Transcriptional inhibitors of immune-related genes

Abstract

The problem addressed by the present invention is to provide a pyrrole imidazole polyamide (PIPA) for transcriptionally inhibiting the expression of cytokines necessary for immune response activation, particularly type I IFNs. More specifically, the present invention is a PIPA that specifically binds to a binding region of an IFN regulatory factor (IRF) family transcription factor. Specifically, said PIPA binds to 5'-GAAAGTG-3 (sequence number 1) or a modified sequence thereof, 5'-GAAAG-3' (sequence number 9) or a modified sequence thereof, 5'-GAAAA-3' (sequence number 15) or a modified sequence thereof, or 5'-GAAAC-3' (sequence number 21) or a modified sequence thereof.

Description

[Technical Field]

[0001] The present invention relates to a means for suppressing the transcription of immune-related genes. More specifically, the present invention relates to pyrrole imidazole polyamides (PIPA) that specifically bind to the binding domains of IRF family transcription factors on genomic DNA and can function as competitive pseudo-transcription factors. [Background technology]

[0002] The immune system has the ability to distinguish between self and non-self structures at the molecular level. It recognizes invading pathogens as non-self and activates an immune response to eliminate them through various mechanisms. Specifically, an immune response is activated specifically against non-self molecules, with the appropriate strength required for their elimination. After the pathogen is eliminated, the immune response quickly ceases, maintaining homeostasis. However, the immune response is not always beneficial to the body; its failure can lead to the onset or exacerbation of various diseases. First, immune response activation is accompanied by inflammation. While inflammation itself is an important response that triggers the accumulation and activation of immune cells, excessive activation can destroy tissue and trigger various inflammatory diseases. Furthermore, while the immune response is activated against non-self molecules, a failure of this discrimination can result in activation of the immune response against self molecules, leading to autoimmune diseases. Furthermore, when eliminating intracellular pathogens such as viruses, both the virus and the cell are inevitably eliminated simultaneously. However, if this elimination response persists without convergence, it can naturally pose a critical threat to the body. Furthermore, in situations other than disease, for example, in the case of mRNA vaccines, if the mRNA vaccine activates the antiviral response too much, the mRNA vaccine itself will be eliminated by the immune system and will not be effective. Because immune responses have various unfavorable aspects, if we can control the response to an appropriate level, it may be possible to treat and prevent various diseases and enhance the effectiveness of mRNA vaccines.

[0003] The immune response is activated when immune cells directly encounter a pathogen. Various responses and cell types are sequentially activated, resulting in a defensive response by innate immune cells, which are less specific but can be activated rapidly, followed by a highly specific response by adaptive immune cells. This immune response activation and intercellular communication are primarily mediated by humoral factors called cytokines, which play an important role in activating, directing, and suppressing immune responses. Immune cells that encounter pathogens produce various cytokines to activate distant cells and, depending on the type of cytokine, activate a response appropriate to the pathogen. Therefore, once the pathogen is successfully eliminated, cytokine production weakens, resulting in the end of the immune response. However, if the pathogen cannot be completely eliminated, immune cells continue to produce more cytokines, further activating the immune response. This negative chain reaction leads to a cytokine storm, in which massive cytokines are released. As is also known from SARS-CoV-2, cytokine storms can cause fatal multiple organ failure due to the activation of excessive immune responses (inflammatory responses), leading to the death of the organism.

[0004] Various cytokines are involved in regulating immune responses. Among them, type I IFNs (interferons) are induced by the recognition of pathogen components or viral infection and play an essential role in activating the immune response (Non-Patent Document 1). Type I IFNs consist of 13 subtypes, including IFN-β, IFN-ε, IFN-κ, IFN-ω, and IFN-α. All of these subtypes induce an antiviral state in cells and play an essential role in suppressing viral proliferation at the cellular level (Non-Patent Documents 1 and 2). Furthermore, they activate various immune cells, such as promoting antibody production from B cells, inducing differentiation into T cells, and maturing dendritic cells, thereby activating the entire immune response and playing an important role in host defense (Non-Patent Document 3). On the other hand, as mentioned above, it is known that failure of this control can cause adverse responses in the body, and it is an important cytokine that is deeply involved in autoimmune and autoinflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, and type I diabetes, as well as the worsening of pathology during viral infections, cytokine storms, and the inhibition of the effectiveness of mRNA vaccines (Non-Patent Documents 1, 4, and 9).

[0005] The production of cytokines, including type I IFNs, is triggered by cells directly exposed to pathogens or pathogen components, which recognize the pathogen components through receptors called pattern recognition receptors (PRRs). When PRRs recognize pathogen components, intracellular signaling pathways are activated, ultimately leading to the activation of transcription factors. The activated transcription factors translocate to the nucleus and bind to the promoter region of target genes, liberating the region and promoting the association of general transcription factors, which then triggers the production of mRNA using DNA as a template. The production of most cytokines is regulated at the transcriptional level. After cytokine mRNA is produced in the cell, it is translated into protein and secreted extracellularly, thereby exerting its function. Cytokine transcription is typically regulated by multiple transcription factors, and three major transcription factors are known to function downstream of PRRs: IFN regulatory factor (IRF) family transcription factors, NF-κB, and AP-1 (Non-Patent Document 1 and Non-Patent Document 5). Both are controlled by the activation of downstream signals from pattern recognition receptors, and translocate to the nucleus after activation, but it is known that the degree of dependence on each transcription factor varies depending on the type of gene.

[0006] Among these, nine IRF transcription factor families, IRF1 to IRF9, have been reported, and IRF transcription factors are known to play a particularly important role in the transcription of type I IFN (Non-Patent Documents 5 and 6). Specifically, it is known that type I IFN induction is highly dependent on IRF transcription factors, and type I IFN induction is significantly inhibited in cells and mice lacking IRF3 or IRF7, which are IRF transcription factors. The promoter regions of all type I IFN genes contain an IRF transcription factor binding sequence (IRF-E), and specific binding of IRF transcription factors to this sequence activates type I IFN gene expression (Non-Patent Documents 7 and 8).

[0007] Furthermore, recent reports have revealed that IRF transcription factor binding sequences are present in the promoter regions of other cytokines, such as IL-6 and TNF-α, demonstrating that IRF transcription factors are important transcription factors for regulating not only type I IFN but also various other cytokines (Non-Patent Document 5). Controlling these IRF transcription factors could potentially enable the treatment of various diseases through the regulation of type I IFN responses and immune responses. However, it is known that some of the nine IRF transcription factors function complementarily, and it has also been reported that different IRF transcription factors are important for different cells and genes. Therefore, a method for specifically and effectively controlling the transcriptional activity of IRF transcription factors has yet to be developed, which remains an important challenge in the field of immunology. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Fernandez-Ruiz and Niewold, J Invest Dermatol, doi:10.1016 / j.jid.2021.11.031 (2022). [Non-patent document 2] Gibbert et al., Br J Pharmacol 168, 1048-1058, doi:10.1111 / bph.12010 (2013). [Non-patent document 3] Ivashkiv et al., Nat Rev Immunol 14, 36-49, doi:10.1038 / nri3581 (2014). [Non-patent document 4] Jefferies, Front Immunol 10, 325, doi:10.3389 / fimmu.2019.00325 (2019). [Non-patent document 5] Negishi et al., Cold Spring Harb Perspect Biol. 10(11):a028423. (2018). [Non-patent document 6] Tamura et al., Annu Rev Immunol. 26:535-84. (2008) [Non-Patent Document 7] Braganca and Civas, Biochimie 80, 673-687, doi:10.1016 / s0300-9084(99)80021-2 (1998). [Non-patent document 8] Honda et al., Int Immunol 17, 1367-1378, doi:10.1093 / intimm / dxh318 (2005). [Non-Patent Document 9] Beuckelaer et al., Mol Ther. 24(11): 2012-2020. (2016) Summary of the Invention [Problem to be solved by the invention]

