RNA interference expression plasmid- cationic liposome-heparin antineoplastic complexes targeting human FAK and PLK1 gene

A technology of liposome complexes and cationic liposomes, which is applied in the direction of DNA / RNA fragments, antineoplastic drugs, liposome delivery, etc., can solve the problems of large amount of siRNA, poor quality of life of patients, and not very ideal , to achieve suitable for clinical use, long-lasting in vivo action time, good effect

Inactive Publication Date: 2009-02-04
SICHUAN UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Among them, the problems that need to be solved urgently are: currently there are too few genes used for tumor gene therapy, and there are not many genes that can inhibit tumor growth, so it is urgent to provide more available genes; in addition, due to the occurrence, development, metastasis, etc. The process is a complex network system involving multiple genes and multiple signaling pathways, so single gene therapy or single therapy often has limited efficacy
[0005] At present, RNA interference has been widely used in tumor treatment research, but most of them use chemically synthesized siRNA to treat tumors, and chemically synthesized siRNA therapy has the following disadvantages: chemically synthesized siRNA requires a large amount of use, high cost of use, and difficulty in industrialization Large; chemically synthesized siRNA has poor stability and short time of action in the body, and requires high technical requirements for operators every time it is used; multiple infusions also have a negative impact on the quality of life of patients
At the same time, judging from literature reports, the RNA interference effect of a single gene is often not very ideal, because the occurrence, development, metastasis, and invasion of tumors are a complex process involving multiple genes and multiple steps, which may involve multiple genes, multiple Therefore, RNA interference therapy targeting multiple target genes may be a better choice, and the effect may be better. At the same time, double-gene or even multi-gene RNA interference therapy is used in combination with chemotherapy and radiotherapy in clinical tumor treatment. It may be more promising, but there is no relevant report so far
In addition, the effective introduction of therapeutic genes has always been an important factor affecting the effect of gene therapy. Although there are many studies on improving the efficiency of therapeutic gene transfer and enhancing targeting, it is still far from finding a widely applicable solution. there is a great distance

Method used

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  • RNA interference expression plasmid- cationic liposome-heparin antineoplastic complexes targeting human FAK and PLK1 gene
  • RNA interference expression plasmid- cationic liposome-heparin antineoplastic complexes targeting human FAK and PLK1 gene
  • RNA interference expression plasmid- cationic liposome-heparin antineoplastic complexes targeting human FAK and PLK1 gene

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Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Construction of an shRNA expression plasmid targeting both FAK and PLK1 genes

[0035] Select the siRNA target sequence (SEQ ID NO.1) on the human FAK gene that can significantly inhibit the expression of the gene: 5'-AACCACCTGGGCCAGTATTAT- 3' (human FAK: GenBank accession no: NM_153831) and the human PLK1 gene that can significantly inhibit The siRNA target sequence (SEQ ID NO.2) for inhibiting the expression of this gene: 5'-CCTTGATGAAGAAGATCAC-3' (human PLK1: GenBank accession no: NM_005030).

[0036] The construction process of the shRNA plasmid expression vector expressing the double-gene RNA interference sequence is as follows:

[0037] 1. Design and synthesize the RNA interference expression framework according to the following structure

[0038] Express frame structure:

[0039] (wherein Loop refers to the connection sequence).

[0040] (1) The RNA interference target sequence of the FAK gene is:

[0041] 5'-AACCACCTGGGCCAGTATTAT-3'

[0042] Syn...

Embodiment 2

[0070] Embodiment two, the preparation of plasmid DNA-cationic liposome-heparin complex

[0071] 1. Preparation of DOTAP-Chol cationic liposomes

[0072] The cationic liposome DOTAP was mixed with the neutral component cholesterol (Chol) at a molar ratio of 1:1, and the mixture was dissolved with HPLC grade chloroform, and placed on a rotary evaporator at 40°C for 1 hour to rotate Form a film and dry overnight in vacuum. The formed film was taken out and dissolved in 5% glucose solution, then shaken in a water bath at 55°C for 1 hour, transferred the mixture to a test tube, and squeezed 3 times through a 220nm polycarbonate film, and finally dissolved the mixture In a 5% glucose solution, a 5 mg / ml DOTAP-Chol cationic liposome suspension was obtained.

[0073] 2. Preparation of pGen-(FAK+PLK1) plasmid DNA

[0074] The pGen-(FAK+PLK1) plasmid DNA constructed in the high-purity embodiment 1 was produced in a pilot scale using the bacteria high-density fermenter of 50L and the...

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Abstract

Belonging to the field of tumor gene therapy, the present invention provides an anti-tumor RNA interference plasmid-lipidosome-heparin complex targeting the human FAK and FLK1 genes. The RNA interference expression vector can express a RNA interference sequence aimed at a target site shown by SEQ ID NO:1 on the FAK gene and a RNA interference sequence aimed at a target site shown by SEQ ID NO:2 on the FLK1 gene in the body. The FAK and PLK1 dual-gene RNA interference plasmid prepared by the present invention can effectively inhibit the growth of lung cancer cells and induce the lung cancer cells to be apoptosed. A tumor inhibition experiment conducted in the body also indicates that the FAK and PLK1 dual-gene RNA interference plasmid-lipidosome-heparin complex can notably inhibit the growth of manifold tumors and prolong the lifetime of tumor-bearing mice. The product of the anti-tumor RNA interference plasmid-lipidosome-heparin complex has obvious efficacy and can reduce dosage and improve the life quality of patients, thus having a good application prospect.

Description

technical field [0001] The invention belongs to the field of tumor gene therapy, and in particular relates to an RNA interference expression plasmid-liposome-heparin anti-tumor complex targeting human FAK and PLK1 genes. Background technique [0002] At present, the gene therapy of tumor has been developed vigorously and rapidly, but so far, there are still many problems to be solved. Among them, the problems that need to be solved urgently are: currently there are too few genes used for tumor gene therapy, and there are not many genes that can inhibit tumor growth, so it is urgent to provide more available genes; in addition, due to the occurrence, development, metastasis, etc. The process is a complex network system involving multiple genes and multiple signaling pathways, so a single gene therapy or a single treatment method often has limited efficacy. Therefore, the focus of future gene therapy development will be to mine and identify genes with clinical therapeutic val...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/11A61K48/00A61K9/127A61K47/36A61K47/28A61K47/24A61P35/00
Inventor 魏于全邓洪新陈俐娟
Owner SICHUAN UNIV
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