RNA regulatory compositions

Abstract

To provide RNA regulatory compositions with Regnase-1.SOLUTION: The RNA regulatory composition comprises an antisense nucleic acid part against a target RNA and a stem loop part that can recruit Regnase-1. The RNA regulatory composition is designed to bind the antisense nucleic acid part to the target RNA and has a structure to which the stem loop part that can recruit Regnase-1 is bound. When the composition binds to a binding site of the target RNA, Regnase-1 is recruited and cleaves the target RNA. Thus, the RNA regulatory composition for an arbitrary RNA can be provided.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a substance that binds to any sequence in the translated and untranslated regions of mRNA by chemically modifying ASO-SL, which combines a stem-loop structure (SL) to which Regnase-1, which is involved in controlling the stability of inflammatory-related mRNA such as IL-6, binds, and an antisense nucleic acid (ASO) that binds to mRNA in a sequence-specific manner, thereby cleaving the mRNA and inhibiting its translation into protein. [Background technology]

[0002] Cytokine storm, the out-of-control proliferation of inflammatory cytokines, is a problem in various diseases. It is believed that inflammatory cytokines such as IL-6 are controlled by Regnase-1, an RNA-degrading enzyme. However, no method for controlling autoimmune diseases targeting Regnase-1 has been developed.

[0003] In view of this problem, Patent Document 1 has proposed a composition for controlling inflammatory cytokines by discovering that Regnase-1 can be increased by disrupting the stem-loop structure in the 3' untranslated region of Regnase-1 mRNA. In this document, a specific sequence of nucleotides is shown as a substance that disrupts the stem-loop structure in the 3' untranslated region of Regnase-1 mRNA.

[0004] The mechanism of action is to destroy the stem-loop structure in the 3' untranslated region of Regnase-1 mRNA, preventing Regnase-1 from destroying itself, resulting in an increase in Regnase-1. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2019 / 182055 [Non-patent literature]

[0006] [Non-Patent Document 1] Fundamentals of CAR-T cell therapy and future clinical developments, Journal of the Japanese Society of Transfusion Medicine, Vol. 65, No. 6, 851-857 [Non-Patent Document 2] Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy.Wei J,LongL[..] Chi H.Nayure.2019-12-11. Summary of the Invention [Problem to be solved by the invention]

[0007] The mechanism of action that promotes the increase of Regnase-1 mRNA by destroying its stem-loop structure is unprecedented, and it is thought that this will play a key role in controlling inflammatory cytokines in the future.

[0008] On the other hand, we hypothesized that the emergence of a method targeting Regnase-1 might enable us to solve the following problems that have been previously considered to be challenges:

[0009] (1) Immune exhaustion caused by CAR-T cell therapy Kymriah (registered trademark) (Novartis Pharma, approved by the Ministry of Health, Labour and Welfare on March 26, 2019), a CAR-T (Chimeric Antigen Receptors) cell therapy drug that utilizes T cells with enhanced ability to attack cancer cells through genome editing technology, is the first cell therapy drug to have proven effective in treating relapsed or refractory CD19-positive B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma, and is also expected to be applied to solid cancers.

[0010] However, CAR-T has the problem of immune exhaustion, which weakens its effectiveness over time (Non-Patent Document 1). On the other hand, it has been reported that CD8-positive T cells in which Regnase-1 has been knocked out maintain their activity without exhaustion (Non-Patent Document 2).

[0011] However, because Regnase-1 cannot be knocked out in vivo, this reporting technology cannot be applied to CAR-T therapy.

[0012] (2) Resistance to immune checkpoint inhibitors In recent years, many immune checkpoint inhibitors have been developed and are being applied to the treatment of various cancers, demonstrating their effectiveness. However, the therapeutic effect of immune checkpoint inhibitors is limited to 30% of patients, and 70% are resistant to immune checkpoint inhibitors. Activating immune function (cytotoxic T cells) against these immune checkpoint inhibitor-resistant cancers remains a challenge.

