CN103275971A - RNA interference targets for hepatitis b virus (HBV) infection treatment - Google Patents

Abstract

The invention relates to 42 different hepatitis b virus (HBV)-targeting RNA interference targets for HBV infection treatment. The RNA interference targets can be used for preparation of a drug for HBV infection treatment. The invention provides recombinant expression vectors for expression of HBV-targeting siRNA and/or miRNA and/or ribozyme and/or antisense oligonucleotide. The invention relates to cells which can inhibit HBV gene expression and can express and/or be introduced with the siRNA and/or the miRNA and/or the ribozyme and/or the antisense oligonucleotide and/or a drug obtained according to the RNA interference targets.

Description

RNA interference targets that can be used in the treatment of hepatitis B virus infection

This application is a divisional application of the application with the same title filed on June 13, 2008 and with application number 200810110686.4.

technical field

The present invention relates generally to the fields of molecular biology, cell biology and gene therapy. More specifically, the present invention relates to 42 targets of RNA interference (RNAi) that can be used in the treatment of hepatitis B virus infection, as well as recombinant expression vectors using these targets and the recombinant expression vectors that can be used in hepatitis B obtained in various ways using these targets. Drugs and methods for the treatment of hepatitis virus infection.

Background technique

Hepatitis B virus infection is one of the most important public health problems worldwide. Related diseases caused by HBV infection include acute or chronic hepatitis B, and liver cirrhosis (HC) and hepatocellular carcinoma (HCC) caused by the further development of chronic hepatitis B. The range of HBV infection is very wide. At present, there are about 400 million HBV infected people in the world, and about 2 billion people have been infected with HBV. A considerable number of chronic HBV carriers develop into CHB, and some patients develop into HC and HCC, and the number of deaths due to HBV infection exceeds 1 million every year (DonGanem et al., New England Medicine, 2004, Volume 35: 1118-1129 Page). my country is a high-incidence area of HBV infection. The HBV infection rate is about 56.7%, and the HBV virus carrier rate is 9.75%. Among various types of viral hepatitis, HBV is the most serious hazard to human health. With the advent of HBV vaccine in 1982, the infection rate of HBV has been significantly reduced, and the emergence of antiviral drugs has also made some progress in the treatment of hepatitis B. However, due to the large number of HBV carriers and the fact that current antiviral drugs cannot cure HBV infection, a large number of HBV carriers will still face the risk of developing liver cirrhosis and hepatocellular carcinoma. Therefore, there is an urgent need to develop new treatments to deal with the threat of HBV.

RNA interference (RNA interference, RNAi) is a mechanism of intracellular sequence-specific inhibition of gene expression mediated by double-stranded RNA (double-stranded RNA, dsRNA). was first proposed in (Fire A et al., Nature, 1998, Vol. 391: pp. 806-811). After that, further research found that RNAi widely exists in higher mammals and almost all eukaryotes such as fungi, Arabidopsis, hydra, planarians, trypanosomes, zebrafish, etc., and is a ubiquitous and conserved inhibitory The mechanism of gene expression can regulate gene expression, resist virus invasion, inhibit transposon activity, etc. At present, the mechanism of RNAi has been basically elucidated: dsRNA molecules produced endogenously or exogenously are cut into small interfering RNA (small interfering RNA, siRNA) by Dicer, which belongs to RNase III, in the cytoplasm. Typical structural features of siRNA are: phosphorylation at the 5' end, 2-3 nt protruding symmetrically at the 3' end with a hydroxyl group, and dsRNA with a length of 19-23 nt. The siRNA molecule binds to the protein complex of the RNA-inducing silencing complex (RISC), which has helicase and endonuclease activities. The siRNA molecule is depolymerized in the complex, in which the antisense strand binds to the target mRNA that can be matched with it according to the principle of base pairing, and guides the RISC bound to it to place the target mRNA at a distance of 5 in the binding region with the antisense strand The position of 10nt at the ' end is enzymatically hydrolyzed, thereby inhibiting the expression of the target gene. At present, the methods for obtaining siRNA mainly include: plasmids and recombinant virus vectors that can express small hairpin RNA (short hairpin RNA, shRNA), chemical synthesis methods, in vitro transcription, etc.

At present, RNAi technology has shown good application prospects in the prevention and treatment of viral diseases including hepatitis B virus infection and tumors. Studies have shown that siRNA targeting HBV mRNA can inhibit HBV replication and viral gene expression in human liver cancer cell lines cultured in vitro (Konishi M et al., Hepatology, 2003, Vol. 38: pp. 842-850). Since the pathogenic mechanism of HBV is relatively complex, it must be able to efficiently inhibit virus replication and gene expression in order to achieve effective treatment. However, not all RNAi targets that meet the conventional design requirements can effectively inhibit the expression of target genes, and the inhibition efficiency varies among different targets. Therefore, the selection of suitable RNAi targets with high inhibitory efficiency has become an important factor for whether RNAi technology can be successfully applied to anti-HBV therapy. Selecting a suitable RNAi target needs to be comprehensively considered in terms of structural characteristics, inhibition efficiency, and non-human gene homology. Auxiliary methods that can be used include the proposed siRNA-assisted design software, RNA molecular structure analysis, nucleic acid sequence analysis and comparison, and experimental experience, etc., and are verified by specific inhibition evaluation experiments.

Based on RNA interference technology, it is expected to develop new and more effective methods for the treatment of hepatitis B virus infection. This type of treatment method needs to provide RNA interference targets that can effectively inhibit HBV replication and expression. The present invention meets this requirement by providing RNA interference targets, recombinant expression vectors and the like that can be used for this purpose.

Contents of the invention

The present invention provides an RNA interference target targeting HBV, which can be used to construct expression plasmids, recombinant virus vectors and cells that contain or introduce the coding nucleic acid sequence of the RNA interference target involved in the present invention, and can be used to obtain the RNA interference target containing the present invention. Involved RNA interference targets to obtain therapeutic drugs.

The RNA interference target provided by the invention can target HBV, and can inhibit HBV replication and viral gene expression. The RNA interference target provided by the present invention is obtained by the following method: selecting and designing the RNA interference target sequence that can target HBV, constructing shRNA by designing suitable primers, and cloning into the expression vector to obtain the corresponding shRNA expression plasmid. The plasmids were co-transfected with the HBV infectious clone plasmids, and the appropriate RNA interference targets were obtained through analysis and screening of HBV protein expression levels and identification of inhibition specificity.

The present invention provides (specifically) RNA interference target sequence targeting HBV, the sequence is selected from:

(1) the sequence shown in any one of SEQ ID NO: 1-42, or

(2) A sequence that is at least 70% (preferably at least 80%, 85%, 90%, 95%, 98% or more) identical to the sequence in (1), or

(3) A nucleotide sequence that can hybridize with the sequence in (1) under stringent conditions or highly stringent conditions, or

(4) A sequence that differs from the sequence in (1) by only 1-3 (preferably 1-2, more preferably 1) nucleotides; or

(5) Fragments or complementary sequences of the above sequences.

In a specific aspect, the RNA interference target sequence can target HBV X, S, P or C genes.

In a preferred embodiment, the RNA interference target is selected from siHBV7 (SEQ ID NO: 7) and siHBV12 (SEQ ID NO: 12).

The RNA interference target provided by the present invention can be a DNA or RNA sequence.

The present invention also provides nucleic acid constructs or vectors such as expression vectors comprising the RNA interference target sequence. The recombinant expression vector containing the RNA interference target sequence can be used to express the HBV-targeted siRNA and/or miRNA and/or ribozyme and/or antisense oligonucleotide of the present invention.

The present invention also provides a recombinant expression vector capable of expressing the HBV-targeting siRNA and/or miRNA and/or ribozyme and/or antisense oligonucleotide of the present invention.

