Antisense oligonucleotide agents for treating conditions associated with NLRP3 / NLRP1 expression or activation

Abstract

This disclosure provides antisense oligonucleotide compounds and compositions (e.g., pharmaceutical compositions) that target NLRP3 and / or NLRP1 mRNA, as well as methods for treating diseases or conditions associated with the expression of NLRP3 and / or NLRP1 mRNA, or the activation of the NLRP3 and / or NLRP1 inflammasome. Exemplary conditions in which these compounds and compositions are used include, among others, neurodegenerative diseases and inflammatory diseases such as autoimmune diseases.

Description

[Technical Field]

[0001] Priority This application claims the benefits and priority of U.S. Provisional Application No. 63 / 449,178, filed on 1 March 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing submitted in XML format via EFS-Web. The XML copy named "MAX-003PC_133279-5003 Sequence Listing," created on February 29, 2024, has a size of 158,135 bytes, and its entire content is incorporated herein by reference. [Background technology]

[0003] Inflammasomes are receptors of the innate immune system that detect pathogenic endogenous and exogenous stressors and activate the highly pro-inflammatory cytokines interleukin-1β and interleukin-18. Inflammasomes are involved not only in inflammation but also in carcinogenesis and tumor progression.

[0004] NLRP3 and NLRP1 are proteins encoded by the NLRP3 and NLRP1 genes. NLRP3 and NLRP1 are components of the innate immune system, functioning as pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs). Danger signals are characteristic of many inflammatory diseases, and these stimuli can function to activate NLRP3 or NLRP1. When activated, NLRP3 or NLRP1 initiates inflammasome assembly. This triggers caspase-1-mediated proteolytic activation of interleukin-1β (IL-1β) family cytokines, resulting in inflammation and cell death. Activation of NLRP3 or NLRP1 contributes to a variety of inflammatory diseases, including several neurodegenerative diseases, inflammatory diseases, and autoimmune diseases. Therefore, treatment is needed to inhibit or reduce abnormal or persistent activation of the NLRP3 or NLRP1 inflammasome. [Modes for carrying out the invention]

[0005] This disclosure provides antisense oligonucleotide compounds and compositions (e.g., pharmaceutical compositions) that target NLRP3 and / or NLRP1 mRNA, as well as methods for treating diseases or conditions associated with the expression of NLRP3 and / or NLRP1 mRNA, or with persistent or abnormal activation of NLRP3 and / or NLRP1. Exemplary conditions in which such compounds and compositions are used include neurodegenerative diseases, inflammatory diseases, and autoimmune diseases.

[0006] In one embodiment, the disclosure provides an antisense oligonucleotide having a length of 10 to 30 nucleotides and comprising at least eight consecutive nucleotides from any one of SEQ ID NOs. 1 to 52, which reduces the expression of NOD-, LRR-, pyrin domain-containing protein 1 (NLRP1) mRNA and / or pyrin domain-containing protein 3 (NLRP3) mRNA. In embodiments, the oligonucleotide reduces the expression of both NLRP3 and NLRP1 mRNA. In various embodiments, the antisense oligonucleotide is complementary to an equal-length portion of human NLRP3 mRNA. In various embodiments, the antisense oligonucleotide is complementary to an equal-length portion of human NLRP1 mRNA (or also complementary to an equal-length portion of human NLRP1 mRNA). Exemplary NLRP3 and NLRP1 transcripts are disclosed in PCT / US2022 / 042394 (the entirety of which is incorporated herein by reference). NLRP3 and NLRP1 transcripts are further provided herein as SEQ ID NOs. 53 to 65. In one embodiment, the antisense oligonucleotide targets the transcript of SEQ ID NO: 53 (for example, complementary to an equal-length portion thereof). In another embodiment, the antisense oligonucleotide targets the transcript of SEQ ID NO: 60 (for example, complementary to an equal-length portion thereof).

[0007] In some embodiments, the oligonucleotide has a length of at least 12 nucleotides or at least 14 nucleotides. In some embodiments, the oligonucleotide has a length of 10 to 24 nucleotides, such as 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, including those shown in Tables 1 and 2, the oligonucleotide has a length of 14 nucleotides. Tables 4 and 5 show embodiments in which the antisense oligonucleotide has a length of 14 to 20 nucleotides.

[0008] In various embodiments, the oligonucleotide comprises at least 10 or at least 12 consecutive nucleic acid bases of any one of SEQ ID NOs: 1 to 52, and the oligonucleotide is substantially (i.e., at least 90%) or entirely complementary to a segment (e.g., a segment of equal length) of NLRP3 mRNA (human) (e.g., mRNA of SEQ ID NOs: 53 to 59). In various embodiments, the oligonucleotide comprises at least 10 or at least 12 consecutive nucleic acid bases of any one of SEQ ID NOs: 1 to 52, and the oligonucleotide is substantially (i.e., at least 90%) or entirely complementary to a segment (e.g., a segment of equal length) of NLRP1 mRNA (human) (e.g., mRNA of SEQ ID NOs: 60 to 65). In embodiments, the oligonucleotide is substantially (i.e., at least 90%) or entirely complementary to segments (e.g., a segment of equal length) of NLRP3 mRNA and NLRP1 mRNA. As used herein, the term “complementary” refers to a Watson-Crick base pairing under conditions in which up to four bases in the oligonucleotide can be substituted with inosine (e.g., deoxyinosine). In certain embodiments, the oligonucleotide has one of the nucleic acid base sequences of SEQ ID NOs. 1 to 52. For brevity, nucleic acid base sequences may be represented herein using DNA nucleotide sequences (i.e., including T nucleic acid bases). From this context, it is understood that if the nucleotide is intended to be RNA (e.g., mRNA transcript), T nucleic acid bases are substituted with U nucleic acid bases. In the context of oligonucleotides, T and U nucleic acid bases are interchangeable. In embodiments, one or more nucleic acid bases represented as T in the oligonucleotide are U nucleic acid bases.

