JP2006223173A - NMT1-SPECIFIC siRNA - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable various diseases such as cancer and virus-infectious disease to be treated by specifically silencing gene expression of each of a human N-myristoyltransferase (hNMT) family catalyzing N-myristoylation of an N-myristoylated protein closely associated with progress of various kinds of the diseases, and various kinds of physiological functions by applying the principle of siRNA. <P>SOLUTION: The shRNA, the dsRNA or the siRNA inhibiting the function of hNMT1 but not inhibiting the function of hNMT2 is provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to shRNA, dsRNA or siRNA specific to NMT1 (N-myristoyltransferase-1), and an anti-HIV drug using the same.

  HIV infection is a progressive disease in which human immunodeficiency virus type-1 (HIV-1) mainly infects CD4-positive lymphocytes and the immune system is gradually destroyed. In non-treated cases, the course of the initial infection stage, asymptomatic stage, and AIDS (acquired immunodeficiency syndrome) onset period is followed. As of March 2002, there are 15 anti-HIV drugs approved in Japan: 6 nucleic acid reverse transcriptase inhibitors, 3 non-nucleic acid reverse transcriptase inhibitors, and 6 HIV-1 protease inhibitors. As a result of the development of highly active antiretroviral therapy (HAART) combining these therapeutic agents, HIV infection is now positioned as a controllable disease. However, HAART has to take as many as a dozen tablets a day and has side effects such as nausea, vomiting, diarrhea, lactic acidosis, fatty liver, diabetes, abnormal body fat distribution, and hyperlipidemia. QOL (quality of life) is bad. And one of the most serious problems is the emergence of drug-resistant viruses associated with treatment failures based on HIV-1 mutability. This therapy only suppresses the growth of HIV-1 and does not eliminate it from the body. Even if blood viral RNA falls below the detection sensitivity due to HAART, viral RNA will be detected again when treatment is stopped. That is not a radical cure. Current anti-HIV drugs can control the progression of the disease, but patients still have to suffer from the side effects of heavy drugs for the rest of their lives and still pay for high medical costs. The ultimate anti-HIV drug is a drug that completely eliminates HIV-1 from the body, but unfortunately, there are no therapeutic drugs or treatments that show such an effect.

  Research and development of anti-HIV drugs is being conducted energetically around the world. Since the discovery of HIV-1 as the causative virus for AIDS (Non-patent Document 1), the elucidation of the virus life cycle has progressed, and target molecules linked to treatment have been identified one after another. An anti-HIV drug has been reported (Non-patent Document 2). Viruses that inhibit the adsorption of viruses to host cells, including the above-mentioned reverse transcription process (i) already applied clinically and the HIV-1 gag protein processing process (ii) by HIV-1 protease Inhibitors that target the coat glycoprotein gp120 (iii), inhibitors that target the chemokine receptors CXCR4 and CCR5 that are used when the virus enters the host cell (iv), and the virus fusion step to the host cell Inhibitors targeting the inhibitory gp41 protein (v), the zinc finger of NCp7, the HIV-1 nucleocapsid protein, and the uncoating and disassembly stages after virus entry into the host cell or before and after the virus budding from the host cell Inhibitors that block the assembly step (vi), inhibitors that target the integration process of proviral DNA into host DNA by integrase (vii), and transcription processes of viral genes Inhibitors (viii), and inhibitors (ix) that block N-Myrization of the amino terminus of HIV-1gag protein, a protein post-translational modification essential for the acquisition of viral infectivity It has been reported as a target / inhibitor for research and development of anti-HIV-1 drugs.

On the other hand, Myristoyl coenzyme A (Myr-CoA): protein N-myristoyltransferase (NMT) (EC 2.3.1.9.7) has Met ₁ -Gly ₂ -X ₃ -X ₄ -X ₅ -Ser / Thr The start Met of a protein having a _6- consensus sequence is an enzyme that catalyzes the transfer of Myr-CoA to Myr-CoA to the α-amino group of exposed Gly ₂ after removal by methionine aminopeptidase during translation, This modification is called N-myrtoylation of the protein. N-Myrization was first discovered by Shoji et al. In 1982 when determining the total primary structure of calf myocardial cAMP-dependent protein kinase catalytic subunit (Non-patent Documents 3 and 4). -Myr protein has been reported (Non-patent Document 5).

