application comprises at least one packaging plasmid, most preferably two packaging plasmids, each carrying a rev gene, a gag gene, and a pol gene.

[0023] The second object of the present application discloses a lentivirus prepared from the plasmid packaging system disclosed in the present application. Further, it uses a lentivirus from Suzhou BoTeng Biological​ of.

[0024] The cells obtained in this invention are used to prepare chimeric antigen receptors for NK cells. The structure of the chimeric antigen receptors for NK cells is determined by the target gene carried by the delivery plasmid. The gene can be expressed as an extracellular ligand-binding domain (scFV), a transmembrane domain, and an intracellular signaling domain. The extracellular ligand-binding domain can specifically target specific antigens on the tumor surface. These specific antigens determine the use of NK cells, with the optimal use being for cancer treatment.

[0025] Compared with the prior art, the present invention has the following technical effects:

[0026] The technical solution of this invention increases the transduction rate of CAR-NK cells to over 80%, far exceeding the publicly disclosed transduction rate of existing technologies. FMC63-CAR-NK cells exhibit a 30%-60% in vitro kill rate. Attached Figure Description

[0027] Figure 1 Using the packaging system of the present invention, wherein the coating plasmid includes VSVG and BaEVRLess; Figure A shows that using the four-plasmid system of the present invention, the CD19 positivity rate of CAR-NK obtained from PMBC of donor 3 is 83.15%; Figure B shows that using the four-plasmid system of the present invention, the CD19 positivity rate of CAR-NK obtained from PMBC of donor 4 is 80.89%; Figure C shows that using the five-plasmid system of the present invention, the CD19 positivity rate of CAR-NK obtained from PMBC of donor 5 is 77.87%.

[0028] Figure 2 Using the packaging system of the present invention, wherein the coating plasmid contains only VSVG; Figure A shows the CD19 positivity rate of CAR-NK obtained from PMBC of donor 3 using the four-plasmid system of the present invention, which is 3.29%; Figure B shows the CD19 positivity rate of CAR-NK obtained from PMBC of donor 4 using the four-plasmid system of the present invention, which is 4.89%; Figure C shows the CD19 positivity rate of CAR-NK obtained from PMBC of donor 5 using the four-plasmid system of the present invention, which is 2.54%.

[0029] Figure 3 Using the packaging system of the present invention, wherein the coating plasmid contains only BaEVRLess; Figure A shows that using the four-plasmid system of the present invention, the CD19 positivity rate of CAR-NK obtained from PMBC of donor 3 is 15.69%; Figure B shows that using the four-plasmid system of the present invention, the CD19 positivity rate of CAR-NK obtained from PMBC of donor 4 is 14.20%; Figure C shows that using the four-plasmid system of the present invention, the CD19 positivity rate of CAR-NK obtained from PMBC of donor 5 is 14.39%.

[0030] Figure 4 Figure A CD19 positive rate of CAR-NK from PMBC of donor 3 using the packaging system of the present application with BaEV TR on the envelope plasmid; Figure B CD19 positive rate of CAR-NK from PMBC of donor 4 using the packaging system of the present application with BaEV TR+VSVG on the envelope plasmid; Figure C CD19 positive rate of CAR-NK from PMBC of donor 5 using the packaging system of the present application with BaEV TR+VSVG on the envelope plasmid.

[0031] Figure 5 Figure A CD19 positive rate of CAR-NK from PMBC of donor 3 using the packaging system of the present application with BaEV TR on the envelope plasmid; Figure B CD19 positive rate of CAR-NK from PMBC of donor 4 using the packaging system of the present application with BaEV TR+VSVG on the envelope plasmid; Figure C CD19 positive rate of CAR-NK from PMBC of donor 5 using the packaging system of the present application with BaEV TR+VSVG on the envelope plasmid.

[0032] Figure 6 Reference for NK cells. Specific embodiments

[0033] The technical means adopted by the present application to achieve the predetermined object of the application are further illustrated in the following drawings and the preparation examples and experiments of the present application.

[0034] Preparation Example 1: Preparation of delivery plasmid

[0035] The delivery plasmid used is FMC63-CAR delivery plasmid from Suzhou BoTeng Bioengineering. The plasmid contains EF-1a promoter, FMC63 scFV, CD8 hinge, CD28 hinge, CD8 TM, CD28 TM, CD28 costimulation, CD137 costimulation, OX40 costimulation, DAP10 costimulation, DAP12 costimulation, CD3z, WPRE, and some elements combined

[0036] Preparation Example 2: Preparation of packaging plasmid

[0037] The packaging plasmid used is pMDLg / pRRE and pRSV-Rev plasmid from Suzhou BoTeng Bioengineering.