[0009] In some embodiments, the oligonucleotides have nucleic acid base sequences selected from Table 1 or Table 4. For example, the oligonucleotides may have nucleic acid base sequences selected from SEQ ID NOs: 1 to 52. Various antisense oligonucleotides based on these sequences (shown in Table 2) showed at least 40%, at least 50%, or at least 60% NLRP3 knockdown efficiencies in THP-1 cells 24 hours after transduction at a concentration of 40 nM. Other antisense oligonucleotides based on these sequences (shown in Table 5) showed at least 40%, at least 50%, or at least 60% NLRP3 and / or NLRP1 knockdown efficiencies in U87-MG cells 24 hours after transduction at a concentration of 25 nM. Various antisense oligonucleotides based on these sequences (shown in Table 5) showed at least 40%, at least 50%, or at least 60% NLRP3 and NLRP1 knockdown efficiencies in U87-MG cells 24 hours after transduction at a concentration of 25 nM. These oligonucleotides include STN-101016, STN-101017, and STN-101020. Such oligonucleotides and other oligonucleotides based on these nucleic acid base sequences may be selected in accordance with this disclosure.

[0010] Additional oligonucleotide sequences and antisense oligonucleotides that may be used in accordance with this disclosure are disclosed in PCT / US2022 / 042394 (which is incorporated herein by reference in its entirety).

[0011] In some embodiments, binding of an antisense oligonucleotide to a target mRNA (e.g., hybridization) degrades the target mRNA or blocks the translation of the target mRNA. In some embodiments, binding of an antisense oligonucleotide to a target mRNA creates a double-stranded nucleic acid molecule, which then recruits an endogenous nuclease that degrades the mRNA. In some embodiments, the antisense oligonucleotide has a stretch of DNA nucleotides sufficient to recruit RNaseH, thereby inducing degradation of the target mRNA. For example, the antisense oligonucleotide may have a stretch of at least six or at least eight DNA nucleotides (e.g., a central sequence), and the stretch may optionally be a stretch of nine or ten DNA nucleotides. In some embodiments, one or more DNA nucleotides contain a 2′ chemical modification independently selected from 2′-fluoro, 2′-methyl, and 2′-ethyl. In embodiments, the DNA nucleotides in the antisense oligonucleotide have no 2′ modifications, i.e., they have 2′-H. For example, an antisense oligonucleotide may be a gapmer having a 5′ segment and a 3′ segment, where the 5′ and 3′ segments each have 2 to 6 nucleotides or 2 to 4 nucleotides, and the 5′ and 3′ segments do not contain DNA nucleotides. In some embodiments, the gapmer is a 3-8-3 gapmer having a central block of 8 DNA nucleotides and 5′ and 3′ segments of 3 RNA nucleotides each. In other embodiments, the gapmer is a 2-10-2 gapmer having a central block of 10 DNA nucleotides and 5′ and 3′ segments of 2 RNA nucleotides each. In embodiments, the gapmer is a 3-8-2 gapmer, a 3-9-2 gapmer, a 2-8-3 gapmer, or a 2-9-3 gapmer.In embodiments, the gapmer is a 3-10-3 gapmer, a 3-10-2 gapmer, a 2-10-3 gapmer, a 3-12-3 gapmer, a 2-12-3 gapmer, a 3-12-2 gapmer, a 2-15-3 gapmer, a 3-15-2 gapmer, a 3-11-3 gapmer, a 3-11-2 gapmer, a 2-11-3 gapmer, a 3-13-3 gapmer, a 2-13-3 gapmer, or a 3-13-2 gapmer. In some embodiments, one or more nucleotides in the 5′ and 3′ segments contain a 2′-O substituent, and optionally, all nucleotides in the 5′ and 3′ segments contain a 2′-O substituent. Exemplary 2′-O substituents are independently selected from 2′-O methyl, 2′-O ethyl, 2′-O methoxyethyl (MOE), and crosslinked nucleotides having 2′-4′ crosslinks (e.g., loc nucleotides or bicyclic nucleotides). In some embodiments, the crosslinked nucleotides have methylene crosslinks (locked nucleic acids or LNA) or restricted ethyl crosslinks (cEt).

[0012] The term “gapmer” refers to an oligonucleotide having a central block of a deoxynucleotide (also referred to herein as “DNA nucleotide”) having 5′ and 3′ segments of an RNA nucleotide (at least two nucleotides). As used herein, the term “DNA nucleotide” refers to a nucleotide that is not an RNA nucleotide. DNA nucleotides typically have a 2′H, but can alternatively have a variety of 2′ chemical modifications, including 2′-halo and 2′-lower alkyl (e.g., C1-4). In some embodiments, the 2′ chemical modifications of the DNA nucleotide are independently selected from 2′-fluoro, 2′-methyl, and 2′-ethyl. “RNA nucleotide” includes a 2′ hydroxyl, or a derivative of a 2′ hydroxyl (i.e., a 2′-O substituent).

