Application of intracellular protein, nucleic acid or carrier

Abstract

The invention discloses application of intracellular protein, nucleic acid or a carrier in preparation of a medicine for treating HBV (Hepatitis B Virus) infection. The intracellular protein is NF-kB induced kinase; the nucleic acid is a nucleic acid for coding NF-kB induced kinase; the vector contains nucleic acid for coding NF-kB-induced kinase. The effect of NF-kappa B induced kinase in inhibition of HBV replication and gene expression is proved through a HepG2 cell HBV plasmid transfection model, a HepG2-NTCP cell HBV virus infection model, a high-pressure tail vein injection animal model and an LNP system delivery model. The NF-kB inducible kinase can activate an interferon pathway and induce expression of interferon stimulated genes so as to inhibit HBV replication and gene expression, and is a host factor for limiting HBV replication in hepatocytes.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a use of an intracellular protein, nucleic acid or carrier. Background Art

[0002] Hepatitis B virus (HBV) infection remains a global health problem affecting more than 257 million people. HBV is an enveloped, partially double-stranded DNA virus that replicates its genome by reverse transcription using a viral RNA template within the capsid. In sodium taurocholate cotransporting polypeptide (NTCP)-mediated infection, the 3.2 kb relaxed circular (rc) viral DNA is transported into the nucleus and converted into a nucleosome-modified covalently closed circular (ccc) DNA minichromosome. Using cccDNA as a transcription template, five viral mRNAs are transcribed. The 3.5 kb pgRNA is the template encoding HBV polymerase (pol) and core protein (HBcAg), and is also the template for HBV reverse transcription. After reverse transcription into rcDNA, the pgRNA is encapsidated by the mature nucleocapsid and secreted into the extracellular space as progeny virions. In addition, the rcDNA synthesized by reverse transcription can circulate into the nucleus to replenish the cccDNA pool, thereby maintaining the steady state of HBV infection.

[0003] At present, antiviral treatment for chronic hepatitis B mainly includes nucleotide analogs and interferon therapy: nucleotide analogs require long-term medication, and the virological recurrence rate is high after discontinuation of medication; interferon treatment has a narrow scope of application and has many adverse reactions after long-term use. If an intracellular protein that can activate the anti-HBV natural immune system can be found to exert an anti-HBV effect and provide a new target for the development of drugs to treat HBV, the above shortcomings can be avoided. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a use of an intracellular protein, a nucleic acid or a vector in the preparation of a drug for treating HBV infection.

[0005] To this end, the present invention provides the following technical solutions:

[0006] Use of an intracellular protein, a nucleic acid or a vector in the preparation of a drug for treating HBV infection, wherein the intracellular protein is NF-κB-induced kinase;

[0007] The nucleic acid is a nucleic acid encoding NF-κB inducing kinase;

[0008] The vector contains a nucleic acid encoding NF-κB inducing kinase.

[0009] NF-κB inducing kinase (NIK) is a key mediator of non-canonical NF-κB signal transduction. NF-κB plays an important role in innate immune response by inducing antiviral genes such as interferon (IFN) and interferon stimulated gene (ISG). The present invention proposes and experimentally confirms the role of NIK and its mediated non-canonical NF-KB signaling pathway in anti-HBV infection.

[0010] Optionally, the amino acid sequence of the NF-κB inducing kinase is shown in Seq ID No.1.

[0011] Optionally, the nucleic acid is mRNA.

[0012] Optionally, the nucleotide sequence of the nucleic acid is as shown in Seq ID No.2.

[0013] Optionally, the vector is selected from a plasmid or LNP.

[0014] Optionally, the plasmid is a PXJ40-HA vector.

[0015] Optionally, the vector is a lentivirus.

[0016] Optionally, the vector is a CV186 vector.

[0017] A pharmaceutical composition for treating HBV infection, comprising the above-mentioned intracellular protein, nucleic acid or vector.

[0018] Optionally, the pharmaceutical composition is an injection.

[0019] Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable solvent.

