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WO2004078181A1 - Procedes bases sur l'interference d'arn et compositions destinees a l'inhibition de l'expression genique hbv - Google Patents

Procedes bases sur l'interference d'arn et compositions destinees a l'inhibition de l'expression genique hbv Download PDF

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Publication number
WO2004078181A1
WO2004078181A1 PCT/CN2003/000718 CN0300718W WO2004078181A1 WO 2004078181 A1 WO2004078181 A1 WO 2004078181A1 CN 0300718 W CN0300718 W CN 0300718W WO 2004078181 A1 WO2004078181 A1 WO 2004078181A1
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sequence
seq
dsrna
cell
hbv
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PCT/CN2003/000718
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English (en)
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Duanqing Pei
Xin Zhao
Fangxun Wang
Yong Guo
Hongying Yin
Jing Cheng
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Capital Biochip Company, Ltd.
Tsinghua University
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Priority to AU2003258452A priority Critical patent/AU2003258452A1/en
Publication of WO2004078181A1 publication Critical patent/WO2004078181A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • RNA interference RNA interference
  • HBV infection is one of the most common viral diseases to human, and is a major cause of chronic viral hepatitis and liver cirrhosis distributed world widely. There are estimated 200-300 million people infected with HBV worldwide. In Southeast Asia and parts of Africa, the carriers are up to 20% of the population.
  • HBV is a partially double-stranded circular DNA virus of 42 nm diameter and belongs to a group of hepatotropic DNA virus (hepadnaviruses).
  • the virus contains an outer coat and an inner core.
  • the viral coat is composed mainly of hepatitis B surface antigen (HBsAg), which plays a central role in the diagnosis of HBV infection.
  • the inner core contains hepatitis B core antigen (HBcAg), DNA polymerase/reverse transcriptase and the genome (Table 1).
  • the genome is 3,200 bp (Blum, H.E., von
  • HBV infection is a major cause of chronic viral hepatitis and liver cirrhosis worldwide, and is a major cause for converting to liver cancer.
  • This virus has been characterized in great detail and can be identified by serological and molecular techniques.
  • numerous mutations in HBV have been detected in patients with both acute and chronic HBV infection, and the mutations have been accumulated over time. This mutation has made the treatment of HBV infection more difficult.
  • RNA interference is the process of sequence-specific, posttranscriptional gene silencing that has become an active field in the antiviral research of mammalians, e.g., HIV (Lee, N,S, Dohjima, T., Bauer, G., Li, H, Li, M. J., Ehsani, A., Dalvaterra, P. and Rossi, J. (2002) Nature Biotechnology, 19, 500-505) and HCV (Kapadia, S. B., Brideau-Andersen, A. and Chisari, F. V. (2003) Proc. Natl. Acad. Sci. USA, 100, 2014-2018).
  • HIV Lee, N,S, Dohjima, T., Bauer, G., Li, H, Li, M. J., Ehsani, A., Dalvaterra, P. and Rossi, J. (2002) Nature Biotechnology, 19, 500-505
  • HCV Kerapadia, S. B., Brideau-Andersen, A. and Chisari, F
  • RNAi is an antiviral mechanism initially found in plants, Caenorhabditis elegans and Drosophila. It is known as post-transcriptional gene silencing (PTGS). RNAi has a highly gene-specific effect that functions through double strand RNA. It specifically reduces the expression of the gene with sequence homology to dsRNA. In this process, dsRNA is initially recognized and cleaved into 21-23-bp fragments called siRNA by an enzyme called Dicer with RNase III activity (Cullen, B. R. (2002) Nat. Immunol. 3, 597-599; Hannon, G. J. (2002) Nature 418, 244-251; Tuschl, T. (2001) Chembiochem 2, 239-245; Sharp, P. A.
  • RNA-induced silencing complex (RISC)
  • the siRNA fragments specifically bind with a multicomponent nuclease and form a complex known as the RNA-induced silencing complex (RISC); finally the RISC hybridizes to the target mR A with a complementary region, and the target mRNA is cleaved in the middle of the 21- to 23 -bp region (Cullen, B. R. (2002) Nat. Immunol. 3, 597-599; Hannon, G. J. (2002) Nature 418, 244-251; Tuschl, T. (2001) Chembiochem 2, 239-245; Sharp, P. A. (2001) Genes Dev. 15, 485-490; Moss, E. G. (2001) Curr. Biol. 11 , R772-R775).
  • RISC RNA-induced silencing complex
  • RNAi also exists in mammalian cells (Cullen, B. R. (2002) Nat. Immunol. 3, 597-599; Yu, J. Y., DeRuiter, S. L. and Turner, D. L. (2002) Proc. Natl. Acad. Sci. USA 99, 6047-6052; Brummelkamp, T. R., Bernards, R. and Agami, R. (2002) Science 296,550-553; Sui, G. 3 Soohoo, C, Affarel, B., Gay, F., Y. and Forrester, W. C. (2002) Proc. Natl. Acad. Sci. USA 99, 5515-5520).