[0013] Locked nucleic acids (LNAs) or “locked nucleotides” are described, for example, in U.S. Patents 6,268,490, 6,316,198, 6,403,566, 6,770,748, 6,998,484, 6,670,461, and 7,034,133 (all of which are incorporated herein by reference). LNAs are modified nucleotides that contain a crosslink between the 2′ and 4′ carbons of the sugar moiety, resulting in a “locked” conformation and / or bicyclic structure. Other suitable locked nucleotides that may be incorporated into oligonucleotides of this disclosure include those described in U.S. Patents 6,403,566 and 6,833,361 (both of which are incorporated herein by reference). In exemplary embodiments, the locked nucleotide is independently selected from 2′-4′ methylene crosslinks and restricted ethyl (cEt) crosslinks (see U.S. Patents 7,399,845 and 7,569,686, which are incorporated herein by reference in their entirety).

[0014] In some embodiments, one or more RNA nucleotides in the compound are unlocked nucleic acids (UNAs) lacking the C2′-C4′ bond typically found in ribonucleosides. UNAs are highly flexible and useful for fine-tuning the specificity and potency of oligonucleotides. In various embodiments, the oligonucleotide contains one, two, three, four, five, or six UNA nucleotides. In some embodiments, the oligonucleotide uses one, two, three, or four UNAs along with the RNA.

[0015] In some embodiments, the compounds are based on FANA antisense oligonucleotides, i.e., fluoroarabinonucleotides (i.e., 2′-deoxy-2′-fluoro-beta-D-arabinonucleotides or FANA). These nucleic acid analogs can regulate gene expression by enzymatic degradation of target RNA, which may be RNase H-mediated RNA cleavage.

[0016] In some embodiments, antisense oligonucleotides have a modified backbone or modified internucleotide linkages. The term “internucleotide linkage” refers to a linkage between two adjacent nucleosides in a polynucleotide molecule. Naturally, internucleotide linkages are phosphodiester bonds, which occur between the two oxygen atoms of a phosphate group and the oxygen atom of a sugar (either at the 3′ or 5′ position), forming two ester bonds that bridge two adjacent nucleosides. Modifications to internucleotide linkages can provide a variety of properties, including, but are not limited to, enhanced stability. For example, phosphorothioate or phosphorodithioate linkages increase the resistance of the internucleotide linkage to nucleases. Another example is phosphoacetate linkage (PACE), which can improve transfection properties and enhance nuclease resistance. Examples of nucleotide linkages and oligonucleotide backbone modifications that may be used in the oligonucleotides herein include, but are not limited to, phosphodiesters, phosphorothioates, phosphorodithioates, methylphosphonates, alkylphosphonates, alkylphosphonothioates, phosphotriesters, phosphoramidates, phosphoramidates, phosphorodiamidates, siloxanes, carbonates, carboalkoxys, acetamidates, carbamates, morpholinos, peptide nucleic acids, boranos, thioethers, cross-linked phosphoramidates, cross-linked methylenephosphonates, cross-linked phosphorothioates, and sulfone nucleoside linkages. In some embodiments, the antisense oligonucleotide comprises one or more phosphorothioates or phosphorodithioate nucleotides.

[0017] In some embodiments, the antisense oligonucleotide comprises one or more phosphorothioate or phosphorodithioate nucleotide linkages. In some embodiments, the phosphorothioate or phosphorodithioate linkage may be introduced between the last 3 to 5 nucleotides at the 5′-terminus and / or 3′-terminus of the oligonucleotide to reduce exonuclease degradation. In some embodiments, the antisense oligonucleotide has a combination of phosphodiester linkages and phosphorothioate / phosphodithioate linkages. In some embodiments, the antisense oligonucleotide contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 phosphorothioate or phosphorodithioate nucleotide linkages. In some embodiments, the antisense oligonucleotide comprises substantially alternating phosphodiester and phosphorothioate nucleotide linkages. In some embodiments, the antisense oligonucleotide is entirely linked by phosphorothioate / phosphodithioate (i.e., all links are either phosphorothioate or phosphorodithioate). In one embodiment, the antisense oligonucleotide is completely linked by a phosphorothioate.

[0018] In some embodiments, antisense oligonucleotides have a morpholino backbone, such as when RNaseH recruitment is undesirable. Morpholino oligonucleotides generally do not induce degradation of their target RNA molecules and may be effective for stereoblocking of target RNA sequences. Morpholino oligonucleotides and their synthesis are generally disclosed in U.S. Patents 11,028,386, 10,947,533, and 10,927,378 (each of which is incorporated herein by reference in whole). In some embodiments, antisense oligonucleotides include thiomorpholino nucleotides and / or other substituted or modified nucleotides, such as those described in International Patent Application Publication WO / 2019 / 060522 and International Patent Application Publication WO / 2018 / 057430 (each of which is incorporated herein by reference in whole). For example, Langner et al. describe a method for synthesizing oligonucleotide analogs called thiophosphoramidate morpholino oligonucleotides (TMOs) that incorporate morpholino nucleosides and phosphorothioate linkages (see "Synthesis and characterization of thiophosphoramidate morpholino oligonucleotides and chimeras." JACS 142.38 (2020):16240-16253, and also Dumbovic, Gabrijela, et al. "Nuclear compartmentalization of TERT mRNA and TUG1 lncRNA is driven by intron retention." Nature Communications 12.1(2021):1-19 (both in their entirety are incorporated herein by reference)). Therefore, the antisense oligonucleotides described herein may include fully or partially TMO-modified nucleotides, or may include chimeras of TMO-modified nucleotides with unmodified nucleotides and / or other nucleotides with different modifications (e.g., LNA).

