Nucleic acid or nucleic acid combination, and use thereof in preparation of drug for treating hepatitis b

Abstract

Provided are a combination comprising a drug for treating hepatitis B and an antiviral agent, and a use thereof. The drug for treating hepatitis B comprises a nucleic acid or nucleic acid combination (e.g., an mRNA combination) encoding an antigen of hepatitis B virus. The combined use of the drug for treating hepatitis B and the antiviral agent can significantly reduce the number of liver HBV Core positive cells in an infected subject, greatly improve the HBV-specific T cell response level in the subject, and shows no obvious rebound of HBsAg after drug withdrawal.

Description

A nucleic acid or combination of nucleic acids and its application in the preparation of drugs for treating hepatitis B. Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a nucleic acid or combination of nucleic acids and its application in the preparation of drugs for treating hepatitis B. Background Technology

[0002] Hepatitis B (also known as hepatitis B virus infection) is a disease caused by infection with the hepatitis B virus (HBV). More than 400 million people worldwide are chronically infected with HBV, and it is estimated to cause 600,000 deaths annually.

[0003] Currently, the two most widely recognized classes of drugs for the prevention and treatment of hepatitis B are interferon and nucleoside analogues. However, both classes of drugs have several drawbacks, such as the tendency to develop drug resistance or limitations in their use. For example, interferon is prone to adverse reactions, and nucleoside analogues have issues with drug resistance and relapse after discontinuation. Lifelong treatment is required to reduce plasma viremia.

[0004] Therapeutic vaccines are natural, synthetic, or genetically engineered products or products that, when introduced into an organism infected with pathogens or suffering from certain diseases, induce a specific immune response to treat the disease or prevent its progression. Before 1995, the medical community generally believed that vaccines were only for disease prevention. With the development of immunological research, a new use for vaccines was discovered: the treatment of some intractable diseases. Since then, vaccines have acquired a dual function of prevention and treatment.

[0005] Therapeutic hepatitis B vaccines belong to specific active immunotherapy. Therapeutic hepatitis B vaccines under development are mainly divided into recombinant protein vaccines, DNA vaccines, DC vaccines, viral vector vaccines, and mRNA vaccines, but these vaccines still have some problems. Reports have indicated that although therapeutic vaccines can effectively induce anti-HBs antibodies in vivo (manifested as, for example, persistently high titers of HBsAb in the blood, decreased HBsAg levels, etc.), the HBV virus in the patient's liver is not cleared. For example, patients with chronic hepatitis B can produce anti-HBs neutralizing antibodies, but even 18 months after HBsAg seroconversion, their livers remain positive for both HBsAg and HBcAg, along with positive HBV DNA and HBeAg in their blood (see, for example, Su, J., et al. Journal of Hepatology 2023. vol. 78, 717–730; Zhang, ZH, et al. Journal of Viral Hepatitis, 2011, 18, 424–433; Galati, G. et al. BMC Gastroenterology 2014, 14:94). Therefore, the production of anti-HBs antibodies is only one important indicator of disease control; it can temporarily clear hepatitis B antigens from the blood, but does not mean the elimination of the hepatitis B virus from the body (see, for example, Zhang, ZH, et al. Journal of Viral Hepatitis, 2011, 18, 424–433; Galati, G. et al. BMC Gastroenterology 2014, 14:94). Hepatitis, 2011, 18, 424–433. With a deeper understanding of hepatitis B treatment, scientists have realized that therapeutic vaccines for hepatitis B, in addition to effectively inducing a long-term and sustained immune response, are more importantly able to effectively and continuously reduce and clear the hepatitis B virus in the body. For example, they can continuously reduce and clear hepatitis B virus DNA in the patient's blood and hepatitis B virus core antigen HBcAg in the patient's liver. This is the key to functional cure of hepatitis B (see, for example, Michler, T. et al. Gastroenterology 2020; 158:1762–1775; Su, J., et al. Journal of Hepatology 2023. vol. 78, 717–730).Especially HBcAg, its core role in the life cycle of HBV has been recognized. For example, it is a component of the HBV cccDNA minichromosome complex, participating in the transcription of cccDNA and the generation of HBV viral proteins; it can inhibit the host's immune response to HBV and slow down the clearance of HBV virus, etc. (see, for example, Bock, C.T., et al. (2001); Pollicino, T., et al. (2006); Viswanathan, U., et al. (2020); Du, J., et al. (2009); Zhao, H.J., et al. (2022), etc.). Therefore, reducing HBcAg plays an important role in the functional cure of hepatitis B. Unfortunately, the current hepatitis B vaccines still cannot achieve both inducing long-term and persistent immune responses and effectively reducing and clearing hepatitis B virus in the body for a long time.

[0006] Another problem with existing therapeutic vaccines is that the initial HBV antigen titer in patients can affect their antiviral effect. It is impossible or difficult to induce an effective immune response in chronic hepatitis B patients with high plasma levels of HBsAg (see, for example, Michler, T. et al. Gastroenterology 2020; 158: 1762–1775; Danming Zhu et al. J Immunol (2016) 196(7): 3079–3087). Usually, high HBV DNA and HBsAg levels to a certain extent correspond to the pathological conditions of chronic hepatitis B patients with more severe clinical conditions. Chronic hepatitis B patients with low levels of HBsAg are more likely to achieve functional cure through existing treatment methods. For example, the expert recommendations issued by the Chinese Medical Association pointed out that after NA treatment, HBV DNA < LOQ, HBeAg negative, HBsAg < 1500 IU / mL or for naïve CHB patients, HBeAg negative, HBV DNA < 1000 IU / mL, HBsAg < 500 IU / mL are all populations with advantages for clinical cure (Chinese Journal of Hepatology, 2024, 32(6): 497 - 503). In addition, existing therapeutic vaccines may cause hepatocyte lysis and have potential serious side effects.

[0007] Small interfering RNA (siRNA) can inhibit or block the expression of target genes in a sequence-specific manner based on the RNA interference (RNAi) mechanism, exerting an inhibitory effect at the level of mRNA translation to protein, thereby achieving the purpose of treating diseases. This requires stabilization modification of siRNA and the use of appropriate delivery systems to target organs and cells to improve metabolic stability.

[0008] Therefore, there is still a need in this field to develop more effective hepatitis B treatment strategies. Summary of the Invention

[0009] To address the aforementioned problems with existing therapeutic vaccines, this application provides a nucleic acid or a combination thereof, and its use in the preparation of a drug for treating hepatitis B.

[0010] In a first aspect, this application provides a nucleic acid or combination of nucleic acids encoding a hepatitis B virus antigen, wherein the antigen is a large S protein, and an S protein and / or a Core protein.

[0011] In some embodiments of this application, a nucleic acid or combination thereof is provided, the nucleic acid or combination thereof comprising a nucleic acid encoding the hepatitis B virus large S protein, and nucleic acids encoding the hepatitis B virus S protein and / or Core protein.

[0012] In some embodiments of this application, a nucleic acid is provided, the nucleic acid comprising a nucleic acid encoding the hepatitis B virus large S protein, the amino acid sequence of the large S protein being shown in any one of SEQ ID NO: 266, 514-518.

[0013] In some embodiments of this application, a nucleic acid or combination thereof is provided, the nucleic acid or combination thereof comprising:

[0014] (a) Nucleic acid encoding the hepatitis B virus large S protein, and nucleic acid encoding the hepatitis B virus S protein; or,

[0015] (b) Nucleic acid encoding the hepatitis B virus large S protein and the hepatitis B virus core protein; or,

[0016] (c) Nucleic acid encoding the hepatitis B virus large S protein, nucleic acid encoding the hepatitis B virus S protein, and nucleic acid encoding the hepatitis B virus Core protein.

[0017] In some embodiments of this application, a nucleic acid combination is provided, the combination comprising:

[0018] (i) a first nucleic acid containing a polynucleotide sequence encoding the hepatitis B virus large S protein; and,

[0019] (ii) a second nucleic acid containing a polynucleotide sequence encoding the hepatitis B virus S protein, and / or a third nucleic acid containing a polynucleotide sequence encoding the hepatitis B virus Core protein.

[0020] In other embodiments of this application, a nucleic acid combination is provided, the combination comprising:

[0021] (i) a first nucleic acid containing a polynucleotide sequence encoding the hepatitis B virus large S protein; and,

[0022] (ii) A second nucleic acid, which contains a polynucleotide sequence encoding the hepatitis B virus S protein.

[0023] In other embodiments of this application, a nucleic acid combination is provided, the combination comprising:

[0024] (i) a first nucleic acid containing a polynucleotide sequence encoding the hepatitis B virus large S protein; and,

[0025] (ii) A second nucleic acid, which contains a polynucleotide sequence encoding the hepatitis B virus Core protein.

[0026] In some preferred embodiments of this application, a nucleic acid combination is provided, the combination comprising:

[0027] (i) The first nucleic acid, which contains a polynucleotide sequence encoding the hepatitis B virus large S protein.

[0028] (ii) A second nucleic acid, which contains a polynucleotide sequence encoding the hepatitis B virus S protein, and,

[0029] (iii) The third nucleic acid contains a polynucleotide sequence encoding the hepatitis B virus Core protein.

[0030] In this application, "first nucleic acid", "second nucleic acid" and "third nucleic acid" are used only to distinguish them as different nucleic acids in the nucleic acid combination described in this application, and do not impose any limitation on the nucleic acids themselves.

[0031] Preferably, the above-mentioned nucleic acid includes a polynucleotide sequence as described in any one of SEQ ID NO: 1-6, 259-263.

[0032] In some embodiments of this application, the genotype of the hepatitis B virus is selected from one or more of A to H. In other embodiments of this application, the serotype of the hepatitis B virus is selected from one or more of ayw, adw, ayr, and adr.

[0033] In some embodiments of this application, the amino acid sequence of the hepatitis B virus large S protein is shown in any one of SEQ ID NO: 266, 514-518. Preferably, the polynucleotide sequence encoding the hepatitis B virus large S protein is shown in any one of SEQ ID NO: 1, 4 or 259-263.

[0034] In some embodiments of this application, the amino acid sequence of the hepatitis B virus S protein is shown in SEQ ID NO: 267. Preferably, the polynucleotide sequence encoding the hepatitis B virus S protein is shown in SEQ ID NO: 2 or 5.

[0035] In some embodiments of this application, the amino acid sequence of the hepatitis B virus Core protein is shown in SEQ ID NO: 268. Preferably, the polynucleotide sequence encoding the hepatitis B virus Core protein is shown in SEQ ID NO: 3 or 6.

[0036] In some embodiments of this application, the nucleic acid further comprises a 5'UTR sequence and / or a 3'UTR sequence.

[0037] In some embodiments of this application, the 5'UTR sequence is as shown in any of SEQ ID NO:7 to 12,264; and / or, the 3'UTR sequence is as shown in any of SEQ ID NO:13 to 17,265.

[0038] Some implementation schemes of this application:

[0039] The 5'UTR sequence is shown in SEQ ID NO:7, and the 3'UTR sequence is shown in SEQ ID NO:13;

[0040] The 5'UTR sequence is shown in SEQ ID NO:8, and the 3'UTR sequence is shown in SEQ ID NO:14;

[0041] The 5'UTR sequence is shown in SEQ ID NO:9, and the 3'UTR sequence is shown in SEQ ID NO:15;

[0042] The 5'UTR sequence is shown in SEQ ID NO:10, and the 3'UTR sequence is shown in SEQ ID NO:15;

[0043] The 5'UTR sequence is shown in SEQ ID NO:11, and the 3'UTR sequence is shown in SEQ ID NO:16;

[0044] The 5'UTR sequence is shown in SEQ ID NO:12, and the 3'UTR sequence is shown in SEQ ID NO:17; or,

[0045] The 5'UTR sequence is shown in SEQ ID NO:264, and the 3'UTR sequence is shown in SEQ ID NO:265.

[0046] In some embodiments of this application, the nucleic acid comprises a nucleotide sequence as shown in any of SEQ ID NO:18-35.

[0047] In some embodiments of this application, the nucleic acid further comprises a promoter and / or a polyA tail; the promoter is preferably a T7 promoter.

[0048] In some embodiments of this application, the nucleic acid comprises a nucleotide sequence as shown in any of SEQ ID NO:36-53, 254-258.

[0049] In some embodiments of this application, the nucleic acid is mRNA.

[0050] In some embodiments of this application, the mRNA further comprises a 5′ cap; and / or, the mRNA further comprises nucleoside modification, such as N1-methylpseuuridine modification.

[0051] In some embodiments of this application, a nucleic acid (e.g., the first nucleic acid described above) comprising a polynucleotide sequence encoding the hepatitis B virus large S protein is included, with a nucleotide sequence as shown in SEQ ID NO: 18, 21, 24, 27, 30, or 33. Preferably, the nucleic acid (e.g., the first nucleic acid described above) comprising a polynucleotide sequence encoding the hepatitis B virus large S protein is included, with a nucleotide sequence as shown in any one of SEQ ID NO: 36, 39, 42, 45, 48, 51, or 254-258.

[0052] In some embodiments of this application, a nucleic acid (e.g., the second nucleic acid described above) is included, which encodes a polynucleotide sequence encoding the hepatitis B virus S protein, and whose nucleotide sequence is shown in SEQ ID NO: 19, 22, 25, 28, 31, or 34. Preferably, the nucleic acid (e.g., the second nucleic acid described above) is included, which encodes a polynucleotide sequence encoding the hepatitis B virus S protein, and whose nucleotide sequence is shown in SEQ ID NO: 37, 40, 43, 46, 49, or 52.

[0053] In some embodiments of this application, a nucleic acid (e.g., the second or third nucleic acid described above) is included, which encodes a polynucleotide sequence encoding the hepatitis B virus Core protein, and whose nucleotide sequence is shown in SEQ ID NO: 20, 23, 26, 29, 32 or 35. Preferably, the nucleic acid (e.g., the second or third nucleic acid described above) is included, which encodes a polynucleotide sequence encoding the hepatitis B virus Core protein, and whose nucleotide sequence is shown in SEQ ID NO: 38, 41, 44, 47, 50 or 53.

[0054] In some further embodiments of this application, the nucleic acid combination comprises: a first nucleic acid containing a polynucleotide sequence as shown in any one of SEQ ID NO:1, 4 or 259-263, and a second nucleic acid containing a polynucleotide sequence as shown in SEQ ID NO:2 or 5 and / or a third nucleic acid containing a polynucleotide sequence as shown in SEQ ID NO:3 or 6.

[0055] In some embodiments of this application, the nucleic acid combination comprises: a first nucleic acid containing a nucleotide sequence as shown in SEQ ID NO:18, 21, 24, 27, 30 or 33; a second nucleic acid containing a nucleotide sequence as shown in SEQ ID NO:19, 22, 25, 28, 31 or 34; and / or a third nucleic acid containing a nucleotide sequence as shown in SEQ ID NO:20, 23, 26, 29, 32 or 35.

[0056] In some embodiments of this application, the nucleic acid combination comprises: a first nucleic acid containing a nucleotide sequence as described in any one of SEQ ID NO: 274, 277, 280, 283, 286, 289, 269-273; a second nucleic acid containing a nucleotide sequence as described in SEQ ID NO: 275, 278, 281, 284, 287 or 290; and / or a third nucleic acid containing a nucleotide sequence as described in SEQ ID NO: 276, 279, 282, 285, 288 or 291.

[0057] In some embodiments of this application, the nucleic acid combination comprises: a first nucleic acid containing a nucleotide sequence as shown in any one of SEQ ID NO:36, 39, 42, 45, 48, 51, 254-258; a second nucleic acid containing a nucleotide sequence as shown in SEQ ID NO:37, 40, 43, 46, 49 or 52; and / or a third nucleic acid containing a nucleotide sequence as shown in SEQ ID NO:38, 41, 44, 47, 50 or 53.

[0058] In some embodiments of this application, the nucleic acid combination includes: a first nucleic acid containing the nucleotide sequence shown in SEQ ID NO:1, and a second nucleic acid containing the nucleotide sequence shown in SEQ ID NO:2 and / or a third nucleic acid containing the nucleotide sequence shown in SEQ ID NO:3.

[0059] In some embodiments of this application, the nucleic acid combination comprises: a first nucleic acid containing the nucleotide sequence shown in SEQ ID NO:21, and a second nucleic acid containing the nucleotide sequence shown in SEQ ID NO:22 and / or a third nucleic acid containing the nucleotide sequence shown in SEQ ID NO:35.

[0060] In some embodiments of this application, the nucleic acid combination comprises: a first nucleic acid containing the nucleotide sequence shown in SEQ ID NO:277, and a second nucleic acid containing the nucleotide sequence shown in SEQ ID NO:278 and / or a third nucleic acid containing the nucleotide sequence shown in SEQ ID NO:291;

[0061] In some embodiments of this application, the nucleic acid combination comprises: a first nucleic acid containing the nucleotide sequence shown in SEQ ID NO:39, and a second nucleic acid containing the nucleotide sequence shown in SEQ ID NO:40 and / or a third nucleic acid containing the nucleotide sequence shown in SEQ ID NO:53.

[0062] It should be understood that the above-described nucleotide or amino acid sequences are merely exemplary sequences for embodiments of this application and are not intended to limit the scope of this application. Regarding nucleotide sequences, although the sequence listing indicates a DNA sequence, the above-described nucleotide sequences can represent either a DNA sequence or an RNA sequence. When representing an RNA sequence, the "T" represents uridine "U".

[0063] Secondly, this application provides a pharmaceutical composition (or "hepatitis B treatment drug") comprising any of the nucleic acids or combinations of nucleic acids as described above.