  • RNAi technology has been widely used in the study of many human diseases especially those caused by viruses.
  • Present studies of RNAi in mammalian cells demonstrated that the expression of both exogenous genes delivered by DNA transfection and endogenous genes can be suppressed by the delivery of siRNA (Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl. T.
  • HCV hepatitis C virus
  • HIV-1 human immunodeficiency virus-1
  • FHV fast house virus
  • Rous sarcoma virus dengue virus and poliovirus et al.
  • the present invention provides a method for attenuating expression of target genes of hepatitis B virus (HBV) in a host cell, which method comprises providing a double stranded RNA (dsRNA) sequence in a host cell in an amount sufficient to attenuate expression of HBV target genes, said dsRNA sequence comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of a HBV target gene.
  • dsRNA double stranded RNA
  • the present invention provides a pharmaceutical composition for preventing or treating HBV infection, which pharmaceutical composition comprises: a) a double stranded RNA (dsRNA) sequence in an amount sufficient to attenuate expression of HBV target genes in a host cell, said dsRNA sequence comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of a HBV target gene; or b) a double stranded DNA (dsDNA) sequence in an amount sufficient to attenuate expression of HBV target genes, said dsDNA sequence comprising a nucleotide sequence that is transcribed in a host cell to become a dsRNA sequence that hybridizes under stringent conditions to a nucleotide sequence of a HBV target gene.
  • dsRNA double stranded RNA sequence in an amount sufficient to attenuate expression of HBV target genes in a host cell
  • dsDNA double stranded DNA
  • kits for preventing or treating HBV infection which kit comprises, an above-described pharmaceutical composition and an instruction for using said pharmaceutical composition for preventing or treating HBV infection in a mammal.
  • the present invention provides an isolated oligonucleotide sequence, which: a) hybridizes, under high stringency, with an oligonucleotide sequence, or a complementary strand thereof, that is set forth in Table 3; or b) has at least 90% identity to an oligonucleotide sequence, or a complementary strand thereof, that is set forth in Table 3.
  • the present invention also provides a vector comprising an isolated oligonucleotide sequence described above.
  • the present invention also provides a cell which comprises a vector of the invention.
  • the present invention also provides a pharmaceutical composition for preventing or treating HBV infection, which pharmaceutical composition comprises an isolated oligonucleotide sequence described herein .
  • the present invention also provides a pharmaceutical composition for preventing or treating HBV infection, which pharmaceutical composition comprises a vector of the invention described herein.
  • the nucleotide sequence of the HBV target gene used for designing a dsRNA is in the coding region of the target gene.
  • Figure 1 illustrates a plasmid map of pBS/U6 for the cloning of siDNA.
  • siDNA was cloned into Apal and EcoBl sites, and GFP cassette was inserted into Smal site of pBS/U6.
  • Figure 2 illustrates a scheme of construction of siDNA-pBS/U6-GFP showing the location of siDNA and GFP, and the design of siDNA oligos forming hairpin structure.
  • Figure 3 illustrates visible (left panel) and fluorescence (right panel) images of
  • Figure 4 illustrates the production of HBsAg from HepG2.2.15, and HepG2.2.15 transfected with pBS/U6-GFP and siDNA-1 — 7, respectively.
  • HBsAg levels produced by cells transfected with siDNA- 1 and -7 were significantly reduced comparing to those by HepG2.2.15 or HepG2.2.15 transfected with pBS/U6-GFP.
  • cells expressing GFP were sorted by FACS and cultivated in two plates from which samples were taken for HBsAg-ELISA measurement. ELISAs were performed in triplicates. Data on siDNA-2 were from an independent experiment.
  • Figure 5 illustrates the mRNA quantification by real-time PCR using cDNA from cells as the PCR template.
  • the siRNA measured were siRNA- 1, -2 and -7;
  • HepG2.2.15 transfected with pBS/U6-GFP was used as the control.
  • cDNAs were prepared by reverse transcription using the total RNA isolated from the cells, which had been treated with RNase-free DNase. To eliminate the contamination of chromosome DNA brought from the RNA which was used to amplify the cDNA, the same amount of RNA were used as the template in real-time PCR. Serial dilutions of templates of both HepG2.2.15 and GAPDH (Gyceraldehyde-3 -Phosphate Dehyrogenase), the housekeeping gene, was used as the standard.
  • the primers used in the real-time PCR were 5'- ATG GAG AAC ATC ACA TCA GGA TTC C -3 5 (upstream) (SEQ ID NO:l) and 5'- AAA GAA AAC AGA AAC CC ATA TGT AAA TT -3' (downstream) (SEQ ID NO:2 for HBV HBsAg; and 5 5 -CGG ATT TGG TCG TAT TGG G-3 5 (upstream) (SEQ ID NO:3) and 5'-TCT CGC TCC TGG AAG ATG G-3' (downstream) (SEQ ID NO:4) for GAPDH.
  • Experiments were performed in duplicates and the inhibition rates were calculated with the data obtained from two independent experiments.