[0019] In some embodiments, antisense oligonucleotides may contain one or more modified bases. In some embodiments, cytosine is substituted with 5-methylcytosine, thereby enhancing base pairing. If necessary, other modified bases (in particular cytosine or guanine) may be used to reduce immunogenicity. Other modified bases are described in U.S. Patent No. 10,064,959 (incorporated herein by reference). In various embodiments, the cytidine nucleic acid base in antisense oligonucleotides is 5-methylcytidine. Therefore, if the sequence contains a cytidine nucleic acid base ("C"), it will be understood by those skilled in the art that the term includes 5-methylC. Furthermore, if the sequence contains a uracil base, it will be understood that the term "U" includes pseudouridine and N1-methylpsoiduridine. Other modified forms of standard bases may also be envisioned in this disclosure.

[0020] In some embodiments, the antisense oligonucleotide comprises one or more inosine bases, the one or more inosine bases being deoxyinosine, which in some embodiments may be used as the central block of DNA nucleotides in the gapmer. Inosine can be used in place of any base, such as guanine, cytosine, adenine, or thymine nucleic acid bases. In embodiments, the oligonucleotide has 1 to 4 inosine nucleic acid bases, e.g., one, two, or three inosine nucleic acid bases. Inosine modifications can, in some embodiments, modulate mRNA cleavage and / or hybridization properties.

[0021] An exemplary antisense oligonucleotide according to the present invention is shown in Table 2 and is a 14-mer oligonucleotide having a 3-8-3 gapmer structure. An exemplary antisense oligonucleotide according to the present invention is also shown in Table 5 and has various lengths and gapmer structures. In one embodiment, the antisense oligonucleotide is a 13-mer oligonucleotide having a 3-8-2 gapmer. In one embodiment, the antisense oligonucleotide is a 14-mer oligonucleotide having a 3-9-2 gapmer. In one embodiment, the antisense oligonucleotide is a 13-mer oligonucleotide having a 2-8-3 gapmer. In one embodiment, the antisense oligonucleotide is a 14-mer oligonucleotide having a 2-9-3 gapmer. In one embodiment, the antisense oligonucleotide is a 15-mer oligonucleotide having a 3-10-2 gapmer. In one embodiment, the antisense oligonucleotide is a 17-mer oligonucleotide having a 2-12-3 gapmer. In one embodiment, the antisense oligonucleotide is a 20-mer oligonucleotide having a 2-15-3 gapmer. In one embodiment, the antisense oligonucleotide is a 17-mer oligonucleotide having a 3-11-3 gapmer. In one embodiment, the antisense oligonucleotide is a 16-mer oligonucleotide having a 3-10-3 gapmer. In another embodiment, the antisense oligonucleotide is a 19-mer oligonucleotide having a 3-13-3 gapmer. In yet another embodiment, the antisense oligonucleotide is an 18-mer oligonucleotide having a 3-12-3 gapmer. In yet another embodiment, the antisense oligonucleotide is an 18-mer oligonucleotide having a 3-13-2 gapmer.

[0022] In the embodiment, the melting temperature of the antisense oligonucleotide hybridized to the target sequence is at least about 35°C. mIt is the temperature at which 50% of the oligonucleotide forms a double strand with its complete complement and 50% is free in solution. Tm can be determined experimentally as a function of temperature by measuring the change in absorbance of the oligonucleotide and its complement. T m can also be estimated using publicly available known T m calculators. In some embodiments, the T of the oligonucleotide hybridized to the target sequence m is at least about 40 °C, or at least about 45 °C, or at least about 50 °C. In some embodiments, the T of the oligonucleotide hybridized to the target sequence m is from about 35 °C to about 60 °C. In some embodiments, the T of the oligonucleotide hybridized to the target sequence m is from about 40 °C to about 60 °C, or from about 50 °C to about 60 °C.

[0023] In some embodiments, the antisense oligonucleotide further comprises a cell targeting moiety or a permeable moiety, which in some embodiments is conjugated directly or indirectly to the 3′ end of the oligonucleotide, optionally via a linker. In some embodiments, the composition further comprises a sterol conjugate (e.g., cholesterol conjugate) or a fatty acid conjugate such as a palmitoyl or stearyl lipid conjugate conjugated, optionally, to the 3′ end of the antisense oligonucleotide. These moieties can enhance cell permeability. See US 9,012,225, which is incorporated herein by reference in its entirety.

[0024] In embodiments, the cell targeting moiety is an N-acetylgalactosamine (GalNAc) ligand that can improve the targeting of compounds to hepatocytes in embodiments. Exemplary GalNAc ligands are described in U.S. Patent Nos. 5,985,826 and 8,552,163, which are incorporated herein by reference in their entirety.