[0009] In view of the above circumstances, an objective of the present invention is to provide a novel means for controlling the transcription of immune-related genes. More specifically, an objective of the present invention is to provide pyrrole imidazole polyamides (PIPAs) that function as competitive pseudo-transcription factors that specifically bind to binding regions of IRF family transcription factors on genomic DNA and suppress the transcription of IRF family transcription factors, a method for producing the PIPAs, and a cytokine transcription inhibitor containing the PIPAs. [Means for solving the problem]

[0010] The inventors focused on the genomic DNA sequences to which IRF transcription factors bind and investigated the use of PIPA as a means of suppressing type I IFN transcription. PIPA is a small organic compound composed of pyrrole and imidazole isolated from antibiotics. It can target and bind to specific genomic DNA sequences. Furthermore, it easily crosses cell membranes. Therefore, if PIPA can be engineered to bind to key promoter sequences with a certain degree of binding affinity, it could inhibit transcription factor binding to the promoter region and control target protein expression at the transcriptional level. Because it can be designed to target any DNA sequence, it is theoretically possible to control the expression of any gene. Furthermore, it has a much higher cellular transduction efficiency than previous gene regulation methods such as siRNA and antisense nucleic acids. However, identifying sequences important for transcription factor binding and designing PIPA (e.g., adjusting the distance between the pyrrole and imidazole) require advanced expertise. Even if the binding sequence can be identified, designing PIPA with the desired binding affinity and specificity is not easy. To date, no PIPA has been developed that can effectively control the activation of early immune responses, including type I IFN responses. IRF family transcription factors are known to bind to specific sequence regions called IRF-E (IRF-responsive element) and ISRE (IFN-stimulated response element). However, the identification of these sequence regions has mainly been based on the analysis of artificial experimental systems at the cellular level. Therefore, it has not yet been possible to narrow down which sequences are physiologically important or which sequences are suitable as drug targets.

[0011] Therefore, the present inventors conducted extensive research to narrow down the predicted binding sequences of IRF family transcription factors based on these sequence information and to design PIPAs that bind to these sequences with the desired binding strength and specificity. Specifically, the present inventors narrowed down the sequences predicted to be most suitable for binding to IRF family transcription factors to four, constructed four PIPAs that bind to each sequence, and examined the effects of these PIPAs on IFN-β transcription. All PIPAs were confirmed to suppress IFN-β transcription. From another perspective, the PIPA produced by the present inventors has the property of binding to sequences to which IRF family transcription factors specifically bind, and can be said to function as a "pseudo-transcription factor" that inhibits transcription and / or activation mediated by IRF family transcription factors via their binding sequences. Herein, the term "pseudo-transcription factor" is broadly defined as a substance that has the property of binding specifically to a specific transcription factor and inhibits transcription and / or activation mediated by the transcription factor via its binding sequence.

[0012] The inventors have demonstrated for the first time that PIPA according to this embodiment, which can function as a pseudo-transcription factor, can suppress or inhibit the transcription of genes that are transcriptionally activated by IRF family transcription factors, and can be used as a medicine for suppressing excessive immune responses and as an adjuvant for mRNA vaccines, thereby completing the present invention. That is, the present invention is the following (1) to (38). (1) Pyrrole imidazole polyamide (PIPA) specifically binds to the binding domain of IRF (IFN regulatory factor) family transcription factors. (2) PIPA according to (1) above, which functions as a pseudo-transcription factor. (3) The PIPA according to (1) above, which has a binding affinity to the binding domain as expressed as a dissociation constant (Kd value) of about 500 nM or less. (4) The PIPA described in (3) above, wherein the binding region is a region containing 5'-GAAAGTG-3' (SEQ ID NO: 1) or a modified sequence thereof, and the modified sequence includes a sequence in which any one base in 5'-GAAAGTG-3' (SEQ ID NO: 1) is deleted or mutated, or a sequence in which one base is added to any position in 5'-GAAAGTG-3' (SEQ ID NO: 1). (5) The PIPA described in (3) above, wherein the binding region is a region containing 5'-GAAAG-3' (SEQ ID NO: 9) or a modified sequence thereof, and the modified sequence includes a sequence in which any one base in 5'-GAAAG-3' (SEQ ID NO: 9) is deleted or mutated, or a sequence in which one base is added to any position in 5'-GAAAG-3' (SEQ ID NO: 9). (6) The PIPA described in (3) above, wherein the binding region is a region containing 5'-GAAAA-3' (SEQ ID NO: 15) or a modified sequence thereof, and the modified sequence includes a sequence in which any one base in 5'-GAAAA-3' (SEQ ID NO: 15) is deleted or mutated, or a sequence in which one base is added to any position in 5'-GAAAA-3' (SEQ ID NO: 15). (7) The PIPA described in (3) above, wherein the binding region is a region containing 5'-GAAAC-3' (SEQ ID NO: 21) or a modified sequence thereof, and the modified sequence includes a sequence in which any one base in 5'-GAAAC-3' (SEQ ID NO: 21) is deleted or mutated, or a sequence in which one base is added to any position in 5'-GAAAC-3' (SEQ ID NO: 21). (8) A PIPA according to any one of (4) to (7) above, represented by the following formula (I), (II), (III) or (IV): [ka] [ka] [ka] [ka] (In formulas (I), (II), (III), and (IV), L represents a C2-6 alkyl linker; R1 and R2 represent a C1-C10 alkyl group which may have a substituent or a C1-C10 alkyl group which may contain an amide bond and / or an amino group; R1 and R2 may combine to form a C2-6 alkyl linker; and X1, X2, X3, X4, X5, X6, X7, and X8, the same or independently, represent a bond or an aliphatic amino acid residue.) (9) The PIPA according to (8) above, which is represented by the following formula (Ia), (IIa), (IIIa) or (IVa): [ka] [ka] [ka] [ka] (10) A composition comprising PIPA that specifically binds to the binding domain of an IRF family transcription factor for inhibiting the function of the IRF family transcription factor. (11) The composition according to (10), wherein the PIPA is the PIPA according to any one of (2) to (7). (12) The composition according to (10), wherein the PIPA is the PIPA according to (8). (13) The composition according to (10) above, wherein the PIPA is the PIPA according to (9) above. (14) A composition comprising PIPA that specifically binds to the binding domain of an IRF family transcription factor, for use as a pseudo-transcription factor. (15) An inhibitor of an IRF family transcription factor, including PIPA, which specifically binds to the binding domain of an IRF family transcription factor. (16) The IRF family transcription factor inhibitor according to (15) above, wherein the PIPA is the PIPA according to any one of (2) to (7) above. (17) The IRF family transcription factor inhibitor according to (15) above, wherein the PIPA is the PIPA according to (8) above. (18) The IRF family transcription factor inhibitor according to (15) above, wherein the PIPA is the PIPA according to (9) above. (19) A pharmaceutical or pharmaceutical composition for suppressing an excessive immune response, comprising, as an active ingredient, PIPA that specifically binds to the binding domain of an IRF family transcription factor. (20) The pharmaceutical or pharmaceutical composition according to (19) above, wherein the PIPA is a PIPA according to any one of (2) to (7) above. (21) The pharmaceutical or pharmaceutical composition according to (19) above, wherein the PIPA is the PIPA according to (8) above. (22) The pharmaceutical or pharmaceutical composition according to (19) above, wherein the PIPA is the PIPA according to (9) above. (23) An adjuvant containing PIPA that specifically binds to the binding domain of an IRF family transcription factor. (24) The adjuvant according to (23) above, wherein the PIPA is a PIPA according to any one of (2) to (7) above. (25) The adjuvant according to (23) above, wherein the PIPA is the PIPA according to (8) above. (26) The adjuvant according to (23) above, wherein the PIPA is the PIPA according to (9) above. (27) A method for producing an IRF family transcription factor inhibitor including PIPA, comprising: The method comprises the steps of providing a binding domain of an IRF family transcription factor and producing a PIPA that specifically binds to the binding domain. (28) A complex comprising PIPA that specifically binds to the binding region of an IRF family transcription factor and a PIPA cofactor, wherein the PIPA cofactor is a substance different from the PIPA, and the PIPA cofactor enhances the function of the PIPA and / or assists the action of PIPA. (29) The complex according to (28) above, wherein the PIPA and the PIPA cofactor are linked via a C1 to C6 linker. (30) The complex described in (28) above, wherein the PIPA cofactor is a substance that enhances the function of the PIPA and is one or more substances selected from the group consisting of hydroxychloroquine, steroids, and FK506. (31) The complex according to (28), wherein the PIPA cofactor is a substance that assists the action of the PIPA and is one or more substances selected from the group consisting of vitamins, cholesterol, and anisamide. (32) The complex according to any one of (28) to (31) above, wherein the PIPA is the PIPA according to any one of (2) to (7) above. (33) The complex according to any one of (28) to (31) above, wherein the PIPA is the PIPA according to (8) above. (34) The complex according to any one of (28) to (31) above, wherein the PIPA is the PIPA according to (9) above. (35) A complex according to any one of (28) to (31) above, which functions as a pseudo-transcription factor. (36) The complex according to (32) above, which functions as a pseudo-transcription factor. (37) The complex according to (33) above, which functions as a pseudo-transcription factor. (38) The complex according to (34) above, which functions as a pseudo-transcription factor. In this specification, the symbol "to" indicates a numerical range including the values ​​on either side of it. [Effects of the Invention]

