JPWO2006090906A1 - New method to overcome RNAi resistant virus strains - Google Patents

Abstract

At least one shRNA (short hairpin RNA) moiety capable of forming a siRNA capable of suppressing a virus with a viral RNA as a target sequence or at least one of the viral RNAs as a target sequence and capable of suppressing the expression of a protein involved in the life cycle of the virus at least one decoy RNA portion that contains a miRNA portion and can bind to a protein involved in the life cycle of the virus and suppress the virus; at least one anti-hybrid that hybridizes to a gene involved in the life cycle of the virus and can suppress the virus Sense RNA part, RNA part composed of at least one ribozyme capable of suppressing the expression of proteins involved in the viral life cycle, small quantity capable of suppressing the expression of proteins involved in the viral life cycle At least one RNA portion selected from the group consisting of at least one tRNase ZL-EGS RNA portion and at least one RNase P-EGS RNA portion capable of suppressing the expression of a protein involved in the life cycle of the virus A chimeric RNA molecule comprising two or more RNA moieties cleavably linked.

Description

  The present invention relates to a method for suppressing viral infection and treating a viral infection using RNAi, decoy RNA and / or antisense RNA.

RNA interference (RNAi) has been shown to be a powerful tool to inhibit various gene functions (Cogoni, C. et al., Antonio Van Leeuwenhoek Int J 1994, 65: 205-2092-14; Baucombe , DC Plant Mol Biol 1996, 32: 79-88; Kennerdell, JR et al, Cell 1998, 95: 1017-1026; Ngo, H. et al, Proc Natl Acad. Sci. 14687-14692; Timmons, L. et al, Nature 1998, 395, 854; Waterhouse, PM et al, Proc Natl Acad Sci USA 1998, 95, 13959-1. Lohmann, JU et al., Dev Biol. 1999, 214: 211-214; Sanchez-Alvarado, A. et al., Proc Natl Acad Sci USA 1999, 96, 5049-5054; A. Singh et al., Nature 2000, 407: 319-320; Wiany, F. et al., Nat Cell Biol 2000, 2, 70-75, Chuang, CF et al, Proc Natl Acad Sci USA 2000. Yang, S. et al., Mol CellBiol 2000, 21, 7807-7816; Svoboda, P., Stein, P., Hayashi, H., and Schul. tz, RM Selective reduction of dominant material mRNAs in mouse oocytes by RNA interference. 2000, 127, 4147-4156). The use of RNAi has also been extended to differentiated cultured cells (Elbashir, SM et al., Nature 2001, 411, 494-498; Rosel, KF et al., Nucleic Acid Res. 2003). 31 (15), 4417-4425). RNAi is caused by siRNA (small interfering RNA) or miRNA (micro RNA) (Lidardi, C. et al., Cell 107, 297-307 (2001); Sijen, T. et al., Cell 107, 465-476 ( 2001)). It has been found that there are five steps in the mechanism involving RNAi. In the first step, dsRNA (greater than 30 bp) is cleaved by an RNase III-like enzyme called Dicer to produce a 19-23 nucleotide fragment (Berstein, E. et al, Nature 2001, 409, 363-6; Ketting, R. F. et al., Genes and Development 2001, 15, 2654-9; Knight, SW et al., Science. 2001, 293, 2269-71). These short duplex siRNAs have special properties essential for the function of a 2 nucleotide 3 ′ overhang (Ebashir, SM et al, Gene and Development 2001, 15, 188-200). . As a result, it is speculated that the ATP-dependent RNA helicase recognizes these short duplexes and turns the siRNA duplex into two single-stranded RNAs (Nykanen, A. et al., Cell 2001, 107, 309- 21). Next, one of the single strands is incorporated into a macromolecular protein complex (Ebashir, SM et al, Gene and Development 2001, 15, 188-200) called RISC (RNA-induced silencing complex). Serve as a guide to guide the cleavage of homologous RNA sequences by components with endonuclease activity not yet identified (Ebashir, SM et al, Gene and Development 2001, 15, 188-200; Hammond, S M. et al., Nature 2001, 404, 293-6). Finally, RISC is reused to leave the cleaved RNA and catalyze the cleavage again. This role of RISC is essential for the silencing effect. It has been shown that siRNA expressed with a DNA template also suppresses gene expression as efficiently as exogenously introduced siRNA (Brummelkamp, TR et al., Science 2002, 296, 550). Paddison, PJ et al, Gene and Development 2002, 16, 948-58; Paul, CP et al, Nature Biotechnology 2002, 20, 505-8; ZengY et al, Molecular Cell 2002. , 9, 1327-33). RNAi effectively suppresses the replication of several different pathogenic viruses in cultured cells. (See Non-Patent Documents 1 to 5). Examples of these viruses include graymyelitis virus (Reomyelitis virus), RSV (respiratory synchronous virus) and HIV. In the report on HIV, silencing by siRNA has been achieved by targeting specific mRNA. Target RNAs include Gag-Pol, Env, Vif and small regulatory proteins Tat and Rev. These reports have shown that RNAi can efficiently target not only viral RNA but also genomic RNA before and after the integration phase of the viral infection cycle (Bitko, V. et al., BMC Microbiology 2001, 1, 34-46: Gitin, L. et al., Nature 2002, 418, 430-434: Coburn, GA et al., Journal of Virology 2002, 76, 9225-31: Jacque, J. M. et al. , Nature 2002, 418, 435-8: Novina, CD et al., Nat Med 2002, 8, 681-6). For efficient delivery of siRNA in animal models and human applications, retroviruses (Brummelkamp, TR et al., Cancer Cell 2002, 2, 243-7), adenovirus (Xia, H. et al., Nat Biotechnol 2002, 20, 1006-10) and lentivirus (Qin, X. et al., Proc. Natl Acad of Sci USA 2003.100, 183-8) based gene therapy vectors As a result, it was possible to stably and substantially express siRNA in various cells and tissues. However, the emergence of resistant virus strains due to mutations induced by siRNA in long-term culture has recently been reported (Atze T. Das et al., J. viol. 78: 2601-2605 (2004): Daniel Boden et al. al., J. viol. 77: 11531-11535 (2003)). This phenomenon appears to hold back the use of siRNA for HIV-1 therapeutic applications.
On the other hand, the use of decoy-type nucleic acid pharmaceutical molecules has been studied for the suppression of virus growth. As an example, a decoy is a short nucleic acid containing the same sequence as a transcription factor binding site on the viral genome, and when administered in the body, it inhibits the transcription factor from binding to the genome and suppresses the function of the gene. It is possible to suppress viral growth in the body by decoy for viral transcription factors. For example, decoy RNA that binds to the Tat transcription growth factor of HIV (R. Yamamoto et al., Genes Cells 5, 371-388 (2000)) or decoy RNA that suppresses NS3 protease of HCV (K. Fukuda et al.,). Eur. J. Biochem 267, 3865-3694 (2000)).

An object of the present invention is to provide a chimeric RNA containing a plurality of functional RNA moieties capable of exerting an action as at least one pharmaceutical of an RNA pharmaceutical such as an RNAi pharmaceutical or a decoy RNA pharmaceutical, and a pharmaceutical containing the chimeric RNA.
The present inventor examined a phenomenon in which the HIV-1 replication inhibitory effect by siRNA targeting HIV-1 RNA is lost due to mutation of HIV-1 gene. After application of siRNA, mutation occurs in HIV-1 RNA, and siRNA cannot recognize HIV-1 RNA. After time, siRNA cannot cleave HIV-1 RNA, but before mutation occurs in HIV-1 RNA, HIV- In view of the fact that the replication of 1 is considerably suppressed and the HIV-1 replication inhibitory effect is exerted to some extent, even when a mutation occurs in HIV-1 RNA, other RNA regions that are not mutated are targeted. It has been found that HIV-1 replication can be suppressed over a long period of time by suppressing HIV-1 RNA replication. That is, various HIV-1 infection treatment methods targeting various HIV-1 genes have been studied conventionally, but they all target one target sequence and attack one target molecule. It has been found that by using a plurality of attack molecules with a plurality of target sequences as targets, it is possible to reliably suppress the growth of HIV-1 even if mutation occurs in HIV-1. It was.
As an example, the present inventors have found that a second generation anti-HIV shRNA encoding a splittable HIV-1 vif shRNA expressed as a chimeric RNA portion and a decoy TAR RNA has a remarkable HIV-1 replication inhibitory effect. . Decoy TAR RNA competitively interacts with HIV-1 Tat protein (protein-RNA interaction), causing down-regulation of transactivation phenomenon from 5 ′ LTR promoter of HIV-1 transcription, and complementary inhibitor Work as (CIF). On the other hand, HIV-1 vif-shRNA is expressed in Jurkat cells stably transfected, and acts on post-transcriptional inhibition of genes originating from HIV-1 by RNA-RNA interaction. Genotypic analysis of the target virus revealed that mutations occurred in the vif shRNA target site of HIV-1 viral RNA in transgenic Jurkat cells expressing vif shRNA and decoy TAR chimeric RNA and shRNA alone. Moreover, the mutations increased with time, and virus mutants were generated one after another. On the other hand, no mutation occurred in the decoy RNA target site of HIV-1 viral RNA. Interestingly, gain of resistance due to mutations involving siRNA was observed only in cultures expressing control vifshRNA alone and random vif shRNA mutant decoy TAR chimeric RNA (vif shRNARan-mTAR). A novel strategy that combines and expresses double HIV-1 antigene molecules, splits them into single RNA molecules in cells, and utilizes both RNA-RNA and protein-RNA interactions is HIV-1 gene therapy Useful for.