In one embodiment, the recombinant expression vector of the present invention has the following characteristics: the nucleic acid sequence encoding the siRNA and/or miRNA and/or ribozyme and/or antisense oligonucleotide comprising the RNA interference target sequence provided by the present invention , these coding nucleic acid sequences are operably linked with expression control sequences, so that the siRNA and/or miRNA targeting HBV can be expressed in animal cells (especially mammalian cells, such as human cells, preferably liver cells and stem cells) and /or ribozymes and/or antisense oligonucleotides.

The recombinant expression vector of the present invention may be a plasmid vector or a virus vector, such as a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, and the like.

The present invention also provides siRNA or miRNA or ribozyme or antisense oligonucleotide obtained according to the above-mentioned RNA interference target sequence and capable of inhibiting the expression of HBV corresponding genes and/or the replication and/or infection of HBV.

The present invention also relates to isolated cells comprising: (1) the RNA interference target sequence of the present invention, or (2) a nucleic acid construct or vector such as an expression vector containing the RNA interference target sequence of the present invention.

The present invention also relates to an isolated cell transformed or transfected or transduced with a recombinant expression vector expressing the HBV-targeting siRNA and/or miRNA and/or ribozyme and/or antisense oligo of the present invention Nucleotides.

The present invention also relates to a modified cell (including animal such as mammalian, preferably human cells, preferably liver cells and stem cells), which can express or contain the siRNA or miRNA or ribozyme or antisense oligonucleotide of the present invention.

The present invention also relates to cells carrying the nucleic acid sequence encoding the RNA interference target involved in the present invention in the genome or outside the genome, including prokaryotic cells (such as bacterial cells, such as Escherichia coli cells) and eukaryotic cells (such as fungal cells, insect cells) , plant cells, animal cells, preferably mammalian cells such as human cells, preferably hepatocytes and stem cells), which contain the coding nucleic acid sequences of the RNA interference targets involved in the present invention (these coding nucleic acid sequences can be operably with expression control sequences Ligated so that said siRNA and/or miRNA and/or ribozyme and/or antisense oligonucleotide can be expressed in the cell).

The present invention also relates to cells, including prokaryotic cells (such as bacterial cells, such as E. ) and eukaryotic cells (such as fungal cells, insect cells, plant cells, animal cells, preferably mammalian cells such as human cells, preferably liver cells and stem cells), which are introduced into cells containing the RNA interference target obtained by the present invention siRNA and/or miRNA and/or ribozyme and/or antisense oligonucleotide.

In a preferred embodiment, after the shRNA expression element containing the nucleic acid sequence encoding the RNA interference target obtained in the present invention is introduced into hepatocytes, the cells can obtain the ability to inhibit HBV replication and viral gene expression.

The invention also relates to tissues and organisms, such as animals, comprising the cells described above. The invention also relates to pharmaceutical compositions comprising the cells of the invention.

On the other hand, the present invention also relates to the method for preparing the modified cells of the present invention, including transforming or transfecting or transducing cells (including animal such as mammalian, preferably human cells, preferably liver cells and stem cells) with the recombinant expression vector of the present invention.

In one embodiment, the method includes transducing mammalian, preferably human, hepatocytes and stem cells with the recombinant viral vector of the present invention (eg, lentiviral vector, such as lentivirus Lenti-siHBV7, etc.).

In the above method, the cells may be isolated (or ex vivo), such as isolated from HBV-infected patients or normal individuals, or in vivo, or cell lines cultured in vitro.

The invention also relates to a combination of DNA sequences comprising or consisting of a first DNA sequence encoding a sense RNA segment comprising RNA encoded by a target sequence of the invention and a second DNA sequence encoding an antisense RNA segment Sequence, antisense RNA fragment and positive sense RNA fragment can form double-stranded RNA, and this double-stranded RNA can inhibit the expression of HBV gene and/or the replication and/or infection of HBV.

The present invention also relates to small-molecule interfering ribonucleic acid (siRNA), which includes sense RNA fragments and antisense RNA fragments, the sense RNA fragments comprise the RNA sequence encoded by the target sequence of the present invention, and the antisense RNA fragments and sense RNA fragments can form Double-stranded RNA, and the double-stranded RNA can inhibit the expression of HBV corresponding genes and/or the replication and/or infection of HBV.

The present invention also relates to the siRNA and/or miRNA and/or ribozyme and/or antisense oligonucleotide obtained from the RNA interference target provided by the present invention in the preparation of medicines and/or pharmaceutical compositions for treating HBV infection or HBV patients the use of.

The present invention also relates to the use of siRNA and/or miRNA and/or ribozyme and/or antisense oligonucleotide obtained from the RNA interference target provided by the present invention in the preparation of drugs and/or pharmaceutical compositions for inhibiting HBV replication or HBV gene expression use in .

The present invention also relates to the use of the RNA interference target sequence of the present invention, or the nucleic acid construct or vector, or the recombinant expression vector in the preparation of medicines for treating HBV infection or HBV patients.

The present invention also relates to the use of the modified cells of the present invention (including animal, such as mammalian, preferably human cells, preferably liver cells and stem cells) in the preparation of medicines and/or pharmaceutical compositions for treating HBV infection or HBV patients.

The present invention also relates to the application of the siRNA target sequence of the present invention in screening anti-HBV drugs.

The present invention also relates to a method for treating HBV infection or HBV patients, comprising administering the RNA interference target sequence, nucleic acid construct or vector, recombinant expression vector, siRNA or miRNA or ribozyme or antisense oligonucleotide of the present invention to individuals in need nucleotides, or cells.

The present invention also relates to the use of the vector and cells of the present invention for treating HBV infection or HBV patients.

The present invention also relates to a method for treating HBV infection or HBV patients, comprising administering to the patient a therapeutically effective amount of the RNA interference target sequence, nucleic acid construct or vector, siRNA or miRNA or ribozyme or antisense oligonucleotide of the present invention , expression vectors, cells, DNA sequence combinations or siRNA.

The present invention also relates to a method for inhibiting HBV replication or HBV gene expression, comprising administering to an individual in need an effective amount of the RNA interference target sequence, nucleic acid construct or vector, siRNA or miRNA or ribozyme or antisense oligo Nucleotides, expression vectors, cells, combinations of DNA sequences, or siRNA.

The present invention also relates to the RNA interference target sequence, nucleic acid construct or carrier, siRNA or miRNA or ribozyme or antisense oligonucleotide, expression vector, cell, DNA sequence combination or siRNA described in the present invention, for treating HBV infection Or for HBV patients, or for inhibiting HBV replication or HBV gene expression.

The present invention will be described in more detail below in conjunction with the accompanying drawings. The above aspects of the invention and other aspects of the invention will be apparent from the following detailed description.

Description of drawings

Figure 1 is a schematic diagram of the construction process of pSUPER-siRNA series expression plasmids.

Figure 2 shows the inhibitory effect of siRNA expression plasmids respectively targeting the 42 RNA interference targets obtained by us in the co-transfection experiment with HBV infectious clone plasmids on HBV gene expression. The results showed that these RNAi targets suppressed HBV.

Figure 3 shows that HepG2-N10 cells transfected with expression vector plasmids pSUPER-siHBV7 and pSUPER-siHBV12 carrying HBV-targeted siRNA expression sequences can show the ability to inhibit HBV expression, and can inhibit the production of HBsAg in HepG2-N10 cells Express.

Figure 4 shows that HepG2-N10 cells transfected with expression vector plasmids pSUPER-siHBV7 and pSUPER-siHBV12 carrying HBV-targeting siRNA expression sequences can show the ability to inhibit HBV replication, which can reduce the expression of HepG2-N10 cell culture supernatant The copy amount of HBV nucleic acid in the

Figure 5 shows that HepG2-N10 cells transduced by recombinant lentiviruses Lenti-siHBV7 and Lenti-siHBV12 carrying HBV-targeting siRNA expression sequences can show the ability to inhibit HBV expression, and can inhibit the production of HBsAg in HepG2-N10 cells Express.

