A pharmaceutical composition and its use in the preparation of a drug for preventing and treating hepatitis B virus

Abstract

The application belongs to the field of biological medicine, and relates to a kind of pharmaceutical composition and its application in preparation of the drug for preventing and treating hepatitis B virus. Including nucleic acid drug and the carrier loaded with the nucleic acid drug, the carrier can specifically target liver kupffer cell, and the nucleic acid drug is mRNA, and the gene sequence coding the mRNA includes the nucleotide sequence shown in SEQ ID NO:1 and SEQ ID NO:2. The pharmaceutical composition provided by the application can specifically activate the Notch signal pathway of kupffer cell, and can play the effect of treating and preventing hepatitis B. Meanwhile, the anti-HBV effect of the pharmaceutical composition depends on the participation of kupffer cell, and becomes a new type of strategy for potential clinical treatment of chronic hepatitis B.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a pharmaceutical composition and its application in the preparation of a drug for the prevention and treatment of hepatitis B virus. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Chronic hepatitis B (CHB) infection remains a global health problem, affecting more than 250 million people worldwide. Current active drugs and antiviral therapies, such as nucleoside analogs and interferon (IFN), have failed to achieve complete virological suppression, accompanied by adverse side effects and relapse after discontinuation. Chronic stimulation by high levels of hepatitis B virus (HBV) antigens leads to the inhibition of HBV-specific CD8 receptors. + T cell depletion eventually leads to impaired HBV-specific humoral and cellular immune responses, resulting in persistent HBV infection. One current treatment for chronic hepatitis B involves inducing activation of innate immune components using Toll-like receptor (TLR) agonists, which may produce both direct and indirect antiviral effects. Kupffer cells (KCs), the largest tissue macrophage population, play a crucial sentinel role in clearing and capturing pathogens and cellular debris, detecting and presenting antigens, and maintaining liver homeostasis. KCs can be further divided into KC1 and KC2 subsets based on function and phenotype, with the KC2 subset exhibiting a stronger antigen cross-presentation capacity, thereby reactivating T cell responses. Given that KCs, especially KC2s, can capture and present antigens released by hepatocytes and initiate immune responses without transporting lymph nodes, KCs play a key role in regulating intrahepatic T cell responses in chronic viral infection. However, chronic HBV infection leads to dysfunction of various cells, including KC cells, promoting the persistent progression of hepatitis B. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a pharmaceutical composition and its application in the preparation of drugs for the prevention and treatment of hepatitis B virus. The pharmaceutical composition provided by the present invention can specifically activate the Notch signaling pathway of Kupffer cells, thereby achieving therapeutic and preventive effects against hepatitis B. Furthermore, the anti-HBV effect of this pharmaceutical composition depends on the participation of Kupffer cells, representing a potential novel strategy for the clinical treatment of chronic hepatitis B.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] In a first aspect, a pharmaceutical composition includes a nucleic acid drug and a carrier loaded with the nucleic acid drug, the carrier being capable of specifically targeting hepatic Kupffer cells, the nucleic acid drug being mRNA, and the gene sequence encoding the mRNA including the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0007] This invention first optimizes the codons based on MFE to obtain mRNAs encoding membrane-anchored Notch ligands DLL1 and DLL4. These mRNAs exhibit high in vivo stability, ensuring relatively long-term sustained antigen expression. Secondly, a vector specifically targeting hepatic Kupffer cells is used to load these mRNAs. This vector targets hepatic Kupffer cells and activates the Notch signaling pathway through the mRNA, thereby specifically rebuilding the immune function of KC cells, reversing the suppressive state of the hepatic immune microenvironment, restoring antiviral immunity, and ultimately achieving the prevention and treatment of hepatitis B.

[0008] In some embodiments, the carrier is a lipid nanoparticle (LNP). The mRNA encapsulated in LNPs can be transcribed and delivered to cells in vitro, translated into proteins, and subsequently stimulate specific humoral and cellular immune responses. LNPs can protect the mRNA from extracellular RNases and promote efficient uptake and intracellular release of the mRNA by target cells. Furthermore, the mRNA itself is not infectious and can be degraded through normal cellular pathways, posing no risk of infection or insertional mutations.

[0009] In some embodiments, the carrier is a mannose-modified LNP. Studies have shown that mannose-modified LNPs can target hepatic Kupffer cells.

