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siRNA sequences for silencing programmed death receptor-ligand expression and their applications

A programmed death and receptor technology, applied in DNA/RNA fragments, recombinant DNA technology, pharmaceutical formulations, etc., can solve problems such as undisclosed siRNA sequences

Active Publication Date: 2021-01-05
WUHAN ZEZHI BIOLOGICAL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention does not disclose the siRNA sequence that can efficiently silence the expression of human PD-L1

Method used

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  • siRNA sequences for silencing programmed death receptor-ligand expression and their applications
  • siRNA sequences for silencing programmed death receptor-ligand expression and their applications
  • siRNA sequences for silencing programmed death receptor-ligand expression and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: RT-qPCR-verified gene silencing efficiency at the RNA level of siPD-L1-1-10.

[0064] Such as figure 1 As shown, according to the known method of cell biology, the human glioma cell line (U87MG) in the logarithmic growth phase was transfected in the cell culture plate, with the assistance of lipofectamine 2000 transfection reagent, 100nM concentration of siPD -L1-1~10 formed lipoplexes with liposomes to transfect cells, and the results of RT-qPCR experiments showed that at the RNA level, siPD-L1-1, siPD-L1-2 siPD-L1-3, siPD-L1 -4, siPD-L1-5 and siPD-L1-8 can effectively silence the expression of PD-L1 / CD274 gene mRNA. Among them, the silencing effect of siPD-L1-8 was the most significant.

Embodiment 2

[0065] Example 2: The surface protein level gene silencing efficiency of siPD-L1-1-10 verified by flow cytometry.

[0066] Such as figure 2 As indicated, according to the well-known method of cell biology, the human glioma cell line (U87MG) in the logarithmic growth phase was transfected in the cell culture plate, with the assistance of lipofectamine 2000 transfection reagent, 100 nM concentration of siPD- L1-1~10 respectively formed liposomes with liposomes to transfect cells. After 48 hours, the cells were collected and flow cytometry was used to detect cell surface proteins. The results showed that at the RNA level, siPD-L1-1, siPD-L1-2, siPD-L1-3, siPD-L1-4, siPD-L1-5 and siPD-L1-8 all silenced cell surface PD-L1 / CD274 protein expression.

Embodiment 3

[0067] Example 3: Flow cytometry verification of the cytotoxic effect of human CD8-positive T cells on glioma cells after PD-L1 / CD274 was silenced.

[0068] Such as image 3 As shown, according to the known method of cell biology, it is known from previous experiments that siPD-L1-3, siPD-L1-5 and siPD-L1-8 can all inhibit the cell surface PD-L1 / CD274 protein by more than 50%. On this basis, the human glioma cell line (U87MG) in the logarithmic growth phase was transfected in the cell culture plate, with the assistance of lipofectamine 2000 transfection reagent, siPD-L1-3 and siPD-L1 at a concentration of 100nM -5 and siPD-L1-8 formed lipoplexes with liposomes to transfect cells, and 24 hours later, human CD8 positive T cells were added for intervention. After continuing to intervene for 24 hours, the apoptosis of glioma cells was detected by flow cytometry. The experimental results showed that siPD-L1-3, siPD-L1-5 and siPD-L1-8 could all induce apoptosis, among which siPD-L...

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Abstract

The invention discloses a siRNA sequence capable of silencing the expression of programmed death receptor-ligand. The siRNA sequence is any one selected from the group consisting of six nucleotide sequences capable of silencing human programmed death receptor-ligand 1; in particular, optimal silencing effect is obtained when a target sequence is GTAGTAGATGTTACAATTT or GGATAAGAACATTATTCAA. The invention can effectively reduce the expression level of PD-L1 / CD274 protein on the surface of tumor cells and promote the killing effect of T lymphocytes on tumor cells by, so tumor growth is inhibited.The siRNA sequence provided by the invention can be applied to designing and preparation of nucleic acid drugs used for preventing and treating various cancers.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an siRNA sequence for silencing gene or protein expression and its application. Background technique [0002] Small interfering RNA (siRNA) can specifically silence the expression level of target mRNA (2006 Nobel Prize in Physiology and Medicine), so siRNA molecules can specifically silence genes that cause or promote the occurrence or development of diseases, and have therapeutic gene overexpression and gene mutation The application prospects of the diseases caused by the drug (Science, 2016, 352(6292): 1417-1420; Nat ChemBiol, 2006, 2(12): 689-700.). Since 2004, the US FDA has approved more than 20 siRNA molecular drug preparations to enter clinical trials, of which nearly 10 phase III clinical trials are underway. In September 2017, Alnylam of the United States (www.alnylam.com) was the first to announce that its siRNA drug Patisiran targeting transthyretin (T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K31/713A61P35/00
CPCA61K31/713A61P35/00C12N15/1135C12N2310/14
Inventor 季爱民岑柏宏
Owner WUHAN ZEZHI BIOLOGICAL PHARMA
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