[0013] The present invention provides PIPA that specifically binds to the binding domain of IRF family transcription factors and suppresses their transcriptional activity. PIPA of the present invention suppresses the expression of various target genes, including cytokines and chemokines, whose transcription is activated by IRF family transcription factors, such as type I IFN, IL-6, and TNF-α. Therefore, pharmaceuticals and the like containing PIPA of the present invention as an active ingredient are effective in preventing and treating excessive immune responses caused by target genes of IRF family transcription factors, including these cytokines, and diseases and the like caused by such excessive immune responses. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the results of an investigation into the effect of PIPA according to this embodiment on IFN-β transcription. Raw264.7 cells, a macrophage cell line, were seeded at 2 × 10 cells / well on a 96-well plate and allowed to adhere for 8 hours. IRF-PIPA was then added to final concentrations of 1, 3, and 10 μM. After 24 hours of culture, LPS was added to a final concentration of 20 ng / mL, and the supernatant was collected after 16 hours of culture. The supernatant was diluted 2-fold with 1% BSA-PBS, and the IFN-β concentration was measured by ELISA. [Figure 2] Figure 2 shows the results of an investigation into the effects of IRF-PIPA3 on the transcription of IFN-β, IL-6, and TNF-α (1). Peritoneal macrophages were collected from wild-type mice and seeded at 2.5 × 10 cells / well in 96-well plates for 2 hours. The cells were then washed twice with PBS. IRF-PIPA3 was then suspended in 10% FBS-RPMI or D-MEM to a final concentration of 3 or 10 μM and added to the wells. After 24 hours of incubation, Poly(I:C) was added to a final concentration of 1 μg / mL, and the cells were incubated for 16 hours. The supernatants were then collected, and the concentrations of IFN-β, IL-6, and TNF-α were measured by ELISA. [Figure 3]Figure 3 shows the effects of IRF-PIPA3 on the transcription of IFN-β, IL-6, and TNF-α (2). Peritoneal macrophages were collected from mice using PBS and seeded at 2.5 × 10 cells / well in 96-well plates for 2 hours. The cells were then washed twice with PBS, and IRF-PIPA3 was added to the cells at a final concentration of 6 μM in 10% FBS-RPMI or D-MEM medium. After 24 hours of culture, Poly(I:C) was added to a final concentration of 1 μg / mL. After 2, 4, and 6 hours of culture, the cells were harvested and suspended in 100 μL / well using TRIsure. The relative expression levels of Ifnb1, Il6, and TNF genes were measured by qRT-PCR. [Figure 4] Figure 4 shows the results of an investigation into the cytotoxic activity of IRF-PIPA3. To verify the cytotoxic activity of IRF-PIPA3, the dsDNA concentration (left) and LDH concentration (right) in the culture supernatant were measured. The same experiment as in Figure 2 was performed, and the culture supernatant was collected. As a positive control, a lysate prepared by disrupting 2.5 x 105 peritoneal macrophages was used, mixed with 90 μL of 10% FBS-RPMI or D-MEM medium and 10 μL of lysis buffer. [Figure 5] Figure 5 shows the results of an investigation into the binding specificity and affinity of IRF-PIPA3 to its target sequence. Using Octet K2, we analyzed the binding of IRF-PIPA3 to the target DNA sequence at each concentration shown in the figure. After mixing the PIPA solution with the target DNA, the binding between the two was measured for 60 seconds. The DNA sequence was then transferred to a solution containing no PIPA, and the dissociation between the two was measured for another 60 seconds. From the time course of binding and dissociation at each concentration, the KD value, which is the dissociation constant indicating the binding strength, was calculated. [Figure 6]Figure 6 shows the results of an in vivo study of the effects of IRF-PIPA3 on the transcription of IFN-β, IL-6, and TNF-α. Wild-type mice were intraperitoneally administered IRF-PIPA3 at 400 μg / mouse, followed 3 hours later by intraperitoneal administration of Poly(I:C) at 250 μg / mouse. Peritoneal macrophages were harvested 2 hours after Poly(I:C) administration, and the relative expression levels of Ifnb1, Il6, and TNF genes were analyzed by qRT-PCR. [Figure 7] Figure 7 shows the results of an investigation into the effects of IRF-PIPA3 on the expression of IFN-β, IL-6, and TNF-α proteins in vivo. Wild-type mice were intraperitoneally administered IRF-PIPA3 at 400 μg / mouse, and 24 hours later, Poly(I:C) was intraperitoneally administered at 250 μg / mouse. Blood was collected 2 hours after Poly(I:C) administration, and the plasma concentrations of IFN-β, IL-6, and TNF-α proteins were measured by ELISA. [Figure 8] Figure 8 shows the effect of IRF-PIPA3 on the transcriptional activation of IFN-β by an mRNA vaccine (mRNA vaccine for SARS-CoV-2). Peritoneal macrophages were collected from wild-type mice and seeded at 2.5 × 105 cells / well in a 96-well plate for 2 hours. The cells were then washed twice with PBS. IRF-PIPA3 was then suspended in 10% FBS-RPMI or D-MEM to a final concentration of 3 or 10 μM and added to the wells. After 24 hours of culture, the mRNA vaccine was added to a final concentration of 1 μg / mL and cultured for 20 hours. RNA was then prepared from the cells and the relative expression level of the Ifnb1 gene was measured by qRT-PCR (left), or the culture supernatant was collected and the IFN-β concentration was measured by ELISA (right). [Figure 9]Figure 9 shows the effects of IRF-PIPA3 on the induction of IFN-β, IL-6, and TNF-α protein expression by an mRNA vaccine (mRNA encoding ovalbumin). Peritoneal macrophages were collected from wild-type mice and seeded at 2.5 × 105 cells / well in a 96-well plate. The cells were then cultured for 2 hours. The cells were then washed twice with PBS. Next, IRF-PIPA3 was suspended in 10% FBS-RPMI or D-MEM to a final concentration of 6 μM and added to the wells. After 16 hours of culture, the mRNA vaccine was added to a final concentration of 0.5 μg / mL, and the cells were cultured for 20–24 hours. The culture supernatants were then collected, and the concentrations of IFN-β, IL-6, and TNF-α proteins were measured by ELISA. [Figure 10] Figure 10 shows the results demonstrating that IRF-PIPA can improve the suppression of vaccine antigen expression by double-stranded RNA. Wild-type mice were administered IRF-PIPA3 at 400 μg / mouse intramuscularly (im) or intraperitoneally (ip). 24 hours later, Moderna's SARS-CoV-2 mRNA vaccine was administered intramuscularly at 1 μg / mouse, and Poly(I:C) was simultaneously administered intraperitoneally or intramuscularly at 250 μg / mouse. One day later, blood was collected from the mice, and plasma RBD concentrations were measured by ELISA. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, embodiments of the present invention will be described. Note that, unless otherwise specified, the term "the present embodiment" refers to all embodiments described in this specification. The first embodiment is a pyrrole imidazole polyamide (PIPA) that specifically binds to the binding domain of an IRF (IFN regulatory factor) family transcription factor (hereinafter also referred to as "PIPA according to this embodiment"). In this embodiment, "PIPA" refers to a low-molecular-weight organic compound isolated from an antibiotic, primarily composed of pyrrole (Py) and imidazole (Im), capable of sequence-specific binding to double-stranded DNA. PIPA penetrates the minor groove of double-stranded DNA and reversibly binds to each base of the DNA via hydrogen bonds. Py binds to A, T, and C residues, and Im binds to G residues. By appropriately configuring the Py and Im sequences, PIPAs can be designed to bind to any DNA sequence. PIPAs generally form a hairpin loop structure so that the Py / Im pair targets CG base pairs and the Py / Py pair targets AT or TA base pairs in the minor groove of double-stranded DNA (see the specific structure of PIPA shown below). However, PIPAs may also be pairs of two independent PIPAs not connected by a hairpin loop, or they may form a ring structure. Appropriate structures can be designed depending on the purpose.

