Compounds for use in inhibiting amanzi

Abstract

This invention relates to ASO (antisense oligonucleotide) compounds capable of inhibiting the transcriptional activity of AMANZI. More specifically, the disclosed ASO comprises a spacer-type ASO containing about 14 to about 25 nucleotide bases; (a) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (b) a spacer region having at least 8 to no more than 19 DNA bases; and (c) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases. Preferably, the spacer is substantially complementary to the region of AMANZI (SEQ ID NO. 1). More preferably, the spacer is substantially complementary to the 14-25 base region of AMANZI.

Description

[0001] Previous related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 542,512, filed October 4, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] sequence declaration

[0004] This application includes a sequence list electronically submitted in XML format, which is incorporated herein by reference in its entirety. The XML copy created on October 3, 2024, is named J1742-00203_SL.xml and has a size of 765,657 bytes. Technical Field

[0005] This invention relates to compounds and methods for regulating the transcriptional activity of AMANZI. Specifically, gapmer-type ASO (antisense oligonucleotide) compounds are provided, which are capable of regulating the transcriptional activity of AMANZI associated with inflammation and activation of the immune system. These compounds comprise: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a gap region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the gap region is substantially complementary to the 14-25 base region on AMANZI (SEQ ID NO. 1). Specifically, within the entire interstitial space, the interstitial nucleotide bases can be linked individually by phosphate thioester (P=S) nucleotide inter-linking bonds; and the modified nucleotide bases can be selected from 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNA), and combinations thereof. Background Technology

[0006] The following content includes information that may help in understanding the invention. This is not an admission that any information, publications, or documents explicitly or implicitly referenced herein are prior art or of importance to the invention described and claimed herein. All publications, patents, related applications, and other written or electronic materials mentioned or confirmed herein are incorporated herein by reference in their entirety. The incorporated information is also part of the text and content of this application at the time of filing, just as all other text and content are repeated in this application, and should be considered part of the text and content of this application at the time of filing.

[0007] Long non-coding RNAs (lncRNAs) are non-coding transcripts that are typically longer than 200 nucleotides (200 nt). lncRNAs can act as scaffolds for the assembly of chromatin remodeling complexes, thereby powerfully influencing gene transcription at specific gene loci. (Mattick, JS et al.) Nat Rev Mol Cell Biol 24, 430–447 (2023). Long non-coding transcripts have been found in many species. DNA (cDNA) sequencing projects (such as FANTOM) have identified the complexity of these transcripts in humans (Carninci P, et al. (September 2005), Science. 309 (5740): 1559–1563).

[0008] Inflammation is characterized by a biphasic cycle consisting of an initial pro-inflammatory phase, which is subsequently resolved by an anti-inflammatory process. Interleukin-1β (IL-1β) is a major regulator of pro-inflammatory activity and is encoded within the same topologically associated domain (TAD) as IL-37, an anti-inflammatory cytokine that antagonizes the function of IL-1β. Summary of the Invention

[0009] The invention described and claimed herein has many attributes and embodiments, including but not limited to those set forth, described, or referenced in this brief summary. This invention is not intended to be exhaustive, and the invention described and claimed herein is not limited to or restricted to the features or embodiments defined in this introduction, which is incorporated by way of illustration only and not by way of limitation.

[0010] A dominant non-coding RNA that antagonizes inflammation ( A MA ster N on coding RNA antagoni Z ing I AMANZI is a lncRNA encoded within the IL-1β promoter. AMANZI is co-transcribed with IL-1β on opposite strands and in opposite directions, and mediates a time-delayed long-range chromatin circularization interaction with the IL-37 promoter, which is normally spatially isolated from the active IL-1β locus. AMANZI is an IL-1β inhibitor.

[0011] This disclosure provides an ASO (antisense oligonucleotide) compound capable of causing IL-1β overexpression. This disclosure also provides a spacer-type ASO (antisense oligonucleotide) capable of inhibiting the transcriptional activity of AMANZI. More specifically, the disclosed ASO includes a spacer-type ASO comprising about 14 to about 25 nucleotide bases; a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; a spacer region having at least 8 to no more than 19 DNA bases; and a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases. Preferably, the spacer is substantially complementary to the 14 to 25 base region of AMANZI (SEQ ID NO. 1). Preferably, each spacer nucleotide base within the spacer is linked by a phosphate thioester (P=S) nucleotide internucleotide bond; and wherein the modified nucleotide base is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNA), and combinations thereof.

[0012] This disclosure provides a spacer-type ASO (antisense oligonucleotide) capable of inhibiting the transcriptional activity of AMANZI, the spacer-type ASO comprising: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to region A of AMANZI (bases 10 to 93 of SEQ ID NO. 1). Preferably, the spacer region comprises a 10-nucleotide DNA sequence of nucleotides 5 to 15 from any one of SEQ ID NO. 2-105 or an octamer of thereon. Preferably, each interstitial nucleotide within the entire interstitial space is linked by a phosphate thioester (P=S) nucleotide internucleotide bond; and the modified nucleotide base modifiers are selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNA), and combinations thereof. Preferably, region A of AMANZI is bases 12 to 27 of SEQ ID NO. 1. More preferably, the interstitial ASO is selected from the group consisting of SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, and combinations thereof. Most preferably, the interstitial ASO is SEQ ID NO. 42.

[0013] This disclosure provides a spacer-type ASO (antisense oligonucleotide) capable of inhibiting AMANZI transcriptional activity, the spacer-type ASO comprising: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to region B of AMANZI (bases 194 to 253 of SEQ ID NO. 1). Preferably, the spacer region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 or an octamer fragment thereof from any one of SEQ ID NO. 78, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84 and combinations thereof. Preferably, each interstitial nucleotide within the interstitial space is linked by a phosphate thioester (P=S) nucleotide internucleotide bond; and the modified nucleotide base is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNA), and combinations thereof. More preferably, the interstitial ASO is selected from the group consisting of SEQ ID NO. 78, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, and combinations thereof. Most preferably, the interstitial ASO is SEQ ID NO. 84.

[0014] This disclosure provides a spacer-type ASO (antisense oligonucleotide) capable of inhibiting the transcriptional activity of AMANZI, wherein the spacer comprises: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to region C of AMANZI (SEQ ID NO. 1 bases 519 to 568). Preferably, the interstitial region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 or an octamer fragment thereof, derived from any one of SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104 and combinations thereof. Preferably, the interstitial nucleotides within the entire interstitial region are each linked by a phosphate thioester (P=S) nucleotide internucleotide bond; and the modified nucleotide base modifiers are selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNA), and combinations thereof. More preferably, the gap-shaped ASO is selected from the group consisting of SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104 and combinations thereof. Most preferably, the gap-shaped ASO is SEQ ID NO. 101.

[0015] This disclosure provides a spacer-type ASO (antisense oligonucleotide) capable of inhibiting the transcriptional activity of AMANZI, wherein the spacer comprises: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to region D of AMANZI (SEQ ID NO. 1 bases 377 to 404). Preferably, the spacer region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 or an octamer fragment thereof from any one of SEQ ID NO. 228-234 and combinations thereof. Preferably, each interstitial nucleotide in the entire interstitial body is linked by a phosphate thioester (P=S) nucleotide interlink; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNAs), and combinations thereof.

[0016] This disclosure provides a spacer-type ASO (antisense oligonucleotide) capable of inhibiting AMANZI transcriptional activity, wherein the spacer comprises: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to region E of AMANZI (SEQ ID NO. 1, bases 574 to 615). Preferably, the spacer region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 or an octamer fragment thereof from any one of SEQ ID NO. 206-215 and combinations thereof. Preferably, each interstitial nucleotide in the entire interstitial body is linked by a phosphate thioester (P=S) nucleotide interlink; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNAs), and combinations thereof.

[0017] This disclosure provides a pharmaceutical composition comprising a spacer-type ASO (antisense oligonucleotide) capable of inhibiting the transcriptional activity of AMANZI and a pharmaceutical excipient, wherein the spacer comprises: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to region D of AMANZI (SEQ ID NO. 1 bases 377 to 404). Preferably, the spacer region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 or an octamer fragment thereof from any one of SEQ ID NO. 228-234 and combinations thereof. Preferably, each interstitial nucleotide in the entire interstitial body is linked by a phosphate thioester (P=S) nucleotide interlink; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNAs), and combinations thereof.

[0018] This disclosure provides a pharmaceutical composition comprising a spacer-type ASO (antisense oligonucleotide) capable of inhibiting the transcriptional activity of AMANZI and a pharmaceutical excipient, wherein the spacer comprises: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to region E of AMANZI (SEQ ID NO. 1 bases 574 to 615). Preferably, the spacer region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 or an octamer fragment thereof from any one of SEQ ID NO. 206-215 and combinations thereof. Preferably, each interstitial nucleotide in the entire interstitial body is linked by a phosphate thioester (P=S) nucleotide interlink; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNAs), and combinations thereof.

[0019] The interstitial nucleotide bases are independently linked by internucleotide bonds throughout the interstitial space, wherein at least one of these internucleotide bonds is a phosphate thioester (P=S) nucleotide bond; and wherein the nucleotide bases include at least one modified nucleotide base variant selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNAs), and combinations thereof. In some aspects, at least one modified internucleotide linker is a phosphate thioester linker. In some aspects, all internucleotide bonds throughout the interstitial space are phosphate thioester bonds. Detailed Implementation

[0020] This disclosure is based on the research finding that inhibitors of AMANZI transcriptional activity can lead to overexpression of IL-1β (β).

[0021] This invention relates to AMANZI transcription regulators and compositions comprising the AMANZI transcription regulator for regulating AMANZI transcription. In some embodiments, the AMANZI transcription regulator is an AMANZI transcription inhibitor.

[0022] Target AMANZI

[0023] (FANTOM CAT No.: FTMT20800006495.1) (SEQ ID NO. 1)

[0024] Human AMANZI sequence:

[0025] definition

[0026] The following terms have the following meanings: "2'-substituted nucleosides" refers to nucleosides containing a 2'-substituted sugar moiety. When referring to the sugar moiety, "2'-substituted" means a sugar moiety containing at least one 2'-substituent other than H or OH.

[0027] "2'-Deoxynucleoside" refers to a nucleoside that contains the 2'-H furanose sugar moiety, as naturally present in deoxyribonucleosides (DNA). 2'-Deoxynucleosides may contain modified nucleotides or may contain RNA nucleotides (e.g., uracil).

[0028] "2'-O-methoxyethyl" (also known as 2'-MOE and 2'-O(CH2)2—OCH3) refers to the O-methoxy-ethyl modification at the 2' position of the furanyl ring. Sugars modified with 2'-O-methoxyethyl are modified sugars.

[0029] "2'-O-methoxyethyl nucleotide" refers to a nucleotide containing a sugar moiety modified with 2'-O-methoxyethyl.

[0030] "5-Methylcytosine" refers to cytosine modified with a methyl group attached to the 5-position. 5-Methylcytosine is a modified nucleobase.

[0031] "Approximately" indicates a value plus or minus 7%.

[0032] "Active target region" or "target region" refers to the region targeted by one or more active antisense compounds. "Active antisense compound" refers to an antisense compound that reduces the transcription of a target gene or the level of the resulting protein.

[0033] "Adjuvant" is defined as any molecule that enhances an antigen-specific adaptive immune response.