Figure 6 shows that HepG2-N10 cells transduced by recombinant lentiviruses Lenti-siHBV7 and Lenti-siHBV12 carrying HBV-targeted siRNA expression sequences can show the ability to inhibit HBV replication, which can reduce the expression of HepG2-N10 cell culture supernatant The copy amount of HBV nucleic acid in the

Figure 7 shows that the expression vector plasmids pSUPER-siHBV7 and pSUPER-siHBV12 carrying the siRNA expression sequence targeting HBV can show the ability to inhibit HBV expression after injection and transduction of the expression vector plasmids pSUPER-siHBV7 and pSUPER-siHBV12, and can inhibit the HBV infectious clone plasmid expression, reducing the level of HBsAg in serum.

Figure 8 shows that the expression vector plasmids pSUPER-siHBV7 and pSUPER-siHBV12 carrying the siRNA expression sequence targeting HBV can show the ability to inhibit HBV replication after injection and transduction of the expression vector plasmids pSUPER-siHBV12, which can inhibit the expression of HBV infectious clone plasmids Expression, reducing the copy amount of HBV nucleic acid in serum.

Fig. 9 shows the inhibitory effect of the synthetic siRNA targeting the RNA interference target of HBV on HBV. HepG2-N10 cells transfected with siR-HBV7 and siR-HBV12 can show the ability to inhibit the expression of HBV, and can inhibit the expression of HBsAg in HepG2-N10 cells.

FIG. 10 shows the inhibitory effect of the synthesized siRNA targeting the RNA interference target of HBV on HBV. HepG2-N10 cells transfected with siR-HBV7 and siR-HBV12 can show the ability to inhibit HBV replication, and can reduce the copy amount of HBV nucleic acid in the culture supernatant of HepG2-N10 cells.

Figure 11 shows the inhibitory effect on HBV of the siRNA targeting the RNA interference target of HBV modified by 2'-Ome (2'-methoxy) and/or phosphorylated and/or sterol-modified. HepG2-N10 cells transfected with siRpo-HBV7, siRpo-HBV12, siRpoC-HBV7, and siRpoC-HBV12 can show the ability to inhibit the expression of HBV, and can inhibit the expression of HBsAg in HepG2-N10 cells.

Figure 12 shows the inhibitory effect on HBV of synthetic and 2'-OMe-modified and/or phosphorylated-modified and/or sterol-modified siRNA targeting the RNA interference target of HBV. HepG2-N10 cells transfected with siRpo-HBV7, siRpo-HBV12, siRpoC-HBV7, and siRpoC-HBV12 can show the ability to inhibit HBV replication, and can reduce the copy amount of HBV nucleic acid in the culture supernatant of HepG2-N10 cells.

Detailed ways

Unless otherwise specified, terms in the present invention have meanings commonly used in the art.

The present invention provides an RNA interference target that can target HBV, comprising the following sequence or a sequence having at least 70% (preferably at least 80%, 85%, 90%, 95%, 98% or higher) identity therewith Any one or several sequences: SEQ ID NO:1-42.

Identity can be calculated according to methods known in the art. A preferred example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST2.0 algorithms described in Altschul et al. (1977) Nucl. Acid. Res. 25:3389-3402 and Altschul et al. (1990 ) J. Mol. Biol. 215:403-410. Using parameters such as those described herein, BLAST and BLAST 2.0 can be used to determine percent sequence identity for polynucleotides and polypeptides of the invention. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information.

In other embodiments, the sequence of the RNA interference target has a polynucleotide sequence that can hybridize to a polynucleotide provided herein, or a fragment thereof, or a complementary sequence thereof under stringent conditions or highly stringent conditions. Hybridization techniques are well known in the field of molecular biology. For the purpose of illustration, the conditions of the hybridization are stringent conditions, for example, the DNA bound to the filter is hybridized at about 45° C. in 6× sodium chloride/sodium citrate (SSC), followed by 0.2×SSC/0.1% One or more washes in SDS at about 50-65°C; highly stringent conditions, such as hybridization of filter-bound nucleic acids at about 45°C in 6×SSC, and then in 0.1×SSC/0.2% SDS at about One or more washes at 68° C.; or other stringent hybridization conditions known to those skilled in the art (see, for example, Ausubel, F.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, pp. 6.3.1-6.3.6 and 2.10.3).

The present invention also relates to a nucleotide sequence capable of hybridizing to any sequence of SEQ ID NO: 1-42, or a fragment thereof, or a complementary sequence thereof under stringent conditions or highly stringent conditions.

In the present invention, siRNAs and/or miRNAs and/or ribozymes and/or antisense oligonucleotides can be designed to target genes or regulatory sequences, such as genes whose expression needs to be inhibited or their regulatory sequences, in order to inhibit or reduce its expression. The targeted gene or its regulatory sequence may be any gene or its regulatory sequence whose expression needs to be suppressed or reduced, such as those from pathogens, or those involved in the formation and progression of cancer, especially targeting HBV. The siRNA, miRNA, ribozyme and antisense oligonucleotide of the present invention can be designed according to conventional methods.

"siRNA, miRNA, ribozyme and antisense oligonucleotide obtained according to the RNA interference target sequence of the present invention" refers to siRNA, miRNA, ribozyme and antisense oligonucleotide obtained by design expression or design synthesis acid, the target sequence (which can be a DNA or RNA sequence) acts on or contains the RNA interference target sequence involved in the present invention.

The conventional design method of siRNA can refer to the literature (such as: Reynolds A et al., Nature Biotechnology, 2004, volume 22: 326-330) or the public information on the websites of companies such as Amhion and Qiagen or the description in Example 1. The conventional design method of miRNA can refer to the literature (Lo HL et al., Gene Therapy, 2007, Volume 14: 1503-1512). The method of selecting the target sequence is similar to the design method of siRNA, for example, the designed sense gene containing the target sequence can be Strand and the corresponding antisense strand are replaced on the pri-microRNA, so that the constructed miRNA can prevent the expression of mRNA containing the target sequence. The conventional design method of ribozyme can refer to the literature (Haseloff J et al., Nature, 1988, Volume 334: 585-591 pages), for example, the nucleotide sequence complementary to the front and rear sequences of the target sequence can be placed in the ribozyme conservative Before and after the sequence of the core (such as the hammerhead structure), the constructed ribozyme can cut the nucleic acid containing the target sequence at the target sequence. Conventional design methods of antisense oligonucleotides can be referred to (Matveeva OV et al., Nucleic Acid Research, 2003, Vol. 31: pp. 4989-4994).

The promoter used in the present invention can be any promoter suitable for expressing a gene of interest in a cell. Can be constitutive or inducible. It can also be a composite promoter, such as a double promoter.

"Operably linked" means that the linked molecules are linked in such a way that the intended function is achieved. For example, the operative linkage of the expression control sequence and the gene coding sequence can realize the expression control effect of the expression control sequence on the gene coding sequence.

"Expression control sequence" is a control sequence required for gene expression, which is well known in the art. Usually a promoter must be included, often a transcription termination sequence is also included, and other sequences, such as enhancer sequences, may also be included. Gene expression refers to transcription for siRNA, miRNA, ribozymes and antisense oligonucleotides, and may also include post-transcriptional processing; for protein coding sequences, it usually refers to transcription and translation to produce mature proteins.

The invention provides an RNA interference target that can target HBV and siRNA, miRNA, ribozyme and antisense oligonucleotide designed according to the target. The siRNA, miRNA, ribozyme and antisense oligonucleotides of the present invention include chemical modification of the phosphate backbone and/or ribose and/or bases and other constituents of the siRNA, miRNA, ribozyme and antisense oligonucleotides Modified products, the modification method is known in the art, can be sulfur modification and/or sterol modification and/or PEG modification and/or sugar modification and/or LNA modification, etc., can refer to literature: Dykxhoorn DM etc., biology Annual Review of Medical Engineering, 2006, Vol. 8: pp. 377-402; Behlke MA et al., Molecular Therapy, 2006, Vol. 13: pp. 644-670.