[0016] The PIPA in this embodiment is a polyamide in which pyrrole and imidazole are primarily linked by amide (carboxamide) bonds, but this amide chain may contain aliphatic amino acid residues, such as glycine, β-alanine, γ-aminobutyric acid, R2,4-diaminobutyric acid, and 5-aminovaleric acid, to adjust its length or degree of curvature. Here, γ-aminobutyric acid can be used to maintain a hairpin loop structure, and β-alanine can be used to optimize hydrogen bonding between adjacent amino acid moieties and nucleotide moieties of a DNA strand.

[0017] The binding region of an IRF family transcription factor to which PIPA according to this embodiment binds includes the following sequences 1 to 4 or modified sequences thereof. Sequence 1 and its modified sequences 5'-GAAAGTG-3' (SEQ ID NO: 1) or A region containing a modified sequence of the sequence represented by SEQ ID NO: 1, wherein the modified sequence is a sequence in which one base in 5'-GAAAGTG-3' (SEQ ID NO: 1)' has been deleted or mutated, or a sequence in which one base has been added to any position in 5'-GAAAGTG-3' (SEQ ID NO: 1). Examples of the modified sequences include 5'-NAAAGTG-3' (SEQ ID NO: 2), 5'-GNAAGTG-3' (SEQ ID NO: 3), 5'-GANAGTG-3' (SEQ ID NO: 4), 5'-GAANGTG-3' (SEQ ID NO: 5), 5'-GAAANTG-3' (SEQ ID NO: 6), 5'-GAAAGNG-3' (SEQ ID NO: 7), and 5'-GAAAGTN-3' (SEQ ID NO: 8) (wherein N is A, T, G, or C).

[0018] Sequence 2 and its modified sequences 5'-GAAAG-3' (SEQ ID NO: 9) or A region containing a modified sequence of the sequence represented by SEQ ID NO: 9, wherein the modified sequence is a sequence in which one base in 5'-GAAAG-3' (SEQ ID NO: 9) is deleted or mutated, or a sequence in which one base is added to any position in 5'-GAAAG-3' (SEQ ID NO: 9). Examples of the modified sequences include 5'-NAAAG-3' (SEQ ID NO: 10), 5'-GNAAG-3' (SEQ ID NO: 11), 5'-GANAG-3' (SEQ ID NO: 12), 5'-GAANG-3' (SEQ ID NO: 13), and 5'-GAAAN-3' (SEQ ID NO: 14) (wherein N is A, T, G, or C).

[0019] Sequence 3 and its modified sequences 5'-GAAAA-3' (SEQ ID NO: 15) or A region containing a modified sequence of the sequence represented by SEQ ID NO: 15, wherein the modified sequence is a sequence in which one base in 5'-GAAAA-3' (SEQ ID NO: 15) is deleted or mutated, or a sequence in which one base is added to any position in 5'-GAAAA-3' (SEQ ID NO: 15). Examples of the modified sequences include 5'-NAAAA-3' (SEQ ID NO: 16), 5'-GNAAA-3' (SEQ ID NO: 17), 5'-GANAA-3' (SEQ ID NO: 18), 5'-GAANA-3' (SEQ ID NO: 19), and 5'-GAAAN-3' (SEQ ID NO: 20) (wherein N is A, T, G, or C).

[0020] Sequence 4 and its modified sequences 5'-GAAAC-3' (SEQ ID NO: 21) or A region containing a modified sequence of the sequence represented by SEQ ID NO: 21, wherein the modified sequence is a sequence in which one base in 5'-GAAAC-3' (SEQ ID NO: 21) is deleted or mutated, or a sequence in which one base is added to any position in 5'-GAAAC-3' (SEQ ID NO: 21). Examples of the modified sequences include 5'-NAAAC-3' (SEQ ID NO: 22), 5'-GNAAC-3' (SEQ ID NO: 23), 5'-GANAC-3' (SEQ ID NO: 24), 5'-GAANC-3' (SEQ ID NO: 25), and 5'-GAAAN-3' (SEQ ID NO: 26) (wherein N is A, T, G, or C).

[0021] Examples of PIPA according to this embodiment include compounds represented by the following formula (I), (II), (III) or (IV). [ka]

[0022] [ka]

[0023] [ka]

[0024] [ka]

[0025] In the above formulas (I), (II), (III) and (IV), L represents a C2-6 alkyl linker, which is not particularly limited, and may contain a γ-aminobutyric acid residue or the like. R1 and R2 are C1 to C10 alkyl groups which may have a substituent or C1 to C10 alkyl groups which may contain an amide bond and / or an amino group, and R1 and R2 may be bonded to form a C2 to C6 alkyl linker. X1, X2, X3, X4, X5, X6, X7, and X8 are the same or independently a bond or an aliphatic amino acid residue, and examples of the aliphatic amino acid residue include, but are not limited to, β-alanine, glycine, γ-aminobutyric acid, and 5-aminovaleric acid.

[0026] More specifically, examples of PIPA according to this embodiment include compounds represented by the following formula (Ia), (IIa), (IIIa), or (IVa). [ka]

[0027] [ka]

[0028] [ka]

[0029] [ka]

[0030] The PIPA of this embodiment can bind to its binding region with good affinity, and the affinity, expressed as a dissociation constant (Kd value), is not particularly limited, but may be, for example, about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, and more preferably about 10 nM or less.

[0031] A second embodiment is a PIPA that specifically binds to the binding domain of an IRF family transcription factor, or the use of the PIPA, or a composition comprising the PIPA, for inhibiting the function of an IRF family transcription factor.

[0032] Furthermore, a third embodiment is an IRF family transcription factor inhibitor (hereinafter also referred to as "the IRF transcription factor inhibitor according to this embodiment") that comprises PIPA that specifically binds to the binding domain of an IRF family transcription factor.