[0034] "Antisense activity" refers to any detectable and / or measurable alteration attributable to the hybridization of an antisense compound with its target nucleic acid. Antisense activity is a reduction in the amount or expression of a target nucleic acid or the protein encoded by that target nucleic acid compared to the level of the target nucleic acid or target protein in the absence of an antisense compound.

[0035] "Antsense compound" refers to an oligomeric compound that can achieve at least one antisense activity.

[0036] An "alkyl" group refers to a saturated aliphatic hydrocarbon group containing 1 to 8 (e.g., 1 to 6 or 1 to 4) carbon atoms. Alkyl groups can be straight-chain or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-heptyl, or 2-ethylhexyl. Alkyl groups can be substituted (i.e., optionally substituted) with one or more substituents such as halogens; alicyclic groups [e.g., cycloalkyl or cycloalkenyl]; heterocyclic groups [e.g., heterocyclic alkyl or heterocyclic alkenyl]; aryl; heteroaryl; alkoxy; aryl; heteroaryl; acyl [e.g., (aliphatic)carbonyl, (aliphatic)carbonyl, or (heterocyclic)carbonyl]; nitro; cyano; amide [e.g., (cycloalkyl)carbonylamino, arylcarbonylamino, arylalkylcarbonylamino, (heterocyclic)carbonylamino, (heterocyclic)carbonylamino, ... [Heteroarylcarbonylamino, heteroarylalkylcarbonylamino, alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, arylaminocarbonyl, or heteroarylaminocarbonyl]; amino [e.g., aliphatic amino, alicyclic amino, or heteroalicyclic amino]; sulfonyl [e.g., aliphatic -S(O)2-]; sulfinyl; thioalkyl; sulfonoxy; urea; thiourea; aminosulfonyl; sulfonamide; oxo; carboxyl; carbamoyl; alicyclic oxy; heteroalicyclic oxy; aryloxy; heteroaryloxy; arylalkoxy; heteroarylalkoxy; alkoxycarbonyl; alkylcarbonyl; or hydroxyl. Without limitation, some examples of substituted alkyl groups include: carboxylalkyl (such as HOOC-alkyl, alkoxycarbonylalkyl and alkylcarbonyloxyalkyl); cyanoalkyl; hydroxyalkyl; alkoxyalkyl; acylalkyl; aralkyl; (alkoxyaryl)alkyl; (sulfonamide)alkyl (such as alkyl-S(O)2-aminoalkyl); aminoalkyl; amamide alkyl; (alicyclic)alkyl; or haloalkyl.

[0037] "alkylene" refers to a difunctional alkyl group.

[0038] A "bifunctional" moiety refers to a chemical group, such as a linker moiety, that is attached to the host chemical structure at two sites. A bifunctional moiety can be attached to the host chemical structure at any two chemically feasible substituted sites. Unless otherwise stated, bifunctional moietyes can be attached in either direction; for example, the bifunctional moiety "NO" can be attached in either the –NO- or –ON- direction.

[0039] A “chemically distinct region” refers to a region of an antisense compound that is chemically different in some way from another region of the same antisense compound. For example, a region containing a 2'-O-methoxyethyl nucleotide is chemically different from a region containing a nucleotide that does not have the 2'-O-methoxyethyl modification.

[0040] "Chimeric antisense compounds" refers to antisense compounds that have at least two chemically distinct regions.

[0041] "Complementarity" refers to the ability of the nucleobases of the first nucleic acid to pair with the nucleobases of the second nucleic acid.

[0042] "Continuous nucleobases" refers to nucleobases that are adjacent to each other.

[0043] "Diluent" refers to a component in a composition that lacks pharmacological activity but is pharmaceutically necessary or desirable. For example, a diluent in an injectable composition can be a liquid, such as a saline solution.

[0044] "Dosage" refers to a specified amount of medicine delivered in a single administration or over a specified period of time. In some embodiments, the dosage may be administered in the form of one, two, or more boluses, tablets, or injections. For example, in some embodiments where subcutaneous administration is desired, the volume required for the desired dosage may be difficult to accommodate with a single injection; therefore, two or more injections may be used to achieve the desired dosage. In some embodiments, the medicine is administered by infusion over an extended period of time or continuously. Dosage may be expressed as the amount of medicine per hour, per day, per week, or per month.

[0045] "Chemical modification" has its meaning as understood in the art when referring to RNA or DNA bases, and includes nucleoside bases selected from the group consisting of: 2'-substituted nucleosides, 2'-O-methoxyethyl (also known as 2'-MOE and 2'-O(CH2)2—OCH3), 2'-deoxynucleosides, 2'-O-methoxyethyl nucleotides, 5-methylcytosine, monocyclic nucleosides, bicyclic nucleosides, 4'-2' bicyclic nucleosides, 4' to 2' bicyclic nucleosides, locked nucleic acids, and nucleoside analogs, all as defined herein.

[0046] "Completely complementary" or "100% complementary" means that each nucleobase in the first nucleic acid has a complementary nucleobase in the second nucleic acid. "Substantially complementary," when referring to a spacer that is "substantially complementary" to a defined region (SEQ ID NO. 1) on its target, means that no more than two nucleobases can form hydrogen bonds with each other when the nucleobase sequences of the oligonucleotide and other nucleic acids are aligned in opposite directions. Complementary nucleobases are nucleobases that can form hydrogen bonds with each other. For example, for a decameric oligonucleotide, a substantially complementary sequence can have 8, 9, or 10 bases complementary to the second nucleic acid. "Substantially complementary" includes "completely complementary" sequences in which there are no mismatched bases with the second nucleic acid within the oligonucleotide sequence.

[0047] Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (mC) and guanine (G). Complementary oligonucleotides and / or nucleic acids do not need to have nucleobase complementarity at every nucleoside. Rather, some mismatches are permissible. "Complete complementarity" or "100% complementarity" when referring to oligonucleotides means that the oligonucleotide is complementary to another oligonucleotide or nucleic acid at every nucleoside of the oligonucleotide.

[0048] In the context of oligonucleotides, "sequential" refers to nucleosides, nucleobases, sugar moieties, or internucleotide bonds that are adjacent to each other. For example, "sequential nucleobases" means nucleobases that are adjacent to each other in the sequence.

[0049] "Interstitial body" refers to a modified oligonucleotide containing an internal "interstitial" region of multiple DNA nucleotides located between an outer region containing one or more nucleotides, wherein the nucleotides constituting the internal region are chemically different from the one or more nucleotides constituting the outer region. The internal region is generally referred to as the "interstitial body," while the outer region is generally referred to as the "wing." Unless otherwise stated, "interstitial body" refers to a glycosylation. Unless otherwise stated, the glycosylation portion of the nucleotide in the central region of the interstitial body is an unmodified 2'-deoxyribonucleotide. Therefore, the term "MOE interstitial body" refers to an interstitial body having a glycosylation of 2'-MOE nucleotides located in both wings and having an interstitial space containing 2'-deoxyribonucleotides. Unless otherwise stated, an MOE interstitial body may contain one or more modified internucleotide linkages and / or modified nucleobases, and such modifications do not necessarily follow the pattern of glycosylated interstitial bodies.

[0050] "Hybridization" refers to the pairing or annealing of complementary oligonucleotides and / or nucleic acids. While not limited to specific mechanisms, the most common hybridization mechanisms involve hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds.

[0051] "Closely adjacent" means that there are no intermediate components between closely adjacent components.

[0052] "Modified nucleotide bases" and "modified nucleosides" are deoxyribonucleotides or ribonucleotides that have been modified to have one or more chemical moieties not present in natural nucleic acids. Examples of modified nucleotide bases and "modified nucleosides" are compounds of formula Ia, Ib, IIa, or IIb as described herein.

[0053] "Non-bicyclic modified sugar moieties" are sugar moieties of modified nucleotide bases where the chemical modification does not involve converting the sugar moieties into bicyclic or polycyclic systems.

[0054] A "monocyclic nucleoside" is a nucleoside that includes a modified sugar moiety that is not a bicyclic sugar moiety. In some embodiments, the sugar moiety of the nucleoside or a sugar moiety analogue may be modified or substituted at any position.

[0055] "2'-modified sugar" refers to a furanyl sugar modified at the 2' position. Such modifications include substituents as described herein.

[0056] “Bicyclic nucleosides” (BNAs) are modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. Synthesization of bicyclic nucleosides has been disclosed, for example, in 7,399,845, WO / 2009 / 006478, WO / 2008 / 150729, US2004-0171570, US Patent 7,427,672, Chattopadhyaya et al. J. Org. Chem. 2009, 74, 118-134, WO 99 / 14226, and WO 2008 / 154401. The synthesis and preparation of methyleneoxy(4'-CH2—O-2')BNA monomers adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil, as well as their oligomerization and nucleic acid recognition properties, have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNA and its preparation methods have also been described in WO98 / 39352 and WO 99 / 14226. Analogs of methyleneoxy(4'-CH2—O-2')BNA and 2'-thio-BNA have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (WO 99 / 14226). Furthermore, the synthesis of 2'-amino-BNA (a novel conformationally restricted high-affinity oligonucleotide analog) has been described in the prior art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-amino and 2'-methylamino-BNA have been prepared, and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported. A carbocyclic bicyclic nucleoside with a 4'-(CH2)3-2' bridge and an alkenyl analog bridge 4'-CH═CH—CH2-2' has been described (Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides, as well as their oligomerization and biochemical studies, have also been described (Srivastava et al., J. Am. Chem. Soc., 2007, 129(26), 8362-8379).

[0057] "4'-2' bicyclic nucleoside" or "4' to 2' bicyclic nucleoside" is a bicyclic nucleoside containing a furanose ring with a bridge connecting the two carbon atoms of the furanose ring (i.e., the 2' and 4' carbon atoms of the ring).

[0058] Locked nucleic acid (LNA) is a modified nucleotide base in which the chemical modification involves converting the sugar moiety into a bicyclic or polycyclic ring system. Two specific examples of locked nucleic acid compounds are β-D-methyleneoxynucleotide (or "restricted methyl" (cMe) nucleotide) and β-D-ethyleneoxynucleotide (or "restricted ethyl" (cEt) nucleotide).

[0059] "Mismatch" or "non-complementarity" means that when the first oligonucleotide and the second oligonucleotide are aligned, the nucleobases of the first oligonucleotide are not complementary to the corresponding nucleobases of the second oligonucleotide or the target nucleic acid.

[0060] "Motif" refers to the pattern of unmodified and / or modified sugar moieties, nucleobases, and / or nucleoside linkages in an oligonucleotide.

[0061] "Nucleobase" refers to an unmodified or modified nucleobase. "Unmodified bases" are adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). "Modified nucleobases" are atomic groups other than the unmodified A, T, C, U, or G that can pair with at least one unmodified nucleobase. "5-Methylcytosine" is a modified nucleobase. A universal base is a modified nucleobase that can pair with any of the five unmodified nucleobases. "Nucleobase sequence" refers to the sequence of consecutive nucleobases in a nucleic acid or oligonucleotide, regardless of any modifications to the sugar or nucleoside linkages.

[0062] "Nucleoside" refers to a compound containing a nucleobase and a sugar moiety. The base and sugar moiety are either unmodified or modified independently. "Modified nucleoside" refers to a nucleoside containing a modified nucleobase and / or a modified sugar moiety. Modified nucleosides include abase-free nucleosides lacking a nucleobase. "Linked nucleosides" are nucleosides linked into a continuous sequence (i.e., there are no additional nucleosides between the linked nucleosides).

