WO2018209848A1

WO2018209848A1 - Sirna molecule for inhibiting hbv and use thereof

Info

Publication number: WO2018209848A1
Application number: PCT/CN2017/101022
Authority: WO
Inventors: 朱远源; 李铁军; 刘永红; 刘刚; 陈建新; 彭薇
Original assignee: 百奥迈科生物技术有限公司
Priority date: 2017-05-19
Filing date: 2017-09-08
Publication date: 2018-11-22
Also published as: CN108929870B; CN108929870A

Abstract

Provided is a siRNA molecule for inhibiting HBV, which is composed of a sense strand and an antisense strand, the sense strand being selected from those having an odd number sequence number n in SEQ ID NOs: 1-159, while the antisense strand being selected from those having an even number n+1 sequence number corresponding to the odd number n above in SEQ ID NOs: 2-160. The siRNA molecule or an expression frame thereof can be used for preparing a anti-HBV medicament.

Description

siRNA molecule inhibiting HBV and its application

Technical field

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.

Background technique

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 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).

According to the World Health Organization, about 2 billion people worldwide are infected with HBV, of which about 2.5-350 million people are 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.

The spread of HBV is mainly caused by mother-to-child transmission, unprotected sexual transmission, blood transfusion and injecting drugs, and the transmission route is the same as that of human immunodeficiency virus (HIV). HBV is 50-100 times more infectious than HIV, and HBV can survive for at least 7 days in vitro.

At present, 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. However, 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.

In recent years, the rapid development of RNA interference (RNAi) technology, 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. On December 23, 2016, 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. RNAi is a post-transcriptional gene silencing triggered by double-stranded RNA (dsRNA). The mechanism of action is: Dicer enzyme of the ribonuclease III family, which binds to dsRNA and is cleaved into 21-23 nt and 3' ends. Prominent siRNA, followed by siRNA binding to RNA-induced silencing complex (RISC), unwinding into a single strand, activated RISC is guided by a single-stranded siRNA, sequence-specifically binds to the target gene The mRNA is cleaved and cleaves to specifically decompose the target mRNA, thereby blocking its expression. RNAi has been widely used as a simple and effective gene knockout technique in functional genomics research and in antiviral and antitumor therapy research.

The present invention provides a method of inhibiting HBV using RNAi technology. Using RNA interference 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.

Summary of the invention

In order to provide a new way to effectively treat hepatitis B, 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.

Accordingly, a first object of the present invention is to provide an HBV-inhibiting siRNA molecule consisting of a sense strand and an antisense strand, wherein the sense strand is selected from the SEQ ID NO of an odd number n (n = 1 - 159). 1-159, the antisense strand is selected from SEQ ID NO: 2-160 of the even n+1 (2-160) sequence number corresponding to the above odd n.

