Low-interfering RNA (ribonucleic acid) medicine for treating chronic hepatitis B and hepatic fibrosis

A technology of liver fibrosis and small interference, applied in DNA/RNA fragments, gene therapy, drug combination, etc., can solve problems such as easy recurrence and drug resistance mutation

Active Publication Date: 2017-01-11
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Interferon therapy is only effective for 10% of the population, and nucleotide analogues are slightly more effect

Method used

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  • Low-interfering RNA (ribonucleic acid) medicine for treating chronic hepatitis B and hepatic fibrosis
  • Low-interfering RNA (ribonucleic acid) medicine for treating chronic hepatitis B and hepatic fibrosis
  • Low-interfering RNA (ribonucleic acid) medicine for treating chronic hepatitis B and hepatic fibrosis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Recombinant vector construction and recombinant virus packaging

[0040] Anneal the oligonucleotide (i.e. the DNA complementary primer corresponding to the small interfering RNA of the present invention, see sequence 3 and sequence 4 in the sequence listing, synthesized by the applicant according to the sequence of the small interfering RNA) to form a double-stranded fragment and clone it into a linearized vector In the pSC-H1-empty vector, the recombinant plasmid pSC-H1-shRNA ( figure 1 ). The brief operation process is as follows:

[0041] Annealed oligonucleotides

[0042] Dissolve oligonucleotides in DNase-free and RNase-free sterile water to a final concentration of 3 mg / mL. The specific calculation formula is as follows:

[0043] The amount of water needed to obtain the final concentration of the oligonucleotide solution=(oligonucleotide quality μg×10 -3 ) / final concentration (X mg / mL)

[0044] To obtain an oligonucleotide solution with a final...

Embodiment 2

[0074] Embodiment 2 in vitro antiviral experiment

[0075] 2.1 Recombinant adeno-associated virus AAV2-shRNA infection of HepG2.2.15

[0076] (1) with 4×10 5 Density of cells / well HepG2.2.15 cells were seeded in a six-well plate, 2 mL of culture medium was added to each well, cultured to a confluence of about 70%, and then infected. Medium components: high glucose DMEM+10%FBS+1% double antibody+G418 (200μg / mL);

[0077] (2) Before infection, aspirate the medium in the wells of the six-well plate, and wash the cells 2-3 times with PBS (1 mL PBS / well). Add MOI=10 respectively 3 , MOI=10 4 , MOI=10 5 The virus (the virus was prepared with PBS to an equal volume of 500 μL) was incubated at 37° C. in 5% CO2 for 3 hours. Aspirate the virus liquid, add 2 mL of complete medium to each well to continue the culture, harvest the supernatant after 24 hours, and add 2 mL of fresh medium to the six-well plate to continue the culture. Subsequently, the supernatant was aspirated every ...

Embodiment 3

[0088] Embodiment 3 in vivo antiviral experiment

[0089] Press 1×10 11 The dose of copy / mouse will inject AAV2-shRNA tail vein into the mouse model of hyperhepatophilic AAV8-1.2HBV hepatitis B chronic infection for 1 month to construct the treatment model, and take serum samples and tissue samples at each time point, Measure each index to evaluate the treatment model.

[0090] The specific scheme is as follows: Dilute AAV8-1.2HBV with PBS to 2×10 11 copy / 200μL PBS, that is, each C57BL / 6 mouse was injected with a volume of 200μL virus, and the virus dose was 2×10 11 copy / only; put the successfully injected mice back into the mouse cage to continue feeding, the feeding conditions were 25°C, humidity 30%-60%, given 12 hours of light and 12 hours of darkness every day. At one month of modeling, the blood of the mice was taken to detect HBV DNA, HBsAg and HBeAg levels, and the mice that were positive for all three were used as mouse models of chronic hepatitis B infection with ...

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Abstract

The invention relates to low-interfering RNA (ribonucleic acid) medicine for treating chronic hepatitis B and hepatic fibrosis. The RNA medicine comprises 19bp oligonucleotides; the shRNA expression is driven by H1 promoters; the medicine is packaged into recombinant viruses by using AAAV2; the transcription, the copying and the expression of HBV (hepatitis B virus) can be obviously inhibited on the HepG2.2.15 cells; the production of the HBV and the occurrence of hepatic fibrosis can be obviously inhibited; the HBV genome in the serum and the liver are obviously reduced; the HBVmRNA in the liver is lowered by more than 90 percent; the surface antigen in the serum is also lowered by more than 10 times; the collagen accumulation in the liver and the collagenous fiber generation can be obviously reduced; the form of the liver cells can be converted into light fibrosis from heavy fibrosis.

Description

technical field [0001] The invention belongs to the field of biological technology inventions, and the invention relates to a small interfering RNA drug, which is a 19bp shRNA mediated and expressed by a recombinant virus AAV, has significant antiviral activity on cell lines in vitro, and also has significant anti-HBV activity and anti- The role of fibrosis. Background technique [0002] HBV infection is the main cause of acute and chronic hepatitis in humans. It is estimated that there are 350 million hepatitis B virus carriers worldwide, and more than 1 million people die each year from HBV-related liver failure, advanced fibrosis (Cirrhosis) and liver cancer (Hepatocellular carcinoma, HCC). Chronic carriers have a 15-25% risk of developing HBV-related disease. HCC accounts for the third place in cancer death, and HBV is the main pathogenic factor of HCC. my country is a high-incidence area of ​​hepatitis B, the prevalence rate in the population is as high as 60%, and 1...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K31/713A61P1/16A61P31/20C12N15/113C12N15/864
CPCA61K31/713A61K48/0008A61K48/005C12N15/113C12N15/86C12N2310/141C12N2750/14143
Inventor 李武平叶磊
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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