[0063] "Nucleoside mimics" include those structures used to replace sugars or sugar and bases at one or more positions in an oligomer compound, but not necessarily the linking bonds, such as nucleoside mimics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic, or tricyclic sugar mimics, for example, non-furanose sugar units. Nucleotide mimics include those structures used to replace nucleosides and linking bonds at one or more positions in an oligomer compound, such as peptide nucleic acids or morpholino (morpholino groups linked by —N(H)—C(═O)—O— or other non-phosphodiester linkages). Sugar substitutions overlap with the slightly broader term nucleoside mimics, but are intended to indicate only the substitution of sugar units (furanose rings). The tetrahydropyranyl ring provided herein illustrates an example of a sugar substitution where the furanose sugar group has been replaced by a tetrahydropyran ring system.

[0064] "Parenteral administration" means administration by injection (e.g., bolus) or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intraperitoneal administration, or intracranial administration, such as intrathecal or intraventricular administration.

[0065] "Pharmaceutically acceptable carrier" refers to a compound carrier that is physiologically and pharmaceutically acceptable. A pharmaceutically acceptable carrier retains the desired biological activity of the parent compound and does not produce undesirable toxicological effects.

[0066] "Thiophosphate linking bond" refers to the linking bond between nucleosides, in which the phosphodiester bond is modified by replacing a non-bridging oxygen atom with a sulfur atom.

[0067] "Reduction or inhibition of amount or activity" refers to a reduction or blockage of transcriptional expression or activity relative to the transcriptional expression or activity in the untreated or control sample, and does not necessarily indicate the complete elimination of transcriptional expression or activity.

[0068] "Self-complementary" in the context of oligonucleotides refers to oligonucleotides that at least partially hybridize with themselves.

[0069] "Side effects" refer to physiological reactions attributable to treatment but not to the desired outcome. Side effects include injection site reactions, abnormal liver function tests, abnormal kidney function, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, myopathy, and malaise. For example, elevated serum aminotransferase levels can indicate hepatotoxicity or abnormal liver function. Similarly, elevated bilirubin levels can indicate hepatotoxicity or abnormal liver function.

[0070] "Single-stranded oligonucleotide" refers to an oligonucleotide that has not hybridized with its complementary strand.

[0071] "Specific hybridization" means that the antisense compound has sufficient complementarity between the antisense oligonucleotide and the target nucleic acid to induce the desired effect, while exhibiting minimal or no effect on non-target nucleic acids under the conditions of desired specific binding (i.e., under physiological conditions in vivo assays and therapeutic treatments).

[0072] The term "glycan" refers to either an unmodified or modified sugar moiety. As used herein, "unmodified sugar moiety" refers to the 2'-OH(H) ribosyl moiety present in RNA ("unmodified RNA sugar moiety") or the 2-H(H) deoxyribosyl moiety present in DNA ("unmodified DNA sugar moiety"). An unmodified sugar moiety has one hydrogen atom at each of the 1', 3', and 4' positions, one oxygen atom at the 3' position, and two hydrogen atoms at the 5' position. As used herein, "modified sugar moiety" or "modified sugar" refers to a modified furanyl sugar moiety or a sugar substitute.

[0073] "Sugar substitute" refers to a modified sugar moiety having a non-furanose portion that can link a nucleobase to another group, such as an internucleotide linker, coupling group, or terminal group in an oligonucleotide. Modified nucleosides containing sugar substitutes can be incorporated into one or more positions within an oligonucleotide, and such oligonucleotides can hybridize to complementary oligomers or target nucleic acids.

[0074] “Targeting” or “targeted” refers to the process of designing and selecting antisense compounds that will specifically hybridize to a target nucleic acid and induce the desired effect.

[0075] "Target region" refers to the nucleotide sequence of the target nucleic acid targeted by the antisense compound. "5' target site" refers to the 5' last nucleotide of the target region. "3' target site" refers to the 3' last nucleotide of the target region.

[0076] "Target nucleic acid" and "target RNA" refer to antisense compounds designed to affect nucleic acids, such as AMANZI.

[0077] "Target region" refers to a portion of the target nucleic acid, to which oligomeric compounds are designed to hybridize.

[0078] "Unmodified nucleotides" refer to nucleotides composed of naturally occurring nucleobases, sugar moieties, and internucleotide bonds. Unmodified nucleotides are either RNA nucleotides (i.e., β-D-ribonucleosides) or DNA nucleotides (i.e., β-D-deoxyribonucleosides).

[0079] Antisense ASO compounds

[0080] This disclosure provides an interstitial antisense oligonucleotide (ASO) compound (“interstitial compound”) that is complementary to a region of AMANZI long non-coding RNA (e.g., about 91% to about 100% complementary, including 100% complementary over the entire length of the interstitial compound) and inhibits the transcription of multiple acute inflammatory genes regulated by AMANZI long non-coding RNA. In various embodiments of this disclosure, the interstitial compound comprises a modified oligonucleotide of 12 to 29 linked nucleosides. The interstitial compound is substantially complementary to a region of AMANZI (SEQ ID NO. 1) (e.g., having no more than one nucleotide mismatch over the entire length of the interstitial compound) and inhibits the transcription of multiple acute inflammatory genes regulated by AMANZI long non-coding RNA. The interstitial compound comprises: (a) a 5' wing sequence having about three to about seven wing-modified nucleotide bases; (b) a central interstitial region sequence having about six to about fifteen 2' deoxynucleotides; and (c) a 3' wing sequence having about three to about seven wing-modified nucleotide bases; wherein the nucleotides of the interstitial compound are each linked along the entire length of the interstitial compound by a thiophosphate nucleotide linker, a phosphothiol nucleotide linker, or a combination thereof; and wherein the modification of the modified nucleotide base is selected from the group consisting of: 2'-methoxyethyl (MOE) modification, locked nucleic acid (LNA) modification (e.g., a modified sugar containing cMe or cEt), 2'F-ANA modification, 2'-O-methoxyethyl (2'OMe) modification), or a combination thereof.

[0081] Chemical modification of antisense oligonucleotides can enhance their resistance to nucleases (resistance, tolerance, resistance) and their ability to enter cells. For example, phosphate-thioester oligonucleotides can be used. Other deoxynucleotide analogs include methylphosphonates, phosphoramide esters, dithiophosphate esters, N3'P5'-phosphonamide esters, and oligoribonucleotide phosphate-thioesters and their 2'-O-alkyl analogs and 2'-O-methylribonucleotide methylphosphonates. Alternatively, mixed backbone oligonucleotides (“MBOs”) can be used. MBOs contain a phosphate-thioester oligodeoxynucleotide fragment and a properly positioned modified oligodeoxynucleotide or oligoribonucleotide fragment. MBOs have a phosphate-thioester linker fragment and other fragments of other modified oligonucleotides, such as methylphosphonates (which are non-ionic and highly resistant to nucleases) or 2'-O-alkyl oligoribonucleotides.

[0082] In some embodiments, the oligonucleotide sugar moiety is a modified sugar moiety. In some embodiments, the modified sugar moiety may be a conformationally restricted sugar moiety. In some embodiments, the conformationally restricted sugar may be a locked nucleotide (locked nucleic acid or LNA). In some embodiments, the locked nucleotide may be selected from one of the following types: 2'-O-CH2-4' (oxy-LNA), 2'-CH2-CH2-4' (methylene-LNA), 2'-NH-CH2-4' (amino-LNA), 2'-N(CH3)-CH2-4' (methylamino-LNA), 2'-S-CH2-4' (thio-LNA), and 2'-Se-CH2-4' (seleno-LNA). In some embodiments, the conformationally restricted sugar may be a bridged nucleic acid (BNA). Some conformationally restricted sugars may be locked nucleic acids as shown in Formulas III and IV of U.S. Patent No. 10,465,188, which is incorporated herein by reference.

[0083] Antisense oligonucleotides can be synthesized. See, for example, Stein CA and Krieg AM (eds), Applied Antisense Oligonucleotide Technology, 1998 (Wiley-Liss).

[0084] Antisense compound motif

[0085] In a spacer, an inner region containing multiple nucleotides or linked nucleosides is positioned between an outer region containing multiple nucleotides or linked nucleosides, the nucleotides or linked nucleosides in the outer region being chemically different from those in the inner region. In the case of antisense oligonucleotides with a spacer motif, the spacer segment typically acts as a substrate for endonuclease cleavage, while the wings contain modified nucleosides. The regions of the spacer (5' wings, spacer sequence, and 3' wings) are distinguished by the type of sugar moiety contained in each distinct region. The types of sugar moiety used to distinguish the regions of the spacer can include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides can contain 2'-MOE and 2'-O-CH3, etc.), and bicyclic sugar-modified nucleosides (such bicyclic sugar-modified nucleosides can include those with 4'-(CH2)). nThose with -O-2' bridges (where n=1 or n=2). Preferably, each distinct region contains a uniform sugar motif. Wing-space-wing motifs are often described as "X—Y—Z", where "X" represents the length of the 5' wing, "Y" represents the length of the spacer, and "Z" represents the length of the 3' wing. Typically, a spacer body described as "X—Y—Z" has a configuration such that the spacer segment is positioned adjacent to each of the 5' and 3' wings. Therefore, there is no intermediate nucleotide between the 5' wing and the spacer segment, or between the spacer segment and the 3' wing. Typically, X and Z are modified nucleobases with the same chemical properties, or they are different. Preferably, Y is 8 to 15 nucleotides. X or Z can be any of 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. Therefore, the interstitial bodies include, but are not limited to, for example, 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 6-8-6, or 5-8-5.

[0086] In a preferred embodiment, the spacer has ten 2'-deoxyribonucleotide spacer segments immediately adjacent to and located between the wings of five chemically modified nucleosides. In some embodiments, the chemical modification in the wings includes 2'-sugar modification. In another embodiment, the chemical modification includes 2'-MOE sugar modification. Preferably, the space-widening antisense oligonucleotide has eight 2'-deoxyribonucleotide spacer segments immediately adjacent to and located between the wings of five chemically modified nucleosides. Alternatively, the chemical modification includes 2'-sugar modification. Alternatively, the chemical modification includes 2'-MOE sugar modification.

[0087] The interstitial body has eight 2'-deoxyribonucleotide segments located adjacent to and between the wings of five to six chemically modified nucleosides. Chemical modifications include 2'-sugar modifications, such as 2'-MOE sugar modifications.

[0088] Hybridization

[0089] Hybridization occurs between the interstitial compound and the target AMANZI nucleic acid (SEQ ID NO. 1). The most common hybridization mechanism involves hydrogen bonding between complementary nucleobases of the nucleic acid molecules (e.g., Watson-Crick, Husstein, or reverse Husstein hydrogen bonding). Hybridization can occur under varying conditions. Strict conditions are sequence-dependent and determined by the properties and composition of the nucleic acid molecule to be hybridized.