Specifically, 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: 21, antisense strand SEQ ID NO: 22; sense strand SEQ ID NO: 23, antisense strand SEQ ID NO: 24; sense strand SEQ ID NO: 25, anti-sense strand SEQ ID NO: 26; sense strand SEQ ID NO: 27, antisense Chain SEQ ID NO: 28; sense strand SEQ ID NO 29:, antisense strand SEQ ID NO: 30; sense strand SEQ ID NO: 31, antisense strand SEQ ID NO: 32; sense strand SEQ ID NO: 33, SEQ ID NO: 34; sense strand SEQ ID NO: 35, antisense strand SEQ ID NO: 36; sense strand SEQ ID NO: 37, antisense strand SEQ ID NO: 38; sense strand SEQ ID NO: 39, Antisense strand SEQ ID NO: 40; 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: 45 , antisense strand SEQ ID NO: 46; sense strand SEQ ID NO: 47, antisense strand SEQ ID NO: 48; sense strand SEQ ID NO: 49, anti-sense strand SEQ ID NO: 50; sense strand SEQ ID NO: 51, antisense strand SEQ ID NO: 52; sense strand SEQ ID NO: 53, antisense strand SEQ ID NO: 54; sense strand SEQ ID NO: 55, antisense strand SEQ ID NO: 56; sense strand SEQ ID NO :57, antisense strand SEQ ID NO: 58; sense strand SEQ ID NO: 59, anti-sense strand SEQ ID N O:60; sense strand SEQ ID NO: 61, antisense strand SEQ ID NO: 62; sense strand SEQ ID NO: 63, antisense strand SEQ ID NO: 64; sense strand SEQ ID NO: 65, anti-sense strand SEQ ID NO: 66; sense strand SEQ ID NO: 67, antisense strand SEQ ID NO: 68; sense strand SEQ ID NO: 69, antisense strand SEQ ID NO: 70; sense strand SEQ ID NO: 71, anti-sense strand SEQ ID NO: 72; sense strand SEQ ID NO: 73, antisense strand SEQ ID NO: 74; sense strand SEQ ID NO: 75, anti-sense strand SEQ ID NO: 76; sense strand SEQ ID NO: 77, antisense SEQ ID NO: 78; sense strand SEQ ID NO: 79, antisense strand SEQ ID NO: 80; sense strand SEQ ID NO: 81, antisense strand SEQ ID NO: 82; sense strand SEQ ID NO: 83, reverse SEQ ID NO: 84; sense strand SEQ ID NO: 85, antisense strand SEQ ID NO: 86; sense strand SEQ ID NO: 87, antisense strand SEQ ID NO: 88; sense strand SEQ ID NO: 89, Antisense strand SEQ ID NO: 90; sense strand SEQ ID NO: 91, antisense strand SEQ ID NO: 92; sense strand SEQ ID NO: 93, antisense strand SEQ ID NO: 94; sense strand SEQ ID NO: 95 , antisense strand SEQ ID NO: 96; sense strand SEQ ID NO: 97, anti-sense strand SEQ ID NO: 98; SEQ ID NO: 99, antisense strand SEQ ID NO: 100; sense strand SEQ ID NO: 101, antisense strand SEQ ID NO: 102; sense strand SEQ ID NO: 103, antisense strand SEQ ID NO: 104; SEQ ID NO: 105, antisense strand SEQ ID NO: 106; sense strand SEQ ID NO: 107, antisense strand SEQ ID NO: 108; sense strand SEQ ID NO: 109, antisense strand SEQ ID NO: 110 ; sense strand SEQ ID NO: 111, antisense strand SEQ ID NO: 112; sense strand SEQ ID NO: 113, antisense strand SEQ ID NO: 114; sense strand SEQ ID NO: 115, antisense strand SEQ ID NO: 116; sense strand SEQ ID NO: 117, antisense strand SEQ ID NO: 118; sense strand SEQ ID NO: 119, antisense strand SEQ ID NO: 120; sense strand SEQ ID NO: 121, antisense strand SEQ ID NO :122; sense strand SEQ ID NO: 123, antisense strand SEQ ID NO: 124; sense strand SEQ ID NO: 125, anti-sense strand SEQ ID NO: 126; sense strand SEQ ID NO: 127, antisense strand SEQ ID NO: 128; sense strand SEQ ID NO 129:, antisense strand SEQ ID NO: 130; sense strand SEQ ID NO: 131, antisense strand SEQ ID NO: 132; SEQ ID NO: 133, antisense strand SEQ ID NO: 134; sense strand SEQ ID NO: 135, antisense strand SEQ ID NO: 136; sense strand SEQ ID NO: 137, antisense strand SEQ ID NO: 138; SEQ ID NO: 139, antisense strand SEQ ID NO: 140; sense strand SEQ ID NO: 141, antisense strand SEQ ID NO: 142; sense strand SEQ ID NO: 143, antisense strand SEQ ID NO: 144; SEQ ID NO: 145, antisense strand SEQ ID NO: 146; sense strand SEQ ID NO: 147, antisense strand SEQ ID NO: 148; sense strand SEQ ID NO: 149, antisense strand SEQ ID NO: 150 ; sense strand SEQ ID NO: 151, antisense strand SEQ ID NO: 152; sense strand SEQ ID NO: 153, antisense strand SEQ ID NO: 154; sense strand SEQ ID NO: 155, anti-sense strand SEQ ID NO: 156; sense strand SEQ ID NO: 157, antisense strand SEQ ID NO: 158; or sense strand SEQ ID NO: 159, antisense strand SEQ ID NO: 160.

Preferably, 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.

Alternatively, 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).

Preferably, 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.

