Connector-modified synthetic RIG-I agonist and method of use thereof

Abstract

The present invention provides a composition comprising a nucleic acid compound capable of inducing interferon production, wherein the nucleic acid compound comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are complementary to each other and hybridize to form double-stranded portions of 8 base pairs or more and less than 20 base pairs; wherein the two nucleic acid molecules are connected via connector elements that bind to nucleotides of the first nucleic acid sequence and nucleotides of the second nucleic acid sequence.

Description

Technical Field

[0001] (Related Applications) This application claims the benefit of U.S. Provisional Application No. 63 / 468,612, filed May 24, 2023. The entire disclosure of the above application is incorporated herein by reference.

Background Art

[0002] (Background of the Present Disclosure) RIG-I (retinoic acid-inducible gene I) is a cytoplasmic pattern recognition receptor (PRR) responsible for the type I interferon (IFN1) response. IFN1 has three main functions: restricting the spread of viruses to nearby cells, promoting innate immune responses including inflammatory responses, and assisting in the activation of the adaptive immune system.

[0003] RIG-I plays an important role in the innate immune system response to infections by foreign organisms such as bacteria or viruses. Exogenous nucleic acids introduced into cells, particularly viral nucleic acids, induce an innate immune response, resulting in, among other things, interferon (IFN) production and cell death. Upon sensing viral RNA, RIG-I-like receptors induce the secretion of type I interferons (IFNs), leading to upregulation of antiviral IFN-induced proteins in infected and neighboring cells, thereby inhibiting viral replication. Further downstream events attract immune cells and initiate an adaptive immune response. Additionally, RIG-I ligands have been reported to induce apoptosis in many different types of tumor cells, but not in normal cells.

[0004] There continues to be a need for further improved compositions and methods for modulating the activity of immunomodulatory proteins. Such agents can be used for cancer immunotherapy and for the prevention and treatment of other conditions such as infectious diseases. Such agents can also be used as adjuvants to enhance the immunogenicity of vaccines against infectious diseases and cancer. Therefore, there is a need to develop improved RIG-I-like receptor ligands for a variety of therapeutic immunomodulatory applications. SUMMARY OF THE INVENTION

[0005] (SUMMARY OF THE DISCLOSURE) The present invention provides a nucleic acid compound capable of inducing interferon production. In one aspect, the nucleic acid compound comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are complementary to each other and hybridize to form a double-stranded portion, wherein the number of base pairs of the double-stranded portion is an integer in the range of 8 to 19; and wherein the 3' end of the first nucleic acid sequence is linked to one end of a linker element, and wherein the other end of the linker element is linked to the 5' end of the second nucleic acid sequence, wherein the element is as defined herein.

[0006] In an embodiment, the RIG-I agonist has the structure of formula I

CHEMICAL

[0007] (Brief explanation of the drawing) Several embodiments of the present invention are described herein, with reference to the accompanying drawings, as merely examples. It is emphasized that the details shown, with specific detail reference to the drawings, are illustrative and intended for explanatory purposes regarding embodiments of the present invention. In this regard, the description accompanied by the drawings will make it clear to those skilled in the art how embodiments of the present invention may be carried out. In the drawing: [Figure 1]Figure 1 shows HPLC and mass spectrometry data of one embodiment of the biotin conjugate according to the present invention. [Figure 2] Figure 2 shows HPLC and mass spectrometry data for one embodiment of the dye-conjugate according to the present invention. [Figure 3] Figure 3 shows the stimulation of RIG-I by the compound according to the present invention. [Figure 4] Figure 4 provides an example illustrating phosphorylation mimics. [Figure 5] Figure 5 shows RIG-I activation in A549 cells. [Figure 6] Figure 6 shows the induction of the chemokine CXCL10. [Modes for carrying out the invention]

[0008] (Detailed explanation) This disclosure provides nucleic acid compounds that can specifically bind to retinoic acid-inducible gene 1 receptor (RIG-I) and activate the interferon response of RIG-I. In one embodiment, this disclosure provides a synthetic RNA molecule that agonizes or activates one or more RIG-Is. In one embodiment, this disclosure provides compositions and methods for inducing the interferon response of RIG-I. In one embodiment, this disclosure provides nucleic acid compounds. Exemplary nucleic acids for use in this disclosure include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), or hybrids thereof. In one embodiment, the nucleic acid is ribonucleic acid (RNA).

[0009] As used herein, the terms “nucleic acid compound” and “polynucleotide molecule” may be used interchangeably to refer to the compounds of formula I or formula II described herein.

[0010] The present invention provides nucleic acid compounds (i.e., RIG-I agonists) capable of inducing interferon production. In one embodiment, the nucleic acid compound comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein the first and second nucleic acid sequences are complementary to each other and hybridize to form a double-stranded portion, where the number of base pairs in the double-stranded portion is an integer in the range of 8 to 19; wherein the 3' end of the first nucleotide sequence is conjugated to one end of a connector element, where the other end of the connector element is ligated to the 5' end of the second nucleotide sequence, where the connector element is as defined herein; wherein the furthest 5' nucleotide of the first nucleic acid sequence comprises a 5' diphosphate or tripphosphate portion, or derivatives or analogs thereof. In the embodiment, the nucleotides of the first and second nucleotide sequences are ribonucleic acid (RNA). The double strands do not need to be perfectly complementary by Watson-Crick base pairing.

[0011] As used herein, the terms “first nucleic acid sequence,” “first nucleic acid molecule,” and “first nucleotide sequence” are interchangeable, and the terms “second nucleic acid sequence,” “first nucleic acid molecule,” and “second nucleotide sequence” are interchangeable.

[0012] In this embodiment, the RIG-I agonist has the structure of formula I. [ka] (In the formula, 5'-P z -(N) b N-3' represents the first nucleic acid sequence; 5'-N(N) b' -3' represents the second nucleic acid sequence; In each case, P is independently a phosphate or an analogue thereof; z is either 2 or 3; N is, in each case, any nucleotide or modified nucleotide or an analog or derivative thereof; b and b' are, independently, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; 5'-(E) y (E)-L-(E)(E) y' -3' represents a linker element, where E is, in each occurrence, independently, any nucleotide, modified nucleotide, or abasic; y and y' are, independently, 0 to 9, provided that y + y' is equal to 0 to 8; L is the structure

Chemical formula

[0013] The sequence of the first nucleic acid molecule 5'-(N) b N-�' is the sequence of the second nucleic acid molecule 3'-(N) b'Complementary to N-5', the first and second nucleic acid molecules hybridize to form a double-stranded segment. In each case, N is any nucleotide or modified nucleotide or its analogue or derivative; and b and b' are independently nucleotides of length 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. In one embodiment, b and b' are independently nucleotides of length 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. In another embodiment, b and b' are independently nucleotides of length 9, 10, 11, 12, 13, 14, or 15. In yet another embodiment, b and b' are independently nucleotides of length 10, 11, 12, or 13.

[0014] In one embodiment, b has a length of 9 nucleotides, and b' has a length of less than, equal to, or greater than 9 nucleotides. In one embodiment, b has a length of 10 nucleotides, and b' has a length of less than, equal to, or greater than 10 nucleotides. In one embodiment, b has a length of 11 nucleotides, and b' has a length of less than, equal to, or greater than 11 nucleotides. In one embodiment, b has a length of 12 nucleotides, and b' has a length of less than, equal to, or greater than 12 nucleotides. In one embodiment, b has a length of 13 nucleotides, and b' has a length of less than, equal to, or greater than 13 nucleotides. In one embodiment, b has a length of 14 nucleotides, and b' has a length of less than, equal to, or greater than 14 nucleotides. In one embodiment, b has a length of 15 nucleotides, and b' has a length less than, equal to, or greater than 15 nucleotides. In another embodiment, b has a length of 16 nucleotides, and b' has a length less than, equal to, or greater than 16 nucleotides. In yet another embodiment, b has a length of 17 nucleotides, and b' has a length less than, equal to, or greater than 17 nucleotides. In yet another embodiment, b has a length of 18 nucleotides, and b' has a length less than, equal to, or greater than 18 nucleotides.

[0015] In this embodiment, b=b'. In this embodiment, the nucleic acid compound includes a blunt end. A blunt end refers to an RNA double helix lacking any protrusions, such as a 3'-dinucleotide protrusion, such that at least one end of the double helix is ​​flat or blunt, as referred to herein, with both the 5' and 3' ends being flat together. The molecule of the present invention may have at least one blunt end. The molecule of the present invention may have two blunt ends.

[0016] In this embodiment, b is not equal to b'. In this embodiment, the nucleic acid compound includes protrusions. As used herein, the term “protrusion” refers to a terminal nonbase-pairing nucleotide resulting from one chain or region that extends beyond the end of the complementary chain forming the double helix. One or more polynucleotides that can form a double helix via hydrogen bonds may have protrusions.

[0017] In one embodiment, when b is smaller than b', the nucleic acid compound has a protrusion at the 3' end of the second nucleic acid molecule. The single-stranded region extending beyond the 3' end of the double helix is ​​called a protrusion. In one embodiment, the 3'-protrusion contains one non-base-pairing nucleotide. In another embodiment, the 3'-protrusion contains two non-base-pairing nucleotides.

[0018] In one embodiment, when b is greater than b', the nucleic acid molecule has a 5'-overhang. In another embodiment, the dsRNA structure gives rise to a 5'-overhang. In yet another embodiment, the 5'-overhang contains a non-base-pairing nucleotide. In yet another embodiment, the 5'-overhang contains two non-base-pairing nucleotides.

[0019] In one embodiment, the double-stranded portion contains one or more mispairing bases. That is, Watson-Crick base pairing is not required for every nucleotide pair.

[0020] In this embodiment, the nucleic acid compound includes a nucleotide insertion that creates a twist in the double-stranded region (see, for example, US Patent No. 20210000856, incorporated herein by reference).

[0021] In this embodiment, the nucleic acid compound may also contain an internal bulge structure in the first nucleic acid sequence, the second nucleic acid sequence, or both.

[0022] As described herein, the nucleic acid compounds of this disclosure are independent of any specific nucleotide sequence. Rather, any nucleotide sequence can be used, provided that the sequence has the ability to form the structures of the nucleic acid compounds described herein.

[0023] Preferably, the first nucleic acid sequence and / or the second nuclear sequence are not antisense oligonucleotides and do not possess antisense activity; that is, the first nucleic acid sequence and / or the second nuclear sequence are not complementary to the (selected) target nucleic acid sequence such that, when introduced into an animal or cell, the first nucleic acid sequence and / or the second nuclear sequence do not bind to RNA, resulting in a decrease in its translation. Preferably, the first nucleic acid sequence and the second nuclear sequence do not possess antisense activity.

[0024] In some embodiments, the disclosure provides nucleic acid compounds wherein the nucleotide sequence comprising the compound is not complementary to a genomic DNA sequence or mRNA sequence, wherein the nucleic acid compound does not participate in RNA interference, and wherein the nucleic acid compound does not silence gene expression.

[0025] In one embodiment, the nuclease resistance of nucleic acid compounds can be enhanced by skeletal modifications (e.g., phosphorothioates), sugar modifications, and 5'-terminal and / or 3'-terminal modifications.

[0026] In one embodiment, the present invention provides a polynucleotide molecule having the structure of formula II. [ka] (In the formula, 5'-P z -Nu-3' represents the first nucleic acid sequence; 5'-Nu'-3' represents the second nucleic acid sequence; In each case, P is independently a phosphate or an analogue thereof. z is 0, 1, 2, or 3; 5'-(E) y (E)-L-(E)(E)y' -3' represents a connector element, where, E is, independently, any nucleotide, modified nucleotide, or debase at each instance; y and y' are independently between 0 and 9, where y + y' is equal to between 0 and 8; L is structure [ka] (Here, X and X' are independently O or S; Y and Y' are independently OR'', SR'', or NRR'; V and V' are independently O, S, or NRR'; q is between 1 and 20; k is between 1 and 20; t is between 1 and 20; M is selected from aliphatic, substituted aliphatic, aryl, substituted aryl, heteroalkyl, heterocyclyl, or substituted heterocyclyl; W is any reactive or conjugation group; and (d is either 0 or 1) (It is a non-nucleotide segment that has [a certain characteristic].)

[0027] In embodiments of Formula II, Nu and Nu' are the sense and antisense strands of the siRNA molecule. In embodiments, Nu is the sense strand of the siRNA molecule, and Nu' is the antisense strand of the siRNA molecule. In embodiments, Nu is the antisense strand of the siRNA molecule, and Nu' is the sense strand of the siRNA molecule.

