CN114767704B - Medicine structure capable of targeting hepatitis B virus and medicine composition - Google Patents
Medicine structure capable of targeting hepatitis B virus and medicine composition Download PDFInfo
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Abstract
The present invention provides compositions and methods for delivering siRNA molecules targeted to Hepatitis B Virus (HBV). The siRNA molecule may be one or more nucleic acids comprising a nucleotide or nucleotide analogue. The siRNA molecules are covalently bound to a peptide docking carrier (PDoV) and further covalently bound to one or more GalNAc ligands.
Description
Cross-reference to related patent applications
The present application is directed to the acquisition of U.S. patent application Ser. No. 63/140,232 (patent name: TARGETED NUCLEIC ACID THERAPY FOR HEPATITITITS B), patent application Ser. No. 2021, 1, 21, and Chinese patent application Ser. No. 2021102899267 (patent name: a pharmaceutical composition and pharmaceutical composition capable of targeting HEPATITIS B virus), patent application Ser. No. 2021, 3, 18, the disclosures of which are incorporated herein by reference in their entireties.
Technical Field
The invention particularly relates to a drug structure and a drug composition capable of targeting hepatitis B virus.
Background
Hepatitis B virus
Hepatitis B Virus (HBV) is one of the most thoroughly and most complex hepatitis viruses with characteristics studied. Infectious particles consist of a viral core and an outer epidermis. The core contains circular portions of double-stranded DNA and DNA polymerase that replicate within the nucleus of an infected hepatocyte. Hepatitis b is caused by HBV and may cause life threatening liver infections. It can cause acute and chronic infections, leading to a high risk of death from cirrhosis and liver cancer. Leung, med.j. Malaysia;60Suppl B:63-6 (2005). The World Health Organization (WHO) estimated that 2.57 million people had chronic hepatitis b worldwide in 2015. Despite the availability of prophylactic vaccines and effective and well-tolerated viral inhibitors, patient numbers continue to rise. However, there is currently no effective treatment to clear HBV infection.
Over 90% of infected infants will develop chronic hepatitis b. Patients may be asymptomatic, and may also exhibit non-specific symptoms such as fatigue and discomfort. Hepatitis b may heal by itself (not commonly) within decades without treatment, may progress rapidly, or slowly to cirrhosis. Remission of symptoms usually begins with a transient increase in disease severity, resulting in conversion of serum from hepatitis b e antigen (HBeAg) to hepatitis b e antigen antibodies (anti-HBe). In addition, co-infection with Hepatitis D Virus (HDV) results in the most severe HBV infection, and cirrhosis can occur in up to 70% of patients if untreated. Chronic HBV infection increases the risk of hepatocellular carcinoma. Christopher et al CLIN LIVER DIS; .23 (3):557-572 (2019)
Acute hepatitis b can last six months (whether or not there are signs), during which an infected person can transmit the virus to other people. HBV in blood can be detected by a simple blood test. Symptoms of acute infections may include loss of appetite, joint and muscle pain, low fever, and stomach pain. Although most people are asymptomatic, they can develop symptoms 60-150 days after infection, on average 90 days or 3 months. Some people may develop more severe symptoms such as nausea, vomiting, jaundice (yellowing of eyes and skin) or gastric swelling.
Current situation of anti-hepatitis b drug development
Immunomodulator drugs have been used to treat severe pneumonia, immunodeficiency and chronic hepatitis b, jiang, vaccine; .30 (4):758-766 (2012). These drugs can enhance the immune response of patients, especially specific immunity to HBV, and may help immune cells to recognize and destroy HBV-infected cells and clear them of hepatitis B virus. Interferon (IFN) is a secreted glycoprotein with antiviral, antiproliferative and immunomodulatory cytokine functions. In addition, thymosin-alpha 1 is another type of immunomodulatory drug that has the primary function of promoting differentiation of T cells to the mature stage and enhancing responses to stimuli such as antigens. In addition, various types of immune cells (e.g., monocytes, macrophages, T cells, B cells, and NK cells) and non-immune cells (e.g., endothelial cells, epidermal cells, and fibroblasts) synthesize and secrete cytokines under stimulation.
Nucleoside and nucleoside analog drugs (collectively: NA) are also used to treat HBV infection. Lamivudine is a pyrimidine nucleoside drug, the first NA approved for the treatment of chronic hepatitis B virus infection. Telbivudine is a specific, selective and oral drug for the treatment of chronic hepatitis b. Other drugs such as entecavir, adefovir and tenofovir are also oral antiviral drugs that can effectively and selectively inhibit HBV infection. These NA are integrated into the viral DNA, preventing the synthetic extension of the viral DNA. Kang et al, viruses;7:4960-77 (2015)
Each of the currently approved clinical intervention therapies for Chronic Hepatitis B (CHB) requires long-term treatment of most patients. Although continuous inhibition of HBV DNA using current therapies is associated with improvement of clinical outcome, excessive risk of hepatocellular carcinoma (HCC) still exists and hepatitis b surface antigen (HBsAg) clearance is unusual.
Treatment of siRNA
Double stranded RNA has been shown to inhibit gene expression by RNA interference (RNAi). Small interfering RNA (siRNA) -induced RNA interference modulation has shown great potential in the treatment of a variety of human diseases, ranging from cancer to other traditional non-drug diseases. However, the introduction of siRNA into tissues required for the human body remains a difficult problem. In particular, there is a need to improve nucleic acid drug targeting to specific cell types or tissues, as well as to develop non-toxic endosomal escape agents, as will be further explained below.
Currently, there are two routes of effective administration commonly used for nucleic acid pharmaceuticals. One approach is to use lipid nanoparticles (liposomes) containing multiple components. Another approach is to target asialoglycoprotein receptor (ASGPR) with conjugates containing GalNAc molecules.
One major challenge of RNA therapy is that all drugs delivered to the cells eventually need to escape from the endosome. ASGPR-targeted GalNAc-siRNA conjugates can achieve delivery of ASO and siRNA to the liver, as the characteristics of ASGPR are very suitable for delivery of macromolecular drugs to hepatocytes. In particular, hepatocytes express millions of ASGPRs on their cell surfaces, which circulate rapidly every 10-15 minutes. These properties enable GalNAc-based delivery methods to achieve significant effects even with endosome escape rates of less than 0.01% as currently assumed. In contrast, efficient delivery of ASO or RNA to other tissues has not been achieved. No other ligand-receptor system expresses receptors at high levels like ASGPR nor circulates as rapidly as ASGPR into endosomes. In fact, most cell surface receptors are expressed in the range 10000-100000 (or less) per cell, with cryptan and clathrin mediated endocytosis typically occurring once every 90 minutes. Juliano, nucleic Acids Res.44,6518-6548 (2016)
Endosomal escape remains a challenge for all RNA-based therapies. In order to target cells or tissues other than hepatocytes, it is necessary to enhance endosomal escape by developing new chemicals and materials. Small molecule endosomolytic agents, such as chloroquine, have been used to destroy or lyse endosomes, but at effective concentrations these agents always lyse all types of endosomes within the cell, resulting in serious toxicity.