[0090] Modified sugar portion

[0091] Antisense compounds may optionally contain one or more nucleosides, wherein the sugar groups have been modified. Such sugar-modified nucleosides can confer enhanced nuclease stability, increased binding affinity, or other beneficial biological properties to the antisense compounds. The nucleosides comprise a chemically modified furanose ring moiety. Examples of chemically modified furanose rings include, but are not limited to: the addition of substituents (including 5' and 2' substituents), bridging non-homogeneous ring atoms to form bicyclic nucleic acids (BNAs), and using S, N(R), or C(R1)(R2) (where R, R1, and R2 are each independently H, C1-C). 12 Alkyl or protecting groups) and combinations thereof replacing the ribosyl epoxy atom. Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleosides (for other disclosed 5',2'-disubstituted nucleosides, see WO2008 / 101157) or ribosyl epoxy atoms substituted with S and further substituted at the 2'-position (US Patent Application 2005 / 0130923) or alternatively 5'-substituted BNA (WO2007 / 134181, wherein LNA is substituted, for example, with 5'-methyl or 5'-vinyl).

[0092] The antisense oligonucleotide may be part of a composition that may contain more than one region of AMANZI (SEQ ID NO: 1). Preferably, an AMANZI transcriptional inhibitor interstitial compound is an oligonucleotide having a sequence that is perfectly matched to or contains one or two base mismatches of SEQ ID NO: 41-46, 63-64, 66, 67 or 101-104.

[0093] When a particular protein is referred to herein, derivatives, variants, and fragments are considered and included. Those skilled in the art fully understand protein derivatives and variants, and this may involve insertional, substitutional, or deletion amino acid sequence variants known in the art.

[0094] Modified nucleotide bases

[0095] The modified nucleotide bases include formula Ia, formula Ib, formula IIa, or formula IIb:

[0096] Formula Ia

[0097] Formula Ib

[0098] Formula IIa

[0099] Formula IIb

[0100] in, Each X is independently O or S, where 0, 1, or 2 instances of X constitute S; Each W is independently H, OH, halogen, or -OC. 1-6 Alkyl group, wherein the alkyl group is optionally surrounded by at most three carbon atoms. 1-4 Alkyl, C 1-4 Alkoxy, halogen, amino, CN, NO 2或 Instance substitution of OH; each Q a Independently a bifunctional C 1-6 Alkylene, optionally with at most two Cs 1-4 Alkyl, C 1-4 Examples of substitution with alkoxy, halogen, or OH; and each Q b Independently a bond or a bifunctional moiety selected from –O-, -S-, -NO-, -N(R)-, -C(O-), -C(O)O-, and -C(O)N(R-), where R is an unsubstituted C 1-4 alkyl.

[0101] In one implementation, each X is O. In another implementation, an instance of X is S.

[0102] In one embodiment, the spacer contains one or more nucleotides of formula Ia or Ib, wherein W is a halogen. In another embodiment, W is fluorine. In yet another embodiment, the spacer contains one or more nucleotides of formula Ia. In yet another embodiment, the spacer contains one or more nucleotides of formula Ib.

[0103] In one embodiment, the spacer comprises one or more nucleotides of formula Ia or Ib, wherein W is -OC. 1-6 Alkyl group, wherein the alkyl group is optionally surrounded by at most three carbon atoms. 1-4 Alkyl, C 1-4 Alkyl, halogen, amino, or OH substitutions. In another embodiment, W is -OC. 1-6 Alkyl group, wherein the alkyl group is optionally C 1-4 Alkyl substitution. In another embodiment, W is unsubstituted -OC. 1-6 Alkyl group. In another embodiment, W is -OC 1-6 Alkyl group, wherein the alkyl group is C 1-4 Alkyl substitution. In another embodiment, W is selected from methoxy and –O-CH2CH2-OCH3. In one embodiment, the spacer contains one or more nucleotides of formula Ia. In another embodiment, the spacer contains one or more nucleotides of formula Ib.

[0104] In one embodiment, the spacer comprises one or more β-D nucleotides of formula IIa or α-L nucleotides of formula IIb, wherein Q a It is an unsubstituted bifunctional C 1-6 Alkylene, and Q b It is a bond or a bifunctional part selected from –O-, –S-, –NO-, and –N(R)-. In another embodiment, Q a Selected from –CH2-, –CH2-CH2-, –CH(CH3)-, –CH2-CH2(CH3)-, and Q b It is a bond or a bifunctional part selected from –O-, -S-, -N(R)-O- and -N(R)-, where R is H or C. 1-6 alkyl.

[0105] In one embodiment of formula IIa or IIb, Q a It is –CH2- and Q b It is –O-. In another embodiment of formula IIa or IIb, Q a It is –CH2-CH2- and Q b It is –O-. In another embodiment of formula IIa or IIb, Q a It is –CH2- and Q b It is -N(R)-O-, where R is H or C. 1-6 Alkyl group. In another embodiment of formula IIa or IIb, Q a It is –CH(CH3)- and Q b It is –O-. In another embodiment of formula IIa or IIb, Q a It is –CH2- and Q b Yes –S-. In another embodiment of formula IIa or IIb, Q a It is –CH2- and Q b It is -N(R)-, where R is H or C. 1-6 Alkyl group. In another embodiment of formula IIa or IIb, Q a It is –CH2-CH(CH3)- and Q b It is a key.

[0106] In some embodiments, the spacer comprises one or more nucleotides selected from the following modified nucleotides:

[0107] Arabica nucleic acid (ANA)

[0108] 2'-Methoxyethyl (MOE)

[0109] 2'-Methoxy(2'-OMe)

[0110] 2'-Fluoroarabinonucleotide (2'F-ANA)

[0111] β-D-methyleneoxy (cMe)

[0112] β-D-Ethyleneoxy (cEt)

[0113] Many other bicyclic and tricyclic sugar substitution ring systems can be used to modify nucleosides to incorporate into antisense compounds (see, for example, the review article: Leumann, Bioorg. Med. Chem. , 2002, 10, 841-854).

[0114] AMANZI transcription regulator

[0115] Any transcription regulator capable of inducing desired repression of protein expression (e.g., transcription) in the AMANZI gene region, as well as the AMANZI transcription inhibitor space body compounds of this disclosure, can be used in embodiments of the invention as described herein. In specific embodiments, any AMANZI transcription inhibitor space body compounds that regulate the AMANZI gene region (e.g., those that regulate, block, or attenuate AMANZI transcription) are also provided.

[0116] application

[0117] The interstitial bodies described herein can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be local, pulmonary (e.g., by inhalation or blowing of powder or aerosol, including via nebulizer), intratracheal, intranasal, epidermal and transdermal, oral, or parenteral. The compounds and compositions described herein can be delivered in a manner targeting specific tissues, such as bone marrow or brain. The compounds and compositions described herein are administered via parenteral administration. “Parenteral administration” means administration by injection or infusion. Parenteral administration includes subcutaneous, intravenous, intramuscular, intra-arterial, intraperitoneal, or intracranial (e.g., intracerebral), intrathecal, intraventricular, intraventricular administration, cerebral intraventricular administration, or cerebral ventricular administration. Administration can be continuous or long-term, or short-term or intermittent.

[0118] Parenteral administration can also be performed via infusion. Infusion can be long-term or continuous, or short-term or intermittent, administered via a pump or injection. In some embodiments, parenteral administration is subcutaneous.

[0119] preparation

[0120] The pharmaceutical compositions of the present invention include, for example, compositions comprising an AMANZI transcriptional inhibitor interstitial compound. The pharmaceutical formulations of the present invention may further comprise one or more pharmaceutically acceptable excipients.

[0121] The pharmaceutical formulations of the present invention may further comprise one or more pharmaceutically acceptable excipients.

[0122] The AMANZI transcription inhibitor interstitial compound can be present in the formulation in a substantially separable form. It should be understood that the product can be mixed with a carrier or diluent that does not interfere with the intended purpose of the product and will still be considered substantially separable. The product of the present invention can also be in a substantially purified form, in which case it will typically contain about 80%, 85%, or 90%, for example at least about 88%, at least about 90%, 95%, or 98%, or at least about 99% oligonucleotides, or the dry weight of the formulation.

[0123] Pharmaceutically acceptable diluents, carriers, and / or excipients include those suitable for both veterinary and human use. For example, diluents, carriers, and / or excipients include solutions, solvents, dispersion media, retardants, polymerizers and liposomes, emulsions, etc. As another example, suitable liquid carriers (especially for injectable solutions) include water, aqueous saline solutions, aqueous glucose solutions, etc., and carriers such as liposomes are also particularly suitable for drug administration.

[0124] Suitable carriers and diluents include buffered aqueous solutions, saline solutions, glucose, glycerol, isotonic saline solutions (e.g., phosphate-buffered saline), isotonic water, and combinations thereof. In some embodiments, the carrier may include propylene glycol, dimethyl isosorbide dimethyl ester, and water, and even more particularly, phosphate-buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols. In some embodiments, pharmaceutically acceptable carriers or diluents may be or include thermosetting poloxamer (which may be a liquid or gel, depending on temperature), carboxycellulose (… For example Carboxymethyl cellulose), collagen ( For example, Type I collagen), collagen raw materials containing procollagen, hyaluronic acid or derived hyaluronic acid and / or oil ( For example, Emu oil). Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers.

[0125] The composition may be in any standard known dosage form, including tablets, pills, capsules, semi-solid dosage forms, powders, sustained-release formulations, solutions, suspensions, elixirs, aerosols, injectable liquids, gels, creams, transdermal delivery devices (e.g., transdermal patches), inserts such as ophthalmic inserts, or any other suitable composition.

[0126] Preferably, the AMANZI transcriptional inhibitor interstitial compound is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition.

[0127] It can also exist in pharmaceutically acceptable salts. For example Inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and organic acid salts such as citrate, acetate, propionate, malonate, benzoate, etc.

[0128] In addition, desired substances such as wetting agents or emulsifiers, stabilizers or pH buffers or preservatives may be present, if desired. In some embodiments, the pharmaceutical compositions of the present invention will comprise suitable ophthalmologically acceptable buffers, such as acetate buffer, citrate buffer, phosphate buffer, borate buffer, and mixtures thereof. In some embodiments, buffers useful in the present invention include boric acid, sodium borate, sodium phosphate (including mono, di, and tribasic phosphates, such as sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate heptahydrate), and mixtures thereof. In some embodiments, the preservative may be stabilized chlorine dioxide, a cationic polymer, or a quaternary ammonium compound. In some embodiments, the pharmaceutical composition may further comprise wetting agents, nutrients, viscosity enhancers, antioxidants, etc., such as disodium ethylenediaminetetraacetate, alkali metal hexametaphosphate, citric acid, sodium citrate, sodium metabisulfite, sodium thiosulfate, N-acetylcysteine, butylated hydroxyanisole, butylated hydroxytoluene, polyvinyl alcohol, polyoxyethylene-polyoxypropylene copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, and mixtures thereof. In some embodiments, the pharmaceutical formulation of the present invention will not contain preservatives. In some embodiments, the AMANZI transcription inhibitor interstitial composition or formulation comprises disodium hydrogen phosphate heptahydrate or potassium dihydrogen phosphate, or both.

[0129] Nucleic acid uptake in mammalian cells can be enhanced using known transfection techniques, including the use of transfection agents. Such techniques can be used in conjunction with certain AMANZI transcriptional inhibitors and interstitial compounds. Examples of useful transfection agents include cationic agents (such as calcium phosphate and DEAE-glucan) and lipid transfection agents (such as lipofectam). TM and transfectam TM ) and surfactants.

[0130] These compositions can be formulated according to standard techniques known in the art, including, for example, those found in works such as Gennaro AR: Remington: The Science and Practice of Pharmacy, 20 th Those are the standard references in Lippincott, Williams & Wilkins, ed., 2000.