[0013] (3) Severe side effects from mRNA vaccines Due to its high effectiveness against the new coronavirus, mRNA has made a great contribution as a savior in overcoming the new coronavirus pandemic. However, due to its high effectiveness, severe side effects such as high fever and swelling at the injection site have also been reported.

[0014] (4) Limitations of antisense nucleic acid medicines and siRNA nucleic acid medicines Nucleic acid medicines use chemically synthesized oligonucleotides to control the expression of specific targeted genes to treat diseases, and many nucleic acid medicines have been approved in recent years. Nucleic acid medicines have several known mechanisms of action, but the most common mechanism for treating diseases is antisense nucleic acid and siRNA nucleic acid, which inhibit the translation of mRNA into protein.

[0015] The function of antisense nucleic acids is improved by chemical modification, but in many cases the activity of RNase H is inhibited, weakening the effect of the antisense nucleic acid. siRNA nucleic acids exert their functions by forming complexes such as Argonaute 2 in cells, but nonspecific off-target effects are a problem, and chemical modification often reduces their effectiveness. [Means for solving the problem]

[0016] The inventors of the present invention came up with an idea to solve these problems in view of Patent Document 1 and the above problems. That is, the present invention focuses on the fact that Regnase-1 is recruited by a specific stem loop and has the ability to cleave the recruited RNA, and provides a composition in which the stem loop is bound to an antisense nucleic acid that binds to any target RNA.

[0017] More specifically, the present invention relates to an antisense nucleic acid portion for a target RNA; Provided is an RNA regulatory composition having a stem-loop portion capable of recruiting Regnase-1. Effect of the Invention

[0018] The composition according to the present invention does not cleave RNA by itself, but in an environment where Regnase-1 is present, such as inside a cell, it can cleave RNA by recruiting Regnase-1. This allows for the following applications:

[0019] (1) Preventing immune exhaustion caused by CAR-T cell therapy By applying this technology to combine ASO-SL, which targets Regnase-1, with CAR-T cell therapy drugs, it is expected that it will be possible to strongly suppress Regnase-1 mRNA, preventing immune exhaustion while maintaining the activity of CAR-T cells.

[0020] (2) Overcoming immune checkpoint inhibitor-resistant cancer By applying this technology to combine ASO-SL, which targets Regnase-1, with immune checkpoint inhibitors, it is expected that it will be possible to strongly suppress Regnase-1 mRNA and prevent immune exhaustion while maintaining the activity of immune checkpoint inhibitors.

[0021] (3) Severe side effects from mRNA vaccines It may be possible to mitigate side effects caused by mRNA vaccines by combining them with chemically synthesized ASO-SLs that target inflammation-related mRNAs.

[0022] (4) A new nucleic acid drug that overcomes the weaknesses of antisense nucleic acid drugs and siRNA nucleic acid drugs This technology is a novel method for controlling the expression of specific genes by inhibiting the translation of mRNA into protein, and utilizes Regnase-1, which originally acts on the 3'UTR (untranslated region) of the mRNA of inflammation-related genes, to act on the coding region (CDS) of the mRNA of cancer-related genes and various other genes, making it possible to control their expression. Furthermore, Regnase-1 has the function of recognizing the stem-loop structure formed in the mRNA and cleaving the mRNA, but by introducing a chemically synthesized stem-loop structure nucleic acid from outside, it has become possible for Regnase-1 to cleave the mRNA, making it possible to easily and artificially control the expression of any gene.