[0028] In embodiments of Formula II, Nu is an antisense oligonucleotide, and Nu' is a nucleic acid sequence that is at least 80% complementary to the antisense oligonucleotide of Nu. In embodiments, the nucleic acid sequence of Nu' is at least 90%, at least 93%, at least 95%, at least 975%, at least 99%, or at least 100% complementary to the antisense oligonucleotide of Nu. In embodiments, the number of nucleotides in the nucleic acid sequence of Nu' is the same as, less than, or more than the number of nucleotides in the antisense oligonucleotide of Nu.

[0029] In embodiments of Formula II, Nu' is an antisense oligonucleotide, and Nu is a nucleic acid sequence that is at least 80% complementary to the antisense oligonucleotide of Nu'. In embodiments, the nucleic acid sequence of Nu is at least 90%, at least 93%, at least 95%, at least 975%, at least 99%, or at least 100% complementary to the antisense oligonucleotide of Nu'. In embodiments, the number of nucleotides in the nucleic acid sequence of Nu is the same as, less than, or more than the number of nucleotides in the antisense oligonucleotide of Nu'.

[0030] (Connector element) A connector element is a divalent linker that connects a first nucleic acid sequence to a second nucleic acid sequence. The connector element comprises a 5' nucleotide portion having 1 to 9 nucleotides, a non-nucleotide segment, and a 3' nucleotide portion having 1 to 9 nucleotides, wherein the non-nucleotide segment has structure L as defined herein, and the total number of nucleotides in the 5' nucleotide portion + 3' nucleotide portion is 2 to 10 nucleotides. In embodiments, the nucleotides of the element are ribonucleic acid (RNA).

[0031] In this embodiment, the connector element is structure 5'-(E) y (E)-L-(E)(E) y' -3' (Here, E is, independently, any nucleotide, modified nucleotide, or debase at each instance; y and y' are independently between 0 and 7, where y+y' is equal to 0 and 8; L is structure [ka] (Here, X and X' are independently O or S; Y and Y' are independently OR'', SR'', or NRR'; V and V' are independently O, S, or NRR'; q is between 1 and 20; k is between 1 and 20; t is between 1 and 20; M is selected from aliphatic, substituted aliphatic, aryl, substituted aryl, heterocyclyl, or substituted heterocyclyl; W is any reactive or conjugation group; and (d is either 0 or 1) : has.

[0032] As discussed in further detail below, the targeting molecule (Tm) may be further bound to W, Y, or Y'. Preferably, Tm is bound to W.

[0033] In this embodiment, q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In this embodiment, q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In this embodiment, q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In this embodiment, q is 1, 2, 3, 4, or 5. In this embodiment, q is 1. In this embodiment, q is 2. In this embodiment, q is 3. In this embodiment, q is 4. In this embodiment, q is 5.

[0034] In one embodiment, k and t are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In another embodiment, k and t are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In yet another embodiment, k and t are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In yet another embodiment, k and t are independently 1, 2, 3, 4, or 5.

[0035] In one embodiment, k is 1. In one embodiment, k is 2. In one embodiment, k is 3. In one embodiment, k is 4. In one embodiment, k is 5.

[0036] In this embodiment, t is 1. In this embodiment, t is 2. In this embodiment, t is 3. In this embodiment, t is 4. In this embodiment, t is 5.

[0037] In this embodiment, k and t are the same. In this embodiment, L is symmetric.

[0038] In this embodiment, k and t are different. In this embodiment, L is asymmetric.

[0039] In this embodiment, d is 0. In this embodiment, d is 1.

[0040] In the embodiment, R, R', and R'' are independently selected from the group consisting of alkyl, aminoalkyl, carboxamide, polyethylene glycol (PEG), aralkyl, heteroaralkyl, heteroalkyl, substituted or unsubstituted cycloalkyl. In the embodiment, the R and R'' groups may contain functional groups such as amino, hydroxy, azide, or thiol, which can optionally be used in the bond that can be used to link to a targeting molecule (Tm) as described herein.

[0041] In one embodiment, the R and R'' groups may be peptide groups. These peptide groups may include a variety of enzymatically cleavable or non-cleavable peptides. The individual amino acid groups of the peptide may be natural or synthetic amino acids.

[0042] In this embodiment, R and R'' are -CH2-O-CO-R 1 (Here, R 1 =Me, isopropyl, t-butyl, -(CH2)nR 2 (Here, R 2 R'' can be selected from aryl, aralkyl, heteroaryl, heteroaralkyl, alkyl, aminoalkyl, carboxamide, polyethylene glycol (PEG), heteroalkyl, substituted or unsubstituted cycloalkyl. Preferred examples of R and R'' are, but are not limited to, those listed below.

[0043] In one embodiment, the reactive group W may be further linked to an alkyl, aminoalkyl, carboxamide, polyethylene glycol (PEG), aralkyl, heteroaralkyl, heteroalkyl, substituted or unsubstituted cycloalkyl group. In the embodiment, the R and R'' groups may contain functional groups such as amino, hydroxy, azide, or thiol, which can be optionally used for binding to the target molecule (Tm).

[0044] In embodiments, M is selected from aliphatic, substituted aliphatic, aryl, substituted aryl, heteroalkyl, heterocyclyl, or substituted heterocyclyl. The terms “aliphatic group” or “aliphatic” refer to a non-aromatic moiety that may contain substituted (e.g., single bonds) or one or more unsaturated units, such as double and / or triple bonds. Aliphatic groups may be linear, branched, or cyclic, may contain carbon, hydrogen, or optionally one or more heteroatoms, and may be saturated or unsaturated. In addition to aliphatic hydrocarbon groups, aliphatic groups include, for example, polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and polyimines. Such aliphatic groups may be further substituted. It is understood that aliphatic groups may include alkyls, substituted alkyls, alkenyls, substituted alkenyls, alkynyls, substituted alkynyls, and substituted or unsubstituted cycloalkyl groups as described herein.

[0045] The term "acyl" refers to carbonyls substituted with hydrogen, alkyl, partially or fully saturated cycloalkyl, partially or fully saturated heterocyclic, aryl, or heteroaryl. For example, acyls include (C1-C6) alkanoyls (e.g., formyl, acetyl, propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.), (C3-C6) cycloalkylcarbonyls (e.g., cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.), and heterocyclic carbonyls (e.g., pyrrolidinyl carbonyl, pyrrolid-2-one-5-carbonyl). Examples of groups include yl, piperidinyl carbonyl, piperadinyl carbonyl, tetrahydrofuranyl carbonyl, etc., aroyl (e.g., benzoyl), and heteroaloyl (e.g., thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl, furanyl-3-carbonyl, 1H-pyrrolyl-2-carbonyl, 1H-pyrrolyl-3-carbonyl, benzo[b]thiophenyl-2-carbonyl, etc.). Furthermore, the alkyl, cycloalkyl, heterocyclic, aryl, and heteroaryl portions of the acyl group may be any one of the groups described in their respective definitions. Where indicated as “optionally substituted”, the acyl group may be unsubstituted, or may be optionally substituted with one or more substituents (usually 1 to 3 substituents) independently selected from the substituent groups listed below in the definition of “substituted”, or the alkyl, cycloalkyl, heterocyclic, aryl, and heteroaryl portions of the acyl group may be substituted as described above in the preferred and more preferred lists of substituents.

[0046] The term "alkyl" is intended to include both branched and linear substituted or unsubstituted saturated aliphatic hydrocarbon radicals / groups having a specified number of carbon atoms. Preferred alkyl groups have about 1 to about 24 carbon atoms ("C1- 24This includes ''). Other preferred alkyl groups include about 1 to about 8 carbon atoms ("C1-C8"), for example, about 1 to about 6 carbon atoms ("C1-C6"), or for example, about 1 to about 3 carbon atoms ("C1-C3"). Examples of C1-C6 alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl, and n-hexyl radicals.

[0047] The term "alkenyl" refers to a linear or branched radical having at least one carbon-carbon double bond. Such radicals preferably have about 2 to about 24 carbon atoms ("C2-C"). 24 It contains ''). Other preferred alkenyl radicals have 2 to about 10 carbon atoms (''C2-C 10 "Lower alkenyl" radicals having the 'C2', for example, ethenyl, allyl, propenyl, butenyl, and 4-methylbutenyl. Preferred lower alkenyl radicals contain 2 to about 6 carbon atoms ("C2-C6"). The terms "alkenyl" and "lower alkenyl" encompass radicals having "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations.

[0048] The term "alkynyl" refers to a linear or branched radical having at least one carbon-carbon triple bond. Such radicals preferably have about 2 to about 24 carbon atoms ("C2-C"). 24 It contains ''). Other preferred alkynyl radicals are “lower alkynyl” radicals having 2 to about 10 carbon atoms, such as propargyl, 1-propynyl, 2-propynyl, 1-butyne, 2-butynyl, and 1-pentynyl. Preferred lower alkynyl radicals contain 2 to about 6 carbon atoms ("C2-C6").

[0049] The term "cycloalkyl" refers to a group of 3 to approximately 12 carbon atoms ("C3- 12The term "cycloalkyl" refers to a saturated carbocyclic radical having 3 to approximately 12 carbon atoms. Examples of such radicals include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0050] The term "cycloalkenyl" refers to a partially unsaturated carbocyclic radical having 3 to 12 carbon atoms. Cycloalkenyl radicals that are partially unsaturated carbocyclic radicals containing two double bonds (which may or may not be conjugated) are called "cycloalkyldienyl" radicals. More preferred cycloalkenyl radicals are "lower cycloalkenyl" radicals having 4 to about 8 carbon atoms. Examples of such radicals include cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0051] As used herein, the term "alkylene" refers to a divalent group derived from a linear or branched saturated hydrocarbon chain having a specified number of carbon atoms. Examples of alkylene groups include, but are not limited to, ethylene, propylene, butylene, 3-methylpentylene, and 5-ethylhexylene.

[0052] As used herein, the term "alkenylene" refers to a divalent group derived from a linear or branched hydrocarbon moiety containing a specified number of carbon atoms having at least one carbon-carbon double bond. Examples of alkenylene groups include, but are not limited to, ethenylene, 2-propenylene, 2-butenylene, and 1-methyl-2-butene-1-ilene.

[0053] As used herein, the term "alkynylene" refers to a divalent group derived from a linear or branched hydrocarbon moiety containing a specified number of carbon atoms having at least one carbon-carbon triple bond. Typical alkynylene groups include, but are not limited to, propynylene, 1-butynylene, and 2-methyl-3-hexynylene.

[0054] The term "alkoxy" refers to a linear or branched oxy-containing radical having an alkyl moiety of 1 to about 24 carbon atoms, or preferably 1 to about 12 carbon atoms. More preferred alkoxy radicals are "lower alkoxy" radicals having 1 to about 10 carbon atoms, and more preferably 1 to about 8 carbon atoms. Examples of such radicals include methoxy, ethoxy, propoxy, butoxy, and tert-butoxy.

[0055] The term "alkoxyalkyl" refers to an alkyl radical having one or more alkoxy radicals bonded to an alkyl radical, i.e., to form monoalkoxyalkyl and diaryxyalkyl radicals.

[0056] The term “aryl,” alone or in combination, refers to an aromatic system containing one, two, or three rings (where such rings may be bonded together in a drooping manner or condensed). The term “aryl” encompasses aromatic radicals such as phenyl, naphthyl, tetrahydronaphthyl, indanfuranyl, quinazolinyl, pyridyl, and biphenyl.

[0057] The terms "heterocyclyl," "heterocycle," "heterocyclic," or "heterocyclo" refer to saturated, partially unsaturated, and unsaturated heteroatom-containing cyclic radicals, which can be correspondingly called "heterocyclyl," "heterocycloalkenyl," and "heteroaryl," respectively, where the heteroatom can be selected from nitrogen, sulfur, and oxygen. Examples of saturated heterocyclyl radicals include saturated 3-6 membered heteromonocyclic groups containing 1-4 nitrogen atoms (e.g., pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl); saturated 3-6 membered heteromonocyclic groups containing 1-2 oxygen atoms and 1-3 nitrogen atoms (e.g., morpholinyl); and saturated 3-6 membered heteromonocyclic groups containing 1-2 sulfur atoms and 1-3 nitrogen atoms (e.g., thiazolidinyl). Examples of partially unsaturated heterocyclyl radicals include dihydrothiophene, dihydropyran, dihydrofuran, and dihydrothiazole. Heterocyclyl radicals, for example, may contain pentavalent nitrogen in tetrazolium and pyridinium radicals. The term "heterocyclic" also includes radicals in which the heterocyclyl radical is condensed with an aryl or cycloalkyl radical. Examples of such condensed bicyclic radicals include benzofuran and benzothiophene.