[0131] Any container suitable for storing and / or administering the pharmaceutical composition can be used in the combination products of this invention. Those skilled in the art will understand suitable containers. For example, such containers include vials and syringes. Containers can be suitably sterilized and airtight.

[0132] Such compositions contain pharmaceutically acceptable solvents (such as water or saline), diluents, carriers, or adjuvants. The pharmaceutical composition can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be local (including ophthalmic and mucosal administration, including vaginal and rectal delivery), pulmonary (by inhalation or blowing of powder or aerosol, including via nebulizer), intratracheal, intranasal, epidermal, and transdermal administration, oral, or parenteral administration. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial (intrathecal or intraventricular) administration.

[0133] These compounds can also be mixed, conjugated, or otherwise linked with other molecules, molecular structures, or mixtures of compounds (such as liposomes, receptor-targeting molecules, or other formulations) to aid in uptake, distribution, and / or absorption.

[0134] The term "pharmaceutically acceptable carrier" refers to a carrier of a physiologically and pharmaceutically acceptable compound, that is, a carrier that retains the desired biological activity of the parent compound and does not produce undesirable toxicological effects. For oligonucleotides, preferred examples of pharmaceutically acceptable carriers and their uses are further described in U.S. Patent 6,287,860, which is incorporated herein by reference. Sodium carriers have proven to be a suitable form for oligonucleotide drugs.

[0135] Formulations include liposome formulations. The term "liposome" refers to a vesicle composed of one or more spherical bilayers of amphiphilic lipids. Liposomes are monolayered or multilayered vesicles having a membrane formed of lipophilic material and an aqueous interior containing the composition to be delivered. Cationic liposomes are positively charged liposomes that are thought to interact with negatively charged DNA molecules to form stable complexes. pH-sensitive or negatively charged liposomes are thought to capture DNA rather than complex with it. Both cationic and non-cationic liposomes have been used to deliver DNA into cells.

[0136] Liposomes also include spatially stable liposomes, which are liposomes containing one or more specialized lipids that, when incorporated into the liposome, result in a longer cycle life compared to liposomes without such specialized lipids. Liposomes and their uses are further described in U.S. Patent 6,287,860, which is incorporated herein by reference.

[0137] Preferred formulations for topical application include those in which oligonucleotides are mixed with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Preferred lipids and liposomes include neutral lipids (e.g., dioleoylphosphatidylethanolamine, dimyristoylphosphatidylcholine DMPC, distearylphosphatidylcholine), negatively charged lipids (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic lipids (e.g., dioleoyltetramethylaminopropane DOTAP and dioleoylphosphatidylethanolamine DOTMA).

[0138] lipid nanoparticles

[0139] LNPs are typically multi-component systems composed of ionizable amino lipids, phospholipids, cholesterol, and polyethylene glycol (PEG)-lipids, all of which contribute to the efficient delivery of nucleic acid drug carriers and particle stability (Schroeder et al., J. Intern. Med. (2010;267:9–21). Cationic lipids aggregate negatively charged RNA into nanoparticles via electrostatic interactions, and the use of positively charged ionizable lipids at acidic pH is thought to enhance endosome escape. Formulations for clinical and non-clinical delivery are primarily based on cationic lipids, such as DLin-MC3-DMA (MC3). (Kanasty et al.) Nat. Mater. 2013;12:967–977; and Xue et al. Curr. Pharm. Des. 2015;21:3140–3147).

[0140] Other LNPs comprise nanoemulsions having: a perfluorinated carbon component (a) consisting of at least one perfluorinated carbon compound; an emulsifying component (b), such as phospholipids and optionally assisting lipids; and an endocytosis-enhancing component (c) comprising at least one compound that induces cellular uptake of the nanoemulsion. The perfluorinated carbon compound of component (a) is preferably selected from compounds having the following structure: C m F 2m+1 X, XC m F 2m X, XC n F 2n OC o F 2o X, N(C) o F 2o X)3 and N(C o F 2o +1)3, where m is an integer from 3 to 10, n and o are integers from 1 to 5, and X is independently selected from Cl, Br, and I each time it appears. Examples of perfluorocarbon compounds are perfluorooctyl bromide and perfluorotributylamine.

[0141] Examples of emulsifiers include phospholipids (such as phospholipid compounds represented by Formula I), or pharmacologically acceptable carriers thereof: (I), Among them, R 1 and R 2 Independently selected from H and C 16-24 An acyl residue, which can be saturated or unsaturated and can carry 1 to 3 residues R. 3 And one or more of the C atoms can be O or NR 4 Substitution, and X is selected from H, —(CH2). p —N(R 4 )3 + —(CH2) p —CH(N(R 4 )3 + —COO - —(CH2) p —CH(OH)—CH2OH and —CH2(CHOH) p —CH2OH (where p is an integer from 1 to 5); R 3 Independently selected from H, lower alkyl groups, F, Cl, CN, and OH; and R 4 It is independently selected from H, CH3 and CH2CH3.

[0142] Following subcutaneous (sc) administration, LNPs and their mRNA carriers are expected to remain largely at the injection site, resulting in high local concentrations. Since LNPs are known to be pro-inflammatory, this is primarily attributed to the ionizable lipids present in LNPs (Sabnis et al.). Mol. Ther. (2018;26:1509–1519), therefore it can be expected that sc administration of LNP-formulated mRNA will be associated with dose-limiting inflammatory responses. Co-administration of dexamethasone with LNP reduced the immune inflammatory response following ivory administration (Abrams et al.). Mol. Ther. 2010;18:171–180). And Chen et al. ( J. Control. Release. (2018;286:46–54.) demonstrated that incorporating a lipophilic dexamethasone prodrug into an LNP-containing nucleic acid reduced the immunostimulatory effect after systemic administration.

[0143] dose

[0144] The optimal dosing regimen is calculated based on measurements of drug accumulation in the patient's body. Optimal dose variations depend on the relative potency of each oligonucleotide and can usually be based on... in vitro and in vivo The EC50, which has been found to be effective in animal models, is used for estimation. Typically, the dose is 0.01 μg to 100 g per kilogram of body weight and can be administered once or more daily, weekly, monthly, or annually, or at desired intervals. Following successful treatment, maintenance therapy may be expected to prevent relapse of the disease state, in which oligonucleotides are administered at a maintenance dose ranging from 0.01 μg to 100 g per kilogram of body weight, once or more daily.

[0145] Therapeutic effective doses include, but are not limited to, the doses described herein. The doses and other therapeutically effective doses are administered according to one or more of the therapeutically effective dose regimens described herein.

[0146] Products / Reagent Kits

[0147] In another embodiment of the invention, an article of manufacture or "kit" is provided that contains materials that can be used to inhibit AMANZI. The kit includes a container having a composition comprising one or more modulators, such as AMANZI modulators, such as AMANZI transcriptional inhibitor interstitial compounds. Suitable containers include... For example Bottles, small bottles wait wait The container can be formed from a variety of materials such as glass or plastic. The kit may also contain a pharmaceutically acceptable carrier. In some embodiments, the kit may also contain components for administering the composition, such as a syringe, needle, or microneedle. wait .

[0148] manufacture

[0149] The oligonucleotides of the present invention can be manufactured using solid-phase chemistry for synthesizing oligonucleotides, chemistry known in the art for synthesizing and preparing peptides and peptide mimics, and chemistry known in the art for synthesizing organic compounds. In one aspect, the formulation of the present invention will comprise a salt of the oligonucleotide of the present invention, such as a sodium salt of the oligonucleotide of the present invention. The kit may also comprise a pharmaceutically acceptable carrier. In one embodiment, the formulation may comprise a sodium salt of an interstitial ASO compound as described herein.

[0150] In some embodiments, the formulations of the present invention are substantially pure. Substantially pure means that the formulation contains less than about 10%, 5%, or 1%, and preferably less than about 0.1%, of any nucleotide or non-nucleotide impurities. In some embodiments, the total impurities, including metabolites of the AMANZI transcriptional inhibitor spacesomal ASO (antisense oligonucleotide) compound, will not exceed 15%. In some embodiments, the total impurities, including metabolites of the AMANZI transcriptional inhibitor spacesomal ASO (antisense oligonucleotide) compound, will not exceed 12%. In some embodiments, the total impurities, including metabolites of the AMANZI transcriptional inhibitor spacesomal ASO (antisense oligonucleotide) compound, will not exceed 11%. In other embodiments, the total impurities, including metabolites of the AMANZI transcriptional inhibitor spacesomal ASO (antisense oligonucleotide) compound, will not exceed 10%.

[0151] The sterile composition comprising the AMANZI transcription inhibitor spacesomal ASO (antisense oligonucleotide) compound of the present invention is prepared by aseptic processing of the AMANZI transcription inhibitor spacesomal ASO (antisense oligonucleotide) compound dissolved in a formulation carrier. In one embodiment, the formulation may also be sterilized by filtration. The excipients used in the manufacture of the formulations of the present invention are widely used in pharmaceuticals and conform to pharmacopoeia standards.

[0152] Example

[0153] Example 1

[0154] This embodiment provides a candidate spacer for inhibiting gene transcription regulated by the long non-coding RNA AMANZI. in vitro Screening system. The effect of candidate spacer compounds on target nucleic acid expression (e.g., messenger RNA) is screened using RT-PCR.

[0155] The THP-1 human monocyte cell line (derived from an acute leukemia patient) was purchased from InvivoGen. THP-1 cells were kept in complete medium containing RPMI 1640, 1% (2 mM) GlutaMAX L-glutamine supplement, 25 mM HEPES, 10% FBS, 100 μg / ml Normocin, Pen-Strep (100 U / ml), Blastidin (10 μg / ml), and Zeocin (100 μg / ml). Before seeding into sieves, the THP-1 monocyte culture was aliquoted at 50% to allow cells to re-enter the exponential growth phase. 250,000 cells were seeded in quadruplicates into each well of a 96-well plate, with 180 μL of complete medium added to each well. Each interstitial compound tested was added to the THP-1 cells at a final concentration of 10 µM and gently mixed. The plate was incubated at 37°C for 24 hours in 5% CO2. Then, LPS (10 ng / mL) was added to each well, and the plate was incubated at 37°C for another 24 hours in 5% CO2.

[0156] The antisense regulation of aMANZI expression on a specified gene was determined by real-time PCR (RT-PCR). RNA analysis was performed on total cellular RNA or poly(A)+ mRNA.

[0157] RNA was isolated and prepared using TRIZOL® reagents (Thermo Fisher Scientific) and the Direct-zol RNA Mini-Preparation Kit (Zymo Research) according to the manufacturer's recommended protocol.

[0158] Quantification of target RNA levels was performed using quantitative real-time PCR via the CFX real-time qPCR detection system (Bio Rad). Prior to real-time PCR, isolated RNA underwent a reverse transcriptase (RT) reaction, producing complementary DNA (cDNA), which was then used as the substrate for real-time PCR amplification. RT reaction reagents and real-time PCR reagents were obtained from Thermo Fisher Scientific, and their usage protocols were provided by the manufacturer. The gene (or RNA) target levels obtained by real-time PCR were normalized using the expression levels of stably expressed internal control genes, such as HPRT or RPL37A. Total RNA was quantified using a Qubit Fluorometer (Invitrogen / Scientific) and the Qubit RNA HS Assay Kit (Thermo Fisher Scientific catalog number Q32852) according to the manufacturer's protocols. The Qubit Fluorometer was calibrated with standards.

