WO2018209848A1 - Molécule de petit arni destinée à inhiber le vhb et utilisation associée - Google Patents

Molécule de petit arni destinée à inhiber le vhb et utilisation associée Download PDF

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WO2018209848A1
WO2018209848A1 PCT/CN2017/101022 CN2017101022W WO2018209848A1 WO 2018209848 A1 WO2018209848 A1 WO 2018209848A1 CN 2017101022 W CN2017101022 W CN 2017101022W WO 2018209848 A1 WO2018209848 A1 WO 2018209848A1
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seq
strand seq
sense strand
antisense strand
antisense
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朱远源
李铁军
刘永红
刘刚
陈建新
彭薇
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百奥迈科生物技术有限公司
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    • 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
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    • 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
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • 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
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Definitions

  • the present invention belongs to the field of molecular biology, and in particular to an siRNA molecule which inhibits hepatitis B virus HBV and its use in the preparation of an anti-HBV drug.
  • Hepatitis B is an infectious disease caused by Hepatitis B virus (HBV) infection and the most serious type of viral hepatitis. It can cause chronic liver disease, leading to an increased risk of liver cirrhosis and hepatocellular carcinoma in patients. Serious threat to human health.
  • HBV Hepatitis B virus
  • HBV is a hepadnavirus that belongs to the hepadnaviridae family.
  • the whole genome is about 3.2 kb in length and is a partially double-stranded circular DNA.
  • the genome has four open reading frames (ORF).
  • the encoded protein includes a surface antigen (S gene), a core antigen (C gene), a polymerase protein (P gene), and an X protein (C gene).
  • HBV chronic hepatitis B infections. It is estimated that 600,000 people die each year from HBV-related liver diseases or hepatocellular carcinoma. HBV can cause acute inflammation, vomiting, and jaundice in the liver, causing severe fulminant disease and death in a few cases. HBV can also cause chronic liver infections, which may later develop into cirrhosis or liver cancer.
  • HBV human immunodeficiency virus
  • hepatitis B vaccine is mainly used to prevent the occurrence of hepatitis B, but it cannot be used for treatment.
  • Several drugs currently inhibit HBV replication by blocking HBV polymerase such as lamivudine, adefovir, entecavir, and telbivudine.
  • the recurrence rate is high after stopping the drug, and long-term use can lead to virus variability. It is easy to produce drug resistance after a period of administration, which makes the clinical anti-viral treatment face great challenges.
  • RNA interference RNA interference
  • some RNA drugs have entered the clinical trial stage, opening up a new therapeutic approach for intractable diseases, especially multi-factor diseases such as cancer and viral infection.
  • the US Food and Drug Administration approved the FDA.
  • the first application for the RNA drug Spinraza (Nusinersen) for the treatment of spinal muscular atrophy marks the official entry of RNA drugs into the drug army, becoming the third largest new drug type after chemical drugs and biological protein drugs.
  • RNAi technology is used to treat diseases by interfering with the expression of specific target gene messenger RNA (mRNA) by small interfering RNA (siRNA), which is an important part of gene therapy.
  • mRNA target gene messenger RNA
  • siRNA small interfering RNA
  • the present invention provides a method of inhibiting HBV using RNAi technology.
  • siRNA targeting HBV gene is used as a targeted drug to interfere with the transcription of HBV genome, effectively inhibiting the expression of HBV protein, thereby inhibiting viral replication.
  • This method is specific, efficient, and has few side effects. Sustainable use of drugs, and can make up for the shortcomings of current hepatitis B treatment drugs, may become a new means of treating hepatitis B in the near future.
  • the present invention designs and screens a series of RNA molecules having HBV activity, which can specifically target HBV genome transcripts and achieve the purpose of inhibiting HBV.