N-Myrization plays roles such as targeting of protein to the cell membrane, association with cell membranes, particularly lipid rafts (Non-patent Document 6), and stabilization of the three-dimensional structure of the protein (Non-patent Document 5) (FIG. 1). ). In recent years, it has been reported that N-Myrization can occur not only during translational modification but also after the protein is completely biosynthesized (Non-patent Document 7). Table 1 shows some of the N-Myr proteins that have been reported to date.

As shown in Table 1, the feature of the peptide substrate recognized by NMT is that Gly at position 2 is absolutely required. From experience, it has been found that Ser / Thr position 7 or 8 is often Lys or Arg at position 6. X-ray crystal structure analysis of NMT (Nmt1p), Myr-CoA analog and substrate peptide complex derived from Saccharomyces cerevisiae reveals that the catalytic mechanism is the following Bi Bi reaction mechanism (non-patented) References 8 and 9). 1) Binding of Myr-CoA to NMT 2) Peptide substrate can bind to NMT. 3) Myristic acid is transferred to the amino terminal Gly of the peptide. At this time, the carboxyl group at the carboxy terminus of NMT deprotonates the α-amino group of the amino terminal Gly residue of the substrate peptide, and then the nitrogen undergoes nucleophilic attack on the carbonyl carbon of Myr-CoA to transfer the acyl group. Happens. 4) CoA is released from the enzyme. 5) N-Myr peptide is released from the enzyme.

  NMT exists more than eukaryotic cells, and 15 to 19 NMTs have been identified to date. From studies using Saccharomyces cerevisiae (Non-patent Document 10), Drosophila (Non-patent Document 11), and Arabidosis thaliana (Non-patent Document 12), NMT is an essential enzyme for cell survival, development, and plant growth. It is understood. In mammals, two NMT genes have been identified to date in humans, mice and cattle (Non-patent Document 13), and human NMT genes have been reported to have hNMT1 gene in chromosome 17 and hNMT2 gene in chromosome 10 They have 77% homology (Figure 2). hNMT2 is detected as a single 65 kDa band on SDS-PAGE, while hNMT1 exhibits a diverse isoform from 49 kDa to 68 kDa. This diversity is suggested to be due to alternative splicing in hNMT1 (Non-patent Document 14). Some NMT isoforms have a sequence containing a large amount of basic amino acids in the N-terminal region, which is considered to be important for targeting to ribosomes. Is suggested (Non-Patent Document 15).

  NMT is one of the targets for the development of antifungal agents against Candida albicans and Cryptococcus neoformans, the causative agents of candidiasis and cryptococcosis, which are systemic mycoses. Inhibitor development utilizing the difference in substrate specificity of the derived NMT is in progress (Non-Patent Documents 16 to 18). In addition, as shown in Table 1, some pathogenic viruses have proteins that undergo N-Myrization, and NMT is considered as one of the targets for the development of antiviral drugs.

In HIV-1, it is known that the viral structural protein p17 ^gag and the viral regulatory protein p27 ^nef have undergone N-Myrization. p17 ^gag is a protein called a matrix that is N-Myrized when translated as Pr55 ^gag , and is processed after being processed by a viral protease. p17 ^gag exists as Pr55 ^{gag in} cells, but its N-Myrization has been shown to be an essential modification for assembly of Pr55 ^{gag into} the cell membrane and for virus particle formation. It is known that inhibition of N-Myrization leads to an anti-HIV effect (Non-patent Documents 19 to 22). There is no report of an infectious virus composed of non-myristolated p17 ^gag in HIV-1 where drug-resistant virus is likely to appear.

De Clercq, E. (2001) Curr. Med. Chem. 13, 1543-1572. Sinoussi, F. B., et al., Science (1983) 220, 868-871. Carr, S.A., et al. (1982) Proc. Natl. Acad. Sci. USA 79, 6128-6131. Shoji, S., et al. (1983) Biochemistry 22, 3702-3709. Resh, M. D. (1999) Biochim. Biophys. Acta. 1451, 1-16. Melkonian, K. A., et al. (1999) J. Biol. Chem. 274, 3910-3917. Zha, J., et al. (2000) Science 290, 1761-1765. Bhatnagar, R.S., et al. (1998) Nature structural biology 5, 1091-1097. Farazi, T.A., et al. (2001) Biochemistry 40, 6335-6343. Duronio, R. J., et al. (1989) Science 243, 796-800. Ntwasa, M., et al. (2001) Exp. Cell. Res. 262, 134-144. Qi, Q., et al. (2000) J. Biol. Chem. 275, 9673-9683. Giang, D. K, et al. (1998) J. Biol. Chem. 273, 6595-6598. McIlhinney, R.A., et al. (1998) Biochem. J. 333. 491-495. Glover, C. J., et al. (1997) J. Biol. Chem. 272, 28680-28689. Devadas, B., et al. (1997) J. Med. Chem. 40, 2609-2625. Nagarajan, S. R., et al. (1997) J. Med. Chem. 40, 1422-1438. Sikorski, J.A., et al. (1997) Biopolymers 43, 43-71. Furuishi, K., et al. (1997) Biochem. Biophys. Res. Commun. 237, 504-511. Shiraishi, T., et al. (2001) Biochem. Biophys. Res. Commun. 282, 1201-1205. Kaminchik, J., et al. (1991) J. Virol. 65, 583-588. Yu, G., et al. (1992) Virology 187, 46-55.