In a preferred embodiment, 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. The purpose of inhibiting the expression of HBV gene is achieved. For this new double-stranded RNA molecule (dsRNA molecule, in order to distinguish it from the aforementioned siRNA molecule, it is referred to herein as "combined dsRNA (molecule)".

The combinatorial dsRNA of the present invention consists of a sense strand comprising two or more sense strands selected from odd sequence numbers SEQ ID NO: 1-159, and correspondingly, an antisense strand complementary to the sense strand. Contains two The antisense strand selected from the even-numbered sequences corresponding to the above odd number is SEQ ID NO: 2-160. Taking a combined dsRNA in which the sense strand comprises two sense strands 1 and sense strand 2, 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. Taking the combined dsRNA in which the sense strand contains three sense strands 1, sense strand 2 and sense strand 3 as an example, 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, and the antisense strand 3 is the antisense strand SEQ ID NO: 150. And so on.

Alternatively, in the combined dsRNA of the present invention, 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.

Alternatively, 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).

Preferably, 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.

In one embodiment, 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. The molecular structure of the RNA expression cassette is: 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 patent template - RNA polymerase type II promoter transcription termination signal; or 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).

In one embodiment, 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). For example, the siRNA molecule can be prepared into the following siRNA expression cassette: U6 promoter-siRNA transcription template-H1 promoter.

Similarly, for a combined dsRNA molecule of the invention, it can be prepared into the following RNA expression cassette: RNA polymerase type III promoter (such as the U6 promoter) - dsRNA transcription template - RNA polymerase type III promoter such as H1 promoter, or 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; or RNA polymerase type III RNA promoter -dsRNA sense strand transcriptional template - circular sequence - dsRNA antisense strand patent template - RNA polymerase type III promoter transcription termination signal (or PolyA tail). For example, a dsRNA molecule can be prepared into the dsRNA expression cassette: U6 promoter-dsRNA transcription template-H1 promoter.

A second object of the present invention is to provide the use of the above siRNA molecule or a combined dsRNA molecule and an RNA expression cassette for the preparation of an anti-HBV drug.

Among them, 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.

In a preferred embodiment, 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.

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". For example, 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: 158. Preferably, 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.

In another preferred embodiment, the above anti-HBV drug comprises one of the above-described combined dsRNA molecules.

In another preferred embodiment, 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.

In another preferred embodiment, the anti-HBV drug comprises an expression cassette of one of the above-described combined dsRNA molecules.

Preferably, the above medicament is an injectable or oral dosage form.

Preferably, the above medicament further comprises an essential adjuvant.

In vitro experiments have demonstrated that 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.

detailed description

The present invention will be further described in detail below in conjunction with specific embodiments. It is understood that the following examples are merely illustrative of the invention and are not intended to limit the scope of the invention.

As used herein, the terms "siRNA", "siRNA sequence", "siRNA molecule", "double-stranded siRNA", or "double-stranded siRNA molecule" are used interchangeably and mean the same meaning and scope. Among them, siRNA is a double-stranded structure formed by annealing the sense strand and the antisense strand.

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. The term "combined dsRNA" refers to a combination of two or more siRNAs that form a new double-stranded RNA molecule in one molecule.

Herein, 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. For example, 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 sense strand of the odd sequence number SEQ ID NO: 3 and the antisense strand of the even sequence number SEQ ID NO 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.

As used herein, the term "RNA expression cassette" includes both siRNA expression cassettes and combinatorial dsRNA expression cassettes. For convenience, for the RNA expression cassette of the present invention, the "U6 promoter-siRNA transcription template-H1 promoter" may be abbreviated herein as "U6-siRNA transcription template-H1" or "U6-siRNA-H1" or "siRNA expression cassettes", which represent the same meaning and scope. Similarly, the "U6 promoter-dsRNA transcription template-H1 promoter" can be abbreviated as "U6-dsRNA transcription template-H1" or "U6-dsRNA-H1" or "dsRNA expression cassette", which means meaning and range the same.

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 preparation of siRNA can be carried out by various methods, such as: chemical synthesis, in vitro transcription, enzymatic cleavage of long-chain dsRNA, vector expression of siRNA, PCR synthesis of siRNA expression elements, etc., and the emergence of these methods provides researchers with an alternative space. Better access to gene silencing efficiency.

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.

