A kind of plasmid and preparation for directional clearance of hbvcccDNA in hepatocytes

A preparation and auxiliary lipid technology, which is applied in the field of biomacromolecular pharmaceutical preparations, can solve the problems of lack of safe and effective preparations for in vivo application, and achieve the effects of reducing the expression level of antigens, overcoming instability in vivo, and high stability

Active Publication Date: 2019-11-08
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, most of the CRISPR / Cas systems in current research are transfected by viral vectors or hydrodynamic injection, and there is a lack of a safe and effective preparation for in vivo application.

Method used

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  • A kind of plasmid and preparation for directional clearance of hbvcccDNA in hepatocytes
  • A kind of plasmid and preparation for directional clearance of hbvcccDNA in hepatocytes
  • A kind of plasmid and preparation for directional clearance of hbvcccDNA in hepatocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Design, synthesis and eukaryotic expression vector construction of sgRNA sequence

[0079] We chose to target different subtypes of HBV genomes: adw (Genebank ID: AF100309.1), adw2 (Genebank ID: X02763), adr (Genebank ID: AF411411), ayw (Genebank ID: V01460.1) as CRISPR / Cas The targeting sequence of the system is used as the targeting sequence of the CRISPR / Cas system, and multiple sgRNAs targeting the conserved regions of the viral genome were designed according to the sgRNA design principles. Compare the obtained sgRNA with the human genome sequence, exclude the sgRNA with high homology, and finally find five new sgRNA targets and target sequences (as shown in Table 1 & as shown in Table 1). figure 1 shown).

[0080] Table 1

[0081]

[0082]According to the selected target sequence, we first synthesized the corresponding sgRNA insert sequence: GN 19, and the GN 19 sequence was annealed and cloned into the linearized pAdeno-U6-sgRNA-CMV-3Flag-hCas9 vect...

Embodiment 2

[0090] Five kinds of CRISPR / Cas9 expression plasmids (sg1, sg2, sg3, sg4, sg5) constructed in Example 1 were co-transfected into SMMC-7721 cells with the pHBV1.3 plasmid, and the virus surface antigen, e antigen, Changes in the cccDNA of hepatitis B virus, and based on this, the effects of five CRISPR / Cas9 systems on inhibiting HBV replication were evaluated.

[0091] (1) Cell transfection After mixing pHBV 1.3 and CRISPR / Cas9 plasmids, SMMC-7721 cells were transfected with lipofectamine 2000, and pHBV 1.3 was mixed with pGl3 plasmids as a control group. Cultured for 48h after transfection, culture medium and cells were collected for subsequent assay experiments.

[0092] (2) Determination of hepatitis B surface antigen and e antigen The above cells were centrifuged, and the cell supernatant was collected. The hepatitis B surface antigen and e antigen in the supernatant were measured with the hepatitis B virus surface antigen diagnostic kit and the hepatitis B virus e antigen...

Embodiment 3

[0100] The preparation of embodiment 3 cationic lipid nucleic acid drug preparation

[0101] Cationic lipids are prepared using DOTAP as an example, and nucleic acid drugs are prepared using CRISPR / Cas9 as an example (sg1, sg2, sg3, sg4, sg5).

[0102] The pH-responsive lipid used in this example is lipid CA, which is a prior art. The structural formula of the lipid CA is as shown in formula II, specifically:

[0103]

[0104] Those skilled in the art can use the method reported in the document "Controlling HBV Replication in Vivo by Intravenous Administration of Triggered PEGylated siRNA-Nanoparticles" to prepare and obtain lipid CA.

[0105] Such as Figure 4 As shown, the lipid CA reacts with PEG whose terminal group is an aldehyde group to obtain PEGylated lipid CA. The structural formula of the PEGylated lipid CA is shown in formula III, specifically:

[0106]

[0107] (1) Preparation of blank liposomes: Take a certain amount of DOTAP / DOPE / CA lipid stock solutio...

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Abstract

The invention belongs to the field of biomacromolecule drug preparations, and in particular relates to a plasmid and a preparation for directional removal of HBV cccDNA in liver cells. The present invention discovers five new CRISPR / Cas systems through extensive and in-depth research, which can effectively remove HBVcccDNA in cells, inhibit the replication of HBV virus and reduce the expression of hepatitis B virus-related proteins. Furthermore, a pH-sensitive PEG-modified cationic lipid carrier was also prepared, which has good stability and high transfection efficiency, and overcomes the in vivo instability of many cationic lipid carriers. The CRISPR / Cas9 cationic lipid carrier preparation was prepared by combining the CRISPR / Cas system with a pH-sensitive PEG-modified cationic lipid carrier, which can effectively inhibit virus replication and reduce antigen expression in a mouse model of acute HBV infection.

Description

technical field [0001] The invention belongs to the field of biomacromolecule pharmaceutical preparations, and in particular relates to a class of DNA, plasmids and preparations for directional elimination of HBV ccc in liver cells. Background technique [0002] Hepatitis B is an infectious disease caused by hepatitis B virus (Hepatitis B virus, HBV) infection that seriously endangers human health. Despite the successful use of preventive vaccines for many years, hepatitis B remains one of the major public health problems worldwide. According to statistics, more than 350 million people in the world are chronically infected with HBV, and the number of HBV infected people in my country is 93 million. About 20% to 40% of HBV-infected patients will eventually develop serious liver diseases, such as liver cirrhosis and liver cancer. HBV infection has become a major problem endangering public health. At present, the commonly used antiviral drugs for hepatitis B mainly fall into...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/88A61K48/00A61K47/69A61K47/59A61K9/127A61P31/20A61P1/16
CPCA61K48/0041A61K48/0058C12N15/1131C12N15/88C12N2310/10C12N2800/80
Inventor 徐宇虹房文慧彭金良刘君
Owner SHANGHAI JIAOTONG UNIV
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