That is, the aspects of the present invention are as follows.
[1] At least one shRNA (short hairpin RNA) portion or miRNA portion capable of forming a siRNA capable of suppressing a virus using a viral RNA as a target sequence, and further binding to a protein involved in the life cycle of the virus to suppress the virus At least one decoy RNA portion capable of suppressing at least one antisense RNA portion capable of suppressing the virus by hybridizing to a gene involved in the viral life cycle, at least one capable of suppressing the expression of a protein involved in the viral life cycle An RNA moiety comprising one ribozyme, an RNA moiety comprising at least one tRNase ZL-EGS capable of suppressing the expression of a protein involved in the viral life cycle, and a tag involved in the viral life cycle A chimeric RNA molecule comprising at least one RNA portion selected from the group consisting of at least one RNA portion consisting of RNase P-EGS capable of suppressing protein expression, wherein two or more RNA portions are cleavably linked .
[2] At least one shRNA or miRNA part capable of forming a siRNA capable of suppressing the virus using the viral RNA as a target sequence, and at least one decoy RNA part capable of suppressing the virus by binding to a protein involved in the life cycle of the virus Chimeric RNA molecules to which are cleavably linked.
[3] The chimeric RNA molecule according to [1] or [2], wherein a UU sequence is present between the shRNA part or the miRNA part and another RNA part.
[4] The chimeric RNA molecule according to any one of [1] to [3], wherein the virus is selected from the group consisting of HIV-1, HIV-2, HCV, cancer and influenza virus.
[5] shRNA part, miRNA part, antisense RNA part, RNA part consisting of ribozyme, RNA part consisting of tRNase ZL-EGS or RNA part consisting of RNase P-EGS is HIV-1 gag, pol, env, tat, [1] The chimeric RNA molecule that suppresses HIV-1 that targets a partial sequence of a region selected from the group consisting of rev, nef, vif, vpr, vpu, vpx, or LTR region.
[6] Target a partial sequence of a region selected from the group consisting of gag, pol, env, tat, rev, nef, vif, vpr, vpu, vpx, or LTR region of HIV-1 The chimeric RNA molecule according to [2], which suppresses HIV-1.
[7] The chimeric RNA molecule according to [5] or [6], wherein the decoy RNA portion has a sequence homologous to the TAR region of HIV-1 and can bind to a Tat protein.
[8] The chimeric RNA molecule according to [7], which has the following secondary structure.
[9] A vector containing the template DNA of the chimeric RNA molecule of any one of [1] to [8] and expressing the chimeric RNA.
[10] The vector according to [9], which is a viral vector.
[11] A prophylactic or therapeutic agent for viral infection and cancer comprising the chimeric RNA molecule according to any one of [1] to [8], which is cleaved into each RNA portion in vivo.
[12] A prophylactic or therapeutic agent for a viral infection comprising the vector according to [9] or [10], wherein each RNA moiety is generated in vivo.
[13] The prophylactic or therapeutic agent for viral infection and cancer according to [11] or [12], which can block viral RNAi resistance based on viral mutation.
[14] A virus comprising at least one shRNA or miRNA capable of forming a siRNA capable of suppressing a virus using a viral RNA as a target sequence and at least one decoy RNA capable of suppressing a virus by binding to a protein involved in the life cycle of the virus Preventive or therapeutic agent for infectious diseases.
[15] shRNA or miRNA capable of forming siRNA capable of suppressing virus using viral RNA as a target sequence is HIV-1, gag, pol, env, tat, rev, nef, vif, vpr, vpu, vpx, or LTR region [14] The preventive or therapeutic agent for viral infections, which targets a partial sequence of a region selected from the group consisting of
[16] The preventive or therapeutic agent for a viral infection according to [14] or [15], wherein the decoy RNA portion has a sequence homologous to the TAR region of HIV-1 and can bind to a Tat protein.
[17] The preventive or therapeutic agent for viral infections and cancer according to [15] or [16], which can block viral RNAi resistance based on viral mutations.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2005-051602, which is the basis for the priority of the present application.

FIG. 1A is a diagram showing the construction of a second generation shRNA expression vector.
FIG. 1B shows the construction of a lentivirus-based vector.
FIG. 2A shows the secondary RNA structure of vif-TAR chimeric RNA analyzed by GENETYX software, with a UU single-strand cleavage site between the 3 ′ end of vif shRNA and the 5 ′ end of TAR RNA .
FIG. 2B is a photograph showing the results of RT-PCR analysis of HeLa CD4 ⁺ cells.
FIG. 2C is a photograph showing Northern blot analysis of expression vector mRNA in HeLa CD4 ⁺ cells.
Figure 2D is a photograph showing a dicer present in HeLa CD4 ⁺ cells.
FIG. 2E shows in vivo cleavage of the chimeric RNA.
FIG. 2F is a photograph showing in vitro cleavage of the chimeric RNA.
FIG. 3A shows the HIV-1 antiviral effect.
FIG. 3B is a photograph showing down-modulation of HIV-1 reporter gene expression.
FIG. 3C is a diagram showing long-term suppression of HIV-1 replication in Jurkat by CS-vif shRNA-TAR.
FIG. 3D shows long-term suppression of HIV-1 replication in RBMCs by CS-vif shRNA-TAR.
FIG. 3E shows long-term suppression of HIV-1 replication in H9 cells by CS-vif shRNA-TAR.
FIG. 3F is a diagram showing the results of genotype analysis in Jurkat cells.
FIG. 3G is a diagram showing the results of genotype analysis in PBMCs.
FIG. 4 is a diagram showing the action of the chimeric RNA of the present invention on HIV-1.

Hereinafter, the present invention will be described in detail.
The chimeric RNA of the present invention comprises at least two functional RNA parts, the RNA part acting as an RNAi drug via the RNAi mechanism, the action as a decoy nucleic acid (RNA) drug via the decoy mechanism, and antisense RNA It exhibits at least one of the following actions as a medicine, an action as a ribozyme RNA medicine, an action as a tRNase ZL-EGS medicine, and an action as an RNase P-EGS medicine. Preferably, one RNA part of two or more RNA parts exerts an action as an RNAi drug via the RNAi mechanism, and the other RNA part is a decoy-type nucleic acid (RNA) drug via the decoy mechanism, Sense RNA drug, ribozyme RNAi drug, tRNase ZL-EGS drug, and RNase P-EGS drug function as at least one drug.
The chimeric RNA of the present invention can be used for virus suppression. Here, virus suppression refers to inhibiting virus replication and proliferation and making the virus unable to survive. For the treatment of viral infections, in order to suppress viral replication, the use of siRNA, antisense nucleic acids and ribozymes targeting genes involved in the viral replication mechanism has been studied. However, in RNA viruses such as HIV, mutations can occur at high frequency in viral genes. Therefore, the virus suppression effect of siRNA, antisense nucleic acid or ribozyme targeting a specific gene sequence disappears due to virus mutation. For example, when siRNA targeting the vif gene of HIV-1 is administered, mutation occurs in the HIV-1 gene in several weeks, the mutation gradually increases, and the gene expression suppression effect of siRNA decreases. Even if a mutation occurs in a gene involved in the life cycle of a virus that attacks to suppress the virus, the chimeric RNA of the present invention causes the virus to escape from the attack by simultaneously attacking the unmutated gene. It is possible to prevent viruses and efficiently and reliably suppress viruses. Here, genes involved in the life cycle of the virus refer to genes necessary for the virus to replicate or propagate, and include viral structural genes, accessory genes, genes encoding transcriptional regulatory factors, and the like. In the present invention, it is said that the siRNA target sequence of a virus is mutated to escape siRNA attack is to acquire siRNA resistance, and the anti-sense nucleic acid target sequence is mutated to escape an antisense nucleic acid attack. It is referred to as a mutation that acquires sense nucleic acid resistance. The same applies to other nucleic acid drugs. The chimeric RNA of the present invention can prevent the virus from acquiring resistance to a nucleic acid drug even if a mutation occurs in the virus.
The virus that suppresses replication by the chimeric RNA of the present invention is not limited, and may be a DNA virus or an RNA virus, but an RNA virus such as a single-stranded RNA virus or a double-stranded RNA virus that acquires resistance to nucleic acid drugs by mutation Is desirable. Such viruses include retroviridae, togaviridae, flaviviridae, coronaviridae, tetraviridae, nodaviridae, astroviridae, caliciviridae, picornaviridae, arenaviridae, bunyaviridae, Examples include viruses belonging to various families such as Orthomyxoviridae, Filoviridae, Rapdoviridae, Paramyxoviridae, Birnaviridae, and Reoviridae. Specifically, HIV-1, HIV-2, hepatitis C virus, HTLV-1, HTLV-2, influenza A virus, influenza B virus, human spumavirus, rubella virus, Sindbis virus, yellow fever Virus, hepatitis A virus, poliovirus, Lassa virus, SARS virus, coronavirus, mumps virus, RS virus, coxsackie virus, echo virus, Marburg virus, Ebola virus, proboscis fever virus, hepatitis G virus and the like.