  • the siRNA molecule of the present invention is a double-stranded RNA molecule consisting of a sense strand and an antisense strand selected from the group consisting of: sense strand SEQ ID NO: 1, antisense strand SEQ ID NO: 2; sense strand SEQ ID NO: 3, antisense strand SEQ ID NO: 4; sense strand SEQ ID NO: 5, antisense strand SEQ ID NO: 6; sense strand SEQ ID NO: 7, antisense strand SEQ ID NO: 8; ID NO: 9, antisense strand SEQ ID NO: 10; sense strand SEQ ID NO: 11, antisense strand SEQ ID NO: 12; sense strand SEQ ID NO: 13, antisense strand SEQ ID NO: 14; SEQ ID NO: 15, antisense strand SEQ ID NO: 16; sense strand SEQ ID NO: 17, antisense strand SEQ ID NO: 18; sense strand SEQ ID NO: 19, antisense strand SEQ ID NO: 20; Chain SEQ ID NO:
  • the siRNA molecule of the present invention is a double-stranded RNA molecule consisting of a sense strand and an antisense strand selected from the group consisting of: sense strand SEQ ID NO: 25, antisense strand SEQ ID NO: 26; sense strand SEQ ID NO :41, antisense strand SEQ ID NO: 42; sense strand SEQ ID NO: 43, antisense strand SEQ ID NO: 44; sense strand SEQ ID NO: 53, antisense strand SEQ ID NO: 54; sense strand SEQ ID NO: 65, antisense strand SEQ ID NO: 66; sense strand SEQ ID NO: 67, antisense strand SEQ ID NO: 68; sense strand SEQ ID NO: 85, antisense strand SEQ ID NO: 86; ID NO: 113, antisense strand SEQ ID NO: 114; or sense strand SEQ ID NO: 157, antisense strand SEQ ID NO: 158.
  • the 3' end of the sense strand of the siRNA molecule provided by the present invention and/or the 3' end of the antisense strand contains 0-2 prominent bases "NN", wherein the two Ns are the same or different, and are each independently Adenine deoxynucleotide (dA), thymidine deoxynucleotide (dT), cytosine deoxynucleotide (dC), guanylate deoxynucleotide (dG), adenine nucleotide (A), Any of uracil nucleotide (U), cytosine nucleotide (C) or guanylate nucleotide (G).
  • N Adenine deoxynucleotide
  • dT thymidine deoxynucleotide
  • dC cytosine deoxynucleotide
  • dG guanylate deoxynucleotide
  • A Any of uracil nucleotide
  • the 3' end of the sense strand of the siRNA molecule provided by the present invention and/or the 3' end of the antisense strand contains 2 thymine deoxynucleotides dTdT.
  • the above siRNA molecules may be combined into two or more, such as two, three, or four, to form a double-stranded RNA molecule (dsRNA molecule), which respectively target different regions of the HBV genome transcript.
  • dsRNA molecule double-stranded RNA molecule
  • the purpose of inhibiting the expression of HBV gene is achieved.
  • dsRNA molecule in order to distinguish it from the aforementioned siRNA molecule, it is referred to herein as "combined dsRNA (molecule)".
  • the combined dsRNA of the present invention is composed of a sense strand and an antisense strand, wherein the sense strand comprises a sense strand 1 and a sense strand 2, a sense strand 1 is SEQ ID NO: 3, sense strand 2 is SEQ ID NO: 71; the antisense strand complementary to sense strand comprises antisense strand 1 and antisense strand 2, and antisense strand 1 is antisense strand SEQ ID NO: 4 , antisense strand 2 is SEQ ID NO:72. And so on.
  • the combined dsRNA of the present invention is composed of a sense strand and an antisense strand, wherein the sense strand contains the sense strand 1 Chain 2 and sense strand 3, sense strand 1 is SEQ ID NO: 3, sense strand 2 is SEQ ID NO: 71, sense strand 3 is SEQ ID NO: 149; antisense strand complementary to sense strand contains antisense strand 1
  • the antisense strand 2 and the antisense strand 3 the antisense strand 1 is the antisense strand SEQ ID NO: 4
  • the antisense strand 2 is SEQ ID NO: 72
  • the antisense strand 3 is the antisense strand SEQ ID NO: 150. And so on.