[0033] The PIPA in the second and third embodiments is the PIPA according to this embodiment. The PIPA according to this embodiment binds specifically and with good affinity to a binding sequence (e.g., an IRF-E sequence or an ISRE sequence) present in the promoter region of a target gene whose transcription is activated by an IRF family transcription factor, thereby suppressing or inhibiting the transcriptional activation of the target gene by the IRF family transcription factor. Genes whose transcription is activated by an IRF family transcription factor, i.e., genes whose promoter region contains a binding sequence for an IRF family transcription factor, include various genes such as type I IFN (Interferon) genes (a collective term for 13 cytokines including IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω), as well as IL-6 and TNF-α genes. Therefore, the IRF transcription factor inhibitor according to this embodiment can be effectively used to suppress or inhibit the transcription of genes whose transcriptional activation is controlled by the IRF family. In this specification, the term "gene" refers to a base sequence or nucleic acid that encodes genetic information. This nucleic acid includes polynucleotides, RNA, DNA, etc., and does not necessarily only include nucleic acids present in living organisms, but also includes nucleic acid sequences (e.g., cDNA) that encode any protein, including artificially synthesized ones. Furthermore, the origin of a gene may be from any biological species.

[0034] The fourth embodiment is a pharmaceutical or pharmaceutical composition (hereinafter also referred to as a "pharmaceutical according to this embodiment" or a "pharmaceutical composition according to this embodiment," or collectively referred to as a "pharmaceutical, etc. according to this embodiment") that contains, as an active ingredient, an IRF family transcription factor inhibitor according to the third embodiment or a PIPA complex according to the eleventh embodiment (i.e., contains, as an active ingredient, a PIPA or a PIPA complex according to this embodiment). Alternatively, the fourth embodiment is use of a PIPA according to this embodiment in the manufacture of a pharmaceutical or pharmaceutical composition. PIPA exists stably in vivo without being degraded by peptidases and the like. Furthermore, because it easily crosses cell membranes, it does not require a means of intracellular introduction, such as a drug delivery system (DDS), making it an excellent substance as an active ingredient for pharmaceuticals and the like. PIPA according to this embodiment can suppress the expression of various cytokine genes, such as type I IFN, IL-6 gene, and TNF-α gene, by suppressing or inhibiting the activity of IRF family transcription factors. Therefore, it is effective as a pharmaceutical for suppressing excessive immune responses caused by abnormal cytokine expression, or as a pharmaceutical for preventing or treating diseases or infectious diseases caused by excessive immune responses. Pharmaceuticals and the like according to this embodiment have the effect of suppressing excessive immune responses and can be used prophylactically or therapeutically. Diseases caused by an excessive immune response include, but are not limited to, autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus (SLE), Goodpasture's syndrome, Graves' disease, Hashimoto's disease, multiple sclerosis, and autoimmune hemolytic anemia, as well as viral infections such as COVID-19 and influenza virus infections that induce excessive immune responses, and infections caused by pathogens such as bacteria, fungi, and protozoa.

[0035] The medicament according to this embodiment may be administered as the active ingredient (PIPA according to this embodiment) itself, but it is generally desirable to administer it in the form of a pharmaceutical composition containing one or more active ingredients as well as one or more formulation additives. Furthermore, the medicament according to this embodiment may also contain other ingredients that are effective in suppressing excessive immune responses.

[0036] Dosage forms of pharmaceuticals and the like according to this embodiment include, but are not limited to, liquid preparations such as injections and drip infusions. Liquid preparations may be dissolved or suspended in water or other suitable solvents at the time of use. When using a liquid preparation as an injection or drip infusion, the active ingredient is prepared by dissolving it in water, but if necessary, it may also be dissolved in physiological saline or a glucose solution, and a buffer or preservative may also be added.

[0037] The type of formulation additive used in the production of the medicament, etc. according to this embodiment, the ratio of the formulation additive to the active ingredient, and the production method of the medicament, etc. can be appropriately selected by those skilled in the art depending on the form of the medicament, etc. As the formulation additive, an inorganic or organic substance, or a solid or liquid substance can be used, and generally, it can be blended in an amount of 1 to 90% by weight based on the weight of the active ingredient. Specific examples of pharmaceutical additives include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, Veegum, titanium oxide, sorbitan fatty acid esters, sodium lauryl sulfate, glycerin, fatty acid glycerin esters, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oils, wax, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, and water.

[0038] To prepare an injection, the active ingredient is dissolved in distilled water for injection, if necessary, together with a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc., and an isotonic agent such as sodium chloride or glucose, and the solution is sterile filtered and filled into ampoules, or mannitol, dextrin, cyclodextrin, gelatin, etc. are added, followed by vacuum freeze-drying to produce an injection that is dissolved just before use. Alternatively, the active ingredient can be emulsified in water with lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, etc. to produce an emulsion for injection.

[0039] The dosage, frequency of administration, and method of administration of the pharmaceutical etc. according to this embodiment are not particularly limited, and can be appropriately selected at the discretion of a physician depending on conditions such as the purpose of preventing and / or treating the worsening or progression of the disease to be prevented or treated, the type of disease, and the weight and age of the patient. Generally, when used as an injection, the daily dose for adults is 0.001 to 1000 mg (weight of active ingredient), which may be administered continuously or intermittently.

[0040] The pharmaceuticals of this embodiment may be formulated as sustained-release formulations, such as implants and microencapsulated delivery systems, using carriers that can prevent immediate elimination from the body. Such carriers include biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials can be easily prepared by those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Liposomes can be prepared as lipid compositions containing, but not limited to, phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanol (PEG-PE), filtered through a filter with an appropriate pore size to obtain a suitable size for use, and then purified by reverse-phase evaporation.

[0041] The pharmaceutical agent according to this embodiment may be provided in the form of a kit together with instructions for administration, etc. The pharmaceutical agent contained in the kit is supplied in a container made of a material that effectively maintains the activity of the components of the therapeutic agent for a long period of time, does not adsorb to the inside of the container, and does not alter the components. For example, a sealed glass ampoule may contain a buffer or the like sealed in the presence of a neutral, non-reactive gas such as nitrogen gas. The kit may also include instructions for use, which may be printed on paper or stored on an electromagnetically readable medium such as a CD-ROM or DVD-ROM and provided to the user.

[0042] The fifth embodiment is an adjuvant containing PIPA according to this embodiment (hereinafter also referred to as "adjuvant according to this embodiment"). Recently, it has been reported that type I IFN inhibits mRNA transcription and induces mRNA degradation, reducing the efficacy of mRNA vaccines (Linares-Fernandez et al., Trends Mol Med 26:311-323 doi: 10.1016 / j.molmed.2019.10.002. 2020; De Beuckelaer et al., Mol Ther. 24:2012-2020 2016.). Because PIPA according to this embodiment suppresses or inhibits type I IFN expression at the transcriptional level, its incorporation into mRNA vaccine formulations is expected to enhance the efficacy of the vaccine. Specifically, PIPA according to this embodiment is thought to suppress type I IFN expression and support or enhance the action of mRNA (antigen expression in vivo), and therefore can also be used as an adjuvant or adjuvant-like additive.

[0043] The adjuvant according to this embodiment may contain, for example, about 0.01 to 99.99% by weight of the PIPA according to this embodiment. The adjuvant according to this embodiment may also contain a component other than the PIPA according to this embodiment that does not inhibit the function of PIPA as an adjuvant. Examples of such components include stabilizers, pH adjusters, preservatives, antiseptics, and buffers. The adjuvant according to this embodiment may also contain a component other than the PIPA according to this embodiment that is contained in adjuvants for existing mRNA vaccines and is known to have immunostimulatory activity. The adjuvant according to this embodiment can be used for all living organisms that have an immune system.

[0044] The sixth embodiment is a vaccine comprising the adjuvant and mRNA according to this embodiment (hereinafter also referred to as "vaccine according to this embodiment"). The mRNA contained in the vaccine of the sixth embodiment is not particularly limited as long as it expresses a protein that induces an immune response, and examples include mRNA that expresses proteins derived from viruses, bacteria, parasites, fungi, rickettsia, chlamydia, prions, cancer cells, normal cells, etc.

