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SiRNA (Small interference ribonucleic acid) as well as medicine composition and pharmaceutical application thereof

A technology of small interfering nucleic acid and composition, applied in recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of poor biological activity and high cytotoxicity, and achieve the effect of inhibiting gene expression, reducing cytotoxicity and increasing serum stability

Active Publication Date: 2013-05-22
SUZHOU RIBO LIFE SCIENCE CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem solved by the present invention is to overcome the shortcomings of high cytotoxicity and poor biological activity of the existing small interfering nucleic acid used to inhibit the expression of hepatitis B virus gene, and to provide a stable serum, good biological activity and low Small Interfering Nucleic Acids for Inhibiting Hepatitis B Virus Gene Expression with Cytotoxic Properties

Method used

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  • SiRNA (Small interference ribonucleic acid) as well as medicine composition and pharmaceutical application thereof
  • SiRNA (Small interference ribonucleic acid) as well as medicine composition and pharmaceutical application thereof
  • SiRNA (Small interference ribonucleic acid) as well as medicine composition and pharmaceutical application thereof

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Experimental program
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preparation example Construction

[0059] Optionally, the preparation method of the small interfering nucleic acid further comprises: phosphorothioate modification of the phosphodiester bond between the -3' terminal dTdT of the sense strand and the antisense strand.

[0060] Alternatively, a biotechnology company specializing in nucleic acid synthesis can also be entrusted to synthesize the siRNA of the present invention according to the nucleotide sequence shown in one of the above D1-D7, such as entrusting Shanghai GenePharma Company to synthesize.

[0061] Generally, the method for synthesizing small interfering nucleic acids includes the following four processes: (1) synthesis of oligoribonucleotides; (2) deprotection; (3) purification and separation; (4) desalting.

[0062] For example, the specific steps are as follows:

[0063] (1) Synthesis of oligoribonucleotides: set synthetic 1 millimolar RNA on an automatic DNA / RNA synthesizer (for example, AppliedBiosystems EXPEDITE8909), and set the coupling time ...

Embodiment 1

[0079] Entrust Shanghai GenePharma Technology Co., Ltd. (GenePharma) to synthesize small interfering nucleic acids with one of the modifications shown in D1-D7 below.

[0080] Table 1

[0081]

[0082]

Embodiment 2

[0084] The small interfering nucleic acids with one of the modifications shown in D1-D7 are respectively detected as follows:

[0085] 1. Serum stability test

[0086] Add 10 μL of small interfering nucleic acid with a concentration of 20 μM to a solution (pH 7.4) containing 10 μL of fetal bovine serum and 80 μL of 1×PBS, incubate the reaction system at 37°C for a certain period of time and then take samples, respectively 0, 2, 4, 6, 8, 10, 12, 24 and 48 hours; take 10 μL samples each time and perform quick freezing in liquid nitrogen to immediately stop the effect of serum nucleases on small interfering nucleic acids, and then store the samples at -80°C for later use .

[0087] Configure a 20% by weight polyacrylamide gel, mix 3 μL of loading buffer (20mM EDTA, 36% by weight of glycerol, 0.06% by weight of bromophenol blue) with the degraded sample of siRNA, and then load the sample at 80mA Electrophoresis was performed under constant current conditions for 60 minutes. Aft...

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Abstract

The invention provides a siRNA (small-interference ribonucleic acid) which has the following sequences: a sense strand of 5'-GAAAGUAUGUCAACGAAUUdTdT-3' and an antisense strand of 5'-AAUUCGUUGACAUACUUUCdTdT-3'; and the siRNA is modified by one of the modes shown in D1 to D7. The invention also provides a medicine composition for preventing and / or treating hepatitis B, which contains the siRNA provided in the invention as active constituents. The invention also provides an application of the siRNA in preparing medicaments for preventing and / or treating the hepatitis B. With specific modification, the purpose of increasing the serum stability of the siRNA can be achieved by only introducing a little modification so that the modified siRNA potential cytotoxicity and influences on biological activity are reduced.

Description

technical field [0001] The present invention relates to a small interfering nucleic acid for inhibiting hepatitis B virus gene expression, a pharmaceutical composition containing the small interfering nucleic acid as an active ingredient, and the preparation of the small interfering nucleic acid for preventing and / or treating hepatitis B. application in medicine. Background technique [0002] Hepatitis B, called hepatitis B for short, is an infectious disease caused by hepatitis B virus (HBV). According to the statistics of the World Health Organization (WHO), there are more than 2 billion hepatitis B virus carriers in the world, of which 360 million chronic hepatitis B patients are getting worse, and about 1 million people die every year from liver failure, cirrhosis and liver disease caused by HBV infection. primary hepatocellular carcinoma. China is the country with the largest number of people infected with HBV in the world, and the infection rate of hepatitis B virus ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113
Inventor 梁子才张鸿雁
Owner SUZHOU RIBO LIFE SCIENCE CO LTD
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