[0038] Preparation Example 3: Preparation of envelope plasmid

[0039] The envelope plasmid used is pMD_VSV-G from Suzhou BoTeng Bioengineering, or pMD_BaEvRless, pMD_BaEvTR, pMD_BaEvRless, pMD_VSV-G_BaEvRless, pMD_VSV-G_BaEvTR,

[0040] Preparation Example 4: NK cells from PBMC of human donor 3.

[0041] Preparation Example 5: Virus packaging and infection titer detection

[0042] Lentivirus packaging, the packaging system is prepared from one or two of the plasmids of Preparation Example 1, the plasmid of Preparation Example 2 and the plasmid of Preparation Example 3. Further use is made of LV-SMART from Suzhou BoTeng Bioengineering. Infection titer detection, the virus prepared is used to infect Jurkat cells by gradient dilution, and FACS detection is used to obtain the results.

[0043] Example 1

[0044] NK cells sorted from PBMC of donor 3 in Preparation Example 4 are expanded and cultured to D14, and 5E5 cells are used for transduction. The cells are plated in a 24-well plate, and the lentivirus packaged by the four-plasmid system including VSVG and BaEVRLess in Preparation Example 5 is used for transduction at MOI = 10, 32°C, 1000x g, 60 min, followed by 37°C, 5% CO2 incubator for 4 h. Fresh medium is replaced, and the culture is continued for 72 h.

[0045] Transduction to D4, 200 μl of cells are used, 800 μl of PBS + 2% FBS, 300Xg, room temperature centrifugation for five minutes, the supernatant is discarded, 100 μl of PBS + 2% FBS is used to resuspend the cells, a flow cytometry antibody targeting FMC63 and a flow cytometry antibody targeting CD56 are added, 4°C incubation for 30 min, PBS + 2% FBS washing for 2 times, and then FACS analysis is performed, and the CD19 positive rate of CAR-NK is 83.15%

[0046] Example 2

[0047] NK cells sorted from PBMC of donor 4 in Preparation Example 4 are expanded and cultured to D14, and 5E5 cells are used for transduction. The cells are plated in a 24-well plate, and the lentivirus packaged by the four-plasmid system including VSVG and BaEVRLess in Preparation Example 5 is used for transduction at MOI = 10, 32°C, 1000x g, 60 min, followed by 37°C, 5% CO2 incubator for 4 h. Fresh medium is replaced, and the culture is continued for 72 h.

[0048] Transduction to D4, take 200ul cells, 800ul PBS+2%FBS, 300Xg, centrifugal five minutes at room temperature, discard supernatant, 100ul PBS+2%FBS resuspend cells, add flow cytometry antibody targeting FMC63 and flow cytometry antibody targeting CD56, 4℃ incubate 30min, PBS+2%FBS wash twice, then carry out FACS analysis, the CD19 positive rate of CAR-NK obtained is 80.89%

[0049] Example 3

[0050] NK cells sorted from PBMC of donor 5 in Preparation Example 4 are expanded and cultured to D14, and 5E5 cells are taken for transduction. The cells are plated in a 24-well plate, and the lentivirus packaged by the five-plasmid system including VSVG and BaEVRLess on the envelope plasmid in Preparation Example 5 is taken at MOI=10, 32℃, 1000x g, 60min, and then 37℃, 5% CO2 incubator is cultured for 4h. Fresh culture medium is replaced, and the culture is continued for 72h.

[0051] Transduction to D4, take 200ul cells, 800ul PBS+2%FBS, 300Xg, centrifugal five minutes at room temperature, discard supernatant, 100ul PBS+2%FBS resuspend cells, add flow cytometry antibody targeting FMC63 and flow cytometry antibody targeting CD56, 4℃ incubate 30min, PBS+2%FBS wash twice, then carry out FACS analysis, the CD19 positive rate of CAR-NK obtained is 77.87%

[0052] Example 4

[0053] NK cells sorted from PBMC of donor 3 in Preparation Example 4 are expanded and cultured to D14, and 5E5 cells are taken for transduction. The cells are plated in a 24-well plate, and the lentivirus packaged by the four-plasmid system including VSVG on the envelope plasmid in Preparation Example 5 is taken at MOI=10, 32℃, 1000x g, 60min, and then 37℃, 5% CO2 incubator is cultured for 4h. Fresh culture medium is replaced, and the culture is continued for 72h.