The chimeric RNA of the present invention can also be used for the prevention and treatment of cancer caused by viral infection by suppressing viruses. The following cancers can be exemplified as a cancer caused by virus infection. Cervical cancer (human papillomavirus type 16, type 18 (HPV-16, 18)), Burkitt lymphoma (EB virus (EBV)), adult T-cell leukemia (human T lymphocyte-loving virus), liver cancer ( Hepatitis B virus (HBV), hepatitis C virus (HCV)), Kaposi sarcoma (Kaposi sarcoma-associated herpesvirus (KSHV)).
In order to exert such an action, the chimeric RNA of the present invention comprises at least one shRNA (short hairpin RNA) portion or miRNA portion, at least one decoy RNA portion, at least one antisense RNA portion, and at least one ribozyme. At least one RNA portion selected from the group consisting of an RNA portion consisting of at least one tRNase ZL-EGS and an RNA portion consisting of at least one RNase P-EGS. Preferably, the chimeric RNA of the present invention comprises at least one shRNA (short hairpin RNA) portion or miRNA portion, and further comprises an RNA portion comprising at least one decoy RNA portion, at least one antisense RNA portion, and at least one ribozyme. , At least one RNA portion selected from the group consisting of an RNA portion consisting of at least one tRNase ZL-EGS and an RNA portion consisting of at least one RNase P-EGS. In the present invention, when the chimeric RNA is composed of two types of RNA portions, it may be referred to as dual type RNA, and when it is composed of three or more types of RNA portions, it may be referred to as multitype RNA. A medicine containing the chimeric RAN of the present invention is called a chimeric RNA medicine or a dual type RNA medicine. In the present invention, the RNA portion refers to RNA having a sequence that is substantially the same as a gene involved in viral replication or a sequence that can hybridize with a gene that is highly complementary to a gene involved in viral replication. The chimeric RNA has a specific sequence, and has at least two RNA sequences of RNA sequences that function as RNAi drugs, decoy drugs, antisense RNA drugs, ribozyme drugs, tRNase ZL-EGS or RNase P-EGS drugs. It has a connected structure. The linked RNA sequences are different RNA sequences, but the combination of the functions of the linked RNA sequences as a drug may be any combination. For example, when there are two types of RNA portions to be linked, RNAi and RNAi, decoy RNA and Decoy RNA, antisense RNA and antisense RNA, ribozyme and ribozyme, tRNase ZL-EGS and tRNase ZL-EGS, RNase P-EGS and RNase P-EGS, RNAi and decoy RNA, RNAi and antisense RNA, RNAi and ribozyme, RNAi and tRNase ZL-EGS, RNAi and RNase P-EGS, decoy RNA and antisense RNA, decoy RNA and ribozyme, decoy RNA and tRNase ZL-EGS, decoy RNA and RNase P-EGS, antisense RNA And ribozyme, antisense RNA and tRNase ZL-EGS, antisense RNA and RNase P-EGS, ribozyme and tRNase ZL-EGS, ribozyme and RNase P-EGS, and tRNase ZL-EGS and RNase P-EGS. Moreover, three or more types of RNA sequences may be linked, and when three types of RNA sequences are linked, any combination of RNA portions that exhibit the above RNA medicine may be used. Preferably, an RNA portion that exhibits an action as an RNAi pharmaceutical is necessarily included. The shRNA or miRNA part, decoy RNA part, antisense RNA, RNA part consisting of ribozyme, RNA part consisting of tRNase ZL-EGS and RNA part consisting of RNase P-EGS contained in the chimeric RNA of the present invention are in vivo. It can be cut by a cutting means such as an enzyme. Therefore, the chimeric RNA of the present invention has an shRNA part or miRNA part, a decoy RNA part, an antisense RNA part, an RNA part consisting of ribozyme, an RNA part consisting of tRNase ZL-EGS and / or an RNA part consisting of RNase P-EGS. It is a chimeric RNA linked in a cleavable manner. A combination of RNAi and other RNA moieties, particularly RNAi and decoy RNA is preferred. This is because when an attempt is made to suppress the virus by the RNAi mechanism, the virus is mutated and the virus acquires RNAi resistance. Since the decoy RNA mechanism does not lose its effect even when the virus is mutated, the RNAi mechanism can attack many viruses first, and if a mutation occurs, the decoy RNA mechanism can further attack the mutated virus. The above combinations are preferred.
An RNAi drug is a drug that can cleave a target mRNA having a specific sequence by RNAi (RNA interference) and suppress the expression of a gene corresponding to the mRNA. In RNAi, siRNA (short interfering RNA) consisting of a sense strand having substantially the same sequence as a specific target mRNA and an antisense strand complementary to the sense strand recognizes the target sequence as a guide RNA, and the target mRNA Is cleaved to suppress gene expression. siRNA is formed from double-stranded RNA (dsRNA) by being processed by Dicer in cells or in vivo. Therefore, the chimeric RNA of the present invention is a chimeric RNA capable of forming an siRNA targeting a specific mRNA. The portion of the chimeric RNA that can generate siRNA targeting a specific mRNA is RNA that has a short hairpin structure (shRNA). The shRNA has a stem-loop structure including a double-stranded part and in which a sense strand and an antisense strand are linked via a loop sequence. The double-stranded structure is formed by a self-complementary RNA strand that contains a sense strand and an antisense strand as reverse sequences in one RNA strand. Short hairpin RNA is processed in cells or in vivo to produce siRNA. Triphosphate (ppp) may be bound to the 5 ′ side. In addition, the antisense strand may have an overhang at the 3 ′ end, and the type and number of bases of the overhang are not limited, and are, for example, 1 to 5, preferably 1 to 3, and more preferably 1. Or the sequence | arrangement which consists of 2 bases is mentioned, For example, UU is mentioned. In the present invention, the term “overhang” refers to a base that is added to the end of one strand of shRNA and that does not have a base that can complementarily bind to the corresponding position of the other strand. The double-stranded portion is complementary to the RNA strand (sense strand) having a sequence capable of hybridizing to the target gene sequence to be knocked down by RNA interference or a specific target sequence contained in the non-coding region, and the sequence. It has a structure in which RNA strands (antisense strands) are complementarily bound.
In the shRNA of the present invention, the 3 ′ end of the sense strand and the 5 ′ end of the antisense strand are linked via a loop (hairpin loop sequence). The hairpin loop sequence is not limited, and examples thereof include a sequence starting with UU consisting of 5 to 12 bases, such as UUCAAGGA (SEQ ID NO: 19). Other loop sequences include Lee NS. et al. (2002) Nat. Biotech. 20, 500-505, Paddison PJ. et al. (2002) Genes and Dev. 16, 948-958, Sui G. et al. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 5515-5520, Paul CP. et al. (2002) Nat. Biotech. 20, 505-508, Kawasaki H. et al. et al. (2003) Nucleic Acids Res. A loop having the sequence described in 31,700-707 or the like can be employed.
The target sequence of the shRNA portion of the chimeric RNA of the present invention is a partial sequence of a gene involved in virus replication. The number of bases of the target gene of the shRNA of the present invention or the specific target sequence of the non-coding region is not limited and is selected in the range of 15 to 500 bases. Preferably it is 15-50, 15-45, 15-40, 15-35 or 15-30 bases, more preferably 20-35 bases, more preferably 19-30 bases, particularly preferably 19-29 bases or 28 bases. is there. For the target gene or the target sequence in the non-coding region, for example, a portion having a large expression suppression effect may be selected as appropriate depending on the target gene. The shRNA of the present invention and the target sequence are desirably the same, but may be substantially the same, that is, a homologous sequence. That is, as long as the sense strand sequence of the shRNA of the present invention and the target sequence are hybridized, one or more, that is, 1 to 10, preferably 1 to 5, more preferably 1 to 3, 2, or 1. Although there may be a mismatch, the gene suppression effect is reduced by the mismatch. The hybridization conditions in this case are in vivo conditions when the shRNA of the present invention is administered in vivo and used as a pharmaceutical, and moderate when the shRNA of the present invention is used as a reagent in vitro. Stringent conditions or highly stringent conditions. Examples of such conditions include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, hybridization conditions at 50 to 70 ° C. for 12 to 16 hours. Can be mentioned. In addition, the sense strand sequence and the target sequence of the shRNA of the present invention have a sequence homology of 90% or more, preferably 95% or more, more preferably 95, 96, 97, 98 or 99% or more.
