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Guide RNA targets for the treatment of hepatitis B virus infection

A target sequence, targeting technology, applied in DNA/RNA fragments, gene therapy, antiviral agents, etc., can solve the problems of HBV reactivation, body intolerance, inability to clear HBV, etc. spectral effect

Active Publication Date: 2017-11-03
鲁凤民 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although nucleotide analogs (Nucleotide, NAs) can effectively inhibit HBV replication by inhibiting the activity of HBV polymerase, they have no effect on HBV covalently closed circular DNA (cccDNA), because cccDNA is HBV The half-life of the replicated template can reach several years to decades, so NAs cannot clear HBV in a short period of time, and factors such as low immunity or drug resistance can induce HBV reactivation and hepatitis recurrence
Although interferon-α (IFN-α) can eliminate HBV in some infected persons, and in vitro experiments have confirmed that high doses of IFN-α can degrade cccDNA, but its clearance rate is low (<8%), and due to high doses of IFN-α has strong side effects, and the body cannot tolerate it
Therefore, HBV cccDNA is a major obstacle in the treatment of chronic hepatitis B (hepatitis B), and there is currently no efficient method for removing HBV cccDNA

Method used

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  • Guide RNA targets for the treatment of hepatitis B virus infection
  • Guide RNA targets for the treatment of hepatitis B virus infection
  • Guide RNA targets for the treatment of hepatitis B virus infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. Design of gRNA sequence

[0056] Download multiple HBV reference sequences of A, B, C and D genotypes from GenBank, analyze the conserved sequences of different genotypes of HBV by MegAlign software, combine the regulatory regions and coding regions that affect HBV replication, according to the gRNA in the CRISPR / Cas9 system According to the design principle, 15 gRNA sequences were designed (Table 1). These gRNAs cover multiple regulatory and coding regions of the HBV genome ( figure 1 ).

[0057] Table 1.15 gRNA target sequence information

[0058]

Embodiment 2

[0059] Example 2. Construction of gRNA expression vector

[0060] According to the pSpCas9(BB)-2A-GFP(p458) (Addgene, 48138) vector backbone, the downstream sequence of the human U6 promoter can be digested with BbsI to form sticky ends of 5”-CACC and 3’-CAAA, as well as the U6 promoter The first base that initiates transcription is the characteristic of "G". Synthesize a pair of oligonucleotides (synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.). The Top sequence is CACCGN (18-20), and the Bottom sequence is AAACN(18-20)C, wherein the N18-20 sequence in the Top oligonucleotide is reverse complementary to the N18-20 sequence in the Bottom. Mix the two synthetic oligonucleotides 1:1, boil and denature for 5 minutes After that, it was slowly annealed to room temperature naturally, and finally the p458 vector (pSpCas9(BB)-2A-GFP) after the annealed oligonucleotide was digested with T4DNA ligase (Takara, D2011A) and BbsI (NEB, R0539S) After ligation at 16°C for 8 hours, ...

Embodiment 315

[0067] Example 3.15 gRNAs inhibit HBV replication result evaluation

[0068] 1) Evaluate the ability of 15 gRNAs to inhibit HBV replication, and evaluate their cytotoxicity. The HBV expression vector and each HBV gRNA expression vector (p458-HBV gRNA) (1.2μg) were co-transfected into Huh7 cells, and the expression levels of HBsAg and HBeAg in the cell culture supernatant were detected. And the cytotoxicity of each gRNA was detected by MTT assay. The results confirmed that all 15 gRNAs could efficiently inhibit the expression level of HBV surface antigen (HBsAg) ( figure 2 A). Most gRNAs can efficiently inhibit the expression level of HBV e antigen (HBeAg) ( figure 2 B). 15 gRNAs had no obvious cytotoxicity ( figure 2 C).

[0069] The HBV expression vector (pBB4.5-HBV1.2) (0.4 μg) and each HBV gRNA expression vector (p458-HBVgRNA) (1.2 μg) were co-transfected into Huh7 cells, and after 72 hours, the cell supernatant was collected and used Time-resolved immunofluoresce...

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Abstract

The invention provides 15 guide RNA (short guide RNA, gRNA) targets for the treatment of hepatitis B virus (Hepatitis B virus, HBV) infection, as well as the medicine for preparing the treatment for HBV infection. The invention efficiently destroys multi-genotype HBV covalently closed circular DNA (Covalently closed circular, cccDNA) through the CRIPSR / Cas system, thereby inhibiting HBV replication. These gRNA targets cover multiple regulatory and coding regions of HBV.

Description

technical field [0001] The present invention relates generally to the fields of molecular biology, cell biology and gene therapy. More specifically, the present invention relates to 15 guide RNA (short guide RNA, gRNA) targets for the treatment of hepatitis B virus (Hepatitis B virus, HBV) infection, the guide RNA obtained by these targets and its recombinant expression vector , and the medicines and methods for the treatment of HBV infection obtained by using these targets and guide RNAs in various ways. Background technique [0002] The current antiviral drugs against HBV are nucleotide analogs and interferon. Although nucleotide analogs (Nucleotide, NAs) can effectively inhibit HBV replication by inhibiting the activity of HBV polymerase, they have no effect on HBV covalently closed circular DNA (cccDNA), because cccDNA is HBV The half-life of the replicated template can reach several years to decades, so NAs cannot clear HBV in a short period of time, and factors such ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/63A61K48/00A61P31/20
Inventor 鲁凤民王杰许中伟
Owner 鲁凤民
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