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Small interfering nucleic acid and medical composite and pharmaceutical applications of nucleic acid

A small interfering nucleic acid and composition technology, applied in the application field of medicine, can solve the problems of high cytotoxicity and poor biological activity, and achieve the effects of reducing cytotoxicity, inhibiting gene expression, and increasing serum stability

Active Publication Date: 2012-12-05
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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  • Small interfering nucleic acid and medical composite and pharmaceutical applications of nucleic acid
  • Small interfering nucleic acid and medical composite and pharmaceutical applications of nucleic acid
  • Small interfering nucleic acid and medical composite and pharmaceutical applications of nucleic acid

Examples

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preparation example Construction

[0053] 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.

[0054] 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-mentioned A1-A5, such as entrusting Shanghai GenePharma Company to synthesize.

[0055] 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.

[0056] For example, the specific steps are as follows:

[0057] (1) Synthesis of oligoribonucleotides: set synthetic 1 millimolar RNA on an automatic DNA / RNA synthesizer (for example, AppliedBiosystems EXPEDITE8909), and set the coup...

Embodiment 1

[0073] Entrust Shanghai GenePharma Technology Co., Ltd. (GenePharma) to synthesize small interfering nucleic acids with one of the modifications shown in A1-A5.

[0074] Table 1

[0075]

[0076]

Embodiment 2

[0078] The small interfering nucleic acids modified in one of the ways shown in A1-A5 in Example 1 were detected as follows:

[0079] 1. Serum stability test

[0080] 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 .

[0081] 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. After ...

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Abstract

The invention provides small interfering nucleic acid of which sequences are as follows: the sequence of the sense strand is 5'-CCUUGAGGCAUACUUCAAAdTdT-3' and the sequence of the anti-sense strand is 5'-UUUGAAGUAUGCCUCAAGGdTdT-3'. The small interfering nucleic acid has the modifications shown from A1 to A5. The invention also provides a medical composite used for preventing and / or curing hepatitis B and the medical composite contains the small interfering nucleic acid provided by the invention and uses the nucleic acid as the active component. The invention also provides the applications of the small interfering nucleic acid in preparations of medicines for preventing and / or curing hepatitis B. Through the specific modifications, the aim of increasing the serum stability of the small interfering nucleic acid can be achieved under the condition of only introducing a small amount of modifications, thus the potential cytotoxicity of the modified small interfering nucleic acid molecule can be reduced and the influence on the biological activity can be lowered.

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): A61K48/00C12N15/113A61P31/20
Inventor 梁子才张鸿雁
Owner SUZHOU RIBO LIFE SCIENCE CO LTD
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