CD19-CAR-T (Chimeric Antigen Receptor T) cell interfering with IFN (interferon)-gama expression and application thereof

Abstract

The invention discloses two shRNAs (short hairpin RNA) interfering with human IFN (interferon)-gama gene expression shown as SEQ ID NO: 1 and SEQ ID NO: 2. The invention further discloses a lentiviralexpression vector. The lentiviral expression vector contains the shRNAs interfering with the human IFN-gama gene expression and a chimeric antigen receptor encoding nucleotide fragment shown as SEQ ID NO: 4. The invention further discloses a CD19-CAR-T cell interfering with the human IFN-gama gene expression. The CD19-CAR-T cell is a T lymphocyte containing the lentiviral expression vector, or aT lymphocyte which is integrated with the shRNAs interfering with the human IFN-gama gene expression and the chimeric antigen receptor encoding nucleotide fragment in a chromosome. The IFN-gama shRNAsare introduced into the CAR-T cell, the shRNAs of IFN-gama are coexpressed while CAR is expressed, and release of the IFN-gama is inhibited from the source by a gene silencing technology, so that influence of CRS (cytokine release syndrome) is reduced and the safety of CAR-T treatment is improved. Important application to preparation of medicines for prevention, treatment and adjuvant treatment of malignant tumors can be expected.

Description

technical field [0001] The invention relates to the preparation and application of chimeric antigen receptor T cells (Chimeric Antigen Receptor T cells, CAR-T) targeting CD19 that interfere with the expression of IFN-gama, and belong to the fields of genetic engineering and cell biology. Background technique [0002] In 1989, the concept of chimeric antigen receptor-modified T cells (CAR-T) was first proposed to establish adoptive cell therapy with tumor-specific recognition capabilities. The design of the first-generation CAR is to simply replace the extracellular region of the TCR (Tcell receptor) with scFv (single-chain antibody variable region fragment) fragments derived from specific monoclonal antibodies, retaining the transmembrane structure and intracellular signal transmission In the CD3ζ region, some research groups also use FcR for the intracellular signal transmission part. However, the results of the first-generation CAR clinical trials were very disappointing....

AI Technical Summary

Problems solved by technology

In early July 2016, Seattle-based Juno Therapeutics reported that in the company's clinical trial JCAR015, three patients (later confirmed to be four) died after receiving CAR-T cell therapy, and the FDA immediately stopped In the clinical trial of JCAR015, Juno later explained that the use of the chemotherapy drug fludarabine caused neurotoxins, but many people speculated that it was due to the strong cytokine storm (CRS) generated by the input T cells that caused the death of the patient

The earliest method of using RNAi technology to study gene function in organisms is to directly inject single-stranded siRNA molecules prepared in vitro into nematodes. Although it was later used in gene function research, this method has relatively short interference timeliness. Increased concentration of siRNA for effective interference is often cytotoxic

The current RNAi technology mostly adopts the method of constructing a specific RNAi expression vector. After the vector is introduced into animal and plant cells, the corresponding shRNA, amiRNA, etc. can be transcribed by the RNA polymerase type II or III promoter, and then interfere with its target gene. expression

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