The viral gene targeted by the shRNA portion of the chimeric RNA of the present invention is not limited as long as it is a gene related to viral replication, and a partial sequence of the gene can be targeted. For example, in the case of HIV-1 virus, LTR (long terminal repeats), 5 ′ untranslated region, splice donor-acceptor site, primer binding site, 3 ′ untranslated region, gag, pol, protease, integrase, env, tat , Rev, nef, vif, vpr, vpu, or vpx region. ShRNA may be designed by using a part of the sequence of these regions as a sense strand. These sequences are known, and a partial sequence may be appropriately selected from known sequence information. In the case of HCV virus, a part of the sequence of each region of the structural protein (core protein; C, coat protein; E1, E2) and non-structural protein (NS2, NS3, NS4A, NS4B, NS5A, NS5B) is sensed. What is necessary is just to design shRNA as a chain | strand. These sequences are known, and a partial sequence may be appropriately selected from known sequence information.
miRNA (microRNA) is a single-stranded RNA of 18 to 25 bases, and is processed by a dicer from a miRNA precursor containing a double-stranded RNA region of about 70 bases. Similarly to siRNA, miRNA also suppresses the expression of a gene containing a target sequence by RNAi. In the chimeric RNA of the present invention, miRNA may be contained as single-stranded RNA having 18 to 25 bases, or may be contained as double-stranded RNA (miRNA precursor) having about 70 bases. In the present invention, the miRNA portion is referred to in any case. When the chimeric RNA is contained as a double-stranded RNA (miRNA precursor) of about 70 bases, a single-stranded RNA of 18 to 25 bases is generated by Dicer and suppresses the expression of the gene containing the target sequence. The target sequence contained in the miRNA part is the same as in the case of siRNA.
A decoy nucleic acid type (RNA) drug (a decoy type nucleic acid drug) is a drug that can bind to a target gene of interest and inhibit the expression of the gene of interest. For example, it can bind to a transcriptional regulatory factor of interest and inhibit gene transcription. The decoy RNA portion of the chimeric RNA of the present invention binds to a protein involved in the viral life cycle and inhibits the expression of the gene. Here, the proteins involved in the viral life cycle include viral transcriptional regulator proteins and enzymes necessary for viral replication and growth.
For example, the decoy RNA portion of the chimeric RNA of the present invention has a primary structure and a secondary structure similar to the region where a transcriptional regulatory factor recognizes and binds. For example, HIV-1 gene expression is regulated by the interaction of cellular factors with the viral transactivator protein Tat, which has specific regulatory elements in the HIV-1 LTR. The HIV-1 regulatory protein Tat binds to one of the regulatory elements of the 59 nucleotide LTR region, called the transactivation response region (TAR). TAR forms a stable hairpin structure and allows Tat binding. In addition to Tat, Rev and Nef act as transcriptional regulators on HIV-1, and the Rev protein binds to the PRE region. For Rev decoys, see, for example, Ding SF et al. , Front Biosci. 2002 Feb 01; 7: a15-28, Kohn DB et al. , Blood. 1999 Jul 1; 94 (1): 368-71, Akkina et al. , Anticancer Res. 2003 May-Jun; 23 (3A): 1997-2005, and can be designed based on these documents. In this case, the decoy RNA portion of the chimeric RNA of the present invention has a three-dimensional structure similar to that of the TAR region or PRE region, and therefore the primary and secondary structures are also similar. Specifically, it is RNA having a sequence that is substantially the same, that is, homologous to the sequence of the TAR region or the sequence of the PRE region. That is, as long as the decoy RNA portion of the chimeric RNA of the present invention and the TAR region or the PRE region hybridize, one or more, that is, 1 to 10, preferably 1 to 5, more preferably 1 to 3, There may be two or one mismatch. The conditions for hybridization in this case are in vivo conditions. The sequence of the decoy RNA portion of the chimeric RNA of the present invention and the TAR region or PRE region has a sequence homology of 90% or more, preferably 95% or more, more preferably 95, 96, 97, 98 or 99% or more. The chimeric RNA portion of the present invention preferably has a loop-stem structure, and the loop-stem structure refers to a structure in which a single-stranded RNA forms a loop and a stem. Examples of the sequence of the decoy RNA portion of the present invention having a structure similar to the TAR region include CAC CAG AUC UGA GCC UGG GAG CUC UCU GGC UUC CUU UUU GAA UUC G (SEQ ID NO: 1), one for the sequence RNA with several mismatches can be used as long as it binds to the Tat protein.
In addition, RNA that binds to NS3 protease or NS3 helicase can be used as the decoy RNA portion of the chimeric RNA of the present invention when suppressing HCV replication. For example, it can design with reference to Unexamined-Japanese-Patent No. 2002-345475.
The decoy RNA portion of the present invention is an RNA that can bind to a specific protein, and is also referred to as an aptamer RNA portion.
An antisense drug is DNA or RNA having a sequence that is complementary to and hybridizes to a target gene of interest, and is a drug that suppresses the expression of the gene. The antisense RNA portion of the chimeric RNA of the present invention is a sequence complementary to the target sequence of the target gene of interest, and is a sequence complementary to the sequence of the region of the gene involved in virus replication. The antisense RNA portion complementarily binds to mRNA of a gene involved in virus replication, inhibits translation, and suppresses gene expression. For example, in the case of HIV-1 virus, LTR (long terminal repeats), 5 ′ untranslated region, splice donor-acceptor site, primer binding site, 3 ′ untranslated region, gag, pol, protease, integrase, env, tat , Rev, nef, vif, vpr, vpu, or vpx region. An antisense RNA complementary to a part of the sequence of these regions may be designed. In the case of HCV virus, it is complementary to a part of the sequence of each region of the structural protein (core protein; C, coat protein; E1, E2) and nonstructural protein (NS2, NS3, NS4A, NS4B, NS5A, NS5B). A specific antisense RNA may be designed. The RNA is 10 to 400 nucleotides in length, preferably 250 or less nucleotides, more preferably 100 or less nucleotides, more preferably 50 or less nucleotides, particularly preferably long. Is between 12 and 28 nucleotides. The antisense RNA site may have a loop structure as appropriate.
Ribozyme refers to RNA having an activity of cleaving nucleic acids. Examples of ribozymes include hammerhead ribozymes, hairpin ribozymes, and ribozymes derived from human delta hepatitis virus. In the present invention, an RNA portion comprising any ribozyme can be included in the chimeric RNA. The ribozyme also has a target sequence, and the ribozyme RNA portion can be designed by a known method. The RNA portion composed of ribozymes (ribozyme RNA portion) recognizes and cleaves mRNA of a gene involved in virus replication and suppresses gene expression. For example, in the case of HIV-1 virus, LTR (long terminal repeats), 5 ′ untranslated region, splice donor-acceptor site, primer binding site, 3 ′ untranslated region, gag, pol, protease, integrase, env, tat , Rev, nef, vif, vpr, vpu, or vpx region.
tRNase ZL-EGS and RNase P-EGS refer to RNA having an activity of cleaving nucleic acids in the same manner as ribozymes, and are described in Nucleic Acids Research, 2005, Vol. 33, no. 1 235-243 and Bioorg. Med, Chem. Lett. 14 (2004) 4941-4944 and the like. Similarly to ribozymes, tRNase ZL-EGS and RNase P-EGS recognize and cleave mRNA of genes involved in virus replication and suppress gene expression.
In the present invention, RNAi medicines, decoy medicines, antisense medicines, ribozyme medicines, tRNase ZL-EGS medicines, and RNase P-EGS medicines all suppress gene expression, so these medicines are called antigene medicines. Sometimes. That is, the chimeric RNA of the present invention contains a plurality of antigenes and can act as an antigene medicine. In addition, since it is an unprecedented gene drug, it may be called a second generation anti-gene drug.
In the chimeric RNA of the present invention, the shRNA part or miRNA part, decoy RNA part, antisense RNA part, RNA part consisting of ribozyme, RNA part consisting of tRNase ZL-EGS and RNA part consisting of RNase P-EGS were linked. It can be synthesized in vitro by chemical synthesis in a form or by a transcription system using a promoter and RNA polymerase. At this time, a terminator or the like is appropriately included in the DNA sequence. For example, a TTTTT sequence or the like may be used as the terminator sequence. As the promoter, in the case of producing in vitro, T3 promoter, T7 promoter, etc. are used. The template DNA of the chimeric RNA of the present invention is introduced into a vector, the vector is administered in vivo, and the chimeric RNA is administered in vivo. When synthesize | combining, PolIII type | system | group promoters, such as U6 promoter and H1 promoter, tRNA promoter, etc. are used. When using a vector, a plasmid vector, a viral vector, etc. can be used as a vector. As a plasmid vector, a pBAsi vector, a pSUPER vector or the like may be used, and as a virus vector, an adenovirus vector, a lentivirus vector, a retrovirus vector or the like can be used. The chimeric RNA of the present invention can be incorporated into a vector in a state where it can be expressed by a known method. When the chimeric RNA of the present invention contains an shRNA portion, the shRNA portion and other RNA portions are cleaved by the action of dicer in vivo. The sequence recognized and cleaved by Dicer is UU present at the 3 ′ overhang site of the shRNA portion. Therefore, a UU sequence may be included between the shRNA portion and other RNA portions. As a result, the chimeric RNA containing each RNA portion is expressed as a single molecule from the vector, cleaved into the portion written by the in vivo dicer or other cutting means, and siRNA or miRNA is formed from the shRNA portion or miRNA portion. Decoy RNA from RNA part, antisense RNA from antisense RNA part, ribozyme from RNA part consisting of ribozyme, tRNase ZL-EGS from RNA part consisting of tRNase ZL-EGS, from RNase P-EGS RNase P-EGS is generated from the RNA part, and each function is exhibited.
