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Difunctional 3p-siRNA inhibiting hepatitis B viruses, and application thereof

A dual-function, anti-viral technology, used in DNA/RNA fragments, anti-viral agents, recombinant DNA technology, etc., to achieve the effect of inhibiting HBV replication

Inactive Publication Date: 2014-01-15
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, this property is only found in RNA molecules

Method used

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  • Difunctional 3p-siRNA inhibiting hepatitis B viruses, and application thereof
  • Difunctional 3p-siRNA inhibiting hepatitis B viruses, and application thereof
  • Difunctional 3p-siRNA inhibiting hepatitis B viruses, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Cell-level toxicity evaluation of 3p-siRNA(s / p)

[0022] 1.HepG2.2.15 cell culture

[0023] HepG2.2.15 cells were cultured in DMEM medium containing 10% fetal bovine serum and 200ug / ml at 37°C and 5% CO2 incubator. Observe that the cells grow well. After culturing to the logarithmic growth phase, inoculate HepG2.2.15 cells in a 96-well plate (1×10 4 Cell / well), continue to culture.

[0024] 2.3p-siRNA transfection

[0025] The cells inoculated into the 96-well plate were cultured for 24 hours. After the cells grew to 40-60% confluence, without G418, use the transfection reagent jetPRIME and follow the instructions to transfect 3p-siRNA (100nM) for 6 hours Then change to normal cell culture medium (DMEM cell culture medium containing 10% FBS), 37℃, 5% CO 2 Continue to cultivate.

[0026] 3.MTT detection

[0027] 3p-siRNA was transfected according to the above method, the concentration was 100nM and 500nM respectively, and the cell control was set up. Five multiple well...

Embodiment 2

[0030] Example 2: 3p-siRNA(s / p) activates RIG-I-mediated interferon pathway

[0031] 1.293T cell and HepG2.2.15 cell culture

[0032] The cells were cultured in DMEM medium containing 10% fetal bovine serum and 200ug / ml at 37°C and 5% CO2 in an incubator. Observe that the cell growth is in good condition. After culturing to the logarithmic growth phase, inoculate the cells in a 24-well plate containing G418-free, 10% FBS, and DMEM medium (3×10 6 Cells / well), continue to culture overnight.

[0033] 2.3p-siRNA transfection

[0034] The cells inoculated into the 24-well plate were cultured for 24 hours. After the cells were 40-60% confluent, the transfection reagent jetPRIME was used to transfect 3p-siRNA (100nM) according to the instructions in a serum-free state for 6 hours Then change to normal cell culture medium (DMEM cell culture medium containing 10% FBS), 37℃, 5% CO 2 Continue to cultivate.

[0035] 3. Dual luciferase reporter gene analysis method to detect interferon pathway act...

Embodiment 3

[0039] Example 3: Inhibition rate of 3p-siRNA(s / p) on HBsAg and HBeAg for 48 hours, 96 hours and 144 hours

[0040] 1.HepG2.2.15 cell culture

[0041] HepG2.2.15 cells were cultured in DMEM medium containing 10% fetal bovine serum and 200ug / ml at 37°C and 5% CO2 incubator. Observe that the cells grow well. After culturing to the logarithmic growth phase, inoculate HepG2.2.15 cells in a 96-well plate (1×10 4 Cell / well), continue to culture.

[0042] 2.3p-siRNA transfection

[0043] The cells inoculated into the 96-well plate were cultured for 24 hours. After the cells were 40-60% confluent, the transfection reagent jetPRIME was used to transfect 3p-siRNA (100nM) according to the instructions in a serum-free state for 6 hours Then change to normal cell culture medium (DMEM cell culture medium containing 10% FBS), 37℃, 5% CO 2 Continue to cultivate.

[0044] 3. ELISA method to detect HBsAg and HBeAg in cell culture fluid

[0045] The cell culture supernatant was collected 48, 96, and 144 ...

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Abstract

The invention relates to a synthesis method of difunctional 5'-ppp-siRNA (3p-siRNA) for efficiently and specifically inhibiting the replication of hepatitis B viruses (HBVs), and an application of the 3p-siRNA. The 3p-siRNA specifically inhibit the HBV target gene expression through an RNAi mechanism, activates the host interferon expression through an RIG-I dependence mechanism, has an antivirus effect through antiviral natural immunoreactions, and is difunctional anti-HBV novel 3p-siRNA simultaneously having the effects of RNAi and the activation of the host antiviral natural immunoreactions; and the 3p-siRNA has a small toxicity, can efficiently activate the RIG-I mediated host natural antiviral interferon (IFN) expression, can substantially inhibit the HBeAg and HBsAg secretion and the DNA replication of the HBVs, and has an obvious dosage dependency. The anti-HBV difunctional 3p-siRNA is prepared through adopting an in-vitro transcription method and an artificial chemical synthesis method, and can be used for the researches and exploitation of novel HBV medicines. The difunctional 3p-siRNA can be used for the researches and exploitation of other important human viral and bacterial nucleic acid novel medicines.

Description

Technical field: [0001] The invention relates to the synthesis and application of a bifunctional 3p-siRNA that inhibits hepatitis B virus. Background technique: [0002] Hepatitis B virus (HBV) is a virus that can infect globally. It has tissue and species specificity. It can cause chronic hepatitis, cirrhosis and hepatocellular carcinoma. It mainly infects adults and seriously affects human health. And life safety. China is a large country with HBV infection, and the annual direct cost of treatment is as high as 50 billion yuan. There is currently no specific treatment for chronic HBV infection and carriers. The HBV genome is small and susceptible to mutations, which limits the application of antiviral drugs that target viruses. Nucleic acid drugs and interferon-α are currently the most commonly used drugs for the treatment of HBV, but they face problems such as "rebound" with drug withdrawal and drug resistance. So far, there is no specific medicine that can completely cure...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P31/20A61P37/04C12N15/113
Inventor 陈忠斌邢雅玲陈晓娟闫飞
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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