[0064] In some embodiments of this application, the pharmaceutical composition is a vaccine or a vaccine composition.

[0065] In some embodiments of this application, the pharmaceutical composition is used to treat and / or prevent diseases caused by hepatitis B virus. Preferably, the disease is hepatitis B. More preferably, the genotype of the hepatitis B virus is D and / or the serotype is ayw.

[0066] In some embodiments of this application, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or delivery carrier; preferably, the delivery carrier is a lipid nanoparticle.

[0067] In some embodiments of this application, the lipid nanoparticles satisfy one or more of the following conditions:

[0068] (1) The N / P ratio of the lipid nanoparticles is 5 to 6;

[0069] (2) The lipid phase of the lipid nanoparticles comprises one or more selected from SM-102, DSPC, cholesterol and DMG-PEG2000;

[0070] (3) The volume ratio of the lipid phase to the aqueous phase of the lipid nanoparticles is 1:2 to 1:4, preferably 1:3; and,

[0071] (4) The lipid nanoparticles are prepared using microfluidic technology.

[0072] Some implementation schemes of this application:

[0073] The N / P ratio of the lipid nanoparticles is 5.5 to 5.7; preferably 5.65 to 5.68, for example 5.67.

[0074] And / or, when using microfluidic technology, the total flow rate of microfluidic synthesis is 11-13 mL / min, preferably 12 mL / min; and / or, the flow rate ratio of the aqueous phase to the lipid phase of the lipid nanoparticles is 2:1-4:1, preferably 3:1.

[0075] Thirdly, this application provides a combination and / or composition of a pharmaceutical composition comprising any of the above-mentioned nucleic acids or combinations of nucleic acids or including them, and an antiviral agent.

[0076] In some embodiments of this application, the above-described nucleic acid encoding the hepatitis B virus large S protein or a pharmaceutical composition comprising thereof, in combination and / or in combination with an antiviral agent, are provided.

[0077] In some preferred embodiments of this application, a combination and / or composition is provided, the combination comprising: a nucleic acid containing the above-described polynucleotide sequence encoding the hepatitis B virus large S protein, and an antiviral agent.

[0078] In some embodiments of this application, the antiviral agent comprises an oligonucleotide that targets a gene of the hepatitis B virus. Preferably, the oligonucleotide is a double-stranded siRNA or an ASO.

[0079] In some embodiments of this application, the oligonucleotide sequence is as shown in SEQ ID NO: 67, 69, 71, 73, 75, 77, 80, 82, 98, 100, 102 or 107.

[0080] In some embodiments of this application, the oligonucleotide sequence is as shown in any one of SEQ ID NO: 67, 69, 71, 73, 75, 77, 80, 98, 100, 109-112 or 113-116.

[0081] In some embodiments of this application, the oligonucleotide further includes one or more of the following modifications: 5'-phosphate modification, 2'-O-methylation modification, 2'-fluoro modification, and phosphorothioate modification.

[0082] In some embodiments of this application, the oligonucleotide sequence is as shown in any one of SEQ ID NO: 68, 70, 72, 74, 76, 78-79, 81, 83-97, 99, 101, 103-106 or 108.

[0083] In some embodiments of this application, the double-stranded siRNA comprises a sense strand and an antisense strand, the sense strand comprising a sequence as shown in SEQ ID NO:67, 69, 71, 73, 75, 77, 80 or 82, and the antisense strand comprising a sequence as shown in SEQ ID NO:98, 100, 102 or 107.

[0084] In some embodiments of this application, the sense chain comprises a sequence as shown in any of SEQ ID NO: 67, 69, 71, 73, 75, 77, 80 or 109-112, and the antisense chain comprises a sequence as shown in any of SEQ ID NO: 98, 100, 113-116 or 107.

[0085] In some embodiments of this application, the positive chain comprises the sequence shown in SEQ ID NO:67, and the negative chain comprises the sequence shown in SEQ ID NO:98; or, the positive chain comprises the sequence shown in SEQ ID NO:69, and the negative chain comprises the sequence shown in SEQ ID NO:98; or, the positive chain comprises the sequence shown in SEQ ID NO:71, and the negative chain comprises the sequence shown in SEQ ID NO:98; or, the positive chain comprises the sequence shown in SEQ ID NO:73, and the negative chain comprises the sequence shown in SEQ ID NO:98; or, the positive chain comprises the sequence shown in SEQ ID NO:75, and the negative chain comprises the sequence shown in SEQ ID NO:98; or, the positive chain comprises the sequence shown in SEQ ID NO:67, and the negative chain comprises the sequence shown in SEQ ID NO:100; or, the positive chain comprises the sequence shown in SEQ ID NO:77, and the negative chain comprises the sequence shown in SEQ ID NO:102 or any one of SEQ ID NO:113-116; or, the positive chain comprises the sequence shown in SEQ ID NO:69, and the negative chain comprises the sequence shown in SEQ ID NO:98. The sequence shown in NO:80, wherein the antisense chain comprises a sequence as shown in any one of SEQ ID NO:102 or 113-116; or, the positive chain comprises a sequence as shown in any one of SEQ ID NO:82 or 109-112, wherein the antisense chain comprises a sequence as shown in any one of SEQ ID NO:102 or 113-116; or, the positive chain comprises a sequence as shown in any one of SEQ ID NO:82 or 109-112, wherein the antisense chain comprises a sequence as shown in SEQ ID NO:107.

[0086] In some embodiments of this application, the positive strand further includes one or more of the following modifications to the nucleotide: 5'-phosphate modification, 2'-O-methylation modification, 2'-fluoro modification, and phosphorothioate modification; the above modifications may be present on nucleotides at different positions of the positive strand, or may be present on nucleotides at the same position of the positive strand.

[0087] In some embodiments of this application, the antisense strand further includes one or more of the following modifications to the nucleotide: glycerol modification, 5'-phosphate modification, 2'-O-methylation modification, 2'-fluoro modification, and phosphorothioate modification; the above modifications may be present on nucleotides at different positions of the antisense strand, or may be present on nucleotides at the same position of the antisense strand.

[0088] In some embodiments of this application, the positive chain includes the following combination of modifications: 2'-O-methylation, 2'-fluorination, and thiophosphate modification.

[0089] In some embodiments of this application, the antisense chain includes one of the following combinations of modifications:

[0090] (1) Glycerol modification, 2'-O-methylation modification, 2'-fluoro modification and thiophosphate modification;

[0091] (2) 5'-phosphate modification, 2'-O-methylation modification, 2'-fluorination modification, and thiophosphate modification; and,

[0092] (3) Glycerol modification, 5'-phosphate modification, 2'-O-methylation modification, 2'-fluorination modification and thiophosphate modification.

[0093] In some embodiments of this application, the modifications to the nucleotides in the sense and antisense strands include one or more of the following:

[0094] (1) The first nucleotide at the 5' end of the antisense strand contains a 5'-phosphate modification;

[0095] (2) One or more adenosine nucleotides of the antisense chain contain glycerol modification;

[0096] (3) The first and second nucleotides at the 5' end of the antisense strand contain phosphate thiophosphate modification;

[0097] (4) The second and third nucleotides at the 3' end of the antisense strand contain a 2'-fluorinated modification; and,

[0098] (5) The first two nucleotides at the 5' end and the first three nucleotides at the 3' end of the positive strand contain 2'-O-methylation modification; or the first two nucleotides at the 5' end and the first four nucleotides at the 3' end of the positive strand contain 2'-O-methylation modification.

[0099] As is known to those skilled in the art, 2'-fluorination and 2'-O-methylation cannot coexist on the same nucleotide.

[0100] In some embodiments of this application, the positive chain comprises the sequence shown in SEQ ID NO: 67 or 68, and the negative chain comprises the sequence shown in SEQ ID NO: 98 or 99; or, the positive chain comprises the sequence shown in SEQ ID NO: 69 or 70, and the negative chain comprises the sequence shown in SEQ ID NO: 98 or 99; or, the positive chain comprises the sequence shown in SEQ ID NO: 71 or 72, and the negative chain comprises the sequence shown in SEQ ID NO: 98 or 99; or, the positive chain comprises the sequence shown in SEQ ID NO: 73 or 74, and the negative chain comprises the sequence shown in SEQ ID NO: 98 or 99; or, the positive chain comprises the sequence shown in SEQ ID NO: 75 or 76, and the negative chain comprises the sequence shown in SEQ ID NO: 98 or 99; or, the positive chain comprises the sequence shown in SEQ ID NO: 67 or 68, and the negative chain comprises the sequence shown in SEQ ID NO: 100 or 101; or, the positive chain comprises the sequence shown in SEQ ID NO: 67 or 68, and the negative chain comprises the sequence shown in SEQ ID NO: 100 or 101; or, the positive chain comprises the sequence shown in SEQ ID NO: 68 or 69. The sequence shown in SEQ ID NO:77 or 78, wherein the antisense chain contains the sequence shown in SEQ ID NO:102 or 103; or, the sequence shown in SEQ ID NO:77 or 79, wherein the antisense chain contains the sequence shown in SEQ ID NO:102 or 103; or, the sequence shown in SEQ ID NO:80 or 81, wherein the antisense chain contains the sequence shown in SEQ ID NO:102 or 103; or, the sequence shown in SEQ ID NO:82 or 83, wherein the antisense chain contains the sequence shown in SEQ ID NO:102 or 103; or, the sequence shown in SEQ ID NO:82 or 84, wherein the antisense chain contains the sequence shown in SEQ ID NO:102 or 103; or, the sequence shown in SEQ ID NO:82 or 85, wherein the antisense chain contains the sequence shown in SEQ ID NO:102 or 103; or, the sequence shown in SEQ ID NO:77 or 79, wherein the antisense chain contains the sequence shown in SEQ ID NO:102 or 103; or, the sequence shown in SEQ ID NO:80 or 81, wherein the antisense chain contains the sequence shown in SEQ ID NO:102 or 103; or, the sequence shown in SEQ ID NO:82 or 85, wherein the antisense chain contains the sequence shown in SEQ ID NO:102 or 103; or, the sequence shown in SEQ ID NO:80 or 81, wherein the antisense chain contains the sequence shown in SEQ ID NO:102 or 103. The sequence shown in NO:82 or 86, wherein the antisense chain comprises the sequence shown in SEQ ID NO:102 or 103; or, the sequence shown in SEQ ID NO:82 or 87, wherein the antisense chain comprises the sequence shown in SEQ ID NO:102 or 103; or, the sequence shown in SEQ ID NO:82 or 88, wherein the antisense chain comprises the sequence shown in SEQ ID NO:102 or 103; or, the sequence shown in SEQ ID NO:82 or 84, wherein the antisense chain comprises the sequence shown in SEQ ID NO:107 or 108.Alternatively, the positive chain may contain a sequence as shown in SEQ ID NO: 82 or 85, and the negative chain may contain a sequence as shown in SEQ ID NO: 107 or 108; or, the positive chain may contain a sequence as shown in SEQ ID NO: 82 or 86, and the negative chain may contain a sequence as shown in SEQ ID NO: 107 or 108; or, the positive chain may contain a sequence as shown in SEQ ID NO: 82 or 87, and the negative chain may contain a sequence as shown in SEQ ID NO: 107 or 108; or, the positive chain may contain a sequence as shown in SEQ ID NO: 82 or 88, and the negative chain may contain a sequence as shown in SEQ ID NO: 107 or 108; or, the positive chain may contain a sequence as shown in SEQ ID NO: 82 or 89, and the negative chain may contain a sequence as shown in SEQ ID NO: 102 or 103; or, the positive chain may contain a sequence as shown in SEQ ID NO: 82 or 90, and the negative chain may contain a sequence as shown in SEQ ID NO: 102 or 103; or, the positive chain may contain a sequence as shown in SEQ ID NO: 82 or 90, and the negative chain may contain a sequence as shown in SEQ ID NO: 102 or 103; or, the positive chain may contain a sequence as shown in SEQ ID NO: 82 or 90, and the negative chain may contain a sequence as shown in SEQ ID NO: 102 or 103. The sequence shown in SEQ ID NO: 82 or 91, wherein the antisense chain contains the sequence shown in SEQ ID NO: 102 or 103; or, the positive chain contains the sequence shown in SEQ ID NO: 82 or 92, wherein the antisense chain contains the sequence shown in SEQ ID NO: 102 or 103; or, the positive chain contains the sequence shown in SEQ ID NO: 82 or 93, wherein the antisense chain contains the sequence shown in SEQ ID NO: 102 or 103; or, the positive chain contains the sequence shown in SEQ ID NO: 82 or 94, wherein the antisense chain contains the sequence shown in SEQ ID NO: 102 or 103; or, the positive chain contains the sequence shown in SEQ ID NO: 82 or 95, wherein the antisense chain contains the sequence shown in SEQ ID NO: 102 or 104; or, the positive chain contains the sequence shown in SEQ ID NO: 82 or 96, wherein the antisense chain contains the sequence shown in SEQ ID NO: 102 or 105; or, the positive chain contains the sequence shown in SEQ ID NO: 82 or 96, wherein the antisense chain contains the sequence shown in SEQ ID NO: 102 or 105; or, the positive chain contains the sequence shown in SEQ ID NO: 82 or 91. The sequence shown in NO:82 or 97, wherein the antisense chain comprises the sequence shown in SEQ ID NO:102 or 106.

[0101] In some embodiments of this application, the sequence of the positive chain is as shown in SEQ ID NO:68, and the sequence of the negative chain is as shown in SEQ ID NO:99; or, the sequence of the positive chain is as shown in SEQ ID NO:70, and the sequence of the negative chain is as shown in SEQ ID NO:99; or, the sequence of the positive chain is as shown in SEQ ID NO:72, and the sequence of the negative chain is as shown in SEQ ID NO:99; or, the sequence of the positive chain is as shown in SEQ ID NO:74, and the sequence of the negative chain is as shown in SEQ ID NO:99; or, the sequence of the positive chain is as shown in SEQ ID NO:76, and the sequence of the negative chain is as shown in SEQ ID NO:99; or, the sequence of the positive chain is as shown in SEQ ID NO:68, and the sequence of the negative chain is as shown in SEQ ID NO:101; or, the sequence of the positive chain is as shown in SEQ ID NO:78, and the sequence of the negative chain is as shown in SEQ ID NO:103; or, the sequence of the positive chain is as shown in SEQ ID NO:68, and the sequence of the negative chain is as shown in SEQ ID NO:103; or, the sequence of the positive chain is as shown in SEQ ID NO:68, and the sequence of the negative chain is as shown in SEQ ID NO:103. As shown in NO:79, the sequence of the antisense chain is as shown in SEQ ID NO:103; or, the sequence of the right chain is as shown in SEQ ID NO:81, and the sequence of the antisense chain is as shown in SEQ ID NO:103; or, the sequence of the right chain is as shown in SEQ ID NO:83, and the sequence of the antisense chain is as shown in SEQ ID NO:103; or, the sequence of the right chain is as shown in SEQ ID NO:84, and the sequence of the antisense chain is as shown in SEQ ID NO:103; or, the sequence of the right chain is as shown in SEQ ID NO:85, and the sequence of the antisense chain is as shown in SEQ ID NO:103; or, the sequence of the right chain is as shown in SEQ ID NO:86, and the sequence of the antisense chain is as shown in SEQ ID NO:103; or, the sequence of the right chain is as shown in SEQ ID NO:87, and the sequence of the antisense chain is as shown in SEQ ID NO:103; or, the sequence of the right chain is as shown in SEQ ID NO:88, and the sequence of the antisense chain is as shown in SEQ ID NO:103. As shown in NO:103; or, the sequence of the positive chain is as shown in SEQ ID NO:84, and the sequence of the negative chain is as shown in SEQ ID NO:108; or, the sequence of the positive chain is as shown in SEQ ID NO:85, and the sequence of the negative chain is as shown in SEQ ID NO:108; or, the sequence of the positive chain is as shown in SEQ ID NO:86, and the sequence of the negative chain is as shown in SEQ ID NO:108; or, the sequence of the positive chain is as shown in SEQ ID NO:87, and the sequence of the negative chain is as shown in SEQ ID NO:108.Alternatively, the sequence of the positive chain is as shown in SEQ ID NO:88, and the sequence of the negative chain is as shown in SEQ ID NO:108; or, the positive chain comprises the sequence shown in SEQ ID NO:89, and the negative chain comprises the sequence shown in SEQ ID NO:103; or, the positive chain comprises the sequence shown in SEQ ID NO:90, and the negative chain comprises the sequence shown in SEQ ID NO:103; or, the positive chain comprises the sequence shown in SEQ ID NO:91, and the negative chain comprises the sequence shown in SEQ ID NO:103; or, the positive chain comprises the sequence shown in SEQ ID NO:92, and the negative chain comprises the sequence shown in SEQ ID NO:103; or, the positive chain comprises the sequence shown in SEQ ID NO:93, and the negative chain comprises the sequence shown in SEQ ID NO:103; or, the positive chain comprises the sequence shown in SEQ ID NO:94, and the negative chain comprises the sequence shown in SEQ ID NO:103; or, the positive chain comprises the sequence shown in SEQ ID NO:89, and the negative chain comprises the sequence shown in SEQ ID NO:103; The sequence shown in SEQ ID NO:95, wherein the antisense chain comprises the sequence shown in SEQ ID NO:104; or, the forward chain comprises the sequence shown in SEQ ID NO:96, and the antisense chain comprises the sequence shown in SEQ ID NO:105; or, the forward chain comprises the sequence shown in SEQ ID NO:97, and the antisense chain comprises the sequence shown in SEQ ID NO:106.