The chimeric RNA of the present invention can be used as a drug for gene suppression, and particularly suppresses viral replication, so that it is a virus inhibitor, a preventive or therapeutic agent for viral infection, and cancer caused by viral infection. It can be used as a preventive or therapeutic drug.
As a method for introducing the chimeric RNA of the present invention, when the subject is a cell or tissue, it can be introduced by culturing simultaneously with the cell or tissue. In addition, a method using calcium ions, an electroporation method, a spheroplast method, a lithium acetate method, a calcium phosphate method, a lipofection method, a microinjection method, and the like can be given. If the subject is an animal individual, it can be administered by the oral route, as well as intravenous, intramuscular, subcutaneous and intraperitoneal injection or parenteral route. Moreover, when administering to a specific site | part, you may deliver to a specific site | part using a drug delivery system. There are various known methods for drug delivery systems, and an appropriate method can be adopted depending on the site to be administered. Examples of drug delivery systems include known methods using liposomes, emulsions, polylactic acid and the like as carriers. It is desirable that administration be performed in admixture with a pharmaceutically acceptable diluent or carrier. Suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline, phosphate buffered saline glucose solution, and buffered saline. The amount of the chimeric RNA to be introduced can be appropriately determined according to the type, severity, age, weight, etc. of the disease to be prevented or treated, but at least one copy per cell in the diseased part. An amount such that chimeric RNA is introduced is preferred. When administering chimeric RNA, the amount of RNA may be about 0.1 μg to 100 mg once to several times a day. Moreover, when using viral vectors, such as a lentiviral vector, about 0.1 microgram-100 mg should just be administered as a vector DNA amount, and what is necessary is just to administer once to several times a day.
For example, when the chimeric RNA of the present invention comprises a shRNA part or miRNA part and a decoy RNA part, siRNA or miRNA and a decoy RNA are generated in vivo when a vector containing the chimeric RNA or DNA encoding the chimeric RNA is administered. . At the beginning of administration, siRNA or miRNA suppresses viral gene expression and suppresses viral replication. However, mutation occurs in the siRNA or miRNA target sequence of the viral gene several weeks after administration, and the mutation increases with time. The siRNA or miRNA cannot recognize the target sequence, and the effect of siRNA or miRNA is lost. Even in this case, since the decoy RNA can suppress viral replication by binding to a transcriptional regulatory factor of the virus regardless of the mutation of the virus, the action of the decoy RNA can be achieved even after the siRNA loses its effect. By this, virus replication can be suppressed.
In addition, when a vector containing a chimeric RNA comprising a shRNA part or miRNA part of the present invention and a decoy RNA part or a vector containing DNA encoding the chimeric RNA is administered, the vector containing the DNA encoding shRNA or miRNA or shRNA or miRNA alone When administered, a decrease in the rate of virus mutation is observed. That is, when a vector containing the chimeric RNA of the present invention or a DNA encoding the chimeric RNA is administered, there is also an effect that the virus mutation rate can be reduced.
Furthermore, when a vector containing a DNA encoding the chimeric RNA of the present invention is administered, the chimeric RNA is generated in the nucleus of the cell, and then transported to the cytoplasm by the action of exportin, so that it functions well in the cytoplasm. There is also an effect.
The present invention also includes a method for administering or treating the above chimeric RNA to a subject, suppressing viral replication in the subject, and preventing or treating a viral infection. Furthermore, the present invention also includes the use of the above chimeric RNA for the production of a viral replication inhibitor, a viral infection preventive agent or a viral infection therapeutic agent.
FIG. 2A shows an example of the chimeric RNA of the present invention. The example shown in FIG. 2A is a chimeric RNA composed of a shRNA portion and a decoy RNA portion having an action as an RNAi drug that suppresses HIV-1 replication and an action as a decoy RNA. The sequence is shown in SEQ ID NO: 2. The shRNA part suppresses the expression of the HIV-1 vif gene, and the decoy RNA part binds to the HIV-1 Tat protein and suppresses the transcription of the TAR region. In HIV-1, the vif gene part is likely to be mutated, and the RNAi drug alone with the vif gene as a target gene loses its effect over time. However, since the chimeric drug of the present invention also contains decoy RNA, even after the vif gene has been mutated and the shRNA part can no longer suppress the expression of the vif gene, HIV- One copy can be suppressed. Therefore, since replication of HIV-1 can be suppressed over a long period of time, it can be used as an effective and reliable preventive or therapeutic agent for HIV-1 infection. The chimeric RNA of the present invention may be produced as a chimeric RNA using a chemically synthesized or in vitro transcription system and administered to a subject in need of prevention or treatment, or the chimeric RNA may be applied to a viral vector such as a lentivirus. A template DNA may be introduced so that the vector can be expressed, and the vector may be administered to a subject. When the vector is used as in the latter case, the chimeric RNA of the present invention is synthesized over a long period of time in the subject, so that the effect of suppressing HIV-1 replication continues. FIG. 4 shows how the chimeric RNA of FIG. 2A of the present invention suppresses HIV-1 replication.
Moreover, as long as it hybridizes with the chimeric RNA having the sequence of FIG. 2, there may be 1 or several, ie, 1-3, 2, or 1 mismatches. The conditions for hybridization in this case are in vivo conditions. The chimeric RNA of the present invention includes RNA having a sequence homology of 90% or more, preferably 95% or more, more preferably 95, 96, 97, 98 or 99% or more with the chimeric RNA of FIG.
Furthermore, the present invention also encompasses a pharmaceutical composition comprising separately shRNA or miRNA acting as an RNAi drug and decoy RNA acting as a decoy RNA drug. In the pharmaceutical composition, the shRNA or miRNA and the decoy RNA are not linked to form a chimeric RNA, and each exists alone. The pharmaceutical composition can be used as a virus replication inhibitor, a viral infection preventive agent or a viral infection therapeutic agent. Furthermore, the present invention also includes a method for preventing or treating a viral infection by suppressing viral replication in a subject, including administering the above-mentioned shRNA or miRNA and decoy RNA to the subject simultaneously or before and after. Include. In this case, shRNA or miRNA may be administered first, and decoy RNA may be administered several days to several weeks later.
The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
In this example, each experiment was performed by the method described below.
Cell culture
HeLa CD4 ⁺ 293T, Jurkat, H9 and MT-4 cells are 10% (v / v) heat-immobilized fetal bovine serum (FBS), L-glutamine (2 mM), penicillin (100 U / mL) and streptomycin (100 μg / mL) Were supplemented with RPMI 1640 medium (Sigma Chemical) or DMEM (Gibco). All cultures should be 37 ° C, 5% CO ₂ Performed under atmosphere.
Human peripheral blood lymphocytes (PBMCs)
Faicol is placed in a test tube for blood separation, Rycosep (Falcon) so that the fresh blood is Faicol: fresh blood = 1: 3. Centrifugation was performed at 1500 rpm for 15 minutes to separate red blood cells and white blood cells. The leukocyte portion was transferred to a 50 ml centrifuge tube, doubled in PBS, and centrifuged at 1500 rpm for 5 minutes. Repeat this operation once more, 1.0X10 ⁶ 10% RPMI was added so as to be / ml. Finally, PHA was added so as to be 1 μmol / ml, and the cells were cultured for 2 to 3 days to be rejuvenated.
Construction of U6 expression plasmid and lentivirus-based vector
The expression plasmid was constructed by a known method. Hairpin siRNA sequences chemically synthesized as two complementary DNA oligonucleotides were mixed in equimolar amounts, heat treated at 95 ° C. for 5 minutes, and slowly cooled in annealing buffer (10 mM Tris-Hcl / 100 mM NaCl). . The resulting duplex was ethanol precipitated and ligated to the KpnI and BamHI cloning sites upstream of the U6 promoter (LeeNs et al., Nat Biotechnol 2002, 20, 500-5) to prepare the following vector.
U6-vif shRNA-TAR vector: both HIV-1 vif shRNA and decoy TAR, a dsRNA sense sequence containing KpnI, EcoRI and BamHI cloning sites (5'-CCA GAT GGC AGG TGA TGA TTG TCC ACA CCA CAC GCC ATC TTG TTA CCA GAT CTG AGC CTG GGAGCT CTC TGG CTT CCT TTT TGA ATT CG-3 'and SEQ ID NO: 3) and antisense sequence (5'-GAT CCG ATA TCA A AA AA AGA AGA TCA GAT CTG GTAACA GAT GGC AGG TGA TGA TTG TGG TGT GGA CAA TCA TCA CCT GCC ATC TG GTA C-3 ', which encodes a SEQ ID NO: 4).