  • the sense strand 1, the sense strand 2 and the sense strand 3 in the above sense strand may be provided with a spacer sequence which is linked to each other; correspondingly, complementary to the sense strand
  • the antisense strand 1, the antisense strand 2 and the antisense strand 3 in the antisense strand may be provided with a spacer sequence which is linked to each other, and these spacer sequences are different from the target gene sequence.
  • the 3' end of the sense strand and/or the 3' end of the antisense strand of the combined dsRNA molecule provided by the present invention contain 0 to 2 overhanging bases "NN", wherein the two Ns are the same or different and are independent Adenine deoxynucleotide (dA), thymidine (dT), cytosine deoxynucleotide (dC), guanylate deoxynucleotide (dG), adenine nucleotide (A Any one of uracil nucleotide (U), cytosine nucleotide (C) or guanylate nucleotide (G).
  • N Adenine deoxynucleotide
  • dT thymidine
  • dC cytosine deoxynucleotide
  • dG guanylate deoxynucleotide
  • A Any one of uracil nucleotide (U), cytosine nucleot
  • the 3' end of the sense strand and/or the 3' end of the antisense strand of the combined dsRNA molecule provided herein comprises 2 thymine deoxynucleotides dTdT.
  • the siRNA molecule or the combined dsRNA molecule described above may be presented in the form of an RNA expression cassette. Accordingly, a second object of the present invention is to provide an expression cassette of the above siRNA molecule or a combined dsRNA molecule which is a DNA molecule.
  • RNA polymerase type III promoter such as U6 promoter
  • RNA transcription template - RNA polymerase type III promoter such as H1 promoter
  • RNA polymerase type II promoter - RNA sense strand transcription template - circular sequence - RNA antisense strand
  • RNA polymerase type III RNA promoter - RNA sense strand transcription template - circular sequence - RNA counter
  • the sense strand patent template-RNA polymerase type III promoter transcription termination signal (or PolyA tail).
  • the siRNA molecule of the invention can be prepared as the siRNA expression described below Box: RNA polymerase type III promoter (eg U6 promoter) - siRNA transcription template - RNA polymerase type III promoter (eg H1 promoter); RNA polymerase type II promoter - siRNA sense strand transcription template - loop Sequence-siRNA antisense strand patent template-RNA polymerase type II promoter transcription termination signal; or RNA polymerase type III RNA promoter-siRNA sense strand transcription template-loop sequence-siRNA antisense strand patent template-RNA polymerase Type III promoter transcription termination signal (or PolyA tail).
  • the siRNA molecule can be prepared into the following siRNA expression cassette: U6 promoter-siRNA transcription template-H1 promoter.
  • RNA polymerase type III promoter such as the U6 promoter
  • RNA polymerase type II promoter such as H1 promoter
  • RNA polymerase type II promoter-dsRNA sense strand transcription template-loop sequence-dsRNA antisense strand patent template-RNA polymerase type II promoter transcription termination signal
  • a dsRNA molecule can be prepared into the dsRNA expression cassette: U6 promoter-dsRNA transcription template-H1 promoter.
  • the siRNA molecule of the present invention a combined dsRNA molecule, an RNA expression cassette, or a plasmid containing an RNA expression cassette can be used as an active ingredient of an anti-HBV drug.
  • the anti-HBV drug comprises one of the above siRNA molecules or a mixture of two or more siRNA molecules. More preferably, one of the above siRNA molecules is contained in the anti-HBV drug.
  • siRNA molecules When two or more siRNA molecules are contained in an anti-HBV drug, a mixture of these siRNA molecules targets different sites of the HBV genome such as the polymerase protein P gene, and thus may be referred to as “multi-target siRNA” or “multi-target siRNA combination”.
