Preparation of PD-1 knocked-out CD19 CAR (Chimeric Antigen Receptor)-T cell

Abstract

The invention discloses preparation of a PD-1 knocked-out CD19 CAR (Chimeric AntigenReceptor)-T cell. The preparation comprises the following steps of mixing a CD19 CAR plasmid, a transposase plasmid thereof, an sgRNA (single guide Ribonucleic Acid) plasmid for knocking out a PD-1 gene, a CAS9 plasmid and an electrotransfection reagent, and adding an obtained mixture into a PBMC (Peripheral Blood Mononuclear Cell) to carry out electrotransfection; enriching a CD3 positive T cell by adopting a magnetic bead which is obtained by coupling CD3 or CD3 plus CD28 antibodies; screening, culturing and amplifying the T cell subjected to culture, amplification and transfection to obtain the PD-1 knocked-out CD19 CAR-T cell. According to the preparation, a PiggyBac transposon system is selected and used to improve transfection efficiency and the expression efficiency and time of transgene; the preparation procedure of a CAR-T cell is simplified; the loading capacity of a system is enhanced. In addition, through the preparation, the use of a viral vector is avoided when the PD-1 gene is knocked out; the safety is higher.

Description

technical field [0001] The present invention relates to the preparation of PD-1 knockout CD19 CAR-T cells using the CRISPR-Cas9 system. Background technique [0002] Leukemia, commonly known as "blood cancer", is a kind of malignant clonal disease originating from hematopoietic stem cells. Clonal leukemia cells continue to proliferate, infiltrate other non-hematopoietic tissues and organs, and inhibit normal hematopoiesis. In China, about tens of thousands of patients are diagnosed with B-lineage acute lymphoblastic leukemia (B-ALL) every year. In the United States, about 80% of people with acute leukemia are B-ALL, and 80% of B-ALL patients are treated with first-line chemotherapy. Remission, but still 60% of B-ALL will relapse. Due to the complexity of leukemia classification and prognosis stratification, for example, according to the priority of onset, it can be divided into acute and chronic leukemia, and according to the type of diseased cells, it includes myeloid and...

AI Technical Summary

Problems solved by technology

However, problems such as poor persistence of CAR-T cells, tumor recurrence caused by antigen escape, and potential safety hazards have also been exposed: (1) short survival time

The survival time of CAR-T cells in vivo is not long enough, and the loss of antigens creates an immune escape mechanism, which eventually leads to relapse; (3) The success rate of preparation is not high

Retroviral systems are commonly used to prepare CAR-T, which can effectively infect host cells, but its loading capacity is limited, and the preparation process of recombinant virus particles is complicated

In addition, virus systems may pose security risks

And because the patient has undergone multiple treatments, the activity of T cells will be too low, and in vitro culture cannot guarantee the amount of reinfused CAR-T

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