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siRNA capability of preventing influenza virus as well as expression vector and pharmaceutical composition of siRNA

A technology for expressing vectors and compositions, applied in DNA/RNA fragments, using vectors to introduce foreign genetic material, antiviral agents, etc.

Active Publication Date: 2013-01-23
ARGORNA PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing flu medicines and flu vaccines have major drawbacks

Method used

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  • siRNA capability of preventing influenza virus as well as expression vector and pharmaceutical composition of siRNA
  • siRNA capability of preventing influenza virus as well as expression vector and pharmaceutical composition of siRNA
  • siRNA capability of preventing influenza virus as well as expression vector and pharmaceutical composition of siRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: siRNA that can prevent and treat influenza

[0049] 1. Determination of the target sequence of influenza virus

[0050] More than 3,000 influenza-related genes were collected from the genkbank database. Using bioinformatics analysis methods, these sequences were classified, analyzed, and compared, and a group of influenza viruses targeting PB2, PB1, and PA were found. Highly conserved regions of , MP, NP, NS genes, so as to find out the target sequence, which is the highly conserved regions of different species of influenza virus or influenza virus pressure type genes PB2, PB1, PA, MP, NP, NS.

[0051] The selected artificial sequence interacts specifically with one or several of the following target sequences, which can inhibit the replication or expression of influenza virus genes that cause influenza in humans or animals. The target sequences are as follows:

[0052] TARGET PB2-1: 5'GCCATAATTAAGAAGTACA 3') (SEQ ID NO.43)

[0053] TARGET PB2-2: 5'GAAGTGCT...

Embodiment 2

[0138] Example 2. Effect of siRNA modification on the interference effect

[0139] Using synthetic 2'-O-methyl ribonucleosides, 2'-fluoropyrimidine nucleosides and locked nucleosides, a series of siRNAs with different chemical modifications were synthesized against the luciferase gene. The siRNA sequence is the sense strand 5'UGAAGAGCCUGAUCAAAUA 3 ', the antisense strand is 3'ACUUCUCGGACUAGUUUAU 5', the different modification methods of the site are as ( figure 1 ). These synthesized and modified siRNA molecules were transfected into different cell lines Hela and MCF-7 cells stably expressing the luciferase gene by liposome transfection. and liposomes, and then mix the two together, let stand at room temperature for 20 minutes, and add the siRNA to the cells to complete the transfection after the liposomes are encapsulated. After a certain period of time, the intensity of luciferase fluorescence was detected to evaluate the silencing effects of different modified siRNAs.

...

Embodiment 3

[0141] Embodiment three, the biological activity assay of influenza virus siRNA

[0142] Influenza siRNA was transferred into canine kidney passage cells (MDCK) at a concentration of 100 nM, avian influenza virus H5N1 was added 5 hours after transfection, and untransfected virus (normal) and transfected with no target siRNA were used as controls. After 45 hours of transfection, crystal violet staining was used to detect the interference effect of siRNA on the virus. The results show that siRNA interference with viral genes can effectively inhibit its proliferation in cells image 3 . In the same way, detect the interference effect of siRNA on the virus after 68 hours of transfection, see Figure 4 , the inhibitory effect of siRNA on the virus after transfection 68h (the last group of pictures is 100X, the next group of pictures is 200X), the results show that after 68 hours, siRNA still has a strong inhibitory effect on the virus ( Figure 4 ).

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Abstract

The invention discloses siRNA capability of preventing influenza, the base composition of which is shown in the specification. The invention also provides an expression vector containing the siRNA and a pharmaceutical composition the active ingredient of which is the siRNA or the expression vector. By the siRNA sequence, the reproduction or expression of influenza virus in human bodies or animal bodies can be inhibited.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to an siRNA capable of preventing and treating influenza virus, its expression vector and a medicinal composition. Background technique [0002] The discovery of RNA interference (RNAi) has revolutionized life science research and the development of medical therapeutics. Small interfering RNA (siRNA) is the fastest way to block gene expression and is one of the most promising new drugs in the current pharmaceutical industry. The research and development of related products is experiencing rapid development and fierce competition in the world. siRNA drugs can achieve the purpose of disease treatment by inhibiting and eliminating disease-related genes. Due to its high efficiency, specific targeting and silencing mechanism, it has become the nemesis of traditional incurable diseases. More than 50 siRNA drugs have been approved by the FDA for preclinical or clinical research. Th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63A61K48/00A61P31/16
Inventor 张必良冯世鹏
Owner ARGORNA PHARM CO LTD