[0023] Moreover, ASOs can use various types of nucleic acid derivatives, such as peptide nucleic acid (PNA), molpolynucleotide, LNA (locked nucleic acid), sugar 2'-modified nucleic acid (2'-OMeRNA, 2'-MOERNA, BNA, ENA, etc.), and phosphorothioate nucleic acid, and can be adjusted to suit the application, such as for stability in the body and affinity to the target. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 is a schematic diagram of the structure of an RNA regulatory composition according to the present invention. [Diagram 2] FIG. 2 is a schematic diagram illustrating the operation of an RNA regulatory composition according to the present invention. [Diagram 3] This is a graph showing the expression level of Regnase-1 expressed in various cells. [Figure 4] 1 is a graph showing how various samples containing the RNA regulatory composition of the present invention change the expression level of KRAS in HeLa cells. [Diagram 5] 1 is a graph showing how various samples containing the RNA regulatory composition of the present invention change the expression level of KRASG12D in PK-45H cells. [Figure 6] This is a graph that examines how various samples containing the RNA regulatory composition of the present invention change the expression level of NRAS in HeLa cells. [Figure 7] This is a graph that examines how various samples containing the RNA regulatory composition of the present invention change the expression level of NRAS in PK-45H cells. [Figure 8] 1 is a graph showing how various samples containing the RNA regulatory composition of the present invention change the expression level of HRAS in HeLa cells. [Figure 9] 1 is a graph showing how various samples containing the RNA regulatory composition of the present invention change the expression level of HRAS in PK-45H cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The RNA regulatory composition according to the present invention will be described below with reference to the drawings and examples. The following description is an example of one embodiment of the present invention and one example, and the present invention is not limited to the following description. The following description can be modified within the scope of the present invention. In addition, the embodiments and examples obtained by appropriately combining the technical means disclosed in the different embodiments and examples are also included in the technical scope of the present invention. In addition, all of the documents described in this specification are incorporated herein by reference.

[0026] The structure of an RNA regulatory composition according to the present invention is shown diagrammatically in Figure 1. RNA regulatory composition 1 according to the present invention is a composition comprising an antisense nucleic acid portion 10 that binds to a target RNA and a stem-loop portion 12 that can recruit Regnase-1. Antisense nucleic acid portion 10 is an antisense nucleic acid against a base sequence at a desired site in the target RNA.

[0027] Antisense nucleic acids are known to have RNaseH-dependent and independent mechanisms of action. Antisense nucleic acids that inhibit translation from mRNA by steric blocking in an RNaseH-independent manner include sugar-modified nucleic acids such as 2'-OMeRNA (2'-O-methyl RNA), 2'-MOERNA (2'-O-methoxyethyl RNA), LNA (locked nucleic acid), BNA (briged nucleic acid), and ENA (N-ethyl BNA), PNA (peptide nucleic acid), morpholino nucleic acid, and oligo-RNA.

[0028] On the other hand, known ASOs that cleave target mRNA in an RNase H-dependent manner include oligoDNA, phosphorothioate nucleic acid, phosphoramidate nucleic acid, and Gapmer ASOs in which these nucleic acids are sandwiched between 2'-OMeRNA, LNA, etc. In the present invention, both types of ASOs can be used as the antisense nucleic acid portion 10 in the technology.

[0029] The stem-loop portion 12 can preferably use a stem-loop structure formed in genes involved in inflammatory action such as IL-6, IL-1β, IL-2, and IL-12p404-7, or in the 3'UTR of Regnase-1 itself. Alternatively, it may be an artificially designed stem-loop structure based on these stem-loop structures. Specific examples are shown in Table 1.

[0030] [Table 1]

[0031] Referring to Table 1, SEQ ID NO: 1 is a stem-loop structure formed in the 3'-UTR of IL-6, and SEQ ID NO: 2 and SEQ ID NO: 3 are stem-loop structures formed in the 3'-UTR portion of Regnase-1 mRNA.

[0032] In addition, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 are artificial stem-loop structures designed from these stem-loop structures. These base structures can be preferably used, but of course structures other than these can also be used as long as they can preferably recruit Regnase-1.

[0033] As for the antisense nucleic acid portion 10 and the stem-loop portion 12, it is preferable that the stem-loop portion 12 is positioned on the 5' side, as will be shown in the Examples described later.

