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Recombinant hbv cccdna, the method to generate thereof and the use thereof

A genome and site technology, applied in the field including HBV genome, can solve the problems of poor physiological correlation

Active Publication Date: 2017-09-26
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

HBV replication is not driven by cccDNA in transduced and HDI mouse models, making them less physiologically relevant

Method used

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  • Recombinant hbv cccdna, the method to generate thereof and the use thereof
  • Recombinant hbv cccdna, the method to generate thereof and the use thereof
  • Recombinant hbv cccdna, the method to generate thereof and the use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0192] Design and production of HBVcircle

[0193] The plasmid pBR322-HBV1.3 containing the 1.3-unit-length genotype D-type HBV genome (GeneBank JN664917.1) and the sequence of the plasmid are listed as SEQ ID NO:7. The parental minicircle DNA vector plasmid pMC.CMV-MCS-SV40polyA was purchased from System Biosciences (catalogue number MN501A1, sequence number is SEQ ID NO: 13).

[0194] For the parental HBVcircle-CMV-HBV1.1 construct, the HBV genome with a length of 1.1 units starting from nucleotides 1805 to 3182 and 1 to 1990 of the genotype D HBV genome was extracted by PCR from pBR322-HBV1.3, and then used The SalI and NheI sites were cloned into pMC.CMV-MCS-SV40polyA vector. The sequence of the parental HBVcircle-CMV-HBV1.1 construct is set forth in SEQ ID NO:8.

[0195] For the parental HBVcircle-HBV1.3 construct, the pMC.CMV-MCS-SV40 polyA vector was digested with SmaI and KpnI (purchased from New England Biolabs Ltd). To generate an HBV genome insert of 1.3 units in...

Embodiment 2

[0203] Evaluation of HBV replication after HBVcircle transfection in vitro

[0204] HBVcircle, HBVcircle-CMV-HBV1.1 and HBVcircle-HBV1.3 and their parental plasmids were transiently transfected into HepG2 cells for virus replication detection. 72 hours after transfection, cell culture supernatants were collected and analyzed by ELISA and qRT-PCR. HBeAg, HBsAg, and HBV DNA were highly abundant in the supernatant, indicating robust viral replication ( figure 2 A, B and C). Cells were lysed and total DNA was extracted, and cccDNA was quantified using real-time PCR with cccDNA-specific primer and probe sets ( figure 2 D). Compared with the parental HBVcircle-HBV1.3 plasmid carrying the traditional 1.3-unit HBV genome ultra-length design, HBVcircle showed at least comparable or higher expression of HBV markers.

[0205] In addition, HBsAg and HBV core (HBc) proteins were readily detectable in HBVcircle transfected cells by immunofluorescent staining ( figure 2 E). In order...

Embodiment 3

[0208] In vitro assessment of cccDNA markers

[0209] The presence of cccDNA in the nucleus is a unique feature of HBV. To determine whether HBVcircle is capable of forming cccDNA in hepatic nuclei, Southern blot and CHIP analysis were performed. For Southern blot analysis, parental HBVcircle or HBVcircle was first transfected into HepG2 cells, and then Hirt DNA was prepared (Cai et al., 2013, Methods Mol Biol, 1030:151-61). Supercoiled thermostable cccDNA bands appeared only on Southern blots of HBVcircle-transfected cells, but not in parental HBVcircle-transfected cells. When EcoRI was linearized, the cccDNA band disappeared ( Figure 4 A, RC: relaxed loop; DSL: double-stranded linear; CCC: cccDNA). CHIP analysis was performed using HBVcircle transfected cells. Consistent with previous publications, epigenetic modifications including trimethylated lysine 9 (H3K9me3) and acetylated lysine 27 (H3K27ac) are associated with cccDNA (Liu et al., 2013, PLoS Pathog, 9:e1003613 ...

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Abstract

The present invention relates to a recombinant HBV cccDNA comprising HBV genome or the fragment or variant thereof and a site-hybrid insert, a method to generate said recombinant HBV cccDNA, a method for establishment of an in vitro or in vivo cccDNA based model for persistently hepatitis B virus replication by using the recombinant HBV cccDNA of the present invention, and a method for anti-HBV drug evaluation.

Description

technical field [0001] The present invention relates to comprise HBV genome, or its fragment or variant; And the recombinant HBVcccDNA of site hybridization insert fragment; Produce the method for described recombinant HBV cccDNA, be used for establishing the persistent type B by using the recombinant HBV cccDNA of the present invention Methods for in vitro or in vivo cccDNA-based models of hepatitis virus replication, and methods for the evaluation of anti-HBV drug products. Background technique [0002] Hepatitis B virus (HBV) is one of the most dangerous human pathogens. Although safe and effective vaccines have been available for more than two decades, about 2 billion people worldwide are infected with HBV, and more than 350 million people are chronically infected (Liaw et al., 2009, Lancet, 373:582-92). Chronic hepatitis B (CHB) infection predisposes to severe liver disease, including cirrhosis and hepatocellular carcinoma. According to the 2010 Global Burden of Disea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/51G01N33/50G01N33/576
CPCC07K14/005C12N7/00G01N33/5088G01N33/5761C12N2730/10122C12N2730/10121C12N2730/10151A61K49/0008C12N2800/30C12N2800/50C12N2800/80A01K2267/0337A01K67/027A01K2207/05A01K2227/105C12P19/34C12Q1/706C12Q2600/136G01N2333/02G01N2500/10
Inventor 高璐胡慧阎志鹏向昆仑于有军曾晶
Owner F HOFFMANN LA ROCHE & CO AG
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