Type T non--viral vector and composite medicament containing the same

A non-viral carrier and drug technology, applied in the direction of using vectors to introduce foreign genetic material, drug combinations, anti-tumor drugs, etc., can solve problems such as high cost, antibody neutralization and inactivation, and allergic reactions

Inactive Publication Date: 2009-05-13
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, they are heterologous toxin proteins of the human body, and there are two problems in their clinical application: one is that their cell killing effect does not have tumor cell specificity, so they can cause serious toxic and side effects to the human body; The source protein has strong immunogenicity, and its introduction into the human blood circulation will not only lead to the rapid generation of antibodies and be neutralized and inactivated, but also cause severe allergic reactions in the human body
There are two outstanding shortcomings of this technical route: one is that the cost of chemically sy

Method used

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  • Type T non--viral vector and composite medicament containing the same
  • Type T non--viral vector and composite medicament containing the same
  • Type T non--viral vector and composite medicament containing the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0162] Example 1 Construction of recombinant plasmid 5×NFBS-SCP-Luc (ie 5×κ B sites-survivin core promoter-luciferase)

[0163] 1.1 Cloning of SCP (survivin core promoter) sequence

[0164] It has been confirmed that the gene expression guided by the SCP with a length of 269 bp located at positions 2543-2811 of the DNA sequence (14796 bp) of the human survivin gene has tumor specificity. To evaluate it using the luciferase reporter gene expression system, the SCP will be inserted into the vector through the Nhe I and Sma I sites in the vector MCS. Firstly, it was inserted into pMD18-T simple vector [purchased from Takara Bioengineering (Dalian) Co., Ltd. (TAKARA)]. Primers were designed according to the DNA sequence of the human survivin gene provided by GenBank, and the PCR reaction was carried out with human genomic DNA as a template to obtain a 287bp target fragment-Nhe I—SCP—Sma I—(see figure 2 ). This fragment was ligated with the linear pMD18-T simple vector. The li...

Embodiment 2

[0170] Example 2 Construction of other various luciferase reporter gene expression recombinants

[0171] 2.1 Construction of recombinant plasmid SCP-Luc-Esv40 (survivin core promoter-luciferase-SV40 enhancer)

[0172] The 277bp fragment Nhe I—SCP—Sma I from Example 1.3 was inserted into the plasmid pGL3-Enhancer through the sites Nhe I and Sma I. It has been confirmed that the correct recombinant plasmid SCP-Luc-Esv40 has been obtained.

[0173] 2.2 Construction of recombinant plasmid 5×NFBS-Luc (ie κ B site-luciferase)

[0174] The 93bp fragment Kpn I-5×NFBS-Sac I from Example 1.4 was inserted into the plasmid pGL3-Basic via the sites Kpn I and Sac I. It has been confirmed that the correct recombinant plasmid 5×NFBS-Luc has been obtained.

[0175] 2.3 Construction of recombinant plasmid 5×NFBS-SCP-Luc-Esv40 (ie κ B sites-survivin core promoter-luciferase-SV40 enhancer)

[0176] The 93bp fragment Kpn I-5×NFBS-Sac I from Example 1.4 was inserted into the recombinant plasmid...

Embodiment 3

[0177] Example 3 The liposome-mediated transient transfection of the luciferase reporter gene expression recombinant was used to evaluate the tumor specificity of the gene expression guided by the regulatory sequence.

[0178] 3.1 A large amount of the above-mentioned luciferase expression vectors were extracted.

[0179] Includes: (1) Psv40-Luc-Esv40 (2) SCP-Luc (3) 5×NFBS-SCP-Luc

[0180] (4)SCP-Luc-Esv40 (5)5×NFBS-SCP-Luc-Esv40 (6)5×NFBS-Luc

[0181] 3.2 Detection of gene expression guided by various forms of regulatory sequences

[0182] Mediated by liposomes, the above-mentioned luciferase expression vectors were respectively applied to three tumor cell lines (human breast cancer cell lines MCF-7 and MDA-MB-231, and human cervical cancer cell line HeLa; all three were NF-κ B + SCP + ); and a non-tumor cell line (human embryonic skin fibroblast cell line CCC-ESF-1, for NF-κB + SCP - ) were transiently transfected; and the harvested cells were detected and analy...

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Abstract

The invention relates to a composite medicine of non-viral vector fused protein TNF alpha m2-SON2-GR and limiting expression bacillus pyocyaneus exotoxin gene killing functional domain high-efficiency mutant type recombinant 5xNFBS-SCP-PEIIImut. In the non-viral vector, the TNF alpha m2 is a strong-efficiency and low-toxicity TNF alpha derivative, SON2 is a DNA combined protein, and GRP is gastrin release peptide; the 5 x NFBS of the toxin gene recombinant is the combined site of NF-kappa B repeatedly cloned for 5 times, SCP is a tumor specificity promoter, and PEIIImut is the bacillus pyocyaneus exotoxin gene killing functional domain high-efficiency mutant type. The non-viral vector can combine and carry the toxin gene recombinant to enter positive cells of a GRP acceptor, but the toxin gene can only be expressed in positive malignant tumor cells of the NF-kappa B<+>SCP <+>GRP acceptor, and further strongly efficient kills the tumor cells insensitive to chemical treatment and radioactive treatment. The non-viral vector can independently kill the positive malignant tumor cells of the GRP acceptor. The invention also relates to application of the composite medicine and the independent non-viral vector in the treatment of the malignant tumor.

Description

technical field [0001] The invention belongs to the field of gene therapy drugs. Specifically, the present invention relates to a composite drug of non-viral vector fusion protein TNFαm2-SON2-GRP and restricted expression of Pseudomonas aeruginosa exotoxin gene killing function domain efficient mutant recombinant 5×NFBS-SCP-PEIIImut. TNFαm2 in non-viral vectors is a potent and low-toxic tumor necrosis factor α (TNFα) derivative, SON2 is a DNA-binding protein, and GRP is a gastrin-releasing peptide. The 5×NFBS on the toxin gene recombinant is the binding site of NF-κB cloned five times, SCP is a tumor-specific promoter, and PEIIImut is a high-efficiency mutant of the Pseudomonas aeruginosa exotoxin gene killing domain. This non-viral vector can combine and carry the above toxin gene recombinant into GRP receptor positive cells, but the toxin gene can only be expressed in NF-κB + SCP + GRP receptor-positive malignant tumor cells are expressed, thereby effectively killing such...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/62C07K14/525C07K14/435A61K48/00A61P35/00
Inventor 卢圣栋史艳晖陈伟京李涛路金芝
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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