Method for preparing general type CAR-T cells by using CRISPR/Cas9+AAV

Abstract

The invention relates to a method for preparing general type CAR-T cells by using CRISPR / Cas9+AAV, and belongs to the field of immunocytotherapy. The method utilizes CRISPR / Cas9+AAV to knock out an immunological checkpoint PD-1, a human leukocyte antigen (HLA) and TCR; multi-gene knockout is employed to prepare the general type CAR-T cells; and a CRISPR / Cas9 gene editing technology is used to perform precise gene knockout, and is used in combination with adeno-associated virus (AAV) to replace lentiviruses for precise gene knock-in and to avoid the potential risk of random integration of the lentiviruses. The method of the invention can realize multi-target gene knock-in and knock-out, construct the general type CAR-T cells for allogeneic treatment, and solve the problems existing in traditional CAR-T cell therapy such as risk of random insertion mutation of lentiviruses, high production cost and long preparation period for autologous CAR-T design and culture, and insufficient cell number and expansion ability.

Description

technical field [0001] The invention relates to a method for preparing universal CAR-T cells by using CRISPR / Cas9+AAV, which belongs to the field of immune cell therapy. Background technique [0002] Chimeric antigen receptor T cells (chimeric antigen receptor T cells, CAR-T) are T cells that have been genetically engineered to obtain single-chain variable regions (scFv) of monoclonal antibodies to target antigens. CAR-T specifically recognizes antigens on the surface of tumor cells through the principle of antigen-antibody binding, so it is not restricted by the major histocompatibility complex (MHC), and it can also avoid immune escape caused by down-regulation or loss of tumor cell MHC. Moreover, by adding co-stimulatory molecular signals during chimeric gene design, the tumor-killing activity of CAR-T cells can be enhanced. After more than 20 years of development, the design of CAR-T has become more and more perfect, and there are more and more methods. [0003] Accord...

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Problems solved by technology

[0004] However, the current CAR-T technology generally has the following problems: (1) It takes a longer time to observe whether the viral vector will bring about harmful insertional mutations: the CAR sequence is the core component of CAR-T cells, and the current manufacture of CAR-T is mainly The method adopted is to use lentivirus as a carrier to randomly integrate the CAR sequence into the T cell genome, thereby realizing the internalization of the exogenous CAR sequence; however, due to the randomness of the integration site of the lentivirus, there is a certain potential risk of random integration , resulting in canceration of transduced T cells, random expression of irrelevant genes or gradual non-expression of antigen receptors, etc.; (2) The cost of CAR-T design and culture preparation is too high: since each patient’s CAR-T Personalized preparation is required, and the cost of treatment is as high as hundreds of thousands of dollars. At the same time, uniform standards cannot be used for quality control; (3) The preparation of autologous CAR-T takes 3-4 weeks, which is very important for cancers where "time is life" It is a very long waiting period for patients; (4) After many patients have undergone multiple rounds of treatment, their autologous T cells have poor activity or limited expansion ability, which directly affects the efficacy of CAR-T, and some patients even cannot Obtain cells capable of expanding and preparing CAR-T

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