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Lentiviral vector for CAR-T preparation and construction method and application of lentiviral vector

A technology of lentiviral vector and recombinant lentivirus, applied in the biological field, can solve the problems of low titer of recombinant virus, difficulty in in vivo application, large lentiviral vector, etc., and achieve the effect of increasing the titer of recombinant virus

Inactive Publication Date: 2016-11-09
SHENZHEN PREGENE BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the titer of the recombinant virus is not high enough, except for the results reported by Naldini et al., the others are all at 10 1 TU / ml~10 3 Between TU / ml, it is difficult to meet the needs of in vivo applications
One of the main reasons for the low titer of the recombinant virus is that the lentiviral vector is too large, and the larger vector will directly affect the titer of the virus and the infection efficiency of T cells

Method used

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  • Lentiviral vector for CAR-T preparation and construction method and application of lentiviral vector
  • Lentiviral vector for CAR-T preparation and construction method and application of lentiviral vector
  • Lentiviral vector for CAR-T preparation and construction method and application of lentiviral vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Design and synthesis of CAR-T with lentiviral vector Pre-Lenti-EF1-MCS

[0034] 1.1 Use the pLVX-IRES-Puro lentiviral vector from Clontech Company as the starting vector, the size of pLVX-IRES-Puro is 8140bp, the vector sequence is shown in SEQ ID 3, and the vector map is attached figure 1 .

[0035] 1.2 Synthesis of EF1-MCS sequence (Sino-American Taihe Biotechnology Co., Ltd.)

[0036] Using the pDRAW32 software to analyze the sequence of the pLVX-IRES-Puro vector, it was found that the single restriction site ClaI (sequence ATCGAT, position 2180) and the single restriction site MluI (sequence ACGCGT, position 4049) can cut the CMV on the starting vector +MCS+IRES+Puro element (a total of 1870bp sequence was excised), and the size of the vector after digestion was 6270bp. Using the sequence of the EF1 promoter, plus the sequence of the multiple cloning site of the pLVX-IRES-Puro vector, the sequence EF1-MCS is obtained, see SEQ ID 2. Synthetic EF1-MCS se...

Embodiment 2

[0074] Example 2 Construction of lentiviral vector Pre-Lenti-EF1-MCS-CD19 expressing CD19CAR

[0075] 2.1. CD19CAR gene synthesis

[0076] The CD19CAR gene sequence is shown in SEQ ID 5.

[0077] 2.2 Primer design

[0078] Using the pDRAW32 software to analyze the CD19CAR gene sequence, it was found that the single restriction sites EcoRI (recognition sequence GAATTC) and BamHI (recognition sequence GGATCC) were suitable for gene cloning, and could match the multiple cloning sites of the Pre-Lenti-EF1-MCS vector, Design upstream and downstream primers as follows,

[0079] The EcoRI-forward primer sequence is shown in SEQ ID 6;

[0080] The BamHI-reverse primer sequence is shown in SEQ ID 7.

[0081] 2.3PCR amplification: After receiving the synthesized sequence and primers, perform PCR amplification. The reaction system is shown below (using Toyobo's high-fidelity KOD Fx enzyme),

[0082]

[0083] The reaction procedure is

[0084]

[0085]

[0086] PCR product ...

Embodiment 3

[0099] Example 3. Packaging of Pre-Lenti-EF1-MCS-CD19 lentivirus

[0100] 3.1 The lentiviral packaging follows the conventional method, roughly as follows:

[0101] 3.1.1 Cell culture 293T cells were cultured at 37°C, 5% CO 2 In the incubator, the medium is DMEM / High Glucose / 10% FBS.

[0102] 3.1.2 One day before planting cell-packaging virus, trypsinize 293T cells, 5×10 6 Cells / well are planted in 10cm culture dish, ready to pack Pre-Lenti-EF1-MCS-CD19 lentivirus.

[0103]3.1.3 Cell transfection When cells are transfected, in addition to using the Pre-Lenti-EF1-MCS-CD19 plasmid, each plasmid also needs to be co-transfected with packaging plasmids (providing viral membrane proteins and structural proteins) psPAX2 and pMD2.0G . in

[0104] 5 μg was used for Pre-Lenti-EF1-MCS-CD19, 3.75 μg for psPAX2, and 1.25 μg for pMD2.0G. For transfection, add the mixture of the above three plasmids to 500μl MEM medium, and put 25μl in another microcentrifuge tube

[0105] Add Lipofec...

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Abstract

The invention discloses a lentiviral vector for CAR-T preparation, a construction method of the lentiviral vector and application of the lentiviral vector in preparation of anti-tumor drugs. A method for detecting viral titer through a fluorescent quantitative PCR is further established, and therefore a detection standard is provided for clinical application. The lentiviral vector has the advantages of being small in length, capable of effectively expressing a CAR gene and packaging a lentivirus with sufficient liter and not capable of expressing marker genes such as GFP which is irrelevant to treatment and is suitable for CAR-T research and clinical application through experimental verification.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a lentiviral vector and its preparation and application. Background technique [0002] Immune cell therapy technology is the latest tumor treatment technology that has emerged in recent years. Compared with existing surgery, radiotherapy, chemotherapy, and targeted therapy, it has its own irreplaceable advantages and development potential. Therefore, it is called tumor therapy. the fifth largest technology. Immune cell therapy mainly includes traditional CIK (Cytokine Induced Killer cells, CIK), DC-CIK (Dendritic Cells, DC) and the latest CAR-T technology. CAR-T technology, the full name is Chimeric Antigen Receptor T cells (CAR-T), the main structure of CAR includes a single-chain antibody domain that binds tumor-associated antigen (Tumor Associated Antigen, TAA) extracellularly And the CD28 co-stimulatory signal and CD3ζ signal of activated T cells in the cell cytoplasm, the sequ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867A61K35/17A61P35/00C12Q1/70C12Q1/68C12R1/93
CPCA61K35/17C12N15/86C12N2740/15043C12N2800/107C12Q1/702
Inventor 何昱
Owner SHENZHEN PREGENE BIOPHARMA CO LTD