[0010] In a second aspect, the use of the pharmaceutical composition described in the first aspect of the present invention in the preparation of a medicament for preventing and treating hepatitis B virus.

[0011] The beneficial effects of this invention are as follows:

[0012] 1. This invention utilizes a vector specifically targeting hepatic Kupffer cells to load mRNA, enabling precise targeting and efficient delivery, thus solving the challenge of cellular accessibility for immune regulation. Specifically, the use of a mannose-modified LNP vector significantly improves delivery efficiency to KC cells expressing the mannose receptor. This active targeted delivery strategy ensures that the therapeutic mRNA is primarily taken up by KCs, greatly improving local bioavailability. Simultaneously, due to its specific targeting of the liver, it effectively avoids potential side effects from systemic non-specific immune activation, laying the foundation for precise and safe immune regulation.

[0013] 2. This invention does not directly deliver the protein, but rather delivers optimized mRNA encoding the membrane-anchored Notch ligands DLL1 / DLL4. First, by performing MFE-based codon optimization on the coding sequence, the in vivo stability of the mRNA is significantly improved, making it resistant to rapid degradation. Second, mRNA delivery enables the target protein (DLL1 / DLL4) to be translated in situ within target cells (KCs) and anchored to the cell membrane, mimicking natural intercellular signaling. Compared to direct protein injection, this strategy achieves longer-term and more stable functional expression of the Notch ligand on the target cell surface, ensuring sustained activation of the Notch pathway.

[0014] 3. The pharmaceutical composition provided by this invention delivers mRNA via a carrier, which is translated in situ within target cells (KCs) and anchored to the cell membrane, specifically activating the Notch signaling pathway in KC cells. This directly targets the key pathological link of KC dysfunction caused by chronic HBV infection. Activation of Notch signaling, particularly the restoration of function of the KC2 subset, which is capable of efficiently cross-presenting antigens, can effectively reverse the immunosuppressive state in the liver, thereby breaking the immune tolerance caused by HBV infection and creating the prerequisites for restoring an effective antiviral immune response.

[0015] 4. The pharmaceutical composition provided by this invention acts on the immune system itself. By restoring the antigen presentation and immune activation functions of KC, it can rebuild the body's specific cellular immune response against HBV from the source. Animal experiments have confirmed that the pharmaceutical composition of this invention can effectively promote HBV-specific T cell responses, significantly reduce the levels of viral markers such as HBsAg in HBV-carrier mice in treatment models, and protect wild-type mice from HBV infection in prevention models. This mechanism of action not only achieves deep virological suppression, but also may provide a novel treatment strategy for pursuing "clinical cure" (i.e., HBsAg clearance) through the restored immune clearance capacity. Especially for individuals with low viral loads, it shows the potential to rapidly clear the virus in a short period of time, and is expected to solve the clinical bottleneck of easy relapse after the discontinuation of existing therapies.

[0016] 5. The pharmaceutical composition provided by this invention has both therapeutic and preventative effects. It can not only treat existing chronic HBV infection, reduce viral load, and restore immune response, but also effectively prevent the establishment of initial HBV infection at the preventative level. This greatly expands its clinical application potential and provides a new candidate strategy for post-exposure prophylaxis in high-risk populations or prophylactic treatment in immunosuppressed patients. Attached Figure Description

[0017] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0018] Figure 1 The images show the membrane expression levels of DLL1 and DLL4 proteins after transfection of HEK-293T cells with the mRNA sequence of this invention. a) shows the membrane expression level of DLL1 protein after transfection of HEK-293T cells with the DLL1 mRNA sequence; b) shows the statistical graph of the mean fluorescence intensity (MFI) values ​​of DLL1 before and after transfection; c) shows the expression level of DLL1 in the cell supernatant; d) shows the membrane expression level of DLL4 protein after transfection of HEK-293T cells with the DLL4 mRNA sequence; e) shows the statistical graph of the mean fluorescence intensity (MFI) values ​​of DLL4 before and after transfection; and f) shows the expression level of DLL4 in the cell supernatant.