In a specific embodiment, the present invention relates to small-molecule interfering ribonucleic acid (siRNA), which includes sense RNA fragments and antisense RNA fragments, said sense RNA fragments comprising RNA sequences encoded by RNA interference targets of the present invention, antisense RNA The fragment and the sense RNA fragment can form double-stranded RNA, and the double-stranded RNA can inhibit the expression of HBV corresponding genes and/or the replication and/or infection of HBV.

In the present invention, the terms "small molecule ribonucleic acid", "small molecule interfering ribonucleic acid" or "siRNA" are used interchangeably, and they all refer to the ability to inhibit the expression of HBV target genes, including sense RNA fragment regions and antisense RNA fragment regions ribonucleic acid (RNA).

Relatedly, the present invention also provides a combination of DNA sequences comprising or consisting of a first DNA sequence encoding a sense RNA segment comprising a target sequence of the invention encoded by a second DNA sequence encoding an antisense RNA segment The RNA sequence, the antisense RNA fragment and the sense RNA fragment can form a double-stranded RNA, and the double-stranded RNA can (by RNA interference) inhibit the expression of HBV genes and/or the replication and/or infection of HBV.

In this aspect of the invention, the sense RNA fragment and the antisense RNA fragment can be present on two different RNA strands or on one RNA strand, for example comprising a sense RNA fragment and an antisense RNA in a single stranded RNA molecule fragment.

For example, the siRNA of the present invention can be a hairpin single-stranded RNA molecule, wherein the region of complementarity between the sense RNA segment and the antisense RNA segment forms a double-stranded RNA region.

The length of the sense RNA fragment and the antisense RNA fragment is preferably 8-50 nucleotides, preferably 10-30 (more preferably 15-27, 19-23, such as 19, 20 or 21) nucleotides. But it can also be longer or shorter.

There are at least 10 (preferably 15, more preferably 18, such as 19, 20 or 21) base pairs in the complementary region of the double-stranded RNA formed by the sense RNA segment and the antisense RNA segment. Preferably, the complementary region between the sense RNA fragment and the antisense RNA fragment contains 19, 20, or 21 complementary base pairs.

In one embodiment, a small number of mismatches, such as 1-5, such as 1 or 2 or 3 or 4 base mismatches are allowed between the sense RNA fragment and the antisense RNA fragment. In a preferred embodiment, the sense RNA segment and the antisense RNA segment are fully complementary.

In one embodiment, the siRNA of the present invention is a double-stranded RNA molecule having 10-30 (preferably, 15-27, more preferably 19-23) base pairs, at least 10 (preferably 15) , more preferably 18) base pairing.

In a preferred embodiment, the GC content of the sense RNA fragment and the antisense RNA fragment is 35%-75%, such as 40-60%, 45-55%, 48-52%, such as about 50%.

In a preferred embodiment, the sense RNA segment and the antisense RNA segment have no significant identity to known human genes and gene expression segments. Significant agreement means at least 60%, such as 70, 80, 90% agreement.

Preferably, the number of nucleotides whose base is guanine (G) and the number of nucleotides whose base is cytosine (C) in the 19 nucleotide sequence starting from the 5' end of the sense RNA fragment The sum accounted for 35%-75% (i.e. G/C ratio) of the 19 nucleotides except TT at the 3' end. There was no significant identity between human genes and gene expression fragments.

In one embodiment of the recombinant expression vector of the present invention, the recombinant expression vector of the present invention comprises the coding nucleic acid sequences of the RNA interference targets involved in the present invention, and these coding nucleic acid sequences are operably linked to expression control sequences, so that they can be expressed in animal cells ( In particular, mammalian cells, such as human cells, such as liver cells or stem cells) express the siRNA and/or miRNA and/or ribozyme and/or antisense oligonucleotides targeting HBV.

Similarly, in the method for preparing the modified cells of the present invention, cells (including animals such as mammals, preferably human cells) can be transformed or transfected or transduced with an expression vector comprising the nucleic acid sequence encoding the RNA interference target involved in the present invention. , preferably liver cells and stem cells) to obtain the modified cells of the present invention, as long as the finally obtained cells contain the coding nucleic acid sequence of the RNA interference target involved in the present invention.

The transformed cells of the present invention can also be obtained by introducing siRNA and/or miRNA and/or ribozymes and/or antisense oligonucleotides obtained from the RNA interference target provided by the present invention into the cells, as long as the obtained It only needs that the cells contain siRNA and/or miRNA and/or ribozyme and/or antisense oligonucleotide obtained from the RNA interference target provided by the present invention.

The recombinant expression vector of the present invention may be a plasmid vector, or a virus vector (such as a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, etc.).

The engineered cells of the present invention are preferably mammalian (preferably human) cells, preferably hepatic cells, preferably stem cells. The cells carry the coding nucleic acid sequences of the RNA interference target involved in the present invention in the genome or outside the genome, and these coding nucleic acid sequences are operably linked to expression control sequences, so that the siRNA and/or miRNA can be expressed in the cell and/or ribozymes and/or antisense oligonucleotides.

The recombinant vector and modified cell of the present invention can be used for the treatment of HBV infection.

In specific embodiments, the following are involved:

1. RNA interference target sequence targeting HBV:

siHBV1: AGGACCCCTGCTCGTGTTACA (SEQ ID NO.1)

siHBV2: GGACCCCTGCTCGTGTTACAG (SEQ ID NO. 2)

siHBV3: GACCCCTGCTCGTGTTACAGG (SEQ ID NO.3)

siHBV4: ACCCCTGCTCGTGTTACAGGC (SEQ ID NO.4)

siHBV5: AGAGTCTAGACTCGTGGTGGA (SEQ ID NO.5)

siHBV6: AGTCTAGACTCGTGGTGGACT (SEQ ID NO. 6)

siHBV7: GAGTCTAGACTCGTGGTGGAC (SEQ ID NO. 7)

siHBV8: GTCTAGACTCGTGGTGGACTT (SEQ ID NO.8)

siHBV9: AGACTCGTGGTGGACTTCTCT (SEQ ID NO.9)

siHBV10: GACTCGTGGTGGACTTCTCTC (SEQ ID NO. 10)

siHBV11: ACTCGTGGTGGACTTCTCTCA (SEQ ID NO. 11)

siHBV12: GATGTGTCTGCGGCGTTTTAT (SEQ ID NO. 12)

siHBV13: GGATGTGTCTGCGGCGTTTTA (SEQ ID NO. 13)

siHBV14: ATGTGTCTGCGGCGTTTTTATC (SEQ ID NO. 14)

siHBV15: GTGTCTGCGGCGTTTTTATCAT (SEQ ID NO. 15)

siHBV16: ATCCTGCTGCTATGCCTCATC (SEQ ID NO. 16)

siHBV17: GCTGCTATGCCTCATCTTCTT (SEQ ID NO. 17)

siHBV18: AAGGTATGTTGCCCGTTTGTC (SEQ ID NO. 18)

siHBV19: AGGTATGTTGCCCGTTTGTCC (SEQ ID NO. 19)

siHBV20: GGTATGTTGCCCGTTTGTCCT (SEQ ID NO. 20)

siHBV21: GTATGTTGCCCGTTTGTCCTC (SEQ ID NO. 21)

siHBV22: ATGTTGCCCGTTTGTCCTCTA (SEQ ID NO. 22)

siHBV23: GCCGATCCATACTGCGGAACT (SEQ ID NO. 23)

siHBV24: GTGTGCACTTCGCTTCACCTC (SEQ ID NO. 24)

siHBV25: GTGCACTTCGCTTCACCTCTG (SEQ ID NO. 25)

siHBV26: GCACTTCGCTTCACCTCTGCA (SEQ ID NO. 26)

siHBV27: ACTTCGCTTCACCTCTGCACG (SEQ ID NO. 27)

siHBV28: GGAGGCTGTAGGCATAAATTG (SEQ ID NO. 28)

siHBV29: GAGGCTGTAGGCATAAATTGG (SEQ ID NO. 29)

siHBV30: AGGCTGTAGGCATAAATTGGT (SEQ ID NO. 30)

siHBV31: AAGCCTCCAAGCTGTGCCTTG (SEQ ID NO. 31)

siHBV32: AGCCTCCAAGCTGTGCCTTGG (SEQ ID NO. 32)

siHBV33: AGAAGAAGAACTCCCTCGCCT (SEQ ID NO. 33)

siHBV34: GAAGAAGAACTCCCTCGCCTC (SEQ ID NO. 34)

siHBV35: AAGAAGAACTCCCTCGCCTCG (SEQ ID NO. 35)

siHBV36: AGAAGAACTCCCTCGCCTCGC (SEQ ID NO. 36)

siHBV37: GAAGAAGAACTCCCTCGCCTC (SEQ ID NO. 37)

siHBV38: AAGAACTCCCTCGCCTCGCAG (SEQ ID NO. 38)

siHBV39: AGAACTCCCTCGCCTCGCAGA (SEQ ID NO. 39)

siHBV40: GAACTCCCTCGCCTCGCAGAC (SEQ ID NO. 40)

siHBV41: GATCCATACTGCGGAACTCCT (SEQ ID NO. 41)

siHBV42: AACTCCCTCGCCTCGCAGACG (SEQ ID NO. 42)