Another approach to endosomal escape is to bind the endosomal lytic peptide or molecule directly to the RNA, but this severely limits the effect of the endosome containing the therapeutic RNA. Because of toxic effects, many clinical trials using a bi-molecular Dynamic Polymer (DPC) system containing cholesterol or soluble melittin to escape endosomes have been forced to terminate. Wooddell, et al, mol. Ter.21, 973-985 (2013); hou et al, biotechnol. Adv.33,931-940 (2015)
SiRNA technology for treating hepatitis B infection
In a mouse model, siRNA molecules can induce RNA interference to inhibit HBV replication in mammalian hepatocytes, lian et al, J Pharmacol Sci;114 (2):147-57 (2010). Chemically synthesized siRNA/shRNA has been shown to be a potential therapeutic approach to treating HBV infection, wu et al, virus Research,112:100-107 (2005). However, a secondary clinical trial of an RNAi-based drug did not show a reduction in viral numbers in some patients. Further analysis confirmed that the viral antigen was produced from HBV transcripts without target sequences.
Cytokines may also lead to RNAi resistance to Dowdy, nature Biotechnology,35:222-229 (2017). This presents challenges for delivering synthetic siRNA and shRNA expression vector systems to specific target cells or tissues in a manner consistent with clinical administration. This challenge is also due to the fact that RNAi molecules are small double-stranded oligonucleotides, with a highly negatively charged hydrophilic phosphate backbone. This prevents them from interacting with and penetrating the cell membrane, allowing them to be rapidly filtered out by renal blood circulation. RNAi molecules that are in contact with the target cells are taken up by endocytosis and retained in the endosome for a prolonged period of time, which is degraded by the nucleic acid cleaving enzyme. Thus, methods are needed to allow the escape of intact RNAi molecules from endosomes into the cytoplasm where the RNA-induced silencing complex (RISC) resides.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide compositions and methods using interfering RNA molecules with enhanced therapeutic benefits.
In order to solve the technical problems, the invention adopts the following technical scheme:
The present invention provides a composition and method for delivering siRNA molecules targeting Hepatitis B Virus (HBV). The siRNA molecule may be one or more nucleic acids comprising a nucleotide or nucleotide analogue. The siRNA molecules are covalently bound to a peptide docking carrier (PDoV) and further covalently bound to one or more GalNAc ligands. The present invention provides methods for treating chronic HBV infection and related diseases.
The present invention provides compositions and methods of using interfering RNA molecules with enhanced therapeutic benefits. The compositions and methods deliver therapeutic compounds (e.g., siRNA molecules) to a subject targeted to cells/tissue by attaching a targeting ligand to the compound. The subject may be an animal or a human.
In some embodiments, a targeting ligand as described herein can be covalently bound to an endosomal release peptide by an orthogonal biological conjugation method. The targeting ligands may be particularly useful for improving delivery of RNAi molecules to a selected target (e.g., liver). In other embodiments, the targeting ligand allows targeted delivery of the RNAi molecules into other tissues, such as skin and brain.
The targeting ligand described herein may be one or more targeting moieties, one or more linkers. The linker is covalently bound to the siRNA by click chemistry, thiol/maleimide chemistry or other bio-orthogonal chemical methods and serves as a targeting ligand. The linker is preferably hydrophilic, for example, may be a water-soluble flexible polyethylene glycol (PEG) that is sufficiently stable and limits potential interactions between one or more targeting groups. PEG has been demonstrated by clinical studies to be safe and compatible, and suitable for clinical treatment. In some embodiments, the linker may be poly (L-lactide) n having a specific molecular weight (where n=5-20), wherein the ester linkage is enzymatically or hydrolytically labile.
The targeting ligand may include one or more targeting moieties, one or more linkers with linked reactive units. They are covalently bound to the siRNA and targeting ligand by click chemistry, thiol/maleimide chemistry or other bio-orthogonal chemical methods. The linker reactive units may be, but are not limited to, thiol-maleimide linkages, triazole linkages formed from the reaction of an alkyne and an azide, and amides formed from amine-succinimidyl ester linkages. Each of these linkers is suitable for covalent attachment of the targeting ligand and the therapeutic compound.
In some embodiments, the disclosed targeted specific RNA compounds can bind directly to endosomal release docking peptides via the 3 'or 5' end of the RNA. The targeting ligand (e.g., n-acetyl-galactosamine) may also be bound to the same docking peptide in a compatible manner.
In some embodiments, the disclosed target-specific RNA compounds can also be directly linked to a targeting ligand (e.g., n-acetyl-galactosamine) through, for example, the 3 'or 5' end of the RNA. In some embodiments, the RNA can comprise one or more modified nucleotides, such as 3' -OMe, 3' -F, or 3' -MOE. In some embodiments, the RNA can be an RNAi agent, e.g., a double stranded RNAi agent. In some embodiments, the targeting ligand disclosed herein is attached to the 5 'or 3' end of the sense strand of a double stranded RNAi agent or the 5 'or 3' end of the antisense strand of a double stranded RNAi agent. The targeting ligand is optionally attached to the 3 '/3', 3'/5' or 5'/5' ends of the sense strand and the antisense strand of the double stranded RNAi agent. The targeting ligand may be covalently bound to the RNA molecule through a phosphate, thiophosphate or phosphonate group at the 3 'or 5' end of the double stranded RNAi agent.
In some embodiments, the disclosed target-specific RNA molecules are siRNA molecules that target and inhibit the expression of HBV mRNA. As used herein, the term "targeting" refers primarily to one or more nucleotide sequences in an HBV gene.
In some embodiments, the target sequence of the selected siRNA is located in a surface gene (HBsAg) in the HBV genome. This region is HBV-specific and highly conserved. The main function of HBV surface antigen proteins is to form HBV envelope.
In some embodiments, the target sequence of the selected siRNA is located in a core protein (HBcAg) gene in the HBV genome. HBcAg forms a dimer, rich in arginine sequences at the C-terminus, and is thought to interact with viral nucleic acids in the nucleocapsid.
In some embodiments, the target sequence of the selected siRNA is located in the E antigen (HBeAg) gene in the HBV genome. The HBeAg ORF encodes an Endoplasmic Reticulum (ER) targeting sequence, which synergistically delivers peptides into the ER where the protein is processed into the final 15kD HBeAg and secreted from HBV infected cells.
In some embodiments, the target sequence of the selected siRNA is located in the X (HBx) gene in the HBV genome. HBx is the only regulatory protein encoded by HBV and plays an important role in HBV replication, as HBx can bind cccDNA; HBx is necessary for cDNA transcription; downstream HBx-mediated effects are necessary for HBV replication.
In some embodiments, the target sequence of the selected siRNA is located in a polymerase (HBp)/Reverse Transcriptase (RT) gene in the HBV genome. The polymerase plays a key role in the life cycle of HBV, and the activities of these two enzymes are critical to HBV replication in the life cycle of HBV proliferation.
In some embodiments, the gene-targeted siRNA of HBV comprises a sense strand and an antisense strand, each sense strand comprising a core sequence of 19-21 bases in length. The lengths of the sense strand and the antisense strand of the siRNA described are 19 to 27 nucleotides in length, respectively.