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. . For example, for an injectable delivery system, the dosage form can be a lyophilized powder. For dermal administration, the ointment may be selected from ointments or lotions.

Optionally, any pharmaceutically acceptable adjuvant (adjuvant) may be included in the above pharmaceutical dosage form as long as it is suitable for the corresponding administration system, and properly maintains the siRNA molecule and/or the combined dsRNA molecule, and the expressed RNA molecule. The activity of the DNA (including the expression cassette and plasmid).

Example

Materials and Method

The RNA molecules and primer sequences herein are synthesized by BioMico Biotechnology Co., Ltd.; Subcloning of Biomax Biotech Co., Ltd.

Example 1 Preparation of HBV genome full-site siRNA molecular library

Obtaining HBV genomic DNA from HepG2 2.2.15 cells, 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).

Example 2 Preparation of siRNA expression cassette and screening of siRNA target

1. Preparation of siRNA expression cassette

1.1 PCR amplification to prepare 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 μl of reaction system: 0.5 μl of template DNA (10-50 ng), 1 μl of 5'U6 primer (10 μM), 1 μl of 3'H1 primer (10 μM), 1 μl of dNTP (10 mM), 0.5 μl of Pfu DNA polymerase, Make up to 50 μl 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).

Primer sequence used:

5'U6 primer: 5'-AAGGTCGGGCAGGAAGAGGGC-3';

3'H1 primer: 5'-TATTTGCATGTCGCTATGTGTTCT-3'.

1.2 Expression cassette PCR product purification: The expression cassette obtained by PCR amplification was separated by 1.0% agarose gel electrophoresis, and the gel product was purified by an agarose gel purification kit. The purified DNA was again subjected to 1.0% agarose gel electrophoresis. The purity and concentration of the purified U6-siRNA transcription template-H1 expression frame were in accordance with the requirements. The concentration of the prepared frame was determined to be about 200 ng by UV spectrophotometer. Ll.

2. siRNA target screening

2.1 Cell culture: HepG2 2.2.15 cells were cultured in DMEM medium (Thermo) containing 10% FBS in a 37 ° C, 5% CO ₂ incubator.

2.2 Cell plating and transfection: 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%.

use

2000 transfection reagent (Thermo), 0.2 μg/well of "U6-siRNA transcription template-H1 expression cassette DNA" was transfected into the cells according to the instructions. Untransfected cells served as a negative control (NC).

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 μl 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 μl reaction system was established: 2 μl of template RNA, 5 μl of 2×One-Step qPCR Mix, 0.2 μl of forward primer (10 μM), 0.2 μl of reverse primer (10 μM), and the system was supplemented to 10 μl 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.

Real-time quantitative PCR primer sequences:

HBV forward primer: 5'-TGTGGTTATCCTGCGTTAATG-3';

HBV reverse primer: 5'-GCGTCAGCAAACACTTGG-3';

GAPDH forward primer: 5'-GAAGGTGAAGGTCGGAGTC-3';

GAPDH reverse primer: 5'-GAAGATGGTGATGGGATTTC-3'.

2.4 Analysis of results: The relative expression levels of HBV genomic transcripts (mRNA) in cells transfected with each siRNA transcription template were analyzed by 2- ^ΔΔct method. The experimental results are shown in Table 1.

Table 1: Inhibition of HBV genome transcript expression levels by siRNA sequences and their transcriptional templates

In the table, "S" stands for Sense strand and "As" stands for Antisense strand.

It can be seen from the data in Table 1 that the relative expression level of the HBV genome transcript (HBV mRNA) is ≤0.5 after transfection of each siRNA transcription template, that is, the silencing effect of the transcription templates of the 80 siRNAs is ≥50%.

Example 3 in vitro screening of siRNA

1. Cell culture: HepG2 2.2.15 cells were cultured in DMEM medium (Thermo) containing 10% FBS in a 37 ° C, 5% CO ₂ incubator.

2. 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%.

use

2000 transfection reagent (Thermo), 50nM 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:

Justice chain: 5'-UUCUCCGAACGUGUCACGUdTdT-3';

Antisense strand: 5'-ACGUGACACGUUCGGAGAAdTdT-3’

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.