[0019] Figure 2 This invention demonstrates the precise targeting of mannose-modified LNPs to cells expressing mannose receptors, particularly KC cells, according to embodiments of the invention. a shows the uptake ratio of mannose-modified LNPs by different immune cells in the liver; b shows the uptake ratio of mannose-modified LNPs by KC cells and their subtypes KC1 and KC2.

[0020] Figure 3 In this embodiment of the invention, mannose-modified mtDLL1+4 mRNA-LNPs promote an increase in the proportion of KC cells, especially KC2 cells; a is a graph showing the uptake ratio of 10 μg mannose-modified mtDLL1+4 mRNA-LNPs by KC cells; b is a graph showing the uptake ratio of 10 μg mannose-modified mtDLL1+4 mRNA-LNPs by KC1 and KC2 cells.

[0021] Figure 4 Mannose-modified mtDLL1+4 mRNA-LNPs enhance the antigen presentation ability of KCs and KC2 cells in this embodiment of the invention; a is the average fluorescence intensity of CD86 and MHC II on DLL1+4 double-positive KC cells; b is the average fluorescence intensity of CD86 and MHC II on DLL1+4 double-positive KC1 and KC2 cells.

[0022] Figure 5 This invention evaluates the effect of mannose-modified mtDLL1+4 mRNA-LNPs on HBV prevention in WT mice; a) shows the expression level of HBsAg in mouse serum after pre-injection of 10 μg of mannose-modified mtDLL1+4 mRNA-LNPs and subsequent HBV infection; b) shows the immunohistochemical map of HBcAg in liver sections.

[0023] Figure 6 This invention presents an analysis of the virological inhibitory effects of mannose-modified mtDLL1+4 mRNA-LNPs on HBsAg and HBcAg in the serum of HBV mice. a is a graph showing the expression level of HBsAg in the serum of HBV mice; b is an immunohistochemical image of HBcAg in liver sections.

[0024] Figure 7 The mannose-modified mtDLL1+4 mRNA-LNPs of this invention specifically target CD8 for HBV mouse antigen. + A graph showing the proportion of T cells.

[0025] Figure 8 The mannose-modified mtDLL1+4 mRNA-LNPs of this invention specifically target CD8 for HBV mouse antigen. + Functional analysis of T cells; a represents liver HBV-specific CD8. + Scatter plot of IFN-γ expression by T cells; b is a scatter plot of TNF-α expression by liver HBV-specific CD8+ T cells; c is a scatter plot of IL-2 expression by liver HBV-specific CD8+ T cells; d is a scatter plot of IL-21 expression by liver HBV-specific CD8+ T cells; e is a statistical plot of a; f is a statistical plot of b; g is a statistical plot of c; h is a statistical plot of d; i is a statistical plot of liver HBV-specific CD8+ T cells. + The diagram shows the proportion of T cells simultaneously expressing the above 1, 2, and 3 cytokines. Detailed Implementation

[0026] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0028] Given that existing treatments for chronic hepatitis B cannot effectively restore the body's specific antiviral immune clearance function, resulting in difficulty in completely eliminating the virus and easy relapse after discontinuation of medication, this invention proposes a pharmaceutical composition and its application in the preparation of a drug for the prevention and treatment of hepatitis B virus.

[0029] In one typical embodiment of the present invention, a pharmaceutical composition is provided, comprising a nucleic acid drug and a carrier loading the nucleic acid drug, the carrier being capable of specifically targeting hepatic Kupffer cells, wherein the nucleic acid drug is mRNA, and the gene sequence encoding the mRNA includes the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0030] In some embodiments, the carrier is a lipid nanoparticle. Specifically, the lipid nanoparticle includes cationic lipids, cofactor phospholipids, sterol lipids, and PEG-modified lipids. More specifically, the lipid nanoparticle is composed of Lipid5, DSPC, cholesterol, PEG, and DSPE-PEG. The molar ratio of Lipid5, DSPC, cholesterol, PEG, and DSPE-PEG is 45~55:9~11:37~40:1:0.45~0.55.