[0020] Optionally, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0021] The technical solution of the present invention has the following advantages:

[0022] The present invention proposes the use of NF-κB induced kinase in the preparation of drugs for treating HBV infection. The present invention confirms the role of NF-κB induced kinase in inhibiting HBV replication and gene expression through HepG2 cell HBV plasmid transfection model, HepG2-NTCP cell HBV virus infection model, high-pressure tail vein injection animal model and LNP system delivery model. NF-κB induced kinase can activate the interferon pathway and induce the expression of interferon-stimulated genes to inhibit HBV replication and gene expression, and is a host factor that limits HBV replication in hepatocytes. This discovery helps to further clarify the interaction between HBV and the host's natural immune system and develop new therapeutic targets for HBV. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The result is that NIK is overexpressed to inhibit the expression levels of intracellular HBV core antigen, core DNA, pgRNA, and HBsAg and HBeAg in the cell culture supernatant.

[0025] Figure 2 The anti-HBV effect of HepG2-NTCP cells overexpressing NIK was established by lentiviral transduction.

[0026] Figure 3 The anti-HBV effect of overexpressing NIK in mouse liver. Figure 3 A is a schematic diagram of the experiment; Figure 3 B is the result of HBsAg level in mouse serum; Figure 3 C is the result of HBeAg level in mouse serum; Figure 3 D is the result of HBV DNA level in mouse serum; Figure 3 E shows the results of immunohistochemical staining of HBV core antigen in the liver and the proportion of positive cells.

[0027] Figure 4 This is the experimental result of using LNP system to deliver NIK mRNA for treating chronic hepatitis B in mice. Figure 4 A is a schematic diagram of the experiment; Figure 4 B is the result of HBsAg level in mouse serum; Figure 4 C is the result of HBeAg level in mouse serum; Figure 4 D is the result of HBV DNA level in mouse serum; Figure 4 E is the result of mouse serum HBsAg positive rate; Figure 4 F is the HBeAg positive rate result of mouse serum; Figure 4 G is the weight result of mice; Figure 4 H is the result of ALT level in mice; Figure 4 I is the T-Bil level result.

[0028] Figure 5 This is a diagram showing that overexpression of NIK in hepatocytes can activate the interferon pathway and induce the expression of interferon-stimulated genes (ISGs). Figure 5 A, the effect of overexpression of NIK on gene expression; Figure 5B is the result of gene enrichment analysis of Hallmark pathway; Figure 5 C is the enrichment score result diagram; Figure 5 D is the result of real-time fluorescence quantitative PCR analysis; Figure 5 E is the result of luciferase reporter gene. DETAILED DESCRIPTION

[0029] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0030] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0031] The NF-κB signaling pathway is well known for its role in proliferation, cell survival, and the induction of inflammatory cytokine responses. It is activated through two different pathways, the canonical and non-canonical NF-κB signaling pathways. Canonical NF-κB signaling activates transcription factors p105 / p50, RelA, and RelB in multiple cell types through toll-like receptors and cytokine receptors. Upon activation of this pathway, the inhibitory molecule IκBα is rapidly phosphorylated and degraded, and the transcription factors are released to mediate signal transduction. Non-canonical NF-κB signaling is activated in a more limited manner. Under physiological conditions or resting states, NF-κB-inducing kinase (NIK), a key mediator of non-canonical NF-κB signaling, can be constitutively degraded by the TRAF complex composed of cIAP1, cIAP2, TRAF2, and TRAF3. Upon stimulation by multiple receptors of the tumor necrosis factor (TNF) superfamily, including CD40, lymphotropin β (LTβ), and B-cell activating factor (BAFF), TRAF3 is ubiquitinated and degraded, allowing NIK to slowly accumulate through de novo protein synthesis. Accumulation of NIK leads to phosphorylation of IκB kinase α (IKKα), which in turn phosphorylates the transcription factor p100, resulting in ubiquitin-mediated processing of p100 into mature p52 subunits and subsequent nuclear translocation of RelB / p52 heterodimers.