2. Expression vectors, preferably viral vectors, can be used to transform liver cells or stem cells.

Recombinant viral vectors capable of expressing single or multiple siRNAs, and/or miRNAs, and/or ribozymes targeting HBV.

The application of the viral vector on hepatocytes and/or stem cells can be used to stably express anti-HBV molecules, such as siRNA that can specifically block HBV replication in hepatocytes and/or stem cells.

3. Transformed cells, such as hepatocytes and stem cells, which contain the nucleic acid sequence encoding the RNA interference target of the present invention, can express the siRNA and/or miRNA and/or ribozyme and/or antisense oligonucleotide or be introduced into the siRNA and/or miRNA and/or ribozyme and/or antisense oligonucleotide obtained by the RNA interference target provided by the present invention.

Sequences of RNA interference targets (SEQ ID NO: 1-42)

Example

Example 1. Design and Construction of siRNA Expression Vector Plasmids Targeting HBV

The design of the RNA interference target sequence targeting HBV: take the HBV reference sequence as the target sequence, select a well-conserved region to walk and design the siRNA sequence; perform a BLAST search on the siRNA sequence obtained from the primary selection in GenBank, select and non- The target sequence has 3 or more bases different sequences as candidate sequences.

Construction of siRNA expression plasmid: In this example, the pSUPER vector (Cat.No VEC-PBS-0001/0002 of oligoengine company) was used as an example to construct an siRNA expression vector. For the specific construction process, please refer to the company's pSUPER vector experiment guide ( www.oligoengine.com), the construction process can be seen in schematic diagram 1. Primers with RNA interference sequences were synthesized, and the complementary primers were annealed and ligated to the pSUPER vector digested with Bgl II and Hind III, and the correct siRNA expression plasmid was obtained through enzyme digestion and sequencing.

Construction of the control siRNA expression plasmid: the siRNA sequence siRNA-luc (the specific sequence is 5'-GTGCGCTGCTGGTGCCAAC-3') specifically targeting the luciferase gene and the irrelevant siRNA sequence siRNA-Nk (the specific sequence is 5'-TGCATCGGAAAATAGATGT-3') as a control. The primers were synthesized by the above method and constructed on the pSUPER vector, and the corresponding siRNA expression plasmids pSUPER-luc and pSUPER-Nk were respectively obtained after enzyme digestion and sequencing identification.

Example 2. Co-transfection experiment screening to obtain RNA interference targets that can inhibit HBV

pN31-N10 plasmid (this plasmid contains about 1.4 times the HBV genome (GenBank accession number: AY707087). The construction method is briefly described as follows: Mammalian cell expression vector plasmid pCDNA3.1(+) (Invitrogen Cat.No V790-20) and After SpeI digestion, the recovered vector was self-ligated to obtain plasmid pN31. Respectively, N1f (ACT AGT GGA TCC TTC GCGGGA CGT CC)/N1r (GAA TTC CAC TGC ATG GCC TGA G), N2f (GAA TTCCAC TGC CTT CCA CC)/N2r (GAT ATC CAC ATT GTG TAA ATG G), N3f (GAT ATC CTG CCT TAA TGC CTT TG)/N3r (GGG CCC ACA AAT TGTTGA CAC C) were used for PCR amplification of HBV genome, and the products were cloned into T The vectors were pTN1, pTN2, and pTN3. The fragment obtained after pTN3 was digested with EcoRV and ApaI was ligated with the vector recovered after pN31 was digested with EcoRV and ApaI to obtain pN31-N3. The fragment recovered from pTN1 was digested with SpeI and EcoRI and SpeI and pN31-N1N3 was obtained by pN31-N1N3 after EcoRI digestion of pN31-N3. pTN2 was digested with EcoRI and EcoRV, and the recovered fragment was ligated with pN31-N1N3 after EcoRI and EcoRV double digestion to obtain pN31-N10) for HBV infection Sexual cloning plasmid, after transfecting suitable mammalian cells (such as human liver-derived cells HepG2 cells, huh7 cells, etc.), has the ability to express HBV viral proteins and virus particles. HBsAg is the membrane protein of HBV. By detecting the content of HBsAg protein in the cell supernatant, it can reflect the expression level of viral protein and virus particles, and it is also positively correlated with the virus titer. Therefore, siRNA expression plasmids can be co-transfected with HBV infectious cloning plasmids (pN31-N10 plasmids, or other HBV infectious cloning plasmids) in Huh-7 cells, and HBsAg in cells after co-transfection can be detected. The protein expression level was used to test the efficiency of different siRNAs in inhibiting HBV expression and replication.

Huh-7 cells (Japanese Collection of Research Bioresources, JCRB0403) were cultured in 24-well cell culture plates, and the medium was DMEM medium (adding 10% FBS, 2mM L-glutamine, 0.1mM MEM Non-Essential Amino Acids and 1% penicillin-streptomycin), the confluence rate was about 70%. After 12 hours, each well of cells was transfected with 0.1 μg pN31-N10 plasmid and 1 μg siRNA expression plasmid. The transfection reagent was Lipofectamine2000 (Invitrogen Cat. No. 11668-027). For the transfection method, refer to the operation guide of the reagent. After 48 hours of co-transfection, the cell culture supernatants were collected separately, and after serial dilution, they were detected by HBsAg protein detection kit (Beijing Wantai, Guoyao Zhunzi S10980090) or HBeAg protein detection kit (Beijing Wantai, Guoyao Zhunzi S10980088) HBsAg or HBeAg protein activity in cell culture supernatants. Taking the activity of HBsAg or HBeAg protein in the culture supernatant of Huh-7 cells co-transfected with the control siRNA expression plasmid and the HBV infectious cloning plasmid as a control, the inhibitory efficiency of each siRNA on HBV was calculated. By comparison, 42 RNA interference targets that can inhibit HBV were obtained. Accompanying drawing 2 shows the inhibition efficiency of HBV viral gene expression after transfection into cells by the siRNA expression plasmids constructed with these 42 RNA interference target sequences respectively.