[0058] The term "heteroaryl" refers to unsaturated aromatic heterocyclyl radicals. Examples of heteroaryl radicals include unsaturated 3-6 membered heteromonocyclic groups containing 1-4 nitrogen atoms, such as pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridadinyl, triazolyl (e.g., 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, etc.), and tetrazolyl (e.g., 1H-tetrazolyl, 2H-tetrazolyl, etc.); Unsaturated condensed heterocyclyl groups containing 1 to 5 nitrogen atoms, e.g., indolyl, isoindolyl, indolidinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl (e.g., tetrazolo[1,5-b]pyridazinyl); unsaturated 3 to 6-membered heteromonocyclic groups containing an oxygen atom, e.g., pyranyl, furyl; unsaturated 3 to 6-membered heteromonocyclic groups containing a sulfur atom, e.g., thienyl; unsaturated 3 to 6-membered heteromonocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, e.g., oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl); Examples include saturated condensed heterocyclyl groups containing 1-2 oxygen atoms and 1-3 nitrogen atoms (e.g., benzoxazolyl, benzoxadiazolyl, etc.); unsaturated 3-6 membered heteromonocyclic groups containing 1-2 sulfur atoms and 1-3 nitrogen atoms, such as thiazolyl, thiadiazolyl (e.g., 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.); and unsaturated condensed heterocyclyl groups containing 1-2 sulfur atoms and 1-3 nitrogen atoms (e.g., benzothiazolyl, benzothiadiazolyl, etc.).

[0059] The term "heterocycloalkyl" refers to heterocyclosubstituted alkyl radicals. More preferred heterocycloalkyl radicals are "lower heterocycloalkyl" radicals, which have 1 to 6 carbon atoms in the heterocyclo radical.

[0060] The term "alkylthio" refers to a radical containing a linear or branched alkyl radical with 1 to about 10 carbon atoms bonded to a divalent sulfur atom. Preferred alkylthio radicals have an alkyl radical with 1 to about 24 carbon atoms, or preferably 1 to about 12 carbon atoms. More preferred alkylthio radicals have an alkyl radical that is a "lower alkylthio" radical with 1 to about 10 carbon atoms. Most preferred are alkylthio radicals having a lower alkyl radical with 1 to about 8 carbon atoms. Examples of such lower alkylthio radicals include methylthio, ethylthio, propylthio, butylthio, and hexylthio.

[0061] The terms "aralkyl" or "arylalkyl" refer to aryl-substituted alkyl radicals such as benzyl, diphenylmethyl, triphenylmethyl, phenylethyl, and diphenylethyl.

[0062] The term "aryloxy" refers to an aryl radical that is bonded to another radical via an oxygen atom.

[0063] The terms "aralkoxy" or "arylalkoxy" refer to aralkyl radicals that are bonded to other radicals via an oxygen atom.

[0064] The term "aminoalkyl" refers to an alkyl radical substituted with an amino radical. Preferred aminoalkyl radicals have an alkyl radical with about 1 to about 24 carbon atoms, or preferably 1 to about 12 carbon atoms. More preferred aminoalkyl radicals are "lower aminoalkyls" having an alkyl radical with 1 to about 10 carbon atoms. Most preferred are aminoalkyl radicals having a lower alkyl radical with 1 to 8 carbon atoms. Examples of such radicals include aminomethyl and aminoethyl.

[0065] The term "alkylamino" refers to an amino group substituted with one or two alkyl radicals. Preferred alkylamino radicals have about 1 to about 20 carbon atoms, or preferably 1 to about 12 carbon atoms. More preferred alkylamino radicals are "lower alkylaminos" having an alkyl radical with 1 to about 10 carbon atoms. Most preferred are alkylamino radicals having a lower alkyl radical with 1 to about 8 carbon atoms. Preferred lower alkylaminos may be monosubstituted N-alkylaminos or disubstituted N,N-alkylaminos, such as N-methylamino, N-ethylamino, N,N-dimethylamino, and N,N-diethylamino.

[0066] The term "substituted" refers to the substitution of one or more hydrogen radicals in a given structure with a radical of a specified substituent, including, but not limited to, halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic substituents. It is understood that substituents may be further substituted.

[0067] For simplicity, the chemical parts defined and referred to throughout may be monovalent (e.g., alkyl, aryl, etc.) or polyvalent parts under appropriate structural circumstances that are obvious to those skilled in the art. For example, the “alkyl” part may refer to a monovalent radical (e.g., CH3-CH2-), or in other examples, the divalent linkage part may be “alkyl,” in which case those skilled in the art will understand that this alkyl is a divalent radical (e.g., -CH2-CH2-) equivalent to the term “alkylene.” Similarly, in situations where a divalent part is required and described as “alkoxy,” “alkylamino,” “aryloxy,” “alkylthio,” “aryl,” “heteroaryl,” “heterocyclic,” “alkyl,” “alkenyl,” “alkynyl,” “aliphatic,” or “cycloalkyl,” those skilled in the art will understand that the terms “alkoxy,” “alkylamino,” “aryloxy,” “alkylthio,” “aryl,” “heteroaryl,” “heterocyclic,” “alkyl,” “alkenyl,” “alkynyl,” “aliphatic,” or “cycloalkyl” refer to the corresponding divalent part.

[0068] As used herein, the terms "halogen" or "halo" refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[0069] Suitable M groups include substituted or unsubstituted C1-C 15 Alkyl, substituted, or unsubstituted C2-C 16 -Alkenylene, or substituted or unsubstituted C2-C 16 Examples include, but are not limited to, alkylylenes, aralkyls, heteroaralkyls, heteroalkyls, and substituted or unsubstituted cycloalkyls.

[0070] In this embodiment, M is optionally substituted W, where W is defined herein.

[0071] For this reason, M is (a) Ethylene glycol linker; and (b) Alkyl linker It may include a non-nucleotide linker selected from the group consisting of:

[0072] In some embodiments, M is a hexaethylene glycol linker. In some embodiments, M is a C9 alkyl linker.

[0073] Non-limiting examples of M include ethylene glycol (-CH2CH2O), peptides, peptide nucleic acids (PNA), alkylene chains (groups based on divalent alkanes), amides, esters, ethers, and any combination thereof.

[0074] In one embodiment, M comprises at least one ethylene glycol group. In another embodiment, M comprises one ethylene glycol group. In yet another embodiment, M comprises two ethylene glycol groups. In yet another embodiment, M comprises three ethylene glycol groups. In yet another embodiment, M comprises four ethylene glycol groups. In yet another embodiment, M comprises five ethylene glycol groups. In yet another embodiment, M comprises six ethylene glycol groups. In yet another embodiment, M comprises seven ethylene glycol groups. In yet another embodiment, M comprises eight ethylene glycol groups. In yet another embodiment, M comprises nine ethylene glycol groups. In yet another embodiment, M comprises ten ethylene glycol groups. In yet another embodiment, M comprises more than ten ethylene glycol groups. In yet another embodiment, M comprises (OCH2CH2)n, where n is an integer between 1 and 10. In yet another embodiment, n is 1. In yet another embodiment, n is 2. In yet another embodiment, n is 3. In yet another embodiment, n is 4. In yet another embodiment, n is 5. In another embodiment, n is 6. In another embodiment, n is 7. In another embodiment, n is 8. In another embodiment, n is 9. In another embodiment, n is 10.

[0075] In one embodiment, M comprises at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, at least ten amino acids, or more than ten amino acids.

[0076] In one embodiment, M is an alkyl or alkylene chain optionally substituted with at least one substituent selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkoxy, -OH, halo, -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)(C1-C6 alkyl), -C(=O)OH, -C(=O)O(C1-C6 alkyl), and -C(=O)O(C3-C8 cycloalkyl), for example, but not limited to C1-C 50 The material comprises an alkyl or alkylene chain, wherein the alkyl or cycloalkyl is optionally substituted with at least one selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkoxy, -OH, halo, -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)(C1-C6 alkyl), -C(=O)OH, -C(=O)O(C1-C6 alkyl), and -C(=O)O(C3-C8 cycloalkyl). In another embodiment, M is -(CH2)-, -(CH2)2-, -(CH2)3-, -(CH2)2-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 10 -,-(CH2) 11 -,-(CH2) 12 -,-(CH2) 13 -,-(CH2) 14 -,-(CH2) 15 -,-(CH2) 16 -,-(CH2) 17 -,-(CH2) 18 -,-(CH2) 19 -, and -(CH2) 20 - Selected from the group consisting of these.

[0077] The non-nucleotide segment attaches to the 5' nucleotide portion and the 3' nucleotide portion of the element either at the 3' position of the sugar or at a phosphorus-containing nucleotide linkage. In this embodiment, the non-nucleotide segment attaches to the 5' nucleotide portion and the 3' nucleotide portion of the element at the 3' position of the sugar. In this embodiment, the non-nucleotide segment attaches to the 5' nucleotide portion and the 3' nucleotide portion of the element at a phosphorus-containing nucleotide linkage. In this embodiment, the non-nucleotide segment attaches to the 3' position of the sugar of the nucleotide of the 5' or 3' nucleotide portion of the element, relative to the phosphorus-containing nucleotide linkage of the other nucleotide portion of the element.

[0078] Oligonucleotide synthesis begins at the 3-terminus of the lower chain sequence (a sequence without a diphosphate or triphosphate group). Therefore, the terminus of this sequence has a "free" 5'-OH group, which is later ligated to a connector via a phosphate group. The connector segment is ligated to the 5'-OH group of the element via a phosphate group. The other terminus of the connector has a hydroxyl group, which is ligated to the 3'-OH group of the terminal nucleotide of the upper chain (or sequence with a 5'-DP or TP group) via a phosphate group (see, for example, the scheme for the synthesis of compound 1).

[0079] In standard oligonucleotides, the nucleotide linkage is via phosphate linkage between the 3' hydroxyl group of one nucleotide and the 5' OH group of the second nucleotide. In non-standard oligonucleotides, the linkage is between the 2' hydroxyl group of one nucleotide and the 5'-terminus of the second nucleotide.

[0080] In some embodiments, the non-nucleotide segment is symmetrically attached to the 5' nucleotide portion of the element and the 3' nucleotide portion of the element. “Symmetrically” refers to an element where y=y' such that the nucleotides of the 5' nucleotide portion and the 3' nucleotide portion of the element are attached to a non-nucleotide segment whose distance from the 3' end of the first nucleic acid sequence is the same (in terms of the number of nucleotides) as the distance from the 5' end of the second nucleic acid sequence. Preferably, y and y' are 0-7. Preferably, y and y' are 0-4. Preferably, y and y' are 0. Preferably, y and y' are 1. Preferably, y and y' are 2. Preferably, y and y' are 3. Preferably, y and y' are 4. Preferably, y and y' are 5. Preferably, y and y' are 6. Preferably, y and y' are 7. Non-limiting examples of symmetric connector elements that attach to the first and second nucleotide sequences of a RIG-I agonist are as follows: [ka] .

[0081] In some embodiments, the non-nucleotide segment is asymmetrically bound to the 5' nucleotide portion and the 3' nucleotide portion of the element. “Asymmetrically” means that y is not equal to y' such that the number of nucleotides in the 5' nucleotide portion of the element is not equal to the number of nucleotides in the 3' nucleotide portion of the element. For example, when y is 0, y' is 1, 2, 3, 4, 5, 6, 7, 8, or 9. Or, for example, when y' is 0, y is 1, 2, 3, 4, 5, 6, 7, 8, or 9. Non-limiting examples of asymmetric connector elements that bind to the first and second nucleotide sequences of a RIG-I agonist are as follows: [ka] .

[0082] (Conjugation base (W)) W is any reactive or conjugation group that can be used to conjugate various small and large targeting molecules (Tm) to the nucleic acid compounds of the present invention. In embodiments, W is selected from the group consisting of alkyl, aminoalkyl, carboxamide, polyethylene glycol (PEG), aralkyl, heteroaralkyl, heteroalkyl, substituted or unsubstituted cycloalkyl. W may contain functional groups such as amino, hydroxy, azide, or thiol that can be used for conjugation to the targeting molecule (Tm).

[0083] In this embodiment, W is a reactive group selected from OR, NRR', SR, or N3, where R and R' are as defined herein.

[0084] In this embodiment, W may be a peptide group. The peptide group may include a variety of enzymatically cleavable or non-cleavable peptides. The individual amino acid groups of the peptide may be natural or synthetic amino acids. In this embodiment, W is -CH2-O-CO-R1 (where R1 = Me, isopropyl, t-butyl, -(CH2)n-R2 (where R2 is selected from aryl, aralkyl, heteroaryl, heteroaralkyl, alkyl, aminoalkyl, carboxamide, polyethylene glycol (PEG), heteroalkyl, substituted or unsubstituted cycloalkyl)). Preferred examples of W are, but are not limited to, those listed below. [ka] TM is the targeting part. [ka]

[0085] W can be a bifunctional connector, where the bifunctional connector group can be a different composition capable of connecting two ends of a nucleic acid chain. Non-limiting examples include: [ka] .