  • the siRNA molecule forming a multi-target siRNA combination is a double-stranded RNA molecule consisting of a sense strand and an antisense strand selected from the group consisting of: sense strand SEQ ID NO: 25, anti-sense strand SEQ ID NO: 26; sense strand SEQ ID NO: 41, antisense strand SEQ ID NO: 42; sense strand SEQ ID NO: 43, antisense strand SEQ ID NO: 44; sense strand SEQ ID NO: 53, antisense strand SEQ ID NO: 54, sense strand SEQ ID NO: 65, antisense strand SEQ ID NO: 66; sense strand SEQ ID NO: 67, antisense strand SEQ ID NO: 68; sense strand SEQ ID NO: 85, antisense strand SEQ ID NO: 86; Chain SEQ ID NO: 113, antisense strand SEQ ID NO: 114; or sense strand SEQ ID NO: 157, antisense strand SEQ ID NO:
  • the siRNA molecule forming the multi-target siRNA combination is a double-stranded RNA molecule consisting of a sense strand and an antisense strand selected from the group consisting of: sense strand SEQ ID NO: 25, anti-sense strand SEQ ID NO: 26; sense strand SEQ ID NO:43, antisense strand SEQ ID NO:44; The sense strand SEQ ID NO: 53, the antisense strand SEQ ID NO: 54; the sense strand SEQ ID NO: 67, the antisense strand SEQ ID NO: 68.
  • the above anti-HBV drug comprises one of the above-described combined dsRNA molecules.
  • the anti-HBV drug comprises one of the above siRNA expression cassettes or a mixture of two or more siRNA expression cassettes. More preferably, the anti-HBV drug comprises one of the above siRNA expression cassettes.
  • the anti-HBV drug comprises an expression cassette of one of the above-described combined dsRNA molecules.
  • the above medicament is an injectable or oral dosage form.
  • the antisense strand of the siRNA molecule or the combined dsRNA molecule provided by the present invention can specifically bind to the HBV genome transcript, thereby degrading, thereby interfering with the post-transcriptional translation process, inhibiting HBV protein translation and viral replication, and achieving The purpose of treating hepatitis B.
  • siRNA is a double-stranded structure formed by annealing the sense strand and the antisense strand.
  • dsRNA As used herein, the terms “dsRNA”, “dsRNA sequence”, “dsRNA molecule”, “double-stranded RNA”, or “double-stranded RNA molecule” are used interchangeably and mean the same meaning and scope, both sense strand and anti- A double-stranded structure formed by annealing the sense strand.
  • combined dsRNA refers to a combination of two or more siRNAs that form a new double-stranded RNA molecule in one molecule.
  • the term "(even) corresponds to the above odd number” means that the even number n+1 and the odd number n form a one-to-one correspondence, wherein the odd number n is selected from 1-159, and accordingly, the even number n+1 is selected from 2 - 160.
  • the sense strand of the odd sequence number SEQ ID NO: 1 corresponds to the antisense strand of the even sequence number SEQ ID NO: 2
  • the corresponding strand of SEQ ID NO: 159 corresponding to the odd-numbered sequence number of SEQ ID NO: 159 corresponds to the antisense strand of SEQ ID NO: 160 of the even-numbered sequence, and so on.
  • the siRNA molecule of the present invention is screened for a siRNA molecular library prepared for the functionally conserved region of the HBV genome.
  • the siRNA molecule library is prepared by the method of the invention with the patent number ZL 200710024217.6, which has the advantages that the prepared siRNA sites are randomly distributed. The length is controllable and can increase the hit rate of effective target sites.
  • the preferred siRNAs screened from the siRNA molecular library of the present invention have a total of 80 siRNAs with a molecular length of 16-31 base pairs (bp). Their sense and antisense strands are odd sequence numbers SEQ ID NO: 1-159 and even sequence numbers SEQ ID NO: 2-160, respectively, the specific sequences of which are listed in Table 1.