In some embodiments, the siRNA sense strand and antisense strand are 21 or 25 nucleotides in length, respectively. The sense and antisense strands of siRNA typically anneal to form a double strand. In the complementary double-stranded region, the positive strand core sequence is 100% complementary to the antisense core sequence.
In some examples, the siRNA may have an asymmetric structure, wherein the sense strand is about 19 nucleotides in length and the antisense strand is about 21 nucleotides in length.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
The drug structure of the invention can remarkably inhibit the expression of HBV target genes, the concentration of the drug acting is far lower than that of other drugs in the prior art, and the drug structure of the invention has low cytotoxicity, can effectively transfer active ingredients into targeted cells, and can enable complete active molecules to escape from endosomes into cytoplasm where RNA-induced silencing complex (RISC) is located.
Drawings
FIG. 1 shows a schematic representation of HBV genome (NC_ 003977.2). The HBV genome is a partially double-stranded DNA of about 3.2kb in a relaxed circular conformation (rcDNA). The HBV genome has four overlapping Open Reading Frames (ORFs), namely surface (S), core (C), pol (P) and X (X). In general, these four HBV ORFs together encode seven different HBV proteins.
FIG. 2 shows the design of [ GalNAc ] peptide docking vector (G-PDoV). Trivalent GalNAc is covalently bound to one binding site a and the oligonucleotide and siRNA are covalently bound to the other or both binding sites B, respectively.
FIG. 3 shows the design of a peptide docking vector (PDoV). It has a (HnKm) oXpYq polypeptide backbone with multiple repeating units histidine (H), lysine (K) and functional units X (amino acids or functional linkers), where n=1-10, m=1-10, o=1-10, p=1-5, q=1-5. HK repeat units have been shown to have good cell penetration, to facilitate endosome release, lysine or various functional units X or Y will be employed as binding sites for binding ligands, and Y will be used as binding sites for binding oligonucleotides via different covalent bonds. For example, site ① only reacts in the presence of a ligand, such as GalNAc or other targeting ligand. Site ③ can only bind to oligonucleotides and siRNAs under specific conditions.
FIG. 4 shows an example of a structure of PDoV comprising one or two oligonucleotide sites and one ligand binding site.
Fig. 5 shows an example of a trivalent GalNAc targeting ligand molecule.
FIG. 6 shows an example of the construction of HBV siRNA-PDoV-ligand compound 1.
FIG. 7 shows an example of the construction of a double HBV siRNA-PDoV-ligand compound 2.
FIG. 8 shows an example of the construction of a double HBV siRNA-PDoV-ligand compound 3.
FIG. 9 shows the result of screening siRNA by clone 1 (psi-P & S & S) fluorescent quantitative expression assay. Most siRNAs have an inhibition rate of more than 80% (asterisk), and some even more than 90% of the target gene expression. NTS00042 represents experimental code, 21 or 25 represents the length of siRNA, and 1# represents the sequence number of 21 or 25 base pair length siRNA.
FIG. 10 shows the results of screening siRNA using the fluorescent quantitative expression assay of clone 2 (psi-P & X & C0 uORF). Only a few siRNAs have good inhibition effect on the expression of target genes. Asterisks indicate inhibition exceeding 80%. NTS00042 represents experimental code, 21 or 25 represents the length of siRNA, and 1# represents the sequence number of 21 or 25 base pair length siRNA.
FIG. 11 shows half maximal effector concentration (EC 50) data for partially screened siRNA in both 293T and A549 cells. The EC 50 of most siRNA was less than 10 pg/. Mu.L, indicating that these siRNA molecules showed strong inhibitory activity at concentrations well below conventional drug molecules.
FIG. 12 shows the inhibition of HBsAg gene expression in the supernatant of HepAD38 cells by partially selected siRNA and modified siRNA.
FIG. 13 shows the inhibition of HBeAg gene and HBsAg gene by partially screened siRNA and modified siRNA.
FIG. 14 shows the inhibition of HBV particle expression by partially screened siRNA and modified siRNA.
FIG. 15 shows the results of detection of the expression level of HBV Core PARTICLES DNA by the partially selected siRNA and the modified siRNA.
FIG. 16 shows the results of detection of expression levels of HBV cccDNA by partially screened siRNA and modified siRNA.
FIG. 17 shows the inhibition effect of chemically modified siRNA molecules on target genes, and the comparison of normal siRNA molecules with unmodified identical sequences. The inhibition efficiency of the modified siRNA molecules on target genes is reduced, but still exceeds 80%, and most of the modified siRNA molecules are kept above 90%.
Figure 18 shows half maximal effector concentration (EC 50) data for partially modified siRNA in both 293T and a549 cells. EC 50 of most modified sirnas was increased compared to unmodified sirnas.
Detailed Description
These and other aspects of the invention are described in more detail below.
Definition:
An "oligonucleotide" as used herein refers to a chemically modified or unmodified nucleic acid molecule (RNA or DNA) that is less than 100 nucleotides in length (e.g., less than 50, 30, or 25 nucleotides). It may be siRNA, microRNA, anti-microRNA, microRNA mimetic, dsRNA, ssRNA, aptamer, triplex forming oligonucleotides. In one embodiment, the oligonucleotide is an RNAi agent.
As used herein, an "siRNA molecule" or "RNAi molecule" is a duplex oligonucleotide, which is a short double-stranded polynucleotide that, when introduced into a cell, can interfere with the expression of a gene in the cell. For example, siRNA molecules are targeted to bind to complementary nucleotide sequences in a single stranded target RNA molecule. In general, when an siRNA molecule is recognized by a particular nucleotide sequence, that sequence refers to the sense strand of the duplex molecule. The ribonucleotide or ribonucleotides comprising the molecule can be chemically modified by techniques known in the art. In addition to being modified at the level of one or more oligonucleotides, the backbone of the oligonucleotide may also be modified. Other modifications include the use of small molecules (e.g., glycosyl), amino acids, peptides, cholesterol, and other macromolecules coupled to siRNA molecules.
"Peptide docking vector" (PDoV) refers to a synthetic peptide having a defined sequence that contains multiple conjugation sites to allow conjugation to one or more targeting ligands, as well as one or more oligonucleotides. It contains a functional group (e.g., a hydrophobic chain or a pH sensitive residue) that aids in the release of the oligonucleotide payload that is encapsulated inside the endosome of the cell after delivery of the conjugated PDoV to the cell.
By "inhibition of expression" is meant the absence or significant reduction in the level of protein and/or mRNA expression products from the target gene. Inhibition need not be absolute, but may be partial, sufficient to produce a detectable or observable change due to administration of the siRNA molecules of the invention. Inhibition may be measured by analysis of a decrease in the level of mRNA and/or protein product corresponding to the gene targeted by the siRNA molecule in the cell, as compared to cells treated without the siRNA molecule, as low as 10%, 50% or absolute (i.e., 100%) inhibition. Inhibition can be determined by examining the extrinsic properties of the cell or organism, i.e., quantitative and/or qualitative phenotypes, and can also include assessing viral load following administration of the siRNA molecules of the invention.