Conventionally, research aimed at anti-HIV effect by inhibiting N-Myr formation, particularly by inhibiting N-Myr formation of p17 ^gag , has been underway. So far, N-Myrization inhibitors are a group of low molecular weight compounds targeting the active center of NMT, and these have inhibited the activity of all hNMT isozymes. The expression of side effects (toxicity) caused by inhibition of N-Myrization of many proteins other than Myrated protein was a major obstacle to practical use.

  Therefore, in the present invention, human N-myristoyltransferase (hNMT) that catalyzes N-myristoylation of N-myristoylated protein closely related to the progression of various diseases such as cancer and viral infection or various physiological functions. ) Making it possible to treat these diseases by silencing gene expression specifically by applying the principle of siRNA to each of the families was an issue to be solved.

  In recent years, it has become clear that there are at least two genes in hNMT, and their gene products are also present in several isozymes. In order to overcome the low specificity that has been a problem with conventional N-myristoylation inhibition strategies, the present inventors aimed to specifically inhibit each of the hNMT isozymes. In particular, each hNMT isozyme specific to each hNMT isozyme is identified by which hNMT isozyme is intracellularly associated with pathogenicity or physiological function regulation, such as carcinogenic protein or viral protein. The purpose of this study was to elucidate the effects of specific inhibition on cancer cell growth, virus replication ability, and various cell functions by using each hNMT isozyme-specific siRNA. As a result of examining in vitro systems using siRNA which hNMT1 or hNMT2 isozyme is important for HIV-1 replication, the present inventors have found that the contribution ratio of hNMT1 is high. It was. As a result, it was found that the development of AIDS therapy targeting hNMT can obtain an anti-HIV-1 effect while further reducing side effects by specifically inhibiting hNMT1. In addition, the present inventors have demonstrated that it is effective to use hNMT1-specific siRNA as a means for specifically suppressing hNMT1. The present invention has been completed based on these findings.

  That is, according to the present invention, there is provided shRNA, dsRNA or siRNA characterized by inhibiting the function of hNMT1 and not inhibiting the function of hNMT2.

  Preferably, the shRNA, dsRNA or siRNA of the present invention has the base sequence represented by SEQ ID NO: 1, 2 or 3 as a target sequence.

Preferably, the shRNA, dsRNA or siRNA of the present invention is any of the following.
(1) siRNA targeting the base sequence described in any one of SEQ ID NOs: 1 to 3;
(2) shRNA consisting of the base sequence set forth in SEQ ID NO: 4;
(3) dsRNA composed of a sense strand RNA consisting of the base sequence set forth in SEQ ID NO: 5 and an antisense strand RNA consisting of the base sequence set forth in SEQ ID NO: 6; or (4) the base sequence set forth in SEQ ID NO: 7 DsRNA composed of a sense strand RNA consisting of and an antisense strand RNA consisting of the base sequence set forth in SEQ ID NO: 8:

  According to another aspect of the present invention, there is provided an expression vector capable of expressing the shRNA, dsRNA or siRNA of the present invention described above.

According to still another aspect of the present invention, there is provided an NMT1 inhibitor comprising the above-described shRNA, dsRNA, siRNA or expression vector of the present invention as an active ingredient.
According to still another aspect of the present invention, an anti-HIV drug comprising the above-described shRNA, dsRNA, siRNA or expression vector of the present invention as an active ingredient is provided.