  • the siRNA molecule of the present invention for the purpose of treating hepatitis B, the siRNA molecule of the present invention, the expression cassette expressing the siRNA molecule, or the plasmid comprising the siRNA expression cassette, the combined dsRNA molecule, the expression cassette expressing the combined dsRNA, or the combination thereof may be used.
  • the plasmid of the dsRNA expression cassette is directly administered as a pharmaceutical active ingredient to a specific part of the subject, such as a lesion tissue.
  • the dosage form of the medicament of the present invention may be in various forms as long as it is suitable for administration of the corresponding disease, and appropriately maintains the activity of the siRNA molecule and/or the combined dsRNA molecule, and the DNA (including the expression cassette and the plasmid) expressing the RNA molecule.
  • the dosage form can be a lyophilized powder.
  • the ointment may be selected from ointments or lotions.
  • RNA molecules and primer sequences herein are synthesized by BioMico Biotechnology Co., Ltd.; Subcloning of Biomax Biotech Co., Ltd.
  • HepG2 2.2.15 cells contain 2 copies of HBV genome, which can stably secrete HBsAg, HBeAg, HBcAg and Dane particles, and can detect the DNA and RNA of HBV in the cell.
  • a replicating body containing a serum subtype of HBV is ayw (GenBank Accession number: U95551), and a siRNA molecular library of the HBV genome was constructed according to the method of the patent (CN100570022C).
  • U6-siRNA transcription template-H1 expression cassette Screening from the siRNA molecular library of the HBV genome prepared in Example 1, using 80 siRNA positive clone plasmids as templates, using Pfu DNA polymerase (Bai DNA) The U6-siRNA transcription template-H1 expression cassette was amplified by PCR.
  • Each PCR reaction system was 50 ⁇ l of reaction system: 0.5 ⁇ l of template DNA (10-50 ng), 1 ⁇ l of 5'U6 primer (10 ⁇ M), 1 ⁇ l of 3'H1 primer (10 ⁇ M), 1 ⁇ l of dNTP (10 mM), 0.5 ⁇ l of Pfu DNA polymerase, Make up to 50 ⁇ l with ddH 2 O.
  • the reaction conditions were: pre-denaturation at 95 ° C for 1 min, denaturation at 95 ° C for 15 sec, annealing at 58 ° C for 30 sec, extension at 72 ° C for 30 sec, 20 cycles.
  • the mixture of PCR products was detected by 1.0% agarose gel electrophoresis, and the fragment size was in accordance with the experimental requirements (not shown).
  • 3'H1 primer 5'-TATTTGCATGTCGCTATGTGTTCT-3'.
  • HepG2 2.2.15 cells were cultured in DMEM medium (Thermo) containing 10% FBS in a 37 ° C, 5% CO 2 incubator.
  • the cells were seeded at a rate of 2.5 ⁇ 10 5 /ml into a 96-well cell culture plate, and cultured overnight at 37 ° C in a 5% CO 2 incubator to a confluence of about 50%.
  • 2.3 mRNA expression level detection Real-time quantitative PCR was used to detect the mRNA level expression level of HBV polymerase gene in each experimental group, and 4 ⁇ l RNA was used as a template for real-time quantitative PCR reaction.
  • the gene-specific primers were used to detect the expression level of HBV polymerase mRNA in the sample, and the housekeeping gene GAPDH was amplified as an internal reference control.
  • Three parallel experiments were performed for each reaction. The following 10 ⁇ l reaction system was established: 2 ⁇ l of template RNA, 5 ⁇ l of 2 ⁇ One-Step qPCR Mix, 0.2 ⁇ l of forward primer (10 ⁇ M), 0.2 ⁇ l of reverse primer (10 ⁇ M), and the system was supplemented to 10 ⁇ l with RNase-free water. Reaction conditions: reverse transcription at 42 ° C for 30 min, pre-denaturation at 95 ° C for 10 min, denaturation at 95 ° C for 20 sec, annealing at 60 ° C for 30 sec, and 35 cycles.