U6-vif shRNA vector: HIV-1 vif sense fragment sequence (5′-CCA GAT GGC AGG TGA TGA TTG TCC ACA CCA CAA TCA TCA CCT GCC ATC TGT TCC TTT TTG-3 ′ sequence No. TAT ACG T (5′-GAT CCG ATT CAA AAA GGA ACA GAT GGC AGG TGA TGA TTG TGG TGT GGA CAA TCA TCA CCT GCC ATC TGG GTA C-3 ′, SEQ ID NO: 6)
U6-vif shRNA-Ran RNA vector: random vif sense sequence (5′-CGG ACG TTG ATT AGT ATG CGG ACC ACA CCT CCG CAT ACT AAT CAA CGT CCT TCC TTT TTG AAT TCG-3 ′ and anti-TCG-3 ′ The sequence (5′-GAT CCG AAT TCA AAA AGG AAG GAC GTT GAT TAG TAT GCG GAG GTG TGG TCC GCA TAC TAA TCA ACG T-3 ′, SEQ ID NO: 8) is encoded.
U6-TAR RNA vector: HIV-1 TAR sense sequence (5'-CAC CAG ATC TGA GCC TGG GAG CTC TCT GGC TTC CTT TTT GAA TTC G-3 ', SEQ ID NO: 9) and antisense sequence (5'-GAT CCG AAT TCA AAA GGA AGC CAG AGA GCT CCC AGG CTC AGA TCT GGT GGT AC-3 ′, SEQ ID NO: 10)
U6-mTAR31-34 RNA vector: Mutant TAR loop sense sequence (5'-CAC CAG AGA GCC TGG GAG CTC TCT GGC TTC CTT TTT GAA TTC G-3 ', SEQ ID NO: 11) and antisense sequence (5'-GAT CCG AAT TCA AAA AGG AAG CCA GAG ACT CCC AGG CTC TCT GGT GGT AC-3 ′, SEQ ID NO: 12)
U6-vif shRNA-Ran-mTAR vector: random vif and mutant TAR sense sequences (5′-CGG ACG TTG ATT AGT ATG CGG ACC ACA CCT CCG CATC ACT AAT CAA CGT CCT TCC ACC TGA ACC It encodes TTG AA T TCG-3 ′, SEQ ID NO: 13) and antisense sequence (5′-GAT CCG AAT TTC AAA AAG GAA GCC AGA GAG CTC-3 ′, SEQ ID NO: 14).
The lentiviral vector was constructed as follows. The EcoRI site upstream of the U6 promoter and the EcoRI cloning site downstream of the insert fragment are digested and cloned into the EcoRI site of a lentiviral vector (CS-CDF-CG-PRE), and CS-vif shRNA-TAR, CS-TAR CS-vif shRNA, CS-vif Ran-MTAR and control transfer vectors were constructed.
RT-PCR analysis of mRNA expression
HeLa CD4 ⁺ Cells (5 × 10 ⁵ Cells) were seeded in 60 mm dishes. After 24 hours, the medium was replaced with fresh medium without antibiotics and transfected with 3 μg of vector DNA using Lipofectamine 2000 transfection reagent optimized by Opti-MEM according to the manufacturer's protocol. 72 hours later, vector transfected cells and untransfected HeLa CD4 ⁺ Total RNA from the cells was extracted using Trizol reagent (Invitrogen). Next, using RNA PCR high-pluskit (manufactured by Toyobo), HeLa CD4 ⁺ Dicer upstream primer (nucleotides 1-24, forward primer F- (5′-CCA GAT GGC AGG TGA TGA TTG TCC TCC-3 ′, SEQ ID NO: 15)) and downstream (in order to detect the presence of endogenous Dicer in the cell) RT-PCR was performed using nucleotides 435-459, reverse primer R- (5′-GGA AGC CAG AGA GCT CCC AGG CTC-3 ′, SEQ ID NO: 16). As an internal control, human control gene (GAPDH) mRNA was amplified using GADPH-F (nucleotides 230-254) and downstream GADPH-R (nucleotides 422-466) primers. All extracted RNA products were treated with 2 μg DNase I without RNase at 37 ° C. for 30 minutes. At this time, non-RT-PCR using extracted vif-TAR RNA was also performed to check the contamination of DNA. The resulting RT-PCR product was fractionated and analyzed by electrophoresis using a 2% agarose gel.
Northern blot analysis
Transiently transfected HeLa CD4 ⁺ Cells 5 × 10 ⁵ Total RNA was extracted 24 hours after the cells, and 30 μg of RNA per lane was electrophoresed using a 20% polyacrylamide / 8M urea gel. After electrophoresis, the RNA band was expressed as Hybond-N. ^TM Transferred to a nylon membrane (Amersham). Bands were detected using a synthetic oligonucleotide complementary to the antisense strand of Vif-shRNA-TAR RNA as a probe. Hybridization was performed at 37 ° C., using 2 × SSPE, washed at 39 ° C., and then washed once at 41 ° C. with 1 × SSPE prior to exposure to autoradiography.
In vitro and in vivo Dicer cleavage assays
Purified vif-TAR dsRNA (60 μg) transcribed from the T7 RNA polymerase promoter using BLOCK-iT RNAi TOPO Transcription Kit (Invitrogen) was prepared from the manufacturer's protocol (BLOCK-iT Dicer RNAi Kit Manual (Invitrogen)) Was cut according to (reaction volume 300 μl). That is, the dicing reaction product was incubated at 37 ° C. for 20 hours, and the resulting dicing product was analyzed on a 20% polyacrylamide gel (manufactured by Invitrogen life technologies). To confirm if the Vif shRNA-TAR chimeric RNA substrate is really cleaved in the cell, HeLaCD4 ⁺ Cells were transfected with 3 μg of vector DNA and total RNA was extracted 24 hours after transfection with Trizol reagent. As a control, vif shRNA-TAR DNA was transcribed into RNA using T7 polymerase. Both transfected and RNA transcribed with T7 were fractionated on a 20% polyacrylamide / 8M urea gel and Hybond-N ^TM The sample was transferred to a nylon membrane and subjected to Northern blot analysis.
Inhibition of target mRNA down-regulation and HIV-1 replication in cells
HeLa CD4 cotransfected with 2 μg vector DNA and 0.2 μg HIV-1 pNLE ⁺ After culturing the cells for 72 hours, total RNA was extracted using Trizol reagent. RNA content was examined using a primer set capable of detecting HIV-1 vif viral mRNA and TAR mRNA. The level of HIV-1 gag 24 antigen production in the culture supernatant containing no collected cells was measured by a fully automated ELISA system (CLEIA) (Lee NS et al., Nat Biotechnol 2002, 20, 500-5). .).
Dose-dependent suppression of HIV-1 replication
To determine a dose-dependent range for inhibition of HIV-1 replication, HeLa CD4 ⁺ Cells 3 × 10 ⁵ The cells were co-transfected with 0.1, 1 and 3 μg of vector DNA and 0.2 μg of HIV 1pNLE DNA. The culture supernatant containing no cells was collected, and the extracellular HIV-1 gag p24 antigen production level was measured as an index of virus replication.
Calcium phosphate precipitation transfection of 293T cells
To produce lentiviral vectors, 293T cells were co-transfected by the calcium phosphate precipitation transfection method with 15 μg of transfer vector cassette DNA, a helper construct encoding gag-pol (15 μg) and 5 μg each of tat, rev and VSV-G. (Ane-Lyse, D. et al, Nucleic Acids Research, 2002, 30, (14): 65-69). At 72 hours after transfection, the culture supernatant was collected, filtered through a 0.45 μm filter disc, and concentrated 100-fold by overnight ultracentrifugation at 6,000 g. The resulting virus pellet was resuspended in serum-free antibiotic-free RPMI medium and stored at −80 ° C. until use. 5 × 10 to determine virus titer ⁵ Cells were transfected into the 293T cells using the virus stock, and after culturing for 72 hours, GFP positive cells were counted by flow cytometer analysis (Ane-Lyse, D. et al., Nucleic Acids Res. 30). 65-69 (2002)).
Jurkat cell gene transfer
3 x 10 for gene transfer into Jurkat cells ⁵ Cells were seeded in 15 mL centrifuge tubes with 1 mL culture medium in the presence of CS-vif shRNA-TAR and control lentiviral vector 20MOI and 8 μg / mL polybrene. After centrifugation at 800 g for 1 hour, the cells were transferred to a 24-well culture plate, and after 24 hours, the culture medium was changed before virus infection.
Challenges with HIV-1 and long-term culture assays
1 × 10 24 hours after gene transfer ⁶ Of Jurkat cells with HIV-1 of 0.01 MOI _NL4-3 Infected with. After overnight incubation, the cells were washed 3 times with serum-free medium and cultured in R10 (RPMI1640 + 10% FBS). Half of the culture volume was collected at the same interval and replaced with the same volume of culture medium. The collected culture was centrifuged, the cell-free medium was used for HIV-1 gag p24 antigen analysis, and the cell pellet was used for cell viability test, GFP expression and RNA extraction.
