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Expression cassette of interleukin-1 receptor antagonist protein and AAV-based gene delivery system

A receptor antagonist protein, gene delivery technology, used in gene therapy, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2022-05-10
SHANGHAI MYGT BIOPHARMACEUTICAL LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although AAV has low immunogenicity, it is often reported clinically that the systemic administration of AAV will trigger a heavier immune response, which is mostly due to the use of higher doses

Method used

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  • Expression cassette of interleukin-1 receptor antagonist protein and AAV-based gene delivery system
  • Expression cassette of interleukin-1 receptor antagonist protein and AAV-based gene delivery system
  • Expression cassette of interleukin-1 receptor antagonist protein and AAV-based gene delivery system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Example 1: Codon optimization and plasmid construction of IL1Ra gene

[0129] First, based on the cDNA gene sequence of human IL1Ra in the NCBI gene bank, the original sequence of IL1Ra (SEQID NO: 6) was codon-optimized to obtain two codon-optimized sequences: IL1Ra optimized sequence one (IL1Ra(1) ) (SEQ ID NO: 7) and IL1Ra optimized sequence two (IL1Ra(2)) (SEQ ID NO: 8).

[0130] Next, construct expression cassettes based on IL1Ra original sequence, IL1Ra optimized sequence 1 and IL1Ra optimized sequence 2:

[0131]With CB as promoter and VH4 as intron, plasmid B93 (SEQ ID NO: 9) was constructed with the original sequence of IL1Ra; with CB as promoter and VH4 as intron, plasmid B94 (SEQ ID NO: 9) was constructed with the optimized sequence of IL1Ra NO: 10); with CB as the promoter, VH4 as the intron, and based on IL1Ra optimized sequence 1, Chi and / or SV40 introns were inserted at the 5' end and 3' end of the transcription unit to construct plasmids B95 to B98 (S...

Embodiment 2

[0133] Example 2: Expression of codon-optimized human IL1Ra-encoding nucleic acid sequence

[0134] Spread Huh7 cells in a 12-well plate at a seeding density of 2E+5 cells / well, and replace serum-free DMEM when the cells increase to 80-90%. A transfection system was prepared, the amount of transfection plasmid was 2.5 μg (plasmid: PEI=1:2), added to each well for 12 hours after transfection, and then replaced with DMEM with 10% fetal bovine serum to continue culturing for 48 hours. Huh7 cells were transfected with 5 plasmids (B94-B98) with the optimized sequence of IL1Ra, and the supernatant was collected after 72 hours to measure the concentration of IL1Ra protein.

[0135] ELISA results showed that the expression of the four plasmids (B95-B98) obtained by inserting Chi and / or SV40 introns into further optimization was higher than that of the B94 plasmid ( Figure 2A ), which indicated that gene expression could be increased by inserting Chi or SV40 introns.

[0136] The ...

Embodiment 3

[0148] Example 3: In vitro biological function of the codon-optimized IL1Ra gene

[0149] Under the stimulation of IL1, C28 / I2 chondrocytes will undergo an inflammatory reaction, which will promote the production of inflammatory factors such as IL1, IL6, and TNFa, which in turn will cause the increase of cartilage matrix degrading enzyme MMP13, leading to the degradation of cartilage matrix. In this embodiment, in order to verify the biological activity of the protein expressed by the transcription and translation of the codon-optimized IL1Ra gene, C28 / I2 chondrocytes were selected for verification.

[0150] C28 / I2 cells were plated in 12-well plates at a seeding density of 2e5 cells / well. When the confluence reaches 80-90%, replace the serum-free medium, and the virus infection MOI is 7.5E+5vg / cell. After 24 hours of infection, the DMEM medium containing 10% fetal bovine serum was replaced. In addition to the blank control wells, a certain amount of IL1 (10ng / ml) was adde...

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Abstract

The invention relates to a nucleic acid molecule for coding interleukin-1 receptor antagonist protein, a transgenic expression cassette containing the nucleic acid molecule, a gene delivery system containing the transgenic expression cassette and application of the gene delivery system. The transgene expression cassette and the gene delivery system can be used for treating various diseases, including malignant tumors and inflammatory diseases, such as joint inflammation, especially osteoarthritis, which can be improved by blocking an IL-1 signal channel.

Description

technical field [0001] The present disclosure relates to a nucleic acid molecule encoding an interleukin-1 receptor antagonist protein, a transgene expression cassette comprising the nucleic acid molecule, a gene delivery system comprising the transgene expression cassette and applications thereof. Background technique [0002] Interleukin-1 (IL-1) is a pleiotropic cytokine that mainly affects inflammation and immune response, and also regulates other physiological functions of the body, which has an important impact on the pathogenesis of diseases. IL-1 levels are elevated in acute inflammatory diseases (such as septic shock), chronic inflammatory diseases (such as rheumatoid arthritis) and malignant tumors. IL-1 is secreted by macrophages and initiates the inflammatory response. In addition, studies have shown that IL-1 plays an important role in tumorigenesis (Krelin Y et al., Cancer Res, 2007;67(3):1062-1071). The IL-1 family mainly includes IL-1α and IL-1β. IL-1β is ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/864C07K14/015A61K38/17A61K48/00A61P19/02A61P19/04A61P29/00A61P35/00
CPCC07K14/7155C12N15/86C07K14/005A61K38/1793A61K48/0058A61K48/0008A61K48/0075A61P29/00A61P35/00A61P19/02A61P19/04C12N2750/14122C12N2750/14143C12N2800/22C12N2830/34C12N2830/50
Inventor 吴侠周凯艺肖啸郑静袁梦孙嘉宝郑浩陈慧蒋威杜增民
Owner SHANGHAI MYGT BIOPHARMACEUTICAL LLC
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