[0032] The balance between viral replication and host immunity determines the course of HBV infection, so the innate immune system is the first line of defense against foreign pathogens, and NF-κB plays an important role in the innate immune response by inducing antiviral genes such as interferon (IFN) and interferon-stimulated genes (ISG). Activation of the NIK-dependent non-canonical NF-κB signaling pathway is involved in the host defense against DNA and RNA virus infection, but its exact role in controlling HBV infection is still unclear. HBV replication in hepatocytes may be associated with NIK-dependent activation of the non-canonical NF-kB pathway. Cell inhibitor of apoptosis proteins (cIAPs) are a class of highly conserved endogenous anti-apoptotic molecules. The degradation of cIAPs can continuously activate the NIK-dependent non-canonical NF-kB signaling pathway. For example, mice with insufficient expression of hepatocyte-specific cIAPs promote the clearance of chronic HBV infection. In addition, birinapant, a chemical inhibitor of cIAPs, can reduce HBV DNA and hepatitis B surface antigen levels in serum and induce apoptosis of hepatocytes positive for hepatitis B core antigen in a mouse model of HBV persistence. Lymphokine receptor is a member of the TNF receptor superfamily and can activate the NIK-dependent non-canonical NF-kB pathway. Activation of this receptor can induce the expression of APOBEC3A / 3B protein, which degrades cccDNA by mediating deamination on the negative strand. However, no direct evidence has been presented in the prior art to prove the role of NIK and its mediated non-canonical NF-KB signaling pathway in anti-HBV infection.

[0033] Example 1

[0034] The present invention confirms the role of NIK protein in inhibiting HBV replication and gene expression through HepG2 cell HBV plasmid transfection model, HepG2-NTCP cell HBV virus infection model, high-pressure tail vein injection animal model and LNP system delivery model. NIK can activate the interferon pathway and induce the expression of interferon-stimulated genes to inhibit HBV replication and gene expression, and is a host factor that limits HBV replication in hepatocytes.

[0035] The amino acid sequence of the NIK protein in the present invention is shown in SEQ ID No.1.

[0036] The present invention utilizes mRNA to express NIK protein in cells, and the nucleic acid sequence of the mRNA is as shown in SEQ ID No.2.

[0037] The invention constructs a lentivirus by synthesizing the coding sequence of MAP3K14-NM_003954 and cloning it into the CV186 vector using the BamHI / AgeI restriction sites. The vector elements are arranged as Ubi-MCS-3FLAG-SV40-Cherry-IRES-puromycin. The vector is from Shanghai Genechem, and the vector description can be found at http: / / www.genechem.com.cn / index / supports / tool_search.html?keywords=CV186.

[0038] The invention constructs an HA-mNik plasmid vector, and the construction method comprises the following steps: synthesizing the coding sequence of Map3k14-NM_016896 by PCR amplification, and cloning the coding sequence into a PXJ40-HA vector by using a BamHI / KpnI restriction site.

[0039] The present invention also constructs an LNP carrier, and the construction method comprises the following steps:

[0040] Step 1: Dissolve SM102, Cholesterol, DSPC, and DMG-PEG2K in anhydrous ethanol at a certain ratio to obtain a lipid-ethanol phase;

[0041] Step 2: dilute the mRNA stock solution with acetate buffer to obtain the mRNA-buffer phase;

[0042] Step 3: The lipid-ethanol phase and the mRNA-buffer phase were mixed at a flow rate ratio of 1:3 through a microfluidic device, and the mixed solution was collected;

[0043] Step 4: Immediately transfer the mixed solution to a dialysis bag and dialyze it against Tris saline buffer to obtain LNPs.