[0086] (Targeting molecule (Tm)) In embodiments of the present invention, the targeting molecule (Tm) can be conjugated to an element via W, Y, or Y'. In embodiments, the targeting molecule (Tm) can be conjugated to an element via W. In embodiments, the targeting molecule (Tm) can be conjugated to an element via Y. In embodiments, the targeting molecule (Tm) can be conjugated to an element via Y'.

[0087] Examples of Tm include, but are not limited to, molecules such as vitamins, biotin, folic acid, peptides, vitamin D, antibodies, proteins such as integrins, fatty acids and esters, cell-permeable peptides, and tissue and cell targeting agents such as N-acetylglucosamine. In the embodiment, Tm may be a group such as a targeted antibody or a targeting moiety. In the embodiment, Tm may be a group such as a fluorescent dye.

[0088] In some embodiments, Tm is an antibody, hormone, hormone derivative, folic acid, folic acid derivative, biotin, small molecule, oligopeptide, σ-2-ligand, or sugar, fatty acid, ionic, nonionic, or ionizable lipid.

[0089] In some cases, Tm may be a dendrimer based on a glycol or alkyldiamine core structure.

[0090] In some embodiments, the antibody is selected from intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments, single-chain Fv(scFv) mutants, multispecific antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing the antigen-determining portion of an antibody, and other modified immunoglobulin molecules containing an antigen-recognition site.

[0091] In some embodiments, the antibody is muromonab-CD3, absiximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab, etanercept, basiliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, alefacept, omalizumab, efalizumab, tocitumomab-I 131 The antibody is selected from cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, lilonacept, certolizumab pegol, romiplostim, belimumab, anti-CD20, tocilizumab, atlizumab, mepolizumab, pertuzumab, tremelimumab, tisilimunab, inotuzumab ozogamicin, aflibercept, catumakisomab, pregovomab, motabizumab, efumgumab, Aurograb®, laxibakumab, and bertuzumab. In some embodiments, the antibody is selected from anti-CD22 antibody or anti-CD79b antibody.

[0092] In some embodiments, the hormone is a steroid. In some embodiments, the hormone is selected from estrogen, testosterone, dihydrotestosterone, and ethisterone.

[0093] In some embodiments, Tm is a sterol. In some embodiments, Tm is cholesterol, β-sitosterol, phytosterol, or any derivative thereof.

[0094] In some embodiments, Tm is folic acid or any derivative thereof. In some embodiments, Tm is biotin. In some embodiments, Tm is a substituted benzodiazepine. In some embodiments, Tm is glutamate-urea-lysine. In some embodiments, Tm is an asparaginyl-glycinyl-aginine oligopeptide.

[0095] In some embodiments, Tm is an integrin ligand. In some embodiments, the integrin ligand is an RGD peptide. In some embodiments, the RGD peptide is an Arg-Gly-Asp oligopeptide.

[0096] In some embodiments, Tm is a σ-2 ligand.

[0097] In this embodiment, Tm is a lipid. In this embodiment, Tm is an ionizable lipid. In this embodiment, Tm is a cationic lipid.

[0098] In this embodiment, Tm is polyethylene glycol (PEG), 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (PEG-DMG), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (e.g., aminolipid SM-102), or any derivative thereof.

[0099] In this embodiment, Tm is 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium propanemethyl sulfate (DOTAP), distearoylphosphatidylcholine (DSPC), or any derivative thereof.

[0100] In some embodiments, Tm is oleic acid or a pharmaceutically acceptable salt thereof.

[0101] In some embodiments, Tm is a sugar. In some embodiments, the sugar is galactose. In some embodiments, the sugar is N-acetylglucosamine.

[0102] (5'-terminated phosphate (P)) The nucleic acid compounds of the present invention contain a 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); a 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); or a phosphoryl analog at the 5' end. The presence of a 5'-triphosphate or a 5'-diphosphate, or analogs thereof, can improve the binding affinity of nucleic acid molecules.

[0103] Suitable analogues include 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-adenosine cap (Appp), any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); Examples include 5'-monodithiophosphates (phosphodithioates; (HO)(HS)(S)PO-5'), 5'-phosphorothiolates ((HO)2(O)PS-5'); any further combinations of oxygen / sulfur-substituted diphosphates and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoamidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, (OH)2(O)P-5'-CH2-), and 5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-).

[0104] In other examples, the terminal group may be -(HO)(O)PXP(HO)(Y)-O-5'), where X and Y are independently S, NR, and O; where R can be C1-C20 alkyl or aralkyl.

[0105] Other terminal groups include mixed carboxyphosphoryls, sulfonylphosphoryls, or other phosphorus mimics known in the art. For example, (OH-CO-OP(O)-(OH)-OP(O)(OH)-O-5'), OH-S(O)(O)-OP(O)-(OH)-OP(O)(OH)-O-5'), (OH-S(O)(O)-OP(O)(OH)-O-5'), (OH-CO-CH2-P(O)-O-CH2-, OH-S(O)(O)-CH2-P(O)(OH)-O-5').

[0106] An exemplary example of a phosphomimicket is shown in Figure 4.

[0107] It should be noted that a single internucleotide phosphorothioate linkage in a composition can exist in two isomeric forms, represented as Rp and Sp. The compounds disclosed in this invention may be in individual isomeric forms or mixed Rp,Sp compositions. Other linkages may exist in isomeric or diastereomeric forms.

[0108] In one embodiment, the nucleic acid molecule comprises a 5'-triphosphate, wherein the phosphate is unmodified. In one embodiment, the nucleic acid molecule comprises a 5'-triphosphate, wherein at least one of the phosphates is a phosphate analog. In one embodiment, the nucleic acid molecule comprises a 5'-triphosphate, wherein two of the phosphates are phosphate analogs. In one embodiment, the nucleic acid molecule comprises a 5'-triphosphate, wherein all three of the phosphates are phosphate analogs.

[0109] In one embodiment, the nucleic acid molecule comprises a 5'-diphosphate, wherein the phosphate is unmodified. In one embodiment, the nucleic acid molecule comprises a 5'-diphosphate, wherein at least one of the phosphates is a phosphate analog. In one embodiment, the nucleic acid molecule comprises a 5'-diphosphate, wherein both of the phosphates are phosphate analogs.

[0110] In this embodiment, the phosphate analog comprises the following structure [ka] (Here, Y is O or S, or CH-R (where R = alkyl, aralkyl, heteroaryl, or cycloalkylamine (e.g., piperazine)), X is either O or S, and Z is OH, SH, or NHR' (where R' is H, alkyl, aralkyl, or heteroaryl).

[0111] In this embodiment, the nucleic acid compound comprises a 5' diphosphate having the following structure: [ka] (Here, Y is O or S, or CH-R (where R = alkyl, aralkyl, heteroaryl, or cycloalkylamine (e.g., piperazine)), X is independently either O or S, and Z is independently OH, SH, or NHR' (where R' is H, alkyl, aralkyl, or heteroaryl).

[0112] In this embodiment, the nucleic acid compound comprises a 5' triphosphate having the following structure: [ka] (Here, Y is independently O or S, or CH-R (where R = alkyl, aralkyl, heteroaryl, or cycloalkylamine (e.g., piperazine)), X is independently either O or S, and Z is independently OH, SH, or NHR' (where R' is H, alkyl, aralkyl, or heteroaryl).

[0113] (Nucleotides and modifications) In this embodiment, any nucleotide or debase within the first nucleotide sequence, the second nucleotide sequence, and / or within the connector element independently comprises a naturally occurring nucleobase or modified nucleobase, a naturally occurring internucleoside linkage or modified internucleoside linkage, a naturally occurring sugar or modified sugar, or a combination thereof.

[0114] In this embodiment, any nucleotide or debase within the first nucleotide sequence, the second nucleotide sequence, and / or within the connector element independently contains a naturally occurring nucleobase or a modified nucleobase.

[0115] As used herein, “nucleic acid,” “oligonucleotide,” “nucleotide sequence,” or “nucleotide moiety” are interchangeable and typically refer to molecules or compounds containing multiple linked nucleosides. In one embodiment, the nucleic acid comprises one or more modified or unmodified ribonucleosides (RNA) and / or one or more modified or unmodified deoxyribonucleosides (DNA). In another embodiment, the nucleic acid comprises one or more modified or unmodified ribonucleosides (RNA). In yet another embodiment, the nucleic acid consists of one or more modified or unmodified ribonucleosides (RNA).

[0116] As used herein, the terms “ribonucleotide” and “ribonucleic acid” (RNA) refer to a modified or unmodified nucleotide or polynucleotide comprising at least one ribonucleotide unit. A ribonucleotide unit comprises an oxygen atom attached to the 2'-position of a ribosyl moiety, which has a nitrogen base attached to the 1'-position of the ribosyl moiety by an N-glycosidic linkage, and a portion that either enables or prevents linkage to another nucleotide.

[0117] In one embodiment, the nucleic acid comprises an unmodified ribonucleoside (RNA). In another embodiment, the nucleic acid comprises one or more modified ribonucleosides.

[0118] As used herein, “modified nucleic acid” means a nucleic acid molecule or compound comprising at least one modified nucleoside and / or at least one modified sugar.

[0119] The term "nucleoside" typically refers to a compound comprising a sugar, usually ribose, deoxyribose, pentose, arabinose, or hexose, and a purine or pyrimidine base. For the purposes of this invention, the base is considered unnatural if it is not guanine, cytosine, adenine, thymine, or uracil, and the sugar is considered unnatural if it is not a β-ribo-furanoside or 2'-deoxyribo-furanoside.

[0120] The term "nucleotide" typically refers to a nucleoside containing a phosphorus-containing group bonded to a sugar. As used herein, "linked nucleosides" may or may not be linked by phosphate linking and therefore include, but are not limited to, "linked nucleotides." As used herein, "linked nucleosides" are nucleosides linked in a contiguous sequence (i.e., there are no further nucleosides between the linked ones).

[0121] As used herein, “nucleobase” means an atomic group that can be linked to a sugar moiety to form a nucleoside that can be incorporated into an oligonucleotide, wherein the atomic group can be linked to a naturally occurring complementary nucleobase of another oligonucleotide or nucleic acid. The nucleobase may be naturally occurring or modified. As used herein, “nucleobase sequence” means a continuous sequence of nucleobases independent of any sugar, linkage, or nucleobase modification.

[0122] As used herein, the terms “unmodified nucleobase” or “naturally occurring nucleobase” mean naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C or 6-methyl C), and uracil (U).

[0123] As used herein, “modified nucleobase” means any nucleobase that is not naturally occurring. Examples of modified nucleobases include 7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, N6 delta 2-isopentenyladenine (6iA), and N6 delta 2-isopentenyl-2-methylthioadenine (2 ms6iA), N2-dimethylguanine (dmG), 7-methylguanine (7mG), inosine, nebularin, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine, isoguanine, 7-deazaguanine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiouracil, 06-methylguanine, N6-methyladenine, 04-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, pyrazolo[3,4-D]pyrimidine (e.g., U.S. Patent Nos. 6,143,877 and 6,127,121 and PCT Publication Application WO Examples include, but are not limited to, indoles such as etenoadenine, nitroindole and 4-methylindole, and pyrroles such as nitropyrrole (see issue 01 / 38584). Specific exemplary nucleotide bases can be found, for example, in Fasman's Literature, 1989, Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca Raton, Fla. and the references cited therein.

[0124] As used herein, “modified nucleoside” means a nucleoside that contains at least one chemical modification compared to a naturally occurring RNA or DNA nucleoside. Modified nucleosides include a modified sugar moiety and / or a modified nucleobase.

[0125] In other embodiments, the nucleobase may be a "universal" nucleobase, for example, but not limited to, nitrosubstituted aromatic molecules, such as 3-nitropyrrole, 4-nitropyrazole, 4-nitroimidazole, or 5-nitroindole.

[0126] In other embodiments, the nucleobase may be a non-natural hydrophobic pyrimidine-like N-nucleoside or a non-natural hydrophobic N-nucleoside, such as isocarbostyryl, 3-methylisocarbostyryl, 5-methylisocarbostyryl, 3,5-dimethyl-2-pyridone, 7-propynyl-3-methylisocarbostyryl, 7-propynylisocarbostyryl, 7-propynyl-3-methyl-2(1H), or a non-natural hydrophobic purine-like nucleoside, such as 7-azaindole, 6-methyl-7-azaindole, imidazole, pyridine, 3-propynyl-7-azaindole, or 3-propynyl-4,7-diazaindole.