[0034] Next, the operation of the RNA regulatory composition 1 according to the present invention will be described. Please refer to FIG. 2(a). The target RNA (hereinafter referred to as "target T10") may be any RNA. In other words, it is not limited to being mRNA. In addition, there is no need to distinguish between the translated region and the untranslated region, and either region may be used. The site where the RNA regulatory composition 1 binds to the target T10 is referred to as the binding site T10s. Of course, the antisense nucleic acid portion 10 of the RNA regulatory composition 1 has an antisense structure to the binding site T10s. Therefore, the antisense nucleic acid portion 10 of the RNA regulatory composition 1 binds to the binding site T10s.

[0035] See Figure 2(b). When RNA regulatory composition 1 binds to the binding site T10s of target T10, Regnase-1 (20) is recruited to stem-loop portion 12. It then cleaves target T10. In this way, RNA regulatory composition 1 can cleave target T10. Therefore, it is assumed that RNA regulatory composition 1 of the present invention is used in an environment where Regnase-1 is present. EXAMPLES

[0036] <Quantitative analysis of Regnase-1 expression in various cells> The RNA control composition 1 according to the present invention requires an environment in which Regnase-1 is present. Therefore, the expression levels of Regnase-1 in various cells were quantified. The quantified cell lines are as follows. HeLa cells (cervical cancer-derived cell line), PK-45H (human pancreatic cancer-derived cell line), PK-1 (human pancreatic cancer-derived cell line), PK-56 (human pancreatic cancer-derived cell line)), T3M-10 (lung large cell carcinoma-derived cell line), HepG2 cells (human liver cancer-derived cell line), K562 (leukemia cell line), MT-1 (leukemia cell line), MT-2 (leukemia cell line), THP-1 cells (human monocyte-derived cell line), U2OS GFP-NUP98 (osteosarcoma cell-derived cell line), 293 (human fetal kidney-derived cell line), 293T (human fetal kidney-derived cell line).

[0037] The expression levels of Regnase-1 in various cells were calculated by a conventional method. The primers used are shown in Table 2.

[0038]

Table 2

[0039] The results are shown in Figure 3. Referring to Figure 3, the horizontal axis represents various cells, and the vertical axis represents the expression level of Regnase-1. The expression level is the relative value with respect to the expression level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) in various cells. In addition, the various cells on the horizontal axis indicate that they were measured twice each.

[0040] Referring to Figure 3, it was confirmed that Regnase-1 is present in cells, regardless of cancer cells or normal cells. Therefore, the RNA control composition 1 according to the present invention can be used for living cells.

[0041] <Selective suppression of the KRAS gene> [HeLa cells] The silencing effects of KRAS-WTm, KRAS-WTm-3SL, and KRAS-WTm-5SL were evaluated in HeLa cells expressing wild-type KRAS. For comparison, the silencing effects of siRNAWT (13) and siRNAG12D (13) were also evaluated. These nucleic acid derivatives were synthesized by the standard phosphoramidite method using a conventional DNA / RNA automatic synthesizer and commercially available reagents. After synthesis, the derivatives were heated in ammonia water at 55°C for 5-6 hours according to the standard method, and then purified by reversed-phase HPLC. The obtained compounds were confirmed to be the desired compounds by reversed-phase HPLC and electrospray ionization mass spectrometry (ESI-MS).

[0042] KRAS-WTm indicates antisense nucleic acids that bind to the wild type of KRAS. The "m" indicates that these antisense nucleic acids are 2'-OMeRNA (2'-O-methyl RNA). This is because the 2' end of the sugar moiety that constitutes the nucleic acid is methylated, which provides structural stability.

[0043] KRAS-WTm-3SL indicates a structure in which a stem-loop portion (-3SL) is bound to the 3'-end of an antisense nucleic acid portion (KRAS-WTm) that binds to the wild-type form of KRAS. Here, the stem-loop structure of SEQ ID NO: 4 was used.