[0102] In some embodiments of this application, the antiviral agent comprises one or more oligonucleotides selected from the following:

[0103] (1) The positive strand contains a double-stranded siRNA containing a sequence as shown in SEQ ID NO:56 and the antisense strand contains a sequence as shown in SEQ ID NO:57;

[0104] (2) The sense strand contains a double-stranded siRNA containing a sequence as shown in SEQ ID NO:58 and the antisense strand contains a sequence as shown in SEQ ID NO:59;

[0105] (3) The positive strand contains a double-stranded siRNA containing a sequence as shown in SEQ ID NO:60 and the antisense strand contains a sequence as shown in SEQ ID NO:61;

[0106] (4) The sense strand contains a double-stranded siRNA containing a sequence as shown in SEQ ID NO:62 and the antisense strand contains a sequence as shown in SEQ ID NO:63;

[0107] (5) The positive strand contains a double-stranded siRNA containing a sequence as shown in SEQ ID NO:64 and the antisense strand contains a sequence as shown in SEQ ID NO:65;

[0108] (6) An ASO containing the sequence shown in SEQ ID NO:66; and,

[0109] (7) ASO AHB-137.

[0110] In some embodiments of this application, the antiviral agent comprises: a double-stranded siRNA having a sense strand containing the sequence shown in SEQ ID NO:60 and an antisense strand containing the sequence shown in SEQ ID NO:61, and a double-stranded siRNA having a sense strand containing the sequence shown in SEQ ID NO:62 and an antisense strand containing the sequence shown in SEQ ID NO:63.

[0111] In some embodiments of this application, the mass ratio of a double-stranded siRNA comprising the sequence shown in SEQ ID NO:60 in the sense strand and the sequence shown in SEQ ID NO:61 in the antisense strand, and a double-stranded siRNA comprising the sequence shown in SEQ ID NO:62 in the sense strand and the sequence shown in SEQ ID NO:63 in the antisense strand, is 2:1 to 4:1, for example, 3:1.

[0112] In some embodiments of this application, the antiviral agent further comprises a pharmaceutically acceptable carrier, delivery carrier, and / or ligand.

[0113] In some embodiments of this application, the pharmaceutically acceptable carrier is water, saline solution, or buffer solution.

[0114] In some embodiments of this application, the buffer solution contains a buffering agent including acetate, citrate, alcohol-soluble gluten, carbonate or phosphate, Tris-hydrochloric acid or any combination thereof; for example, phosphate.

[0115] In some embodiments of this application, the delivery vector is selected from LNPs or exosomes, and the ligand is selected from carbohydrates and their derivatives. The carbohydrates include monosaccharides (e.g., GalNAc (N-acetylgalactosamine molecule)), disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides.

[0116] In some embodiments of this application, the double-stranded siRNA is attached to carbohydrates and their derivatives via linkers that are monovalent, divalent, or trivalent.

[0117] In some embodiments of this application, the double-stranded siRNA described herein is conjugated with bivalent and trivalent branched linkers, including structures shown in any one of formulas (IV)–(VII):

[0118] in:

[0119] q 2A q 2B q 3A q 3B q4 A q 4B q 5A q 5B and q 5C Each occurrence independently represents 0-20, and the repeating units can be the same or different;

[0120] P 2A P 2B P 3A P 3B P 4A P 4B P 5A P 5B P 5C T 2A T 2B T 3A T 3B T 4A T 4B T 5A T 5B T 5C Each occurrence is independently represented by the following: non-existent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O.

[0121] Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5C Each time it appears, it is independently absent, alkylene, or substituted alkylene, wherein one or more methylene groups may be O, S, S(O), SO2, or N(R). N One or more of the following can be interrupted or terminated: C(R') = C(R”), C≡C, or C(O);

[0122] R 2A R 2B R 3A R 3B R 4A R 4B R 5A R 5B R5C Each occurrence is independently represented by the following: non-existent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R). a -C(O), -C(O)-CH(R) a )-NH-, CO, CH=NO,

[0123] Or heterocyclic group;

[0124] L 2A L 2B L 3A L 3B L 4A L 4B L 5A L 5B and L 5C The term "ligand" indicates that each instance independently represents a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide.

[0125] R a It is H or an amino acid side chain.

[0126] Trivalent branched GalNAc derivatives are particularly useful for use with siRNAs described in this application to suppress the expression of target genes, such as those of formula (VII):

[0127] Where L 5A L 5B and L 5C It refers to monosaccharides, such as GalNAc derivatives.

[0128] Examples of suitable GalNAc derivatives with conjugated divalent and trivalent branched linker groups include, but are not limited to, the following compounds:

[0129] In some embodiments of this application, the siRNA preparation comprises ligand 1.

[0130] In some embodiments of this application, the carbohydrate contained in the ligand is L96 GalNAc, for example, L96 GalNAc conjugated by a trivalent branched linker group. It can also be a ligand in ligand 1 where GalNAc is replaced with L96 GalNAc, with the structure shown below:

[0131] In some embodiments of this application, the oligonucleotides of this disclosure are directly or indirectly conjugated to monovalent GalNAc. In some embodiments, the oligonucleotides are directly or indirectly conjugated to more than one monovalent GalNAc (i.e., conjugated to 2, 3, or 4 monovalent GalNAc moieties, and typically conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, the oligonucleotides of this disclosure are conjugated to one or more divalent, trivalent, or tetravalent GalNAc moieties.

[0132] In some embodiments of this application, one or more nucleotides (e.g., 1, 2, 3, 4, 5, or 6) of the oligonucleotide are each conjugated to a GalNAc moiety. In some embodiments, the targeting ligand is conjugated to 2-4 nucleotides at either end of the sense or antisense strand (e.g., the ligand is conjugated to a 2-4 nucleotide overhang or extension at the 5' or 3' end of the sense or antisense strand), such that the GalNAc moiety resembles the bristles of a toothbrush, and the oligonucleotide resembles the toothbrush. In some embodiments, the GalNAc moiety is conjugated to a nucleotide of the sense strand. For example, four GalNAc moieties can be conjugated to nucleotides in a tetracycle of the sense strand, wherein each GalNAc moiety is conjugated to one nucleotide.

[0133] In some embodiments, the oligonucleotide of this application comprises a monovalent GalNac linked to a guanidine nucleotide, referred to as [ademG-GalNAc] or 2'-aminodiethoxymethanol-guanidine-GalNAc.

[0134] In some embodiments of this application, the oligonucleotide of this application comprises a monovalent GalNac linked to an adenine nucleotide, referred to as [ademA-GalNAc] or 2'-aminodiethoxymethanol-adenine-GalNAc.

[0135] In this application, the types of adapters, delivery vectors, and the methods of ligation with siRNA can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.

[0136] Fourthly, this application provides the use of any of the above-mentioned nucleic acids or combinations of nucleic acids in the preparation of medicaments for the prevention and / or treatment of diseases caused by hepatitis B virus.

[0137] In some embodiments of this application, the disease is hepatitis B. More preferably, the genotype of the hepatitis B virus is D and / or the serotype is ayw, and / or the genotype of the hepatitis B virus is C.

[0138] Fifthly, this application provides the use of any of the above-mentioned nucleic acids or combinations of nucleic acids in the preparation of a medicament for the prevention and / or treatment of diseases caused by hepatitis B virus, wherein the medicament is used in combination with the antiviral agent as described above.

[0139] In addition, the use of any of the above-mentioned nucleic acids or combinations of nucleic acids and the antiviral agents as described above in the combined preparation of medicaments for the prevention and / or treatment of diseases caused by hepatitis B virus is also provided.

[0140] In some embodiments of this application, the disease is hepatitis B; and / or, the genotype of the hepatitis B virus is type D and / or the serotype is ayw, and / or, the genotype of the hepatitis B virus is type C.

[0141] Sixthly, this application provides a method for preventing and / or treating diseases caused by hepatitis B virus, the method comprising administering to a subject in need an effective amount of any of the aforementioned nucleic acids or combinations of nucleic acids, or a pharmaceutical composition comprising the same.

[0142] In addition, a method for preventing and / or treating disease caused by hepatitis B virus (or "combination therapy") is provided, the method comprising administering to a subject in need an effective amount of any of the nucleic acids or combinations of nucleic acids as described above or a pharmaceutical composition containing therefrom, and an antiviral agent as described above.

[0143] In some preferred embodiments, this application also provides a method for preventing and / or treating diseases caused by hepatitis B virus, the method comprising administering to a subject in need an effective amount of a combination and / or composition of a pharmaceutical composition comprising any of the above-described nucleic acids or combinations thereof or including thereof and an antiviral agent.

[0144] In some embodiments of this application, the methods of administering any of the aforementioned nucleic acids or combinations of nucleic acids or pharmaceutical compositions containing them, as well as the aforementioned antiviral agents, include, but are not limited to, administering the nucleic acid or combination of nucleic acids or pharmaceutical compositions containing them at specific time intervals, at specific times, or separately, or administering the antiviral agent. Preferably, for example, as described in Example 5 of this application. The specific method of administration depends on certain factors, including the subject's body size, body surface area, age, the specific combination to be administered, the active ingredient in the combination, the time and route of administration, overall health status, and concurrent administration with other drugs.

[0145] In some embodiments of this application, the nucleic acids or combinations thereof, or pharmaceutical compositions or antiviral agents described herein, are administered to the subject via enteral (e.g., via a gastric feeding tube, a duodenal feeding tube, a gastrostomy, or the rectum), parenteral (e.g., subcutaneous injection, intravenous injection or infusion, intra-arterial injection or infusion, intraosseous infusion, intramuscular injection, intracerebral injection, intraventricular injection, intrathecal injection), local (e.g., epidermal, inhalation, via eye drops, or via mucous membranes), or by direct injection into a target organ (e.g., the liver of the subject). Typically, the nucleic acids or combinations thereof, or pharmaceutical compositions or antiviral agents described herein, are administered intravenously or subcutaneously.

[0146] In some embodiments of this application, the subject to be treated is a human or a non-human primate or other mammal subject. Other exemplary subjects include domestic animals, such as dogs and cats; livestock, such as horses, cattle, pigs, sheep, goats, and chickens; and animals, such as mice, rats, guinea pigs, and hamsters.

[0147] In some embodiments of this application, the disease is hepatitis B; and / or, the genotype of the hepatitis B virus is D and / or the serotype is ayw.

[0148] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.

[0149] All reagents and raw materials used in this application are commercially available.

[0150] The positive and progressive effects of the nucleic acid or combination of nucleic acids in this application, or the pharmaceutical composition containing the above-mentioned nucleic acid or combination of nucleic acids, include:

[0151] (i) Effectively induces a long-lasting immune response, for example, generating sustained high levels of antibody titers while maintaining HBsAg at low levels for an extended period; and

[0152] (ii) It can effectively and continuously reduce and clear hepatitis B virus in the body. For example, it can significantly reduce and clear hepatitis B virus core antigen HBcAg in the liver of HBV-infected subjects in the long term, significantly reduce the number of HBV Core positive cells in the liver in the long term, continuously reduce and clear hepatitis B virus DNA in the blood of patients, and greatly improve the HBV-specific T cell response level of subjects, and no obvious rebound was observed after drug withdrawal.

[0153] (iii) It can effectively and continuously reduce and eliminate hepatitis B virus of various genotypes.

[0154] In addition, the nucleic acids or combinations of nucleic acids in this application have low liver toxicity and high safety. Attached Figure Description

[0155] Figure 1 shows the results of Western blot analysis of mRNA expression in cells; where M represents Marker, NC represents negative control, and the numbers correspond to the sequences in Table 1 (e.g., "3" represents sequence #3 in Table 1).

[0156] Figure 2 shows the signal intensity of in vivo mouse imaging.

[0157] Figure 3 shows the results of in vivo imaging of mice.

[0158] Figure 4 shows the HBsAb titer results.

[0159] Figure 5 shows the HBcAb titer results.

[0160] Figure 6 shows the results of HBV DNA content detection in mouse plasma.

[0161] Figure 7 shows the results of HBsAg content detection in mouse plasma.

[0162] Figure 8 shows the results of HBsAg content detection in mouse plasma.

[0163] Figure 9 shows the results of detecting the number of HBV Core positive cells in mouse liver. Figure 9A shows the results on day 69, and Figures 9B and 9C show the results on day 119.

[0164] Figure 10 shows the results of HBV-specific T cell responses in mouse spleen.

[0165] Figure 11 shows the structure of NAG37.

[0166] Figure 12 shows the structure of MePhosphonate-4O-mU.

[0167] Figure 13 shows the results of HBV DNA content detection in the plasma of mice in each treatment group.

[0168] Figure 14 shows the results of ALT content detection in the plasma of mice in each drug administration group.

[0169] Figure 15 shows the inhibition results of neutralizing antibody on HBV DNA.

[0170] Figure 16 shows the inhibition results of neutralizing antibodies on HBsAg.

[0171] Figure 17 shows the inhibition results of neutralizing antibodies against HBeAg.

[0172] Figure 18 shows the results of HBsAg content detection in mouse plasma.

[0173] Figure 19A shows the results of HBsAg content detection in mouse plasma.

[0174] Figure 19B shows the results of HBsAg content detection in mouse plasma.

[0175] Figure 20 shows the results of HBsAg content detection in mouse plasma.

[0176] Figure 21 shows the results of HBV DNA content detection in mouse plasma.

[0177] Figure 22 shows the results of HBsAg content detection in mouse plasma.

[0178] Figure 23 shows the results of HBV DNA content detection in mouse plasma.

[0179] Figure 24 shows the results of in vitro immunoassay of PBMCs.

[0180] Figure 25 shows the results of HBsAg content detection in mouse plasma.

[0181] Figure 26 shows the results of HBV DNA content detection in mouse plasma.

[0182] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational procedures used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all routine procedures widely used in their respective fields. To better understand this application, definitions and explanations of relevant terms are provided below:

[0183] Hepatitis B virus

[0184] Hepatitis B virus (HBV) is the pathogen that causes hepatitis B (HBV for short). Under an electron microscope, HBV can appear as three types of particle structures: large spherical particles with a diameter of about 42 nm, small spherical particles with a diameter of about 22 nm, and tubular particles. Among them, the large spherical particles (Dane particles) are complete viral particles, composed of an envelope and a nucleocapsid. The envelope contains HBsAg, glycoproteins, and cellular lipids, while the core particle contains core protein (HBcAg), circular double-stranded HBV-DNA, and HBV-DNA polymerase. This is the complete form of the virus and it is infectious.

[0185] Eight HBV genotypes, designated A through H, have been identified, each with distinct geographical distributions. This virus is non-cytopathic and possesses virus-specific cellular immunity, which is a major determinant of the consequences of HBV exposure (acute infection 6 months after liver disease remission; or chronic HBV infection frequently associated with progressive liver damage). The term "HBV" as used in this article includes any one of the eight HBV genotypes (A through H). Reference amino acid sequences and complete coding sequences of the HBV genome can be found, for example, in GenBank accessions U95551.1, GI:21326584, and GI:3582357.

[0186] Based on a common antigenic determinant "a" and two subtype determinants "d / y" and "w / r" on the HBV envelope protein, HBV serotypes are divided into four subtypes: ayw, adw, ayr, and adr. "w" is further divided into "w1-4" and "q" into "q" and "q-", expanding to a total of ten serotypes: ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq, adrq-, and adw4q-.

[0187] The HBV genome contains four open reading frames, designated S, C, P, and X regions. The S region is further divided into three segments: the S gene, the preS1 gene, and the preS2 gene, which encode the S protein, preS1 protein, and preS2 protein, respectively. These three proteins are collectively known as the large S protein. The C gene in the C region encodes the Core protein, also known as the nucleocapsid protein. The wild-type or natural genome sequences of HBV for each serotype and genotype, as well as the amino acid sequences of each protein, are known and can be obtained from publicly available databases such as GenBank, UniProt, and OMIM.

[0188] In this application, large S protein, S protein, or Core protein includes wild-type or naturally occurring large S protein, S protein, or Core protein, i.e., large S protein, S protein, or Core protein having wild-type or naturally occurring amino acid sequences, preferably, these amino acid sequences are described in publicly available databases or literature. In addition, large S protein, S protein, or Core protein also includes proteins having amino acid sequences with the following characteristics (1)-(3): (1) having an amino acid sequence length similar to that of wild-type or naturally occurring large S protein, S protein, or Core protein (e.g., a sequence length difference of no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, preferably no more than 5%); (2) having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% sequence identity with the amino acid sequence of wild-type or naturally occurring large S protein, S protein, or Core protein; and (3) exhibiting antigenicity similar to that of wild-type or naturally occurring large S protein, S protein, or Core protein in in vitro or in vivo experiments. Such amino acid sequences are artificially designed based on wild-type or naturally occurring amino acid sequences, for example, conserved or shared sequences of wild-type or naturally occurring Large S protein, S protein, or Core protein. These conserved or shared sequences can be obtained using methods illustrated, for example, those shown in Liao, F. et al., *Replication and Expression of the Consensus Genome of Hepatitis B Virus Genotype C from the Chinese Population. Viruses 2023, 15, 2302*. A conservative sequence (or shared sequence) generally refers to a sequence consisting of the most frequently occurring (i.e., highest frequency) bases or amino acids at each position in a set (e.g., at least 20, 30, 40, 50, 100, 150, 200, 500, 1000, 2000) of DNA, RNA, or protein sequences that are very similar but not identical.Specifically, for example, for the Large S protein, S protein, or Core protein in this application, the conserved sequence or common sequence is preferably selected from several (e.g., at least 20, 30, 40, 50, 100, 150, 200, 500, 1000, 2000) amino acid sequences of the Large S protein, S protein, or Core protein disclosed in a public database (or wild-type or naturally occurring), and for each residue position, the amino acid sequence composed of the amino acid residues that appear most frequently at that position is selected.