The following are the RNA interference target sequences that we have obtained that can be used to inhibit HBV:

siHBV1: AGGACCCCTGCTCGTGTTACA (SEQ ID NO.1)

siHBV2: GGACCCCTGCTCGTGTTACAG (SEQ ID NO.2)

siHBV3: GACCCCTGCTCGTGTTACAGG (SEQ ID NO.3)

siHBV4: ACCCCTGCTCGTGTTACAGGC (SEQ ID NO.4)

siHBV5: AGAGTCTAGACTCGTGGTGGA (SEQ ID NO.5)

siHBV6: AGTCTAGACTCGTGGTGGACT (SEQ ID NO. 6)

siHBV7: GAGTCTAGACTCGTGGTGGAC (SEQ ID NO. 7)

siHBV8: GTCTAGACTCGTGGTGGACTT (SEQ ID NO.8)

siHBV9: AGACTCGTGGTGGACTTCTCT (SEQ ID NO. 9)

siHBV10: GACTCGTGGTGGACTTCTCTC (SEQ ID NO. 10)

siHBV11: ACTCGTGGTGGACTTCTCTCA (SEQ ID NO. 11)

siHBV12: GATGTGTCTGCGGCGTTTTAT (SEQ ID NO. 12)

siHBV13: GGATGTGTCTGCGGCGTTTTA (SEQ ID NO. 13)

siHBV14: ATGTGTCTGCGGCGTTTTTATC (SEQ ID NO. 14)

siHBV15: GTGTCTGCGGCGTTTTTATCAT (SEQ ID NO. 15)

siHBV16: ATCCTGCTGCTATGCCTCATC (SEQ ID NO. 16)

siHBV17: GCTGCTATGCCTCATCTTCTT (SEQ ID NO. 17)

siHBV18: AAGGTATGTTGCCCGTTTGTC (SEQ ID NO. 18)

siHBV19: AGGTATGTTGCCCGTTTGTCC (SEQ ID NO. 19)

siHBV20: GGTATGTTGCCCGTTTGTCCT (SEQ ID NO. 20)

siHBV21: GTATGTTGCCCGTTTGTCCTC (SEQ ID NO. 21)

siHBV22: ATGTTGCCCGTTTGTCCTCTA (SEQ ID NO. 22)

siHBV23: GCCGATCCATACTGCGGAACT (SEQ ID NO. 23)

siHBV24: GTGTGCACTTCGCTTCACCTC (SEQ ID NO. 24)

siHBV25: GTGCACTTCGCTTCACCTCTG (SEQ ID NO. 25)

siHBV26: GCACTTCGCTTCACCTCTGCA (SEQ ID NO. 26)

siHBV27: ACTTCGCTTCACCTCTGCACG (SEQ ID NO. 27)

siHBV28: GGAGGCTGTAGGCATAAATTG (SEQ ID NO. 28)

siHBV29: GAGGCTGTAGGCATAAATTGG (SEQ ID NO. 29)

siHBV30: AGGCTGTAGGCATAAATTGGT (SEQ ID NO. 30)

siHBV31: AAGCCTCCAAGCTGTGCCTTG (SEQ ID NO. 31)

siHBV32: AGCCTCCAAGCTGTGCCTTGG (SEQ ID NO. 32)

siHBV33: AGAAGAAGAACTCCCTCGCCT (SEQ ID NO. 33)

siHBV34: GAAGAAGAACTCCCTCGCCTC (SEQ ID NO. 34)

siHBV35: AAGAAGAACTCCCTCGCCTCG (SEQ ID NO. 35)

siHBV36: AGAAGAACTCCCTCGCCTCGC (SEQ ID NO. 36)

siHBV37: GAAGAAGAACTCCCTCGCCTC (SEQ ID NO. 37)

siHBV38: AAGAACTCCCTCGCCTCGCAG (SEQ ID NO. 38)

siHBV39: AGAACTCCCTCGCCTCGCAGA (SEQ ID NO. 39)

siHBV40: GAACTCCCTCGCCTCGCAGAC (SEQ ID NO. 40)

siHBV41: GATCCATACTGCGGAACTCCT (SEQ ID NO. 41)

siHBV42: AACTCCCTCGCCTCGCAGACG (SEQ ID NO. 42)

Through the above experiments, we have proved that the 42 RNA interference targets of the present invention can be used to inhibit the expression of HBV genes.

In the following experiments, we selected siHBV7 and siHBV12 from the above-mentioned RNA interference targets as examples, and further constructed recombinant virus vectors capable of expressing siRNA targeting siHBV7 and siHBV12. We take the construction of a recombinant lentivirus capable of expressing siRNA targeting siHBV7 and siHBV12 as an example. See Example 3 and Example 4 for the construction method.

Example 3. Construction of recombinant viral expression vector expressing siRNA

We take the construction of a recombinant lentiviral expression vector that can express siRNA targeting siHBV7 and siHBV12 as an example.

Expression vector: In this example, the expression vector pDEST-MR of the lentiviral system we used (patent application number: 200510112917.1; publication number: CN1948475) contains an expression cassette that can be used to express siRNA controlled by the H1 promoter.

The construction method of expression vector pDEST-siHBV7, pDEST-siHBV12:

Synthesize siHBV7 and siHBV12 gene fragments respectively (the RNA interference target sequence siHBV7 (SEQ ID NO: 7) and siHBV12 (SEQ ID NO: 12) shown in Example 2 are included here as examples, but other inventions can also be included RNA interference target sequence provided), the 5' end of the fragment was added with an Age I site, and the 3' end was added with a Sma I site; the gene fragment was digested with Age I and Sma I and then digested with the pDEST-MR plasmid Connection, construction of expression vectors pDEST-siHBV7, pDEST-siHBV12.

Example 4. Construction of recombinant virus expressing siRNA

Let's take the construction of recombinant lentivirus that can express siRNA targeting siHBV7 and siHBV12 as an example.

In addition to the expression vector plasmid that can express siRNA targeting HBV, other plasmids required for the construction of recombinant lentivirus are pLP1, pLP2, and VSVG, purchased from Invitrogen, the product name is: pLenti4/V5-DEST Gateway Vector Kit, product number: V469- 10.

Preparation method of recombinant lentivirus:

(1) Use the cesium chloride-ethidium bromide density gradient centrifugation method to extract a large number of four plasmids pVSVG, pLP1, pLP2, and expression vector plasmids (such as the pDEST-siHBV7 plasmid and pDEST-siHBV12 plasmid used as examples in this example) ( For the extraction method, please refer to "Molecular Cloning Experiment Guide", written by J. Sambrook and D.W. Russell, Science Press, 2002);

(2) 293FT cells (Invitrogen, Catalog#R700-07) were cultured in DMEM medium (adding 10% FBS, 2mM L-glutamine, 0.1mM MEM Non-Essential Amino Acids and 1% penicillin-streptomycin);

(3) Culture 293FT cells on a cell culture plate with a diameter of 10 cm, and the confluence rate is about 70%. After 12 hours, the calcium phosphate transfection method was used to mediate the co-transfection of 4 plasmids including 10 μg pLP1, 10 μg pLP2, 10 μg pVSVG, and 20 μg expression vector plasmid (for the method, refer to "Molecular Cloning Experiment Guide", written by J. Sambrook D.W. Russell , Science Press, 2002);

(4) Collect the cell culture supernatant 48 hours after transfection, filter it through a 0.45 μm filter membrane; centrifuge at 25,000 rpm at 4°C for 90 minutes with a SW28 rotor (BECKMAN);

(5) Discard the supernatant, add 500 μL PBS to dissolve the precipitate;

(6) Aliquot the virus collection solution and store it at -80°C for later use.

Thus, recombinant lentiviruses capable of expressing siRNAs targeting siHBV7 and siHBV12 were obtained, respectively called Lenti-siHBV7 and Lenti-siHBV12.

Embodiment 5. Inhibitory effect of siRNA targeting HBV introduced into HepG2-N10 cell line through expression vector plasmid

The HepG2-N10 cell line (Pan Jinshui et al., World Chinese Journal of Digestion, 2006, Volume 14: 1172-1177) is derived from human hepatocytes, carries the HBV genome, and can express HBV antigenic proteins and HBV virions. We applied HepG2-N10 cells to verify the inhibitory effect of siRNA on HBV.

We take the expression vector plasmids that can express siRNA targeting HBV as an example (in this example, we take the expression vector plasmids pSUPER-siHBV7 and pSUPER-siHBV12 that can express siRNA targeting siHBV7 and siHBV12 respectively as an example, but also Can include other expression vector plasmids that can express siRNA targeting the RNA interference target provided by the present invention), indicating that the siRNA targeting HBV is transfected with the expression vector plasmid to transform the cells so that the cells can obtain inhibition of HBV gene expression and replication Ability.

