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cationic liposome complex encapsulating plasmid dna

A technology of cationic liposomes and complexes, applied in liposome delivery, drug combination, genetic material components, etc., can solve the problems of low expression, weak tumor immunogenicity, immune escape, etc., and achieve low immunogenicity , the effect of improving the cure rate

Active Publication Date: 2022-04-22
CHENGDU NUOEN BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The difference between tumor antigens and normal cell surface proteins is very small, produced by somatic cell mutations, and even only a few amino acids are different, and the expression level is low, so its immunogenicity is very weak, and it is difficult to induce an effective anti-tumor immune response in the body
Tumor cells with strong immunogenicity can activate the body's effective immune recognition response and be eliminated early, while tumors with relatively weak immunogenicity can escape the surveillance of the immune system and multiply and expand. Some tumor cells can express a large number of tumor cells. Embryonic antigen is a normal component in the embryonic stage of a multi-lineage body. The body has innate immune tolerance to it, and it cannot effectively stimulate the body's immune response.
After selection on mutating tumors, surviving tumors become less and less immunogenic, eventually leading to immune escape
[0014] 2. Abnormal expression of MHC antigens
[0021] 7. Abnormality of TIL or abnormality of signal transduction
At present, the commonly used treatment methods for promoting immune response, such as injection of GM-csf factor, are mostly administered through the whole body, which lacks tumor tissue specificity, causes great side effects, and also reduces the curative effect on tumors

Method used

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  • cationic liposome complex encapsulating plasmid dna
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  • cationic liposome complex encapsulating plasmid dna

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Embodiment 1: The recombinant construction of pNE-ORF gene expression plasmid

[0104] The present invention carries out genetic engineering transformation on the basis of the Escherichia coli plasmid pUC19 base sequence (GenBank: M77789.2) template, and designs a minimal exogenous gene expression plasmid containing only 5 necessary functional regions: pNE-ORF, see the structure Figure 1a , the pNE-ORF plasmid DNA has the nucleotide sequence shown in SEQ ID NO:1. The pUC backbone provides the pUC origin of replication for propagation in E. coli and the f1 origin for single-stranded DNA production. Using the pUC origin of replication, when the plasmid is smaller than 3.0 kb, the yield of the plasmid is greatly reduced. Most of the genes expressed in the present invention are cytokines, which are small molecular proteins, such as the ORF of mouse GM-csf is 423bp, and the formed plasmid is close to 3.0kb, which is not easy to produce the plasmid. Adding the f1 fragment ...

Embodiment 2

[0110] Example 2: Expression and secretion of pNE-hGM-csf in H1299 cells

[0111] Four kinds of pNE-ORF expression plasmids: pNE-GFP, pNE-hGM-csf, pNE-sFlt1, pNE-hBax can express GFP, hGM-csf, sFlt1 and hBax proteins in eukaryotic cells accordingly. The cultured H1299 cells were transfected with these four kinds of plasmid DNA, and the content of human GM-csf protein in the cell culture supernatant and cell lysate was determined by ELISA assay to confirm the yield and secretion of the target protein. The specific method is: use a 6-well plate, inoculate 2.4×10 4 cell.

[0112] Plasmid DNA with a concentration of 0.9 μg / μl-1.00.45 μg / μl is mixed with cDLL liposomes in equal volumes to form a cDLL:DNA complex, wherein the concentration of DOTAP is 4 mM, and the concentration of lecithin is 1.8 mM, see Table 2

[0113] Table 2 The particle size and dispersion index of the complexes of different plasmid DNA and cDLL

[0114] cDLL cDLL: mGM-csf cDLL: Flt1-KDR3 ...

Embodiment 3

[0122] Example 3: cDLL: DNA complexes on the growth of in vitro cultured cells

[0123] In this experiment, the effects of GM-csf, IL-2 and other cytokine plasmids wrapped by cDLL on cell growth were detected. The cell lines are normal mouse fibroblast 3T3 cells, non-small cell lung cancer cells: H1299, A549 and H460, and liver cancer cells HepG2. The cells were cultured in RMPI1640 containing 10% FBS. After the cells were digested with trypsin-EDTA, counted, 5000 cells per well were plated on a 96-well plate, and cultured overnight. Plasmid DNA and cDLL were mixed, diluted with RMPI1640 containing 10% FBS to contain 0.2 μg (low concentration) and 0.4 μg DNA (high concentration) per 50 μl of culture medium, and 50 μl of culture medium containing cDLL:DNA complex was added to each well , for cell transfection. On the second day after transfection, replace the medium with 100 μl of fresh medium, continue to cultivate until the third day, add 20 μl cck-8, and incubate at 37°C f...

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Abstract

The invention discloses a cationic liposome complex for encapsulating plasmid DNA, which is composed of cationic liposome and the encapsulating plasmid DNA, wherein the cationic liposome is composed of DOTAP and auxiliary lipid, and the plasmid DNA It is a plasmid carrier for at least one cytokine that promotes immune response; the principle of immunotherapy is applied in the present invention, and through the protection of the liposome, the recombinant gene of the pro-immune cytokine and anti-angiogenic protein can be transported in the blood, and the The liposome complex has the function of causing ischemic necrosis on tumor cell tissue and helping the body to recognize tumor antigens. The liposome GM csf complex of the present invention has a cure rate of 100% for early EMT6 breast cancer cell lung metastases, and a cure rate of 33% for late EMT6 cell lung metastases; the combination of GM csf gene and recombinant VEGFR gene The combined application of liposome complex can increase the cure rate to 50% for advanced EMT6 cell lung metastases.

Description

technical field [0001] The invention relates to the technical field of tumor gene therapy, in particular to a cationic liposome complex encapsulating plasmid DNA and a preparation method thereof. Background technique [0002] Cell division is a normal process of body development and tissue repair. The mother cell divides into two equal daughter cells, which may further differentiate, assemble new tissue, or replace aging cells or damaged tissue. When the body no longer needs more cells, healthy cells stop dividing through contact inhibition, cytokines, growth hormone regulation and other means. When a series of genetic mutations occur in healthy cells, cell division is no longer regulated by the body, thus forming tumor cells. Tumor cells are constantly dividing cells that form tumor masses and can metastasize to distant tissues through the blood. Generally speaking, "cancer" generally refers to all malignant tumors. Malignant tumors have biological characteristics such ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K9/127A61K47/18A61P35/00
CPCA61K47/186A61K48/0025A61P35/00A61K9/127
Inventor 徐凯罗徳伦唐放吴秀景张耀艺
Owner CHENGDU NUOEN BIOLOGICAL TECH
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