[0044] Similarly, KRAS-WTm-5SL indicates a structure in which a stem-loop portion (-5SL) is bound to the 5'-end side of an antisense nucleic acid portion (KRAS-WTm) that binds to the wild-type form of KRAS. These base sequences are shown in Table 3 as SEQ ID NOs: 9-11. An introduction to these is also shown in Table 4.

[0045] [Table 3]

[0046] [Table 4]

[0047] The siRNAWT(13) used for comparison knocks out the wild type of KRAS using siRNA (small interfering RNA). The siRNAG12D(13) knocks out a mutant of KRAS in which the 12th amino acid is mutated from G (glycine) to D (aspartic acid). The base sequences of the two siRNAs are shown in Table 5 as SEQ ID NOs: 12 and 13.

[0048] [Table 5]

[0049] The 13th base from the 5' end of siRNAWT(13) and siRNAG12D(13) is changed. In Table 5, the difference between cytosine and uracil is indicated by an asterisk (*) on the right superscript.

[0050] The results are shown in Figure 4. Referring to Figure 4, the horizontal axis indicates the type of sample, and the vertical axis indicates the relative value of the KRAS expression level to 18S. Referring to the horizontal axis, "None" represents ···, "LP2000" represents the value of KRAS mRNA quantified when only a gene transfer reagent called LipofectamineTM2000 (manufactured by Thermo Fisher Scientific) was added to the cell culture medium, and Control represents EGFP (enhanced green fluorescence protein).

[0051] In addition, "WT(13)" represents the case where siRNAWT(13) was used, and "G12D(13)" represents the case where siRNAG12D(13) was used. "WTm", "3SL", and "5SL" represent the cases where KRAS-WTm, KRAS-WTm-3SL, and KRAS-WTm-5SL were used, respectively.

[0052] In the following, the silencing effect (%) was calculated by setting the value of LP2000 as 100%, and the value of the bar falling below 100 was considered to be the silencing effect (%).

[0053] siRNAWT (13) targeting wild-type KRAS suppressed KRASWT mRNA almost concentration-dependently, with approximately 75% silencing effect at 10 nM and 100 nM, whereas siRNAG12D (13) targeting mutant KRASG12D mRNA had no silencing effect at all.

[0054] In addition, the antisense sequence KRAS-WTm against wild-type KRAS mRNA showed no silencing effect, but KRAS-WTm-3SL, which has a Regnase-1 binding RNA motif attached to the 3'-end of KRAS-WTm, suppressed KRASWT mRNA in a concentration-dependent manner, showing 50% silencing effect at 100 nM and 75% silencing effect at 1000 nM. However, KRAS-WTm-5SL did not show a significant silencing effect.

[0055] These results strongly suggested that KRAS-WTm-3SL recruited Regnase-1, which exerted its silencing effect by cleaving KRASWT mRNA 3' to its binding site.

[0056] [PK-45H cells] The silencing effect was evaluated in PK-45H cells expressing mutant KRASG12D. The results are shown in Figure 5. Referring to Figure 5, the horizontal axis indicates the sample type, and the vertical axis indicates the expression level of KRASG12D. Each sample type is the same as in Figure 4.

[0057] The control siRNA-WT(13) showed a weak silencing effect of 25-50%. Since wild-type KRASWT was not expressed in PK-45H cells, it was found that siRNA-WT(13) also targeted mutant KRASG12DmRNA.

[0058] In other words, siRNA-WT(13) was unable to distinguish the single nucleotide mutation in the target. siRNAG12D(13) showed a silencing effect in a dose-dependent manner, with silencing efficiency of 75% at 10 nM and 80% at 100 nM.