[0127] In other embodiments, the nucleobase may be a C-nucleoside, such as 3,5-dimethylphenyl-C-nucleoside, 1,4-dimethylnaphthalene-C-nucleoside, or other C-nucleosides derived from trimethylbenzene, dimethylbenzene, dimethylnaphthalene, 3-methyl-2-naphthalene, 1-methyl-3-naphthalene, or 2-naphthalene.

[0128] (Nucleoside linkage) In the embodiment, any nucleotide or debase within the first nucleotide sequence, the second nucleotide sequence, and / or within the connector element independently includes naturally occurring internucleoside linkages or modified internucleoside linkages.

[0129] As used herein, “nucleoside linkage” means a covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein, “naturally occurring nucleoside linkage” means a 3'-to-5' phosphodiester linkage. As used herein, “modified nucleoside linkage” means any nucleoside linkage other than naturally occurring nucleoside linkages. The nucleoside residues of the nucleic acid compounds of the present invention can be coupled to one another by any of the many known nucleoside linkages. The two main classes of nucleoside linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing nucleoside linkages include, but are not limited to, phosphates (which contain phosphodiester links ("P=O") (also called unmodified or naturally occurring linkages)), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates ("P=S"), and phosphorodithioates ("HS-P=S"). Typical non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiesters, thionocarbamates (-OC(=O)(NH)-S-), siloxanes (-O-SiH2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linking groups are well known to those skilled in the art.

[0130] Examples of such nucleoside linkages include, but are not limited to, phosphodiesters, phosphorothioates, phosphorodithioates, methylphosphonates, alkylphosphonates, alkylphosphonothioates, phosphotriesters, phosphoramidates, siloxanes, carbonates, carbolalkoxys, acetamidates, carbamates, morpholino, borano, thioethers, cross-linked phosphoramidates, cross-linked methylenephosphonates, cross-linked phosphorothioates, and sulfone nucleoside linkages. In some embodiments, the nucleic acid compounds of the present invention may include combinations of nucleotide linkages. In some embodiments, the nucleic acid compounds of the present invention may include combinations of phosphorothioate nucleotide linkages and phosphodiester nucleotide linkages. In some embodiments, more than half but not all of the nucleotide linkages are phosphorothioate nucleotide linkages. In some embodiments, all of the nucleotide linkages are phosphorothioate nucleotide linkages.

[0131] In this embodiment, the nucleic acid compound comprises one or more peptide nucleic acids (PNAs). The peptide nucleic acid (PNA) comprises a polypeptide skeleton to which nucleic acid bases are attached as side chains. In this embodiment, the PNA comprises a polyamide skeleton supporting a plurality of ligands at spaced intervals along the skeleton, each ligand independently being a naturally occurring nucleobase, a non-naturally occurring nucleobase, or a nucleobase-binding group, each ligand directly or indirectly bonded to a nitrogen atom in the skeleton, and each ligand mainly supports nitrogen atoms separated from each other in the skeleton by 4 to 8 intercalating atoms.

[0132] (sugar group) In this embodiment, any nucleotide or debase within the first nucleotide sequence, the second nucleotide sequence, and / or within the connector element independently contains a naturally occurring sugar or a modified sugar.

[0133] Modified RNA can include modifications to all or some of the sugar groups of ribonucleic acid. For example, the 2'-hydroxyl group (OH) can be modified or substituted with several different "oxy" or "deoxy" substituents. While not theoretically bound, improved stability is expected because the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion. The 2'-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker-phosphorus atom. While not wishing to be theoretically bound, introducing modifications that make alkoxide formation at the 2'-position impossible may be desirable for some embodiments.

[0134] Examples of "oxy"-2'-hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CH2O). n CH2CH2OR; "Locked" nucleic acids (LNAs) in which the 2'-hydroxyl group is linked to the 4'-carbon of the same ribose sugar by, for example, methylene or ethylene crosslinking (e.g., 2'-4'-ethylene crosslinked nucleic acids (ENA)); amino, O-amines (amines = NH2; alkylaminos, dialkylaminos, heterocyclyls, arylaminos, diarylaminos, heteroarylaminos, diheteroarylaminos, ethylenediamines, polyaminos), and aminoalkoxys, O(CH2) n Examples include amines (e.g., amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino). It is noteworthy that oligonucleotides containing only a methoxyethyl group (MOE) (OCH2CH2OCH3, PEG derivative) exhibit nuclease stability comparable to those modified with robust phosphorothioate modifications.

[0135] Deoxy modifications include hydrogen (i.e., deoxyribose sugar); halo (e.g., fluoro); amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH) n CH2CH2-amines (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino), -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano; mercapto; alkylthioalkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl groups, which can be optionally substituted with amino functional groups, for example. Preferred substituents are 2'-methoxyethyl, 2'-OCH3, 2'-O-allyl, 2'-C-allyl, and 2'-fluoro.

[0136] The sugar group may also contain one or more carbon atoms having a stereochemical configuration opposite to that of the corresponding carbon in ribose. Therefore, modified RNA can contain, for example, nucleotides containing arabinose as the sugar.

[0137] Modified RNA may also contain "debased" sugars lacking the C-1' nucleobase. These debased sugars may also contain modifications in one or more of their constituent sugar atoms.

[0138] To maximize nuclease resistance, the 2' modification can be used in combination with one or more phosphate linker modifications (e.g., phosphorothioates). So-called "chimeric" oligonucleotides are those that contain two or more different modifications.

[0139] Modification can also involve large-scale substitution of the ribose structure with another entity (SRMS) at one or more sites of the nucleic acid agent.

[0140] Modified RNA can also contain one or more morpholino nucleotides.

[0141] (Compounds and Compositions) In this embodiment, the first nucleic acid sequence and the second nucleic acid sequence, together with the connector element, constitute a RIG-I agonist. The RIG-I agonist can induce interferon production.

[0142] In one embodiment, the RIG-I agonist of the present invention has a double-stranded portion of 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 base pairs. In one embodiment, the double-stranded portion contains approximately 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 base pairs. The nucleic acid compound can be any nucleic acid sequence.

[0143] The RIG-I agonist of the present invention comprises elements that bind to a first nucleotide sequence and a second nucleotide sequence of a compound.

[0144] In one embodiment, the double-stranded portion contains one or more mispairing bases. That is, Watson-Crick base pairing is not required for each and all nucleotide pairs.

[0145] In some embodiments, the RIG-I agonist includes a nucleotide insertion in either a first or second nucleotide sequence that maintains a non-pairing state in the double-stranded structure and generates twisting. The nucleotide insertion may be a nucleotide insertion of 1-2 nucleotides, preferably a single nucleotide. In some embodiments, the nucleotide insertion is a nucleotide insertion in the first nucleotide sequence.

[0146] Non-limiting examples of nucleic acid compounds of the present invention, in which a first nucleic acid sequence and a second nucleic acid sequence are connected via a connector element, including an exemplary targeting molecule, are as follows: [ka] TIFF2026519524000018.tif189170TIFF2026519524000019.tif226170TIFF2026519524000020.tif104170

[0147] (Pharmaceutical compositions and preparations) In another embodiment, the present invention relates to a composition comprising at least one nucleic acid compound according to the present invention and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. Such a composition may comprise one species of such nucleic acid compound or a plurality of different nucleic acid compounds according to the present invention.

[0148] The composition may be a pharmaceutical composition, for example, an immunostimulatory composition or an antiviral or anticancer composition. The immunostimulatory composition may be a vaccine composition further comprising a vaccine in which a nucleic acid compound is an adjuvant. If the composition is an antiviral composition, it may further comprise additional active antiviral agents. If the composition is an anticancer composition, it may further comprise additional active anticancer agents.

[0149] In some embodiments, the acceptable formulation materials are preferably non-toxic to the recipient at the dosage and concentration used. In some embodiments, the formulation materials are for sc and / or IV administration. In some embodiments, the pharmaceutical composition may contain formulation materials to modify, maintain, or sustain, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition.In one embodiment, suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, tris-HCl, citrates, phosphates, or other organic acids); fillers (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); and preservatives. Examples include, but are not limited to, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide; solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronics, PEG, sorbitan esters, polysorbates, e.g., polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients, and / or pharmaceutical adjuvants (Remington's Medicinal Chemistry). Pharmaceutical Sciences), 18th edition, AR Gennaro, ed., Mack Publishing Company (1995).In one embodiment, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and / or 10 mM NAOAC, pH 5.2, 9% sucrose. In one embodiment, the optimal pharmaceutical composition is determined by those skilled in the art, for example, depending on the intended route of administration, the form of delivery, and the desired dosage. See, for example, Remington's Pharmaceutical Sciences, above. In one embodiment, such a composition may affect the physical state, stability, in vivo release rate, and / or in vivo clearance rate of the nucleic acid compound.

[0150] In some embodiments, the main vehicle or carrier in the pharmaceutical composition may be either aqueous or non-aqueous. For example, in some embodiments, preferred vehicles or carriers are water for injection, saline solution, or artificial cerebrospinal fluid, which may be supplemented with other materials common to parenteral administration compositions. In some embodiments, the saline solution includes isotonic phosphate-buffered saline. In some embodiments, neutral buffered saline or saline solution mixed with serum albumin are further exemplary vehicles. In some embodiments, the pharmaceutical composition includes Tris buffer at approximately pH 7.0–8.5, or acetate buffer at approximately pH 4.0–5.5, which may further include sorbitol or a preferred substitute thereof. In some embodiments, compositions containing nucleic acid compounds can be prepared for storage by mixing a selected composition having the desired purity with any formulation agent (Remington's Pharmaceutical Sciences, above) in the form of a lyophilized cake or aqueous solution. Furthermore, in one embodiment, a composition containing a nucleic acid compound can be formulated as a lyophilized product using a suitable excipient such as sucrose.

[0151] In some embodiments, the pharmaceutical composition may be selected for parenteral delivery. In some embodiments, the composition may be selected for delivery via the gastrointestinal tract, such as by inhalation or oral administration. The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art.

[0152] In one embodiment, the formulation components are present at a concentration accessible to the administration site. In another embodiment, a buffer is used to maintain the composition at or slightly below the physiological pH, typically within a pH range of about 5 to about 8.

[0153] In some embodiments, where parenteral administration is envisioned, the therapeutic composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing a nucleic acid compound in a pharmaceutically acceptable vehicle. In some embodiments, the vehicle for parenteral injection is sterile distilled water, in which the nucleic acid compound is formulated as a sterile isotonic solution and is appropriately stored. In some embodiments, the preparation may involve formulation of the desired molecule using a delivery vehicle or drug, e.g., microspheres for injection, biodegradable particles, polymer compounds (e.g., polylactic acid, polyglycolic acid, or polyethyleneimine (e.g., JetPEI®)), beads, or liposomes), which can result in controlled or sustained release of the product that can later be delivered via depot injection. In some embodiments, hyaluronic acid may also be used, which may have the effect of promoting a sustained duration in circulation. In some embodiments, the desired molecule can be introduced using an implantable drug delivery device.

[0154] In one embodiment, the pharmaceutical composition may be formulated for inhalation. In one embodiment, the nucleic acid compound may be formulated as a dry powder for inhalation. In one embodiment, an inhalation solution containing the nucleic acid compound may be formulated together with a propellant for aerosol delivery. In one embodiment, the solution may be sprayed. Lung administration is further described in PCT application number PCT / US94 / 001875, which describes pulmonary delivery of chemically modified proteins.

[0155] In some embodiments, the formulation is intended to be administered orally. In some embodiments, the nucleic acid compound administered in this manner may be formulated with or without a carrier commonly used in the formulation of solid dosage forms such as tablets and capsules. In some embodiments, the capsule may be designed to release the active portion of the formulation at a point in the gastrointestinal tract where bioavailability is maximized and degradation before systemic circulation is minimized. In some embodiments, at least one additional agent may be included to enhance the absorption of the nucleic acid compound. In some embodiments, diluents, flavorings, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0156] In one embodiment, the pharmaceutical composition may contain an effective amount of nucleic acid compound in a mixture with a non-toxic excipient suitable for the manufacture of tablets. In one embodiment, the solution can be prepared in unit dose form by dissolving the tablets in sterile water or another suitable vehicle. In one embodiment, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate, or sodium bicarbonate, lactose, or calcium phosphate; binders such as starch, gelatin, or gum arabic; or lubricants such as magnesium stearate, stearic acid, or talc.

[0157] In this embodiment, the pharmaceutical composition is an aqueous liquid pharmaceutical preparation suitable for topical administration to the lungs or nose, comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) a polynucleotide molecule according to formula I or formula II. In this embodiment, the polynucleotide molecule is a polynucleotide molecule according to formula I. In this embodiment, the polynucleotide molecule is a polynucleotide molecule according to formula II.