HIV 1 _NL4-3 Sequence analysis of Vif RNA target region
Viral RNA was isolated from the cell-free culture supernatant using the QIAamp viral RNA kit (Qiagen) according to the manufacturer's protocol. 5 μL of viral RNA was mixed with Powerscript reverse transcriptase (Clontech), 1 mM deoxynucleotide triphosphate, 1 × first strand buffer (Clontech), 200 ng random hexamer (Promega) and 10 U. RNasin (manufactured by Promega) and used for the reverse transcriptase reaction. Reverse transcription was performed at 42 ° C. for 1 hour, followed by heat inactivation of reverse transcriptase at 70 ° C. for 15 minutes. Next, 2 μl of cDNA was added to 1 × Qiagen Taq PCR buffer, 1.5 mM MgCl. ₂ , 20 pmol of sense primer vif F: (5′-ATG GAA AAC AGA TGG CAG GTG AT-3 ′, SEQ ID NO: 17) and antisense primer vifR: (5′-CTA GTG TCC ATT CAT TGT ATG GCT-3 ′, SEQ ID NO: 18) was added to 48 μl of a PCR mixture containing 1 mM deoxynucleotide triphosphate and 2.5 U Taq polymerase (Qiagen). PCR was performed using a gradient PCR thermal cycler (manufactured by ASTEC) at 95 ° C. for 1 minute, 95 ° C. for 15 seconds for 35 cycles, 58 ° C. for 30 seconds, 72 ° C. for 30 seconds, and 72 ° C. for 5 minutes. PCR products were fractionated, analyzed on a 1% seekem gel and purified using a QIAEX II gel extraction kit (Qiagen). Nucleotide cycle sequencing was performed using Dye-labeled compounds.
The following results were obtained by this example.
Vector design, target site and structure for U6 plasmid and CS lentiviral vector are shown in FIGS. FIG. 1A is a diagram showing the construction of a second generation shRNA expression vector, showing an enlarged 5 ′ LTR representing the HIV-1 genome and representing the decoy TAR RNA target and vif shRNA target position for the tat protein. FIG. 1B shows a control expression cassette having a vif shRNA-TAR transcribed with a human U6 promoter and a terminator signal sequence. A lentivirus-based expression cassette is made by digesting the EcoRI site upstream of the U6 promoter and the EcoRI cloning site downstream of the terminator sequence from the U6 vector, and then into the EcoRI site in the CS-CDF-CG-PRE expression cassette. Cloned to make a lentiviral vector. All constructs were made by standard molecular biology techniques and the sequences were determined by nucleotide sequencing. The RNA secondary structure of the Vif shRNA-TAR chimeric molecule was analyzed using GENETYX software (FIG. 2A) and according to the manufacturer's protocol, HeLa CD4 ⁺ Cells (5 × 10 ⁵ ) U6 vif shRNA-TAR and control mRNA expression was measured. That is, 3 μL of DNA was mixed with 47 μl of Opti-MEM to optimize DNA complex formation. Since the expression of HIV-1 antigene mRNA is essential for the degradation of HIV-1 gene mRNA or cellular protein interaction, the transfection efficiency of HIV-1 antigene mRNA expression was increased. Vif-TAR mRNA was also detected by PCR and subcellular localization was determined by Northern blot analysis (FIG. 2B), resulting in a complete profile of expression of all vectors (FIG. 2C). FIG. 2B shows the intracellular localization of the second generation HIV-1 vif shRNA (vif shRNA-TAR chimeric RNA). Cells were transfected with the vif shRNA-TAR construct. RT-PCR products 24 hours after transfection extracted from nuclear and cytoplasmic RNA indicate that the chimeric RNA is probably located in the cytoplasm rather than the nucleus with the help of the export factor exportin. FIG. 2C shows HeLa CD4 ⁺ Figure 5 shows Northern blot analysis of expression vector mRNA in cells. Cells were transfected with various vector constructs and the mRNA expression efficiency in the cells was examined.
The vif shRNA-TAR molecule was localized more strongly in the cytoplasm than in the nucleus. This makes it possible to determine where the biological function is exerted.
Since the RNAi mechanism is strongly associated with the activity of an endogenous RNAase III-like enzyme called Dicer in the cell, transfected HeLa CD4 ⁺ Endogenous Dicer expression in the cells was examined by RT-PCR using specific Dicer primers. That is, this was done to establish evidence that vif shRNA-TAR RNA molecules are processed into siRNA and exert both RNAi and decoy RNA TAR effects in cells. Interestingly, high level expression of Dicer was observed in the cells (FIG. 2D). FIG. 2D shows HeLa CD4 ⁺ Dicer present in cells. Total RNA in cells is HeLa CD4 ⁺ It was recovered from the cells and amplified using specific Dicer detection primers. This is responsible for the dual type of anti-HIV-1 effect that works against viruses in both transiently transfected and infected stable expressing cells.
HeLa CD4 ⁺ To confirm cleavage of U6 vif-TAR mRNA molecules by Dicer in cells, U6 vif-TAR DNA was transfected or transcribed with T7 polymerase in vitro as an internal control sample and obtained 24 hours after transfection. The resulting cleavage products were fractionated on a 20% polyacrylamide gel and subjected to Northern blot analysis. The results showed that the molecule was cleaved into vif siRNA and decoy TAR RNA components (FIG. 2E). FIG. 2E shows in vivo cleavage. HeLa CD4 for confirming intracellular processing of chimeric RNA ⁺ The results of Northern blot analysis of vif shRNA-TAR RNA extracted 72 hours after transfection of cells are shown. Each lane shows the following.
Lane 1: vif shRNA-TAR DNA I transcribed into T7 RNA polymerase in the absence of endonuclease and Dicer effect, lanes 2 and 3: HeLa CD4 ⁺ 2 shows vif shRNA-TAR and TAR RNA expressed in cells. The human control gene G3PDH was run simultaneously for standardization of input RNA. Detection of both vif shRNA-TAR and TAR RNA bands in lane 2 indicates in vivo cleavage of the chimeric RNA in the cells. The TAR RNA in lane 3 is a control to check the migration of the cleaved TAR RNA component of the vif shRNA-TAR substrate.
Lane 1 is a control synthetic oligonucleotide transcribed by T7 RNA polymerase, with no endogenous dicer present. Lane 2 shows the vif shRNA-TAR substrate extracted 24 hours after transfection from transfected cells expressing endogenous dicer. TAR RNA components were detected simultaneously with the remaining uncut vif shRNA-TAR mRNA. This indicates in vivo cleavage within the cell. Lane 3 shows transfected HeLa CD4 as an internal control marker for the cleaved TAR RNA component of the vif shRNA-TAR chimeric RNA. ⁺ Shown is the expressed TAR mRNA extracted from the cells. As further evidence of the high cleavage affinity of the vif shRNA-TAR molecule, the substrate was transcribed, added to a Dicer reaction mixture containing human recombinant dicer, incubated for 20 hours at room temperature, and Northern blot analysis was performed. As a result, it was shown that the complex of dicer and vif shRNA-TAR was cleaved more than 90% with respect to the vif shRNA-TAR product not cleaved by Dicer (FIG. 2F). FIG. 2F shows in vitro cleavage. FIG. 6 shows Northern blot analysis of vif shRNA-TAR transcribed with T7-RNA polymerase treated with human recombinant dicer for 20 hours or more at room temperature. Each lane is as follows. Lane 1: reaction mixture without dicer and sample (negative control), lane 2: reaction mixture with vif shRNA-TAR RNA but without dicer, lane 3: reaction mixture with dicer and vif shRNA-TAR RNA. Thus, two separate mRNA molecules (vif siRNA mRNA and TAR mRNA) exert a dual anti-HIV-1 effect through RNAi and decoy TAR RNA pathways, and HIV replication in cells (Elbashir, SM et a; Nature). 2001, 411, 494-498; Rosel, KF et al, Nucleic Acid Res. 2003, 31 (15), 4417-4425) are expected to be enhanced.
To examine the potential and inhibitory effects of vif shRNA-TAR molecules, HeLa CD4 ⁺ Cells were co-transfected with various amounts of vector DNA (0.1, 1.0, 3.0 μg). As a result, it was found that the HIV-1 replication effect was dose-dependent. An inhibitory effect of more than 90% was shown with 3 μg of DNA (data is expressed as the measured value ± SD of the supernatant of three independent experiments). Interestingly, the results of suppression correlated with the degree of HIV-1 vif mRNA down-modulation (FIG. 3A). FIG. 3A shows the HIV-1 antiviral effect. HeLa CD4 ⁺ Cells were 0.2 μg at various DNA concentrations (0.1, 1.0, and 3.0 μg) of U6-vif shRNA-TAR, vif shRNA, shRNA Random, Decoy TAR, Decoy M-TAR and control U6 vectors. Of HIV-1 pNLE. Culture supernatants were collected 72 hours after transfection and HIV-1 p24 assay was performed using an automated ELISA system. HIV-1 mRNA analysis from co-transfected cells showed down regulation of viral mRNA expression. Further, as shown in the panel showing the vif shRNA-TAR chimeric RNA, vif shRNA and decoy TAR RNA, HIV-1 reporter gene expression (compared to the panel showing HIV-1 pNLE, vif shRNA Ran and M-TAR ( There was a significant decrease in EGFP) (FIG. 3B). FIG. 3B shows suppression of HIV-1 reporter gene expression. Cells were transferred to microscope slides 72 hours after co-transfection of cells and examined for EGFP expression. Panel shows suppression of HIV-1 reporter gene mediated by vif-TAR, vif shRNA and TAR decoy. The panel shows that the vif shRNA random and the mutant TAR maintain the same level of expression compared to the panel showing HIV-1 pNLE.