[0044] Example 2

[0045] To determine the antiviral activity of NIK against HBV replication, in this example, flag-tagged NIK and pHBV1.3 were co-transfected into HepG2 cells. The transfection steps were as follows:

[0046] HepG2 cells were digested with trypsin, resuspended and counted, then plated in 6-well and 12-well plates and cultured at 37°C until the cell confluence reached 90% before transfection. Transfection was performed using reduced serum medium Opti-MEM (Cat. No.: 31985070, Brand: ThermoFisher), and transfection reagent used was lipo3000 Lipofectamine TM 3000 (Cat. No.: L3000015 Brand: Invitrogen&trade);

[0047] 12-well plate transfection system preparation:

[0048] Solution A: Opti-MEM 65ul+Lipofectamine 3000reagent 3ul

[0049] Solution B: Opti-MEM 65ul+P3000 TM Reagent 3ul+plasmid 1.5ug (HBV1.3 plasmid 0.75ug+flag-NIK 0-0.75ug, complete with empty vector pcDNA-flag)

[0050] Mix the A / B solution gently, let it stand for 10-15 minutes, and then add it dropwise into the well plate.

[0051] The transfection system of a 6-well plate is twice that of a 12-well plate, and the amount of plasmid used is also twice that of a 12-well plate.

[0052] 48 hours after transfection, the supernatant was collected from 12-well plates to detect the expression levels of HBsAg and HBeAg in the supernatant, cellular proteins were collected for Western detection of HBcAg and NIK, and cellular mRNA was collected for real-time fluorescence quantitative PCR to detect HBV pgRNA. 96 hours after transfection, HBV nucleocapsid DNA was extracted from 6-well plates, and HBV core DNA was detected by Southern blot.

[0053] like Figure 1 As shown, overexpressed NIK dose-dependently inhibited the expression levels of intracellular HBV core antigen, core DNA, pgRNA, and HBsAg and HBeAg in the cell culture supernatant.

[0054] Example 3

[0055] In order to further verify the effect of NIK on HBV infection, this example established HepG2-NTCP cells that overexpress NIK by lentiviral transduction. The specific steps include: HepG2-NTCP cells were digested, resuspended and counted, then plated in a 6-well plate and cultured at 37°C until the cell confluence was 20-30%. The amount of virus was calculated according to MOI=10, and virus dyeing solution was added at the same time, and added to the cells in the form of culture medium replacement. Cultured at 37°C for 12-16 hours, replaced with regular culture medium, and continued to culture. After 72 hours of infection, the cell infection efficiency was observed, and subsequent HBV infection experiments were performed. Figure 2 As shown in Figure 2, NIK overexpression led to a significant downregulation of cccDNA and pgRNA levels as well as HBcAg and HBV core DNA levels. Similarly, HBeAg and HBsAg levels in the supernatant were also reduced. In vitro experimental results confirmed that NIK overexpression can inhibit HBV replication and gene expression.

[0056] Example 4

[0057] In this example, a mouse chronic hepatitis B model was constructed by high-pressure tail vein injection of 6 μg pAAV-HBV1.2 plasmid, and at the same time, 6 μg HA-mNik plasmid was injected into the tail vein at high pressure to overexpress NIK in the mouse liver. The specific method is to add 6 μg pAAV-HBV1.2 plasmid plus 6 μg HA-mNik plasmid or control empty vector PXJ40-HA plasmid to PBS, and the amount of PBS used is 10% of the mouse body weight. It should be pointed out that the NIK overexpressed in the mouse liver in this example is a fragment of the mouse NF-κB-induced kinase. According to previous reports, the 311-796 amino acid fragment of the mouse NF-κB-induced kinase has better stability in vivo. The amino acid sequence of this fragment is shown in SEQ ID No.4, and the corresponding nucleic acid sequence is shown in SEQ ID No.3. The expression levels of HBsAg, HBeAg, and HBV DNA in the mouse serum were continuously observed. The research results are shown in Figure 3 The results showed that the expression levels of HBsAg, HBeAg, and HBV DNA in the serum of mice injected with high-pressure tail vein on days 1 / 4 / 7 / 14 were significantly lower in the NIK overexpression group than in the control group. On days 1 / 4 / 7 of modeling, the livers of three mice in each group were collected for immunohistochemistry to detect the expression of HBV core antigen in the liver. The results showed that the expression of HBcAg in the liver of the NIK overexpression group was significantly lower than that of the control group, and the difference was statistically significant.

