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Medicine T-VISA-PEA15 capable of effectively and specifically killing breast cancer cells

A T-VISA-PEA15, breast cancer cell technology, applied in the field of medicine and biology, can solve the problems of no tissue specificity of the promoter, low promoter activity, and large toxic and side effects, and achieve no liver and kidney toxicity and high gene expression. , the effect of low toxicity

Active Publication Date: 2014-04-16
SUN YAT SEN UNIV CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cytomegalovirus promoter (CMV) widely used in tumor gene therapy is currently the most active promoter, but the promoter has no tissue specificity and highly expresses the target gene in normal cells. The currently developed clinical trial product Although it has a certain curative effect, it cannot be approved by SFDA (China Food and Drug Administration) and U.S. FDA (Food and Drug Administration) for clinical use due to its large toxicity and side effects.
Some researchers also used tumor-specific promoters instead of CMV for gene therapy research, and found that the toxic and side effects of gene therapy were greatly reduced, but due to the low activity of the promoter, its curative effect became very limited

Method used

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  • Medicine T-VISA-PEA15 capable of effectively and specifically killing breast cancer cells
  • Medicine T-VISA-PEA15 capable of effectively and specifically killing breast cancer cells
  • Medicine T-VISA-PEA15 capable of effectively and specifically killing breast cancer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Construction of T-VISA-PEA15 therapeutic vector

[0019] (1) pCRII-TOPO-hTERT plasmid cloning

[0020] 1. The DNA of breast cancer MCF-7 cells (purchased from ATCC, USA) was extracted by conventional methods.

[0021] 2. Using the DNA as a template, design PCR primers for amplifying hTERT, hTERT PCR upstream (Forward) and downstream (Reverse) primers:

[0022] Forward: 5′-ata tct aga ggc ccc tcc ctc ggg tta ccc cac agc-3′(XbaI)

[0023] Reverse: 5′-ata gat ctt atg cgg ccg ccc acg tgc gca gca gga cgc agc gc-3′.

[0024] 3. Using breast cancer MCF-7 cell DNA as a template, hTERT PCR primers (Forward: 5′-ata tct aga ggc ccc tcc ctc ggg tta ccc cac agc-3′; Reverse: 5′-ata gat ctt atg cgg ccg ccc acg tgc gca gca gga cgc agc gc-3'), Pfu DNA polymerase, dNTP and PCR reaction solution, amplified to obtain hTERT promoter (-416 to +1) product, its sequence is shown in SEQ ID NO.2. Its PCR reaction system: 10×PCR buffer 5 μl, upstream (Forward) and downstream (Rever...

Embodiment 2

[0080] Embodiment 2: the preparation of liposome:

[0081] Lipids were taken out from the refrigerator (DOTAP was stored at -20°C, cholesterol was stored at -4°C) and returned to room temperature. Two rotary evaporators were heated in a water bath to 30°C and 50°C respectively. Weigh 68.75mg of cholesterol and put it into a 1000ml round bottom flask. Into a round bottom flask was added 100 mg DOTAP and 25 mg Chloroform. Swirl the flask to mix well. Rotate the round-bottom flask in a water bath at 30°C for 2 minutes to make it evenly mixed and form a film on the wall of the flask. Turn on the vacuum aspirator, 30min at 30°C. Add 8.9ml of preheated 5% glucose solution to dissolve the dried film, and rotate rapidly at 105 rpm for 45min at 50°C. Then lower the temperature to 35 °C and rotate for 10 min. Seal the flask with plastic wrap (or paraffin), and store it overnight at room temperature, away from light. Measure the volume, add double distilled water to 8.9ml. The fl...

Embodiment 3

[0082] Embodiment 3: the preparation of T-VISA-PEA15 liposome

[0083] T-VISA-PEA15 treatment carrier is dissolved in 5% glucose solution, makes its final concentration 1ug / ul, simultaneously the liposome of the embodiment 2 of storage concentration (20mM) is diluted to working concentration (8mM) with 5% glucose solution . Add 1μg DNA / ul T-VISA-PEA15 therapeutic carrier slowly into 8mM liposomes (therapeutic carrier: liposome = 20ug: 50ul), let stand at room temperature for 20 minutes, react to form T-VISA-PEA15 lipid plastid.

[0084] Then test, biological characteristic detection and quality control such as endotoxin, aseptic packaging. Finished product testing, packaging, storage at 4-8°C; put at room temperature for 20 minutes before use, can be used directly for in vitro research, can also be used for in vivo research, and can also be used after dilution with 5% glucose solution.

[0085] 2. Effect experiment:

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Abstract

The invention discloses a medicine T-VISA-PEA15 capable of effectively and specifically killing breast cancer cells. The sequence of a T-VISA-PEA15 therapy vector capable of effectively and specifically killing the breast cancer cells is shown as SEQ ID NO.1. A liposome of the medicine T-VISA-PEA15 liposome capable of effectively and specifically killing the breast cancer cells comprises a liposome and the T-VISA-PEA15 therapy vector coated by the liposome, wherein the nucleotide sequence of the T-VISA-PEA15 therapy vector is shown as SEQ ID NO.1. After being coated by the liposome and transferred, the T-VISA-PEA15 therapy vector is enriched on the breast cancer part, so as to effectively target the breast cancer ERK (extracellular signal-regulated kinase) target point and inhibit the breast cancer ERK target point from entering the nucleus, thereby promoting the apoptosis of tumor cells, but not killing the normal cells. The medicine T-VISA-PEA15 can be applied to a whole body and has of the advantages of high gene expression amount, long gene expression time, high gene expression efficiency, definite efficacy, low toxicity and side effect, no toxicity to liver and kidneys and wide prospect in the treatment of the breast cancer.

Description

Technical field: [0001] The invention belongs to the field of medical biology, and in particular relates to a drug T-VISA-PEA15 capable of killing breast cancer cells with high efficiency and specificity. Background technique: [0002] Breast cancer is the most common malignant tumor in women, and its incidence is increasing year by year, seriously endangering human physical and mental health. Patients with middle-advanced and recurrent metastatic breast cancer are prone to tolerance to existing radiotherapy and chemotherapy, and their prognosis is poor. Therefore, there is an urgent need to study a new drug with high efficiency targeting to kill breast cancer. [0003] Gene therapy has a development history of more than 20 years, and people hope that it can become an effective alternative therapy besides surgery, radiotherapy and chemotherapy, and targeted therapy. The cytomegalovirus promoter (CMV) widely used in tumor gene therapy is currently the most active promoter, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63A61K48/00A61K9/127A61P35/00
Inventor 谢小明谢新华李来胜郭姣丽伍民庆孔亚楠韦尉东肖祥胜唐军王曦刘鹏
Owner SUN YAT SEN UNIV CANCER CENT
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