  Conventionally, since NMT inhibitors have inhibited all NMT activities in cells, the problem of the possibility of developing side effects (toxicity) due to the inhibition of N-Myr formation which has not been aimed at has been pointed out. By inhibiting NMT-specific target protein related to pathogenicity by inhibiting N-Myr by Izozyme-specific NMT inhibition, a therapeutic effect with less toxic expression is expected. In addition, specific inhibition of hNMT1 is effective for AIDS treatment. That is, according to the present invention, it has become possible to identify hNMT isozymes as molecular targets for control of cancer, viral infections, etc., and to develop new therapeutic strategies using siRNA and the like.

Hereinafter, embodiments of the present invention will be described in detail.
The present invention relates to shRNA, dsRNA or siRNA characterized by inhibiting the function of hNMT1 and not inhibiting the function of hNMT2. When the shRNA, dsRNA or siRNA of the present invention is administered to cells, the expression of NMT1 can be specifically suppressed by the RNAi effect. That is, when shRNA or dsRNA is introduced into a cell, an RNAi phenomenon occurs and RNA having a homologous sequence is degraded. Such RNAi phenomenon is a phenomenon observed in nematodes, insects, protozoa, hydra, plants, vertebrates (including mammals).

  Here, “siRNA” is an abbreviation for short interfering RNA, which is artificially chemically synthesized or biochemically synthesized, synthesized in an organism, or about 40 bases. This is a double-stranded RNA of 10 base pairs or more produced by degrading the above double-stranded RNA in the body. The length of siRNA is generally about 10 to 30 bases, preferably about 15 to 25 bases, more preferably about 19 to 23 bases. siRNA usually has a 5′-phosphate, 3′-OH structure, and the 3 ′ end protrudes by about 2 bases. A protein specific to the siRNA is bound to form RISC (RNA-induced-silencing-complex). This complex recognizes and binds to mRNA having the same sequence as siRNA, and cleaves mRNA at the center of siRNA by RNaseIII-like enzyme activity. As described above, siRNA can suppress its expression by degrading mRNA of a target gene (in the present invention, NMT1 gene).

  It is preferable that the sequence of shRNA or dsRNA and the sequence of mRNA that is cleaved as a target match 100%. However, in the case where the bases at positions deviating from the center of the siRNA do not match, the cleavage activity by RNAi often remains partially, so it does not necessarily have to match 100%.

  The region having homology between the nucleotide sequence of shRNA or dsRNA and the nucleotide sequence of NMT1 gene whose expression should be suppressed preferably does not include the translation initiation region of NMT1 gene. This is because various transcription factors and translation factors are expected to bind to the translation initiation region, and therefore siRNA cannot effectively bind to mRNA, and the effect is expected to be reduced. Therefore, the sequence having homology is preferably 20 bases away from the translation initiation region of the NMT1 gene, and more preferably 70 bases away from the translation initiation region of the NMT1 gene. A specific example of a sequence having homology is shown in FIG. The target sequences of NMT1 shown in FIG. 3 are shown in SEQ ID NOs: 1 to 3 in the sequence listing.

  In the present invention, siRNA may be used, and shRNA (short hairpin RNA), dsRNA (double strand RNA) or an expression vector capable of expressing them can be used. When the shRNA, dsRNA or their expression vector of the present invention is administered to a cell, siRNA is produced in the cell (FIG. 3).

  The shRNA, dsRNA, siRNA or expression vector that expresses it used in the present invention may be in any form as long as it can cause RNAi.

  According to an example of the present invention, shRNA can be used. shRNA refers to a molecule of about 20 base pairs or more that has a double-stranded structure in a molecule and has a hairpin-like structure by including a partially palindromic base sequence in a single-stranded RNA. . Such shRNA, after being introduced into the cell, is degraded to a length of about 20 bases (typically, for example, 21 bases, 22 bases, 23 bases) in the cell, and causes RNAi similarly to siRNA. be able to. As described above, shRNA causes RNAi similarly to siRNA, and thus can be used effectively in the present invention.

  The shRNA preferably has a 3 ′ overhang. The length of the double-stranded part is not particularly limited, but is preferably about 10 nucleotides or more, more preferably about 20 nucleotides or more. Here, the 3 ′ protruding end is preferably DNA, more preferably DNA of at least 2 nucleotides, and further preferably DNA of 2 to 4 nucleotides.

  According to another aspect of the present invention, for example, dsRNA having about 20 bases or more can be used. For example, at least about 70%, preferably 75% or more, more preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% with respect to a part of the nucleic acid sequence of the NMT1 gene. As described above, RNA containing a double-stranded portion or a variant thereof containing a sequence having 100% homology can be used most preferably. The sequence portion having homology is usually at least about 15 nucleotides or more, preferably at least about 19 nucleotides, more preferably at least about 20 nucleotides in length, and even more preferably at least about 21 nucleotides in length.

