Preparation and application of safety-improved lentiviral vector for expressing codon-optimized Anti-MART-1 TCR gene

Abstract

The invention discloses the preparation and application of a lentiviral vector that improves safety and expresses the codon-optimized Anti-MART-1 TCR gene. The safety of this vector is achieved by replacing the WPRE element of the original lentiviral vector with an OPRE element, the sequence of which is shown in SEQ ID No.: 1, and inserting codon-optimized Anti-MART into the vector's multiple cloning site -1 TCR nucleotide sequence, the sequence is shown in SEQ ID No.:2. The OPRE element has no significant impact on the transfection efficiency and packaging titer of lentivirus compared with WPRE; the homology between the codon-optimized Anti-MART-1 TCR gene and the wild-type gene is 79%, and in T cell genes The expression level of Anti-MART-1 TCR can be significantly increased during modification; after T cells genetically modified to express Anti-MART-1 TCR are co-cultured with melanoma cell lines, their IFN-γ secretion levels are also significantly increased; this T cell The cells can also significantly extend the survival time of tumor-bearing mice implanted with melanoma and inhibit the growth of melanoma. The transformed pRRLSIN.cPPT.MSCV‑CoAnti‑MART‑1 / GFP.OPRE vector can be used for the construction of TCR‑T cells in cell therapy.

Description

Technical field [0001] The present invention relates to the fields of gene therapy and cell therapy, and specifically relates to a lentiviral vector that improves safety and is used to express the codon-optimized Anti-MART-1TCR gene and its application. Background technique [0002] Melanoma is a highly malignant tumor that produces melanin. It is more common in adults over 30 years old and often occurs in the skin, mucous membranes and internal organs. Although the incidence of melanoma in China is low, it is highly malignant, metastasizes early, and most of the prognosis is poor. Lymphatic and blood metastasis may occur in the late stage, and the mortality rate is high. Therefore, early diagnosis and early treatment are very important. [0003] For the treatment of melanoma, in addition to traditional surgery, radiotherapy and chemotherapy, cell therapy is also an important method. At present, domestic cell therapy methods for melanoma mainly include CTL (cytotoxic T lymp...

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Problems solved by technology

At present, domestic cell therapy methods for melanoma mainly include CTL (cytotoxic T lymphocytes) and TIL (tumor infiltrating lymphocytes), but there are some problems: although CTL itself has a good effect on targeting and killing melanoma, However, its difficulty in expansion makes it difficult to exert an excellent effect in the treatment of melanoma; TIL is a lymphocyte with anti-melanoma activity isolated from melanoma tissue, which has a good ability to kill melanoma The activity and good expansion of the tumor make it have a good therapeutic effect; however, TIL needs to be isolated from the melanoma tissue of the patient after surgical resection, and the separation process is relatively complicated, so the use of this method is also restricted. many restrictions

[0007] In the prior art, there is no report on improving the safety of lentiviral vectors for WPRE; in addition, although there have been reports that the Anti-MART-1 TCR gene has been introduced into T cells and produced anti-melanoma effects, but The effects of these reports are far from optimal, and there are no reports on the optimization of Anti-MART-1 TCR to improve its effect.

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