4. 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 μl 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 μl reaction system was established: 2 μl of template RNA, 5 μl of 2×One-Step qPCR Mix, 0.2 μl of forward primer (10 μM), 0.2 μl of reverse primer (10 μM), and the system was supplemented to 10 μl 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.

5. Analysis of results: The relative expression levels of HBV polymerase gene transcripts (HBV mRNA) after transfection of each siRNA were analyzed by 2- ^ΔΔCt method. The experimental results are shown in Table 1.

Table 2: Inhibition of siRNA molecules on the expression level of HBV polymerase gene transcripts

In the table, "S" stands for Sense strand and "As" stands for Antisense strand.

It can be seen from the data in Table 2 that after transfection of cells with each siRNA, 38 siRNAs resulted in a relative expression level of HBV mRNA ≤ 0.4, ie, a silencing effect ≥ 60%. Among them, 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. HBV1614; 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: 151 and the antisense strand is SEQ ID NO: 152 of HBV1676.

Among the siRNA molecules screened by the present invention, 4 siRNA molecules 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.

Example 4 Multi-target siRNA combination

use

2000 transfection reagent (Thermo), 50 nM siRNA mixture (molar concentration ratio of two siRNA molecules was 1:1. The molar ratio of three siRNA molecules was 1:1:1) was transfected into the cells according to the instructions. Negative control (NC) selects siRNAs that have no homology to human genes.

Detection of HBV polymerase mRNA expression levels in samples using gene-specific primers, and amplification of housekeeping genes GAPDH was used as an internal control. Three parallel experiments were performed for each reaction. The following 10 μl reaction system was established: 2 μl of template RNA, 5 μl of 2×One-Step qPCR Mix, 0.2 μl of forward primer (10 μM), 0.2 μl of reverse primer (10 μM), and the system was supplemented to 10 μl 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.

5. Analysis of results: The relative expression levels of HBV polymerase gene transcripts (HBV mRNA) after transfection of each siRNA mixture were analyzed by 2- ^ΔΔCt method. The experimental results are shown in Table 3.

Table 3 Inhibition of HBV polymerase gene transcript expression levels by multi-target siRNA combination

多靶siRNA组合Multi-target siRNA combination	mRNA相对表达水平Relative expression level of mRNA
HBV1622+HBV1627HBV1622+HBV1627	0.160.16
HBV1622+HBV1634HBV1622+HBV1634	0.240.24
HBV1627+HBV1634HBV1627+HBV1634	0.140.14
HBV1622+HBV1627+HBV1634HBV1622+HBV1627+HBV1634	0.180.18
HBV1613+HBV1627+HBV1634HBV1613+HBV1627+HBV1634	0.150.15

It can be seen from the data in Table 3 that four combinations of HBV1613, HBV1622, HBV1627 and HBV1634 targeting different sites of HBV genome form 5 combinations, and the siRNA mixture of each combination will result in the relative expression level of HBV mRNA after transfecting cells. ≤ 0.24, that is, the silencing effect ≥ 76%, indicating that the five combinations formed by the four siRNA molecules can effectively inhibit the HBV polymerase gene, and have the application prospect of co-administration.