[0031] In some embodiments, the carrier is mannose-modified lipid nanoparticles. Specifically, the mannose-modified lipid nanoparticles include cationic lipids, cofactor phospholipids, sterol lipids, and lipids co-modified with mannose and PEG. More specifically, the lipid nanoparticles are composed of Lipid5, DSPC, cholesterol, PEG, and DSPE-PEG-mannose. The molar ratio of Lipid5, DSPC, cholesterol, PEG, and DSPE-PEG-mannose is 45~55:9~11:37~40:1:0.45~0.55. Studies have shown that the nucleic acid drug loaded on the mannose-modified lipid nanoparticles of this invention has the following advantages: its targeting effect is superior to ordinary LNP; it can prevent HBV infection in WT mice, and depends on the participation of KC; it can significantly reduce HBsAg levels in HBV-carrier mice and promote HBV-specific T cell responses; it can avoid the side effects of systemic immune activation; it can not only prevent and treat hepatitis B virus infection, but is also effective in mice with low viral loads, and can rapidly clear HBV in a short time.

[0032] In some embodiments, the average particle size of the carrier loaded with nucleic acid drugs is 10-500 nm; preferably 80-120 nm.

[0033] In some embodiments, the pharmaceutical composition is in the form of an injection.

[0034] Another embodiment of the present invention provides the use of the above-described pharmaceutical composition in the preparation of a drug for preventing and treating hepatitis B virus.

[0035] In some embodiments, the drug is a drug for treating chronic hepatitis B.

[0036] In some embodiments, the drug is administered to humans or non-human mammals. Non-human mammals as described in this invention include mice, rats, rabbits, dogs, pigs, etc.

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0038] Example 1: Design of mannose-modified mtDLL1+4 mRNA-LNP sequences

[0039] Activation of the Notch signaling pathway depends on direct physical contact between signaling and receiving cells. Core components: Notch receptors: Four Notch receptors exist in mammals (Notch1, Notch2, Notch3, Notch4), which are single-transmembrane glycoproteins. After synthesis, these receptors undergo enzymatic cleavage and exist on the cell membrane as non-covalently linked heterodimers. Notch ligands: Ligands belong to the DSL (Delta / Serrate / Lag-2) family, and in mammals, they mainly include Jagged1, Jagged2, Delta-like-L (DLL), DLL3, and DLL4. These ligands are also transmembrane proteins expressed on the surface of signaling cells. This invention selects DLL1 and DLL4 as target sequences and adds membrane-binding regions to both sequences, enabling them to anchor on the cell membrane as transmembrane proteins after successful expression.

[0040] Because exogenous proteins are expressed at low levels in mammals, codon optimization is employed to increase their expression. The optimization scheme is primarily based on two parameters: the Codon Adaptation Index (CAI) and the Minimum Free Energy (MFE). The CAI parameter is related to codon bias. In organisms, any amino acid corresponds to multiple different codons, but during translation, one codon often dominates in frequency of use. When using CAI as a parameter for sequence optimization, the preferred codon will be used as much as possible, thereby increasing the translation rate and accelerating protein synthesis. The MFE parameter is related to the stability of mRNA secondary structure; the lower the MFE, the more stable the secondary structure of the mRNA molecule and the longer its half-life. Optimizing codons based on MFE will improve the in vivo stability of the mRNA molecule, ensuring relatively long-term sustained expression of the antigen. Two sequences were obtained after optimization of DLL1 and DLL4.

[0041] The sequence of DLL1 is:

[0042]

[0043] The sequence of DLL4 is:

[0044]

[0045] Example 2: mRNA preparation and characterization

[0046] Full gene synthesis of template plasmid: The codon-optimized DLL1 and DLL4 gene sequences are tandemly linked with the T7 promoter sequence, 5'UTR sequence, 3'UTR sequence and polyA sequence, respectively, and then the full gene is synthesized using Puc57 as a vector to obtain the template plasmid.

[0047] The codon-optimized DLL4 gene sequence, concatenated with the T7 promoter sequence, 5'UTR sequence, 3'UTR sequence, and polyA sequence, respectively, yields the following sequences:

[0048]

[0049] The codon-optimized DLL1 gene sequence concatenated with the T7 promoter sequence, 5'UTR sequence, 3'UTR sequence, and polyA sequence are as follows:

[0050]

[0051] Obtaining the transcribed template DNA sequence by PCR: Using the linearized template plasmid as a template, polyT long primers, high-fidelity DNA polymerase (purchased from Thermo Scientific Pharmacy), dNTPs, and other raw materials, the transcribed template DNA can be obtained by using a suitable program on a PCR instrument. The upstream primer of the polyT long primer is: TAATACGACTCACTATAAGGTTGTTGATTA, as shown in SEQ ID NO: 5; the downstream primer of the polyT long primer is: TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGTCATATGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACCAGGATCCCAAAGTTTTAAT, as shown in SEQ ID NO: 6.