[0045] The seventh embodiment is a method for preventing or treating an excessive immune response, a disease caused by an excessive immune response, and / or an infectious disease, which comprises administering the medicament or the like according to this embodiment to a patient. Here, "treatment" means preventing or alleviating the progression and worsening of the pathology caused in a mammal that has developed the disease to be treated. "Prevention" means preventing the onset of the disease to be prevented in a mammal that is at risk of developing the disease to be prevented. The "mammal" to be prevented or treated means any animal classified as a mammal, and includes, but is not limited to, humans, pet animals such as dogs, cats, rabbits, ferrets, mice, and hamsters, and livestock animals such as cows, pigs, sheep, and horses. A particularly preferred "mammal" is humans.

[0046] An eighth embodiment is a method for producing an IRF family transcription factor inhibitor including PIPA, comprising: The method comprises the steps of providing a binding domain of an IRF family transcription factor, and producing a PIPA that specifically binds to the binding domain. Here, the process of producing a PIPA that specifically binds to the binding region of an IRF family transcription factor may include the steps of designing a pyrrole and / or imidazole polymer selected to correspond to a nucleotide sequence present in the binding region, and, if necessary, designing a polymer in which an aliphatic amino acid residue such as β-alanine or γ-aminobutyric acid is inserted into the pyrrole and / or imidazole polymer, and actually synthesizing the polymer.

[0047] A ninth embodiment is a PIPA that specifically binds to the binding region of an IRF family transcription factor for use as a pseudo-transcription factor, or a composition comprising a PIPA that specifically binds to the binding region of an IRF family transcription factor for use as a pseudo-transcription factor. As described above, PIPA according to this embodiment has the property of binding to a genomic DNA sequence to which an IRF family transcription factor specifically binds, and can inhibit transcription and / or activation of the IRF family transcription factor via the binding sequence. Thus, PIPA according to this embodiment functions as a substance that inhibits transcription and / or activation of the IRF family transcription factor via the binding sequence, i.e., as a "pseudo-transcription factor." Therefore, PIPA according to this embodiment can also be used as a pseudo-transcription factor. For details about PIPA, see the descriptions in other embodiments.

[0048] A tenth embodiment is a method for using PIPA as a pseudo-transcription factor in a subject, the method comprising the step of applying to the subject an effective amount of PIPA that specifically binds to the binding domain of an IRF family transcription factor. Here, the term "subject" refers to any animal classified as a mammal, including, but not limited to, humans, pet animals such as dogs, cats, rabbits, ferrets, mice, and hamsters, and livestock animals such as cows, pigs, sheep, and horses. A particularly preferred "mammal" is a human. For details about PIPA, see the description in other embodiments.

[0049] An eleventh embodiment is a complex (conjugate) comprising a PIPA according to this embodiment (i.e., a PIPA that specifically binds to the binding domain of an IRF family transcription factor) and a PIPA cofactor (hereinafter also referred to as a "PIPA complex according to this embodiment"). The "PIPA cofactor" according to this embodiment is a substance different from PIPA, and is a substance that enhances the function of the PIPA according to this embodiment. The PIPA according to this embodiment has the function of suppressing an excessive immune response by suppressing the expression of genes whose transcription is activated by IRF family transcription factors. Therefore, the "PIPA cofactor" according to this embodiment includes a substance that enhances the function of the PIPA (e.g., immune response suppression function). Furthermore, the "PIPA cofactor" according to this embodiment includes a substance that supports the action of PIPA, such as, but not limited to, a substance that improves the stability of PIPA in vivo, a substance that enhances the transport of PIPA to target organs or target cells, and a substance that enhances the introduction of PIPA into cells.

[0050] In this embodiment, any substance can be used as the PIPA cofactor as long as it binds to PIPA and does not inhibit the intracellular delivery of PIPA. Such PIPA cofactors preferably have a low molecular weight (about 500 to about 2,000), and more preferably have a molecular weight that does not affect the efficiency of intracellular delivery of PIPA, for example, about 500 to about 1,000. By forming a complex with the PIPA of this embodiment, such PIPA cofactors enhance the function of the PIPA and / or support the action of the PIPA. Therefore, the PIPA cofactor of this embodiment exhibits preventive and / or therapeutic effects against, for example, diseases or infectious diseases caused by an excessive immune response.

[0051] Substances that enhance the function of PIPA according to this embodiment include, but are not limited to, immunosuppressants such as hydroxychloroquine and FK506, anti-inflammatory substances such as various steroids, etc. Furthermore, substances that support the action of PIPA according to this embodiment include, for example, vitamins (e.g., vitamin E, vitamin A), lipids such as cholesterol, anisamide, etc.

[0052] The PIPA and the PIPA cofactor according to this embodiment may be directly bound to form a complex, or may be linked via a linker. Any linker can be used as such a linker, as long as it can exert the function of the PIPA complex according to this embodiment, for example, the function as a drug delivery system (DDS). Examples of such linkers include, but are not limited to, alkyl linkers having 1 to 6 carbon atoms (C1 to C6). The linker may contain a functional group for binding the PIPA and the PIPA cofactor according to this embodiment, such as a maleimide group, a thiol group, an amino group, a carboxyl group, a hydroxyl group, or an azide group. The site of PIPA to which the PIPA cofactor is bound is not particularly limited, and may be, for example, the terminal portion of PIPA (e.g., R1 or R2 in formulas (I), (II), and (III)).

[0053] When this specification is translated into English and includes the singular words "a," "an," and "the," it is intended to include the plural as well as the singular, unless the context clearly indicates otherwise. Also, in this specification, "about" or "to the extent" means a numerical range of ±10%. The present invention will be further explained below by showing examples, but these examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention. [Example]

[0054] 1. Materials and Methods 1-1.Cells The cell line Raw264.7 cells were purchased from the RIKEN Cell Bank and cultured in D-MEM (Nacalai Tesque) containing 10% FBS (SIGMA-ALDRICH) and penicillin-streptomycin mixed solution (Nacalai Tesque). Mouse peritoneal macrophages were collected from 6-week-old CL57B / 6 mice (CLEA Japan) by peritoneal lavage with 7 mL of PBS (Nacalai Tesque). After 2 hours of incubation, cells adhered to culture plates and were used for the experiments. Cultures were performed using RPMI or D-MEM medium (Nacalai Tesque) containing 10% FBS (Sigma-Aldrich) and penicillin-streptomycin mixed solution (Nacalai Tesque). CL57B / 6 mice were housed in an SPF environment.

[0055] 1-2. Determination of IRF binding sequence and synthesis of PIPA IRF family transcription factors bind to the consensus sequence 5'-AANNGAAA-3' (SEQ ID NO: 27). Based on this consensus sequence, we selected 5'-GAAAGTG-3' (SEQ ID NO: 1), 5'-GAAAG-3' (SEQ ID NO: 9), 5'-GAAAA-3' (SEQ ID NO: 15), and 5'-GAAAC-3' (SEQ ID NO: 21) as binding sequences for designing PIPAs. We commissioned Peptide Institute, Inc. to synthesize PIPAs that bind to these sequences.