[0159] A series of tested interstitial compounds are shown in Table 1. These interstitials were designed to target different regions of the human AMANZI long non-coding RNA (SEQ ID NO. 1). The interstitial compounds in Table 1 are chimeric oligonucleotides (“interstitials”) with different configurations. For example, an interstitial compound with a configuration of 20 (5-10-5) nucleotides consists of a central “interstitial” region of ten 2’-deoxynucleotides, which is flanked by “wings” of five nucleotides on both sides (in the 5’ and 3’ directions). These wings are composed of nucleosides modified with 2’-methoxyethyl (2’-MOE) sugars. The internucleotide (backbone) linkages are phosphate thioesters throughout the oligonucleotide sequence. Cytidine residues are 5-methylcytidine residues, and in this case, they are cytidine residues unless otherwise specified. The spacer body, with a conformation of 16 (3-10-3) nucleotides in length, consists of a central "spacer" region containing ten 2'-deoxynucleotides, flanked by three nucleotide "wings" on either side (in the 5' and 3' directions). In some embodiments, the wings are composed of nucleosides modified with locked nucleic acids (LNAs) using cMe locked nucleic acid modification. The internucleotide (backbone) linkages are phosphate thioesters throughout the oligonucleotide sequence. Cytidine residues are 5-methylcytidine residues, and unless otherwise specified, they are cytidine residues in this case.

[0160] The interstitial compounds used are shown in Table 1. Abbreviations for nucleoside modifications in Table 1: M = 2'-methoxyethyl (2'-MOE) modified nucleoside; L = locked nucleic acid (LNA) modified nucleoside (cMe); d = 2'-deoxynucleoside.

[0161]

[0162] Table 1.

[0163] The effects of interstitial compounds on IL1B transcription in THP1 cells were analyzed using quantitative real-time PCR. Similarly, the effects of interstitial compounds on cytotoxicity and Toll-like receptor (TLR) signaling activation were analyzed by detecting TNFRSF10B gene transcription and secreted embryonic alkaline phosphatase (SEAP), respectively. Data are the average of four replicate experiments in which THP1 cells were treated with the interstitial compounds listed in Table 1.

[0164] Table 2 shows the fold changes in the expression of IL1B, TNFRSF10B, and SEAP genes in THP1 cells in the presence of the interstitial compounds listed in Table 1. Data were normalized by the expression of the housekeeping gene RPL37A and expressed as fold changes relative to negative controls (SEQ ID NO. 106 served as a negative control for MOE interstitial structures, and SEQ ID NO. 107 served as a negative control for LNA interstitial structures). An expression value < 1.0 indicates that transcription of the gene is repressed, and an expression value > 1.0 indicates that transcription of the gene is induced. For example, a value of 0.25 indicates that gene transcription is repressed by 75%.

[0165]

[0166] Table 2.

[0167] Interstitial bodies SEQ ID NOs 41-47, 51, 63-64, 66-67, 69, 82-84, and 101-104 showed at least a two-fold upregulation of human IL1B expression in this assay. Among them, interstitial body SEQ ID NO. 42 showed more than a three-fold upregulation of human IL1B expression in this assay.

[0168] Based on the screening data in Tables 1 and 2, it was found that three regions (A, B, and C) in the human AMANZI sequence (SEQ ID NO. 1) were effectively targeted by spacer bodies SEQ ID NO. 41-46, 63-68, 83-84, and 101-104. Table 3 provides the locations of the AMANZI target regions (AMANZI target regions A, B, and C) in the human AMANZI sequence (SEQ ID NO. 1) and the average upregulation of IL1B by spacer bodies targeting each region.

[0169]

[0170] Table 3.

[0171] The interstitial spaces of regions A, B, and C in human AMANZI (SEQ ID NO. 1) showed an average upregulation of IL1B gene expression of more than 2.3-fold. Interstitial spaces targeting region A were selected from the group consisting of SEQ ID NO. 41-46. Interstitial spaces targeting region B were selected from the group consisting of SEQ ID NO. 78 and 81-84. Interstitial spaces targeting region C were selected from the group consisting of SEQ ID NO. 63, 64, 66, 67, and 101-104.

[0172] Based on the screening data in Table 3, a first group of supplementary chimeric phosphate thioester interstitial compounds targeting A, B, and C regions of human AMANZI (SEQ ID NO. 1) were synthesized. Table 4 provides the configuration, chemical modifications, and sequences of the supplementary chimeric phosphate thioester interstitial compounds. Abbreviations for nucleoside modifications in Table 4: M = 2'-methoxyethyl (2'-MOE) modified nucleoside; L = locked nucleic acid (LNA) modified nucleoside (cMe); d = 2'-deoxynucleoside, 2'Md = 2'OMe modified deoxynucleoside.

[0173]

[0174] Table 4.

[0175] The effects of the first supplemental chimeric phosphate thioester interstitial compounds listed in Table 4 on the transcription of IL1B, TNFRSF10B, and SEAP in THP1 cells were analyzed by quantitative real-time PCR. Table 5 shows the induction of IL1B, TNFRSF10B, and SEAP gene expression by the first supplemental chimeric phosphate thioester interstitial compounds (SEQ ID NO. 42, 46, 67, 83, 101) targeting the AMANZI target regions (regions A, B, and C) in the human AMANZI sequence (SEQ ID NO. 1). Data were normalized by the expression of the housekeeping gene RPL37A and expressed as fold changes relative to the negative controls (SEQ ID NO. 106 served as the negative control for the MOE interstitial compound, and SEQ ID NO. 107 served as the negative control for the LNA interstitial compound). An expression value < 1.0 indicates that the transcription of the gene is repressed, and an expression value > 1.0 indicates that the transcription of the gene is induced. For example, a value of 0.25 indicates that gene transcription is repressed by 75%.

[0176]

[0177] Table 5.

[0178] Based on the screening data in Tables 4 and 5, another group of LNA interstitial compounds (second supplement) targeting the human AMANZI sequence (SEQ ID NO. 1) was synthesized and tested. Table 6 provides the configuration, nucleoside modifications, and sequences of the second supplemental interstitial compounds used in the embodiments and implementations of the present invention described herein. Abbreviations for nucleoside modifications in Table 6: L = locked nucleic acid (LNA) modified nucleoside (cMe); d = 2'-deoxynucleoside.

[0179]

[0180] Table 6.

[0181] The effects of the second supplemental LNA spacer compound in Table 6 on IL1B, TNFRSF10B, and SEAP transcription in THP1 cells were similarly analyzed by quantitative real-time PCR. Table 7 shows the induction of IL1B gene expression by the second supplemental LNA spacer compound SEQ ID NO. 163-238 targeting the human AMANZI sequence (SEQ ID NO. 1). Data were normalized by the expression of the housekeeping gene RPL37A and expressed as fold changes relative to the negative controls (SEQ ID NO. 106 served as the negative control for MOE spacer compounds, and SEQ ID NO. 107 served as the negative control for LNA spacer compounds). An expression value < 1.0 indicates that transcription of the gene is repressed, and an expression value > 1.0 indicates that transcription of the gene is induced.

[0182] Interstitial bodies SEQ ID NO. 173, 201-208, 226, 223-229 and 235 showed at least a three-fold upregulation of human IL1B expression in this assay.

[0183]

[0184] Table 7.

[0185] Based on the screening data in Tables 6 and 7, it was found that two other regions (regions D and E) in the human AMANZI sequence (SEQ ID NO. 1) were effectively targeted by spacer bodies SEQ ID NO. 201-208 and SEQ ID NO. 223-229. Table 8 provides the locations of the AMANZI target regions (AMANZI target regions D and E) and the average upregulation of IL1B by spacer bodies targeting each region.

[0186]

[0187] Table 8.

[0188] The interstitial spaces of regions D and E in human AMANZI (SEQ ID NO. 1) showed an average upregulation of IL1B gene expression of more than 3.5-fold. The interstitial spaces targeting region D were selected from the group consisting of SEQ ID NO. 228-234. The interstitial spaces targeting region B were selected from the group consisting of SEQ ID NO. 206-213.

[0189] Table 5 shows the results of AMANZI inhibition and the corresponding gene activation. Values ​​less than 1 indicate inhibition.

[0190] THP1 monocytes are heterozygous for rs16944 (G>A) at nucleotide 268 of the human AMANZI sequence (SEQ ID NO. 1). To understand the effect of allele specificity on IL-1β transcript induction during training immunization, allele-specific MOE spacer compounds targeting the region covering rs16944 were synthesized. Table 6 provides the configuration, nucleoside modifications, and sequence of the spacer compounds. Abbreviations for nucleoside modifications in Table 9: M = 2'-methoxyethyl (2'-MOE) modified nucleoside; d = 2'-deoxynucleoside.

[0191]

[0192] Table 9.

[0193] Example 2: Inhibition of AMANZI in the β-glucan-trained monocyte cell line THP1

[0194] Training immunization was induced in THP1 monocytes as follows: THP1 monocytes were grown in RPMI 1640 (Gibco) medium supplemented with 10% fetal bovine serum (FBS) (Gibco), GlutaMAX (Gibco), and 50 nM 2-mercaptoethanol (Gibco). THP1 monocytes were then seeded in flat-bottomed 96-well plates (Corning, NY, USA) and incubated at 37°C for 24 hours with or without 2 μg / ml β-glucan supplemented, along with 10 μM test spacer (SEQ ID NO. 239, 240) or MOE negative control spacer (SEQ ID NO. 106). Cells were washed once with 200 μL warm PBS and incubated for 5 days. The medium was changed on day 3. On day 6, cells were stimulated again for 24 hours with 200 μl RPMI or 10 ng / ml LPS (serum type O55:B5; Sigma), and the gene expression of IL1B was quantified by RT-qPCR.

[0195] Table 10 shows the gene expression of IL1B when THP1 monocytes were trained in the presence of interstitial compounds SEQ ID NO. 239, 240, or the MOE negative control interstitial compound (SEQ ID NO. 106). Data were normalized by the expression of the housekeeping gene RPL37A and are expressed as fold changes relative to the MOE negative control interstitial compound (SEQ ID NO. 106).

[0196]

[0197] Table 10.

[0198] Table 10 shows the differential effects of interstitial compounds SEQ ID NO. 239 and 240 on IL1B gene expression in training immunization of THP1 monocytes. Interstitial compound SEQ ID NO. 239 induced 5.8-fold IL1B transcripts, while interstitial compound SEQ ID NO. 240 slightly inhibited IL1B transcription compared with the negative control interstitial compound (SEQ ID NO. 107).