  • HBV reverse primer 5'-GCGTCAGCAAACACTTGG-3';
  • GAPDH forward primer 5'-GAAGGTGAAGGTCGGAGTC-3';
  • GAPDH reverse primer 5'-GAAGATGGTGATGGGATTTC-3'.
  • Table 1 Inhibition of HBV genome transcript expression levels by siRNA sequences and their transcriptional templates
  • HepG2 2.2.15 cells were cultured in DMEM medium (Thermo) containing 10% FBS in a 37 ° C, 5% CO 2 incubator.
  • Cell plating and transfection The cells were seeded at 2.5 ⁇ 10 5 /ml into 96-well cell culture plates, and cultured overnight at 37 ° C in a 5% CO 2 incubator to a confluence of about 50%.
  • siRNA was transfected into the cells according to the instructions, and the negative control (NC) selected siRNAs with no homology to the human gene.
  • the specific sequence is:
  • Antisense strand 5'-ACGUGACACGUUCGGAGAAdTdT-3’
  • RNA extraction 48 hours after transfection, the cells were washed several times with cold PBS, centrifuged to remove the supernatant, and the RNA in the cells was extracted with RISO RNA extraction reagent (Byomacco) and operated according to the reagent instructions.
  • mRNA expression level detection Real-time quantitative PCR was used to detect the mRNA level of HBV polymerase gene (P gene) in each experimental group, and 4 ⁇ l RNA was used as a template for real-time quantitative PCR reaction.
  • the gene-specific primers were used to detect the expression level of HBV polymerase mRNA in the sample, and the housekeeping gene GAPDH was amplified as an internal reference control.
  • Three parallel experiments were performed for each reaction.
  • the following 10 ⁇ l reaction system was established: 2 ⁇ l of template RNA, 5 ⁇ l of 2 ⁇ One-Step qPCR Mix, 0.2 ⁇ l of forward primer (10 ⁇ M), 0.2 ⁇ l of reverse primer (10 ⁇ M), and the system was supplemented to 10 ⁇ l with RNase-free water. Reaction conditions: reverse transcription at 42 ° C for 30 min, pre-denaturation at 95 ° C 10 min, denaturation at 95 ° C for 20 sec, annealing at 60 ° C for 30 sec, cycle 35 times.
  • HBV polymerase gene transcripts HBV polymerase gene transcripts (HBV mRNA) after transfection of each siRNA were analyzed by 2- ⁇ Ct method. The experimental results are shown in Table 1.
  • siRNAs resulted in a relative expression level of HBV mRNA ⁇ 0.4, ie, a silencing effect ⁇ 60%.
  • 12 siRNA molecules lead to the relative expression level of HBV mRNA ⁇ 0.2, that is, the silencing effect ⁇ 80%, which includes: the sense strand is SEQ ID NO: 3, the opposite The sense strand is HBV1602 of SEQ ID NO: 4; the sense strand is SEQ ID NO: 25, the antisense strand is HBV1613 of SEQ ID NO: 26; the sense strand is SEQ ID NO: 27, and the antisense strand is SEQ ID NO: 28.
  • sense strand is SEQ ID NO:43, antisense strand is HBV1622 of SEQ ID NO:44; sense strand is SEQ ID NO:49, antisense strand is SEQ ID NO:50, HBV1625; sense strand is SEQ ID NO:71, the antisense strand is HBV1636 of SEQ ID NO:72; the sense strand is SEQ ID NO:81, the antisense strand is HBV1641 of SEQ ID NO:82; the sense strand is SEQ ID NO:93, and the antisense strand is HBV1647 of SEQ ID NO: 94; sense strand is SEQ ID NO: 99, antisense strand is HBV1650 of SEQ ID NO: 100; sense strand is SEQ ID NO: 109, antisense strand is SEQ ID NO: 110 of HBV1655; The sense strand is SEQ ID NO: 149, the antisense strand is HBV1675 of SEQ ID NO: 150; and the sense strand is SEQ ID NO:
  • siRNA molecules screened by the present invention can cause the relative expression level of HBV mRNA ⁇ 0.2, and the transcription template thereof results in the relative expression level of HBV mRNA ⁇ 0.4, which includes: the sense strand is SEQ ID NO: 3
  • the antisense strand is HBV1602 of SEQ ID NO: 4; the sense strand is SEQ ID NO: 71, the antisense strand is HBV1636 of SEQ ID NO: 72; the sense strand is SEQ ID NO: 93, and the antisense strand is SEQ ID NO: HBV1647 of 94; or HBV1675 of SEQ ID NO: 149 and the antisense strand of SEQ ID NO: 150.