[0056] 1-3. Measurement of cytokine concentrations 2×10 4Raw264.7 cells were seeded onto plates and treated with IRF-PIPA1, IRF-PIPA2, IRF-PIPA3, or IRF-PIPA4 for 24 hours, then stimulated with LPS (Lipopolysaccharide, SIGMA-ALDRICH) for 16 hours, and the culture supernatant was collected. Cytokine concentrations in the culture supernatant were measured using an ELISA kit (R&D SYSTEMS) according to standard protocols. The culture supernatant was diluted with 1% BSA-PBS as needed and analyzed. Mouse peritoneal macrophages were cultured at 2.5 × 10 5 Cells were plated and treated with IRF-PIPA3 for 16 or 24 hours, followed by stimulation with Poly(I:C) (InvivoGen) for 16 hours or mRNA vaccine (Moderna) for 20–24 hours. Culture supernatants were collected. Cytokine concentrations in the culture supernatants were measured using an ELISA kit (R&D SYSTEMS) according to standard protocols. Furthermore, plasma cytokine concentrations were measured by intraperitoneally administering 400 μg / mouse of IRF-PIPA3 to wild-type mice, followed 24 hours later by intraperitoneal administration of 250 μg of Poly(I:C). Blood was collected 2 hours later, and plasma cytokine concentrations were measured using an ELISA kit (R&D SYSTEMS) according to standard protocols.

[0057] 1-4. Measurement of mRNA levels 2.5×10 5 Peritoneal macrophages were plated and treated with IRF-PIPA3 for 24 hours. Cells were harvested 2, 4, or 6 hours after stimulation with Poly(I:C) (InvivoGen) or 20 hours after stimulation with mRNA vaccine (Moderna). RNA was extracted from the extracted RNA using TRIsure (Nihon Genetics Co., Ltd.) and an RNA extraction kit (ZYMO RESEARCH) according to standard protocols. 5x PrimeScript was used. tRTcDNA was prepared using Master Mix (Takara Bio Inc.), and qPCR was performed using Sybr green (BioLabs) and primers specific to each gene (FASMAC Co., Ltd.). qPCR was performed using a Light Cycler 480 (Roche, Switzerland). Primer sequences were standard sequences used in previous publications (Proc Natl Acad Sci US A. 2019 Nov 19;116(47):23653-23661. doi: 10.1073 / pnas.1915326116).

[0058] 1-5. Measurement of dsDNA concentration and LDH activity The dsDNA concentration in the culture supernatant was measured using Nano Drop One (Thermo Fisher Scientific). LDH assay was performed using the Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories, Inc.) according to the standard protocol. The positive control was 2.5 × 10 5 Each peritoneal macrophage was mixed with 90 μL of 10% FBS-RPMI or D-MEM medium and 10 μL of lysis buffer to disrupt the cells, and the lysate was used.

[0059] Measurement of SARS-CoV-2 spike protein receptor binding domain (RBD) concentration Wild-type mice were injected intramuscularly (im) or intraperitoneally (ip) with 400 μg / mouse of IRF-PIPA3. 24 hours later, Moderna's SARS-CoV-2 mRNA vaccine was administered intramuscularly at 1 μg / mouse, and Poly(I:C) was simultaneously administered intraperitoneally or intramuscularly at 250 μg / mouse. One day later, blood was collected from the mice, and plasma RBD concentrations were measured by ELISA.

[0060] 1-7. Analysis of intermolecular interactions Analysis was performed using an Octet K2 (ForteBio) according to standard protocols. A biotin-labeled target sequence (FASMAC) was bound to a streptavidin-conjugated sensor (ForteBio). The binding and dissociation of IRF-PIPA3 from 10 to 320 nM to the target sequence were analyzed for 60 seconds, respectively, according to standard protocols. KD values ​​were calculated from these results using Data Analysis (Sartorius Japan Co., Ltd.).

[0061] 2.Results 2-1. Identification of PIPA that suppresses type I IFN induction Although in vitro analyses have identified IRF transcription factor binding sequences, no studies have examined the physiological significance of these sequences using promoter knock-in mice or other methods. Therefore, we first investigated which sequences PIPA targets to demonstrate potent inhibitory effects. Based on previous studies, we narrowed down the predicted IRF binding sequences to four: 5'-GAAAGTG-3' (SEQ ID NO: 1), 5'-GAAAG-3' (SEQ ID NO: 9), 5'-GAAAA-3' (SEQ ID NO: 15), and 5'-GAAAC-3' (SEQ ID NO: 21). We then constructed PIPAs that bind to each of these sequences and tested their ability to suppress IFN-β, whose expression is induced by IRF transcription factors, using the macrophage cell line Raw264.7. The structures of PIPA bound to 5'-GAAAGTG-3' (SEQ ID NO: 1), 5'-GAAAG-3' (SEQ ID NO: 9), 5'-GAAAA-3' (SEQ ID NO: 15), and 5'-GAAAC-3' (SEQ ID NO: 21) are shown below as formulas (Ia), (IIa), (IIIa), and (IVa), respectively. In this specification, PIPA represented by formulas (Ia), (IIa), (IIIa), and (IVa) are also referred to as IRF-PIPA3, IRF-PIPA1, IRF-PIPA2, and IRF-PIPA4, respectively.

[0062] [ka]

[0063] [ka]

[0064] [ka]

[0065] [ka]

[0066] Macrophage cell line Raw264.7 cells were pretreated with IRF-PIPA1-4 and then further stimulated with a pathogen component that induces IFN-β production. The IFN-β concentration in the culture supernatant was measured by ELISA. The inhibitory effect of PIPA on IFN-β production was confirmed for all PIPAs (Figure 1). While no concentration-dependence was observed for IRF-PIPA1, IRF-PIPA2, or IRF-PIPA4, IRF-PIPA3 demonstrated a concentration-dependent inhibition, and was the most potent of the four PIPAs. Therefore, IRF-PIPA3 was used in the following experiments, suggesting that it suppresses IFN-β production via the expected transcriptional inhibitory mechanism.

[0067] 2-2. Verification of the inhibitory effect of IRF-PIPA3 on type I IFN induction Next, we examined the inhibitory effect of IRF-PIPA3 on IFN-β production in peritoneal macrophages from mice. After pre-treating peritoneal macrophages with IRF-PIPA3, we stimulated them with double-stranded RNA (Poly(I:C)), a pathogen component that induces IFN-β production, and measured cytokine concentrations in the culture supernatants by ELISA (Fig. 2). We confirmed that IRF-PIPA3 strongly suppressed IFN-β production in a concentration-dependent manner in peritoneal macrophages. Furthermore, we also confirmed that IRF-PIPA3 suppressed IFN-β production in peritoneal macrophages, despite its weaker suppression than IFN-β, in IL-6 and TNF-α, cytokines known to be dependent on IRF transcription factors, similar to IFN-β (Fig. 2). These results suggest that IRF-PIPA3 broadly suppresses responses dependent on IRF transcription factors.

[0068] Furthermore, to verify whether IRF-PIPA3 suppresses IFN-β production through the assumed mechanism, we performed the same experiment using peritoneal macrophages and analyzed the relative expression levels of mRNA using qRT-PCR to evaluate the effect on transcription (Figure 3). As a result, IRF-PIPA3 suppressed IFN-β production in a concentration-dependent manner. Ifnb1 It also strongly suppressed the induction of mRNA expression. Il6 and TNF Similar suppression of IRF-PIPA3 mRNA was observed. These results suggest that IRF-PIPA3 inhibits IRF-dependent transcription.

[0069] 2-3. Verification of the cytotoxicity of IRF-PIPA3 In mRNA analysis, the expression level of the target mRNA is calculated relative to the mRNA expression level of a gene that is typically stable (in this example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)). This allows the mRNA level of the target gene to be verified without being affected by factors such as cell death. Therefore, while the mRNA analysis results shown in Figure 3 strongly suggest that IRF-PIPA3 has inhibitory activity, it is not possible to assess whether cell death occurs in addition to the inhibitory effect. While transcription factors target specific sequences, they also bind to numerous target sequences in non-essential regions. This is likely true for PIPA. This suggests that behind the binding of numerous target sequences in the genome and the inhibition of IRF transcription factors, numerous meaningless binding events occur. From this perspective, it is extremely important to verify the on-target side effects of PIPA, and verifying its cytotoxicity as an output is considered to be of great significance. Therefore, we next examined the cytotoxic activity of IRF-PIPA3 using peritoneal macrophages. We performed the same experiment as in Figure 2 and analyzed DNA release and lactate dehydrogenase (LDH) protein release from dead cells into the culture supernatant as indicators of cytotoxicity. The results showed that PIPA treatment did not increase the amount of DNA in the culture supernatant at all (Figure 4, left). Furthermore, to verify LDH release, we measured LDH activity in the culture supernatant, and similarly found that PIPA treatment did not increase LDH activity at all (Figure 4, right). These results suggest that IRF-PIPA3 does not exhibit cytotoxic activity, at least within the concentration range in which IRF-PIPA3 exerts a potent inhibitory effect.