[0158] By definition, as used herein, “fatty acid” typically refers to a carboxylic acid molecule containing a carboxylic acid group bonded to an aliphatic hydrocarbon “tail” that is 4 to 24 carbon atoms long. For example, the aliphatic hydrocarbon “tail” may have a carbon atom length of 4 to 22, e.g., 4 to 20, e.g., 4 to 18, e.g., 4 to 16, e.g., 4 to 14, e.g., 4 to 12, e.g., 4 to 10, e.g., 4 to 8, e.g., 4 to 6 carbon atoms long. Alternatively, the aliphatic hydrocarbon “tail” may have a carbon atom length of 6 to 24, e.g., 8 to 24, e.g., 10 to 24, e.g., 12 to 24, e.g., 14 to 24 carbon atoms long. For example, the aliphatic hydrocarbon “tail” may have a carbon atom length of 6 to 22, e.g., 6 to 20, e.g., 8 to 20, e.g., 8 to 18, e.g., 10 to 18 carbon atoms long. In one embodiment, the aliphatic hydrocarbon "tail" has a length of 4 to 6 carbon atoms, i.e., the fatty acid is a short-chain fatty acid such as butyric acid (4 carbon atoms). Alternatively, the aliphatic hydrocarbon "tail" has a length of 6 to 12 carbon atoms, i.e., the fatty acid is a medium-chain fatty acid such as caprylic acid (8 carbon atoms) and capric acid (10 carbon atoms). Most preferably, the aliphatic hydrocarbon "tail" has a length of 14 to 24 carbon atoms, i.e., the fatty acid is a long-chain fatty acid such as oleic acid (18 carbon atoms), stearic acid (18 carbon atoms), and arachidic acid (20 carbon atoms). The aliphatic hydrocarbon "tail" can be saturated or unsaturated. In the case of unsaturation, the aliphatic hydrocarbon "tail" may contain, for example, 1, 2, 3, 4, 5, or 6 C=C double bonds, particularly 1 or 2, especially 1 C=C double bond. Fatty acids can be further classified based on the length and saturation of the aliphatic hydrocarbon "tail".

[0159] Exemplary fatty acids typically have a molar mass of about 150 g / mol to about 400 g / mol, for example, about 200 g / mol to about 350 g / mol. These include, but are not limited to, arachidic acid, arachidonic acid, lauric acid, linoleic acid, linolenic acid, myristic acid, myristoleic acid, oleic acid, palmitic acid, palmitoleic acid, sapienic acid, stearic acid, and vaccenic acid. In particular, oleic acid is a notable fatty acid.

[0160] Preferably, the aqueous liquid pharmaceutical formulation contains a single fatty acid as part of the surfactant component. Alternatively, it may contain, for example, a mixture of two (or more) fatty acids as part of the surfactant component.

[0161] Exemplary nonionic surfactants typically have a molar mass of about 100 g / mol to about 10,000 g / mol, and in particular, about 100 g / mol to about 2,000 g / mol. Exemplary nonionic surfactants typically contain one or more polyoxyalkylene moieties, such as polyoxyethylene and / or polyoxypropylene moieties.

[0162] Examples of nonionic surfactants include polyoxyalkylenes, particularly poloxamers such as poloxamer 188, poloxamer 407, poloxamer 171, and poloxamer 185.

[0163] Further exemplary nonionic surfactants include alkyl ethers of polyethylene glycol, such as those known by the trade names Brij 52, Brij 93, Brij 97, Brij L4, Brij 30, and Brij 78.

[0164] Further exemplary nonionic surfactants include alkylphenyl ethers of polyethylene glycol, such as those known by the trade name Triton X-100.

[0165] Examples of specific nonionic surfactants include fatty acid esters, such as fatty acid esters of polyols. Such fatty acid esters may contain one or more fatty acid chains, for example, one, two, or three fatty acid chains, for example, one fatty acid chain. A specific example is polyoxyethylene sorbitan fatty acid ester. In particular, the nonionic surfactant is polyoxyethylene sorbitan fatty acid ester. Preferred polyoxyethylene sorbitan fatty acid esters include polysorbate 80 (e.g., Tween 80), polysorbate 120, polysorbate 85, polysorbate 65, polysorbate 60, polysorbate 40, and polysorbate 20, and in particular polysorbate 80.

[0166] Preferably, the aqueous liquid pharmaceutical formulation comprises a single nonionic surfactant as part of the surfactant component. Alternatively, it comprises, for example, a mixture of two (or more) nonionic surfactants as part of the surfactant component.

[0167] Preferably, the surfactant component is selected from the group consisting of (a) a mixture of oleic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (b) a mixture of lauric acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (c) a mixture of linoleic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (d) a mixture of linolenic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (e) a mixture of palmitic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (f) a mixture of stearic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (g) a mixture of oleic acid or a pharmaceutically acceptable salt thereof and polyoxyalkylene such as poloxamer, (h) a mixture of oleic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol, and (i) a mixture of oleic acid or a pharmaceutically acceptable salt thereof and an alkylphenyl ether of polyethylene glycol.

[0168] Most preferably, the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, particularly a polyoxyethylene sorbitan fatty acid ester selected from polysorbate 80 and polysorbate 20. In particular, the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof, particularly oleic acid and polysorbate 80.

[0169] Pharmaceutically acceptable salt forms of fatty acids that can be used include sodium salts, potassium salts, and ammonium salts, with sodium salts being particularly important. Preferably, the fatty acid is used as a free acid, i.e., in the form of a free acid.

[0170] The aqueous liquid pharmaceutical formulation of the present invention should preferably form a stable colloidal emulsion. Typically, a stable colloidal emulsion contains stable colloidal particles having an average particle size of about 50 to about 1000 nm, for example, about 50 to about 500 nm, for example, about 50 to about 100 nm, or about 100 to about 250 nm, or about 250 to about 500 nm. Thus, for example, the average particle size is about 100 to 200 nm (see Biophysical Example 1). The aforementioned particle size refers to the hydrodynamic diameter (Z-average size) which can be measured as described in Biophysical Example 1.

[0171] Such formulations are preferably achieved when a surfactant component is used, which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant. Such formulations can be more preferably achieved when the fatty acid is present at or below (but ideally close to) the critical micelle concentration of the fatty acid, as defined below in this invention. Such formulations can be more preferably achieved when the nonionic surfactant is water-miscible when present in the concentration range defined below in this invention. Physical measurements are preferably performed at a temperature of 23°C and 1 standard atmospheric pressure.

[0172] Typically, surfactant components can be present in the formulation at concentrations of 0.2 to 30,000 μg / mL, for example, 1 to 30,000 μg / mL, for example, 1 to 20,000 μg / mL, for example, 5 to 20,000 μg / mL, for example, 5 to 15,000 μg / mL, for example, 5 to 10,000 μg / mL, for example, 5 to 5,000 μg / mL (meaning the total concentration of surfactant components). Preferably, surfactant components are present in the formulation at concentrations of 1 to 3,000 μg / mL, for example, 1 to 2,000 μg / mL, for example, 5 to 2,000 μg / mL, for example, 5 to 1,500 μg / mL, for example, 5 to 1,000 μg / mL, for example, 5 to 500 μg / mL. In one embodiment, the surfactant component is present in the formulation at a concentration of 50-200 μg / mL, for example, 75-150 μg / mL, for example, 90-120 μg / mL, or about 100 μg / mL. In an alternative embodiment, the surfactant component is present at a concentration of 500-2000 μg / mL, for example, 750-1500 μg / mL, for example, 900-1200 μg / mL, or about 1000 μg / mL.

[0173] Preferably, fatty acids may be present in the formulation at concentrations of 0.2 to 30,000 μg / mL, for example, 1 to 30,000 μg / mL, for example, 1 to 20,000 μg / mL, for example, 5 to 10,000 μg / mL, and nonionic surfactants may be present in the formulation at concentrations of 0.2 to 20,000 μg / mL, for example, 1 to 20,000 μg / mL, for example, 1 to 15,000 μg / mL, for example, 5 to 5,000 μg / mL. More preferably, fatty acids may be present in the formulation at concentrations of 10-100 ug / mL, for example, 20-80 μg / mL, for example, 25-75 μg / mL, for example, 40-60 μg / mL, or about 50 μg / mL, and nonionic surfactants may be present in the formulation at concentrations of 10-100 ug / mL, for example, 20-80 μg / mL, for example, 25-75 μg / mL, for example, 30-60 μg / mL, for example, 40-50 μg / mL. Alternatively, in another preferred embodiment, fatty acids may be present in the formulation at concentrations of 100-1000 ug / mL, for example, 200-800 μg / mL, for example, 250-750 μg / mL, for example, 400-600 μg / mL, or about 500 μg / mL, and nonionic surfactants may be present in the formulation at concentrations of 100-1000 ug / mL, for example, 200-800 μg / mL, for example, 250-750 μg / mL, for example, 300-600 μg / mL, for example, 400-500 μg / mL.

[0174] Typically, surfactant components can be present in a formulation at concentrations of 0.00002% (w / w) to 3% (w / w), for example, 0.0001% (w / w) to 3% (w / w), for example, 0.0001% (w / w) to 2% (w / w), for example, 0.0005% (w / w) to 2% (w / w), for example, 0.0005% (w / w) to 1.5% (w / w), for example, 0.0005% (w / w) to 1% (w / w), for example, 0.0005% (w / w) to 0.5% (w / w), where the weight percentage is relative to the total weight of the formulation. Preferably, the surfactant component is present in the formulation at a concentration of 0.0001% (w / w) to 0.3% (w / w), for example, 0.0001% (w / w) to 0.2% (w / w), for example, 0.0005% (w / w) to 0.2% (w / w), for example, 0.0005% (w / w) to 0.15% (w / w), for example, 0.0005% (w / w) to 0.1% (w / w), for example, 0.0005% (w / w) to 0.05% (w / w), where the weight percentage is relative to the total weight of the formulation. In one embodiment, the surfactant component is present in the formulation at a concentration of 0.005% (w / w) to 0.02% (w / w), for example, 0.0075% (w / w) to 0.015% (w / w), for example, 0.009% (w / w) to 0.012% (w / w), or about 0.01% (w / w), where weight % is relative to the total weight of the formulation. In another embodiment, the surfactant component is present in the formulation at a concentration of 0.05% (w / w) to 0.2% (w / w), for example, 0.075% (w / w) to 0.15% (w / w), for example, 0.09% (w / w) to 0.12% (w / w), or about 0.1% (w / w), where weight % is relative to the total weight of the formulation.

[0175] Preferably, fatty acids may be present in the formulation at a concentration of 0.00002% (w / w) to 3% (w / w), for example, 0.0001% (w / w) to 3% (w / w), for example, 0.0001% (w / w) to 2% (w / w), for example, 0.0005% (w / w) to 1% (w / w), and nonionic surfactants may be present in the formulation at a concentration of 0.00002% (w / w) to 2% (w / w), for example, 0.0001% (w / w) to 2% (w / w), for example, 0.0001% (w / w) to 1.5% (w / w), for example, 0.0005% (w / w) to 0.5% (w / w), where the weight percentage is relative to the total weight of the formulation. More preferably, fatty acids may be present in the formulation at concentrations of 0.001% (w / w) to 0.01% (w / w), for example, 0.002% (w / w) to 0.008% (w / w), for example, 0.0025% (w / w) to 0.0075% (w / w), for example, 0.004% (w / w) to 0.006% (w / w), or about 0.005% (w / w), and nonionic surfactants may be present at 0.001% ( It may be present in the formulation at concentrations of w / w ~ 0.01% (w / w), for example, 0.002% (w / w) ~ 0.008% (w / w), for example, 0.0025% (w / w) ~ 0.0075% (w / w), for example, 0.003% (w / w) ~ 0.006% (w / w), for example, 0.004% (w / w) ~ 0.005% (w / w), where wt% is relative to the total weight of the formulation. Alternatively, in another preferred embodiment, the Fatty acids may be present in the formulation at concentrations of 0.01% (w / w) to 0.1% (w / w), for example, 0.02% (w / w) to 0.08% (w / w), for example, 0.025% (w / w) to 0.075% (w / w), for example, 0.04% (w / w) to 0.06% (w / w), or approximately 0.05% (w / w), while nonionic surfactants may be present at 0.01% (w / w). It may be present in the formulation at concentrations of ~0.1% (w / w), for example, 0.02% (w / w) to 0.08% (w / w), for example, 0.025% (w / w) to 0.075% (w / w), for example, 0.03% (w / w) to 0.06% (w / w), for example, 0.04% (w / w) to 0.05% (w / w), where the weight % is relative to the total weight of the formulation.