  • Figure 6 illustrates the melting temperature of the real-time PCR product analyzed by LightCycler (Roche Applied Sciences, Mannheim, Germany). Single peak demonstrated the specificity of the PCR and reliability of the data to be used for the calculation of inhibition rate.
  • RNA interference RNA interference
  • dsRNA double stranded RNA sequence that is homologous to a HBV target gene, when expressed in a host cell, attenuates expression of HBV genes expressed in the host cell.
  • the methods and compositions provided by the invention can be used to prevent or treat HBV infections.
  • a "double stranded RNA (dsRNA) sequence” or “siRNA” refers a double stranded RNA (dsRNA) molecule that is capable of attenuating the expression of a gene with which it shares homology.
  • the dsRNA molecule is a hairpin comprising a sense region, a loop region and an antisense region complementary to the sense region. The stem segment of the hairpin contains sequences that share homology with the target gene.
  • the dsRNA comprises two distinct RNA molecules that are non-covalently associated to form a duplex.
  • hairpin structure refers to a polynucleotide or nucleic acid that contains a double-stranded stem segment and a single-stranded loop segment wherein the two polynucleotide or nucleic acid strands that form the double-stranded stem segment is linked and separated by the single polynucleotide or nucleic acid strand that forms the loop segment.
  • the "hairpin structure” can further comprise 3' and/or 5' single-stranded region(s) extending from the double-stranded stem segment.
  • Attenuating expression refers to reduction or decrease in the level of mRNA and/or protein product from a target gene by at least about 10%, e.g., about 20%, about 40%, about 60%, about 70%, about 80%, about 90%, or more, as compared to a control.
  • a target gene refers a targeted nucleic acid sequence, the expression of which is attenuated in the present invention by dsRNA.
  • stringency of hybridization in determining percentage mismatch is as follows:
  • medium stringency 0.2 x SSPE (or 1.0 x SSC), 0.1% SDS, 50°C (also referred to as moderate stringency); and 3) low stringency: 1.0 x SSPE (or 5.0 x SSC), 0.1% SDS, 50°C.
  • the present invention is directed to a method for attenuating expression of a target gene of hepatitis B virus (HBV) in a host cell, which method comprises providing a double stranded RNA (dsRNA) sequence in a host cell in an amount sufficient to attenuate expression of HBV target genes, said dsRNA sequence comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of said HBV a target gene.
  • dsRNA double stranded RNA
  • the double-stranded structure may be formed by a single self-complementary RNA strand (a hairpin structure) or two complementary RNA strands. RNA duplex formation may be initiated either inside or outside the cell.
  • the RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g. , at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition; lower doses may also be useful for specific applications.
  • the dsRNA can be synthesized in vitro or in vivo and delivered to the host cell using any known techniques capable of resulting in the desired attenuation of the target gene expression.
  • the dsRNA used to attenuate expression of a target gene of HBV may be directly introduced into the host cell, for example, injected into the cell.
  • the dsRNA sequence is provided in the host cell by introducing a double stranded DNA (dsDNA) sequence into the host cell and the dsDNA sequence is transcribed into a dsRNA sequence in the host cell.
  • dsDNA double stranded DNA
  • the dsDNA sequence can be constructed in an expression vector which is delivered into the host cell.
  • expression vector can be a plasmid or a viral based vector. Any known viral or non- viral based delivery of the expression vector can be used.
  • Viral-based vectors such as retrovirus vectors and adeno-associated virus vectors can be used as a delivery system for the transfer of exogenous genes into a host cell. These vectors provide efficient delivery of genes into the host cell, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host cell.
  • Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art.
  • Exemplary viral-based vehicles include recombinant retroviruses (see, e.g., PCT Publication Nos.
  • alphavirus-based vectors e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis vims (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)
  • adenovirus-derived vectors see, e.g., Berkner et al. (1998) BioTechniques 6:616; Rosenfeld et al. (1991) Sciencen 252:431-434; and Rosenfeld et al.
  • adeno-associated virus see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655).
  • Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.
  • Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA(see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Patent No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed.
  • Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Patent No. 5,580,859.
  • Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.
  • a marker gene contained in the vector such as a green fluorescent protein (GFP) or any antibiotics or drug resistant genes can be used.
  • GFP green fluorescent protein
  • the double stranded RNA and the marker gene may be expressed in the host cell transiently or stably after the vectors are introduced into the host cell.
  • the level of expression dsRNA can be controlled by regulatory sequences, and the vectors of the invention can include any regulatory sequences known in the art to act in a host cell, such as a mammalian cell.
  • exemplary regulator sequences include promoters, enhancers, and other expression control elements.
  • the regulatory sequences may be inducible promoters that depend on the presence of certain factors or tissue specific promoters (e.g., liver specific).
  • the host cell which expresses a target gene of hepatitis B virus may be derived from or contained in any organism (e.g. , a mammal).
  • the host cell can be an isolated cell or a cultured cell.
  • the host cell may be comprised in a whole mammal. Examples of mammals include non-human mammal (e.g., dog, cat, horse, pig, sheep, cattle, goat, rodent, hamster, mouse, rat, and primate) and human.
