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A vector construction method integrating microrna and car functions

A construction method, CD19-CAR technology, applied in retro RNA viruses, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as attacking normal tissues, multiple organ failure in patients, etc., and achieve high safety. and effectiveness

Active Publication Date: 2021-08-06
马晓冬
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  • Claims
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Problems solved by technology

Although CAR-T cell immunotherapy has achieved good results in the treatment of various malignant tumors, it also brings many adverse reactions while curing tumor patients. For example, CAR-T cells will also attack normal cells while clearing tumors. During the treatment, a large number of cytokines will be produced at the same time, leading to multiple organ failure, etc. There have been clinical reports of fatal cases

Method used

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  • A vector construction method integrating microrna and car functions
  • A vector construction method integrating microrna and car functions
  • A vector construction method integrating microrna and car functions

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Embodiment Construction

[0021] The technical solutions of the present invention will be further introduced below in conjunction with the accompanying drawings and specific embodiments.

[0022] This embodiment is a method for constructing a pFIV-miR-301a-CD19CAR system based on a pFIV vector that co-expresses hsa-miR-301a mimics or repressors and expresses CD19-CAR, and the steps are as follows:

[0023](1) Find the sequence of hsa-miR-301a (AccessionID: MIMAT0000688) in the miRBase data, optimize and synthesize the mimetic and repressor of hsa-miR-301a by design; among them, pre-miR-301a promotes the expression of hsa-miR-301a elevated, sponge-miR-301a inhibits hsa-miR-301a expression, and its design optimizes gene sequences such as SEQ ID NO.1 and SEQ ID NO.2.

[0024] (2) Phosphorylation and annealing extension of two single strands of the synthetic hsa-miR-301a mimic (repressor), the reaction system is: hsa-miR-301a mimic (repressor) single strand 1, hsa- miR-301a mimetic (repressor) single chai...

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Abstract

The present invention relates to a vector construction method integrating microRNA and CAR functions, which can express CD19-CAR-based pFIV-microRNA (miR ‑301a)‑CD19CAR system; by introducing microRNA mimics or repressors into the vector carrying the CAR sequence, the simultaneous expression of the microRNA gene regulation function and the CAR of the tumor-specific antigen CD19 is achieved, thereby achieving dual gene editing and Modification function, so as to prepare a new generation of CAR with higher safety and efficacy, which is more suitable for clinical research.

Description

technical field [0001] The present invention relates to pFIV-miR-301a-CD19CAR system construction technology, in particular to a pFIV-miR-301a-CD19- CAR system construction method. Background technique [0002] Chimeric Antigen Receptor (CAR) technology uses gene editing technology to enable T cells to express tumor-specific chimeric antigen receptors, which bind to tumor antigens in a non-major histocompatibility complex (MHC)-restricted manner. Tumors play a killing role. The structure of CAR mainly includes four parts: the extracellular antibody single-chain variable region (which can specifically bind to tumor-associated antigens), the extracellular hinge region, the transmembrane binding region and the intracellular signaling region for activated T cells. The activation of T cells mediated by the first-generation CAR is accomplished through the tyrosine activation motif of the CD3ζ chain or FcRγ, but the first-generation CAR cannot fully activate T cells, and it is di...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/66C12N15/867
CPCC12N15/66C12N15/86C12N2740/15043
Inventor 马晓冬刘明黄行许
Owner 马晓冬
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