[0052] In vitro transcription to prepare mRNA (using a 40 μL reaction system as an example): The prepared IVT template was mixed with T7 RNA polymerase (purchased from NEB), N1 methylpseudouridine (Synthgene), and other raw materials according to the manufacturer's instructions. The co-transcription capping method was used, and the transcription reaction was carried out at 37°C for 2 hours. After the transcription reaction, the IVT template was digested using DNase (purchased from Novizan) to reduce the risk of residual DNA template.

[0053] Purification: The two mRNAs from the IVT reaction were purified using an RNA purification kit (NEB). The purified mRNAs were dissolved in TE buffer and ready for subsequent formulation coating.

[0054] The expression levels of DLL1 and DLL4 proteins were verified by transfecting the mRNA sequence into HEK-293T cells.

[0055] 293T cells were resuscitated and seeded into 12-well plates when they reached 80-100% confluency. After 12 hours, when the cells reached 70% confluency, the culture medium was replaced with serum-free medium, and mRNA was transfected into the 293T cells using a Lipofectamine transfection kit. Untransfected cells served as a negative control. Cells were harvested 48 hours after transfection. 48 hours post-transfection, cells were collected and transferred to flow cytometry tubes. DLL1 and DLL4 antibody staining was performed at room temperature. After 30 minutes, the cells were washed with PBS, centrifuged at 400 rpm for 5 minutes, and the protein expression of DLL1 and DLL4 on the cell surface was detected using BD FACSymphony A3.

[0056] Experimental results:

[0057] High levels of DLL1 and DLL4 protein expression were detected on the cell surface after transfection of HEK-293T cells with the DLL1+4-mRNA sequence. Figure 1 As shown in Figures a, b, d, and e. However, no expression of DLL1 and DLL4 was detected in the cell supernatant, as... Figure 1 As shown in c and f.

[0058] Example 3

[0059] Test method:

[0060] The experimental group received intravenous injections of 5 μg LNP-GFP and mannose-modified LNP-GFP, while the blank control group received the same volume of PBS. The targeting ability of mannose-modified LNPs to KC cells was evaluated by detecting the proportion of KC cells in GFP-positive cells.

[0061] Experimental results:

[0062] Compared with the LNP-GFP group, the proportion of hepatocytes taking up GFP was significantly reduced in the mannose-modified LNP-GFP group; in both the LNP-GFP and mannose-modified LNP-GFP groups, the number of GFP-positive cells was F4 / 80. + KC cells ( Figure 2 (a) in particular, KC2 cells ( Figure 2 (b) This indicates that mannose-modified LNPs significantly improve the delivery efficiency of LNPs to KC cells expressing the mannose receptor.

[0063] Example 4: Detection of mRNA Encapsulated by Liposome Nanoparticles and Their Particle Size

[0064] Preparation of lipid solution: Lipid5:DSPC:cholesterol:PEG:PEG:DSPE-PEG-mannose was dissolved in ethanol solution in a molar ratio of 50:10:38.5:1:0.5.

[0065] mRNA solution preparation: A certain mass of DLL1 and DLL4 mRNA were mixed and dissolved in 10mM citrate buffer solution at pH=4.0 to prepare an aqueous phase (the concentrations of DLL1 mRNA and DLL4 mRNA were both 25 μg / mL).

[0066] Preparation of lipid nanoparticles: 1 mL of aqueous phase and 3 mL of lipid solution were respectively drawn into a syringe and inserted into a microfluidic chip. The microfluidic flow rate, injection volume and harvest volume were adjusted. After automatic microfluidic injection, the lipid nanoparticle solution was obtained by mixing.

[0067] Ultrafiltration equilibration: The lipid nanoparticle solution was added to an ultrafiltration tube and centrifuged for ultrafiltration. The solution was replaced multiple times with phosphate buffer to obtain the final product.

[0068] Prepare a 2% Triton reagent and lyse the formulation at 37°C for 10 min. Detect the concentration of mRNA-LNP using the Qubit™ RNA High Sensitivity (HS) kit according to the instructions.