[0059] On the other hand, when the silencing effects of KRAS-WTm, KRAS-WTm-3SL, and KRAS-WTm-5SL were evaluated, none of them showed any silencing effect at all. The sequence of KRAS-WTm has a mismatch in the center with KRASG12DmRNA (see Table 4), and it is thought that the silencing effect was not shown due to the reduced thermodynamic stability of the pairing. KRAS-WTm-3SL shows a very high ability to discriminate single base mutations.

[0060] Furthermore, it was demonstrated that the method had no effect on the HRAS and NRAS genes of the RAS family (Figures 6 to 9). In other words, the gene silencing method using the ASO-SL motif is promising as a novel nucleic acid drug with high specificity, and is particularly promising as a nucleic acid drug that targets mutated cancer genes by identifying single-base mutations.

[0061] Figure 6 shows the results of NRAS in HeLa cells, and Figure 7 shows the results of NRAS in PK-45H cells. Figure 8 shows the results of HRAS in HeLa cells, and Figure 9 shows the results of HRAS in PK-45H cells. In both cases, it can be seen that KRAS-WTm-3SL does not show any silencing effect.

[0062] <Effects of the invention> 1. By introducing a chemically synthesized ASO-SL, which is a combination of a chemically modified antisense oligonucleotide (ASO) targeting the KRAS gene and a Regnase-1 binding sequence (SL), into cervical cancer-derived HeLa cells, we succeeded in controlling KRAS mRNA expression in a concentration-dependent manner. This is expected to be applied to new nucleic acid medicines as a new method for controlling gene expression.

[0063] 2. When the above ASO-SL was introduced into pancreatic cancer-derived PK-45H cells, the amount of expressed G12D mutant KRAS gene mRNA was hardly affected. In other words, ASO-SL recognized a single base mutation (35G>A) in the KRAS gene and specifically suppressed the target gene.

[0064] 3. The ASO-SL did not affect the amount of HRAS or NRAS mRNA in either HeLa or PK-45H cells, i.e., ASO-SL selectively regulated only wild-type KRA mRNA in HeLa cells.

[0065] 4.Regnase-1 originally acts on the untranslated region (3'UTR) of mRNA of inflammation-related genes, but by using chemically synthesized ASO-SL, it can be made to act on any gene expressed in any cell. Moreover, it is now possible to target the translation region (coding region) of mRNA, enabling highly versatile direct translation control.

[0066] 5. By using chemically synthesized ASO-SL, it became possible to stably control gene expression in vivo.

[0067] 6. The combination of CAR-T cells and a chemically synthesized ASO-SL that targets Regnase-1 may be a way to resolve immune exhaustion, which gradually weakens the efficacy of CAR-T cell therapy.

[0068] 7. Combining immune checkpoint inhibitors with chemically synthesized ASO-SL targeting Regnase-1 may alleviate T cell exhaustion after immune checkpoint inhibitor treatment.

[0069] 8. It may be possible to mitigate side effects caused by mRNA vaccines by combining them with chemically synthesized ASO-SLs that target inflammation-related mRNAs.

[0070] 9. It may be possible to suppress the proliferation of cancer cells using chemically synthesized ASO-SLs that target cancer-related gene mRNA.

[0071] 10. It may be possible to treat diseases fundamentally using chemically synthesized ASO-SLs that target disease-related genes. [Industrial Applicability]

[0072] This technology is expected to be useful for selectively suppressing the expression of cancer genes, preventing immune exhaustion when used in combination with CAR-T cell therapy or immune checkpoint inhibitors, and suppressing side effects of mRNA vaccines. [Explanation of symbols]

[0073] 1. RNA Regulatory Composition 10 Antisense Nucleic Acid Division 12 Stem loop section

Claims

1. An antisense nucleic acid portion against a target RNA; An RNA regulatory composition having a stem-loop portion capable of recruiting Regnase-1.

2. The RNA regulatory composition according to claim 1, wherein the stem-loop portion is provided on the 5' side of the antisense nucleic acid portion.

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