SiRNA molecules can target active genes directly with minimal off-target events. An "off-target event" refers to a significant decrease in the expression of a particular nucleic acid that is not a target of an siRNA molecule. For HBV infection, the minimal off-target event provides a unique opportunity to meet the clinical therapeutic needs of HBV that have not been met. Thus, in one aspect of the present invention, there is provided an HBV DNA-specific RNA interference formulation for inhibiting expression of one or more target sequences in an HBV gene.
PDoV structure
The present invention provides an endosome escape peptide (PDoV) that enhances escape of macromolecular substances (e.g., siRNA molecules) into the cytoplasm in a non-toxic manner. Various examples of PDoV platforms are shown in fig. 4. In PDoV, the endosome escape peptide serves as both a docking site linker for RNA and a targeting ligand, and multiple RNA molecules can be coupled to the same structure to achieve co-introduction of siRNA molecules against different target mrnas, thereby providing synergistic benefits for silencing genes associated with multiple diseases. Histidine and lysine rich polypeptides or histidine and lysine rich linear peptides have been shown to be effective cell penetration and endosomal release agents in RNA drug delivery. The peptide contains a histidine-rich domain in which the imidazole ring of the histidine residue is protonated at lower pH values (pH <.about.6) and acts as a proton sponge in the endosome, resulting in cleavage of the endosome lipid bilayer, releasing the RNA molecule. The conjugation site on PDoV is described in detail in the lower section.
FIG. 2 is a schematic representation of [ GalNAc ] peptide docking vector design. Trivalent GalNAc ligands are covalently conjugated to one docking site ① of the peptide, while oligonucleotides or sirnas are conjugated to the other or both docking sites ③, respectively.
RNAi formulations
HBV has a small circular DNA genome, about 3.2kb long, containing 4 genes with partially overlapping Open Reading Frames (ORFs). These ORFs encode polymerase proteins (P genes); core antigen and e antigen (C gene); large, medium, small surface antigen proteins (S genes); and protein X (X gene). The structure of HBV is schematically shown in FIG. 1, and NCBI accession number is NC_003977.2.HBV genome exists in two forms: circular covalent closed DNA (cccDNA) and relaxed circular DNA (rcDNA). The genetic organization of HBV viral DNA is highly conserved among all viral subtypes, well suited to the use of gene silencing techniques to inhibit its replication.
The siRNA molecules designed and selected according to the present invention can target the sequence of at least seven HBV strains to cover as many HBV mutants as possible. siRNA molecules use two lengths: 21 base pair length (19+dTdT) and 25 base pair length.
Double-stranded siRNA may be unmodified, or may be chemically modified at the 2' -OCH 3, 2' -F, or 2' -OMe at the 2' position and/or-p2=s, -p2=o at the 5' position. Other chemical modifications, such as pegylation or lipid functionalization, can be used to improve the overall stability and bioavailability of RNAi.
The siRNA molecule is selected to inhibit expression of HBV nucleic acid sequences. Preferably, the HBV target gene is a nucleic acid sequence expressed as a polymerase (P) gene. Thus, in one embodiment, the siRNA formulation inhibits expression of one or more target sequences in HBV viral P gene. The HBV genome has overlapping open reading frames and thus, a particular sequence of a targeted polymerase gene may also target other identical sequences in the overlapping gene. Thus, the siRNA molecules of the invention are capable of targeting multiple genes with a single effector sequence. Thus, in each of the embodiments described below, at least the polymerase gene is targeted. Since genes of HBV genome overlap between four ORFs in most regions, the siRNA molecules of the present invention are also able to target C, S and X genes of HBV. As shown in Table 1, 52 siRNA's were designed according to the present invention for 11 HBV strains.
TABLE 1 siRNA sequences conservatively designed for HBV viral genes:
targeting ligands
The targeting ligand moiety disclosed herein is N-acetyl-galactosamine (GalNAc), galactose, galactosamine, N-formyl-galactosamine, N-propionyl-galactosamine or N-butyryl galactosamine. The targeting ligand may be covalently coupled to the peptide. One, two or three targeting ligands may be used and may have a structure as shown in fig. 5. In this structure, coupling to the remainder of PDoV is achieved by reaction between the cysteine residue of the peptide and the maleimide on the targeting ligand.
The invention further couples the engineered siRNA molecules to GalNAc to form GalNAc-siRNA conjugates, wherein the GalNAc ligand can bind to hepatocyte-expressed asialoglycoprotein receptor (Asialoglycoprotein receptor, ASGPR) and target the siRNA to hepatocytes. Studies have shown that each hepatocyte expresses 0.5-1 million copies of ASGPR, has high conservation among species, and the receptors are rapidly internalized, can circulate to the surface of the hepatocyte in about 15min, and are ideal receptors for targeting the liver. Thus, galNAc-siRNA can be used to develop a variety of liver-targeted delivery nucleic acid drugs, including siRNA molecules that inhibit replication of hepatitis b virus. After GalNAc binds to ASGPR highly expressed on hepatocytes, siRNA molecules are delivered into cells by rapid endocytosis, exerting efficient and specific viral gene expression silencing effects.
Endosome release docking peptides
The peptide docking vector (PDoV) has one ligand conjugation site and multiple oligonucleotide sites. PDoV a peptide backbone having the general structure: (HnKm) oXpYq, a plurality of repeat units having histidine (H), lysine (K) and functional units X and Y (wherein X or Y is an amino acid or amino acid derivative), wherein: n=1 to 10; m=1 to 10; o=1-10, p=1-5, q=1-5. HK repeat units have been shown to have a function to promote endosomal release. Lysine residues or functional units X can be used as a docking site for ligand conjugation, while Y provides a docking site for oligonucleotide conjugation through a different covalent bond. Fig. 3 shows a schematic diagram of how PDoV can be combined. For example, site ① is only capable of reacting in the presence of a ligand such as GalNAc or other targeting ligand; and site ③ can only be conjugated to oligonucleotides and siRNAs under selected conditions.
Using PDoV coupled with siRNA, the liver targeting function of GalNAc can be exploited to deliver siRNA into hepatocytes. It is known that siRNA is released from endosomes into cytoplasm after entering cells, and thus, it is only released from endosomes to exert a nucleic acid interfering effect. Studies have shown that the efficiency of release of GalNAc-siRNA from endosomes into the cytoplasm is very low (even below 0.1%), and therefore, if the endosome release efficiency can be improved, the therapeutic effect will be greatly improved. PDoV the butt joint peptide contains histidine component, histidine is a basic amino acid, unsaturated nitrogen atom on imidazole ring in molecular structure can accept external environment proton under weak acid condition, and generates protonation, so that histidine oligopeptide generates hydrophobic to hydrophilic conversion. This transformation facilitates endosomal expansion, releases siRNA molecules, exerts RNAi effects, and inhibits expression of HBV genes, thus blocking viral life cycle.
Specific example 1 selection of targets based on HBV genomic structure and protein function
The invention selects HBV gene conservation sequence as candidate target area of siRNA design, thereby obtaining siRNA molecule capable of inhibiting all types of HBV strains. Eleven HBV mutants were included together for siRNA molecule design (NCBI accession nos. :NC_003977.2,KY003230.1,AB933282.1,KR013949.1,KR014081.1,AF090840.1,AF325900.1,U95551.1,X02763.1,X70185.1 and X75311.1, respectively). These strains were chosen because they covered as many HBV mutants as possible.