Experimental method: The siRNA expression plasmids pSUPER-siHBV7 and pSUPER-siHBV12 were transfected in HepG2-N10, and the HBsAg and HBeAg protein activities and HBV DNA copy numbers in the supernatant of the transfected cells were detected to inhibit HBV with different siRNAs. The efficiency of expression replication was tested. HepG2-N10 was cultured in a 24-well cell culture plate in DMEM medium (supplemented with 10% FBS, 2mM L-glutamine, 0.1mM MEM Non-Essential Amino Acids and 1% penicillin-streptomycin), and the confluence rate was about 80%. After 12 hours, each well of cells was transfected with 2 μg of siRNA expression plasmid. The transfection reagent was Lipofectamine2000 (Invitrogen Cat. No11668-027). For the transfection method, refer to the operation guide of the reagent. After 48 hours of transfection, the cell culture supernatants were collected respectively, and after serial dilution, the HBsAg protein activity in the cell culture supernatant was detected by the HBsAg detection kit, and the hepatitis B virus (HBV) nucleic acid amplification (PCR) fluorescent quantitative reagent was used The copy amount of HBV DNA in the cell culture supernatant was detected with a kit (Shanghai Kehua Bioengineering Co., Ltd., S20030059). Experimental controls were untransfected HepG2-N10 cells and HepG2-N10 cells transfected with the control siRNA expression plasmid pSUPER-Nk.

The results are shown in Figure 3 and Figure 4, HepG2-N10 cells transfected with pSUPER-siHBV7 and pSUPER-siHBV12 can all show the ability to inhibit the expression and replication of HBV. This indicated that the anti-HBV siRNA was expressed in the cells transfected with the expression vector plasmid carrying the HBV-targeted siRNA expression sequence, thereby inhibiting the expression and replication of HBV.

Embodiment 6. Inhibitory effect of siRNA targeting HBV introduced into HepG2-N10 cell line by recombinant virus

Let’s take the recombinant lentivirus that can express siRNA targeting HBV as an example (in this example, we take the recombinant lentivirus Lenti-siHBV7 and Lenti-siHBV12 that can express siRNA targeting siHBV7 and siHBV12 respectively as an example, but also Can include other recombinant viruses that can express siRNA targeting the RNA interference target provided by the present invention), indicating that the siRNA targeting HBV transduces cells through recombinant viruses, transforming cells so that cells obtain the ability to inhibit HBV gene expression and replication .

Experimental method: HepG2-N10 cells were transduced with lentiviruses Lenti-siHBV7 and Lenti-siHBV12 at moi=40, centrifuged at 600g for 60min and then changed; the control recombinant lentivirus Lenti-luc with siRNA expression elements targeting luciferase gene ( The sequence of the siRNA expression element targeting the lucifezase gene is the same as that in Example 1; the control virus was prepared according to the methods in Examples 3 and 4) HepG2-N10 cells were transduced at moi=40, centrifuged at 600g for 60min and then changed; the transduced HepG2 -N10 cells were cultured at 37° C., and the cell culture supernatant was collected after 72 hours, and the HBsAg protein content in the cell culture supernatant was detected with an HBsAg protein detection kit. At the same time, the hepatitis B virus nucleic acid amplification (PCR) fluorescent quantitative kit was used to detect the copy amount of HBV DNA in the cell culture supernatant. Experimental controls were untransduced HepG2-N10 cells and HepG2-N10 cells transduced by recombinant lentivirus Lenti-luc.

The results are shown in Figure 5 and Figure 6, HepG2-N10 cells transduced by recombinant lentiviruses Lenti-siHBV7 and Lenti-siHBV12 can all show the ability to inhibit HBV expression and replication. This indicated that the anti-HBV siRNA was expressed in the cells transduced by the recombinant lentivirus carrying the HBV-targeting siRNA expression sequence, thereby inhibiting the expression and replication of HBV.

Embodiment 7. Inhibitory effect of siRNA targeting HBV on HBV in mouse model

Experimental animals: SPF grade Balb/c mice, 7-8 weeks old, weighing about 18-22 g. 6 in each group, repeat 3 times.

In this example, we take the expression vectors pSUPER-siHBV7 and pSUPER-siHBV12, which can respectively express siRNA targeting siHBV7 and siHBV12, as an example to illustrate that after the siRNA targeting HBV is introduced into living mouse cells, the cells can obtain the ability to inhibit HBV Ability.

Experimental method: siRNA expression plasmids pSUPER-siHBV7 (40 μg/mouse), pSUPER-siHBV12 (40 μg/mouse), control plasmid pSUPER-Nk (40 μg/mouse) and pN31-N10 plasmid (20 μg/mouse) were passed through the mouse tail Intravenous high-pressure injection method (hydrodynamic transfection) for co-injection, the total injection volume is about 2ml, and the injection is completed within 5 seconds. Collect mouse serum every day before and after injection, use HBsAg protein detection kit to detect the content of HBsAg protein in mouse serum, and use hepatitis B virus nucleic acid amplification (PCR) fluorescent quantitative kit to detect HBV DNA in mouse serum copy amount. Experimental controls were normal Balb/c mice, Balb/c mice injected only with pN31-N10 plasmid, and Balb/c mice co-injected with pN31-N10 plasmid and control siRNA expression plasmid pSUPER-Nk.

The test results (Figure 7, Figure 8) showed that injection of expression plasmids expressing siRNA targeting HBV into mice could inhibit the expression of HBsAg, and also inhibited the HBV DNA load in mouse serum. This indicates that anti-HBV siRNA produced in in vivo cells transduced with an expression vector carrying an HBV-targeted siRNA expression sequence can inhibit the expression and replication of HBV.

Example 8. The inhibitory effect of chemically synthesized siRNA on HBV

We selected siHBV7 and siHBV12 from the above RNA interference targets as examples (the RNA interference target sequences siHBV7 (SEQ ID NO: 7) and siHBV12 (SEQ ID NO: 12) shown in Example 2 are included here as examples , but can also include other RNA interference target sequences provided by the present invention), synthesized siRNAs that can target siHBV7 and siHBV12 respectively, and the positive-sense RNA fragments of the siRNAs respectively contain the target sequences siHBV7 (SEQ ID NO: 7) and siHBV12 of the present invention (SEQ ID NO: 12) encoded RNA sequence, the antisense RNA fragment can be complementary to the sense RNA fragment to form double-stranded RNA (in this embodiment, the antisense strand is completely complementary to the sense strand, but it is also possible to allow the antisense strand and The sense strand has a small number of mismatches, such as 1 or 2 or 3 or 4), and dTdT is added to the 3' ends of the sense RNA fragment and the antisense RNA fragment, respectively. The above siRNAs were named siR-HBV7 and siR-HBV12, respectively. At the same time, siRNA (siR-Nk) targeting an irrelevant RNA interference target siRNA-Nk that does not match HBV and human genes was synthesized (siRNA-Nk sequence is the same as that in Example 1) as a control.

The synthesis method of siRNA is briefly described as follows: the sense RNA fragment and the antisense RNA fragment of the siRNA are synthesized respectively by using the β-acetonitrile phosphorous acid amine chemical synthesis method, and the synthesized sense RNA fragment and the antisense RNA fragment are synthesized by using an automatic DNA synthesizer. The molar ratio is mixed, and the desired siRNA is obtained through denaturation and annealing processes on a PCR machine.

Experimental method: The synthetic siR-HBV7, siR-HBV12 and siR-Nk were transfected into HepG2-N10 cells respectively, and different siRNAs were inhibited by detecting the HBsAg and HBeAg protein activities and the HBV DNA copy number in the supernatant of the transfected cells. The efficiency of HBV expression replication was tested. HepG2-N10 cells were cultured in 24-well cell culture plates with a confluence of about 80%. After 12 hours, each well of cells was transfected with 50pM siRNA, and the transfection reagent was Lipofectamine2000. For the transfection method, refer to the operation guide of the reagent. The cell culture supernatants were collected 48 hours after transfection, and the activity of HBsAg protein in the cell culture supernatant was detected by the HBsAg detection kit, and the HBsAg protein activity in the cell culture supernatant was detected by the hepatitis B virus nucleic acid amplification (PCR) fluorescence quantitative kit. The copy amount of HBV DNA. Experimental controls were untransfected HepG2-N10 cells and HepG2-N10 cells transfected with control siR-Nk.