  • the host cell expressing the target gene may be from the germ line or somatic, totipotent or pluripotent, dividing or 0 non-dividing, parenchyma or epithelium, immortalized or transformed, or the like.
  • the host cell may be a stem cell or a differentiated cell.
  • the host cell may be a cell derived from the liver of a mammal.
  • the method of the invention can be used to prevent or treat HBV infection in the mammal.
  • Exemplary HBV target genes include pre-Surface 1 , per-Surface 2, surface genes 5 (encoding HBsAg), pre-Core, Core genes (encoding HBeAg), Core gene (encoding HBcAg), P gene and X gene (Table 1).
  • Exemplary nucleotide sequence of a HBV target gene that can be used for designing a dsRNA nucleotide sequence includes conserved HBV sequences set forth in Table 2 (SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:l 1). 0
  • HBV AY090458 GenBank accession number
  • P polyraerase 1-1626 (P-l) and 2310-3215 (P-2)
  • HBsAg surface antigen (LHBS, MHBS, HBsAg) , 1-838 and 2851-3215
  • HBeAgCore core antigen 1817-2455
  • X-protein 1377-1841.
  • the dsRNA sequence comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence set forth in Table 2 (SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11).
  • the dsRNA sequence comprises a pair of nucleotide sequences selected from the group consisting of siHBV-1, siHBV-2, siHBV-3, siHBV-4, siHBV-5, siHBV-6, siHBV-7 set forth in Table 3 (SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18), and their complementary sequences.
  • HBeAg e antigen, 1817-2455; HBcAg: core, 1907-2455; X-protein: 1377-1841.
  • the dsRNA sequence of the invention does not comprise nucleotide sequences set forth in Table 4 (SEQ ID NOS : 19-29).
  • HBeAg e antigen, 1817-2455; HBcAg: core antigen; 1907-2455; X-protein: 1377-1841.
  • the expression vector (for example, a plasmid) that can be transcribed into the dsRNA sequence may further comprise a detectable marker in the host cell.
  • detectable markers is a green fluorescent protein (GFP).
  • the dsRNA or the dsDNA encoding the dsRNA comprise a nucleotide sequence of at least about 19, 20, or 21 nucleotides in length of the target sequence. In some embodiments, the dsRNA or the dsDNA encoding the dsRNA are at least about 25, 50, 100, 200, 300, or 400 nucleotides in length of the target sequence.
  • the dsRNA encoded by a dsDNA forms a hairpin structure.
  • the expression of HBV target genes is attenuated by at least about 20%, 40%, 60%, 80%, 90%, or more, relative to the untreated host cell or a host cell treated with a dsDNA which does not encode a dsRNA correspond to a target gene.
  • the expression of the HBV target genes can be measured at RNA level and/or protein level by any biochemical techniques such as RNA hybridization, PCR, Western blotting, enzyme linked immunosorbent assay (ELISA), and fluorescence activated cell sorting analysis (FACS).
  • the present invention also provides an isolated oligonucleotide sequence, which: a) hybridizes, under high stringency, with an oligonucleotide sequence, or a complementary strand thereof, that is set forth in Tables 3; or b) has at least about 90% identity to an oligonucleotide sequence, or a complementary strand thereof, that is set forth in Tables 3.
  • the oligonucleotide sequence includes a nucleotide sequence at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to the oligonucleotide sequence, or a complementary strand, that is set forth in Table 3. In some embodiments, the oligonucleotide sequence comprises a nucleotide sequence , that is set forth in Table 3.
  • the oligonucleotide sequence of the invention comprises DNA, RNA, PNA, or a derivative thereof.
  • the invention also provides an isolated double stranded oligonucleotide sequence, which comprises a pair of complementary oligonucleotide sequences of the isolated oligonucleotide sequence described herein.
  • the isolated double stranded oligonucleotide sequence may comprise a pair of nucleotide sequences set forth in Table 3 and its complementary sequences.
  • the double stranded oligonucleotide sequence is a dsDNA or a dsRNA.
  • the present invention also provides a vector comprising an isolated oligonucleotide sequence describe herein.
  • the vector can be a plasmid or a viral vector.
  • the vector can be introduced into a host cell and integrated into the host genome.
  • the vector can also be constructed to permit it to be an extrachromosomal plasmid after being introduced into a host cell.
  • the vector further comprises regulatory sequences that control the expression of the isolated oligonucleotide sequence.
  • the vector further comprises a transcription terminator.
  • the vector further comprises a nucleotide sequence encoding a detectable marker (e.g., a green fluorescent protein (GFP)) in the host cell.
  • a detectable marker e.g., a green fluorescent protein (GFP)
  • the present invention also provides a cell, which cell comprises a vector described herein.
  • the cell can be an isolated cell or a cultured cell.
  • the cell is comprised in a non-human whole mammal.
  • the cell stably expresses siRNA and/or other sequence(s) encoded by the vector.
  • the cell is a HepG2.2.15 cell.