Example 2 siRNA design targeting HBV key genes
The open reading frame P encodes the reverse transcriptase domain of HBV polymerase, which represents the target of antiviral agents (e.g., nucleoside/nucleotide analogs and acyclic nucleotide analogs). Since this reading frame overlaps with the S reading frame, inhibition of the reverse transcriptase domain can also simultaneously inhibit the expression of the S gene.
The open reading frame S encodes three envelope proteins (large, medium and small envelope proteins, respectively) that determine viral assembly and viral attachment to hepatocytes. The large protein is the substrate to which the viral receptor attaches, the function of the medium protein is not known, and the small protein is commonly referred to as HBsAg or australian antigen. Small, medium and large proteins were detected as HBsAg. The S region is a hepatocyte binding site, also associated with occult HBV status. Furthermore, the S region affects HBsAg expression, synthesis and secretion. The S region deletion inhibits the progression of liver disease.
The open reading frame C may encode one structure of two proteins, HBcAg being the nucleocapsid forming protein and HBeAg being the secreted protein. HBeAg is a marker of HBV replication and infectivity. In the natural course of HBV chronic infection, the loss of HBeAg expression and the appearance of antibodies thereto (anti-HBe) generally represent the end of viral replication and the regression of hepatitis. Mutations in the pre-core and core regions (core) lead to chronic hepatitis B that is HBeAg negative, accompanied by anti-HBe, where replicative infection is still continuing and HBV viral DNA is still detectable.
Open reading frame X encodes a number of multifunctional nonstructural proteins whose function is not yet clear and has been shown to play a role in establishing infection and viral replication. In recent years, it has been speculated that the X gene plays an important role in HBV carcinogenesis.
The basic principle of the siRNA design of the present invention is that the designed siRNA sequence covers as many HBV mutants as possible among 7 selected HBV strains. The invention selects siRNA molecules with two base pair lengths (mer): 21mer (19+dTdT) and 25mer.
The siRNA molecule according to the present invention can inhibit the expression of HBV viral nucleic acid sequences. Preferably, the HBV target gene is the polymerase (P) gene. Thus, in one embodiment, the siRNA molecule inhibits expression of one or more target sequences in the HBV viral P gene. The HBV genome has overlapping open reading frames. Thus, targeting a specific sequence of the polymerase P gene can also target the same sequence in other genes in the overlap region. Thus, the siRNA molecules of the invention are capable of targeting multiple genes with a single effector sequence. However, in each preferred embodiment, at least the polymerase P gene is targeted. Since genes of HBV genome overlap between four ORFs in most regions, the siRNA molecules of the present invention can also target C, S and X genes of HBV.
Example 3 binding of siRNA to multivalent GalNAc (galactosamine)
The siRNA can bind to group 3 (multivalent GalNAc-linker) of PDoV via the 2 'and 5' nucleotide positions, as well as via the 5 'and 2' nucleotide positions shown below.
Specific example 4 screening for potent anti-HBV siRNA molecules based on cell culture
To identify the most effective siRNA to silence HBV genes in A549 and 293T cell culture experiments, experiments were performed using recombinant psiCHECK-2 plasmid with DNA fragment of HBV gene sequence. HepG2 cells were infected with authentic HBV to examine the anti-HBV infection activity of the selected siRNA.
To investigate the role of selected siRNA in degrading targeted HBV genes, a dual luciferase reporter vector psiCHECK-2 with surface, core, E, X, polymerase, reverse transcriptase gene fragments was used. The psiCHECK-2 vector is intended to provide a quantitative and rapid method for the initial optimization of RNA interference (RNAi). These vectors are capable of monitoring changes in target gene expression fused to a reporter gene. In the study, DNA fragments of HBV genome were synthesized first and cloned into multiple cloning sites of psiCHECK-2 vector. The vector takes the Renilla luciferase as a main reporter gene, and the siRNA targeting gene is positioned at the downstream of a Renilla luciferase gene translation termination codon.
A549 or 293T cells were seeded in 96-well plates and incubated for 12 hours. Reporter plasmid (recombinant vector) psi-P & S & X, psi-P & X & C0 uORF and siRNA candidate gene were co-transfected into A549 or 293T cells with Lipofectamine 2000 in DMEM without fetal bovine serum. Blank psi plasmid vector served as negative control. After 6 hours of transfection, the medium was replaced with DMEM supplemented with 10% fetal bovine serum. The activity of firefly luciferase and Renilla luciferase per well was measured using a double luciferase kit at 18, 24, 36 and 48 hours post transfection. The siRNA candidate significantly reduced luciferase activity, indicating that siRNA can significantly inhibit expression of HBV target genes. These candidate genes were selected for in vitro HBV infection assays.
The effect of the designed siRNA on inhibition of target gene expression in both cells was determined by the above-described fluorescent quantitative expression analysis method (a 549 or 293T, see fig. 9 and 10). The results indicate that multiple siRNAs can inhibit the expression of target genes, including NTS00042-21-1#、NTS00042-21-3#、NTS00042-21-6#、NTS00042-21-7#、NTS00042-21-9#、NTS00042-21-10#、NTS00042-21-11#、NTS00042-21-22#、NTS00042-25-1#、NTS00042-25-2#、NTS00042-25-3#、NTS00042-25-4#、NTS00042-25-6#、NTS00042-25-7#、NTS00042-25-16# and the like. The siRNA with the optimal inhibition effect, such as NTS00042-21-1#, NTS00042-21-3#, NTS00042-21-11#, NTS00042-25-3# has obvious inhibition effect on target genes, and reaches 90% or even more than 95%.
Further, the half maximal effect concentration (concentration causing 50% gene silencing effect, EC 50) of the part of siRNA molecules, NTS00042-21-1#、NTS00042-21-3#、NTS00042-21-6#、NTS00042-21-7#、NTS00042-21-9#、NTS00042-21-11#、NTS00042-21-22#、NTS00042-25-1#、NTS00042-25-3#、NTS00042-25-6#、NTS00042-25-16#siRNA molecules, inhibiting target gene expression in both 293T and a549 cells was determined, and it can be seen from fig. 11 that EC 50 of all siRNA is less than 10pg/μl, wherein the two relatively better effective sirnas are: EC 50 of siRNA # 21-6 (P gene and S gene targeting HBV) in 293T and A549 cells was 0.3 pg/. Mu.L (FIG. 11A) and 1.1 pg/. Mu.L (FIG. 11D), respectively; EC 50 in 293T and A549 cells of siRNA # 21-11 (P gene targeting HBV) was 0.7 pg/. Mu.L (FIG. 11A) and 0.9 pg/. Mu.L (FIG. 11D), respectively. These results indicate that the selected siRNA of the present invention exhibits good inhibitory activity against the target gene, and the concentration of drug acting is much less than other conventional drug molecules.
