rAAV8-CRISPR-SaCas9 system and application of system in preparing hepatitis B therapeutics

A drug, hepatitis B virus technology, applied in the field of gene mutation and genetic engineering, can solve the problem of not being able to effectively inhibit HBV replication and so on

Active Publication Date: 2017-12-08
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, less than 10% of patients treated with interferon-alpha produced a sustained therapeutic effect
[0007] However, the current research on the efficient inhibition of HBV replication and expressio

Method used

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  • rAAV8-CRISPR-SaCas9 system and application of system in preparing hepatitis B therapeutics
  • rAAV8-CRISPR-SaCas9 system and application of system in preparing hepatitis B therapeutics
  • rAAV8-CRISPR-SaCas9 system and application of system in preparing hepatitis B therapeutics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of the CRISPR-SaCas9 system with different targets

[0044] 1. CRISPR-SaCas9 target selection

[0045] Find the region with the "NNGRRT" PAM original in the HBV sequence, and try to stay away from the GC-rich region to avoid the possible impact of CpG methylation on gRNA recognition. Select the first 21-23bp of the PAM original as the gRNA sequence, and the first base is G is the best (see the structure of the gRNA figure 1 ). Add a Bsa I restriction site before the screened gRNA sequence: add AAAC at the 3' end and CCAC at the 5' end.

[0046] According to the principle of target site screening, the target site of CRISPR-SaCas9 was finally determined by comparing the HBV sequences of each genotype and searching for the "NNGRRT" PAM motif in the conserved region. Through screening, we obtained 21 target sites located in different genes of the HBV genome. The sequence information of each site is shown in Table 1. The position of each site on the...

Embodiment 2

[0104] Example 2. Observation of the Inhibition Efficiency of CRISPR-SaCas9 Systems with Different Targets to Intracellular HBV

[0105] The 21 sets of CRISPR-SaCas9 systems containing different targets obtained in Example 1 were co-transfected into 293T cells with the HBV expression vector pGL3-HBV1.2, and the cell culture supernatant was collected on the 3rd day after transfection, and tested by ELISA The content of HBsAg and HBeAg in the cell supernatant was detected by the method. The detection results of HBsAg are as follows: Figure 5 As shown, compared with the control group transfected with gRNA-empty, five sets of systems including Sa-4, Sa-6, Sa-10, Sa-16 and Sa-20 among the 21 sets of CRISPR-Cas9 systems were able to express the cells on The average content of HBsAg in the serum was reduced by more than 2 / 3.

[0106] The detection results of HBeAg content in the cell supernatant are as follows: Figure 6 As shown, compared with the control group transfected with ...

Embodiment 3

[0107] Example 3. CCK-8 method to observe the effect of CRISPR-SaCas9 system with different targets on cell activity

[0108] In order to detect whether the inhibition of HBV by the above 8 sets of CRISPR-SaCas9 systems is caused by the inhibition of cell activity, the cell activity was detected by the CCK-8 method. The experiments were divided into 11 groups: transfection of the CRISPR-SaCas9 expression vector px601 without the target, 8 sets of CRISPR-SaCas9 systems that could effectively inhibit HBsAg or HBeAg in the cell supernatant in the previous experiment, and only transfection reagent LTX group and no treatment group, each set three parallel wells. After 48 hours of transfection, the absorbance value was detected at a wavelength of 450nm, and the results were as follows: Figure 7 shown. Compared with the control group, except for Sa-6 and Sa-20, other targets had no significant effect on cell viability (p>0.05).

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Abstract

The invention discloses a gRNA sequence. The sequence is capable of editing a DNA sequence in the manner of taking a hepatitis B viral genome specific locus as a target sequence. The invention also discloses a CRISPR-SaCas9 system containing the gRNA sequence and a recombinant adeno-associated virus packaged with the system. The system and the packaging virus show higher HBV scavenging activity in cells and in transgenic mice body. On the 38th day after twice continuous high dose injection, the contents of HBsAg and HBeAg in experimental group mice serum, compared with the control group, are respectively reduced by 62.96+/-7.59% and 65.18+/-3.08%; the HBV DNA content in the serum, compared with the control group, is reduced by 92.82+/-3.67%; the liver and other visceral organs of the experimental mice are all free from any pathologic change, the off-target effect is also not detected and the application prospects of the gRNA sequence and the corresponding CRISPR-SaCas9 system provided by the invention in preparing the hepatitis B therapeutics are shown.

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 (hepatitis B for short) is a serious global public health problem. Since 1982, the application of hepatitis B vaccine has prevented the transmission of hepatitis B to a large extent, but the number of people with chronic hepatitis B infection (hepatitis B surface antigen HBsAg positive for at least 6 months) remains high, and by 2016 The global HBsAg positive number reached 240 million. It is estimated that more than 688,000 people worldwide die each year from complications of chronic hepatitis B, including liver cancer and cirrhosis. Hepatitis B is caused by hepatitis B virus (Hepatitis B virus, HBV) infection. The HBV genome is a partially double-stranded closed circular DNA virus that replicates through an ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/864C12N7/01A61K48/00A61P1/16A61P31/20
CPCA61K48/0008A61K48/0058C12N7/00C12N15/113C12N15/86C12N2310/10C12N2750/14121C12N2750/14143
Inventor 宋宏彬李浩邱少富生春雨杨超杰刘鸿博王珊贾雷立谢靖王立贵李鹏孙岩松
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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