[0199] Example 3: AMANZI inhibition in LPS-stimulated CD14-positive monocytes

[0200] Percoll mononuclear cells from healthy donors (n=3) were cultured in 24-well plates (Sarstedt) at a rate of 1×10⁻⁶. 6 Cells were seeded in serum-free RPMI (Gibco) medium containing 1% Pen / Strep (Gibco) and incubated at 37°C for 1 hour at 5% CO2. The plated cells were washed with serum-free RPMI and treated with RPMI (ss) containing the spacer compound of SEQ 24 or SEQ ID 107 + 10% mixed human serum (ss), and incubated overnight at 37°C at 5% CO2. Each treatment was performed in duplicate. The plated cells were then treated with 10 ng / ml ultrapure LPS (Invitrogen) and incubated overnight at 37°C at 5% CO2. The spacer-treated cells were then centrifuged at 400 rpm for 5 min at room temperature, and RNA was isolated using a MagMAX RNA Total RNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's recommended protocol. Total RNA was quantified using a NanoDrop® (Thermo Fisher Scientific) manufacturer's recommended protocol. Total RNA was reverse transcriptase (RT) using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's recommended reagents and protocol to produce complementary DNA, which would serve as the substrate for quantitative real-time polymerase chain reaction (RT-qPCR). Target RNA levels were quantified by RT-qPCR using the CFX Real-Time PCR Detection System (Bio-Rad) according to the manufacturer's recommended protocol, utilizing the SsoAdvanced Universal SYBR Green assay. The target amount obtained by RT-qPCR was normalized using the expression of the stably expressed housekeeping gene RPL37A.

[0201] Table 11 shows the fold change in IL1B gene expression in LPS-treated monocytes (n=3 donors) in the presence of the interstitial compound (SEQ ID NO. 42). Data were normalized by the expression of the housekeeping gene RPL37A and are expressed as fold changes relative to LPS-treated monocytes with the control interstitial compound (SEQ ID NO. 107). An expression value < 1.0 indicates that transcription of the gene is repressed, and an expression value > 1.0 indicates that transcription of the gene is induced. For example, a value of 0.25 indicates that gene transcription is repressed by 75%.

[0202]

[0203] Table 11.

[0204] Example 4: AMANZI inhibition in PBMCs treated with LPS and R848

[0205] Peripheral blood mononuclear cells (PBMCs) from four healthy donors with a mean age of 56.3 ± 14.9 years were purchased cryopreserved from CTL Europe. The cryopreserved PBMCs were thawed in pre-warmed wash medium (RPMI (Dutch modified) + 20% FBS + 2 mM GlutaMAX + 1 mM sodium pyruvate + 1% Pen / Strep). The PBMCs were then washed three times in pre-warmed wash medium before centrifugation at 500 rpm for 10 min at room temperature. Centrifugation was repeated twice before resuspending the cells in pre-warmed medium (RPMI (Dutch modified) + 10% FBS + 2 mM GlutaMAX + 1 mM sodium pyruvate + 1% Pen / Strep). The cells were then counted at approximately 1 x 10⁻⁶ cells / cm². 5 Cells were seeded and added to each well of a 96-well U-bottom tissue culture plate (CellSTAR) containing a TLR mixture of 1 μg / mL ultrapure LPS (Invitrogen) and 5 μg / mL remiquimod (Invitrogen) (with or without the interstitial compound (SEQ ID 111)). At a low dose (5 μg / mL... M) or high dose (30) M) Add interstitial compounds. Each treatment was performed twice, and plated cells were incubated at 37°C for 48 hours in 5% CO2. The plated cells were then centrifuged at 400 rpm for 5 min at room temperature, and RNA was isolated using the MagMAX RNA Total RNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's recommended protocol. Total RNA was then quantified using the NanoDrop® (Thermo Fisher Scientific) manufacturer's recommended protocol. Total RNA was then reverse transcriptase (RT) to produce complementary DNA using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's recommended reagents and protocol. The complementary DNA was used as a substrate for quantitative real-time polymerase chain reaction (RT-qPCR) using SsoAdvanced Universal SYBR Green according to the manufacturer's recommended protocol. Target RNA levels were quantified by RT-qPCR using the CFX Real-Time PCR Detection System (Bio-Rad). The target amount obtained by RT-qPCR was normalized using the expression of the stably expressed housekeeping gene RPL37A.

[0206] Table 12 shows the fold change in IL1B gene expression in TLR-stimulated PBMCs in the presence of the interstitial compound (SEQ ID 111). Data were normalized by the expression of the housekeeping gene RPL37A and are expressed as fold changes relative to PBMCs treated with a TLR mixture without the interstitial compound. An expression value < 1.0 indicates that transcription of the gene is repressed, and an expression value > 1.0 indicates that transcription of the gene is induced. For example, a value of 0.25 indicates that gene transcription is repressed by 75%.

[0207]

[0208] Table 12

[0209] Example 5: Inhibition of IL-1β eRNA in the prostate adenocarcinoma cell line PC-3

[0210] PC-3 cells (ATCC) were spaced at 6 x 10⁶ cells per well. 4Cells were seeded in 24-well plates (Sarstedt) and RPMI 1640 containing GlutaMAX (Gibco), 10% FBS (Gibco), 1% Pen / Strep (Gibco), and the interstitial compound SEQ ID42 was added. The interstitial compound was added at a concentration of 200 nM. Each treatment was performed in duplicate. The plated cells were incubated at 37°C for 8 hours in 5% CO2. The interstitial-treated cells were then centrifuged at 400 rpm for 5 min at room temperature, and RNA was isolated using the MagMAX RNA miRVana Total RNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's recommended protocol. Total RNA was quantified using the NanoDrop® (Thermo Fisher Scientific) manufacturer's recommended protocol. Total RNA was reverse transcriptase (RT) using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's recommended reagents and protocol to produce complementary DNA for use as a substrate in quantitative real-time polymerase chain reaction (RT-qPCR). The target RNA level was quantified by RT-qPCR using the CFX real-time PCR detection system (Bio-Rad) and the SsoAdvanced Universal SYBR Green according to the manufacturer's recommended protocol. The target amount obtained by RT-qPCR was normalized using the expression of the stably expressed housekeeping gene RPL37A.

[0211] Table 13 shows the fold change in IL1B gene expression in PC-3 cells in the presence of interstitial compounds. Data were normalized by the expression of the housekeeping gene RPL37A and are expressed as fold changes relative to untreated PC-3 cells. An expression value < 1.0 indicates that transcription of the gene is repressed, and an expression value > 1.0 indicates that transcription of the gene is induced. For example, a value of 0.25 indicates that gene transcription is repressed by 75%.

[0212]

[0213] Table 13

[0214] Example 6: Inhibition of IL-1β eRNA in the TNF-α-treated pancreatic cancer cell line MIA PaCa2

[0215] MIA PaCa-2 cells (ATCC) were planted at a density of 1 × 10⁻⁶ cells per well. 6Cells were seeded in 24-well Sarstedt plates in RPMI 1640 + 10% FBS + 1% Pen / Strep and incubated at 37°C for 24 hours at 5% CO2. The plated cells were then treated with medium containing 10 ng / ml TNF and incubated at 37°C for 24 hours at 5% CO2. Cells were then treated with medium containing SEQ ID 111 or control SEQ ID 106 and incubated at 37°C for 24 hours at 5% CO2. Each treatment was performed in triplicate. Cells treated with interstitial bodies were then centrifuged at 400 rpm for 5 min at room temperature, and RNA was isolated using the MagMAX RNA Total RNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Total RNA was quantified using the NanoDrop® (Thermo Fisher Scientific) manufacturer's recommended protocol. Total RNA was reverse transcriptase (RT) to produce complementary DNA using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's recommended reagents and protocol. Complementary DNA was used as a substrate for quantitative real-time polymerase chain reaction (RT-qPCR). Target RNA levels were quantified by RT-qPCR using the CFX Real-Time PCR Detection System (Bio-Rad) according to the manufacturer's recommended protocol, utilizing Sso Advanced Universal SYBR Green. The target amount obtained by RT-qPCR was normalized using the expression of the stably expressed housekeeping gene RPL37A.

[0216] Table 14 shows the effects of TNF in the presence of the interstitial compound (SEQ ID NO 111). fold change in IL1B gene expression in treated MIAPaCa-2 cells. Data were normalized by the expression of the housekeeping gene RPL37A and expressed as a percentage relative to the control interstitial compound (SEQ ID NO. 106) via TNF. The fold change in treated cells is represented. An expression value < 1.0 means that the transcription of the gene is repressed, and an expression value > 1.0 means that the transcription of the gene is induced. For example, a value of 0.25 means that gene transcription is repressed by 75%.

[0217]

[0218] Table 14

[0219] This disclosure provides the following implementation methods: A1. A space-body type ASO (antisense oligonucleotide) capable of inhibiting the transcriptional activity of AMANZI, the space-body type ASO comprising: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a space region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the space body is substantially complementary to the 14-25 base region of AMANZI (SEQ ID NO. 1).

[0220] A2. The interstitial compound that specifically inhibits AMANZI according to Embodiment 1, wherein each interstitial nucleotide base in the entire interstitial body is linked by a phosphate thioester (P=S) nucleotide inter-bond; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotide, locked nucleic acid nucleotide (LNA) and combinations thereof.

[0221] A3. A space-body type ASO (antisense oligonucleotide) capable of inhibiting the transcriptional activity of AMANZI, the space-body type ASO comprising: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a space region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the space body is substantially complementary to region A (bases 10 to 93) of AMANZI.

[0222] A4. The spacer-type ASO capable of inhibiting AMANZI according to Embodiment 3, wherein the spacer region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 from any one of SEQ ID NO. 41-46 or an octamer fragment thereof.

[0223] A5. The spacer-type ASO capable of inhibiting AMANZI according to Embodiment 3, wherein each spacer nucleotide in the entire spacer is linked by a phosphate thioester (P=S) nucleotide inter-link; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotide, locked nucleic acid nucleotide (LNA) and combinations thereof.

[0224] A6. The interstitial ASO capable of suppressing AMANZI according to Embodiment 3, wherein region A of AMANZI is base 10 to base 93 of SEQ ID NO. 1.

[0225] A7. The spacer-type ASO capable of suppressing AMANZI according to Embodiment 3, wherein the spacer-type ASO is selected from the group consisting of SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46 and combinations thereof.

[0226] A8. The gap-shaped ASO capable of suppressing AMANZI according to Embodiment 7, wherein the gap-shaped ASO is SEQ ID NO. 42.

[0227] A9. A spacer-type ASO (antisense oligonucleotide) capable of inhibiting the transcriptional activity of AMANZI, wherein the spacer comprises: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to region B of AMANZI (SEQ ID NO. 1, bases 194 to 253).

[0228] A10. The spacer-type ASO capable of inhibiting AMANZI according to Embodiment 9, wherein the spacer region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 or an octamer fragment thereof from any one of SEQ ID NO. 78, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84 and combinations thereof.

[0229] A11. The spacer-type ASO capable of inhibiting AMANZI according to Embodiment 9, wherein each spacer nucleotide in the entire spacer is linked by a phosphate thioester (P=S) nucleotide inter-link; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotide, locked nucleic acid nucleotide (LNA) and combinations thereof.

[0230] A12. The interstitial ASO capable of suppressing AMANZI according to Embodiment 9, wherein region B of AMANZI is base 194 to base 253 of SEQ ID NO. 1.

[0231] A13. The spacer-type ASO capable of suppressing AMANZI according to Embodiment 9, wherein the spacer-type ASO is selected from the group consisting of SEQ ID NO. 78, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84 and combinations thereof.

[0232] A14. The gap-shaped ASO capable of suppressing AMANZI according to embodiment 13, wherein the gap-shaped ASO is SEQ ID NO. 84.

[0233] A15. A spacer-type ASO (antisense oligonucleotide) capable of inhibiting the transcriptional activity of AMANZI, wherein the spacer comprises: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to region C of AMANZI (SEQ ID NO. 1 bases 519 to 568).