[0025] In various embodiments, the targeting or cell-penetrating moiety includes an antibody or antigen-binding fragment thereof, an aptamer, a peptide, a biological ligand (e.g., including a glycoconjugate), a lipid, a sterol, cholesterol or a derivative thereof, an integrin, an RGD peptide, or a cell-penetrating peptide (CPP). More specifically, the targeting or cell-penetrating moiety may be selected from a single domain antibody, a single chain antibody, a bispecific antibody, a recombinant heavy chain only antibody (VHH), a single chain antibody (scFv), a shark heavy chain only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin, a tetranectin, an affibody, a transbody, an anticalin, an adnectin, an affilin, a microbody, a filomer, a stradbody, a maxibody, an evibody, a finomer, an armadillo repeat protein, a knotted domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troibody, a pepbody, a wacibody, a unibody, a duoibody, an Fv, a Fab, a Fab′, an F(ab′)2, and a peptidomimetic molecule. Various ligand binding platforms are described in U.S. Patent No. 7,417,130, U.S. Patent Application Publication No. 2004 / 132094, U.S. Patent No. 5,831,012, U.S. Patent Application Publication No. 2004 / 023334, U.S. Patent No. 7,250,297, U.S. Patent No. 6,818,418, U.S. Patent Application Publication No. 2004 / 209243, U.S. Patent No. 7,838,629, U.S. Patent No. 7,186,524, U.S. Patent No. 6,004,746, U.S. Patent No. 5,475,096, U.S. Patent Application Publication No. 2004 / 146938, U.S. Patent Application Publication No. 2004 / 157209, U.S. Patent No. 6,994,982, U.S. Patent No. 6,794,144, U.S. Patent Application Publication No. 2010 / 239633, U.S. Patent No. 7,803,907, U.S. Patent Application Publication No. 2010 / 119446, and / or U.S. Patent No. 7,166,697, the entire contents of which are incorporated herein by reference.

[0026] In some embodiments, the cell-targeting moiety is an aptamer, which may be an aptamer described in U.S. Patent No. 9,464,293, U.S. Patent No. 10,550,394, or U.S. Patent No. 11,261,449 (the entire contents of which are incorporated herein by reference). In embodiments, the cell-targeting moiety targets an antisense oligonucleotide to the pancreas.

[0027] In another embodiment, the Disclosure provides pharmaceutical compositions comprising the compounds of the Disclosure and a pharmaceutically acceptable carrier. In some embodiments, the compositions comprise antisense oligonucleotides encapsulated in particles such as liposomes, polymer nanoparticles, lipid nanoparticles (LNPs), or exosomes. Exemplary polymer nanoparticles may be formed from PLA, PLGA, or PEG copolymers thereof. In some embodiments, the particles comprise poly(β-aminoester) polymers. In various embodiments, the particles comprise diblock and triblock copolymers, as described, for example, in U.S. Patent No. 9,476,063, U.S. Patent No. 9,505,867, and U.S. Patent Application Publication No. 2017 / 0049801 (all of which are incorporated herein by reference). In various embodiments, the LNPs comprise cationic or ionized lipids, neutral lipids, structural lipids, and PEGylated lipids.

[0028] In some embodiments, the compounds are incorporated into LNPs containing cationic or ionized lipids, such as, but not limited to, those known as ALC-059 or SM-102. Other exemplary lipid and LNP compositions are described in US9,708,628 and US2021 / 0023008 (these in whole are incorporated herein by reference).

[0029] Exemplary structural lipids may be selected from one or more of the following: cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and tocopherol (e.g., alpha-tocopherol). In some embodiments, the structural lipid is cholesterol.

[0030] In some embodiments, the LNP comprises one or more phospholipids. Exemplary phospholipids are selected from cardiolipin, sterol-modified lipids (modification by a cholesterol moiety attached to the sn-2 carbon of the glycerol backbone), mixed acylglycerophospholipids, and symmetric acylglycerophospholipids. Examples of the acylglycerophospholipid head group include phosphatidic acid, lysophosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphoinositides, and phosphatidylserine.Exemplary phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-diundecane. Noyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 Selected from PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

[0031] In some embodiments, the lipid nanoparticles further comprise one or more PEG lipids. PEG lipids are lipids modified with polyethylene glycol. Exemplary PEG lipids are selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. PEG lipids may be selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-cholesterol, PEG-tocopherol, and PEG-DSPE lipids.

[0032] Lipid particle formulations used in embodiments of this disclosure include those described in US9,738,593, US10,221,127, and US10,166,298 (all of which are incorporated herein by reference). In some embodiments, the liposome or nanoparticles further include a targeting moiety (e.g., described) that targets LNPs or other particles to a desired tissue or cell type. In embodiments, the cell targeting moiety is conjugated to a portion of a PEG group.

[0033] In various embodiments, antisense oligonucleotides are contained within exosomes for delivery. Exemplary exosomes and their preparation and / or use are described in U.S. Patent No. 10,851,372, U.S. Patent Application Publication No. 2018 / 0193270, and U.S. Patent Application Publication No. 2020 / 00317746 (both incorporated herein by reference).

[0034] In various embodiments, the pharmaceutical composition is formulated for parenteral administration. For example, the composition may be formulated for a route selected from intravenous, subcutaneous, intradermal, intralymphatic, intramuscular, intratumoral, or intrathecal administration, or for other suitable parenteral routes. In some embodiments, but not limited to, other routes are used, including routes for local delivery closer to the site of injury or inflammation, such as intranasal administration, topical administration, and pulmonary administration.

[0035] In another embodiment, the Disclosure provides a method for treating a disease or condition associated with the expression of NLRP3 and / or NLRP1 or the activation of the NLRP3 and / or NLRP1 inflammasome. In various embodiments, the method comprises administering the antisense oligonucleotide or pharmaceutical composition of the Disclosure to a subject, which is generally a mammal such as a human. In embodiments, the disease or condition manifests as inflammation of one or more tissues, organs, or systems, such as the central nervous system (e.g., brain), skeletal muscle, liver, pancreas, gastrointestinal system, lungs, heart, skin, eyes, ears, mucous membranes, joints, or adipose tissue. The composition can be administered systemically or topically according to methods known in the art.