  • Experiments have shown that their silencing effect on the HBV gene is outstanding.
  • HepG2 2.2.15 cells were cultured in DMEM medium (Thermo) containing 10% FBS in a 37 ° C, 5% CO 2 incubator.
  • Cell plating and transfection The cells were seeded at 2.5 ⁇ 10 5 /ml into 96-well cell culture plates, and cultured overnight at 37 ° C in a 5% CO 2 incubator to a confluence of about 50%.
  • NC Negative control
  • RNA extraction 48 hours after transfection, the cells were washed several times with cold PBS, centrifuged to remove the supernatant, and the RNA in the cells was extracted with RISO RNA extraction reagent (Byomacco) and operated according to the reagent instructions.
  • mRNA expression level detection Real-time quantitative PCR was used to detect the mRNA level of HBV polymerase gene (P gene) in each experimental group, and 4 ⁇ l RNA was used as a template for real-time quantitative PCR reaction.
  • HBV polymerase gene transcripts HBV polymerase gene transcripts (HBV mRNA) after transfection of each siRNA mixture were analyzed by 2- ⁇ Ct method. The experimental results are shown in Table 3.
  • Multi-target siRNA combination Relative expression level of mRNA HBV1622+HBV1627 0.16 HBV1622+HBV1634 0.24 HBV1627+HBV1634 0.14 HBV1622+HBV1627+HBV1634 0.18 HBV1613+HBV1627+HBV1634 0.15

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Abstract

L'invention concerne une molécule de petit ARNi destinée à inhiber le VHB, qui est composée d'un brin sens et d'un brin antisens, le brin sens étant choisi parmi ceux ayant un numéro de séquence impair n dans SEQ ID NO: 1 à 159, tandis que le brin antisens est choisi parmi ceux ayant un numéro de séquence pair n+1, correspondant au numéro impair n ci-dessus, dans SEQ ID NO: 2 à 160. La molécule de petit ARNi ou un cadre d'expression correspondant peut être utilisé pour préparer un médicament anti-VHB.
PCT/CN2017/101022 2017-05-19 2017-09-08 Molécule de petit arni destinée à inhiber le vhb et utilisation associée WO2018209848A1 (fr)

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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
US11633482B2 (en) 2017-12-29 2023-04-25 Suzhou Ribo Life Science Co., Ltd. Conjugates and preparation and use thereof
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
US11896674B2 (en) 2018-09-30 2024-02-13 Suzhou Ribo Life Science Co., Ltd. SiRNA conjugate, preparation method therefor and 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

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CN101126176A (zh) * 2007-07-23 2008-02-20 百奥生物技术（南通）有限公司 PCR高通量构建siRNA全位点分子库制备方法
CN103370415A (zh) * 2010-10-28 2013-10-23 本尼特生物制药有限公司 Hbv治疗

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CN101126176A (zh) * 2007-07-23 2008-02-20 百奥生物技术（南通）有限公司 PCR高通量构建siRNA全位点分子库制备方法
CN103370415A (zh) * 2010-10-28 2013-10-23 本尼特生物制药有限公司 Hbv治疗

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
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
US11633482B2 (en) 2017-12-29 2023-04-25 Suzhou Ribo Life Science Co., Ltd. Conjugates and preparation and 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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