[0070] 2-4. Verification of binding between IRF-PIPA3 and target sequence These results suggest that PIPA3 binds to target sequences in the promoters of target genes of IRF transcription factors and represses transcription. Furthermore, to confirm that IRF-PIPA3 actually binds to the target DNA sequence, we verified the binding of PIPA3 to the target DNA sequence in vitro. For the verification, we used Octet K2, which can verify the interaction between two molecules, and bound the DNA sequence (GAAAGTG C) immobilized on the sensor. GAAAGTG The binding of IRF-PIPA3 to the target sequence (C GAAAGTG C: SEQ ID NO: 28) was detected. As a result, a signal indicating the binding of IRF-PIPA3 to the target sequence was detected in a concentration-dependent manner, and the binding strength was 2.27 × 10 in terms of KD value. -8 These results demonstrated that IRF-PIPA3 indeed binds to the predicted target sequence.

[0071] 2-5. Verification of the efficacy of IRF-PIPA3 at the biological level We examined the transcriptional repression effect of IRF-PIPA3 in vivo. IRF-PIPA3 was administered intraperitoneally to mice, and three hours later, poly(I:C) was administered intraperitoneally to the same mice as a pathogen component. Two hours after poly(I:C) administration, peritoneal macrophages were collected from the mice, and the relative mRNA expression levels were analyzed using qRT-PCR (Figure 6). Consistent with the in vitro results, IRF-PIPA3 was Ifnb1 , Il6 and TNF The mRNA expression level of IRF-PIPA3 was significantly suppressed, indicating that IRF-PIPA3 has a suppressive effect in vivo, at least locally.

[0072] Next, IRF-PIPA3 was administered intraperitoneally to mice, and then 24 hours, 1 day, 4 days, or 7 days later, poly(I:C) was administered intraperitoneally as a pathogen component. Two hours after poly(I:C) administration, blood was collected from the mice, and plasma IFN-β, IL-6, and TNF-α protein concentrations were measured by ELISA. The results are shown in Figure 7A (24 hours after IRF-PIPA3 administration) and Figure 7B (1 day, 4 days, or 7 days after IRF-PIPA3 administration). IRF-PIPA3 significantly suppressed the expression of IFN-β, IL-6, and TNF-α proteins. The maximum inhibitory effect was observed 1 day after IRF-PIPA3 administration, and the effect persisted, albeit at a reduced level, for up to 7 days after administration. These results demonstrate that IRF-PIPA3 effectively suppresses the expression of IRF family proteins throughout the body.

[0073] 2-6. Verification of the effect of IRF-PIPA3 mRNA vaccine on immune response As mentioned above, it has been reported that type I IFN inhibits mRNA transcription and induces mRNA degradation, reducing the effectiveness of mRNA vaccines. Therefore, we investigated whether IRF-PIPA3 can suppress IFN-β transcription after administration of Moderna's SARS-CoV-2 mRNA vaccine. Peritoneal macrophages were pretreated with IRF-PIPA3 (3 μM or 10 μM), then treated with the mRNA vaccine (Moderna), and cultured for 20 hours. After culture, RNA was prepared from the collected cells using TRIsure, and qRT-PCR was performed. Ifnb1 The relative expression levels of the genes were measured (Figure 8, left). After culturing, cytokine concentrations in the culture supernatant were measured by ELISA (Figure 8, right). The results confirmed that IRF-PIPA3 strongly suppressed IFN-β transcription in peritoneal macrophages after the addition of the mRNA vaccine in a concentration-dependent manner.

[0074] Next, peritoneal macrophages were pretreated with IRF-PIPA3 (6 μM) and then treated with an mRNA vaccine (LNP-mRNA) containing OVA (ovalbumin:OVA)-encoding mRNA encapsulated in Moderna's lipid nanoparticles (LNPs) instead of the Moderna SARS-CoV-2 mRNA vaccine. The cells were then cultured for 20–24 hours. After culture, cytokine concentrations in the culture supernatant were measured by ELISA (Figure 9). The results confirmed that IRF-PIPA3 strongly suppressed the expression of IFN-β, IL-6, and TNF-α proteins in peritoneal macrophages after the addition of the mRNA vaccine in a concentration-dependent manner.

[0075] It is known that double-stranded RNA (dsRNA) is generated as a contaminant during the LNP-mRNA manufacturing process (Linares-Fernandez et al., 2020 Mar Trends Mol Med 26:311-323 doi: 10.1016 / j.molmed.2019.10.002.). Because dsRNA induces type I IFN expression, it is possible that it may reduce vaccine efficacy. Therefore, we investigated whether PIPA could improve the efficacy of mRNA vaccines due to the presence of dsRNA as a contaminant. Wild-type mice were administered IRF-PIPA3 intramuscularly (im) or intraperitoneally (ip) at 400 μg / mouse. 24 hours later, Moderna's SARS-CoV-2 mRNA vaccine was administered intramuscularly at 1 μg / mouse, and 250 μg / mouse of double-stranded RNA Poly(I:C) was simultaneously administered intraperitoneally or intramuscularly as a contaminant. One day later, blood was collected from the mice, and plasma RBD (Receptor Binding Domain; the region that binds to the SARS-CoV-2 spike protein receptor) concentrations were measured using ELISA. In the absence of IRF-PIPA3, the addition of Poly(I:C) as a co-conjugate during mRNA vaccination reduced blood RBD protein levels, whereas the presence of IRF-PIPA3 suppressed the reduction in RBD protein levels caused by Poly(I:C) (Figure 10).

[0076] These results indicate that IRF-PIPA3 suppresses the transcription of IFN-β after administration of an mRNA vaccine, suggesting that IRF-PIPA3 functions as an adjuvant that supports or enhances the efficacy of mRNA vaccines. [Industrial Applicability]

[0077] The present invention provides PIPA, which transcriptionally suppresses the expression of cytokines essential for immune response activation (e.g., type I IFN, IL-6, TNF-α, etc.), and is therefore expected to be useful in the pharmaceutical and medical fields.

Claims

1. Pyrrole imidazole polyamide (PIPA) represented by the following formula (Ia), (IIa), (IIIa) or (IVa): 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】

2. A composition comprising the PIPA of claim 1 for inhibiting the function of an IRF family transcription factor.

3. An IRF family transcription factor inhibitor comprising the PIPA described in claim 1.

4. A medicine or pharmaceutical composition for suppressing excessive immune responses, comprising the PIPA described in claim 1 as an active ingredient.

5. An adjuvant comprising the PIPA described in claim 1.

6. A complex comprising the PIPA described in claim 1 and a PIPA cofactor, wherein the PIPA cofactor is a substance different from the PIPA, and the PIPA cofactor enhances the function of the PIPA and / or assists the action of PIPA.

7. The complex of claim 6, wherein the PIPA and the PIPA cofactor are linked by a C1 to C6 linker.

8. The complex of claim 6, wherein the PIPA cofactor is a substance that enhances the function of PIPA and is one or more substances selected from the group consisting of hydroxychloroquine, steroids, and FK506.

9. The complex of claim 6, wherein the PIPA cofactor is a substance that assists the action of the PIPA and is one or more substances selected from the group consisting of vitamins, cholesterol, and anisamide.

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