[0069] Using Qubit TM The RNA High Sensitivity (HS) kit was used to detect the encapsulation efficiency of mRNA-LNP.

[0070] The particle size and PDI (penetration density) of the vaccine formulation were analyzed and tested using a particle size analyzer.

[0071] Experimental results: The particle size and polydispersity index (Table 1) of mannose-modified mtDLL1+4 mRNA-LNPs and ordinary mtDLL1+4 mRNA-LNPs were both within the standard range.

[0072] Table 1. Particle size and polydispersity index of mannose-modified mtDLL1+4 mRNA-LNPs

[0073]

[0074] Example 5: Construction of the HBV-carrier mouse model

[0075] Construction of the rAAV / HBV 1.3 mouse model: Normal adult C57BL / 6J mice, aged 5-6 weeks, were intravenously injected with recombinant adeno-associated virus serotype 8 (rAAV8) carrying 1.3 copies of the HBV genome (genotype D, subtype ayw) at a dose of 1×10⁻⁶ per mouse. 10 One viral genome (vg). Six weeks after injection, serum HBsAg levels were quantified, and mice with serum HBsAg levels exceeding 500 IU / mL were classified as HBV carrier mice.

[0076] Example 6

[0077] Test method:

[0078] The experimental groups received intravenous injections of 1 μg or 10 μg of LNP-OVA, mtDLL1+4 mRNA-LNPs, and mannose-modified mtDLL1+4 mRNA-LNPs, while the blank control group received the same volume of PBS. The promoting effect of mannose-modified mtDLL1+4 mRNA-LNPs on antigen presentation and proliferation of KC / KC2 ​​cells was evaluated by detecting the ratio of KC cells to KC2 cells and the MFI values ​​of CD86 and MHC II molecules on these cells.

[0079] Experimental results:

[0080] Compared with the 1 μg and 10 μg LNP-OVA and mtDLL1+4 mRNA-LNPs groups, the 10 μg mannose-modified mtDLL1+4 mRNA-LNPs group significantly promoted the growth of KC cells ( Figure 3 (a) in particular, the proportion of KC2 cells increased ( Figure 3 (b) and enhance KC cells ( Figure 4 a) In particular, the antigen-presenting ability of KC2 cells ( Figure 4 (b) In summary, the above results show that mannose-modified mtDLL1+4 mRNA-LNPs can efficiently and specifically target KCs and promote their differentiation and proliferation into the KC2 subtype, which has strong antigen presentation and immune activation functions. Simultaneously, it can safely and effectively eliminate HBV in CHB mice. In other words, Example 6 demonstrates that mannose-modified mtDLL1+4 mRNA-LNPs significantly promote the differentiation and antigen presentation function of KC cells, especially KC2 cells.

[0081] Example 7: LNP immunization strategy

[0082] WT mice in the experimental group (one group of WT mice was injected with chlorophosphate liposomes three days in advance to clear KC cells) were pre-injected intravenously with 10 μg of mtDLL1+4 mRNA-LNPs and mannose-modified mtDLL1+4 mRNA-LNPs, respectively. The blank control group was injected with the same volume of PBS. After 24 hours, they were infected with HBV virus. HBV-carrier mice in the experimental group were intravenously injected with 10 μg of mtDLL1+4 mRNA-LNPs and mannose-modified mtDLL1+4 mRNA-LNPs, respectively. The blank control group was injected with the same volume of PBS.

[0083] Example 8

[0084] Test method:

[0085] WT mice in the experimental group (one group of WT mice was injected with chlorophosphate liposomes three days in advance to eliminate KC cells) were pre-injected intravenously with 10 μg of mtDLL1+4 mRNA-LNPs and mannose-modified mtDLL1+4 mRNA-LNPs, respectively. The blank control group was injected with the same volume of PBS. After 24 hours, the mice were infected with HBV. The level of HBsAg in peripheral blood was measured to evaluate the preventive effect of mannose-modified mtDLL1+4 mRNA-LNPs against HBV and whether this anti-HBV effect depended on the presence of KC cells.