Specific examples of shRNA, dsRNA or siRNA that can be used in the present invention include:
(1) siRNA targeting the base sequence described in any one of SEQ ID NOs: 1 to 3
(2) shRNA consisting of the base sequence set forth in SEQ ID NO: 4;
(3) dsRNA composed of a sense strand RNA consisting of the base sequence set forth in SEQ ID NO: 5 and an antisense strand RNA consisting of the base sequence set forth in SEQ ID NO: 6; or (4) the base sequence set forth in SEQ ID NO: 7 DsRNA composed of a sense strand RNA consisting of and an antisense strand RNA consisting of the base sequence set forth in SEQ ID NO: 8:
Etc.

  As described above, in the present invention, shRNA, dsRNA, siRNA or an expression vector thereof can be used as a factor capable of suppressing NMT1 expression by RNAi. The advantages of siRNA are as follows: (1) Since RNA itself is not integrated into the chromosome of normal cells even when introduced into cells, it is not a treatment that causes mutations transmitted to offspring, and is highly safe, and ( 2) Short double-stranded RNA is relatively easy to chemically synthesize and is more stable when double-stranded. Further, as an advantage of shRNA, when treatment is performed by suppressing gene expression for a long period of time, a vector that transcribes shRNA in a cell can be prepared and introduced into the cell.

  The shRNA, dsRNA, or siRNA used in the present invention may be artificially chemically synthesized, or the RNA of hairpin structure in which the DNA sequences of the sense strand and the antisense strand are linked in the reverse direction is ligated in vitro with T7 RNA polymerase. It can also be produced by synthesis. For synthesis in vitro, antisense and sense RNAs can be synthesized from template DNA using T7 RNA polymerase and T7 promoter. When these are annealed in vitro and then introduced into cells, RNAi is triggered and NMT1 expression is suppressed. Here, such RNA can be introduced into cells using, for example, the calcium phosphate method or various transfection reagents (eg, oligofectamine, Lipofectamine, lipofection, etc.).

  Furthermore, according to the present invention, there is provided an expression vector comprising a nucleic acid sequence encoding shRNA, dsRNA or siRNA capable of suppressing NMT1 expression by RNAi. Furthermore, according to the present invention, a cell containing the above-described expression vector is provided. The cell of the present invention may transiently or stably express a factor that causes RNAi. The expression vector and cell type described above are not particularly limited, but are preferably those that can be used for therapy.

  The shRNA, dsRNA, siRNA or their expression vector of the present invention can specifically inhibit NMT1 by producing an RNAi effect in the cell. That is, the shRNA, dsRNA, siRNA or their expression vector of the present invention is useful as an NMT1 inhibitor.

  Furthermore, since the shRNA, dsRNA, siRNA or their expression vector of the present invention can specifically inhibit NMT1, it can be used for suppression of HIV-1. More specifically, suppression of HIV-1 includes prevention of HIV-1 infection, suppression of HIV-1 proliferation, etc., and clinically includes all prevention and / or treatment of HIV infection. It means to do. As described above, the shRNA, dsRNA, siRNA or their expression vector of the present invention is useful as an anti-HIV drug. Hereinafter, the NMT1 inhibitor and the anti-HIV drug of the present invention are collectively referred to as the drug of the present invention.

  The drug of the present invention can be used in combination with other anti-HIV drugs. In such cases, the agents of the present invention can be administered together with certain anti-HIV drugs or separately before and after. By administering another anti-HIV drug as described above, it is possible to effectively exert a therapeutic or prophylactic effect on an HIV-1 strain that has been resistant to such an anti-HIV drug.

  The anti-HIV drug that can be used in combination with the anti-HIV drug of the present invention may be any of a nucleic acid reverse transcriptase inhibitor, a non-nucleic acid reverse transcriptase inhibitor, or an HIV-1 protease inhibitor. However, the anti-HIV drug of the present invention can be used in combination.

  The method of administering the agent of the present invention includes oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, intravaginal administration, local administration to the affected area, Skin administration, etc.).

  When the agent of the present invention is used as a pharmaceutical composition, a pharmaceutically acceptable additive can be blended as necessary. Specific examples of pharmaceutically acceptable additives include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles. , Diluents, carriers, excipients and / or pharmaceutical adjuvants and the like.