[0036] The dosage, frequency, and duration of administration can be adjusted according to the needs of the subject. In some embodiments, the composition is administered about daily, about weekly, about every two months (i.e., about every week), about monthly, or about quarterly. In some embodiments, the dosage and frequency are determined based on the patient's condition or response. In some embodiments, the composition is administered at least once a month or at least once a week. In some embodiments, the subject receives at least four, or at least eight, or at least twelve, doses of the composition. The composition may be administered according to the routes already described (either systemic or topical).

[0037] In various embodiments, the diseases or conditions associated with NLRP3 and / or NLRP1 expression or activation of the NLRP3 and / or NLRP1 inflammasome are inflammatory diseases, autoimmune diseases, or neurodegenerative diseases. For example, in some embodiments, the subjects suffer from neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, or multiple sclerosis.

[0038] In other embodiments, the disease or condition is an inflammatory disease or condition such as migraine, traumatic brain injury, stroke (e.g., acute ischemic stroke), myocardial infarction, viral or bacterial meningitis, or nerve injury or inflammation (e.g., neuritis). In various embodiments, nerve injury includes peripheral nerve injury and / or spinal cord injury.

[0039] In yet another embodiment, the disease or condition is an autoimmune disease. Exemplary autoimmune diseases include, but are not limited to, diabetes mellitus, inflammatory bowel syndrome (IBD) (ulcerative colitis or Crohn's disease), arthritis (e.g., rheumatoid arthritis or osteoarthritis), multiple sclerosis, lupus, and dermatomyositis.

[0040] Other conditions or diseases that can be treated in accordance with this disclosure include diseases or conditions related to the immune system, cardiovascular system, endocrine system, gastrointestinal tract, renal system, respiratory system, central nervous system, cancer, or pathogenic infection (e.g., infection by viruses, bacteria, protists, helminths, or fungi).

[0041] Non-exclusive examples of viruses include alphaviruses such as influenza virus, cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus (HIV), chikungunya virus, and Ross River virus, flaviviruses such as dengue virus and Zika virus, or papillomavirus. In embodiments, the virus is SARS-CoV-2 (for example, in embodiments, the subject may have long coronavirus). Non-limiting examples of pathogenic bacteria include Staphylococcus aureus, Helicobacter pylon, Bacillus anthracis, Bordatella pertussis, Corynebacterium dipthenae, Clostridium tetani, Clostridium botulinum, Streptococcus pneumoniae, Streptococcus pyogenes, Listeria monocytogenes, Hemophilus influenzae, Pasteuria multicida, Shigella dysenteriae, Mycobacterium tuberculosis, Mycobacterium leprae, Mycoplasma pneumoniae, Mycoplasma hominis, Neisseria meningitidis, Neisseria gonorrhoeae, Rickettsia rickettsii, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Propionibacterium Examples include acnes, Treponema pallidum, Chlamydia trachomatis, Vibrio cholerae, Salmonella typhimurium, Salmonella typhi, Borrelia burgdorferi, or Yersinia pestis.Non-specific examples of protists include Plasmodium (e.g., cerebral malaria), Babesia, Giardia, Entamoeba, Leishmania, or Trypanosomas. Non-specific examples of helminths include schistosomes, roundworms, tapeworms, or trematodes. Non-specific examples of fungi include Candida or Aspergillus species.

[0042] In some embodiments, the disease or condition is at least partially caused by a constitutively active type of inflammation, such as cryopyrin-associated periodic syndromes (CAPS), Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FCAS), or neonatal-onset multiorgan inflammatory disease (NOMID).

[0043] In some embodiments, the disease or condition is an autoinflammatory disease such as familial Mediterranean fever (FMF), TNF receptor-associated periodic fever syndrome (TRAPS), mevalonate kinase deficiency (MET), hyperimmuneglobulin D deficiency and periodic fever syndrome, interleukin-1 receptor antagonist deficiency (DIRA), Magid syndrome, suppurative arthritis, pyoderma gangrenosum and acne (PAPA), A20 haploinsufficiency (HA20), granulomatous arthritis of children (PGA), PLCG2-associated antibody deficiency immunodeficiency (PLAID), PLCG2-associated autoinflammatory disease, antibody deficiency and immunodysregulation (APLAID), B-cell immunodeficiency, periodic fever and sideroblastic anemia with developmental delay (SIFD), Sweet's syndrome, chronic nonbacterial osteomyelitis (CNO), chronic relapsing multiple osteomyelitis (CRMO), synovitis, acne, pustulosis, hyperostosis, or osteitis syndrome (SAPHO).

[0044] In some embodiments, the disease or condition is an autoimmune disease such as multiple sclerosis (MS), type 1 diabetes, psoriasis, rheumatoid arthritis, Behçet's disease, Sjögren's syndrome, or Schnitzler syndrome.

[0045] In some embodiments, the disease or condition is a respiratory disease such as idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), asthma, asbestosis, neutrophilic asthma, or silicosis and cystic fibrosis. In some embodiments, the disease or condition is neutrophilic asthma.

[0046] In some embodiments, the disease or condition is a metabolic disorder such as type II diabetes, atherosclerosis, obesity, metabolic syndrome, gout, or pseudogout.

[0047] In some embodiments, the disease or condition is an eye disease such as ophthalmoechiitis, age-related macular degeneration (AMD) (wet or dry), diabetic macular edema, corneal infection, uveitis, or dry eye.

[0048] In some embodiments, the disease or condition is a kidney disease such as chronic kidney disease, oxalate nephropathy, or diabetic nephropathy.