[0054] Transduction to D4, take 200ul cells, 800ul PBS+2%FBS, 300Xg, centrifugal five minutes at room temperature, discard supernatant, 100ul PBS+2%FBS resuspend cells, add flow cytometry antibody targeting FMC63 and flow cytometry antibody targeting CD56, 4℃ incubate 30min, PBS+2%FBS wash twice, then carry out FACS analysis, the CD19 positive rate of CAR-NK obtained is 3.29%

[0055] Example 5

[0056] NK cells sorted from PBMC of donor 4 in Preparation Example 4 were expanded to D14, and 5E5 cells were taken for transduction. Cells were plated in 24-well plate, and lentivirus packaged by four-plasmid system including VSVG on envelope plasmid in Preparation Example 5 was taken, MOI = 10, 32℃, 1000x g, 60min, then 37℃, 5% CO2 incubator for 4h. Fresh medium was replaced, and culture was continued for 72h.

[0057] Transduction to D4, 200ul cells were taken, 800ul PBS+2%FBS, 300Xg, centrifuged for five minutes at room temperature, the supernatant was discarded, 100ul PBS+2%FBS resuspended the cells, added flow cytometry antibody targeting FMC63 and flow cytometry antibody targeting CD56, 4℃ incubated for 30min, washed twice with PBS+2%FBS, then FACS analysis was performed, and the CD19 positive rate of CAR-NK obtained was 4.89%

[0058] Example 6

[0059] NK cells sorted from PBMC of donor 5 in Preparation Example 4 were expanded to D14, and 5E5 cells were taken for transduction. Cells were plated in 24-well plate, and lentivirus packaged by four-plasmid system including VSVG on envelope plasmid in Preparation Example 5 was taken, MOI = 10, 32℃, 1000x g, 60min, then 37℃, 5% CO2 incubator for 4h. Fresh medium was replaced, and culture was continued for 72h.

[0060] Transduction to D4, 200ul cells were taken, 800ul PBS+2%FBS, 300Xg, centrifuged for five minutes at room temperature, the supernatant was discarded, 100ul PBS+2%FBS resuspended the cells, added flow cytometry antibody targeting FMC63 and flow cytometry antibody targeting CD56, 4℃ incubated for 30min, washed twice with PBS+2%FBS, then FACS analysis was performed, and the CD19 positive rate of CAR-NK obtained was 4.89%

[0061] Example 7

[0062] NK cells sorted from PBMC of donor 3 in Preparation Example 4 were expanded to D14, and 5E5 cells were taken for transduction. Cells were plated in 24-well plate, and lentivirus packaged by four-plasmid system including BaEVRLess on envelope plasmid in Preparation Example 5 was taken, MOI = 10, 32℃, 1000x g, 60min, then 37℃, 5% CO2 incubator for 4h. Fresh medium was replaced, and culture was continued for 72h.

[0063] Transduction to D4, take 200ul cells, 800ul PBS+2%FBS, 300Xg, centrifugal five minutes at room temperature, discard supernatant, 100ul PBS+2%FBS resuspend cells, add flow cytometry antibody targeting FMC63 and flow cytometry antibody targeting CD56, 4C incubate 30min, PBS+2%FBS wash twice, then FACS analysis, the CD19 positive rate of CAR-NK obtained is 15.69%

[0064] Example 8

[0065] NK cells sorted from PBMC of donor 4 in Preparation Example 4 were expanded and cultured to D14, and 5E5 cells were taken for transduction. The cells were plated in a 24-well plate, and the lentivirus packaged by the four-plasmid system including BaEVRLess on the envelope plasmid in Preparation Example 5 was taken at MOI = 10, 32C, 1000xg, 60min, and then 37C, 5% CO2 incubator for 4h. Fresh medium was replaced, and the culture was continued for 72h.

[0066] Transduction to D4, take 200ul cells, 800ul PBS+2%FBS, 300Xg, centrifugal five minutes at room temperature, discard supernatant, 100ul PBS+2%FBS resuspend cells, add flow cytometry antibody targeting FMC63 and flow cytometry antibody targeting CD56, 4C incubate 30min, PBS+2%FBS wash twice, then FACS analysis, the CD19 positive rate of CAR-NK obtained is 14.20%

[0067] Example 9

[0068] NK cells sorted from PBMC of donor 5 in Preparation Example 4 were expanded and cultured to D14, and 5E5 cells were taken for transduction. The cells were plated in a 24-well plate, and the lentivirus packaged by the four-plasmid system including BaEVRLess on the envelope plasmid in Preparation Example 5 was taken at MOI = 10, 32C, 1000xg, 60min, and then 37C, 5% CO2 incubator for 4h. Fresh medium was replaced, and the culture was continued for 72h.