Claims

An HBV-inhibiting siRNA molecule consisting of a sense strand and an antisense strand, wherein the sense strand is selected from the SEQ ID NOs: 1-159 of the odd-numbered n-SEQ ID NO: 1-159, and the antisense strand is selected from the even-numbered n corresponding to the odd-numbered n above SEQ ID NO: 2-160 of the +1 sequence number.
The siRNA molecule according to claim 1, wherein the siRNA molecule 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, anti-sense strand SEQ ID NO: 2; sense strand SEQ ID NO: 3, antisense strand SEQ ID NO: 4; sense strand SEQ ID NO: 25, antisense strand SEQ ID NO: 26; sense strand SEQ ID NO: 27, antisense strand SEQ ID NO:28; sense strand SEQ ID NO:39, antisense strand SEQ ID NO:40; sense strand SEQ ID NO:41, antisense strand SEQ ID NO:42; sense strand SEQ ID NO:43, antisense SEQ ID NO: 44; sense strand SEQ ID NO: 45, antisense strand SEQ ID NO: 46; sense strand SEQ ID NO: 49, antisense strand SEQ ID NO: 50; sense strand SEQ ID NO: 53, reverse SEQ ID NO: 54; sense strand SEQ ID NO: 63, antisense strand SEQ ID NO: 64; 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: 71, anti-sense strand SEQ ID NO: 72; sense strand SEQ ID NO: 75, anti-sense strand SEQ ID NO: 76; sense strand SEQ ID NO: 77 , antisense strand SEQ ID NO: 78; sense strand SEQ ID NO: 79, anti SEQ ID NO: 80; sense strand SEQ ID NO: 81, antisense strand SEQ ID NO: 82; sense strand SEQ ID NO: 83, antisense strand SEQ ID NO: 84; sense strand SEQ ID NO: 85, Antisense strand SEQ ID NO: 86; sense strand SEQ ID NO: 87, antisense strand SEQ ID NO: 88; sense strand SEQ ID NO: 91, antisense strand SEQ ID NO: 92; sense strand SEQ ID NO: 93 , antisense strand SEQ ID NO: 94; sense strand SEQ ID NO: 97, antisense strand SEQ ID NO: 98; sense strand SEQ ID NO: 99, anti-sense strand SEQ ID NO: 100; sense strand SEQ ID NO: 103, antisense strand SEQ ID NO: 104; sense strand SEQ ID NO: 109, antisense strand SEQ ID NO: 110; sense strand SEQ ID NO: 111, antisense strand SEQ ID NO: 112; sense strand SEQ ID NO : 113, antisense strand SEQ ID NO: 114; sense strand SEQ ID NO: 115, antisense strand SEQ ID NO: 116; sense strand SEQ ID NO: 125, anti-sense strand SEQ ID NO: 126; sense strand SEQ ID NO: 127, antisense strand SEQ ID NO: 128; sense strand SEQ ID NO: 133, antisense strand SEQ ID NO: 134; sense strand SEQ ID NO: 145, antisense strand SEQ ID NO: 146; ID NO: 147, antisense strand SEQ ID NO: 148; sense strand SEQ ID NO 149, antisense strand SEQ ID NO: 150; sense strand SEQ ID NO: 151, antisense strand SEQ ID NO: 152; or sense strand SEQ ID NO: 157, antisense strand SEQ ID NO: 158.
The siRNA molecule according to claim 1, wherein said siRNA molecule is selected from the group consisting of a double-stranded RNA molecule consisting of the sense strand and the antisense strand of the lower group: sense strand SEQ ID NO: 25, antisense strand SEQ ID NO: 26; sense strand SEQ ID NO: 41, antisense strand SEQ ID NO: 42; The sense strand SEQ ID NO: 43, the antisense strand SEQ ID NO: 44; the sense strand SEQ ID NO: 53, the anti-sense strand SEQ ID NO: 54; the sense strand SEQ ID NO: 65, the anti-sense 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; sense strand SEQ ID NO: 113, anti-sense strand SEQ ID NO: 114; or sense strand SEQ ID NO: 157, antisense strand SEQ ID NO: 158.
The siRNA molecule according to claim 1, wherein the 3' end of the sense strand of the siRNA molecule and/or the 3' end of the antisense strand contains 0 to 2 overhanging bases "NN", wherein the two N are the same Or different, and each independently is adenine deoxynucleotide (dA), thymidine (dT), cytosine deoxynucleotide (dC), guanylate deoxynucleotide (dG), gland Any one of purine nucleotide (A), uracil nucleotide (U), cytosine nucleotide (C) or guanylate nucleotide (G).
The siRNA molecule according to claim 4, wherein the 3' end of the sense strand of the siRNA molecule and/or the 3' end of the antisense strand contains two thymine deoxynucleotides dTdT.
A double-stranded RNA molecule comprising two or more siRNA molecules as claimed in claim 1, each of which targets a different region of a HBV genomic transcript.
An RNA expression cassette for expressing the siRNA molecule of claim 1 or the double-stranded RNA molecule of claim 6 in a cell.
The RNA expression cassette according to claim 7, which is in the form of a U6 promoter-RNA transcription template-H1 promoter.
Use of the siRNA molecule according to any one of claims 1 to 5, the double-stranded RNA molecule of claim 6, and the RNA expression cassette of claim 7 for the preparation of an anti-HBV drug.
The use according to claim 9, wherein the medicament comprises two or more siRNA molecules according to any one of claims 1 to 5.