  Although the formulation form of the chemical | medical agent of this invention is not specifically limited, For example, a liquid agent, an injection, a sustained release agent etc. are mentioned. The solvent used for formulating the drug of the present invention as the above-mentioned preparation may be either aqueous or non-aqueous.

  Injections can be prepared by methods well known in the art. For example, after dissolving in an appropriate solvent (physiological saline, buffer solution such as PBS, sterilized water, etc.), sterilized by filtration with a filter, and then filled into a sterile container (eg, ampoule) Can be prepared. This injection may contain a conventional pharmaceutical carrier, if necessary. Administration methods using non-invasive catheters can also be used. Examples of the carrier that can be used in the present invention include neutral buffered physiological saline or physiological saline mixed with serum albumin.

  Furthermore, shRNA, dsRNA, or siRNA that is an active ingredient of the drug of the present invention can be administered in the form of a non-viral vector or a viral vector. Such administration forms are known in the art, and include, for example, separate experimental medicine “Basic technology of gene therapy” Yodosha, 1996; separate experiment medicine “Gene transfer & expression analysis experimental method” Yodosha, 1997, etc. It is described in.

  In the case of a non-viral vector form, a method of introducing nucleic acid molecules using liposomes (liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method, etc.), microinjection method, gene gun (Gene Gun) ) And a method of transferring nucleic acid molecules into cells together with carriers (metal particles). Examples of expression vectors include pCAGGS, pBJ-CMV, pcDNA3.1, pZeoSV (available from Invitrogen or Stratagene).

  When lipofection is used, for example, Lipofectamine 2000, oligofectamine (silencer siRNA Transfection Kit, Gene Silencer siRNA Transfection Reagent) and the like can be used.

  The HVJ-liposome method includes encapsulating a nucleic acid molecule in a liposome made of a lipid bilayer, and fusing the liposome with an inactivated Sendai virus (Hemagglutinating virus of Japan, HVJ). The HVJ-liposome preparation method is described in, for example, a separate volume of experimental medicine “Basic technology of gene therapy” Yodosha, 1996; a separate volume of experimental medicine “Gene transfer & expression analysis experimental method” Yodosha, 1997.

  When shRNA, dsRNA, or siRNA capable of suppressing the expression of NMT1 by RNAi is administered to a living body using a viral vector, a viral vector such as a recombinant adenovirus or a retrovirus can be used. Suppresses expression of NMT1 by RNAi in detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40 A gene can be introduced into a cell or tissue by introducing a DNA that expresses a possible factor and infecting the cell or tissue with the recombinant virus.

  Furthermore, shRNA, dsRNA, siRNA or expression vectors thereof that can suppress the expression of NMT1 by RNAi can be directly injected into an organ or tissue of a living body.

  The dosage of the drug of the present invention is determined in consideration of the purpose of use, the severity of the disease, the patient's age, weight, sex, medical history, or the active ingredient shRNA, dsRNA, siRNA or the type of their expression vector. Can be determined by those skilled in the art.

  The dose of the active ingredient shRNA, dsRNA, siRNA or their expression vector is not particularly limited, and is, for example, about 0.1 ng to about 100 mg / kg, preferably about 1 ng to about 10 mg. When administered as a viral vector or a non-viral vector, the dose is usually 0.0001-100 mg, preferably 0.001-10 mg, more preferably 0.01-1 mg per adult.

The administration frequency of the drug of the present invention can be administered, for example, once a day to once every several months (for example, once a week to once a month). RNAi is generally effective for 1 to 3 days after administration. Therefore, it is preferable to administer daily to once every 3 days. When using an expression vector, it can be administered about once a week.
The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1 Influence of Silencing Effect of Human NMT Gene on HIV-1 Infection FIG. 3 shows the structures of shRNA and dsRNA used in Examples. FIG. 4 shows the base sequences of the hNMT1 gene and the hNMT2 gene containing the siRNA target site. Further, FIG. 5 shows an outline of the procedure of the present embodiment and the measurement result of MAGIC-5 aasay.

(1) System using shRNA expression plasmid (NMT1 siRNA target site 1, NMT2 siRNA target site 1)
The shRNA expression plasmid expressing the NMT1 site1 shRNA (base sequence is SEQ ID NO: 4 in the sequence listing) and the shRNA expression plasmid expressing NMT2 site1 shRNA (base sequence is SEQ ID NO: 12 in the sequence listing) shown in FIG. 3 were used. .