[0176] Preferably, for example, the ratio of the amount of fatty acid or a pharmaceutically acceptable salt thereof, each measured in μg / mL, to the amount of nonionic surfactant is about 5:1 and about 1:5, for example, about 5:1 and about 1:2, for example, about 4:1 and about 1:2, for example, about 2:1 and about 1:2. More preferably, for example, the ratio of the amount of fatty acid or a pharmaceutically acceptable salt thereof, each measured in μg / mL, to the amount of nonionic surfactant is about 3:2 and about 2:3, for example, about 6:5 and about 1:1, for example, about 10:9 or about 11:10.

[0177] The aqueous liquid pharmaceutical formulation of the present invention contains water as a solvent. Examples of water include, but are not limited to, sterile water or purified water, sterile water for injection, RNase-free water, or bacteriostatic water for injection.

[0178] Preferably, aqueous liquid pharmaceutical formulations are substantially free of any solvent or co-solvent other than water. In particular, aqueous liquid pharmaceutical formulations are free of organic solvents or co-solvents such as ethanol, acetone, dimethyl sulfoxide (DMSO), dichloromethane (DCM), N-methylpyrrolidinone (NMP), N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, and benzyl benzoate. As used herein, the term “substantially free” means that the formulation contains less than 2% (w / w), for example, less than 1% (w / w), for example, less than 0.5% (w / w) (where wt% is relative to the total weight of the formulation). Preferably, the formulation does not contain any solvent or co-solvent other than water.

[0179] The aqueous liquid pharmaceutical formulation according to the present invention may further include, but is not limited to, pharmaceutically acceptable excipients, including antioxidants, buffers, diluents, emulsifiers, lubricants, preservatives, solvents, stabilizers, suspending agents, thickeners, tonicity modifiers (osmotic pressure modifiers), vehicles, and wetting agents.

[0180] Suitable antioxidants include, but are not limited to, ascorbic acid (vitamin C), glutathione (reduced form), lipoic acid, uric acid, carotenes including β-carotene, and retinol (vitamin A), cc-tocopherol (vitamin E), ubiquinol (coenzyme Q), butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate, tert-butylhydroquinone, monothioglycerol, lutein, selenium, manganese, zeaxanthin, or combinations thereof.

[0181] The aqueous liquid pharmaceutical formulation of the present invention may contain one or more buffering agents. Suitable buffering agents include, but are not limited to, citrate, borate, formate, glycine, alanine, acetate, aspartate, malate, glyoxylate, gluconate, lactate, glycolate, oxalate, histidine, tartarate, and succinate buffering agents. As used herein, a reference to a "citrate" buffering agent will be understood to refer to a mixture of citrate and a corresponding acid as a buffering agent in a ratio corresponding to the target pH, i.e., the pH to which the aqueous liquid pharmaceutical formulation is intended to be buffered. For example, the buffering agent may include sodium citrate dihydrate and citric acid monohydrate. In particular, the buffering agent may be based on a weak organic acid, for example, the buffering agent may be citrate, acetate, lactate, or formate.

[0182] Suitable diluents for pharmaceutical use include, but are not limited to, isotonic saline (0.9% w / v), isotonic dextrose (5% w / v), isotonic mixtures of physiological saline and dextrose (e.g., physiological saline (0.45% w / v) and dextrose (2.5% w / v)), sterile water or purified water, sterile water for injection, or bacteriostatic water for injection. In particular, diluents should be sterile water or purified water, sterile water for injection, RNase-free water, or bacteriostatic water for injection.

[0183] Suitable preservatives include, but are not limited to, edetate and its alkali salts, such as disodium edetate (also known as "EDTA disodium") or calcium edetate (also known as calcium EDTA), phenol, m-cresol, chlorocresol, benzyl alcohol, propylparaben, methylparaben, butylparaben, chlorobutanol, phenylethyl alcohol, benzalkonium chloride, thimerosal, propylene glycol, sorbic acid, benzoic acid derivatives, and combinations thereof.

[0184] Suitable suspending agents include, but are not limited to, gum arabic (rubber), sodium alginate, starch and starch derivatives, xanthan gum, pectin, methylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose (Avicel RC591), microcrystalline cellulose, hypromellose, hyaluronic acid, and combinations thereof. Particularly preferred suspending agents include microcrystalline cellulose, sodium carboxymethylcellulose (Avicel RC591), hyaluronic acid, and combinations thereof.

[0185] The properties of certain suspending agents can further enhance their suitability as thickeners and / or wetting agents. Therefore, suitable thickeners and / or wetting agents include, but are not limited to, the suspending agents listed above. In particular, suitable thickeners and / or wetting agents include microcrystalline cellulose, sodium carboxymethylcellulose (Avicel RC591), hyaluronic acid, and combinations thereof.

[0186] Suitable tonicity-adjusting (osmotic pressure) agents include, but are not limited to, polyols such as sugars and sugar alcohols, for example, erythritol, glycerol, lactose, maltitol, mannitol, sorbitol, trehalose, and xylitol, as well as salts, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride. Glycerol is a particularly suitable tonicity-adjusting (osmotic pressure) agent.

[0187] The pH of the aqueous liquid pharmaceutical formulation according to the present invention is preferably about 4.0 to about 9.0, for example, about 4.0 to about 8.0, for example, about 4.0 to about 7.0, or about 5.0 to about 8.0. In particular, the pH is preferably about 4.0 to about 6.0, for example, about 4.0 to about 5.5. For example, the pH of the aqueous liquid pharmaceutical formulation is about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0. Alternatively, the pH is preferably about 5.5 to about 8.0, for example, about 6.0 to about 8.0, for example, about 6.5 to about 7.5, or about 7.0 to about 80. For example, the pH of aqueous liquid pharmaceutical formulations is approximately 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. The pH of such pharmaceutical compositions may be adjusted by pH adjusters, which include acidifying agents such as hydrochloric acid, tartaric acid, citric acid, succinic acid, phosphoric acid, ascorbic acid, acetic acid, lactic acid, sulfuric acid, formic acid, and mixtures thereof; or alkaline buffering agents such as ammonium hydroxide, ethylamine, dipropylamine, triethylamine, alkanediamine, ethanolamine, polyalkylene polyamine, heterocyclic amine, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkaline earth metal hydroxides such as magnesium hydroxide and calcium hydroxide, and basic amino acids such as L-arginine, lysine, alanine, leucine, isoleucine, oxylysine, and histidine, and mixtures thereof.

[0188] Those skilled in the art will understand that an aqueous liquid pharmaceutical formulation suitable for topical administration to the nose may preferably have a pH of about 4.0 to about 9.0. Those skilled in the art will further understand that an aqueous liquid pharmaceutical formulation suitable for topical administration to the lungs may preferably have a pH of about 5.5 to about 8.0, for example, about 6.0 to about 8.0, for example, about 7.0 to about 8.0.

[0189] Preferably, the aqueous liquid pharmaceutical formulation according to the present invention does not contain proteins. Furthermore, preferably, the aqueous liquid pharmaceutical formulation according to the present invention does not contain nanoparticles, particularly lipid nanoparticles (LNPs), or liposomes. Furthermore, preferably, the aqueous liquid pharmaceutical formulation according to the present invention is substantially free of nanoparticles, particularly LNPs, and liposome components. For example, preferably, the aqueous liquid pharmaceutical formulation according to the present invention does not contain neutral lipids. In particular, the pharmaceutical formulation does not contain cholesterol or its analogues. Furthermore, for example, preferably, the aqueous liquid pharmaceutical formulation according to the present invention does not contain lipids other than fatty acids present therein. In one embodiment where the fatty acid is oleic acid, preferably, the aqueous liquid pharmaceutical formulation does not contain lipids other than oleic acid.

[0190] The aqueous liquid pharmaceutical formulation of the present invention is suitable for local administration to the lungs or nose. Therefore, the aqueous liquid pharmaceutical formulation of the present invention is suitable for administration by inhalation, for example, for local administration to the lungs by oral inhalation or for intranasal administration. In one embodiment, the aqueous liquid pharmaceutical formulation of the present invention is administered locally to the lungs or nose. Therefore, in one embodiment, the aqueous liquid pharmaceutical formulation of the present invention is administered by inhalation or intranasal administration.

[0191] It should be noted that the aqueous liquid pharmaceutical formulation of the present invention, which is suitable for local administration to the lungs or nose, may, when administered locally to the lungs via oral inhalation or locally to the nose, result in administration to the pharynx.

[0192] It will be understood that formulations suitable for topical administration to the lungs may contain different excipients that are acceptable as pharmaceuticals compared to formulations suitable for topical administration to the nose. For example, formulations suitable for topical administration to the nose may contain suspending agents and / or wetting agents and / or thickening agents, such as microcrystalline cellulose, sodium carboxymethylcellulose (Avicel RC591), hyaluronic acid, or mixtures thereof, while formulations suitable for topical administration to the lungs may not contain these.

[0193] Preferably, the aqueous liquid pharmaceutical formulation of the present invention is suitable for administration to mammals. More preferably, the aqueous liquid pharmaceutical formulation of the present invention is suitable for administration to humans. In one embodiment, the aqueous liquid pharmaceutical formulation of the present invention is administered to mammals. In particular, the aqueous liquid pharmaceutical formulation of the present invention is administered to humans.

[0194] Preferably, the aqueous liquid pharmaceutical formulations disclosed herein can be administered to a patient or subject once or more times a day, for example, twice, three times, four times, or five times a day. Such treatment can be extended for several weeks or months.

[0195] Further pharmaceutical compositions comprising formulations containing nucleic acid compounds in sustained or controlled delivery formulations are obvious to those skilled in the art. In some embodiments, various other sustained or controlled delivery means, e.g., liposome carriers, biodegradable microparticles or porous beads, and techniques for formulating depot injections are also known to those skilled in the art. See, for example, PCT application number PCT / US93 / 00829, which describes the controlled release of porous polymer microparticles for the delivery of pharmaceutical compositions. In some embodiments, the sustained-release preparation may include a semipermeable polymer matrix in the form of a molded article, e.g., a film, or microcapsules. Examples of sustained-release matrices include polyester, hydrogel, polylactide (US Patent No. 3,773,919 and EP No. 058,481), copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., above), or poly-D(-)-3-hydroxybutyrate (EP No. 133,988). In one embodiment, the sustained-release composition may be a liposome that can be prepared by any of several methods known in the art. For example, Eppstein et al., Proc. See Natl. Acad. Sci. USA, 82:3688-3692(1985); EP 036,676; EP 088,046, and EP 143,949.

[0196] Pharmaceutical compositions intended for in vivo administration are typically sterile. In some embodiments, this can be achieved by filtration through a sterile filtration membrane. In some embodiments, if the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. In some embodiments, compositions for parenteral administration can be stored in lyophilized form or in solution. In some embodiments, parenteral compositions are typically placed in containers with a sterile access port, such as intravenous solution bags or vials with a stopper that can be punctured with a subcutaneous needle.

[0197] In one embodiment, once a pharmaceutical composition has been formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In another embodiment, such a formulation can be stored either in a ready-to-use form or in a form that is reconstituted (e.g., lyophilized) before administration.

[0198] In some embodiments, the effective amount of a pharmaceutical composition containing a nucleic acid compound to be used therapeutically is determined, for example, by the context and purpose of the treatment. Those skilled in the art will understand that the appropriate dosage level for treatment according to a particular embodiment will vary considerably, in part, by the molecules being delivered, the indication in which the nucleic acid compound is used, the route of administration, and the patient's size (body weight, body surface, or organ size) and / or condition (age and overall health). In some embodiments, the clinician may increase or decrease the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0199] In one embodiment, the frequency of administration takes into account the pharmacokinetic parameters of the nucleic acid compounds in the formulation used. In one embodiment, the clinician administers the composition until a dosage is reached that achieves the desired effect. In one embodiment, the composition can therefore be administered as a single dose, or as two or more doses over time (which may or may not contain equal amounts of the desired molecule), or as a continuous infusion via an implantable device or catheter. Further refinement of appropriate dosages is routinely performed by those skilled in the art and is within the scope of routine practice. In one embodiment, appropriate dosages can be verified using appropriate dose-response data.

[0200] In some embodiments, the route of administration of the pharmaceutical composition follows known methods, such as by oral, intravenous, intraperitoneal, intracerebral (intraparum), intraventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal, or intrafocal route of injection; by a continuous-release system or by an implantable device. In some embodiments, the composition may be administered by bolus injection, continuously by infusion, or by an implantable device. In some embodiments, the individual elements of a combination therapy may be administered by different routes.