[0069] Transduction to D4, take 200ul cells, 800ul PBS+2%FBS, 300Xg, centrifugal five minutes at room temperature, discard supernatant, 100ul PBS+2%FBS resuspend cells, add flow cytometry antibody targeting FMC63 and flow cytometry antibody targeting CD56, 4C incubate 30min, PBS+2%FBS wash twice, then FACS analysis, the CD19 positive rate of CAR-NK obtained is 14.39%

[0070] Example 10

[0071] NK cells sorted from PBMC of donor 3 in Preparation 4 were expanded to D14, and 5E5 cells were used for transduction. Cells were plated in 24-well plate, and the lentivirus packaged by four-plasmid system including BaEV TR on the envelope plasmid in Preparation 5 was used for transduction at MOI = 10, 32°C, 1000x g, 60 min, and then 37°C, 5% CO2 incubator for 4 h. Fresh medium was replaced, and the culture was continued for 72 h.

[0072] Transduction was performed to D4, 200 μl cells were taken, centrifuged at 800 μl of PBS + 2% FBS, 300Xg, at room temperature for five minutes, the supernatant was discarded, 100 μl PBS + 2% FBS was used to resuspend the cells, and flow cytometry antibodies targeting FMC63 and CD56 were added, 4°C incubation for 30 min, PBS + 2% FBS was washed twice, and then FACS analysis was performed, and the CD19 positive rate of CAR-NK obtained was 0.89%

[0073] Example 11

[0074] NK cells sorted from PBMC of donor 4 in Preparation 4 were expanded to D14, and 5E5 cells were used for transduction. Cells were plated in 24-well plate, and the lentivirus packaged by four-plasmid system including BaEV TR on the envelope plasmid in Preparation 5 was used for transduction at MOI = 10, 32°C, 1000x g, 60 min, and then 37°C, 5% CO2 incubator for 4 h. Fresh medium was replaced, and the culture was continued for 72 h.

[0075] Transduction was performed to D4, 200 μl cells were taken, centrifuged at 800 μl of PBS + 2% FBS, 300Xg, at room temperature for five minutes, the supernatant was discarded, 100 μl PBS + 2% FBS was used to resuspend the cells, and flow cytometry antibodies targeting FMC63 and CD56 were added, 4°C incubation for 30 min, PBS + 2% FBS was washed twice, and then FACS analysis was performed, and the CD19 positive rate of CAR-NK obtained was 0.89%

[0076] Example 12

[0077] NK cells sorted from PBMC of donor 5 in Preparation Example 4 were expanded to D14, and 5E5 cells were taken for transduction. Cells were plated in 24-well plate, and the lentivirus packaged by four-plasmid system including BaEV TR on envelope plasmid in Preparation Example 5 was taken, MOI = 10, 32℃, 1000xg, 60min, then 37℃, 5% CO2 incubator for 4h. Fresh medium was replaced, and the culture was continued for 72h.

[0078] Transduction to D4, 200ul cells were taken, 800ul PBS+2%FBS, 300Xg, centrifuged for five minutes at room temperature, the supernatant was discarded, 100ul PBS+2%FBS resuspended the cells, added flow cytometry antibody targeting FMC63 and flow cytometry antibody targeting CD56, 4℃ incubated for 30min, washed twice with PBS+2%FBS, then FACS analysis was performed, and the CD19 positive rate of CAR-NK obtained was 1.62%

[0079] Example 13

[0080] NK cells sorted from PBMC of donor 3 in Preparation Example 4 were expanded to D14, and 5E5 cells were taken for transduction. Cells were plated in 24-well plate, and the lentivirus packaged by four-plasmid system including VSVG+BaEV TR on envelope plasmid in Preparation Example 5 was taken, MOI = 10, 32℃, 1000xg, 60min, then 37℃, 5% CO2 incubator for 4h. Fresh medium was replaced, and the culture was continued for 72h.

[0081] Transduction to D4, 200ul cells were taken, 800ul PBS+2%FBS, 300Xg, centrifuged for five minutes at room temperature, the supernatant was discarded, 100ul PBS+2%FBS resuspended the cells, added flow cytometry antibody targeting FMC63 and flow cytometry antibody targeting CD56, 4℃ incubated for 30min, washed twice with PBS+2%FBS, then FACS analysis was performed, and the CD19 positive rate of CAR-NK obtained was 1.62%

[0082] Example 14

[0083] NK cells sorted from PBMC of donor 4 in Preparation Example 4 were expanded to D14, and 5E5 cells were taken for transduction. Cells were plated in 24-well plate, and the lentivirus packaged by four-plasmid system including VSVG+BaEV TR on envelope plasmid in Preparation Example 5 was taken, MOI = 10, 32℃, 1000xg, 60min, then 37℃, 5% CO2 incubator for 4h. Fresh medium was replaced, and the culture was continued for 72h.