  • the present invention provides a pharmaceutical composition for preventing or treating HBV infection, which pharmaceutical composition comprises: a) a double stranded RNA (dsRNA) sequence in an amount sufficient to attenuate expression of HBV target genes in a host cell, said dsRNA sequence comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of a HBV target gene; or b) a double stranded DNA (dsDNA) sequence in an amount sufficient to attenuate expression of HBV target genes, said dsDNA sequence comprising a nucleotide sequence that is transcribed in a host cell to become a dsRNA sequence that hybridizes under stringent conditions to a nucleotide sequence of a HBV target gene.
  • dsRNA double stranded RNA sequence in an amount sufficient to attenuate expression of HBV target genes in a host cell
  • dsDNA double stranded DNA
  • the present invention provides a pharmaceutical composition for preventing or treating HBV infection, which pharmaceutical composition comprises an isolated oligonucleotide sequence, which: a) hybridizes, under high stringency, with an oligonucleotide sequence, or a complementary strand thereof, that is set forth in Tables 3; or b) has at least 90% identity to an oligonucleotide sequence, or a complementary strand thereof, that is set forth in Tables 3.
  • the present invention provides a pharmaceutical composition for preventing or treating HBV infection, which pharmaceutical composition comprises a vector comprising an isolated oligonucleotide sequence described herein.
  • the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier or excipient.
  • Pharmaceutically acceptable excipients are known in the art, and are relatively inert substances that facilitate administration of a pharmacologically effective substance.
  • an excipient can give form or consistency, or act as a diluent.
  • Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. Excipients as well as formulations for parenteral and nonparenteral drug delivery are set forth in Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).
  • the present invention provides a kit for preventing or treating HBV infection, which kit comprises any pharmaceutical composition described herein and an instruction for using said pharmaceutical composition for preventing or treating HBV infection in a mammal.
  • a double strand DNA which is homologous to conserved part of HBV mRNA and can form hairpin structure after transcription, was used as siDNA and cloned into plasmid pBS/U6.
  • a GFP cassette containing nucleic-location-site (NLS) was also cloned into this siRNA expressing vector to monitor plasmid transfection. After transfecting these vectors into a HBV-positive stable cell line HepG2.2.15, the cells were sorted by fluorescence activated cell sorting (FACS) technique and tested for the expression of HBV surface antigen (HBsAg).
  • FACS fluorescence activated cell sorting
  • the control cells used to calculate the RNAi inhibition rate on HBsAg expression were HepG2.2.l5 cells transfected with pBSU6-GFP plasmid, which did not contain siRNA sequences. After culturing the cells for 120 h, the HBsAg production of HepG2.2.15 by siRNA- 1 , -2, -3, -4, -5, -6, and -7 (measured by ELISA) were suppressed by 71.8%, 26.3% (result from an independent experiment), 20.2%, 35.2%, 26.8%, 17.0%, and 88.2%, respectively when compared to control cells.
  • Real-time PCR was performed to quantitate the mRNA level of HBV gene transcribed in HepG2.2.15, and the inhibition rates of HBV mRNA by RNAi were 60.8%, 25.3% and 84.7% for siRNA- 1, -2 and -7, respectively, using HepG2.2.15 transfected with pBSU6-GFP as the negative control. These inhibition results were consistent with those of HBsAg assay (by ELISA) for siRNA-1, -2 and -7, respectively.
  • the oligos of the invention can be formulated as a drug to be delivered to the target tissue (liver) in human body as a gene therapy treatment.
  • HBV sequences from 19 individuals were retrieved from GenBankTM. Their GenBank accession numbers are AB076679, AB076678, AY090461 , AY090460,
  • Plasmids that contain siDNA were constructed based on the plasmid pBS/U6 and expression of siRNA was under the control of U6 promoter ( Figure 1). siDNA was inserted between Apal and EcoRl sites of this plasmid ( Figure 1).
  • GFP green fluorescent protein
  • a nucleic-location-site was located in front of GFP, and its sequence was 5'-ACC ATG CAC CGC AGG AAG AAG AAG AGG AGA ACC-3' (SEQ ID NO:30), which was synthesized from BioAsia Biotechnology Ltd. (Shanghai, China).
  • the constructed plasmid was designated siDNA-pBS/U6-GFP.
  • E. coli DH5 ⁇ was used as the siDNA cloning host. Ampicillin at 0.1 g/1 was added for selected screening of cells containing pBS/U6 or its derivatives. Cell culture
  • HepG2.2.15 was used as the HBV-positive cell line for the transcription of pBS/U6 and its derivatives.
  • Cells were cultured in DMEM (GIBCO) supplemented with 10% heat inactivated FBS at 37°C in a 5% CO2 incubator.
  • HBV surface antigen Supernatant of cell culture medium was used for HBsAg quantitation with HBsAg Quantitation Kit S-01 (YingKe Corp., Xiamen, China). Assays were performed in duplicates.
  • RNA were isolated using RNeasy Mini Kit (Qiagen, MD, USA), and then treated with RNase-free DNase I (Amplification grade, Invitrogen, CA, USA).