[0189] Nucleic acid

[0190] As used herein, “nucleic acid” refers to a nucleotide chain of any length and includes both DNA and RNA. The terms “nucleotide sequence,” “nucleic acid,” or “polynucleotide” include oligonucleotides (i.e., short polynucleotides). It also refers to synthetic and / or non-naturally occurring nucleic acid molecules (e.g., those containing nucleotide analogs or modified backbone residues or bonds). The term also refers to deoxyribonucleotides or ribonucleotide oligonucleotides in single-stranded or double-stranded form, modified nucleotides or bases, and / or their analogs, or any substrate capable of being incorporated into the chain by DNA or RNA polymerases. The term covers nucleic acids containing natural nucleotide analogs. The term also covers nucleic acid-like structures having a synthetic backbone. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses its conserved modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Molecular Cell Probes 8:91-98 (1994)).

[0191] Nucleic acid combination

[0192] In this application, the "nucleic acid combination" comprises at least one nucleic acid as defined in one of the combinations described in this application (containing an antigen encoding HBV, such as large S, S, or Core). In some embodiments, the "nucleic acid combination" comprises two or more nucleic acids as defined in one of the combinations described in this application (encoding the same or different types of HBV antigens). In some embodiments, the two or more nucleic acids contain antigens encoding different HBV antigens (e.g., large S+S, large S+Core, S+Core, or large S+S+Core). In some embodiments, the "nucleic acid combination" comprises three nucleic acids as defined in one of the combinations described in this application (encoding the same or different types of HBV antigens). In some embodiments, the three nucleic acids each contain one of three different antigens encoding HBV: large S, S, or Core.

[0193] UTR sequence

[0194] UTR, or Untranslated Region, is located at the 5' and 3' ends of mature mRNA (the 5' UTR is upstream of the translation start codon, and the 3' UTR is downstream of the translation stop codon). It does not encode proteins but has translation regulatory functions and participates in regulating mRNA stability and intracellular localization.

[0195] Hepatitis B treatment drugs (e.g., mRNA vaccines)

[0196] In this application, the "hepatitis B treatment drug" comprises nucleic acid (e.g., mRNA) or a combination of nucleic acids (e.g., a combination of mRNAs) encoding antigens of the hepatitis B virus. The hepatitis B treatment drug described in this application can be a vaccine, such as an mRNA vaccine, which is a third-generation vaccine following inactivated vaccines, live attenuated vaccines, subunit vaccines, and viral vector vaccines. An mRNA vaccine involves introducing mRNA containing an antigen protein into the subject, where it is directly translated to form the corresponding antigen protein, thereby inducing a specific immune response in the body to achieve immune prevention or treatment. mRNA vaccines utilize the viral gene sequence rather than the virus itself; therefore, mRNA vaccines do not contain viral components and pose no risk of infection. In vitro transcribed (IVT) mRNA mimics the structure of endogenous mRNA and has five parts, from 5′ to 3′: 5′cap, 5′UTR, open reading frame encoding the antigen, 3′UTR, and PolyA tail. mRNA vaccines typically contain modified nucleosides, such as pseudouridine, N1-methylpseudouridine, or other nucleoside analogs. The use of modified nucleosides, especially modified uridines, prevents pattern recognition receptors from recognizing the protein, ensuring that the translation process produces sufficient levels of protein.

[0197] lipid nanoparticles

[0198] Lipid nanoparticles (LNPs) are a key technology in lipid delivery systems and represent a significant advancement in oligonucleotide-based therapeutics. LNP formulations consist of four types of lipids: ionizable cationic lipids, phospholipids, cholesterol or cholesterol derivatives, and polyethylene glycol (PEG)-lipids. Oligonucleotides (e.g., mRNA) encapsulated within the lipid nanoparticles are protected from enzymatic degradation during delivery and are efficiently delivered into cells. Within the cells, the contents of the lipid nanoparticles (e.g., mRNA) are released and translated into therapeutic proteins. Those skilled in the art can prepare lipid nanoparticles using conventional methods of the prior art, such as using Moderna's formulations combined with microfluidic (MF) synthesis processes. The "N / P ratio" refers to the molar ratio of ionizable lipid amine to oligonucleotide phosphate (N / P) in the LNP, representing the charge balance between the ionizable cationic lipid cationic tertiary amine and the oligonucleotide backbone anionic phosphate groups.

[0199] Pharmaceutically acceptable carriers

[0200] In this application, suitable pharmaceutically acceptable carriers include pharmaceutical excipients, such as those known in the art, pharmaceutical carriers, pharmaceutical excipients, including buffers. "Pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Water is a preferred carrier when administered intravenously. Saline solutions and aqueous dextran and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The preparation can be made by mixing nucleic acids or combinations of nucleic acids encoding the antigen of hepatitis B virus (HBV) of this application, or oligonucleotides targeting the HBV gene, with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution. Sustained-release formulations can also be prepared, suitable examples of which include a semi-permeable matrix of a solid hydrophobic polymer containing nucleic acids or combinations of nucleic acids encoding the antigen of HBV of this application, or oligonucleotides targeting the HBV gene, said matrix being in the form of shaped articles, such as films or microcapsules.

[0201] Effective dose

[0202] In this application, the term "effective amount" means the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "effective amount" means the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. Within its scope, the term also includes amounts that effectively enhance normal physiological function.

[0203] antiviral agents

[0204] In this application, the antiviral agent is capable of inhibiting HBV replication and assembly. In some embodiments, the antiviral agent of this application includes broad-spectrum antiviral agents (e.g., nucleoside antiviral agents, nucleocapsid inhibitors (GLS4, ALG-000184), HBV antibodies, mRNA destabilizers, or interferons) and / or specific anti-HBV viral activators (e.g., oligonucleotides specifically targeting HBV). In some embodiments, the term "oligonucleotide" in this application refers to short nucleic acids, such as those less than 100 nucleotides in length. Oligonucleotides can be single-stranded or double-stranded. Oligonucleotides may or may not have a double-stranded region. As a set of non-limiting examples, oligonucleotides can be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer substrate interfering RNA (dsiRNA), antisense oligonucleotides (ASO), short siRNA, or single-stranded siRNA. In some embodiments, the double-stranded oligonucleotide is an RNAi oligonucleotide. In some embodiments, the oligonucleotides of this application further include nucleotide modifications.

[0205] In some embodiments, nucleoside antiviral agents include, but are not limited to: ribavirin, acyclovir, ganciclovir, entecavir, and tenofovir; in some embodiments, nucleocapsid inhibitors include, but are not limited to: GLS4 and ALG-000184; in some embodiments, mRNA destabilizers include, but are not limited to: GSK3965193. In some embodiments, siRNAs include, but are not limited to: ALN-HBV02, RG-6346, JNJ-73763976, JNJ-73763924, and ARB-270729; in some embodiments, ASOs include, but are not limited to: bepirovirsen and AHB-137.

[0206] Nucleotide modification

[0207] As used herein, the term "nucleotide modification" refers to having one or more chemical modifications compared to a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. In some embodiments, the nucleotide with the nucleotide modification is a non-naturally occurring nucleotide. In some embodiments, the modified nucleotide has one or more chemical modifications in its sugar, nucleotide, and / or phosphate groups. In some embodiments, the modified nucleotide has one or more chemical moieties conjugated to the corresponding reference nucleotide. Generally, the modified nucleotide imparts one or more desired properties to the nucleic acid in which the modified nucleotide is present. For example, the modified nucleotide can improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, and reduced immunogenicity, etc.

[0208] In some embodiments, 5'-phosphate modification of the oligonucleotide enhances its interaction with Argonaut 2. However, oligonucleotides containing 5'-phosphate modification may be readily degraded by phosphatases or other enzymes, which limits their bioavailability in vivo. In some embodiments, the oligonucleotide contains an analog of the 5'-phosphate ester that is resistant to such degradation. In some embodiments, the phosphate ester analog may be hydroxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In some embodiments, the 5' end of the oligonucleotide chain is attached to a chemical moiety (phosphate ester analog) that mimics the electrostatic and steric properties of the natural 5'-phosphate ester group (see, for example, Prakash et al. (2015), Nucleic Acids Res., Nucleic Acids Res. 2015 Mar 31; 43(6):2993–3011, the contents of which relate to the phosphate ester analog are incorporated herein by reference). Numerous phosphate ester analogs that can be attached to the 5' end have been developed (see, for example, U.S. Patent No. 8,927,513, the contents of which relating to phosphate ester analogs are incorporated herein by reference). Other modifications have been developed for the 5' end of oligonucleotides (see, for example, WO 2011 / 133871, the contents of which relating to phosphate ester analogs are incorporated herein by reference). In some embodiments, a hydroxyl group is attached to the 5' end of the oligonucleotide.

[0209] Modified sugars (also referred to herein as sugar analogs) include modified deoxyribose or ribose moieties, for example, where one or more modifications occur at the 2', 3', 4', and / or 5' carbon positions of the sugar. In some embodiments, modified sugars may also include non-natural alternative carbon structures, such as those present in locked nucleic acids (LNAs) (see, for example, Koshkin et al. (1998), Tetrahedron 54, 3607-3630), unlocked nucleic acids (UNA) (see, for example, Snead et al. (2013), Molecular Therapy–Nucleic Acids, 2, e103), and bridged nucleic acids (BNAs) (see, for example, Imanishi and Obika (2002), The Royal Society of Chemistry, Chem. Commun., 1653-1659). The disclosures by Koshkin et al., Snead et al., and Imanishi and Obika concerning sugar modifications are incorporated herein by reference.

[0210] In some embodiments, the nucleotide modification of the sugar includes 2'-modification. The 2'-modifying group can be 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinonucleotide. Typically, the modifying group is 2'-fluoro, 2'-O-methyl, or 2'-O-methoxyethyl. In some embodiments, the modification of the sugar includes modification of the sugar ring, which may include modification of one or more carbons of the sugar ring. For example, the sugar modification of the nucleotide may include the 2'-oxygen of the sugar being linked to the 1'-carbon or 4'-carbon of the sugar, or the 2'-oxygen being linked to the 1'-carbon or 4'-carbon via an ethylene or methylene bridge. In some embodiments, the modified nucleotide has an acyclic sugar lacking 2'-carbon to 3'-carbon bonds. In some embodiments, the modified nucleotide has a thiol group at, for example, the 4' position of the sugar.

[0211] In some embodiments, phosphate modification or substitution can produce oligonucleotides comprising at least one (e.g., at least 1, at least 2, at least 3, or at least 5) modified internucleotide bonds. In some embodiments, any oligonucleotide disclosed herein comprises 1 to 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3, or 1 to 2) modified internucleotide bonds. In some embodiments, any oligonucleotide disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide bonds.

[0212] The modified internucleotide bond can be a thiophosphate bond, a thiophosphate bond, a phosphate trimer bond, a thioalkylphosphonate bond, a thioalkylphosphonate bond, a phosphoramide bond, a phosphonate bond, or a borate phosphate bond. In some embodiments, at least one modified internucleotide bond of any oligonucleotide disclosed herein is a thiophosphate bond.

[0213] GalNAc is a high-affinity ligand for the desialoglycoprotein receptor (ASGPR), which is primarily expressed on the sinusoidal surface of hepatocytes and plays a major role in the binding, internalization, and subsequent clearance of circulating glycoproteins containing terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins). Conjugation (indirectly or directly) of the GalNAc moiety to oligonucleotides of this disclosure can be used to target these oligonucleotides to ASGPRs expressed on these hepatocytes.

[0214] Combination of hepatitis B treatment drugs and antiviral agents

[0215] In this application, the application scenario involving the combination of hepatitis B treatment drugs and antiviral agents can be the combined use of hepatitis B treatment drugs (e.g., HBV mRNA vaccines) and antiviral agents (e.g., HBV-specific oligonucleotides, such as siRNA and / or ASO). mRNA and siRNA or ASO have different mechanisms; mRNA can enhance the host's immune response or provide antiviral proteins, siRNA can reduce viral protein expression, and ASO can inhibit viral replication. The inventors of this application have discovered for the first time that, in the treatment of hepatitis B, a combination therapy combining hepatitis B treatment drugs and antiviral agents can simultaneously intervene in HBV treatment from multiple angles, and unexpectedly discovered that this combination therapy can produce a synergistic effect, significantly improving treatment efficacy.

[0216] In some embodiments of this application, the combination of hepatitis B treatment drugs and antiviral agents can be any combination of hepatitis B treatment drugs and any antiviral agents as shown in this application, such as a combination of mRNA and siRNA, a combination of mRNA and ASO, a combination of mRNA and a broad-spectrum antiviral agent (e.g., entecavir, tenofovir), a combination of mRNA and CpAM, a combination of mRNA and HBV antibody, or a combination of mRNA and interferon. In some embodiments of this application, the combination of hepatitis B treatment drugs and antiviral agents can be one or more combinations of any hepatitis B treatment drugs and any antiviral agents as shown in this application, such as a combination of mRNA, siRNA, and entecavir, or a combination of mRNA, ASO, and entecavir.

[0217] In this application, although the sequence listing indicates a DNA sequence, the nucleotide sequence in the sequence listing can represent either a DNA sequence or an RNA sequence. When it represents an RNA sequence, the "T" represents uridine "U".

[0218] The present application is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present application. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected in accordance with the product instructions.

[0219] Unless otherwise specified, if the mRNA-LNP composition in the embodiments of this application contains multiple sequences, the mRNA mass corresponding to each sequence is equal.

[0220] In some embodiments, the core sequence in this application refers to a nucleic acid sequence encoding the corresponding antigen amino acid, such as an RNA sequence.

[0221] The detection methods described in the embodiments of this application, such as methods for detecting indicators such as HBsAg and HBV DNA, can be regarded as general detection methods in this application unless otherwise specified, and can be directly referenced as needed in different specific embodiments.

[0222] Preparation Examples

[0223] 1. Plasmid preparation:

[0224] The gene fragment containing the core sequence (SEQ ID NO: 1-6) was bound to both ends with 5'-UTR (SEQ ID NO: 7-12) and 3'-UTR (SEQ ID NO: 13-17), respectively, to obtain a fragment containing the core sequence + UTR (SEQ ID NO: 18-35). After adding components such as the T7 promoter and polyA tail, a complete DNA fragment (i.e., the full-length sequence, SEQ ID NO: 36-53) was obtained. This fragment was cloned into a suitable plasmid (pcDNA3.1, conventional in the art), and a large number of plasmids containing the target sequence were amplified in Escherichia coli Stbl3. The structures and nucleotide sequences of sequences #1-#15 and #18-#20 constructed in this application are summarized in Table 1.

[0225] The core sequence mentioned in the embodiments of this application refers to the coding sequence (CDS) of the corresponding antigen; the mRNA sequence refers to the complete messenger RNA sequence, including 5'UTR + core sequence + 3'UTR + poly A, i.e., the sequence in the mRNA-LNP; the full-length sequence refers to the sequence containing the promoter sequence + mRNA coding sequence (i.e., containing promoter + 5'UTR + core sequence + 3'UTR + poly A). Therefore, those skilled in the art will understand that expressions such as "mRNA-LNP composition including sequence #4" in the embodiments mean that the mRNA sequence in the mRNA-LNP is the mRNA sequence corresponding to #4, i.e., SEQ ID NO: 277.

[0226] Table 1

[0227] 2. Linearization reaction of target plasmid DNA:

[0228] 1) The above plasmids were digested using the restriction endonuclease PacI, and the system is shown in Table 2 below:

[0229] Table 2. Plasmid linearization reaction system

[0230] 2) After enzyme digestion at 37℃ for 2 hours, take 1 μL of the reaction product and add 4 μL of enzyme-free water and 1 μL of 6× loading buffer, mix well. Linearization is detected by agarose gel electrophoresis (1% agarose gel, 150V, 20 min).

[0231] Use commercially available reagent kits PCR & DNA Cleanup Kit for DNA purification:

[0232] 3) Dilute the DNA sample to be purified with DNA Cleanup Binding Buffer containing isopropanol.

[0233] 4) Insert the adsorption column into the collection tube, transfer all the sample into the adsorption column and cover it, centrifuge at 13000 rpm for 30 s, and discard the flow-through.

[0234] 5) Reinsert the adsorption column into the collection tube, add 200 μL of DNA Wash Buffer, centrifuge at 13000 rpm for 30 seconds, and discard the flow-through.

[0235] 6) Repeat step 5 above.

[0236] 7) Reinsert the adsorption column into the collection tube and centrifuge at 13,000 rpm for 30 seconds to remove residual ethanol. Transfer the adsorption column to a clean 1.5 mL centrifuge tube (ensuring the adsorption column does not come into contact with the flow-through).

[0237] 8) Add 20 μL of DNA Elution Buffer to the center of the adsorption column membrane. Let stand at room temperature for 1 min, then centrifuge at 13000 rpm for 1 min to elute the DNA.

[0238] 9) The concentration of linearized template DNA was determined using Nanodrop One.

[0239] 3. In vitro IVT synthesis of mRNA:

[0240] 1) Use a commercially available IVT kit (HighYield T7 mRNA Synthesis Kit (me 1 The in vitro IVT reaction was performed using Ψ-UTP (Jenabioscience), and the specific system is shown in Table 3 below:

[0241] Table 3 IVT System

[0242] 2) Dilute the DNA to 125 ng / μL with enzyme-free water as template DNA (not higher than 125 ng / μL). Incubate at 37°C for 2 hours.