All of the following culture operations were performed aseptically. 293 cells were seeded on a 6-well plate at 2.5 × 10 ⁵ / well and cultured in DMEM medium containing 2 ml of 5% FCS. The next day, 4 μg of HIV-1 expression plasmid pNL4-3 dissolved in 250 μL of serum-free DMEM medium was mixed with 250 μL of serum-free DMEM medium containing 10 μL of transfection reagent lipofectamine 2000. 500 μL of the solution was allowed to stand at room temperature for 20 minutes and then added to 293 cells seeded on a 6-well plate. After 12 hours, remove the culture supernatant, add 1.5 ml of DMEM medium containing 5% FCS, and then add 4 μg of psiRNA-hH1zeo (control plasmid), psiRNA-hNMT1 (hNMT1-specific shRNA expression plasmid) or psiRNA- 250 μL of serum-free DMEM medium containing hNMT2 (hNMT 2-specific shRNA expression plasmid) and 250 μL of serum-free DMEM medium containing 10 μL lipofectamine 2000 were mixed, and the resulting 500 μL solution was mixed at room temperature for 20 minutes. After standing, it was added to the 293 cells. Further, the infectious titer of HIV-1 contained in the culture supernatant obtained after 48 hours of culture was evaluated by MAGIC-5 assay.

(2) System using dsRNA (NMT1 siRNA target site 2, NMT2 siRNA target site 2)
As an NMT1-specific dsRNA, a dsRNA composed of a sense strand RNA consisting of the base sequence shown in SEQ ID NO: 5 and an antisense strand RNA consisting of the base sequence shown in SEQ ID NO: 6 is used, and as an NMT2-specific dsRNA, A dsRNA composed of a sense strand RNA consisting of the base sequence shown in SEQ ID NO: 13 and an antisense strand RNA consisting of the base sequence shown in SEQ ID NO: 14 was used.

All of the following culture operations were performed aseptically. 293 cells were seeded on a 6-well plate at 2.5 × 10 ⁵ / well and cultured in DMEM medium containing 2 ml of 5% FCS. The next day, 4 μg of HIV-1 expression plasmid pNL4-3 dissolved in 250 μL of serum-free DMEM medium was mixed with 250 μL of serum-free DMEM medium containing 10 μL of transfection reagent lipofectamine 2000. 500 μL of the solution was allowed to stand at room temperature for 20 minutes and then added to 293 cells seeded on a 6-well plate. After 48 hours of incubation, mix 100 pmol of dsRNA (NMT1-specific or NMT2-specific) in 250 μL of serum-free DMEM medium and 250 μL of serum-free DMEM medium containing 5 μL of lipofectamine 2000. The obtained 500 μL solution was allowed to stand at room temperature for 20 minutes and then further added to 293 cells seeded on a 6-well plate. After further culturing for 24 hours, the culture supernatant was removed, and a fresh DMEM medium containing 2 ml of 5% FCS was added, followed by further culturing for 24 hours. The infectious titer of produced HIV-1 contained in the obtained culture supernatant was evaluated by MAGIC-5 assay.

(3) MAGIC-5 assay
The MAGIC-5 assay is shown below. Remove the culture supernatant of MAGIC-5 cells seeded at 7.5 x 10 ³ cells / well in a 96-well plate the day before and add 10 μl of virus solution containing DEAE dextran (20 μg / ml) to the wells for 2 hours. After adsorption, 160 μl of DMEM contg. 2.5% FCS was added and incubated at 37 ° C for 48 hours. After incubation, the supernatant was removed, 50 μl of fixing solution was added, and incubated at room temperature for 5 minutes. After removing the fixing solution, the plate was washed twice with PBS (-), 50 μl of the staining solution was added, and incubation (37 ° C, 5% CO2) was performed for 50 minutes. After removing the Staining solution, it was washed twice with PBS (-), 1 mg / ml sodium azide solution was added for storage, and the infected cells stained blue were counted with a microscope.