  • cDNA was prepared by reverse transcription using M-MLV Reverse Transcriptase kit (Invetrogen, CA, USA). The dNTPs and Oligo(dT) 6 used in the reverse transcription were both from Promega (WI, USA). cDNA was quantitated by the absorbance at 260 nm.
  • siDNA candidates The genome sequences of HBV from different individuals vary a lot. To choose siDNA sequences targeting HBV, homology analysis was performed on HBV genome sequences from 19 individuals published in GenBank and obtained 8 conserved sequences that were over 19 nucleotides in length. We used seven of them as the siDNA candidates (Table 5).
  • HBV AY090458 GenBank accession number
  • P polymerase 1-1626 (P-l) and 2310-3215 (P-2);
  • HBsAg surface antigen (LHBS, MHBS, HBsAg), 1-838 and 2851-3215;
  • HBeAgCore core antigen 1817-2455;
  • X-protein 1377-1841.
  • oligos of 19-nt which had G/C 45-65%, were selected from the sequences in Table 5, and used to design 4 oligos, la, lb, 2a and 2b (Table 6) for each siDNA.
  • "Oligo la” and “Oligo lb” formed "GG" at 5'-end as a part of U6 promoter and a Hindl ⁇ l site at its 3 '-end
  • "Oligo 2a” and "Oligo 2b” formed a Hindl ⁇ l site at 5 '-end and a "7 ⁇ " followed by an EcoRl site at its 3 '-end.
  • oligo la/lb was inserted between Apal and Hindl ⁇ l sites of pBS/U6, and Oligo 2a/2b inserted between Hindlll and EcoRl sites (as shown in Figure 2).
  • these two pairs of oligos were joined by Hindlll site, and contained a Pol III transcription terminator "TTTTT" at the 3' end of "Oligo 2a/2b".
  • TTTTTT Pol III transcription terminator
  • siHBV-1 l. siHBV-1-la (sense) 5' GGCTGCTATGCCTCATCTTCTA 3' (SEQ ID NO:31)
  • siHBV-1-2 a (sense) 5' AGCTTAGAAGATGAGGCATAGCAGCCCTTTTTG 3' (SEQ ID NO:33) 4.
  • siHBV-1 -2 b ( antisense ) 5 ' AATTCAAAAAGGGCTGCTATGCCTCATCTTCTA 3 ' (SEQ ID NO:34)
  • siHBV-2-la sense
  • siHBV-2-lb antisense
  • siHBV-2-2a (sense) 5' AGCTTTGAGGCCCACTCCCATAGGCCCTTTTTG 3' (SEQ ID NO:37)
  • siHBV-2-2b (antisense) 5' AATTCAAAAAGGGCCTATGGGAGTGGGCCTCAA 3' (SEQ ID NO:38)
  • siHBV3-la (sense) 5' GGAAGCCTCCAAGCTGTGCCTA 3' (SEQ ID NO:39)
  • siHBV-3-lb antisense 5' AGCTTAGGCACAGCTTGGAGGCTTCC 3' (SEQ ID NO:40)
  • siHB V-3 -2a (sense) 5 ' AGCTTAGGC AC AGCTTGGAGGCTTCCCTTTTTG 3 ' (SEQ ID NO.-41)
  • siHBV-3-2b (antisense) 5' AATTCAAAAAGGGAAGCCTCCAAGCTGTGCCTA 3' (SEQ ID NO:42) siHBV-4:
  • siHBV-5-2a (sense) 5' AGCTTAGAGAAGTCCACCACGAGTCCCTTTTTG (SEQ ID NO:49)
  • siHBV-5-2b (antisense) 5' AATTCAAAAAGGGACTCGTGGTGGACTTCTCTA (SEQ ID NO:50)
  • siHBV-6 21 siHBV-6-la (sense) 5' GGCCAAGTGTTTGCTGACGCAA (SEQ ID NO:51)
  • siHBV-6-lb antisense 5' AGCTTTGCGTCAGCAAACACTTGGCC (SEQ ID NO:52)
  • siHBV-6-2a (sense) 5' AGCTTTGCGTCAGCAAACACTTGGCCCTTTTTG (SEQ ID NO:53)
  • siHBV-6-2b antisense 5' AATTCAAAAAGGGCCAAGTGTTTGCTGACGCAA (SEQ ID NO:54)
  • siHBV-7-la (sense) 5' GGCTCCTCTGCCGATCCATACA (SEQ ID NO:55)
  • siHBV-7-lb (antisense) 5' AGCTTGTATGGATCGGCAGAGGAGCC (SEQ ID NO:56)
  • siHBV-7-2a (sense) 5' AGCTTGTATGGATCGGCAGAGGAGCCCTTTTTG (SEQ ID NO:57)
  • siHBV-7-2b (antisense) 5' AATTCAAAAAGGGCTCCTCTGCCGATCCATACA (SEQ ID NO:58)
  • siDNA-pBS/U6-GFP would express siDNA as well as GFP in nuclei as an intracellular marker for selection.