[0243] 3) Add 1 μL of TURBO™ DNase to each reaction system (20 μL). Continue the reaction at 37 °C for 15 min.

[0244] 4. IVT mRNA purification:

[0245] 1) Use The RNA Cleanup Kit (NEB) is used to purify IVT products.

[0246] 2) Add twice the volume of RNA Cleanup Binding Buffer to the sample to be purified. Add an equal volume of anhydrous ethanol and mix thoroughly by pipetting or gently tapping the tube.

[0247] 3) Centrifuge at 12000 rpm for 1 min, discard the flow-through. Reinsert the adsorption column into the collection tube. Add 500 μL of RNA Cleanup Wash Buffer, centrifuge at 12000 rpm for 1 min, discard the flow-through.

[0248] 4) Repeat step 3.

[0249] 5) Reinsert the adsorption column into the collection tube and centrifuge at 12,000 rpm for 1 min to remove residual ethanol.

[0250] 6) Add 100 μL of enzyme-free water to the center of the membrane of the adsorption column and let it stand at room temperature for 5 min. Elute the RNA by centrifugation at 12000 rpm for 1 min.

[0251] 7) Detection of purified products by agarose gel electrophoresis.

[0252] 5. IVT mRNA capping reaction:

[0253] 1) Heat the RNA to 65°C for 5 min to denature it, then immediately transfer the RNA to ice and let it stand for 2 min.

[0254] 2) Perform the in vitro capping reaction (Vaccinia Capping System) according to Table 4:

[0255] Table 4 Capping System

[0256] 3) Mix thoroughly and react at 37°C in the dark for 1 hour.

[0257] 6. mRNA-LNP encapsulation:

[0258] 1) The formulation of mRNA-LNP: The dosages of HBV-Large S, HBV-S, HBV-core, and Luc (Luciferase mRNA, commercially available: 17101ES80, Yisheng Biotechnology) were 400 μg, 400 μg, 400 μg, and 200 μg, respectively. LNP was formulated using Moderna's formulation, with an N / P ratio of 5.67. The formulation composition is shown in Table 5 below:

[0259] Table 5. Composition of mRNA-LNP formulation

[0260] 2) Preparation of buffer solutions:

[0261] 50mM citrate buffer: Weigh 443.58 mg of citrate monohydrate and dissolve it in 21.10 mL of ultrapure water, and dissolve 607.55 mg of sodium citrate dihydrate in 20.65 mL of ultrapure water. Mix 13.1 mL of the citrate solution, 6.9 mL of the sodium citrate solution, and 20 mL of water to obtain the 50mM citrate buffer. The pH was measured to be 3.83.

[0262] 8% sucrose-20mM Tris-HCl buffer: Weigh 80g sucrose and 910g ultrapure water, stir until the sucrose is completely dissolved, then add 10mL of 2M Tris-HCl, pH 7.5 solution, stir well to obtain 1L of 8% sucrose Tris-HCl buffer.

[0263] 3) Preparation of LNP lipid phase: According to Table 6, SM-102, DSPC, cholesterol, and DMG-PEG2000 were mixed with ethanol respectively, and vortexed to completely dissolve the lipids, obtaining ethanol solutions of the four lipids. Then, 1.533 mL of each lipid ethanol solution was taken and mixed evenly to obtain the lipid phase.

[0264] Table 6. LNP lipid phase formulation

[0265] 4) Preparation of the aqueous phase for mRNA: According to the formula in Table 7, i.e., the volume ratio of lipid phase to aqueous phase is 1:3 and the amount of each mRNA, dilute the mRNA with 50mM citrate buffer (pH 3.83). The concentration of the diluted aqueous phase mRNA is 0.119mg / mL. LNP blank is an LNP negative control without mRNA.

[0266] Table 7. Preparation of mRNA in aqueous phase

[0267] 5) Microfluidic Synthesis of mRNA-LNP: mRNA-LNP was synthesized using a microfluidic device. The lipid phase was drawn into a 5 mL or 1 mL syringe, and the aqueous phase into a 5 mL syringe, with air expelled from the syringes. The microfluidic chip was mounted onto the microfluidic instrument, and the lipid and aqueous phase syringes were connected to the chip respectively. The total flow rate was set to 12 mL / min, and the flow rate ratio of the aqueous to lipid phases was 3:1. The two phases were mixed to complete the synthesis of mRNA-LNP. Detailed process parameters are shown in Table 8.

[0268] Table 8 Microfluidic process parameters

[0269] 6) Post-processing of mRNA-LNP: Immediately after mRNA-LNP synthesis, the LNP was diluted 2-fold with 8% sucrose-20mM Tris-HCl buffer. The sample was then added to a dialysis chamber (Thermo Scientific) for dialyzing, with the dialysate volume being 100 times the sample volume. The buffer was changed every 3 hours, for a total of 20 hours. Each dialysate volume was 100 times the sample volume, and a total of 3 dialyses were performed over a total of 20 hours. After dialysis, the sample was transferred to a 50mL centrifuge tube and diluted 2-fold with 8% sucrose-20mM Tris-HCl buffer. The sample was then filtered through a 0.22μm microporous membrane for sterilization. Afterwards, the sample was centrifuged at 10K... Ultrafiltration concentration was performed using an Ultra-15 centrifugal filter (EMD Millipore) to collect the sample.

[0270] Example 1: Validation of mRNA expression in cells

[0271] Cell transfection: HBV-Large S mRNA (corresponding to #4, #7, #10, and #18 in Table 1), HBV-S mRNA (corresponding to #11, #19, and #5 in Table 1), and HBV-core mRNA (corresponding to #3, #6, #12, and #20 in Table 1) were added to 293T cells that had grown to approximately 70% confluence. The mRNA concentration was approximately 0.5 μg / well. Lipofectamine MessengerMAX transfection was performed according to the manufacturer's instructions (catalog number LMRNA001, Thermo Fisher Scientific). Stable 293T cells were seeded in six-well plates, and transfection was performed when the cells reached 70-80% confluence. Prepare solutions A and B; Solution A: Dilute 4 μg mRNA with 125 μL Opti-MEM; Solution B: Dilute 3.75 μL Lipofectamine MessengerMAX with 125 μL Opti-MEM. Gently mix solutions A and B separately, let stand for 5 min, add solution B to solution A, gently mix, let stand at room temperature for 5 min, add to each well and incubate at 37℃ for 48 h.

[0272] Western blot (WB) for identifying cellular mRNA expression:

[0273] The expression of target proteins in the cell culture supernatant and cell lysate obtained from transfected cells was identified by Western blotting. After adding 6× loading buffer to the cell culture supernatant and cell lysate, polyacrylamide gel electrophoresis was performed after boiling for 5 min. The proteins were then transferred to PVDF via gel electrotransfer. The cells were blocked with 1% BSA at room temperature for 60 min and washed three times with TBST. After incubation with anti-HBsAg or anti-HBcAg antibodies at room temperature for 2 h, followed by three washes with TBST, and incubation with goat anti-mouse-HRP for 1 h at room temperature, the cells were developed using an enhanced chemiluminescence assay kit (ECL, catalog number 32134, Thermo Fisher Scientific), and the results are shown in Figure 1.

[0274] Example 2: In vivo imaging experiment of luciferase mRNA

[0275] This embodiment uses luciferase mRNA to characterize whether the mRNA vaccine prepared in the preparation embodiment can be successfully delivered into cells or animals.

[0276] Female BALB / c mice aged 6-8 weeks without specific pathogens were injected intramuscularly with luciferase mRNA-LNP particle (Luc mRNA) prepared in the preparation example on day 0. At 6, 9, 12, 24, and 48 hours prior to the injection, 150 mpk of D-Luciferin (catalog number 88293, Thermo Fisher Scientific) was injected intraperitoneally. The animals were anesthetized with isoflurane 3 minutes prior to the injection and placed in the IVIS Spectrum imaging chamber for bioluminescence imaging, and the total luminescence was calculated.

[0277] The results showed that mice had high signal intensity 6-12 hours after injection of luciferase mRNA-LNP particle (Figure 2). Regarding tissue distribution, 6 hours after injection, the signal was strongly expressed in both the liver and muscle (Figure 3). This indicates that the mRNA-LNP particle prepared in the examples can be well delivered and expressed in the mouse model.

[0278] Example 3: mRNA vaccine induces the production of HBsAb and HBcAb antibodies.

[0279] Laboratory animals: 6-8 week old, specific pathogen-free female BALB / c mice were divided into 9 groups and administered intramuscular injections (IM) on days 0 and 21 according to the substances and dosages listed in Table 9 below, with dosages expressed as mRNA weight. Serum samples were collected via the submandibular vein at -3, 14, and 35 days (based on the first injection) and frozen at -80°C. The obtained samples were then analyzed for anti-HBs and anti-HBc antibody titers and neutralizing antibody efficacy.

[0280] Table 9

[0281] Anti-HBs antibody titer test:

[0282] 1) Prepare the ELISA kit (Hepatitis B virus surface antibody detection kit (chemiluminescence method), autobio) according to the operating instructions.

[0283] 2) Set the temperature of the incubator to 37℃ and set it aside for use.

[0284] 3) Mix the luminescent substrates A and B in equal proportions to 10 mL.

[0285] 4) Add 50 μL of enzyme conjugate to each well and shake on a shaker for 60 seconds to mix thoroughly. Incubate at 37°C for 60 minutes.

[0286] 5) After washing the plate 6 times with the washing solution, add 50 μL of the mixed substrate to each well and react at room temperature in the dark for 10 minutes.

[0287] 6) Use a Gene5 microplate reader to detect the luminescence intensity.

[0288] 7) The HBsAb titer in the sample was calculated using a double log-linear regression fitting method.

[0289] The results are shown in Figure 4.

[0290] Anti-HBc antibody titer test:

[0291] 1) Prepare the time-resolved fluorescence immunoassay kit (Diagnostic Kit for the Determination of Hepatitis B core Antibody (Time-resolved Immunofluorometric Assay), PerkinElmer) according to the operating instructions provided by the supplier.

[0292] 2) Add 50 μL of anti-HBc calibrator to the microwells in sequence, then add 10 μL of sample processing solution to the wells of the sample to be tested (no sample processing solution is needed in the calibrator wells), and then add 50 μL of the sample to be tested to the wells of the wells where sample processing solution has been added.

[0293] 3) Add 50 μL of neutralizing antigen to each well and cover with sealing paper.

[0294] 4) Incubate the microporous reaction strips at room temperature using a shaker on low speed for 40 minutes.

[0295] 5) After washing the plate four times with washing solution, add 100 μL of europium-labeled working solution to each well and attach a sealing sheet. Incubate on a shaker at low speed for 40 minutes.

[0296] 6) After washing the plate 6 times with the working washing solution, add 100 μL of enhancement solution to each well and attach a sealing sheet. Incubate on a shaker at low speed for 5 minutes.

[0297] 7) Use Envision instruments for testing. Specific testing conditions are shown in Table 10 below.

[0298] Table 10 Detection conditions and information

[0299] 8) The HBcAb titer in the sample was calculated using a double log-linear regression fitting method. The results are shown in Figure 5.

[0300] Conclusion: As can be seen from Figures 4 and 5, the titers of HBsAb and / or HBcAb were significantly increased in the examples containing mRNA compared with those without mRNA. This shows that the mRNA in the examples of this application effectively induced the production of HBsAb and HBcAb antibodies.

[0301] Neutralizing antibody efficacy test:

[0302] The technology platform used: Frozen PHH (batch number RAS) was provided by Shanghai WuXi AppTec New Drug Development Co., Ltd. PHH was revived in InvitroGRO CP medium (Bioreclamation IVT, S03316) containing 10% fetal bovine serum (ExCell Bio, FSP500) and 1% penicillin-streptomycin (Hyclone, SV30010), and then infected with HBV using William's medium E (Gibco, A12176-01) complete medium; subsequent cultures were conducted in DMEM (Gibco, 11965-092) complete medium containing 10% fetal bovine serum, 2% DMSO (Sigma, D2650), and 1% penicillin-streptomycin.

[0303] Procedure: Cell resuscitation: Resuscitate the frozen PHH and seed it into 48-well plates at 1.32E+5 cells per well. One day later, mouse serum from Day 3 and Day 14 was serially diluted 10-fold to the initial concentration, then serially diluted 3-fold to 7 concentrations in duplicate. Mouse serum or HBIG from Day 35 was diluted to a similar concentration (close to 300 mIU / mL) as the initial dilution, then serially diluted 3-fold to 7 concentrations in duplicate. PBS-treated mouse serum served as the negative control, and HBIG served as the positive control. Both were mixed with type D HBV virus (800 GE / cell) and incubated at room temperature for 1 hour before being added to the PHH. On days 2, 4, 6, and 8, the culture medium was replaced with fresh medium (without test serum and control antibodies). On day 10, the supernatant was collected, and HBV DNA levels were detected using qPCR, HBsAg and HBeAg levels were detected using ELISA, and cell viability was assessed using CellTiter-Glo.

[0304] The corresponding results are shown in Figures 15-17. From the three indicators of anti-HBV DNA, HBsAg and HBeAg, compared with Engerix-B HBV vaccine, the neutralizing antibodies produced by the mRNA in the mice in this application example have a good inhibitory effect on the above indicators.

[0305] Example 4: Pharmacological test of mRNA inhibiting HBV in mice

[0306] 4.1 In vivo experimental protocol:

[0307] Male C57BL / 6 mice (5 weeks old) were divided into 6 groups and intravenously injected with rAAV-1.3HBV (1×10⁻⁶). 12 viral genome (vg) / mL diluted to 2.5 × 10⁻⁶ in sterile PBS. 10 vg / mL. 200 μL was injected into each mouse, which is equivalent to 5 × 10⁻⁶ vg / mL per mouse. 9 (vg, genotype D, serotype ayw). 28 days after injection, the animals were given intramuscular injections (IM) of the substances listed in Table 11 (i.e., #4, #5, and #20), designated as day 0. Subsequent injections were given on days 14, 28, and 42, for a total of four injections (the dosage was based on the total weight of mRNA, with the three mRNAs in a 1:1:1 ratio). Blood samples were collected via the submandibular vein at various time points before and after injection to detect HBV marker levels. Dosage schedule: Intramuscular injection, 100 μL / animal, administered once each on days 0, 14, 28, and 42, for a total of four injections. Blood collection times: -14, -7, -1, 7, 14, 21, 28, 35, 46, 53, 60, 67, 74, 81, and 88. Detection indicators: All plasma: HBsAg; plasma on day 88: additional HBV DNA detection.

[0308] Table 11

[0309] 4.2 Detection Method

[0310] The reagents and kits used in the embodiments of this application are shown in Table 12A below.

[0311] Table 12A

[0312] a. Quantitative PCR detection of HBV DNA content in mouse plasma:

[0313] Plasma DNA was extracted using the QIAamp 96DNA Blood Kit, and the qPCR reaction system was prepared according to Table 12B below (reaction conditions: 95℃, 10 min; 95℃, 15 s; 60℃, 1 min; 40 cycles):

[0314] Table 12B qPCR reaction composition table

[0315] In Table 12B, the forward primer sequence is SEQ ID NO:54, 5'-GCCCCTATCCTATCAACACTTCCGG-3'; the reverse primer sequence is SEQ ID NO:55, 5'-TTCGTCTGCGAGGCGAGGGA-3'.

[0316] b. ELISA method for detecting HBsAg levels in mouse plasma:

[0317] Experimental procedure: The HBsAg ELISA kit (Antu Bio, CL 0310) was used for detection: the plasma sample was diluted 600 times, added to the coated plate, and incubated with the enzyme conjugate (37℃, 60 minutes). The plate was washed 5 times, the luminescent substrate was added, and the reaction was carried out at room temperature in the dark for 10 minutes. The luminescence intensity was detected by microplate reader.

[0318] 4.3 Data and Results:

[0319] The obtained data are expressed as mean ± standard error for each group of mouse samples, and statistical analysis was performed using Student's t-test. Figures 6 and 7 show that after four injections, the mRNA vaccine demonstrated a significant HBsAg-lowering effect, with a maximum reduction of 1-2 log. On day 81, HBsAg levels in mouse plasma remained at a low level without significant rebound, indicating that the mRNA vaccine described in this study can effectively reduce HBsAg levels in the long term. Furthermore, the mRNA vaccine also showed a long-term effective reduction in HBV DNA, with a maximum reduction of approximately 1 log, indicating that the mRNA vaccine in this application can sustainably reduce and clear hepatitis B virus from patients' blood. Figure 18 shows that groups 2-4 (mRNA group encoding Large S+S+core antigen) had a better and more sustained ability to reduce HBsAg levels compared to groups 2-b (mRNA group encoding S+core antigen) and 2-a (mRNA group encoding core antigen).

[0320] Example 5: Antiviral agents used in combination with mRNA vaccines

[0321] 5.1 The antiviral agents used in this embodiment are siRNA or ASO, the sequences of which are listed in Table 13.

[0322] Table 13 shows the siRNAs and their sequences used in this embodiment.

[0323] Table 14 Sequences and modifications of siRNA-1 to 29

[0324] The sequence arrangement in Table 14 is shown in Table 15, and the specific representation of its nucleotide modifications is shown in Table 16.

[0325] Table 15 Nucleotide sequences of siRNA-1 to 29

[0326] In Tables 13-15 above, the structure of NAG37 is shown in Figure 11; mu is MePhosphonate-4O-mU, the structure of which is shown in Figure 12, and the abbreviations of other nucleotides are shown in Table 16.