[0049] In some embodiments, the disease or condition is a liver disease such as non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ASH), hepatitis (e.g., HCV), or cirrhosis.

[0050] In some embodiments, the disease or condition is an inflammatory reaction of the skin, such as contact dermatitis or sunburn.

[0051] In some embodiments, the disease or condition is an inflammatory response of the joints, such as osteoarthritis, systemic juvenile idiopathic arthritis, adult-onset Still's disease, or relapsing polychondritis.

[0052] In some embodiments, the disease or condition is polymyositis, stroke, myocardial infarction, graft-versus-host disease, hypertension, colitis, celiac disease, abdominal aortic aneurysm, wound healing, depression, psychological stress, pericarditis including Dressler syndrome, or ischemia-reperfusion injury.

[0053] In some embodiments, the disease or condition is a solid tumor or a cancer such as a hematological malignancy (e.g., leukemia such as AML).

[0054] As used herein, the term “approximately” means ±10% of the relevant value, unless the context should interpret it otherwise.

[0055] All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as any individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference.

[0056] Other aspects and embodiments of this disclosure will be revealed by the following examples. [Examples]

[0057] Example 1: Suppression of NLRP3 in THP-1 human monocytes after ASO treatment. A 14-mer antisense oligonucleotide containing an LNA modification and a fully phosphorothioated backbone was synthesized (Integrated DNA Technologies, Inc.). In 96-well format, Lipofectamine RNAiMax (Thermo-Fisher) was used at 0.4 uL per well to transfect each oligo into THP-1 human monocyte cell line (ATCC, pre-differentiated with 300 ng / mL phorbol 12-myristate 13-acetate) at final concentrations of 100 nM, 40 nM, and 16 nM. After 24 hours, for gene expression assays, cells were lysed with 0.05 mL of QUANTIGENE lysis mixture (Life Technologies) per well. After capturing RNA using the QUANTIGENE SINGLEPLEX assay kit (Life Technologies) according to the manufacturer's protocol, the expression of NLRP3 and the housekeeping gene PPIB was measured by signal amplification and detection by chemiluminescence reaction. The relative gene expression detected per well was quantified as relative light units (RLU) on a Spark luminometer (Tecan). The degree of NLRP3 knockdown is determined by the following formula: %KD 配列X =1 - [(RLU NLRP3 配列X / RLU PPIB 配列X ) / (RLU NLRP3 モックコントロール / RLU PPIB モックコントロール )]

[0058]

Table 1-1

Table 1-2

[0059]

Table 2-1

Table 2-2

[0060] [Table 3-1] [Table 3-2]

[0061] Example 2: Suppression of NLRP1 and NLRP3 in U87-MG cell line after ASO treatment Antisense oligonucleotides (disclosed in Table 5) containing LNA and a fully phosphorothioate-treated backbone were synthesized by Integrated DNA Technologies, Inc. Each oligonucleotide contained 1–3 deoxyinosine bases. Each oligonucleotide was transfected into U87-MG glioblastoma cell line (ATCC) at final concentrations of 100 nM, 25 nM, and 6.25 nM using 0.4 μL of Lipofectamine RNAiMax (Thermo-Fisher) per well in a 96-well format. After 24 hours, cells were lysed with 0.05 mL of Quantigene lysis mixture (Life Technologies) per well for gene expression assays. Using a Quantigene singleplex assay kit (Life Technologies), RNA was captured according to the manufacturer's protocol, and the expression of NLRP1, NLRP3, and the housekeeping gene PPIB was measured by signal amplification and detection via chemiluminescence. Relative gene expression detected per well was quantified as relative light units (RLU) on a Spark luminometer (Tecan). The degree of knockdown of NLRP1 (or NLRP3) is determined by the following formula: %KD 配列X =1-[(RLU NLRP 配列X / RLU PPIB 配列X ) / (RLU NLRP モックコントロール / RLU PPIB モックコントロール )]

[0062] Tables 5 and 6 show the degree of reduction in NLRP1 and NLRP3 mRNA in the U87-MG cell line after antisense oligonucleotide treatment.

[0063] [Table 4]

[0064] [Table 5]

[0065] [Table 6]

[0066] [Table 7]

[0067] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] Table 8-10 Table 8-11 Table 8-12 Table 8-13 Table 8-14 Table 8-15 Table 8-16 Table 8-17 Table 8-18 Table 8-19 Table 8-20 Table 8-21 Table 8-22 Table 8-23 Table 8-24 Table 8-25 Table 8-26 Table 8-27 Table 8-28 Table 8-29 Table 8-30 Table 8-31 Table 8-32 Table 8-33 Table 8-34 Table 8-35 Table 8-36 Table 8-37 Table 8-38 Table 8-39 Table 8-40 Table 8-41 Table 8-42 Table 8-43

Claims

1. A compound comprising an antisense oligonucleotide having a length of 10 to 30 nucleotides and containing at least eight consecutive nucleotides from any one of SEQ ID NOs. 1 to SEQ ID NOs. 52, which inhibits the expression of NLRP3 and / or NLRP1 mRNA.

2. The compound according to claim 1, wherein the antisense oligonucleotide is complementary to segments of equal length of human NLRP3 and / or NLRP1 mRNA, provided that the antisense oligonucleotide may contain 1 to 4 inosine bases.