  • the successful cloning of siDNA into pBS/U6 was verified by DNA sequencing, and the cloning of GFP was confirmed by the visualization of green fluorescence under fluorescence microscopy.
  • RNAi effect on HBV expression cells collected from FACS were cultivated and the supernatant was used in ELISA for HBsAg quantitation. Both HepG2.2.15 and HepG2.2.15 transfected with pBS/U6-GFP (did not contain siDNA) were used as the negative control for RNAi.
  • the production of HBsAg was monitored every 24 h from cells transfected with each plasmid ( Figure 4), and ELISA for HBsAg assay was performed in duplicates. Data in Figure 4 were the average of two independent transfection experiments.
  • Figure 4 showed HBsAg production from cells transfected with different siDNAs including pBS/U6-GFP.
  • RNAi real-time PCR was performed to quantitate HBV mRNA transcribed in the presence of siRNA, and compared with the control.
  • Figure 5 showed the mRNA quantification by real-time PCR using the cDNA of HBsAg as the template to amplify HBsAg fragment.
  • the samples assayed were total RNA isolated from HepG2.2.15 cells transfected with siRNA- 1, 2 and -7; HepG2.2.15; HepG2.2.15 transfected with pBS/U6-GFP.
  • the signals of siRNA-7 and -1 showed up later in cycles than those of the controls and the rest of siRNAs, indicating lower levels of mRNA (of HBsAg).
  • the signal of the negative control using only RNA (not cDNA) as the template did not show up until after 30 cycles, indicating no chromosome DNA contamination in the RNA used for preparing cDNA, the template of real-time PCR.
  • RNAi-7 had the highest the inhibition rate on the expression of HBsAg showing the effect of RNAi technology used in the inhibition of HBV expression in human liver cells.
  • a stable cell line expressing siRNA cells collected from FACS were plated in fresh medium, and sorted by FACS again after 2-3 generations. After several times sorting, all the cells contained GFP by fluorescence microscopy.
  • This cell line can be considered to be a GFP stable cell line, as well as siDNA containing stable cell lines.
  • the active synthesized siDNA was usually 21-nt-long with symmetrical 2- to 3-nt 3' overhangs (Elbashir, S.M., Lendeckel, W. and Tuschl, T. (2001) Genes Dev. 15, 188-200).
  • the siRNA could be either long dsRNA or a hairpin diRNA (Tavernarakis, N., Wang, S.L., Dorovkov, M., Ryazanov, A. and Driscoll, M. (2000) Nat. Genet. 24, 180-183; Kennerdell, J.R. and Carthew, R.W, (2000) Nat. Biotechnol. 18, 96-898).
  • GG 55 was added in the front of each 19-nt to resume "GGG 55 as a part of the transcription initiation site of U6 promoter and the resulted mRNA would form a hairpin.
  • the transcription of different genes of HBV results in only one mRNA for different proteins. Since the delivery of siDNA into liver cell results in the activation of mRNA degradation and inhibition of protein synthesis, the location of the conserved sequences in HBV genome for siDNA design should have no effect on RNAi. However, in our results in Figures 4 and 5, we observed clear differences in levels of inhibition of HBsAg production in the seven siDNA transfected cells. This indicated that the location of siDNA in each coding region of HBV may play roles in effectiveness of RNAi activity.
  • RNAi effective siRNA- 1 and -7 can be generally applied to the knockdown of HBV from different individials.
  • Real-time PCR is a newly developed method to examine the amount of mRNA transcribed from certain genes. It can also be used to evaluate the HBsAg mRNA left in the cell after being digested by RNAi effect.
  • RNAi-7 had the highest degree of inhibition on the expression of HBsAg showing the effect of RNAi technology in inhibiting of HBV expression in human liver cells.

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Abstract

La présente invention concerne des procédés permettant d'atténuer un gène cible du virus de l'hépatite B (HBV) dans une cellule hôte à l'aide d'un ARN double brin (dsRNA) qui comporte une séquence nucléotidique qui s'hybride, dans des conditions sévères, à une séquence nucléotidique du gène cible HBV.