Specific example 5 preliminary screening of anti-HBV siRNA molecules inhibiting Gene expression of intracellular HBV
The siRNA with better inhibition effect on target gene in the part screened in the example 4, namely NTS00042-21-3#(21-3)、NTS00042-21-6#(21-6)、NTS00042-21-9#(21-9)、NTS00042-21-11#(21-11)、NTS00042-21-22#(21-22)、NTS00042-25-3#(25-3)、NTS00042-25-16#(25-16),, is modified by 2' -O-methyl to obtain modified siRNA molecules, namely 21-3-x, 21-6-x, 21-9-x, 21-11-x, 21-22-x, 25-3-x and 25-16-x.
This example was used to examine the cell viability inhibition effect of part of the siRNA molecules selected in example 4, namely NTS00042-21-1#、NTS00042-21-3#、NTS00042-21-6#、NTS00042-21-7#、NTS00042-21-9#、NTS00042-21-11#、NTS00042-21-22#、NTS00042-25-1#、NTS00042-25-3#、NTS00042-25-6#、NTS00042-25-16#(, namely, siRNA molecules represented by 21-1, 21-3, 21-6, 21-7, 21-9, 21-11, 21-22, 25-1, 25-3, 25-6, 25-16), and modified siRNA molecules on hepatoma cell line HepAD 38. The specific experimental steps are as follows:
HepAD38 cells in the logarithmic growth phase were seeded at 1X 10 4 cells/well in 96-well plates with 100. Mu.L of cell suspension per well and cultured overnight. Transfection following LipofectaminTM3000,3000 instructions transfection, siRNA at 50 nM/well, 3 duplicate wells per sample were prepared, 6h later, and the solution was changed and transfected for 96h. Cell supernatants were collected, and the levels of HBsAg gene expression in the cell supernatants were detected according to the procedure of ELISA detection kit (Shanghai Kochia Biotechnology Co., ltd.) using NC-siRNA as a negative control.
As shown in FIG. 12, it can be seen that the selected siRNA molecules can significantly inhibit the expression of the HBsAg gene in the supernatant of HepAD38 cells compared with NC-siRNA, wherein the siRNA molecules represented by 21-7, 21-9, 21-11, 25-1, 25-6, 21-22, 25-16, 21-9-x and 21-11-x have higher inhibition rate on the target gene, and then re-screening is performed.
Specific example 6 rescreening Gene expression and replication of anti-HBV siRNA molecules on liver cancer cell lines
This example was used to examine the cell viability inhibition effect of the siRNA molecules screened in example 5 on liver cancer cell lines. The specific experimental steps are as follows:
HepAD38 cells in the logarithmic growth phase were seeded at 2X 10 5 cells/well in 6-well plates with 2mL of cell suspension per well and cultured overnight. Transfection following LipofectaminTM3000,3000 instructions transfection, siRNA at 50 nM/well, 3 duplicate wells per sample were prepared, and after 6h, the solution was changed and transfected for 7 days.
(1) Detecting the expression level of HBsAg and HBeAg genes:
Cell supernatants were collected, and the levels of HBsAg and HBeAg gene expression in the cell supernatants were detected according to the procedure of ELISA detection kit (Shanghai Kochia Biotechnology Co., ltd.) and NC-siRNA was used as a negative control.
As shown in FIG. 13A and FIG. 13B, the results of the detection of HBeAg and HBsAg show that the siRNA molecules indicated by 25-16 and 21-11-x have high inhibition rate to the target gene, the inhibition rate to the HBeAg gene exceeds 40% and the inhibition rate to the HBsAg exceeds 50%.
(2) Real-time quantitative PCR detection of HBV DNA copy number:
HBV DNA copy number was detected by real-Time fluorescent quantitative PCR (Quantitative Real-Time PCR), and cell supernatants were added to the prepared PCR tubes while NC-siRNA control group was established. The following 10. Mu.L reaction system was established: 4. Mu.L of template, 5. Mu.L of SYBR premix Ex Taq, 0.5. Mu.L of PCR forward primer (10. Mu.M), 0.5. Mu.L of PCR reverse primer (10. Mu.M). Amplification was performed according to the procedure shown in Table 2, in which PCR primers for amplification and GAPDH as an internal reference gene are shown in Table 3.
Pcr program settings:
TABLE 3 PCR primers
The results of the detection of HBV DNA copy number in cell supernatant are shown in FIG. 14, and it can be seen from the graph that compared with NC-siRNA, the selected siRNA can significantly inhibit the expression of HBV particles in supernatant, the inhibition rate is 35.437% -81.225%, the inhibition rate except 21-11 is 35.437%, and the inhibition rate of the rest samples is over 40.000%.
(3) Real-time quantitative PCR detection of expression level of HBV Core PARTICLES DNA:
HBV Core PARTICLES DNA was extracted from cells for real-time fluorescent quantitative PCR detection, core PARTICLES DNA was added to the prepared PCR reaction tube while NC-siRNA control group was established. The following 10. Mu.L reaction system was established: 4. Mu.L of template, 5. Mu.L of SYBR premix Ex Taq, 0.5. Mu.L of PCR forward primer (10. Mu.M), 0.5. Mu.L of PCR reverse primer (10. Mu.M). Amplification was performed according to the procedure shown in Table 2, in which PCR primers for amplification and GAPDH as an internal reference gene are shown in Table 3.
As shown in FIG. 15, the results of the detection of the expression level of HBV Core PARTICLES DNA show that the selected siRNA can significantly inhibit HBV Core particles expression compared with NC-siRNA, wherein samples 21-7, 21-9, 25-16, 21-9-x and 21-11-x have significant differences compared with NC-siRNA of the control sample.
(4) Real-time quantitative PCR detection of expression level of HBV cccDNA:
The cccDNA is extracted from the cells for real-time fluorescence quantitative PCR detection, and is added into a prepared PCR reaction tube, and an NC-siRNA control group is established. The following 10. Mu.L reaction system was established: 4. Mu.L of template, 5. Mu.L of SYBR premix Ex Taq, 0.5. Mu.L of PCR forward primer (10. Mu.M), 0.5. Mu.L of PCR reverse primer (10. Mu.M). Amplification was performed according to the procedure shown in Table 2, in which PCR primers for amplification and GAPDH as an internal reference gene are shown in Table 3.
As shown in FIG. 16, the results of the detection of the expression level of HBV cccDNA showed that the selected siRNA significantly inhibited the expression of HBV cccDNA compared with NC-siRNA, and that 25-6 and 21-11-x showed the best inhibition.
Example 7 silencing of HBV target Gene by chemically modified siRNA molecules
The invention further adopts chemical groups such as 2' -O-methyl and the like to modify siRNA so as to improve the stability of siRNA and the efficiency of entering cells and reduce Off-Target effect. The inhibition effect of these 2' -O-methyl modified siRNAs on target genes was measured in two cells, and the results show (FIG. 17) that the inhibition activity of the modified siRNAs on target genes was reduced, but the inhibition efficiency on gene expression was 80%; taking 21-6# siRNA with good inhibition effect as an example, in 293T cells, the inhibition efficiency of unmodified siRNA reaches 98%, the inhibition rate of modified siRNA is 97% (FIG. 17A), and in A549 cells, the inhibition efficiency of modified siRNA is reduced from 96% to 95% (FIG. 17C).