(result)
The results of the MAGIC-5 assay described above are shown on the right side of FIG. The infectious titer of HIV-1 released from cells in which hNMT1 gene was suppressed by transfection of this HIV-1 expression plasmid and shRNA expression plasmid (NMT1 siRNA target site 1) was reduced to about 50% of the control. On the other hand, there was no change in the infectious titer of HIV-1 released from cells that suppressed the hNMT2 gene (NMT2 siRNA target site 1). Moreover, as a result of examining the suppressive effect by dsRNA treatment, the infectious titer of HIV-1 released from the cells (NMT1 siRNA target site 2) in which hNMT1 gene was suppressed was reduced to about 40% of the control. On the other hand, the infectious titer of HIV-1 released from cells that suppressed the hNMT2 gene (NMT2 siRNA target site 2) decreased to about 60% of the control. The above results indicate that hNMT1 is important for HIV-1 replication and infectivity.

FIG. 1 shows N-milstoylation of proteins. FIG. 2 shows a comparison of the amino acid sequences of human NMT1 and human NMT2. FIG. 3 shows the degradation mechanism of target mRNA by siRNA, and specific examples of target sites and shRNA and dsRNA that can be used in the present invention. FIG. 4 shows the target site of siRNA. FIG. 5 shows the results of measuring the effect of hNMT1 or hNMT2-specific siRNA on HIV-1 replication.

SEQUENCE LISTING
<110> Kumamoto University
<120> NMT1 specific siRNA
<130> A51128A
<160> 16
<210> 1
<211> 19
<212> DNA
<213> human
<400> 1
gacagctggg ctgcgacca 19
<210> 2
<211> 25
<212> DNA
<213> human
<400> 2
gcgaccaatg gaaacaaagg acatt 25
<210> 3
<211> 25
<212> DNA
<213> human
<400> 3
ggacggcaac ctgcagtatt acctt 25
<210> 4
<211> 47
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Transcribed RNA
<400> 4
gacagcuggg cugcgaccau ucaagagaug gucgcagccc agcuguc 47
<210> 5
<211> 25
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Transcribed RNA
<400> 5
gcgaccaaug gaaacaaagg acauu 25
<210> 6
<211> 25
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Transcribed RNA
<400> 6
cgcugguuac cuuuguuucc uguaa 25
<210> 7
<211> 25
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Transcribed RNA
<400> 7
ggacggcaac cugcaguauu accuu 25
<210> 8
<211> 25
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Transcribed RNA
<400> 8
ccugccguug gacgucauaa uggaa 25
<210> 9
<211> 19
<212> DNA
<213> human
<400> 9
gtcagactcg gcatctgat 19
<210> 10
<211> 25
<212> DNA
<213> human
<400> 10
gctcaaggag ttatacacgt tgtta 25
<210> 11
<211> 25
<212> DNA
<213> human
<400> 11
gccacatgca gatactggca tcgat 25
<210> 12
<211> 46
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Transcribed RNA
<400> 12
gucagacucg gcaucugaug agaacuuauc agaugccgag ucugac 46
<210> 13
<211> 25
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Transcribed RNA
<400> 13
gcucaaggag uuauacacgu uguua 25
<210> 14
<211> 25
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Transcribed RNA
<400> 14
cgaguuccuc aauaugugca acaau 25
<210> 15
<211> 25
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Transcribed RNA
<400> 15
gccacaugca gauacuggca ucgau 25
<210> 16
<211> 25
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Transcribed RNA
<400> 16
cgguguacgu cuaugaccgu agcua 25

Claims (6)

shRNA, dsRNA, or siRNA characterized by inhibiting hNMT1 function and not hNMT2 function. The shRNA, dsRNA, or siRNA according to claim 1, wherein the base sequence represented by SEQ ID NO: 1, 2, or 3 is a target sequence. Any of the following shRNA, dsRNA, or siRNA.
(1) siRNA targeting the base sequence described in any one of SEQ ID NOs: 1 to 3;
(2) shRNA consisting of the base sequence set forth in SEQ ID NO: 4;
(3) dsRNA composed of a sense strand RNA consisting of the base sequence set forth in SEQ ID NO: 5 and an antisense strand RNA consisting of the base sequence set forth in SEQ ID NO: 6; or (4) the base sequence set forth in SEQ ID NO: 7 DsRNA composed of a sense strand RNA consisting of and an antisense strand RNA consisting of the base sequence set forth in SEQ ID NO: 8: An expression vector capable of expressing the shRNA, dsRNA or siRNA according to any one of claims 1 to 3. An NMT1 inhibitor comprising the shRNA, dsRNA or siRNA according to any one of claims 1 to 3 or the expression vector according to claim 4 as an active ingredient. An anti-HIV drug comprising the shRNA, dsRNA or siRNA according to any one of claims 1 to 3 or the expression vector according to claim 4 as an active ingredient.

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