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Small hairpin RNA (Ribonucleic Acid) recombination oncolytic adenovirus carrying target mitotic phase phosphoprotein 1 gene

An oncolytic adenovirus, mitotic-phase technology, applied in the field of biopharmaceuticals, can solve problems such as adverse consequences, increase the dosage of drugs, and difficulty in drug aggregation, and achieve the effects of inhibiting the occurrence and development, preventing mitosis, and reducing drug resistance.

Inactive Publication Date: 2014-12-10
WUHAN BAIAOJING BIOLOGICAL PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Second, the current viral vectors of such drugs lack a strong tumor targeting effect, making it difficult for the drugs to form aggregation in the target area. To increase the local concentration of the drug, the dosage of the drug needs to be increased, which is likely to cause the body's immune response and cause adverse consequences; third. , because its carrier is a non-replicating viral vector, the expression time of exogenous anti-cancer genes after infecting tumor cells is relatively short, and it is often cleared by the body before the expression of anti-cancer proteins has formed an effective anti-cancer concentration, resulting in the effect of gene therapy bad
This result has not been reported in the literature at home and abroad before

Method used

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  • Small hairpin RNA (Ribonucleic Acid) recombination oncolytic adenovirus carrying target mitotic phase phosphoprotein 1 gene
  • Small hairpin RNA (Ribonucleic Acid) recombination oncolytic adenovirus carrying target mitotic phase phosphoprotein 1 gene
  • Small hairpin RNA (Ribonucleic Acid) recombination oncolytic adenovirus carrying target mitotic phase phosphoprotein 1 gene

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Construction method of recombinant adenovirus Ad-shMPP1

[0032] Construction of plasmid pSP-Δ55 / shMPP1

[0033] 1) with Xba I and BamThe pXC2 plasmid was digested with HI restriction endonuclease and double-digested, and the 1170 kp fragment was recovered and cloned into the pZD55 vector that was also double-digested to construct the plasmid pXC2-Δ55.

[0034] 2) Using HEK293 cell genomic DNA as a template, the primers are as follows:

[0035] Forward: 5’-ATG AAT TCC GCG TTC TTT GAA AGC AG-3’,

[0036] Reverse: 5’-AAT CTC GAG TGC CGC CGC CGC CAC CT-3’,

[0037] After PCR amplification, a partial fragment of the promoter containing human 269 bp survivin gene was obtained, and cloned into pTG19-T vector to form plasmid pTG19-SP.

[0038] 3) pTG19-SP via xho I and Sal I double digestion, recovered 280 bp fragment and cloned into pXC2-Δ55 Sal I restriction site, after identifying the direction of the survivin gene promoter, the plasmid pSP-Δ55 was o...

Embodiment 2

[0049] Example 2: Ad-shMPP1 inhibits the occurrence of liver cancer

[0050] In an animal orthotopic liver cancer tumor suppressor model, a total of 2 × 10 9 PFU Ad-shMPP1 can significantly inhibit the occurrence of liver cancer xenografts within two weeks ( figure 2 ).

Embodiment 3

[0051] Example 3: Ad-shMPP1 repairs liver damage

[0052] In an animal orthotopic liver cancer tumor suppressor model, a total of 2 × 10 9 PFU Ad-shMPP1 can significantly improve animal liver damage and restore liver function within two weeks, and the values ​​of aspartate aminotransferase and albumin in plasma have been significantly improved ( image 3 ).

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Abstract

The invention belongs to the field of biopharmacy and relates to a small hairpin RNA (Ribonucleic Acid) recombination oncolytic adenovirus carrying a target human kinesin (mitotic phase phosphoprotein 1) gene and a preparation method of the recombination oncolytic adenovirus. The invention provides a brand-new liver cancer drug target, namely mitotic phase phosphoprotein 1 which has a broad spectrum that the traditional gene therapy target p53 gene does not have; the invention further provides the preparation method of the small hairpin RNA recombination oncolytic adenovirus for expressing the mitotic phase phosphoprotein 1; and at the same time, the invention provides an application of the recombination oncolytic adenovirus in preparing a cancer treatment drug.

Description

[0001] technical field [0002] The present invention relates to the field of biopharmaceuticals, in particular to a recombinant oncolytic adenovirus carrying a small hairpin RNA targeting human kinesin-cleavage phase phosphoprotein 1 gene and a preparation method thereof. Background technique [0003] There are approximately 260,000 cases of primary liver cancer each year worldwide. The incidence of liver cancer in my country accounts for more than 40% of the world's total, with more than 100,000 new cases each year. The mortality rate of liver cancer in my country ranks second among all cancer deaths, and about 130,000 patients die of liver cancer every year. Traditional surgical resection and minimally invasive surgery are the main treatment methods at present. However, due to the large size of the liver tumor, poor location, and insufficient liver function reserve, the surgery often fails, coupled with the high postoperative recurrence rate, the surgical treatment of l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/861A61K48/00A61P35/00
Inventor 黄昆刘欣然郑凌吴琼
Owner WUHAN BAIAOJING BIOLOGICAL PHARMA CO LTD
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