The results are shown in Fig. 9 and Fig. 10, after the synthetic siR-HBV7 and siR-HBV12 were transfected into HepG2-N10 cells, both of them could inhibit the expression and replication of HBV.

Example 9. The inhibitory effect of chemically synthesized and modified siRNA on HBV

We selected siHBV7 and siHBV12 from the above RNA interference targets as examples (the RNA interference target sequences siHBV7 (SEQ ID NO: 7) and siHBV12 (SEQ ID NO: 12) shown in Example 2 are included here as examples , but can also include other RNA interference target sequences provided by the present invention), synthesized siRNAs that can target siHBV7 and siHBV12 respectively, and the positive-sense RNA fragments of the siRNAs respectively contain the target sequences siHBV7 (SEQ ID NO: 7) and siHBV12 of the present invention (SEQ ID NO: 12) encoded RNA sequence, antisense RNA fragments and sense RNA fragments are complementary to form double-stranded RNA (in this embodiment, the antisense strand is completely complementary to the sense strand, but it is also possible to allow the antisense strand and sense strand has a small number of mismatches, such as 1 or 2 or 3 or 4), add dTdT to the 3' ends of the sense RNA fragment and the antisense RNA fragment, respectively. At the same time, different modification methods were used in the synthesis process. Among them, siRpo-HBV7 and siRpo-HBV12 are siRNA targeting siHBV7 and siHBV12 respectively after 2'-OMe modification (2'-methoxy modification) and phosphorylation modification (synthetic method: using β-acetonitrile phosphoramidite chemistry Synthesis method, using a fully automatic DNA synthesizer to synthesize the sense RNA fragment and antisense RNA fragment of siRNA respectively, in which the three bases at the 5' end of the sense RNA fragment and the antisense RNA fragment and the three bases in front of the 3' end dTdT Use 2'-OMe modified single nucleotide synthesis, phosphorylate the 5' terminal base of the antisense RNA fragment. The synthesized positive sense RNA fragment and antisense RNA fragment are mixed in equimolar ratio and denatured on the PCR machine , annealing process to obtain the required siRNA); siRpoC-HBV7 and siRpoC-HBV12 are siRNAs targeting siHBV7 and siHBV12 respectively after 2'-OMe modification, phosphorylation modification and sterol modification (synthetic method is the same as above, different places When synthesizing positive-sense RNA fragments, the three bases in front of dTdT at the 3' end are not modified with 2'-OMe, and the Glass carrier containing cholesterol-aminocaproic-acid-pyrrolidine (cholesterol-aminocaproic-acid-pyrrolidine) linker is used as Synthesize the support, and the base in front of dTdT at the 3' end is connected to the cholesterol group through phosphorothioate. The rest of the steps are the same as the method for synthesizing siRpo-HBV7 and siRpo-HBV12 above). At the same time, the same modified siRNAs (siRpo-Nk and siRpoC-Nk) targeting irrelevant RNA interference target siRNA-Nk that did not match HBV and human genes were synthesized as controls. This includes the 2'-OMe modification and/or phosphorylation of the synthetic siRNAs targeting the RNA interference target sequences siHBV7 (SEQ ID NO: 7) and siHBV12 (SEQ ID NO: 12) shown in Example 2, respectively. Bl modification and/or sterol modification are used as examples, but may also include modifications in other different ways to synthetic siRNA targeting other RNA interference target sequences provided by the present invention.

Experimental method: The synthetic siRpo-HBV7, siRpo-HBV12, siRpoC-HBV7, siRpoC-HBV12, siRpo-Nk, siRpoC-Nk were transfected into HepG2-N10 cells respectively, and the HBsAg and HBeAg in the supernatant of the transfected cells were detected Protein activity and HBV DNA copy number were used to test the efficiency of different siRNAs in inhibiting HBV expression and replication. HepG2-N10 cells were cultured in 24-well cell culture plates with a confluence of about 80%. After 12 hours, each well of cells was transfected with 50pM siRNA, and the transfection reagent was Lipofectamine2000. For the transfection method, refer to the operation guide of the reagent. The cell culture supernatants were collected 48 hours after transfection, the HBsAg activity in the cell culture supernatant was detected with the HBsAg detection kit, and the HBV in the cell culture supernatant was detected with the hepatitis B virus nucleic acid amplification (PCR) fluorescence quantitative kit The number of copies of DNA. Experimental controls were untransfected HepG2-N10 cells and HepG2-N10 cells transfected with control siRpo-Nk and siRpoC-Nk.

The results are shown in Fig. 11 and Fig. 12. After the synthetic siRpo-HBV7, siRpo-HBV12, siRpoC-HBV7, and siRpoC-HBV12 were transfected into HepG2-N10 cells, they all inhibited the expression and replication of HBV.

Those skilled in the art will appreciate that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications can be made therein without departing from the spirit and scope of the invention. Therefore, the specific embodiments and examples of the invention should not be considered as limiting the scope of the invention. The invention is limited only by the appended claims. All documents cited in this application are hereby incorporated by reference in their entirety.

Claims (21)

1. The RNA interference target of targeting HBV, its sequence is selected from: (1) the sequence of SEQ ID NO.12; or (2) The complementary sequence of the above sequence. 2. A nucleic acid construct comprising the RNA interference target of claim 1. 3. A vector comprising the RNA interference target of claim 1. 4. obtain according to the RNA interference target spot described in claim 1, can suppress the expression of HBV corresponding gene and/or the replication of HBV and/or the siRNA of infection, its sequence is GAUGUGUCUGCGGCGUUUUAU UUCAAGAGA AUAAAACGCCGCAGACACAUC UU. 5. A recombinant expression vector capable of expressing the siRNA of claim 4. 6. The recombinant expression vector according to claim 5, which comprises coding nucleic acid sequences of siRNA targeting HBV, and these coding nucleic acid sequences are operably linked with expression control sequences, so that said siRNA can be expressed in animal cells. 7. The recombinant expression vector of claim 6, wherein said animal cell is a mammalian cell. 8. The recombinant expression vector of claim 7, wherein said mammalian cells are human cells. 9. The recombinant expression vector of claim 6, wherein said animal cells are hepatocytes. 10. The recombinant expression vector of claim 5, which is a plasmid vector or a viral vector. 11. The recombinant expression vector of claim 10, wherein said viral vector is a retroviral vector. 12. The recombinant expression vector of claim 11, wherein the retroviral vector is a lentiviral vector. 13. An isolated cell transformed or transfected or transduced with the recombinant expression vector of any one of claims 5-12. 14. An isolated engineered cell expressing or comprising the siRNA of claim 4. 15. The transformed cell of claim 14, which carries the coding nucleic acid sequence of the RNA interference target site described in claim 1 in the genome or outside the genome, and these coding nucleic acid sequences are operably linked with the expression control sequence, so that the The siRNA is expressed in the cells. 16. The engineered cell of claim 15, wherein said cell is a mammalian cell. 17. The engineered cell of claim 16, wherein said mammalian cell is a human cell. 18. The engineered cell of claim 15, wherein said cell is a hepatocyte. 19. A method for preparing the cell according to any one of claims 13-18, comprising transforming or transfecting or transducing the cell with the recombinant expression vector according to any one of claims 5-12. 20. Use of the siRNA according to claim 4, or the recombinant expression vector according to any one of claims 5-12, or the cell according to any one of claims 13-18 in the preparation of medicines for treating HBV infection or HBV patients. 21. Use of the siRNA according to claim 4, or the recombinant expression vector according to any one of claims 5-12, or the cell according to any one of claims 13-18 in the preparation of drugs for inhibiting HBV replication or HBV gene expression.

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