To further investigate the potential suppressive capacity and stability of the vif shRNA-TAR construct, lentiviral vectors were generated and chimeric RNA (CS-vif shRNA-TAR), CS-vif shRNA, CS-TAR and control expression cassettes. Increased delivery. Transfected Jurkat, H9 cells and PBMCs stably expressing HIV-1 antigene were infected with HIV-1 NL4-3 and cultured for 8 weeks or more. The culture supernatant without sampled cells is analyzed for HIV-1 gag p24 antigen to assess inhibition of HIV-1 replication (measured as a change in gag p24 antigen in the virus replication assay) and anti-HIV-1 replication Gene's anti-HIV-1 effect was evaluated. As a result, it was found that the inhibitory effect of the second generation anti-HIV molecule was stably prolonged in the transfected gene-transfected cells (FIGS. 3C, D and E). Figures 3C, D and E show long-term suppression of HIV-1 replication by CS-vif shRNA-TAR. Transgenic Jurkat, H9 cells and PBMCs are HIV-1 _NL4-3 For 9 weeks or more. A significant decrease in the amount of HIV-1 p24 was observed in vif shRNA-TAR expressing cells. The HIV-1 p24 antigen production level of vif shRNA gradually increased from the 4th week in Jurkat cells and reached the same level as that of control HIV-1 NL4-3 and vif shRNA Ran-M-TAR (FIG. 3C). PBMCs gradually increased from week 2 and reached the same level as controls HIV-1 NL4-3 and vif shRNARan-M-TAR (FIG. 3D). Furthermore, H9 cells gradually increased from the third week and reached the same level as that of control HIV-1 NL4-3 and vif shRNA Ran-M-TAR (FIG. 3E).
To examine viral mutations and defects in cultures expressing CS-vif shRNA, HIV-1 _NL4-3 Viral RNA was isolated on days 28, 35 and 48 from cells infected with CS-vif shRNA-TAR or CS-vif shRNA in infected cells (Jurkat and PBMCs), and HIV occurs by vif-shRNA and TAR target sites -1 _NL4-3 Mutations and deletions in the vif and Tat genes were analyzed. Sequencing revealed mutations and deletions in the CS-vif shRNA target site in both CS-vif shRNA-TAR and CS-vif shRNA (alone). However, no mutation or deletion was observed in the Tat target region of HIV-1 viral RNA. CS-vif-shRNA has a faster mutation and deletion rate in cultures expressing CS-vif shRNA-TAR, whereas the mutation and deletion rate is faster (FIGS. 3F and G). 3F and G show the results of gene analysis in Jurkat cells and PBMCs, respectively. Viral RNA was extracted from CS-vif shRNA and CS vif shRNA-TAR culture supernatants and sequenced for siRNA mutations at 2, 3, 4, 5 and 6 weeks in Jurkat cells. On the other hand, PBMCs were analyzed for siRNA-mediated mutations and deletions at 2, 3, 4, 5, 6 and 8 weeks. Mutations and deletions in the HIV-1NL4-3 vif shRNA target site were detected in all cultured cells expressing either vif shRNA or vif shRNA-TAR. The protein-RNA interaction between TAR-RNA and tat protein is important for the control of RNAi resistance observed in vif shRNA-TAR cultured cells. Tat protein and TAR RNA specific interactions (protein-RNA interactions) are important for combined gene therapy strategies with siRNA to assist in high antiviral activity under RNAi resistant strains observed in long-term assays.
That is, in this example, the expression of dual or multi-HIV-1 antigene expressed through a single RNA molecule that can be cleaved in cells can enhance the suppression efficiency of HIV-1 replication over a long period of time. Thus, the competitive interactive role of HIV-1 decoy TAR RNA enhances the inhibitory effect of vif siRNA on HIV-1 replication (over 98%) and vif in HIV-1 infected cells over a long period of time, such as 9 weeks. Decoy TAR RNA continues to block HIV-1 production in the presence of shRNA variants and defects. Thus, a new strategic method of expressing dual or multi-HIV-1 antigenes can be a promising HIV-1 gene therapy that can be applied in the prevention and treatment of HIV / AIDS.

The chimeric RNA of the present invention suppresses the expression of a decoy RNA part that suppresses the action of a gene transcription factor by acting as a decoy with an shRNA (short hairpin RNA) part or miRNA part capable of forming an siRNA that suppresses gene expression. An anti-sense RNA portion, an RNA portion consisting of a ribozyme, an RNA portion consisting of tRNase ZL-EGS and an RNA portion consisting of RNase P-EGS. It acts as at least one of a sense RNA drug, a ribozyme drug, a tRNase ZL-EGS drug, and an RNase P-EGS drug. The chimeric RNA of the present invention is effective for virus suppression such as prevention of virus replication that can cause mutation and acquire RNAi resistance, and can be used as a preventive or therapeutic agent for viral infections. Even if the RNAi drug loses its effect due to the mutation of the virus, the effect of the nucleic acid drug that exerts other actions or the effect of the nucleic acid drug that exerts the same action directed to other target sequences is suppressed. By doing so, it is possible to suppress the virus efficiently and reliably.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

SEQ ID NOs: 1 to 8: Synthesis [Sequence Listing]

Claims (17)

It contains at least one shRNA (short hairpin RNA) part or miRNA part capable of forming a siRNA capable of suppressing the virus using the viral RNA as a target sequence, and further binds to a protein involved in the life cycle of the virus and can suppress the virus. From one decoy RNA portion, at least one antisense RNA portion that can hybridize to a gene involved in the viral life cycle and suppress the virus, and at least one ribozyme that can suppress the expression of a protein involved in the viral life cycle An RNA portion comprising at least one tRNase ZL-EGS capable of suppressing the expression of a protein involved in the viral life cycle and a protein involved in the viral life cycle At least one comprises an RNA portion, two or more chimeric RNA molecules the RNA portion is linked to a cleavable selected from at least one of the group consisting of RNA portion consisting of RNase P-EGS capable of suppressing the expression of. Cleavage of at least one shRNA or miRNA part capable of forming a siRNA capable of suppressing a virus using a viral RNA as a target sequence and at least one decoy RNA part capable of suppressing a virus by binding to a protein involved in the life cycle of the virus A chimeric RNA molecule linked to. The chimeric RNA molecule according to claim 1 or 2, wherein a UU sequence is present between the shRNA part or the miRNA part and the other RNA part. The chimeric RNA molecule according to any one of claims 1 to 3, wherein the virus is selected from the group consisting of HIV-1, HIV-2, HCV, cancer and influenza virus. shRNA part, miRNA part, antisense RNA part, RNA part consisting of ribozyme, RNA part consisting of tRNase ZL-EGS or RNA part consisting of RNase P-EGS is HIV-1 gag, pol, env, tat, rev, nef The chimeric RNA molecule according to claim 1, which inhibits HIV-1 targeting a partial sequence of a region selected from the group consisting of, vif, vpr, vpu, vpx, or LTR region. the shRNA or miRNA portion targets a partial sequence of a region selected from the group consisting of gag, pol, env, tat, rev, nef, vif, vpr, vpu, vpx, or LTR region of HIV-1. The chimeric RNA molecule according to claim 2, which suppresses HIV-1. The chimeric RNA molecule according to claim 5 or 6, wherein the decoy RNA portion has a sequence homologous to the TAR region of HIV-1 and can bind to a Tat protein. The chimeric RNA molecule according to claim 7, which has the following secondary structure.
A vector for expressing a chimeric RNA comprising the template DNA of the chimeric RNA molecule according to any one of claims 1 to 8. The vector according to claim 9, which is a viral vector. A preventive or therapeutic agent for a viral infection and cancer comprising the chimeric RNA molecule according to any one of claims 1 to 8, which is cleaved into each RNA portion in vivo. A preventive or therapeutic agent for a viral infection comprising the vector according to claim 9 or 10, wherein the preventive or therapeutic agent for a viral infection in which each RNA moiety is generated in vivo. 13. The preventive or therapeutic agent for viral infections and cancer according to claim 11 or 12, which can block viral RNAi resistance based on viral mutations. A viral infectious disease comprising at least one shRNA or miRNA capable of forming a siRNA capable of suppressing a virus using a viral RNA as a target sequence and at least one decoy RNA capable of inhibiting the virus by binding to a protein involved in the life cycle of the virus. Prophylactic or therapeutic agent. A group of shRNA or miRNA that can form a siRNA capable of suppressing a virus using a viral RNA as a target sequence is HIV-1 gag, pol, env, tat, rev, nef, vif, vpr, vpu, vpx, or LTR region The prophylactic or therapeutic agent for a viral infection according to claim 14, which targets a partial sequence of a region selected from: The agent for preventing or treating viral infection according to claim 14 or 15, wherein the decoy RNA portion has a sequence homologous to the TAR region of HIV-1 and can bind to a Tat protein. The preventive or therapeutic agent for viral infections and cancer according to claim 15 or 16, which can block viral RNAi resistance based on viral mutations.