PCT/CN2003/000718 2003-03-05 2003-08-25 Procedes bases sur l'interference d'arn et compositions destinees a l'inhibition de l'expression genique hbv WO2004078181A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992006693A1 (fr) * 1990-10-22 1992-04-30 Fox Chase Cancer Center Produit de recombinaison d'adn destine a la therapie par l'arn
WO1999020641A1 (fr) * 1997-10-21 1999-04-29 Shanghai Institute Of Biochemistry, Chinese Academy Of Sciences Nouveaux oligonucleotides formant une triple helice et leur utilisation dans la lutte contre le virus de l'hepatite b
WO2002083908A1 (fr) * 2001-03-30 2002-10-24 Fundacion Para La Investigacion Medica Aplicada Procede d'inhibition reversible de l'expression genique a l'aide de ribonucleoproteines modifiees

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992006693A1 (fr) * 1990-10-22 1992-04-30 Fox Chase Cancer Center Produit de recombinaison d'adn destine a la therapie par l'arn
WO1999020641A1 (fr) * 1997-10-21 1999-04-29 Shanghai Institute Of Biochemistry, Chinese Academy Of Sciences Nouveaux oligonucleotides formant une triple helice et leur utilisation dans la lutte contre le virus de l'hepatite b
WO2002083908A1 (fr) * 2001-03-30 2002-10-24 Fundacion Para La Investigacion Medica Aplicada Procede d'inhibition reversible de l'expression genique a l'aide de ribonucleoproteines modifiees

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JP2019527694A (ja) * 2016-08-04 2019-10-03 アローヘッド ファーマシューティカルズ インコーポレイテッド Ｂ型肝炎ウイルス感染のためのＲＮＡｉ薬
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US11590156B2 (en) 2016-08-04 2023-02-28 Arrowhead Pharmaceuticals, Inc. RNAi agents for hepatitis B virus infection
US11324820B2 (en) 2017-04-18 2022-05-10 Alnylam Pharmaceuticals, Inc. Methods for the treatment of subjects having a hepatitis b virus (HBV) infection
US11273173B2 (en) 2017-10-20 2022-03-15 Dicerna Pharmaceuticals, Inc. Methods for treating hepatitis B infection
US11052104B2 (en) 2017-10-20 2021-07-06 Dicerna Pharmaceuticals, Inc. Methods for treating hepatitis B infection
US10799524B2 (en) 2017-10-20 2020-10-13 Dicerna Pharmaceuticals, Inc. Methods for treating hepatitis B infection
US11052105B2 (en) 2017-10-20 2021-07-06 Dicerna Pharmaceuticals, Inc. Methods for treating hepatitis B infection
KR20200083494A (ko) * 2017-10-20 2020-07-08 다이서나 파마수이티컬, 인크. B형 간염 감염의 치료 방법
RU2780021C2 (ru) * 2017-10-20 2022-09-19 Дайсерна Фармасьютикалз, Инк Способы лечения инфекции гепатита в
KR102783206B1 (ko) 2017-10-20 2025-03-20 다이서나 파마수이티컬, 인크. B형 간염 감염의 치료 방법
WO2019079781A3 (fr) * 2017-10-20 2019-05-23 Dicerna Pharmaceuticals, Inc Méthodes de traitement des infections de type hépatite b
US11492620B2 (en) 2017-12-01 2022-11-08 Suzhou Ribo Life Science Co., Ltd. Double-stranded oligonucleotide, composition and conjugate comprising double-stranded oligonucleotide, preparation method thereof and use thereof
EP3718572A4 (fr) * 2017-12-01 2021-09-08 Suzhou Ribo Life Science Co., Ltd. Acide nucléique, composition et conjugué contenant un acide nucléique, procédé de préparation et utilisation
US11414665B2 (en) 2017-12-01 2022-08-16 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof
JP2021503941A (ja) * 2017-12-01 2021-02-15 スーチョウ リボ ライフ サイエンス カンパニー、リミテッドＳｕｚｈｏｕ Ｒｉｂｏ Ｌｉｆｅ Ｓｃｉｅｎｃｅ Ｃｏ．， Ｌｔｄ． 核酸、当該核酸を含む組成物および複合体ならびに調製方法と使用
JP7273417B2 (ja) 2017-12-01 2023-05-15 スーチョウ リボ ライフ サイエンス カンパニー、リミテッド 核酸、当該核酸を含む組成物および複合体ならびに調製方法と使用
US11660347B2 (en) 2017-12-01 2023-05-30 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate containing same, preparation method, and use thereof
US12428642B2 (en) 2017-12-01 2025-09-30 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate comprising the same, preparation method and use thereof
US12274752B2 (en) 2017-12-01 2025-04-15 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate containing same, preparation method, and use thereof
US12084661B2 (en) 2017-12-01 2024-09-10 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof
US12083142B2 (en) 2017-12-01 2024-09-10 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof
TWI856002B (zh) * 2017-12-01 2024-09-21 大陸商蘇州瑞博生物技術股份有限公司 核酸、含有該核酸的藥物組合物與綴合物及其用途與試劑盒
US11633482B2 (en) 2017-12-29 2023-04-25 Suzhou Ribo Life Science Co., Ltd. Conjugates and preparation and use thereof
US11492623B2 (en) 2018-08-13 2022-11-08 Alnylam Pharmaceuticals, Inc. Hepatitis B virus (HBV) dsRNA agent compositions and methods of use thereof
US12173289B2 (en) 2018-08-13 2024-12-24 Alnylam Pharmaceuticals, Inc. Hepatitis B virus (HBV) dsRNA agent compositions and methods of use thereof
US11918600B2 (en) 2018-08-21 2024-03-05 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, pharmaceutical composition and conjugate containing nucleic acid, and use thereof
US11896674B2 (en) 2018-09-30 2024-02-13 Suzhou Ribo Life Science Co., Ltd. SiRNA conjugate, preparation method therefor and use thereof

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