[0084] Transduction to D4, take 200ul cells, 800ul PBS+2%FBS, 300Xg, room temperature centrifugation for five minutes, discard the supernatant, 100ul PBS+2%FBS resuspended cells, add FMC63 targeting flow antibody and CD56 targeting flow antibody, 4C incubation for 30min, PBS+2%FBS wash 2 times, then FACS analysis, the CD19 positive rate of CAR-NK obtained is 4.11%

[0085] Example 15

[0086] NK cells sorted from PBMC of donor 5 in Preparation Example 4 were expanded and cultured to D14, and 5E5 cells were taken for transduction. The cells were plated in a 24-well plate, and the five-plasmid system packaged lentivirus including VSVG+BaEVTR in Preparation Example 5 was taken, MOI=10, 32C, 1000xg, 60min, then 37C, 5%CO2 incubator for 4h. Fresh medium was replaced, and the culture was continued for 72h.

[0087] Transduction to D4, take 200ul cells, 800ul PBS+2%FBS, 300Xg, room temperature centrifugation for five minutes, discard the supernatant, 100ul PBS+2%FBS resuspended cells, add FMC63 targeting flow antibody and CD56 targeting flow antibody, 4C incubation for 30min, PBS+2%FBS wash 2 times, then FACS analysis, the CD19 positive rate of CAR-NK obtained is 4.11%

[0088] Example

[0089] Enveloped plasmid Plasmid system Source Example 1 VSVG + BaEV RLess Four-plasmid system Donor 3 Example 2 VSVG + BaEV RLess Four-plasmid system Donor 4 Example 3 VSVG + BaEV RLess Four-plasmid system Donor 5 Example 4 VSVG Four-plasmid system Donor 3 Example 5 VSVG Four-plasmid system Donor 4 Example 6 VSVG Four-plasmid system Donor 5 Example 7 BaEV RLess Four-plasmid system Donor 3 Example 8 BaEV RLess Four-plasmid system Donor 4 Example 9 BaEV RLess Four-plasmid system Donor 5 Example 10 BaEV TR Four-plasmid system Donor 3 Example 11 BaEV TR Four-plasmid system Donor 4 Example 12 BaEV TR Four-plasmid system Donor 5 Example 13 VSVG + BaEV TR Four-plasmid system Donor 3 Example 14 VSVG + BaEV TR Four-plasmid system Donor 4 Example 15 VSVG + BaEV TR Five-plasmid system Donor 5

[0090] The above only is the preferred embodiment of the present application, not any form of the present application is limited, although the present application has been disclosed as above, however, not in order to limit the present application, any skilled in the art, without departing from the scope of the present application, when the above disclosed technical content can be made some changes or modifications of equivalent changes, but any simple modification, equivalent changes and modification of the above examples, as long as it does not deviate from the scope of the present application, according to the technical essence of the present application, all belong to the scope of the present application.

Claims

1. A packaging system characterized by, Comprising: a transfer plasmid, a packaging plasmid and an envelope plasmid, the envelope plasmid comprises a nucleotide sequence of a gene encoding a vesicular stomatitis virus glycoprotein and a nucleotide sequence of a gene encoding a baboon endogenous virus envelope.

2. The packaging system of claim 1, wherein, the envelope plasmid comprises at least one plasmid.

3. The packaging system of claim 2, wherein, the nucleotide sequence of the gene encoding a vesicular stomatitis virus glycoprotein and the nucleotide sequence of the gene encoding a baboon endogenous virus envelope are on the same envelope plasmid.

4. The packaging system of claim 2, wherein, the nucleotide sequence of the gene encoding a vesicular stomatitis virus glycoprotein and the nucleotide sequence of the gene encoding a baboon endogenous virus envelope are on different envelope plasmids.

5. The packaging system of claim 4, wherein, the packaging system is a four-plasmid system.

6. The packaging system of claim 5, wherein, the packaging system is a five-plasmid system.

7. The packaging system of claim 6, wherein, the nucleotide sequence of the gene encoding a baboon endogenous virus envelope is BaEvRless.

8. A lentivirus prepared from the plasmid packaging system of any one of claims 1-7.

9. A cell transfected with the lentivirus of claim 8.

10. Use of the cell of claim 9 for the preparation of a chimeric antigen receptor for NK cells.

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