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Method for preparing clinic-level CAR-T cell preparation by minicircle DNA transfection T cells

A cell preparation, -CAR-T technology, applied in the biological field, can solve problems such as no experimental confirmation

Pending Publication Date: 2017-10-10
蔡子琪
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whether the microcircle DNA can carry the target gene to realize the transfection and modification of human T cells has not yet been experimentally confirmed

Method used

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  • Method for preparing clinic-level CAR-T cell preparation by minicircle DNA transfection T cells
  • Method for preparing clinic-level CAR-T cell preparation by minicircle DNA transfection T cells
  • Method for preparing clinic-level CAR-T cell preparation by minicircle DNA transfection T cells

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Embodiment Construction

[0085] The present invention is further illustrated below through specific examples, but not as a limitation of the present invention.

[0086] The technology for preparing clinical-grade PSCA-CAR-T cell preparations by transfecting T cells with microcircular DNA, the steps are as follows:

[0087] 1. Design and synthesis of PSCA-CAR gene sequence:

[0088] 1.1 Apply the biological software DNAstar8.0 to design the anti-PSCA Fab, VH and VL sequences, which are used to design the scFv sequence of CAR; design the signal peptide sequence IgGkappa to be placed in the front of the PSCA-scFv sequence; according to the CD8, CD28, CD137 and CD3ζ gene sequences are used to synthesize the framework structure of the third-generation CAR, and EcoR1 and BamH1 restriction sites are set at the front and rear ends of the CAR, respectively. According to the above design, the whole gene sequence of PSCA-CAR was synthesized (attached figure 1 ).

[0089] 1.2 Subcloning the PSCA-CAR gene seque...

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Abstract

The invention discloses a method for preparing a clinic-level CAR-T cell preparation by minicircle DNA transfection T cells. The method comprises a technology of preparing the clinic-level CAR-T cell preparation by using non-viral minicircle DNA (minicircle DNA, mcDNA) vector to efficiently transfect T cells. The mcDNA transfection technology comprises the design and synthesis of a chimeric antigen receptor (CAR) carried with a target gene, the construction of target gene-CAR parent vector plasmids, the extraction of target gene-CAR-mcDNA vector plasmids, the target gene-CAR-mcDNA vector plasmid transfection T cells, and the clinic-level CAR-T cell preparation prepared by using the mcDNA transfection technology. The invention further discloses a use of the mcDNA transfection technology. The mcDNA transfection technology has the characteristics of high transfection efficiency to mammal T cells, stable expression, stable function, and no effect to cytogene feature. The prepared CAR-T cell preparation has the characteristics of strong targeting property, high killing efficiency and clinic safety. The mcDNA transfection technology can be used for preparing the clinic-level CAR-T cell preparation for treating various solid tumors and blood and lymphatic system tumors.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing clinical-grade CAR-T cell preparations by applying microcircle DNA transfection technology. Background technique [0002] Tumor biotherapy has been recognized as the fourth major means after the three traditional treatment means of surgery, radiotherapy and chemotherapy. Biological therapy mainly includes two categories: cellular immunotherapy and antibody-targeted drugs. In recent years, cellular immunotherapy has developed rapidly. On the basis of good clinical efficacy of CTL / TIL, a large number of new cell preparations such as immune checkpoint blocking CTL / TIL, TCR-T, and CAR-T have entered clinical trials. CD-19 The clinical trial represented by CD20-targeted CAR-T treatment of B-cell lymphoma has achieved a killing efficiency of more than 90%, which has become a milestone breakthrough. Therefore, CAR-T cell immunotherapy may become an impor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N5/10A61K35/17A61P35/00
CPCC12N5/0636C12N15/66C12N15/85C12N2510/00A61K2239/31A61K39/464493A61K2239/38A61K2239/58A61K39/4631A61K39/4611
Inventor 蔡守锋蔡子琪韩锦胜杜萍萍叶学帅
Owner 蔡子琪
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