[0058] Example 5

[0059] This example further studies the therapeutic effect of NF-κB-induced kinase in patients with chronic HBV infection. A mouse chronic hepatitis B model is constructed by high-pressure tail vein injection of 6 μg pAAV-HBV1.2 plasmid. Blood is collected on the 10th day of modeling to detect the expression levels of HBsAg, HBeAg, and HBV DNA in mouse serum, and the mice are grouped according to the expression levels of HBsAg, HBeAg, and HBV DNA in mouse serum. The NIK mRNA packaged with empty LNP, 0.1 mg / kg, and 0.2 mg / kg LNP is used for treatment through the tail vein. The specific method is to use LNP balance buffer to dilute the packaged NIK mRNA to 10 ng / ml and 20 ng / ml, respectively, and inject it into the tail vein at 10 ul per gram of body weight; inject once a week for a total of 5 times. It should be pointed out that the NIK overexpressed in the mouse liver in this example is the NF-κB-induced kinase of the mouse, and the amino acid sequence is shown in SEQ ID No.4, and the corresponding nucleic acid sequence is shown in SEQ ID No.3. Figure 4A is the experimental flow chart. After two weeks of LNP-NIK administration, the expression levels of HBsAg, HBeAg, and HBV DNA in the serum of mice in the administration group were significantly lower than those in the control group, and the decrease in the 0.2 mg / kg group was more significant than that in the 0.1 mg / kg group ( Figure 4 BD). After 5 weeks of administration, the HBsAg clearance rate in the 0.2 mg / kg group reached 100%, and the HBsAg clearance rate in the 0.1 mg / kg group reached 71.4%, while the HBsAg positive rate in the control group remained at 57.1% ( Figure 4 E). The 0.2 mg / kg group had complete clearance of HBeAg in the serum after 2 weeks of administration, and the 0.1 mg / kg group had complete clearance of HBeAg in the serum after 3 weeks of administration, while 28% of the mice in the control group had not achieved HBeAg clearance until the 5th week after administration ( Figure 4 F). And the experimental group showed some improvement compared with the control group ( Figure 4 G~I).

[0060] Pathway analysis based on the above experiments showed that overexpression of NIK in hepatocytes can activate the interferon pathway and induce the expression of interferon-stimulated genes (ISGs) ( Figure 5 ), the differentially regulated genes upregulated by NIK overexpression were mainly interferon regulatory factors, ISGs and interferon signaling pathway-related genes ( Figure 5 A), the most significant difference in Hallmark pathway enrichment was the interferon response pathway ( Figure 5 BC), real-time fluorescence quantitative PCR and luciferase reporter gene confirmed that overexpression of NIK promoted the expression of IFN and ISGs ( Figure 5 DE).

[0061] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of an intracellular protein, nucleic acid or vector in the preparation of a drug for treating HBV infection, characterized in that: The intracellular protein is NF-κB inducing kinase; The nucleic acid is a nucleic acid encoding NF-κB inducing kinase; The vector contains a nucleic acid encoding NF-κB inducing kinase.

2. The use according to claim 1, characterized in that The amino acid sequence of the NF-κB inducing kinase is shown in SeqID No.

1.

3. The use according to claim 1, characterized in that The nucleic acid is mRNA.

4. The use according to claim 1, characterized in that The nucleotide sequence of the nucleic acid is shown in Seq ID No.

2.

5. The use according to claim 1, characterized in that: The vector is selected from a plasmid or LNP.

6. The use according to claim 1, characterized in that: The vector is a lentivirus.

7. A pharmaceutical composition for treating HBV infection, characterized in that: The pharmaceutical composition comprises the intracellular protein, nucleic acid or vector described in claim 1.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition is an injection.

9. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition also contains a pharmaceutically acceptable solvent.

10. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition also contains pharmaceutically acceptable excipients.

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