[0327] Table 16 Nucleotide Abbreviations

[0328] 5.2 In vivo experimental protocol

[0329] siRNA: ALN-HBV02 siRNA, working solution at a concentration of 1.12 mg / mL (dosage dose of 6 mpk), for subcutaneous injection in mice.

[0330] Male C57BL / 6 mice (5 weeks old) were grouped according to Table 17 and intravenously injected with rAAV-1.3HBV (1×10⁻⁶). 12 viral genome (vg) / mL, diluted to 2.5 × 10⁻⁶ with sterile PBS. 10 vg / mL. 200 μL was injected into each mouse, which is equivalent to 5 × 10⁻⁶ vg / mL per mouse. 9 A mouse model of HBV infection was constructed using mice with genotype D (vg, serotype ayw). After 28 days (day 0), mice in each group were intramuscularly injected with either (IM) LNP or mRNA-LNP five times, according to the protocol in Table 17, at days 0, 14, 28, 42, and 56. Simultaneously, from days 0 to 55, mice were fed Entecavir (1 μg / mL) via drinking water and subcutaneously injected with anti-HBV siRNA (ALN-HBV02, i.e., antiviral agent number 2 in Table 13, administered on days 0, 14, and 35, for a total of three administrations). Blood and tissue samples were collected via the submandibular vein at each time point before and after injection to detect HBV biomarker levels and immune responses.

[0331] Table 17

[0332] 5.3 Tissue Sample Collection

[0333] a. Plasma sample: Collect whole blood into an EDTA-K2 coated anticoagulant tube, centrifuge at 4°C, 7000g for 10 minutes, and collect the supernatant.

[0334] b. Liver sample collection: The left middle lobe liver of mice was taken and placed in a 50 mL centrifuge tube containing 10% formalin. After being fixed on a shaker for 48 hours, it was transferred to PBS for storage, dehydrated, and embedded in paraffin.

[0335] c. Spleen sample collection: Mouse spleens were collected and immersed in 5% FBS + 1% penicillin antibody + PBS buffer. After collection, the samples were immediately transported to the in vitro environment on wet ice for ELISpot and ICS detection.

[0336] 5.4 Virus biomarker detection

[0337] a. ELISA detection of HBsAg content in mouse plasma: The experimental procedure was performed according to the HBsAg ELISA (Antu Bio, CL 0310) kit instructions. The method is briefly described below: The plasma sample was diluted 600-fold and added to a coated plate. The plate was incubated with the enzyme conjugate at 37°C for 60 minutes. The plate was washed 5 times. The luminescent substrate was added, and the reaction was carried out at room temperature in the dark for 10 minutes. The luminescence intensity was then detected using a microplate reader.

[0338] b. Detection of HBV Core Positive Cell Count in Mouse Liver (IHC, Immunohistochemistry): Liver samples were collected from experimental mice on days 69 and 119, and fixed in formalin for 72 hours. After cleaning, the samples were embedded in paraffin, sectioned into 4 μM sections, and subjected to routine immunohistochemical staining methods including dewaxing, antigen retrieval, blocking, incubation with anti-HBc primary and secondary antibodies, and DBA staining. Images were acquired and observed using a bright-field microscope.

[0339] c. Detection of HBV DNA levels in mouse blood: On day 119, the HBV DNA level in the blood was detected. The detection method is described in Section 4.2.a.

[0340] d. RT-qPCR detection of pgRNA content in mouse plasma: Viral RNA was extracted from plasma, and the experimental procedure was performed according to PureLink. TM Pro 96Viral RNA / DNA Kit Instruction Manual. The method is briefly described below:

[0341] (1) Add 25 μL of proteinase K to each well, followed by 20 μL and 180 μL of DPBS, and finally 200 μL of buffer lysis (containing 5.6 μg carrier RNA). Mix by pipetting up and down 3-5 times. Seal the plate with sealing film and incubate at 56°C for 15 minutes, then centrifuge at 3000g for 1 minute. Add 250 μL of anhydrous ethanol to each well and mix by pipetting up and down 3-5 times. Transfer the lysis buffer to PureLink. TM96-well Viral Filter Plate. Place the Viral Filter Plate on a 96-well deep-well plate and centrifuge at 3000g for 2 minutes. Remove the liquid from the deep-well plate and place the Viral Filter Plate back on the plate. Add 500 μL of Wash buffer II, centrifuge at 3000g for 5 minutes, and wash twice. Centrifuge at 3000g for 10 minutes. Add 100 μL of RNase-free water and incubate at 25°C for 1 minute. Place the Viral Filter Plate on a clean deep-well plate and centrifuge at 3000g for 2 minutes. The collected RNA can be used for subsequent experiments or stored at –80°C.

[0342] (2) Reverse transcription. The extracted RNA was reverse transcribed to obtain cDNA, following these steps: Take 8 μL of the extracted RNA sample, add 2 μL of 5×gDNA Buffer, mix well, and incubate at 42℃ for 3 min on a PCR instrument. Prepare the following RT-PCR mixture: 10×King RT Buffer, 2 μL; FastKing RT Enzyme Mix, 1 μL; RT primer (3' Race-long primer) (10 μM), 2 μL; RNase-Free ddH2O, 5 μL. The 3' Race-long primer contains: an HBV-specific sequence, an anchor sequence, and a poly-T sequence. Then, add 10 μL of the RT mixture to the mixture after removing genomic DNA and mix well. Place the sample in a PCR instrument and run the following program: 42℃, 15 min; 95℃, 3 min. Store the cDNA temporarily at 4℃.

[0343] (3) qPCR detection of pgRNA content in mouse plasma: The following qPCR mixture was prepared: Universal PCR Master Mix, 10 μL / well; probe (10 μM), 0.4 μL / well; forward primer (10 μM), 0.8 μL / well; reverse primer (10 μM), 0.8 μL / well; DEPC-Treated water, 6 μL / well. 18 μL of the above qPCR mixture was added to each well of a 384-well qPCR plate. 2 μL of cDNA sample or serially diluted plasmid standard (pAAV-HBV1.3 plasmid, synthesized by Genewiz. The plasmid was diluted to 10⁸ copies / μL and then serially diluted 10-fold with AE buffer to generate a standard gradient from 10⁸ to 10 copies / μL) was added to the corresponding well of the qPCR plate. Reaction conditions: 95℃, 10 minutes; 95℃, 15 seconds, 60℃, 2 minutes, 45 cycles.

[0344] e. HBeAg detection: Refer to the HBeAg ELISA kit instructions (Antu Bio, CL 0312). The method is briefly described below: Dilute the plasma sample 60-fold, add it to the coated plate, and incubate with the enzyme conjugate (37℃, 60 minutes). Wash the plate 5 times, add the luminescent substrate, and react at room temperature in the dark for 10 minutes. Detect the luminescence intensity using a microplate reader (Molecular Devices, SpectraMaxiD3).

[0345] 5.5 Data Processing and Analysis

[0346] The HBV RNA titer (copies / μL) for each sample was calculated based on the standard curve, and then adjusted according to the specific dilution factor of the sample to obtain the original HBV copies / μL value.

[0347] For samples with HBV RNA titers lower than LLOQ, LLOQ should be used instead when analyzing the data.

[0348] The results of detecting HBsAg and HBV DNA levels in mouse plasma are shown in Figures 20 and 21, respectively. Figure 20 shows that in the groups using mRNA in combination with siRNA or ASO (groups 5-3, 5-5, 5-6, 5-7, and 5-8), HBsAg levels did not rebound after drug withdrawal. In contrast, in the groups using siRNA or ASO alone (groups 5-10 and 5-11), HBsAg levels rebounded rapidly after drug withdrawal. This indicates that the mRNA used in this application, when combined with different siRNAs or ASOs, can achieve a long-term effect of preventing HBsAg rebound after drug withdrawal. As shown in Figure 21, the HBV DNA levels in all groups using mRNA in combination with siRNA were significantly lower than those in the siRNA groups, indicating that the mRNA vaccine helps clear HBV virus from the body and can maintain HBV DNA levels at a low level for a long period.

[0349] Example 6: Preliminary in vivo verification of the combined therapeutic effect of mRNA vaccine and antiviral agent

[0350] This embodiment preliminarily verifies the in vivo efficacy of the aforementioned mRNA-LNP composition (taking an mRNA-LNP composition containing sequences #4, #5, and #20 as an example) in combination with an antiviral agent (taking siRNA as an example).

[0351] 6.1 In vivo experiments:

[0352] Materials preparation:

[0353] ETV: Weigh an appropriate amount of ETV powder, dissolve it in DMSO, vortex and sonicate until clear, prepare a 10 mg / mL solution and dispense it into 0.4 mL / tubes, store at -20℃ until used.

[0354] siRNA: ALN-HBV02 siRNA, working solution at a concentration of 1.12 mg / mL (dosage dose of 6 mpk), for subcutaneous injection in mice.

[0355] Male C57BL / 6 mice (5 weeks old) were divided into 4 groups and intravenously injected with rAAV-1.3HBV (1×10⁻⁶). 12 viral genome (vg) / mL, diluted to 2.5 × 10⁻⁶ with sterile PBS. 10 vg / mL. 200 μL was injected into each mouse, which is equivalent to 5 × 10⁻⁶ vg / mL per mouse. 9 A mouse model of HBV infection was established using mice with genotype D and serotype ayw (vg). After 28 days (day 0), mice in each group were intramuscularly injected with either LNP or mRNA-LNP (listed in Table 23) five times on days 0, 14, 28, 42, and 56. Simultaneously, from days 0 to 55, mice were fed Entecavir (1 μg / mL) via drinking water and subcutaneously injected with anti-HBV siRNA (ALN-HBV02, i.e., antiviral agent number 2 in Table 13, administered three times on days 0, 14, and 35). Blood and tissue samples were collected via the submandibular vein at each time point before and after injection to detect HBV biomarker levels and immune responses. Specific experimental groups are shown in Table 18 (mice in all groups in Table 18 were fed Entecavir as described above).

[0356] Table 18

[0357] 6.2 Tissue Sample Collection:

[0358] a. Plasma sample: Collect whole blood into an EDTA-K2 coated anticoagulant tube, centrifuge at 4°C, 7000g for 10 minutes, and collect the supernatant.

[0359] b. Liver sample collection: The left middle lobe liver of mice was taken and placed in a 50 mL centrifuge tube containing 10% formalin. After being fixed on a shaker for 48 hours, it was transferred to PBS for storage, dehydrated, and embedded in paraffin.

[0360] c. Spleen sample collection: Mouse spleens were collected and immersed in 5% FBS + 1% penicillin antibody + PBS buffer. After collection, the samples were immediately transported to the in vitro environment on wet ice for ELISpot and ICS detection.

[0361] 6.3 Virus biomarker detection:

[0362] a. ELISA detection of HBsAg content in mouse plasma: The experimental procedure followed the instructions for the HBsAg ELISA kit (Antu Bio, CL 0310). The method is briefly described below: The plasma sample was diluted 600-fold and added to a coated plate. It was incubated with the enzyme conjugate at 37°C for 60 minutes. The plate was washed 5 times. The luminescent substrate was added, and the reaction was carried out at room temperature in the dark for 10 minutes. The luminescence intensity was detected using a microplate reader. The results are shown in Figure 8.

[0363] b. Detection of HBV Core-positive cell count in mouse liver (IHC, immunohistochemistry): Liver samples from experimental mice were collected on days 69 and 119, and fixed in formalin for 72 hours. After cleaning, the samples were embedded in paraffin, sectioned into 4 μM sections, and subjected to routine immunohistochemical staining methods including dewaxing, antigen retrieval, blocking, incubation with anti-HBc primary and secondary antibodies, and DBA staining. The results were observed and photographed using a bright-field microscope. The results are shown in Figure 9A (day 69), Figure 9B, and Figure 9C (day 119), respectively.

[0364] c. Detection of HBV DNA levels in mouse blood: On day 119, the HBV DNA level in the blood was measured. The detection method is described in Section 4.2.a. The results are shown in Figure 13.

[0365] 6.4 Immune response detection:

[0366] HBV-specific T-cell response in mouse spleen (ELISPOT): Mouse spleen cells were placed in 96-well ELISPOT plates containing anti-IFNγ. Culture medium (RPMI 1640 (Gibco)) and the corresponding stimulating antigens (Medium (medium added only as a blank control), Core, Pre S, HBsAg (these three stimulating antigens are peptides provided by Genscript Biotech, sequences are shown in Table 19) and ConA (concanavalin A, commercially available C2272 (Sigma)) were added. The cells were incubated in a 5% CO2 incubator for approximately 48 hours. The cultured cells were washed away, blocking solution was added, and then anti-IFNγ-HRP was added and incubated for 1 hour. The cells were washed three times with washing buffer, and chromogenic solution was added. The reaction was continued in the dark until appropriately sized dark spots appeared, at which point the reaction was terminated. The number of dark spots in each well was counted using software for final effect analysis. The corresponding results are shown in Figure 10.

[0367] Table 19 Sequences of stimulating antigens Core, PreS, and HBsAg

[0368] 6.5 Results and Conclusions

[0369] As shown in Figure 8, in the siRNA+mRNA vaccine treatment group, HBsAg did not rebound significantly after siRNA treatment was discontinued and remained at a low level until 56 days after discontinuation; while in the siRNA group, a rebound occurred in the first week after discontinuation and the rebound became more and more obvious over time, rebounding by about 1 log from the bottom by the 56th day after discontinuation.

[0370] As shown in Figures 9A, 9B, 9C, and 10, on days 69 and 119 after the first immunization, the number of HBc+ cells in the liver of the siRNA+mRNA group was significantly reduced compared to that of the siRNA group, and all immune indicators were significantly improved compared to the siRNA group. The siRNA+mRNA group had a better inhibitory effect on HBV than the siRNA group.

[0371] As shown in Figure 13, the HBV DNA level in the siRNA+mRNA vaccine treatment group was still significantly lower than that in the siRNA group until day 119, indicating that the mRNA vaccine helps to clear HBV virus from the body.

[0372] Example 7: In vivo safety and efficacy verification of mRNA vaccine and its combination therapy with antiviral agents.

[0373] This embodiment further verifies the in vivo safety of the mRNA-LNP composition of the present invention, and its safety in combination with antiviral agents (taking siRNA as an example).

[0374] 7.1 In vivo experiments

[0375] Materials preparation and the construction of the HBV-infected mouse model are described in Example 6.1. On day 28 after model establishment (day 0), mice were grouped and administered drugs according to Table 20 below. siRNA was administered subcutaneously (SC), while the mRNA-LNP combination and LNP were administered intramuscularly (IM). Mice in groups 4-10, 4-11, and 4-12 were fed 0.1 μg / mL ETV via drinking water from day 0 to day 55 after drug administration. The administration regimens for the remaining groups are shown in Table 20.

[0376] Table 20

[0377] 7.2 Organize sample collection: See Example 6.2 for specific procedures.

[0378] 7.3 Viral biomarkers and biochemical detection

[0379] ALT assay: Plasma samples were collected from all groups on days -1, 14, 28, 42, and 56. ALT levels were measured according to the ALT Activity Assay Kit (Sigma, MAK052-1KT) instructions. In short: Plasma samples were diluted 4-fold and added to 96-well plates, with 100 μl of reaction mixture added to each well. The samples were then analyzed using a microplate reader (Molecular devices SpectraMax M2e). The results are shown in Figure 14.

[0380] HBsAg detection: HBsAg was detected in mice in groups 4-1 and 4-4 using a method similar to that used in other examples. The results are shown in Figure 19A.

[0381] 7.4 Results and Conclusions

[0382] As shown in Figure 14, neither the mRNA vaccine treatment group nor the siRNA+mRNA vaccine treatment group in this application showed an increase in ALT within 56 days after the first administration, indicating that it did not cause significant liver damage and has high safety.

[0383] As shown in Figure 19A, the mRNA encoding the Large S+S antigen (group 4-4) in this application also showed a good and long-term sustained effect in reducing hepatitis B virus HBsAg.

[0384] Example 8: In vivo efficacy verification of mRNA vaccine and its combination therapy with antiviral agents - 1

[0385] Male C57BL / 6 mice (5 weeks old) were divided into 4 groups and intravenously injected with AAV-HBV (1×10⁻⁶) of genotypes B and C, respectively. 12 viral genome (vg) / mL, diluted to 2.5 × 10⁻⁶ with sterile PBS. 10 vg / mL. 200 μL was injected into each mouse, which is equivalent to 5 × 10⁻⁶ vg / mL per mouse. 9 HBV infection mouse models were constructed using mice with genotype C (vg, C). These models were divided into B-type and C-type mouse models. On day 28 (day 0) after model establishment, mice were grouped and administered medication according to Table 21 below. siRNA was administered subcutaneously (SC), while the mRNA-LNP combination and LNP were administered intramuscularly (IM). Mice in groups 6-13 were fed 0.1 μg / mL ETV via drinking water from day 0 to day 55 post-administration. The administration regimens for the remaining groups are shown in Table 21 below.

[0386] Table 21

[0387] Tissue samples were collected and HBsAg and HBV DNA were detected according to the methods described in Examples 6 and 7. The pgRNA content in mouse plasma was detected by RT-qPCR according to the method described in Example 5. The corresponding results are shown in Figure 19B.

[0388] As can be seen from Figure 19B, the mRNA composition in the embodiments of this application still exhibits the ability to reduce HBsAg in the C-type mouse model.

[0389] The cellular immune response in the spleen or liver of immunized mice was analyzed by detecting intracellular cytokine staining (ICS).

[0390] 8.1 Isolation of immune cells from mouse spleen.