3. The compound according to claim 1 or 2, wherein the oligonucleotide has a length of at least 12 nucleotides.

4. The compound according to claim 3, wherein the oligonucleotide has a length of at least 14 nucleotides.

5. The compound according to claim 2, wherein the oligonucleotide has a length of 10 to 24 nucleotides.

6. The compound according to claim 5, wherein the oligonucleotide has a length of 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

7. The compound according to claim 6, wherein the oligonucleotide has a length of 14 nucleotides.

8. The compound according to any one of claims 1 to 7, wherein the oligonucleotide comprises at least 10, at least 12, or at least 14 consecutive nucleic acid bases of any one of Sequence ID No. 1 to 52.

9. The compound according to claim 8, wherein the oligonucleotide has one nucleic acid base sequence from sequence numbers 1 to 52.

10. The compound according to claim 8 or 9, wherein the oligonucleotide recruits an endogenous nuclease when hybridizing with RNA.

11. The compound according to claim 10, wherein the antisense oligonucleotide has a stretch of at least six DNA nucleotides sufficient to recruit RNaseH, and optionally has a stretch of at least eight DNA nucleotides.

12. The compound according to claim 11, wherein one or more DNA nucleotides independently comprise a 2' chemical modification selected from 2'-fluoro, 2'-methyl, and 2'-ethyl.

13. The compound according to claim 11 or 12, wherein the antisense oligonucleotide is a gapmer having a 5' segment and a 3' segment, the 5' segment and the 3' segment each having 2 to 6 nucleotides or 2 to 4 nucleotides, and the 5' segment and the 3' segment do not contain DNA nucleotides.

14. The compound according to claim 13, wherein the 5' segment and the 3' segment each have a length of 3 nucleotides and sandwich an internal sequence of 8 DNA nucleotides.

15. The compound according to claim 14, wherein one or more nucleotides of the 5' segment and the 3' segment contain a 2'-O substituent, and optionally all nucleotides of the 5' segment and the 3' segment contain a 2'-O substituent.

16. The compound according to claim 15, wherein the 2'-O substituent is independently selected from 2'-O methyl, 2'-O ethyl, 2'-O methoxyethyl (MOE), and a crosslinked nucleotide having a 2'-4' crosslink.

17. The compound according to claim 16, wherein the crosslinked nucleotide has a methylene crosslink (LNA) or a restricted ethyl crosslink (cEt).

18. The compound according to any one of claims 1 to 17, wherein at least one nucleotide is an unlocked nucleic acid (UNA).

19. A compound according to any one of claims 1 to 10, which is a FANA antisense oligonucleotide.

20. The antisense oligonucleotide is a compound according to any one of claims 1 to 19, having a modified skeleton.

21. The compound according to claim 20, wherein the antisense oligonucleotide comprises one or more phosphorothioates or phosphorodithioate nucleotides.

22. The compound according to claim 21, wherein the oligonucleotide is completely linked by a phosphorothioate or phosphorodithioate.

23. The antisense oligonucleotide is a compound according to any one of claims 1 to 9, having a morpholino or thiomorpholino skeleton.

24. The compound according to any one of claims 1 to 23, wherein the cytidine nucleic acid base in the antisense oligonucleotide is 5-methylcytidine.

25. The compound according to any one of claims 1 to 24, wherein the antisense oligonucleotide optionally has 1 to 3 inosine nucleic acid bases which are deoxyinosine.

26. The oligonucleotide is the compound according to claim 1, as shown in Table 2 or Table 5.

27. The compound according to any one of claims 1 to 26, further comprising a cell-targeting portion or a cell-penetrating portion.

28. The compound according to claim 27, wherein the cell-targeting portion or cell-penetrating portion is optionally conjugated directly or indirectly to the 3' end of the oligonucleotide via a linker.

29. A pharmaceutical composition comprising a compound according to any one of claims 1 to 28, further comprising a pharmaceutically acceptable carrier.

30. The pharmaceutical composition according to claim 29, wherein the antisense oligonucleotide is encapsulated in particles.

31. The pharmaceutical composition according to claim 30, wherein the particles are liposomes, polymer nanoparticles, lipid nanoparticles, or exosomes.

32. The pharmaceutical composition according to claim 31, wherein the particles are a polymer, and optionally a diblock or triblock copolymer.

33. The pharmaceutical composition according to claim 31, wherein the particles are lipid nanoparticles.

34. The pharmaceutical composition according to claim 31, wherein the particles are exosomes.

35. A pharmaceutical composition according to any one of claims 29 to 34, formulated for parenteral administration.

36. The pharmaceutical composition according to claim 35, formulated for intravenous, subcutaneous, intradermal, intramuscular, intratumoral, or intrathecal administration.

37. A method for treating a subject having a disease or condition related to the expression of NLRP3 and / or NLRP1 or the activation of the NLRP3 and / or NLRP1 inflammasome, comprising administering to the subject a compound according to any one of claims 1 to 28 or a composition according to any one of claims 29 to 36.

38. The method according to claim 37, wherein the disease or condition is an inflammatory disease, an autoimmune disease, a respiratory disease, or a neurodegenerative disease.

39. The method according to claim 37, wherein the disease or condition is, optionally, a neurodegenerative disease which is amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, multiple sclerosis, or Huntington's disease.

40. The method according to claim 37, wherein the disease or condition includes nerve damage.

41. The method according to claim 40, wherein the nerve injury includes peripheral nerve injury or spinal cord injury.

42. The method according to claim 37, wherein the disease or condition is a migraine, traumatic brain injury, myocardial infarction, or stroke.

43. The method according to claim 37, wherein the disease or condition is diabetes mellitus or inflammatory bowel syndrome (IBD).

44. The method according to claim 37, wherein the disease or condition is neutrophilic asthma.

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