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General type CAR-T cell and preparation method and use thereof

A general-purpose, cell-based technology, applied in the fields of medicine, immunotherapy, biological cytology and molecular biology, can solve problems such as high cost and high price

Active Publication Date: 2019-05-07
GUANGZHOU BIO GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, since CAR-T therapy is individualized for each patient, it can be costly and therefore expensive

Method used

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  • General type CAR-T cell and preparation method and use thereof
  • General type CAR-T cell and preparation method and use thereof
  • General type CAR-T cell and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Construction of Single Gene Knockout Lentiviral Vector

[0083] a. Design Oligo DNA primers: for the first exon sequence of T cell receptor TRAC (α chain) and TRBC (β chain) gene, PD1 receptor gene signal peptide region and Ig-like V-Type region, B2M Oligo DNA primers were designed for the second exon of the gene, and the primer sequences are shown in Table 2:

[0084] Table 1 sgRNA sequence list for single gene knockout

[0085]

[0086] Table 2 Oligo DNA primers

[0087]

[0088]

[0089] b. Anneal and phosphorylate Oligo1 and Oligo2 respectively to form double-stranded DNA. The annealing and phosphorylation system is as follows:

[0090]

[0091] Step down program setting: 37°C for 30min, 95°C for 5min, 90°C for 1min, 85°C for 1min, 80°C for 1min, 75°C for 1min, 70°C for 1min, 65°C for 1min, 60°C for 1min, 55°C for 1min , 50°C 1min, 45°C 1min, 40°C 1min, 35°C 1min, 30°C 1min, 25°C 1min.

[0092] c. The reaction system was diluted 200 times a...

Embodiment 2

[0097] Example 2 Construction of Multiple Gene Knockout Lentiviral Vectors

[0098] The PTG (Polycistronic tRNA-gRNA) structure is a sequence structure in which multiple gRNAs are spaced in series, and the PTG structure can be inserted downstream of the U6 promoter in the LentiCRISPRv2 vector. The PTG sequence of two sgRNAs in series and three sgRNAs in series is inserted into the gene knockout lentiviral vector LentiCRISPRv2, so as to obtain a gene knockout vector that expresses multiple sgRNAs at the same time, thereby achieving the purpose of knocking out multiple genes at the same time. figure 2 Map and elements for the Lenti-PTG-4 lentiviral vector.

[0099] Table 4 The sgRNA sequence information table contained in the PTG of multiple gene knockout

[0100]

[0101] Table 5 PTG sequence list of multiple gene knockout

[0102]

[0103]

[0104] Table 6 Multiple gene knockout lentiviral vectors

[0105]

Embodiment 3

[0106] Example 3 Construction of anti-CD19CAR lentiviral vector

[0107] The anti-CD19 chimeric antigen receptor (CAR) structure includes a CD19 antigen-binding region (derived from the single-chain variable region scFv of the murine monoclonal CD19 antibody FMC63), a CD8α extracellular hinge region, a CD8α transmembrane region, and a 4-1BB intracellular co-stimulatory domain and a CD3ζ activation signal domain, its amino acid sequence is shown in SEQ ID NO: 16, and its nucleic acid sequence is shown in SEQ ID NO: 17. The full length of the sequence is 1458bp, with a total of 486 amino acids. The amino acid sequence of the CAR fusion protein was optimized by the codons of the human species to obtain a nucleic acid sequence, including the N-terminal kozak sequence (GCCGCCACC) and the C-terminal TAA stop codon. The CAR fusion gene sequence is located between the restriction endonucleases EcoRI and BamHI to obtain the full plasmid nucleic acid sequence (7905bp) of the lentiviral...

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Abstract

The invention provides sgRNA or PTG (Polycistronic tRNA-gRNA), through combination with a CRISPR-Cas9 technique, a T cell antigen receptor (TCR) can be knocked out, or one or more of genes relevant tomajor histocompatibility complex (MHCI) and immunosuppression molecules can be knocked out. The invention also provides a general type CAR-T cell which can express chimeric antigen receptor (CAR) butcannot express TCR, and a preparation method of the general type CAR-T cell. The general type CAR-T cell prepared by the preparation method is suitable for heterogeneity antigen, has generality and has higher killability at the same time.

Description

technical field [0001] The invention belongs to the fields of medicine, biological cytology and molecular biology, and relates to the field of immunotherapy. Specifically, it involves the use of sgRNA or PTG (Polycistronic tRNA-gRNA) to knock out one or more genes of T cells and introduce chimeric antigen receptors (CAR) to obtain general-purpose CAR-T cells with enhanced functions. Background technique [0002] In recent years, tumor immunotherapy has entered a stage of rapid development, and CAR-T therapy, as one of the important methods, has achieved very encouraging results in the treatment of hematological malignancies. At present, more and more domestic enterprises, hospitals, and academic institutions are joining in this research field to jointly promote the development of CAR-T therapy and explore a wider range of potential application values. [0003] At present, in the field of chimeric antigen receptor T cell (CAR-T) therapy, autologous CAR-T cells are mainly use...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867C12N15/90C12N5/10A61K35/17A61P35/00A61P35/02A61P37/02
Inventor 李光超罗敏莫文俊周兆邱玉信丁雯
Owner GUANGZHOU BIO GENE TECH CO LTD
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