[0391] Mouse spleens were ground on a 70 μm cell sieve. Cells were washed with Dulbecco's phosphate-buffered saline (DPBS) containing 5% fetal bovine serum (FBS), centrifuged, and then subjected to erythrocyte lysis buffer. After complete lysis, DPBS containing 5% FBS was added to terminate the lysis. Cells were resuspended in RPMI-1640 medium (10% FBS and 1% penicillin-streptomycin, P / S) after centrifugation, and counted using a cell counter for subsequent IFN-γ ELISpot or ICS experiments. After removing enough cells for experiments, the remaining cells were resuspended in cryopreservation buffer (90% FBS + 10% dimethyl sulfoxide, DMSO) and frozen.

[0392] 8.2 Isolation of immune cells from mouse spleen.

[0393] Mouse livers were ground on a 70 μm cell sieve. Cells on the sieve were washed with DPBS containing 5% FBS. After centrifugation, the supernatant was aspirated, and the precipitate was resuspended in 40% Percoll and plated onto an 80% Percoll layer. After centrifugation, immune cells from the white membrane layer were aspirated from the Percoll interface, collected, and washed twice with DPBS. After centrifugation, the cells were resuspended in RPMI-1640 medium (containing 10% FBS and 1% P / S), and the cells were counted using a cell counter. The cell concentration was adjusted for IFN-γ ELISApot or ICS experiments.

[0394] 8.3 Evaluation of HBV-specific T cell responses in mice using ICS

[0395] Immune cells from mice were divided into three groups and treated with RPMI-1640 medium (negative control), Phorbol 12 millistate 13 acetate (PMA) + ionomycin (positive control), and the stimulating antigen from Example 6 for 2 hours, respectively. Then, protein transport inhibitors were added, and incubation continued for 16-18 hours. Afterward, L / D dye was added for live / dead staining, and Fc receptor blockers were added simultaneously. Cells were incubated at 4°C in the dark for 25 minutes. Cells were then washed twice with staining buffer, and cell surface marker antibodies (CD3, CD8, and NK1.1) were added. Cells were incubated at 4°C in the dark for 30 minutes. BD Cytofix / Cytoperm was then added. TM The cells were fixed and incubated in the solution at 4°C in the dark for 30 minutes. Next, the cells were washed twice with washing buffer, and intracellular antibodies (CD4, IFN-γ, IL-2, and TNF-α) were added. The cells were then incubated at 4°C in the dark for 30 minutes. The fluorescent groups carried by the antibodies are shown in Table 22 below. The stained samples were then analyzed by flow cytometry. The percentages of different T cell populations (CD4, CD8, NKT) were calculated based on the results.

[0396] Table 22

[0397] Example 9: In vivo efficacy verification of combined mRNA vaccine and antiviral therapy - 2

[0398] In this embodiment, several Large S sequences (with amino acid sequences shown in SEQ ID NO: 514-518) were designed based on known wild-type or naturally occurring Large S proteins, and CDS were designed based on these amino acid sequences to prepare corresponding mRNA vaccines. The relevant nucleic acid and amino acid sequences are shown in Table 23 below. For the specific construction process, please refer to the preparation example.

[0399] Table 23

[0400] Mouse modeling was performed in the same manner as in Example 7. On day 28 after mouse modeling, the drugs were administered according to the administration methods shown in Table 24 below. siRNA was administered via subcutaneous injection (SC), and the mRNA-LNP combination and LNP were administered via intramuscular injection (IM).

[0401] Table 24

[0402] Tissue samples were collected and HBsAg and HBV DNA were detected according to the methods described in Examples 6 and 7. The results are shown in Figures 22-23.

[0403] As shown in Figures 22 and 23, compared with the siRNA-only group, the mRNA-siRNA combination groups maintained low levels of HBsAg and HBV DNA for a longer period after drug withdrawal, and none rebounded. This example demonstrates that mRNAs encoding different Large S antigen sequences, when used in combination with siRNA, all exhibit similar effects to the aforementioned examples in preventing HBsAg rebound and reducing HBV DNA.

[0404] Example 10: Broad-spectrum immunogenicity of mRNA vaccine

[0405] Experimental animals: 6-8 week old C57Bl / 6 mice without specific pathogen grade were divided into 3 groups. The administration methods are shown in Table 25 below. The mRNA-LNP composition and LNP were administered by intramuscular injection (IM).

[0406] Table 25

[0407] Tissue samples were collected from the mice 35 days later:

[0408] a. Plasma sample: Collect whole blood into an EDTA-K2 coated anticoagulant tube, centrifuge at 4°C, 7000g for 10 minutes, and collect the supernatant.

[0409] b. Liver sample collection: The left middle lobe liver of mice was taken and placed in a 50 mL centrifuge tube containing 10% formalin. After being fixed on a shaker for 48 hours, it was transferred to PBS for storage, dehydrated, and embedded in paraffin.

[0410] c. Spleen sample collection: Mouse spleens were collected and immersed in 5% FBS + 1% penicillin antibody + PBS buffer. After collection, the samples were immediately transported to the in vitro environment on wet ice for ELISpot and ICS detection.

[0411] Immune response detection:

[0412] HBV-specific T-cell response in mouse spleen (ELISPOT): Mouse spleen cells were placed in 96-well ELISPOT plates containing anti-IFNγ, and the corresponding stimulating antigen was added. The cells were incubated in a 5% CO2 incubator for about 48 hours. The cultured cells were washed away, blocking solution was added, and then anti-IFNγ-HRP was added and incubated for 1 hour. The cells were washed three times with washing buffer, and chromogenic solution was added. The reaction was stopped in the dark until dark spots of appropriate size appeared. Medium was used to add culture medium only. The stimulating antigen was a polypeptide corresponding to different HBV genotypes (provided by GenScript Biotech, and the amino acid sequences of the corresponding genotypes and stimulating antigens are shown in Table 26). In Table 26, D(1) and D(2) are two stimulating antigens with different sequences but corresponding to the D genotype. AD refers to a conserved sequence polypeptide derived from all sequences of the AD genotype, which can be used to characterize the broad-spectrum immunity of mRNA against different genotype strains.

[0413] The number of dark spots in each well was counted using software for the final effect analysis. The corresponding results are shown in Table 27.

[0414] Table 26 Different Stimulating Antigen Sequences

[0415] Table 27 Immunostimulatory effects of different stimulating antigens A: 50~500, B: 5~50, C: 1~5, D: ≤1

[0416] As shown in the table, sequences #25 and #4 exhibit broad-spectrum T-cell immune stimulation against different HBV genotypes, indicating their broad-spectrum immune capacity against various HBV genotypes. Furthermore, sequence #25 can effectively induce anti-HBs in mice, demonstrating effects close to or even superior to sequence #4.

[0417] Example 11 mRNA in vitro immunoassay

[0418] The reagents used are shown in Table 28:

[0419] Table 28

[0420] Prepare the corresponding mRNA-LNP compositions according to Table 29:

[0421] Table 29

[0422] The steps are as follows:

[0423] 1. mDC induction and transfection treatment:

[0424] a) Take frozen human PBMC cells, thaw them, remove the supernatant, and use CD14 MicroBeads,human to sort out Monocytes from PBMCs (follow the instructions).

[0425] b) The sorted mononuclear cells were resuspended in complete culture medium (including RPMI-1640, penicillin, and fetal bovine serum), and 50 ng / mL Recombinant Human IL-4 and 100 ng / mL Recombinant Human GM-CSF were added. After mixing, the cells were transferred to culture flasks and incubated at 37°C in a 5% CO2 incubator for 5 days to induce dendritic cells (DCs).

[0426] c) DC cells were stimulated with 1.0 μg / mL bacterial lipopolysaccharide (LPS) for 1 day to induce them to become mDCs.

[0427] d) Collect mDC cells and stain a portion of the cells for flow cytometry identification (mDC: CD11c+CD83+CD209). The remaining cells were divided into 7 groups, placed in 96-well plates, and treated with jetMESSENGER transfection reagent to transfect the mRNA of each example into the corresponding cells (1 μg per well). After culturing for 1 day, the transfected mDCs were obtained.

[0428] 2. Priming of memory T cells:

[0429] Take the frozen PBMCs and thaw them, removing the supernatant. Mix the PBMCs separately with the transfected mDC cells from step 1, and culture them in complete medium supplemented with Recombinant Human IL-2. Incubate at 37°C and 5% CO2 for 7 days, adding medium and IL-2 during induction. After culture, collect all PBMC cells and isolate memory T cells from the PBMCs using the Pan T Cell Isolation Kit (human).

[0430] 3. Boost phase mDC induction and transfection:

[0431] a) Take the frozen PBMCs mentioned above and prepare mDCs according to step ac in step 1.

[0432] b) Group the cells according to the examples, place them in 96-well plates, treat them with jetMESSENGER transfection reagent, and transfect the mRNA of each example into the corresponding cells (1ug per well). Continue culturing for 1 day to obtain transfected mDCs.

[0433] 4. Detection of IFN-γ secreting cells in the reaction system:

[0434] The Human IFN-gamma ELISpotPRO kit (HRP) (MabTech, 3420-2HPT-10) was used to detect IFN-γ secreting cells in the reaction system. The procedure was described in the manufacturer's instructions, and is briefly outlined below: Memory T cells were adjusted to a density of 5.0 E5 / mL and added to 100 μL / well of a 96-well plate (coated with anti-IFN-γ). 1.0 μg / mL of PHA was added to 1-2 wells of the 96-well plate as a positive control. Boost-transfected mDCs from the above groups were collected, adjusted to a density of 5.0 E5 / mL, and added to the remaining wells of the 96-well plate at 100 μL / well. After mixing, the cells were incubated at 37°C with 5% CO2 for 5 days. The cultured cells were washed away, blocking solution was added, followed by incubation with anti-IFNγ-HRP for 1 hour. The cells were washed three times with washing buffer, and chromogenic solution was added. The reaction was stopped in the dark until appropriately sized dark spots appeared. The number of dark spots in each well is counted using software for final effect analysis.

[0435] The results are shown in Figure 24. The results show that in the immunomodulatory activity assay of PBMCs, the groups with different coding sequences (groups 9-2, 9-4, 9-5, 9-8, and 9-9) all exhibited a very strong ability to activate T cells. This indicates that for the mRNA combinations of this application, using different specific mRNA sequences without changing the antigen combination will not significantly affect the activity, and these mRNA combinations can still produce the expected effect.

[0436] Example 12: In vivo efficacy verification of combined mRNA vaccine and antiviral therapy - 3

[0437] Mouse modeling: VS104027 mice (HBV transgenic mice, corresponding to HBV genotype A) were fed for 7 days to complete modeling (referred to as day 0). The modeled mice were grouped and administered drugs according to Table 30 below. siRNA was administered subcutaneously (SC), and the mRNA-LNP combination and LNP were administered intramuscularly (IM).

[0438] Table 30

[0439] Tissue samples were collected and HBsAg and HBV DNA were detected using other methods described elsewhere. The results are shown in Figures 25 and 26, respectively. As can be seen from the figures, compared with the siRNA-only group (10⁻² group), the combination of mRNA and siRNA in this application showed a decrease in both HBsAg and HBV over a longer period after drug withdrawal, and neither rebounded.

[0440] The amino acid and nucleotide sequences of this application are as follows:

[0441] It should be understood that these sequences are merely exemplary sequences for embodiments of this application and are not intended to limit the scope of this application. Although DNA sequences are shown in the sequence listing, the nucleotide sequences in the sequence listing of this application may represent either DNA or RNA sequences. When representing an RNA sequence, the "T" represents uridine "U".

[0442] ①Core sequence

[0443] ②5'UTR

[0444] ③3'UTR

[0445] ④ Core sequence + UTR

[0446] ⑤ Full-length sequence

[0447] SEQ ID NO:254 (#21 full-length sequence)

[0448] SEQ ID NO: 255 (#22 full-length sequence)

[0449] SEQ ID NO: 256 (#23 full-length sequence)

[0450] SEQ ID NO: 257 (#24 full-length sequence)

[0451] SEQ ID NO: 258 (#25 full-length sequence)

[0452] SEQ ID NO: 259 (#21 Large S CDS, core sequence)

[0453] SEQ ID NO: 260 (#22 Large S CDS, core sequence)

[0454] SEQ ID NO: 261 (#23 Large S CDS, core sequence)

[0455] SEQ ID NO: 262 (#24 Large S CDS, core sequence)

[0456] SEQ ID NO: 263 (#25 Large S CDS, core sequence)

[0457] SEQ ID NO: 266 (Large S protein)

[0458] SEQ ID NO: 267 (S protein)

[0459] SEQ ID NO: 268 (Core protein)

[0460] SEQ ID NO:514 (#21 Large S amino acid sequence)

[0461] SEQ ID NO:515 (#22 Large S amino acid sequence)

[0462] SEQ ID NO:516 (#23 Large S amino acid sequence)

[0463] SEQ ID NO:517 (#24 Large S amino acid sequence)

[0464] SEQ ID NO:518 (#25 Large S amino acid sequence)

Claims

1. A nucleic acid or a combination of nucleic acids encoding hepatitis B virus antigens, the antigens being large S protein, and S protein and / or Core protein, the nucleic acid being mRNA.

2. The nucleic acid or the combination of nucleic acids of claim 1, comprising: (a) a nucleic acid encoding hepatitis B virus large S protein, and a nucleic acid encoding hepatitis B virus S protein; or, (b) a nucleic acid encoding hepatitis B virus large S protein, and a nucleic acid encoding hepatitis B virus Core protein; or, (c) a nucleic acid encoding hepatitis B virus large S protein, a nucleic acid encoding hepatitis B virus S protein, and a nucleic acid encoding hepatitis B virus Core protein.

3. The combination of nucleic acids of claim 1, comprising: (i) a first nucleic acid comprising a polynucleotide sequence encoding hepatitis B virus large S protein; and, (ii) a second nucleic acid comprising a polynucleotide sequence encoding hepatitis B virus S protein, and / or, a third nucleic acid comprising a polynucleotide sequence encoding hepatitis B virus Core protein.

4. The combination of nucleic acids of claim 1, comprising: (i) a first nucleic acid comprising a polynucleotide sequence encoding hepatitis B virus large S protein, (ii) a second nucleic acid comprising a polynucleotide sequence encoding hepatitis B virus S protein, and, (iii) a third nucleic acid comprising a polynucleotide sequence encoding hepatitis B virus Core protein. the nucleic acid comprising a polynucleotide sequence as set forth in any one of SEQ ID NOs: 1-6, 259-263. the nucleic acid further comprising a 5’ UTR sequence and / or a 3’ UTR sequence; 5. The nucleic acid or combination of nucleic acids of any one of claims 1 to 4, wherein preferably, the 5’ UTR sequence is as set forth in any one of SEQ ID NOs: 7-12, 264; and / or, the 3’ UTR sequence is as set forth in any one of SEQ ID NOs: 13-17, 265; 6. The nucleic acid or combination of nucleic acids of claim 5, wherein more preferably: the 5’ UTR sequence is as set forth in SEQ ID NO: 7, and the 3’ UTR sequence is as set forth in SEQ ID NO: 13; the 5’ UTR sequence is as set forth in SEQ ID NO: 8, and the 3’ UTR sequence is as set forth in SEQ ID NO: 14; the 5’ UTR sequence is as set forth in SEQ ID NO: 9, and the 3’ UTR sequence is as set forth in SEQ ID NO: 15; the 5’ UTR sequence is as set forth in SEQ ID NO: 10, and the 3’ UTR sequence is as set forth in SEQ ID NO: 15; the 5’ UTR sequence is as set forth in SEQ ID NO: 11, and the 3’ UTR sequence is as set forth in SEQ ID NO: 16; the 5’ UTR sequence is as set forth in SEQ ID NO: 12, and the 3’ UTR sequence is as set forth in SEQ ID NO: 17; or, the 5’ UTR sequence is as set forth in SEQ ID NO: 264, and the 3’ UTR sequence is as set forth in SEQ ID NO:

265. ​ ​ Further more preferably, the nucleic acid comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 18-35.

7. The nucleic acid or combination of nucleic acids of claim 5 or 6, wherein The nucleic acid further comprises a promoter and / or a polyA tail; the promoter is preferably a T7 promoter; Preferably, the nucleic acid comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 36-53, 254-258.

8. The nucleic acid or combination of nucleic acids of any of claims 1 to 7, wherein The mRNA comprises a 5' cap; and / or, the mRNA further comprises a modification of nucleosides, such as N1-methyl pseudouridine modification.

9. The nucleic acid or combination of nucleic acids of any one of claims 1-4, wherein The nucleic acid combination comprises: a first nucleic acid comprising a polynucleotide sequence as set forth in any one of SEQ ID NOs: 1, 4, or 259-263, and, a second nucleic acid comprising a polynucleotide sequence as set forth in SEQ ID NO: 2 or 5 and / or a third nucleic acid comprising a polynucleotide sequence as set forth in SEQ ID NO: 3 or 6; Preferably, the nucleic acid combination comprises: a first nucleic acid comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 18, 21, 24, 27, 30, or 33, and, a second nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 19, 22, 25, 28, 31, or 34 and / or a third nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 20, 23, 26, 29, 32, or 35; More preferably, the nucleic acid combination comprises: a first nucleic acid comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 274, 277, 280, 283, 286, 289, 269-273, and a second nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 275, 278, 281, 284, 287, or 290 and / or a third nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 276, 279, 282, 285, 288, or 291; More preferably, the nucleic acid combination comprises: a first nucleic acid comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 36, 39, 42, 45, 48, 51, 254-258, and, a second nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 37, 40, 43, 46, 49, or 52 and / or a third nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 38, 41, 44, 47, 50, or 53.

