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Application of CRISPR-Cas9 system based on new gRNA (guide ribonucleic acid) sequence in preparing drugs for treating hepatitis B

A DNA sequence and sequence technology, applied in gene therapy, DNA/RNA fragments, digestive system, etc., can solve the problems of not involving HBV DNA destruction and clearance, difficult to determine the course of treatment, and HBV drug resistance mutation.

Pending Publication Date: 2016-06-08
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nucleoside analogs (Nucleostide Analogues, NAs) can inhibit the replication of hepatitis B virus by inhibiting the activity of DNA reverse transcriptase; Treatment or even life-long treatment, and with the extension of treatment time, the risk of HBV resistance mutations also increases
VyasRamanan, EdwardM.Kennedy, XingLiu, DongC, LuFM, etc. also successively used CRISPR-Cas9 to target the HBV genome, and achieved the inhibition of the expression of HBV cccDNA and HBV-related antigens in HBV cells and HBV hydrodynamic mouse models; Madina Karimova et al. used CRISPR-Cas9 to target the S and X regions of the HBV genome to inhibit HBV replication and expression, and confirmed the destruction of the integrated HBV genome by this system in HeLa and HEK293 cells, but not in Destruction or complete elimination of integrated HBV DNA in HBV stably transferred liver cancer cell models and in HBV transgenic mouse models
However, existing studies have also shown that the inhibitory effects of gRNAs targeting different regions of HBV on HBV replication are also quite different.
In addition, none of the existing studies involved the destruction and clearance of integrated HBV DNA by the CRISPR-Cas9 system
Moreover, none of the above studies have clarified the impact of off-target effects and cytotoxicity of CRISPR-Cas9 on the results

Method used

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  • Application of CRISPR-Cas9 system based on new gRNA (guide ribonucleic acid) sequence in preparing drugs for treating hepatitis B
  • Application of CRISPR-Cas9 system based on new gRNA (guide ribonucleic acid) sequence in preparing drugs for treating hepatitis B
  • Application of CRISPR-Cas9 system based on new gRNA (guide ribonucleic acid) sequence in preparing drugs for treating hepatitis B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Design and synthesis of gRNA sequences targeting HBV conserved sequences of four different serotypes, and construction and screening of corresponding CRISPR-Cas9 systems

[0060] 1. Selection and design of specific gRNA

[0061] Taking the HBV sequence of adr serotype (GenebankID: AB299858) as the reference sequence, the CRISPR online design tool ( http: / / crispr.mit.edu / ) to find a target sequence with a higher score, the form of the target sequence is 5'-N(20)NGG-3' or 5'-CCNN(20)-3'. Then we compared the gene sequences of the main four different serotypes of HBV, including: adr (GenBankaccessionnumber.HQ638218), ayw (U95551), ayr (NC003977), adw (EF103278) and adr (AB299858), from Among the selected target sequences, 8 target sequences located in the conserved region of HBV were selected ( figure 1 , figure 2 ).

[0062] Table 1. Candidate gRNA sequences

[0063]

[0064]

[0065] 2. Synthesis of target sequence and construction of CRISPR-Cas9 system

...

Embodiment 2

[0154] Example 2: Evaluation of the effect of gRNA-S4 target on inhibiting HBV replication

[0155] 1. Detection of HBV surface antigen, HBV DNA and HBV replication intermediates after transfection of gRNA-S4 into HBV stably transfected cells

[0156] a. Cells transfected with CRISPR-Cas9 system with S4 target and CRISPR-Cas9 system without target (control)

[0157] 1) Transfection is performed when the cells adhere to the wall and grow to about 90% of the cell culture dish with a diameter of 10 cm.

[0158] 2) Transfection system (per well):

[0159] Medium500μl+ LTX Reagent 22.5 μl

[0160]

[0161] 3) Mix the above two systems and incubate at room temperature for 30 minutes.

[0162] 4) Add the reaction system to a cell culture dish with a diameter of 10 cm, and add puro screening after 48 hours of culture to improve transfection efficiency

[0163] 5) Divide the screened two groups of cells into 2×10 cells per well 4 The cells were divided into three 24-well pl...

Embodiment 3

[0218] Example 3: Detection of HBV replication in HBV stably transfected cells after S4 site deletion

[0219] 1. Screening of S4 site mutation monoclonal cell lines

[0220] 1.1 Cell transfection with CRISPR-Cas9 system with S4 target and CRISPR-Cas9 system without target (control)

[0221] 1) Transfection is performed when the cells adhere to the wall and grow to about 90% of the cell culture dish with a diameter of 10 cm.

[0222] 2) Transfection system (per well):

[0223] Medium500μl+ LTX Reagent 22.5 μl

[0224]

[0225] 3) Mix the above two systems and incubate at room temperature for 30 minutes.

[0226] 4) Add the reaction system to a cell culture dish with a diameter of 10 cm, and add puro screening after 48 hours of culture to improve transfection efficiency

[0227] 5) Divide the screened two groups of cells into ten 96-well plates with 1 cell per well, subculture to 48-well plates, and extract the total DNA of cells in each well.

[0228] 6) Amplify wi...

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Abstract

The invention discloses a gRNA (guide ribonucleic acid) sequence. The sequence can be used for DNA (deoxyribonucleic acid) sequence edition by using a hepatitis B virus genome S gene conserved region site as a target sequence, can successfully destroy the HBV (hepatitis B virus) cccDNA and integrated-state HBV DNA in a HBV stable cell model and an HBV hydrodynamic mouse model, and obtains obvious effects on inhibiting HBV replication and expression. The invention also discloses application of the CRISPR-Cas9 system containing the gRNA sequence in preparing drugs for treating hepatitis B.

Description

technical field [0001] The invention relates to a gRNA sequence and a technology for gene editing of hepatitis B virus through a CRISPR-Cas9 system, belonging to the technical fields of gene mutation and genetic engineering. Background technique [0002] Hepatitis B is a serious infectious disease caused by Hepatitis B Virus (HBV). A total of 2 billion people in the world have been infected with hepatitis B virus, of which about 400 million people are chronically infected with hepatitis B virus. Persistent HBV infection will cause some patients to deteriorate into liver cirrhosis or even liver cancer. The number of people who die of liver-related diseases caused by HBV reaches 100 every year. million people. my country is a big country with hepatitis B, and there are about 93 million HBV-infected people, including about 20 million chronic hepatitis B patients. The direct cost of hepatitis B treatment exceeds 100 billion yuan each year, and hepatitis B virus infection has b...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63A61K48/00A61K31/713A61P1/16A61P31/20
CPCA61K31/713C12N15/1138C12N15/63C12N2310/11C12N2810/10C12N2999/007
Inventor 宋宏彬邱少富李浩刘鸿博生春雨李鹏戚丽华王立贵谢靖贾雷立郝荣章苏文莉
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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