[0234] A16. The spacer-type ASO capable of inhibiting AMANZI according to Embodiment 15, wherein the spacer region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 or an octamer fragment thereof from any one of SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104 and combinations thereof.

[0235] A17. The spacer-type ASO capable of inhibiting AMANZI according to embodiment 15, wherein each spacer nucleotide in the entire spacer is linked by a phosphate thioester (P=S) nucleotide inter-link; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotide, locked nucleic acid nucleotide (LNA) and combinations thereof.

[0236] A18. The interstitial ASO capable of suppressing AMANZI according to Embodiment 15, wherein region C of AMANZI is bases 519 to 568 of SEQ ID NO. 1.

[0237] A19. The gap-body type ASO capable of suppressing AMANZI according to embodiment 15, wherein the gap-body type ASO is selected from the group consisting of SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104 and combinations thereof.

[0238] A20. The gap-shaped ASO capable of suppressing AMANZI according to embodiment 15, wherein the gap-shaped ASO is SEQ ID NO. 101.

[0239] A21. A spacer-type ASO (antisense oligonucleotide) capable of inhibiting the transcriptional activity of AMANZI, wherein the spacer comprises: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to region D of AMANZI (SEQ ID NO. 1 bases 377 to 404).

[0240] A22. The spacer-type ASO capable of inhibiting AMANZI according to embodiment 21, wherein the spacer region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 or an octamer fragment thereof from any one of SEQ ID NO. 223, SEQ ID NO. 224, SEQ ID NO. 225, SEQ ID NO. 226, SEQ ID NO. 227, SEQ ID NO. 228, SEQ ID NO. 229, S and combinations thereof.

[0241] A23. The spacer-type ASO capable of inhibiting AMANZI according to embodiment 21, wherein each spacer nucleotide in the entire spacer is linked by a phosphate thioester (P=S) nucleotide inter-link; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotide, locked nucleic acid nucleotide (LNA) and combinations thereof.

[0242] A24. The interstitial ASO capable of suppressing AMANZI according to embodiment 21, wherein region D of AMANZI is bases 377 to 404 of SEQ ID NO. 1.

[0243] A25. The gap-body type ASO capable of suppressing AMANZI according to embodiment 21, wherein the gap-body type ASO is selected from the group consisting of SEQ ID NO. 223, SEQ ID NO. 224, SEQ ID NO. 225, SEQ ID NO. 226, SEQ ID NO. 227, SEQ ID NO. 228, SEQ ID NO. 229 and combinations thereof.

[0244] A26. The gap-shaped ASO capable of suppressing AMANZI according to embodiment 21, wherein the gap-shaped ASO is SEQ ID NO. 226.

[0245] A27. A spacer-type ASO (antisense oligonucleotide) capable of inhibiting the transcriptional activity of AMANZI, wherein the spacer comprises: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to region E of AMANZI (SEQ ID NO. 1 bases 574 to 615).

[0246] A28. The spacer-type ASO capable of inhibiting AMANZI according to embodiment 27, wherein the spacer region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 or an octamer fragment thereof from any one of SEQ ID NO. 201, SEQ ID NO. 202, SEQ ID NO. 203, SEQ ID NO. 204, SEQ ID NO. 205, SEQ ID NO. 206, SEQ ID NO. 207, SEQ ID NO. 208 and combinations thereof.

[0247] A29. The spacer-type ASO capable of inhibiting AMANZI according to embodiment 27, wherein each spacer nucleotide in the entire spacer is linked by a phosphate thioester (P=S) nucleotide inter-link; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotide, locked nucleic acid nucleotide (LNA) and combinations thereof.

[0248] A30. The interstitial ASO capable of suppressing AMANZI according to embodiment 27, wherein region E of AMANZI is bases 574 to 615 of SEQ ID NO. 1.

[0249] A31. The gap-body type ASO capable of suppressing AMANZI according to embodiment 27, wherein the gap-body type ASO is selected from the group consisting of SEQ ID NO. 201, SEQ ID NO. 202, SEQ ID NO. 203, SEQ ID NO. 204, SEQ ID NO. 205, SEQ ID NO. 206, SEQ ID NO. 207, SEQ ID NO. 208 and combinations thereof.

[0250] A32. The gap-shaped ASO capable of suppressing AMANZI according to embodiment 27, wherein the gap-shaped ASO is SEQ ID NO. 207.

[0251] All patents, publications, scientific articles, websites, and other documents and materials cited or referenced herein indicate the skill level of a person skilled in the art to which this invention pertains, and each such cited document and material is incorporated herein by reference to the extent that it has been separately incorporated herein by reference in its entirety or is set forth herein in its entirety. The applicant reserves the right to physically incorporate any and all materials and information from any such patents, publications, scientific articles, websites, electronically available information, and other cited materials or documents into this specification. References to any application, patent, and publication in this specification should not be construed as, nor should they be interpreted as, an admission or implication of any kind that they constitute valid prior art or are part of common general knowledge in any country of the world.

[0252] The specific methods and compositions described herein represent preferred embodiments and are exemplary, and are not intended to limit the scope of the invention. Other objects, aspects, and embodiments will emerge when considered by those skilled in the art and are included within the spirit of the invention as defined by the claims. Those skilled in the art will readily recognize that various substitutions and modifications can be made to the invention disclosed herein without departing from its scope and spirit. The invention exemplarily described herein can be suitably practiced without any one or more elements or limitations not specifically disclosed herein as necessary. Thus, for example, in each instance herein, in embodiments or examples of the invention, any one of the terms “comprising,” “substantially consisting of,” and “consisting of” can be replaced in the specification with any of the other two terms. Furthermore, the terms “comprising,” “including,” “containing,” etc., should be interpreted broadly and without limitation. The methods and processes exemplarily described herein can be suitably implemented in different sequences of steps, and they are not necessarily limited to the sequence of steps shown herein or in the claims. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Under no circumstances should a patent be construed as limited to the specific examples, embodiments, or methods disclosed herein. Under no circumstances should a patent be construed as being limited by any statement made by any examiner or any other person or employee of the Patent and Trademark Office, unless the applicant expressly and unreservedly adopts that statement in writing. Furthermore, titles, headings, etc., are provided to enhance the reader's understanding of this document and should not be construed as limiting the scope of the invention. Examples of any aspect, implementation, or component of the invention mentioned herein should be considered non-limiting.

[0253] The terms and expressions used herein are for descriptive purposes and not for limitation, and their use is not intended to exclude any equivalents of the features shown and described or portions thereof. However, it should be recognized that various modifications are possible within the scope of the claimed invention. Therefore, it should be understood that although the invention has been specifically disclosed through preferred embodiments and optional features, modifications and variations of the concepts disclosed herein can be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

[0254] This invention has been described broadly and generally herein. Each narrower group of species and subgenus falling within the general scope of disclosure also constitutes part of this invention. This includes the genus description of the invention, but with a prerequisite or negative limitation that excludes any subject matter from that genus, whether or not the excluded material is expressly mentioned herein.

[0255] Other embodiments are within the scope of the appended claims. Furthermore, when features or aspects of the invention are described using the Markush group, those skilled in the art will recognize that the invention is therefore also described using any single member or subgroup of the Markush group.

Claims

1. A spacer-type ASO (antisense oligonucleotide) capable of inhibiting the transcriptional activity of AMANZI, comprising: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to the 14-25 base region of AMANZI (SEQ ID NO. 1).

2. The interstitial compound that specifically inhibits AMANZI according to claim 1, wherein each interstitial nucleotide base in the entire interstitial body is linked by a phosphate thioester (P=S) nucleotide inter-bond; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNAs), and combinations thereof.

3. A space body type ASO (antisense oligonucleotide) capable of inhibiting AMANZI transcriptional activity, comprising: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a space body having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the space body is substantially complementary to region A (bases 12 to 92 of SEQ ID NO. 1) of AMANZI.

4. The spacer-type ASO capable of inhibiting AMANZI according to claim 3, wherein the spacer region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 from any one of SEQ ID NO. 2-105 or an octamer fragment thereof.

5. The spacer-type ASO capable of inhibiting AMANZI according to claim 3, wherein each spacer nucleotide in the entire spacer is linked by a phosphate thioester (P=S) nucleotide inter-link; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNAs), and combinations thereof.

6. The interstitial ASO capable of suppressing AMANZI according to claim 3, wherein region A of AMANZI is base 12 to base 27 of SEQ ID NO.

1.

7. The gap-body type ASO capable of suppressing AMANZI according to claim 3, wherein the gap-body type ASO is selected from the group consisting of SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46 and combinations thereof.

8. The gap-shaped ASO capable of suppressing AMANZI according to claim 7, wherein the gap-shaped ASO is SEQ ID NO.

42.

9. The spacer-type ASO capable of inhibiting AMANZI according to claim 3, wherein (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to region B of AMANZI (SEQ ID NO. 1 bases 519 to 568).

10. The spacer-type ASO capable of inhibiting AMANZI according to claim 9, wherein the spacer region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 or an octamer fragment thereof from any one of SEQ ID NO. 63, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104 and combinations thereof.

11. The spacer-type ASO capable of inhibiting AMANZI according to claim 9, wherein each spacer nucleotide in the entire spacer is linked by a phosphate thioester (P=S) nucleotide inter-link; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNAs), and combinations thereof.

12. The gap-body type ASO capable of suppressing AMANZI according to claim 9, wherein the gap-body type ASO is selected from the group consisting of SEQ ID NO. 63, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104 and combinations thereof.

13. The gap-body type ASO capable of suppressing AMANZI according to claim 12, wherein the gap-body type ASO is SEQ ID NO.

101.

14. A spacer-type ASO (antisense oligonucleotide) capable of inhibiting AMANZI transcriptional activity, wherein the spacer comprises: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to region D of AMANZI (SEQ ID NO. 1 bases 377 to 404).

15. The spacer-type ASO capable of inhibiting AMANZI according to claim 14, wherein the spacer region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 or an octamer fragment thereof from any one of SEQ ID NO. 228-234 and combinations thereof.

16. The spacer-type ASO capable of inhibiting AMANZI according to claim 14, wherein each spacer nucleotide in the entire spacer is linked by a phosphate thioester (P=S) nucleotide inter-link; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNAs), and combinations thereof.

17. A spacer-type ASO (antisense oligonucleotide) capable of inhibiting AMANZI transcriptional activity, wherein the spacer comprises: (a) about 14 to about 25 nucleotide bases; (b) a 3' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; (c) a spacer region having at least 8 to no more than 19 DNA bases; and (d) a 5' wing region (3' to 5') having 3 to 7 chemically modified RNA bases; wherein the spacer is substantially complementary to region E of AMANZI (SEQ ID NO. 1 bases 574 to 615).

18. The spacer-type ASO capable of inhibiting AMANZI according to claim 17, wherein the spacer region comprises a 10-nucleotide DNA sequence of nucleotide 5 to nucleotide 15 or an octamer fragment thereof from any one of SEQ ID NO. 206-215 and combinations thereof.

19. The spacer-type ASO capable of inhibiting AMANZI according to claim 17, wherein each spacer nucleotide in the entire spacer is linked by a phosphate thioester (P=S) nucleotide inter-link; and wherein the modified nucleotide base modifier is selected from the group consisting of 2'-methoxyethyl (MOE) nucleotides, locked nucleic acid nucleotides (LNAs), and combinations thereof.

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