10. The nucleic acid or combination of nucleic acids of claim 9, wherein The nucleic acid combination comprises: a first nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 1, and, a second nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 2 and / or a third nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 3; Preferably, the nucleic acid combination comprises: a first nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 21, and, a second nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 22 and / or a third nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 35; Preferably, the nucleic acid combination comprises: a first nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 277, and, a second nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 278 and / or a third nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 291; Preferably, the nucleic acid combination comprises: a first nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 39, and, a second nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 40 and / or a third nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:

53.

11. A pharmaceutical composition comprising the nucleic acid or nucleic acid combination of any one of claims 1-10, and a pharmaceutically acceptable carrier and / or a delivery vehicle. Preferably, the delivery vehicle is, for example, a lipid nanoparticle. Preferably, the lipid nanoparticle satisfies one or more conditions selected from the following: (1) the N / P ratio of the lipid nanoparticle is 5-6; (2) the lipid phase of the lipid nanoparticle comprises one or more selected from SM-102, DSPC, cholesterol and DMG-PEG2000; (3) the volume ratio of the mixing of the lipid phase and the aqueous phase of the lipid nanoparticle is 1:2-1:4, preferably 1:3; and, (4) the lipid nanoparticle is prepared by a microfluidic process; Further more preferably: the N / P ratio of the lipid nanoparticle is 5.5-5.7; preferably 5.65-5.68, for example 5.67; and / or, when the microfluidic process is used, the total flow rate of the microfluidic synthesis is 11-13 mL / min, preferably 12 mL / min; and / or, the flow rate ratio of the aqueous phase and the lipid phase of the lipid nanoparticle is 2:1-4:1, preferably 3:

1.

12. A combination comprising the nucleic acid or combination of nucleic acids of any one of claims 1-10, or the pharmaceutical composition of claim 11, with an antiviral agent, wherein, The antiviral agent is an oligonucleotide targeting the hepatitis B virus gene; preferably, the oligonucleotide is a double-stranded siRNA or ASO.

13. The combination of claim 12, wherein the nucleic acid is a nucleic acid comprising a polynucleotide sequence encoding a hepatitis B virus large S protein; preferably, the polynucleotide sequence is as set forth in any one of SEQ ID NOs: 1, 4 or 259-263; Further preferably, the nucleic acid comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 18, 21, 24, 27, 30 or 33; More further preferably, the nucleic acid comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 274, 277, 280, 283, 286, 289, 269-273; More further preferably, the nucleic acid comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 36, 39, 42, 45, 48, 51, 254-258.

14. The combination of claim 12 or 13, wherein, The oligonucleotide sequence is as set forth in SEQ ID NO: 67, 69, 71, 73, 75, 77, 80, 82, 98, 100, 102 or 107; Preferably, the oligonucleotide has a sequence as set forth in any one of SEQ ID NOs: 67, 69, 71, 73, 75, 77, 80, 98, 100, 109-112, or 113-116; More preferably, the oligonucleotide further comprises one or more of the following modifications: 5'-phosphate modification, 2'-O-methylation modification, 2'-fluoro modification, and phosphorothioate modification; Further more preferably, the oligonucleotide has a sequence as set forth in any one of SEQ ID NOs: 68, 70, 72, 74, 76, 78-79, 81, 83-97, 99, 101, 103-106, or 108.

15. The combination of claim 12 or 13, wherein, The double-stranded siRNA comprises a sense strand and an antisense strand, the sense strand comprising a sequence as set forth in any one of SEQ ID NOs: 67, 69, 71, 73, 75, 77, 80, or 82, and the antisense strand comprising a sequence as set forth in any one of SEQ ID NOs: 98, 100, 102, or 107; Preferably, the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 67, 69, 71, 73, 75, 77, 80, or 109-112, and the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 98, 100, 113-116, or 107; More preferably, the sense strand comprises a sequence as set forth in SEQ ID NO: 67, and the antisense strand comprises a sequence as set forth in SEQ ID NO: 98; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 69, and the antisense strand comprises a sequence as set forth in SEQ ID NO: 98; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 71, and the antisense strand comprises a sequence as set forth in SEQ ID NO: 98; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 73, and the antisense strand comprises a sequence as set forth in SEQ ID NO: 98; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 75, and the antisense strand comprises a sequence as set forth in SEQ ID NO: 98; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 67, and the antisense strand comprises a sequence as set forth in SEQ ID NO: 100; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 77, and the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 102 or 113-116; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 80, and the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 102 or 113-116; or, the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 82 or 109-112, and the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 102 or 113-116; or, the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 82 or 109-112, and the antisense strand comprises a sequence as set forth in SEQ ID NO:

107.

16. The combination of claim 15, wherein, the sense strand further comprises one or more of the following modifications to the nucleotides: 5'-phosphate modification, 2'-O-methylation modification, 2'-fluoro modification, and phosphorothioate modification; and / or, the antisense strand further comprises one or more of the following modifications to the nucleotides: glycol modification, 5'-phosphate modification, 2'-O-methylation modification, 2'-fluoro modification, and phosphorothioate modification; Preferably, the sense strand comprises a combination of the following modifications: 2'-O-methylation modification, 2'-fluoro modification, and phosphorothioate modification; and / or, the antisense strand comprises one of the following combinations of modifications: (1) glycol modification, 2'-O-methylation modification, 2'-fluoro modification, and phosphorothioate modification; (2) 5'-phosphate modification, 2'-O-methylation modification, 2'-fluoro modification, and phosphorothioate modification; and, (3) glycol modification, 5'-phosphate modification, 2'-O-methylation modification, 2'-fluoro modification, and phosphorothioate modification; More preferably, the modifications to the nucleotides in the sense strand and the antisense strand comprise one or more of the following: (1) the first nucleotide at the 5' end of the antisense strand comprises a 5'-phosphate modification; (2) one or more of the adenosine nucleotides of the antisense strand comprise a glycol modification; (3) the 5'-terminal first and second nucleotides of the antisense strand comprise phosphorothioate modification; (4) the 3'-terminal second and third nucleotides of the antisense strand comprise 2'-fluoro modification; and, (5) the first two 5'-terminal nucleotides and the first three 3'-terminal nucleotides of the sense strand comprise 2'-O-methylation modification; or the first two 5'-terminal nucleotides and the first four 3'-terminal nucleotides of the sense strand comprise 2'-O-methylation modification.

17. The combination of claim 16, wherein, the antisense strand comprises a sequence as set forth in SEQ ID NO: 98 or 99; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 69 or 70, the antisense strand comprises a sequence as set forth in SEQ ID NO: 98 or 99; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 71 or 72, the antisense strand comprises a sequence as set forth in SEQ ID NO: 98 or 99; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 73 or 74, the antisense strand comprises a sequence as set forth in SEQ ID NO: 98 or 99; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 75 or 76, the antisense strand comprises a sequence as set forth in SEQ ID NO: 98 or 99; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 67 or 68, the antisense strand comprises a sequence as set forth in SEQ ID NO: 100 or 101; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 77 or 78, the antisense strand comprises a sequence as set forth in SEQ ID NO: 102 or 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 77 or 79, the antisense strand comprises a sequence as set forth in SEQ ID NO: 102 or 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 80 or 81, the antisense strand comprises a sequence as set forth in SEQ ID NO: 102 or 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 82 or 83, the antisense strand comprises a sequence as set forth in SEQ ID NO: 102 or 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 82 or 84, the antisense strand comprises a sequence as set forth in SEQ ID NO: 102 or 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 82 or 85, the antisense strand comprises a sequence as set forth in SEQ ID NO: 102 or 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 82 or 86, the antisense strand comprises a sequence as set forth in SEQ ID NO: 102 or 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 82 or 87, the antisense strand comprises a sequence as set forth in SEQ ID NO: 102 or 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 82 or 88, the antisense strand comprises a sequence as set forth in SEQ ID NO: 102 or 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 82 or 84, the antisense strand comprises a sequence as set forth in SEQ ID NO: 107 or 108;or the sense strand comprises a sequence as shown in SEQ ID NO: 82 or 85, and the antisense strand comprises a sequence as shown in SEQ ID NO: 107 or 108; or the sense strand comprises a sequence as shown in SEQ ID NO: 82 or 86, and the antisense strand comprises a sequence as shown in SEQ ID NO: 107 or 108; or the sense strand comprises a sequence as shown in SEQ ID NO: 82 or 87, and the antisense strand comprises a sequence as shown in SEQ ID NO: 107 or 108; or the sense strand comprises a sequence as shown in SEQ ID NO: 82 or 88, and the antisense strand comprises a sequence as shown in SEQ ID NO: 107 or 108; or the sense strand comprises a sequence as shown in SEQ ID NO: 82 or 89, and the antisense strand comprises a sequence as shown in SEQ ID NO: 102 or 103; or the sense strand comprises a sequence as shown in SEQ ID NO: 82 or 90, and the antisense strand comprises a sequence as shown in SEQ ID NO: 102 or 103; or the sense strand comprises a sequence as shown in SEQ ID NO: 82 or 91, and the antisense strand comprises a sequence as shown in SEQ ID NO: 102 or 103; or the sense strand comprises a sequence as shown in SEQ ID NO: 82 or 92, and the antisense strand comprises a sequence as shown in SEQ ID NO: 102 or 103; or the sense strand comprises a sequence as shown in SEQ ID NO: 82 or 93, and the antisense strand comprises a sequence as shown in SEQ ID NO: 102 or 103; or the sense strand comprises a sequence as shown in SEQ ID NO: 82 or 94, and the antisense strand comprises a sequence as shown in SEQ ID NO: 102 or 103; or the sense strand comprises a sequence as shown in SEQ ID NO: 82 or 95, and the antisense strand comprises a sequence as shown in SEQ ID NO: 102 or 104; or the sense strand comprises a sequence as shown in SEQ ID NO: 82 or 96, and the antisense strand comprises a sequence as shown in SEQ ID NO: 102 or 105; or the sense strand comprises a sequence as shown in SEQ ID NO: 82 or 97, and the antisense strand comprises a sequence as shown in SEQ ID NO: 102 or 106.

18. The combination of claim 17, wherein, the sequence of the sense strand is set forth in SEQ ID NO: 68, the sequence of the antisense strand is set forth in SEQ ID NO: 99; or the sequence of the sense strand is set forth in SEQ ID NO: 70, the sequence of the antisense strand is set forth in SEQ ID NO: 99; or the sequence of the sense strand is set forth in SEQ ID NO: 72, the sequence of the antisense strand is set forth in SEQ ID NO: 99; or the sequence of the sense strand is set forth in SEQ ID NO: 74, the sequence of the antisense strand is set forth in SEQ ID NO: 99; or the sequence of the sense strand is set forth in SEQ ID NO: 76, the sequence of the antisense strand is set forth in SEQ ID NO: 99; or the sequence of the sense strand is set forth in SEQ ID NO: 68, the sequence of the antisense strand is set forth in SEQ ID NO: 101; or the sequence of the sense strand is set forth in SEQ ID NO: 78, the sequence of the antisense strand is set forth in SEQ ID NO: 103; or the sequence of the sense strand is set forth in SEQ ID NO: 79, the sequence of the antisense strand is set forth in SEQ ID NO: 103; or the sequence of the sense strand is set forth in SEQ ID NO: 81, the sequence of the antisense strand is set forth in SEQ ID NO: 103; or the sequence of the sense strand is set forth in SEQ ID NO: 83, the sequence of the antisense strand is set forth in SEQ ID NO: 103; or the sequence of the sense strand is set forth in SEQ ID NO: 84, the sequence of the antisense strand is set forth in SEQ ID NO: 103; or the sequence of the sense strand is set forth in SEQ ID NO: 85, the sequence of the antisense strand is set forth in SEQ ID NO: 103; or the sequence of the sense strand is set forth in SEQ ID NO: 86, the sequence of the antisense strand is set forth in SEQ ID NO: 103; or the sequence of the sense strand is set forth in SEQ ID NO: 87, the sequence of the antisense strand is set forth in SEQ ID NO: 103; or the sequence of the sense strand is set forth in SEQ ID NO: 88, the sequence of the antisense strand is set forth in SEQ ID NO: 103; or the sequence of the sense strand is set forth in SEQ ID NO: 84, the sequence of the antisense strand is set forth in SEQ ID NO: 108; or the sequence of the sense strand is set forth in SEQ ID NO: 85, the sequence of the antisense strand is set forth in SEQ ID NO: 108; or the sequence of the sense strand is set forth in SEQ ID NO: 86, the sequence of the antisense strand is set forth in SEQ ID NO: 108; or the sequence of the sense strand is set forth in SEQ ID NO: 87, the sequence of the antisense strand is set forth in SEQ ID NO: 108; or the sequence of the sense strand is set forth in SEQ ID NO: 88, the sequence of the antisense strand is set forth in SEQ ID NO: 108; or the sequence of the sense strand is set forth in SEQ ID NO: 84, the sequence of the antisense strand is set forth in SEQ ID NO: 109; or the sequence of the sense strand is set forth in SEQ ID NO: 85, the sequence of the antisense strand is set forth in SEQ ID NO: 109; or the sequence of the sense strand is set forth in SEQ ID NO: 86, the sequence of the antisense strand is set forth in SEQ ID NO: 109; or the sequence of the sense strand is set forth in SEQ ID NO: 87, the sequence of the antisense strand is set forth in SEQ ID NO: 109; or the sequence of the sense strand is set forth in SEQ ID NO: 88, the sequence of the antisense strand is set forth in SEQ ID NO: 109.NO: 88 and a sequence of the anti-sense strand as set forth in SEQ ID NO: 108; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 89, the anti-sense strand comprises a sequence as set forth in SEQ ID NO: 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 90, the anti-sense strand comprises a sequence as set forth in SEQ ID NO: 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 91, the anti-sense strand comprises a sequence as set forth in SEQ ID NO: 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 92, the anti-sense strand comprises a sequence as set forth in SEQ ID NO: 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 93, the anti-sense strand comprises a sequence as set forth in SEQ ID NO: 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 94, the anti-sense strand comprises a sequence as set forth in SEQ ID NO: 103; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 95, the anti-sense strand comprises a sequence as set forth in SEQ ID NO: 104; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 96, the anti-sense strand comprises a sequence as set forth in SEQ ID NO: 105; or, the sense strand comprises a sequence as set forth in SEQ ID NO: 97, the anti-sense strand comprises a sequence as set forth in SEQ ID NO:

106.

19. The combination of claim 12 or 13, wherein, The antiviral agent comprises one or more oligonucleotides selected from the group consisting of: (1) a double-stranded siRNA comprising a sense strand having a sequence as set forth in SEQ ID NO: 56 and an antisense strand having a sequence as set forth in SEQ ID NO: 57; (2) a double-stranded siRNA comprising a sense strand having a sequence as set forth in SEQ ID NO: 58 and an antisense strand having a sequence as set forth in SEQ ID NO: 59; (3) a double-stranded siRNA comprising a sense strand having a sequence as set forth in SEQ ID NO: 60 and an antisense strand having a sequence as set forth in SEQ ID NO: 61; (4) a double-stranded siRNA comprising a sense strand having a sequence as set forth in SEQ ID NO: 62 and an antisense strand having a sequence as set forth in SEQ ID NO: 63; (5) a double-stranded siRNA comprising a sense strand having a sequence as set forth in SEQ ID NO: 64 and an antisense strand having a sequence as set forth in SEQ ID NO: 65; (6) an ASO comprising a sequence as set forth in SEQ ID NO: 66; and, (7) ASO AHB-137; Preferably, the antiviral agent comprises: a double-stranded siRNA comprising a sense strand having a sequence as set forth in SEQ ID NO: 60 and an antisense strand having a sequence as set forth in SEQ ID NO: 61 and a double-stranded siRNA comprising a sense strand having a sequence as set forth in SEQ ID NO: 62 and an antisense strand having a sequence as set forth in SEQ ID NO: 63; More preferably, the mass ratio of a double-stranded siRNA comprising a sense strand having a sequence as set forth in SEQ ID NO: 60 and an antisense strand having a sequence as set forth in SEQ ID NO: 61 and a double-stranded siRNA comprising a sense strand having a sequence as set forth in SEQ ID NO: 62 and an antisense strand having a sequence as set forth in SEQ ID NO: 63 is 2:1 to 4:1, for example, 3:

1.

20. The combination of any one of claims 12 to 19, wherein, The antiviral agent further comprises a pharmaceutically acceptable carrier, a delivery vehicle and / or a ligand; Preferably, the delivery vehicle is selected from the group consisting of LNP or exosome, and the ligand is selected from the group consisting of a carbohydrate and derivatives thereof, the carbohydrate including monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide; More preferably, the double-stranded siRNA is attached to a carbohydrate and derivatives thereof via a linker branched by monovalency, divalency or trivalency.

21. Use of the combination according to any one of claims 12 to 20 in the preparation of a medicament for preventing and / or treating a disease caused by hepatitis B virus; Preferably, the disease is hepatitis B.

22. Use of the nucleic acid or the combination of nucleic acids according to any one of claims 1 to 10, or the pharmaceutical composition of claim 11, for the manufacture of a medicament for the prevention and / or treatment of a disease caused by a hepatitis B virus; preferably, the medicament is administered in combination with an antiviral agent as described in any one of claims 12 to 20.

23. A method for the prevention and / or treatment of a disease caused by a hepatitis B virus, the method comprising administering to a subject in need thereof an effective amount of the nucleic acid or the combination of nucleic acids according to any one of claims 1 to 10, or the pharmaceutical composition of claim 11.

24. The method of claim 23, further comprising administering to the subject in need thereof an effective amount of an antiviral agent as described in any one of claims 12 to 20.

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