As a result of measurement of EC 50 data of modified siRNA, it can be seen from fig. 18 that EC 50 of most modified siRNA was increased (consistent with the decrease in inhibition efficiency of target gene) compared to EC 50 of unmodified siRNA, for example, no. 21-6# siRNA was 0.3pg/μl in 293T cells, and the modified EC 50 was increased to 0.9pg/μl (fig. 18A). There were also individual siRNAs with modified EC 50 reduced, e.g., siRNA No. 21-22 (targeting HBV X gene), with unmodified EC 50 in 293T and A549 cells at 3.9 and 2.64 pg/. Mu.L, respectively, and modified siRNA with EC 50 reduced to 0.67 and 0.3 pg/. Mu.L, respectively (FIGS. 18B and 18D). In general, the EC 50 concentration of the modified siRNA is still lower than 10 pg/MuL, and good inhibition efficiency is still maintained.
While certain embodiments of the compositions and methods have been described herein, and many details have been set forth for purposes of illustration, it will be apparent to those of ordinary skill in the art that the compositions and methods are susceptible to additional embodiments and that certain details can be varied from the embodiments described herein without departing from the basic principles of the disclosure.
Sequence listing
<110> Sanno pharmaceutical Co
Shenno biomedical technology (Guangzhou) Co., ltd
<120> A pharmaceutical structure and pharmaceutical composition capable of targeting hepatitis B virus
<150> 63/140,232
<151> 2021-01-21
<150> 2021102899267
<151> 2021-03-18
<160> 106
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<400> 17
guugacaaga auccucaca 19
<210> 18
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 18
ugugaggauu cuugucaac 19
<210> 19
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 19
gacaagaauc cucacaaua 19
<210> 20
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 20
uauugugagg auucuuguc 19
<210> 21
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 21
cuagacucgu gguggacuu 19
<210> 22
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 22
aaguccacca cgagucuag 19
<210> 23
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 23
cgcagucccc aaccuccaa 19
<210> 24
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 24
uuggagguug gggacugcg 19
<210> 25
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 25
ccaaccuccu guccuccaa 19
<210> 26
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 26
uuggaggaca ggagguugg 19
<210> 27
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 27
guccugguua ucgcuggau 19
<210> 28
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 28
auccagcgau aaccaggac 19
<210> 29
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 29
ccucuucauc cugcugcua 19
<210> 30
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 30
uagcagcagg augaagagg 19
<210> 31
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 31
cugcugcuau gccucaucu 19
<210> 32
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 32
agaugaggca uagcagcag 19
<210> 33
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 33
gacgcaaccc ccacuggcu 19
<210> 34
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 34
agccaguggg gguugcguc 19
<210> 35
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 35
cucucguccc cuucuccgu 19
<210> 36
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 36
acggagaagg ggacgagag 19
<210> 37
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 37
gagauuaggu uaaaggucu 19
<210> 38
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 38
agaccuuuaa ccuaaucuc 19
<210> 39
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 39
gcauaaauug gucugcgca 19
<210> 40
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 40
ugcgcagacc aauuuaugc 19
<210> 41
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 41
gcgcaccagc accaugcaa 19
<210> 42
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 42
uugcauggug cuggugcgc 19
<210> 43
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 43
ccucugccua aucaucucu 19
<210> 44
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 44
agagaugauu aggcagagg 19
<210> 45
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 45
cacuuccgga aacuacugu 19
<210> 46
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 46
acaguaguuu ccggaagug 19
<210> 47
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 47
cccucaggcu cagggcaua 19
<210> 48
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 48
uaugcccuga gccugaggg 19
<210> 49
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 49
cgaggcaggu ccccuagaa 19
<210> 50
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 50
uucuagggga ccugccucg 19
<210> 51
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 51
gggaaucuca auguuagua 19
<210> 52
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 52
uacuaacauu gagauuccc 19
<210> 53
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 53
ggaaucucaa uguuaguau 19
<210> 54
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 54
auacuaacau ugagauucc 19
<210> 55
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 55
caucacauca ggauuccua 19
<210> 56
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 56
uaggaauccu gaugugaug 19
<210> 57
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 57
gggcuuuccc ccacuguuu 19
<210> 58
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 58
aaacaguggg ggaaagccc 19
<210> 59
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 59
ccuauugauu ggaaaguau 19
<210> 60
<211> 19
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 60
auacuuucca aucaauagg 19
<210> 61
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 61
gaacauggag aacaucacau cagga 25
<210> 62
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 62
uccugaugug auguucucca uguuc 25
<210> 63
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 63
ggagaacauc acaucaggau uccua 25
<210> 64
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 64
uaggaauccu gaugugaugu ucucc 25
<210> 65
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 65
gaacaucaca ucaggauucc uagga 25
<210> 66
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 66
uccuaggaau ccugauguga uguuc 25
<210> 67
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 67
cuuguugaca agaauccuca caaua 25
<210> 68
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 68
uauugugagg auucuuguca acaag 25
<210> 69
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 69
cagagucuag acucguggug gacuu 25
<210> 70
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 70
aaguccacca cgagucuaga cucug 25
<210> 71
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 71
gagucuagac ucguggugga cuucu 25
<210> 72
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 72
agaaguccac cacgagucua gacuc 25
<210> 73
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 73
gucuagacuc gugguggacu ucucu 25
<210> 74
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 74
agagaagucc accacgaguc uagac 25
<210> 75
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 75
gcagucccca accuccaauc acuca 25
<210> 76
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 76
ugagugauug gagguugggg acugc 25
<210> 77
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 77