[0086] Experimental results:

[0087] Compared with the untreated group, the mtDLL1+4 mRNA-LNPs group, and the KC cell depletion group, the mannose-modified mtDLL1+4 mRNA-LNPs group reduced HBsAg levels in peripheral blood serum of CHB mice. Figure 5 (a) and the expression level of HBcAg in the liver ( Figure 5 (b) Based on the above results, it can be seen that mannose-modified mtDLL1+4 mRNA-LNPs have a preventive effect against HBV, and that this anti-HBV effect depends on the presence of KC cells.

[0088] Example 9

[0089] Test method:

[0090] The experimental group received intravenous injections of 10 μg of mtDLL1+4 mRNA-LNPs and mannose-modified mtDLL1+4 mRNA-LNPs, while the blank control group received the same volume of PBS. The anti-HBV efficacy of mannose-modified mtDLL1+4 mRNA-LNPs was evaluated by detecting the level of HBsAg in peripheral blood.

[0091] Experimental results:

[0092] Compared with the untreated group and the mtDLL1+4 mRNA-LNPs group, the mannose-modified mtDLL1+4 mRNA-LNPs group reduced HBsAg levels in peripheral blood serum of CHB mice. Figure 6 (a) and the expression level of HBcAg in the liver ( Figure 6 (b) Based on the above results, it can be seen that immunization with mannose-modified mtDLL1+4 mRNA-LNPs can safely and effectively clear HBV in CHB mice.

[0093] Example 10

[0094] Test method:

[0095] The experimental group received intravenous injections of 10 μg of mtDLL1+4 mRNA-LNPs and mannose-modified mtDLL1+4 mRNA-LNPs, while the blank control group received the same volume of PBS. After 24 hours, mouse liver mononuclear cells were isolated, and HBV-specific CD11a was detected by flow cytometry. hi CD8α lo Differences in cell proportions; detection of HBV-specific CD11a by flow cytometry hi CD8α lo The secretion levels of cytokines IFN-γ, IL-2, and TNF-α on cells.

[0096] Experimental results:

[0097] Studies have found that antigen-specific CD11a hi CD8α lo The proportion of cells was significantly increased in the livers of mice in the LNPs group. Figure 7 Furthermore, LNPs can restore cytokines such as IFN-γ (…). Figure 8 a and e), TNF-α ( Figure 8 (b and f), IL-2 ( Figure 8 c and g), IL-21 ( Figure 8 The expression of d and h) in mtDLL1+4 mRNA-LNPs, and the promotion of antigen-specific CD11a by mannose-modified mtDLL1+4 mRNA-LNPs. hi CD8α lo Cells have a stronger ability to simultaneously produce one, two, or three cytokines than mtDLL1+4 mRNA-LNPs ( Figure 8 The results suggest that mannose-modified mtDLL1+4 mRNA-LNPs can enhance antigen-specific CD8+ in HBV-carrier mice, thereby reversing the immune exhaustion state caused by chronic HBV infection. + To restore the function of T cells and reverse their depleted state.

[0098] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition, characterized by, The nucleic acid drug and the carrier loaded with the nucleic acid drug, the carrier being capable of specifically targeting liver Kupffer cells, the nucleic acid drug being mRNA, a gene sequence encoding the mRNA comprising a nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2; The carrier is a mannose-modified lipid nanoparticle.

2. The pharmaceutical composition of claim 1, wherein The mannose-modified lipid nanoparticle comprises a cationic lipid, an auxiliary phospholipid, a solid sterol lipid, a mannose and PEG co-modified lipid.

3. The pharmaceutical composition of claim 1, wherein The mannose-modified lipid nanoparticle is composed of Lipid5, DSPC, cholesterol, PEG, DSPE-PEG-mannose according to a molar ratio of 45-55:9-11:37-40:1:0.45-0.

55.

4. The pharmaceutical composition of claim 1, wherein The average particle size of the carrier loaded with the nucleic acid drug is 10-500 nm.

5. The pharmaceutical composition of claim 1, wherein The dosage form of the pharmaceutical composition is injection.

6. Use of the pharmaceutical composition of any one of claims 1-5 in the preparation of a drug for preventing and treating hepatitis B virus.

7. Use according to claim 6, wherein the compound is ###0002### The drug is a drug for treating chronic hepatitis B.

8. The use according to claim 6, characterized in that The administration subject of the drug is a human or non-human mammal.

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