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Gene medicine for treating hepatitis B and preparation method thereof

A technology of gene medicine and hepatitis B, which is applied in the field of gene medicine for the treatment of hepatitis B and its preparation, and can solve the problems of being unable to directly treat hepatitis B

Inactive Publication Date: 2016-05-11
BIOTOWNTEK CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In view of the above deficiencies in the prior art, the purpose of the present invention is to provide a gene drug for the treatment of hepatitis B and its preparation method, aiming to solve the problem that the prior art cannot directly treat hepatitis B

Method used

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  • Gene medicine for treating hepatitis B and preparation method thereof
  • Gene medicine for treating hepatitis B and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Plasmid vector loaded with CRISPR / Cas9 to treat hepatitis B

[0071] As the most commonly used carrier for expressing genes or RNA, plasmids are widely used. In this example, the most common pcDNA3.1 vector is used including the backbone plasmid vector. After the entire vector is constructed, the HBV-infected human body is injected, and Cas9 can cut the cccDNA of HBV in the liver, thereby eliminating the HBV virus.

[0072] First, the Cas9 gene needs to be constructed into the pcDNA3.1 vector. The construction process is as follows: synthesize the Cas9 gene, and the two ends of the synthesized Cas9 sequence contain EcoRV and NotI double enzyme cleavage sites; use EcoRV and NotI to perform double enzyme digestion on the pcDNA3.1 vector, and then use the gel recovery kit for gel recovery; Mix the pcDNA3.1 vector recovered from the gel with the synthesized Cas9 gene fragment at a ratio of 1:10 (20 μl system), then add T4 DNA ligase buffer solution, and then heat shock in...

Embodiment 2

[0078] Adenovirus loaded with CRISPR / Cas9 to treat hepatitis B

[0079] Adenovirus is also the most commonly used gene therapy vector, which can efficiently express genes or RNA, and is widely used in the field of research and disease treatment. In this example, adenovirus was used to mediate treatment against HBV. After constructing the entire adenovirus vector, co-transfect the 293A cell line with the helper plasmid to package the adenovirus, and then inject the purified adenovirus into a HBV-infected human body. In the liver, Cas9 can cut the cccDNA of HBV, thereby eliminating HBV virus.

[0080] First, the Cas9 gene needs to be constructed into the adenoviral vector pAd-track plasmid vector. The construction process is as follows: the Cas9 gene is synthesized, and the two ends of the synthesized Cas9 sequence contain KpnI and NotI double restriction sites respectively; the pAd-track plasmid vector is double-digested with KpnI and NotI, and then the gel recovery kit is us...

Embodiment 3

[0087] Adeno-associated virus loading CRISPR / Cas9 to treat hepatitis B

[0088] Adeno-associated virus (AAV) is also the most commonly used gene therapy vector, which can efficiently express genes or RNA, and is widely used in the field of research and disease treatment. In this example, AAV virus was used to mediate the treatment against HBV. After constructing the entire AAV virus vector, co-transfect the AAV-293 cell line with the helper plasmid to package the AAV virus, and then inject the purified AAV virus into the HBV-infected human body, Cas9 can cut the cccDNA of HBV in the liver, Thereby eliminating the HBV virus.

[0089] First, the Cas9 gene needs to be constructed into the AAV virus vector pAAV plasmid vector. The construction process is as follows: synthesize the Cas9 gene, and the two ends of the synthesized Cas9 sequence contain EcoRI and BamHI double restriction sites respectively; use EcoRI and BamHI to perform double digestion on the pAAV plasmid vector, a...

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Abstract

The invention discloses gene medicine for treating hepatitis B and a preparation method thereof. The medicine is composed of a carrier, a U6 promoter and a sequence element expressing Cas9 protein and a sequence element expressing sgRNA, wherein the carrier is used for loading the U6 promoter, a Cas9 sequence and a sgRNA sequence and making the U6 promoter, the Cas9 sequence and the sgRNA sequence be expressed in cells. The Cas9 protein expressed in the gene medicine conducts shearing on cccDNA of HBV under the guidance of sgRNA and further destroys cccDNA, the purpose of being clear about HBV is further achieved, and the problem that a current HBV therapeutic method cannot eradicate HBV is solved.

Description

technical field [0001] The invention relates to the field of gene medicine, in particular to a gene medicine for treating hepatitis B and a preparation method thereof. Background technique [0002] Hepatitis is inflammation of the liver, often caused by a viral infection. Among them, viral hepatitis can be divided into A, B, C, D and E types. Although the types of viruses are different, they are all enough to pose serious harm to humans. Among them, hepatitis B and C can lead to liver cirrhosis and liver cancer. It brings a serious disease burden to patients and families. [0003] Especially hepatitis B (Hepatitis B) is very widely distributed in our country. According to authoritative statistics, the infection rate of hepatitis B virus (HBV) in my country is about 60%-70%, and the carrier rate of hepatitis B surface antigen accounts for about 7.18% of the total population. Based on this estimate, there are about 93 million people in the country who carry hepatitis B virus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N15/867C12N15/864C12N15/861C12N15/85A61P1/16A61P31/20
CPCA61K48/005A61K48/0091C12N15/85C12N15/86C12N2710/10041C12N2740/15041C12N2750/14141C12N2800/107C12N2800/22C12N2800/60C12N2800/80C12N2810/10
Inventor 陈光风史秀娟刘小青
Owner BIOTOWNTEK CO LTD
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