guccccaacc uccaaucacu cacca 25
<210> 78
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 78
uggugaguga uuggagguug gggac 25
<210> 79
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 79
ccaaccucca aucacucacc aaccu 25
<210> 80
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 80
agguugguga gugauuggag guugg 25
<210> 81
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 81
guccugguua ucgcuggaug ugucu 25
<210> 82
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 82
agacacaucc agcgauaacc aggac 25
<210> 83
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 83
cucuucaucc ugcugcuaug ccuca 25
<210> 84
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 84
ugaggcauag cagcaggaug aagag 25
<210> 85
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 85
guugcccguu uguccucuaa uucca 25
<210> 86
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 86
uggaauuaga ggacaaacgg gcaac 25
<210> 87
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 87
gugcacuucg cuucaccucu gcacg 25
<210> 88
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 88
cgugcagagg ugaagcgaag ugcac 25
<210> 89
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 89
gcauggagac caccgugaac gccca 25
<210> 90
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 90
ugggcguuca cgguggucuc caugc 25
<210> 91
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 91
cuguaggcau aaauuggucu gcgca 25
<210> 92
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 92
ugcgcagacc aauuuaugcc uacag 25
<210> 93
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 93
gcauaaauug gucugcgcac cagca 25
<210> 94
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 94
ugcuggugcg cagaccaauu uaugc 25
<210> 95
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 95
gccuugggug gcuuuggggc augga 25
<210> 96
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 96
uccaugcccc aaagccaccc aaggc 25
<210> 97
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 97
gaauuuggag cuacugugga guuac 25
<210> 98
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 98
guaacuccac aguagcucca aauuc 25
<210> 99
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 99
cuccaguuca ggaacaguaa acccu 25
<210> 100
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 100
aggguuuacu guuccugaac uggag 25
<210> 101
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 101
ccugagcauu gcucaccuca ccaua 25
<210> 102
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 102
uauggugagg ugagcaaugc ucagg 25
<210> 103
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 103
ccaccaaucg gcagucagga aggca 25
<210> 104
<211> 25
<212> RNA
<213> Synthesis (rengonghecheng)
<400> 104
ugccuuccug acugccgauu ggugg 25
<210> 105
<211> 1314
<212> DNA
<213> Synthesis (rengonghecheng)
<400> 105
ctcgagcttg gccaaaattc gcagtcccca acctccaatc actcaccaac ctcttgtcct 60
ccaacttgtc ctggttatcg ctggatgtgt ctgcggcgtt ttatcatctt cctcttcatc 120
ctgctgctat gcctcatctt cttgttggtt cttctggact atcaaggtat gttgcccgtt 180
tgtcctctaa ttccaggatc ctcaacaacc agcacgggac catgccggac ctgcatgact 240
actgctcaag gaacctctat gtatccctcc tgttgctgta ccaaaccttc ggacggaaat 300
tgcacctgta ttcccatccc atcatcctgg gctttcggaa aattcctatg ggagtgggcc 360
tcagcccgtt tctcctggct cagtttacta gtgccatttg ttcagtggtt cgtagggctt 420
tcccccactg tttggctttc agttatatgg atgatgtggt attgggggcc aagtctgtac 480
agcatcttga gtcccttttt accgctgtta ccaattttct tttgtctttg ggtatacatt 540
taaaccctaa caaaacaaag agatggggtt actctctaaa ttttatgggt tatgtcattg 600
gatgttatgg gtccttgcca caagaacaca tcatacaaaa aatcaaagaa tgttttagaa 660
aacttcctat taacaggcct attgattgga aagtatgtca acgaattgtg ggtcttttgg 720
gttttgctgc cccttttaca caatgtggtt atcctgcgtt gatgcctttg tatgcatgta 780
ttcaatctaa gcaggctttc actttctcgc caacttacaa ggcctttctg tgtaaacaat 840
acctgaacct ttaccccgtt gcccggcaac ggccaggtct gtgccaagtg tttgctgacg 900
caacccccac tggctggggc ttggtcatgg gccatcagcg catgcgtgga accttttcgg 960
ctcctctgcc gatccatact gcggaactcc tagccgcttg ttttgctcgc agcaggtctg 1020
gagcaaacat tatcgggact gataactctg ttgtcctatc ccgcaaatat acatcgtttc 1080
catggctgct aggctgtgct gccaactgga tcctgcgcgg gacgtccttt gtttacgtcc 1140
cgtcggcgct gaatcctgcg gacgaccctt ctcggggtcg cttgggactc tctcgtcccc 1200
ttctccgtct gccgttccga ccgaccacgg ggcgcacctc tctttacgcg gactccccgt 1260
ctgtgccttc tcatctgccg gaccgtgtgc acttcgcttc acctctgcgg ccgc 1314
<210> 106
<211> 1896
<212> DNA
<213> Synthesis (rengonghecheng)
<400> 106
ctcgaggcac gtcgcatgga gaccaccgtg aacgcccacc aaatattgcc caaggtctta 60
cataagagga ctcttggact ctcagcaatg tcaacgaccg accttgaggc atacttcaaa 120
gactgtttgt ttaaagactg ggaggagttg ggggaggaga ttaggttaaa ggtctttgta 180
ctaggaggct gtaggcataa attggtctgc gcaccagcac catgcaactt tttcacctct 240
gcctaatcat ctcttgttca tgtcctactg ttcaagcctc caagctgtgc cttgggtggc 300
tttggggcat ggacatcgac ccttataaag aatttggagc tactgtggag ttactctcgt 360
ttttgccttc tgacttcttt ccttcagtac gagatcttct agataccgcc tcagctctgt 420
atcgggaagc cttagagtct cctgagcatt gttcacctca ccatactgca ctcaggcaag 480
caattctttg ctggggggaa ctaatgactc tagctacctg ggtgggtgtt aatttggaag 540
atccagcgtc tagagaccta gtagtcagtt atgtcaacac taatatgggc ctaaagttca 600
ggcaactctt gtggtttcac atttcttgtc tcacttttgg aagagaaaca gttatagagt 660
atttggtgtc tttcggagtg tggattcgca ctcctccagc ttatagacca ccaaatgccc 720
ctatcctatc aacacttccg gagactactg ttgttagacg acgaggcagg tcccctagaa 780
gaagaactcc ctcgcctcgc agacgaaggt ctcaatcgcc gcgtcgcaga agatctcaat 840
ctcgggaatc tcaatgttag tattccttgg actcataagg tggggaactt tactgggctt 900
tattcttcta ctgtacctgt ctttaatcct cattggaaaa caccatcttt tcctaatata 960
catttacacc aagacattat caaaaaatgt gaacagtttg taggcccact cacagttaat 1020
gagaaaagaa gattgcaatt gattatgcct gccaggtttt atccaaaggt taccaaatat 1080
ttaccattgg ataagggtat taaaccttat tatccagaac atctagttaa tcattacttc 1140
caaactagac actatttaca cactctatgg aaggcgggta tattatataa gagagaaaca 1200
acacatagcg cctcattttg tgggtcacca tattcttggg aacaagatct acagcatggg 1260
gcagaatctt tccaccagca atcctctggg attctttccc gaccaccagt tggatccagc 1320
cttcagagca aacaccgcaa atccagattg ggacttcaat cccaacaagg acacctggcc 1380
agacgccaac aaggtaggag ctggagcatt cgggctgggt ttcaccccac cgcacggagg 1440
ccttttgggg tggagccctc aggctcaggg catactacaa actttgccag caaatccgcc 1500
tcctgcctcc accaatcgcc agtcaggaag gcagcctacc ccgctgtctc cacctttgag 1560
aaacactcat cctcaggcca tgcagtggaa ttccacaacc ttccaccaaa ctctgcaaga 1620
tcccagagtg agaggcctgt atttccctgc tggtggctcc agttcaggaa cagtaaaccc 1680
tgttctgact actgcctctc ccttatcgtc aatcttctcg aggattgggg accctgcgct 1740
gaacatggag aacatcacat caggattcct aggacccctt ctcgtgttac aggcggggtt 1800
tttcttgttg acaagaatcc tcacaatacc gcagagtcta gactcgtggt ggacttctct 1860
caattttcta gggggaacta ccgtgtgtgc ggccgc 1896
Claims (1)
1. A nucleic acid molecule for targeted inhibition of HBV, wherein the nucleic acid molecule is a double-stranded siRNA comprising a sense strand consisting of CUAGACUCGUGGUGGACUUdTdT sequences and an antisense strand consisting of AAGUCCACCACGAGUCUAGdTdT sequences, or wherein the nucleic acid molecule is a modified siRNA obtained by 2' -O-methyl modification of the double-stranded siRNA.
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