[0201] In some embodiments, the composition can be administered topically via implantation of a membrane, sponge, or other suitable material in which the desired molecule is absorbed or encapsulated. In some embodiments, if an implantation device is used, the device can be implanted in any suitable tissue or organ, and the delivery of the desired molecule can be by diffusion, time-release bolus, or continuous administration. In some embodiments, it may be desirable to use a pharmaceutical composition containing a nucleic acid compound in an ex vivo manner. In such cases, cells, tissues, and / or organs extracted from a patient are exposed to the pharmaceutical composition containing the nucleic acid compound, and then the cells, tissues, and / or organs are subsequently re-implanted in the patient.

[0202] In certain embodiments, the nucleic acid compound can be delivered by implanting specific cells that have been genetically modified using methods such as those described herein to express and secrete an agonist. In certain embodiments, such cells can be animal or human cells and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized. In certain embodiments, the cells can be encapsulated to avoid infiltration into surrounding tissues in order to reduce the chance of an immune response. In certain embodiments, the encapsulating material is typically a biocompatible semipermeable polymer encapsulation or membrane that allows for release of the protein product but prevents destruction of the cells by the patient's immune system or other harmful agents from the surrounding tissue.

[0203] In some aspects, the present disclosure provides a pharmaceutical composition comprising a nucleic acid compound according to the present invention and a pharmaceutically acceptable carrier for stimulating an immune response, treating or delaying the progression of cancer, or reducing or inhibiting tumor growth in a subject that requires it. In some embodiments, the nucleic acid compound is formulated in a polyethyleneimine (PEI) carrier. In some embodiments, the PEI carrier is JetPEI®.

[0204] In some embodiments, the nucleic acid compound of the present invention comprises a sequence motif in the first nucleotide sequence and / or the second nucleotide, wherein the sequence motif is (i) GT-repeat motif; (ii) GA-repeat motif; (iii) AUCG-repeat motif; (iv) AU-repeat motif; (v) Dipyrimidine motif; (vi) Dipurine motif; (vii) Pyrimidine triplet motif; (viii) Purine triplet motif; (ix) Palindromic sequence motif; and (x)(i) to (ix) any combination : selected from the group consisting of.

[0205] In some embodiments, the nucleic acid compound of the present invention has at least one improved biological activity, wherein the improved biological activity is (i) increased cytokine production via RIG-I; (ii) increased expression of interferon-stimulated genes via RIG-I; (iii) increased intracellular signaling via RIG-I; (iv) increased binding affinity for RIG-I; and (v) any combination of (i) to (iv) : selected from.

[0206] In some embodiments, the nucleic acid compound of the present invention contains a sequence motif, wherein the sequence motif is a GT-repeat motif containing <19, about 15 - 18, about 15, about 10 - 15, about 10, about 5 - 10, about 5, about 4, about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and thymine nucleotides, or a sequence of derivatives or analogs thereof. In some embodiments, the GT-repeat motif is [GT] n where n = 2 - 9. In some embodiments, the GT-repeat motif is [GT]7. In some embodiments, the GT-repeat motif is [GT]3, where after the GT-repeat motif, a purine triplet and UCG follow, respectively. In some embodiments, the purine triplet is GGA.

[0207] In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of <19, approximately 15-18, approximately 15, approximately 10-15, approximately 10, approximately 5-10, approximately 5, approximately 4, approximately 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the GA-repeat motif is [GA] n Here, n=2 to 9. In some embodiments, the GA-repeat motif is [GA]7.

[0208] In some embodiments, the nucleic acid compounds of the present invention comprise a sequence motif, wherein the sequence motif is an AUCG-repeat motif comprising sequences of <19, about 16, about 12-16, about 12, about 8-12, about 6, about 16, 12, and 8 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof.

[0209] In some embodiments, the AUCG-repeat motif is [AUCG] n Here, n=2 to 4. In some embodiments, the AUCG-repeat motif is [AUCG]3.

[0210] In some embodiments, there is a CG or dipyrimidine motif before the AUCG-repeat motif. In some embodiments, there is a CG before the AUCG-repeat motif. In some embodiments, the dipyrimidine motif is CC. In some embodiments, there is a diprine motif before the AUCG-repeat motif. In some embodiments, the diprine motif is GA. In some embodiments, the diprine motif is GG.

[0211] In some embodiments, the nucleic acid compound of the present invention comprises an AUCG-repeat motif in which one or more uridine nucleosides (U) are substituted with a modified nucleoside. In some embodiments, the modified nucleoside is ribothymidine (T). In some embodiments, the AUGC-repeat motif is [AUCG]3, where one or more uridine nucleosides (U) comprising the AUCG-repeat motif are substituted with a modified nucleoside, where the modified nucleoside is ribothymidine (T). In some embodiments, the AUGC-repeat motif is [AUCG]3, where one or more uridine nucleosides (U) comprising the AUCG-repeat motif are substituted with a modified nucleoside, where the modified nucleoside is ribothymidine (T), and there is GG before the AUGC-repeat motif.

[0212] In some embodiments, the nucleic acid compound of the present invention comprises an AUCG-repeat motif in which one or more guanosine nucleosides (G) are substituted with a modified nucleoside. In some embodiments, the modified nucleoside is inosine (I). In some embodiments, the AUGC-repeat motif is [AUCG]3, where one or more guanosine nucleosides (G) comprising the AUCG-repeat motif are substituted with a modified nucleoside, where the modified nucleoside is ribothymidine (T), and where GG precedes the AUGC-repeat motif.

[0213] In some embodiments, the nucleic acid compound of the present invention comprises an AUCG-repeat motif, where IG precedes the motif. In some embodiments, the AUCG-repeat motif is [AUCG]3, and IG precedes it.

[0214] In some embodiments, the nucleic acid compounds of the present invention comprise an AUCG-repeat in which one or more guanosine nucleosides (G) are substituted with inosine (I), where inosine (I) precedes the AUCG-repeat. In some embodiments, the guanosine nucleoside (G) comprising the AUCG-repeat is substituted with inosine (I), where inosine (I) precedes the AUCG-repeat, and where the most 5' nucleotide of the first polynucleotide is inosine (I).

[0215] In some embodiments, the furthest 5' nucleotide of the first alkyl group contains inosine (I).

[0216] In some embodiments, the nucleic acid compound of the present invention comprises an AUCG-repeat sequence motif, where the AUCG-repeat motif is [AUCG]2. In some embodiments, there is a diprine motif preceding the AUCG-repeat motif. In some embodiments, the diprine motif is GG. In some embodiments, there is a print riplet preceding the AUCG-repeat motif. In some embodiments, the print riplet is GGG. In some embodiments, there is CCCCCG preceding the AUCG-repeat motif. In some embodiments, there is TCGUCG preceding the AUCG-repeat motif.

[0217] In some embodiments, the nucleic acid compound of the present invention comprises a palindromic sequence, wherein the palindromic sequence comprises a sequence of <19, about 15-18, about 15, about 10-15, about 10, about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides, or derivatives or analogs thereof, linked in any order that produces a palindrome.

[0218] In some embodiments, the linker is sandwiched between AUs. In some embodiments, the linker is sandwiched between AU-repeat motifs, where the AU-repeat motif is [AU] nwhere n = 2 - 3. In some embodiments, the AU-repeat motif is [AU]2.

[0219] In some embodiments, the present disclosure provides a nucleic acid compound that specifically binds to RIG-I, wherein the agonist comprises a blunt-ended hairpin RNA comprising at least one nucleotide containing inosine that forms a base pair with cytidine.

[0220] In other embodiments, the present disclosure provides a synthetic RIG-I-like receptor agonist that specifically binds to a RIG-I-like receptor, wherein the agonist comprises a blunt-ended hairpin RNA comprising a non-nucleotide linker.

[0221] In some embodiments, when present, inosine forms a base pair with cytidine.

[0222] In some embodiments, the nucleic acid compounds of the present invention have the following properties: (a) specifically binds to RIG-I; (b) increases cytokine production via RIG-I; (c) increases the expression of interferon-stimulated genes (ISGs) via RIG-I; (d) increases RIG-I-dependent intracellular signaling; (e) increases duplex stability; (f) increases binding affinity for RIG-I; (g) decreases off-target binding; (h) increases biological half-life; (i) increases in vivo distribution and bioavailability; (j) increases and / or enhances uptake into cells and / or tissues; (k) decreases immunogenicity; and (l) any combination of (a)-(k) exhibit at least one or more of.

[0223] In some embodiments, the nucleic acid compound of the present invention is a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist includes blunt-ended agonists, wherein the nucleic acid compound includes at least one inosine nucleoside, wherein the inosine nucleoside forms a base pair with cytidine in hairpin RNA.

[0224] In some embodiments, the nucleic acid compounds of the present invention include modified nucleotides, modified nucleosides, or modified nucleobases, or combinations thereof. In some embodiments, the agonist includes modifications to internucleotide links or to the polynucleotide backbone.

[0225] (Method for producing nucleic acid compounds according to the present invention) The nucleic acid compounds of the present invention can be produced by means available in the art, including, but not limited to, in vitro transcription (IVT) and synthesis methods. Enzymatic (IVT), solid-phase, liquid-phase, complex synthesis, micro-region synthesis, and ligation methods can be used. In one embodiment, the nucleic acid compound is prepared using an IVT enzymatic synthesis method. A method for preparing polynucleotides by IVT is known in the art and is described in International Application PCT / US2013 / 30062, which is incorporated fully herein by reference. Accordingly, this disclosure also includes polynucleotides, such as DNA, constructs, and vectors, that can be used to in vitro transcribe the nucleic acid compounds described herein.

[0226] Non-naturally modified nucleobases can be introduced into polynucleotides, such as RNA, during or after synthesis. In some embodiments, the modifications may be in nucleoside linkages, purine or pyrimidine bases, or sugars. In certain embodiments, the modifications may be introduced at the end of a polynucleotide chain or elsewhere within a polynucleotide chain; either by chemical synthesis or by polymerase enzymes. Examples of modified nucleic acids and their synthesis are disclosed in PCT application number PCT / US2012 / 058519. The synthesis of modified polynucleotides is also described in the literature by Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

[0227] Polynucleotides or their regions can be conjugated with various functional parts, such as targeting agents or delivery agents, fluorescent labels, liquids, or nanoparticles, using either enzymatic or chemical ligation methods. Conjugation of polynucleotides and modified polynucleotides is outlined in Goodchild's literature, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990). The synthesis of oligonucleotides, polynucleotides, and their conjugations and ligations is further described in Taskova et al. (2017) Chembiochem 18(17):1671-1682; Gooding et al. (2016) Eur J Pharm Biopharm 107:321-40; Menzi et al. (2015) Future Med Chem 7(13):1733-49; Winkler J. (2013) Ther Deliv.(7):791-809; Singh et al. (2010) Chem Soc Rev 39(6):2054-70; and Lu et al. (2010) Bioconjug Chem 21(2):187-202.

[0228] (Application) The compositions described herein can be used for diagnostic and therapeutic applications. For example, detectably labeled nucleic acid compounds can be used in assays to detect the presence or amount of a target protein in a sample (e.g., a biological sample). The compositions can be used in in vitro assays to study the inhibition of a target function (e.g., RIG-I-mediated cellular signaling or response). For example, in some embodiments in which the composition binds to a target (e.g., a protein or polypeptide) and activates it, the composition can also induce the activity of the target protein or polypeptide and / or in other ways can be used as a positive control in assays designed to identify further novel compounds useful for treating disorders associated with the target protein or polypeptide. For example, a RIG-I activating composition can be used as a positive control in assays to identify further compounds (e.g., small molecules, aptamers, or antibodies) that induce, enhance, or stimulate RIG-I function. The compositions can also be used in therapeutic methods such as those detailed below.

[0229] The present invention further encompasses the use of the nucleic acid compounds of the present invention as adjuvants, antiviral agents, or anticancer agents. The nucleic acid compounds or compositions of the present invention are also envisioned for use in methods of stimulating the immune system, treating / preventing viral infections, or treating or preventing cancers of subjects requiring such treatment or prevention. Nucleic acid compounds can act as adjuvants to activators or can be used for their own antiviral or anticancer activity. In some embodiments, nucleic acid compounds can be added as adjuvants, either formulated with or added separately to vaccines for infectious diseases or cancer vaccines or other vaccines used to stimulate the immune system.

[0230] Another aspect of the present invention is a method for stimulating the immune system of a subject in need, comprising administering to the subject an effective amount of a nucleic acid compound or composition of the present invention. Another method of the present invention is for treating or preventing a viral infection in a subject in need, comprising administering to the subject an effective amount of a nucleic acid compound or composition of the present invention. A further method of the present invention is for treating or preventing cancer in a subject in need, comprising administering to the subject an effective amount of a nucleic acid compound or composition of the present invention.

[0231] (How to use) The compositions of the present invention have numerous in vitro and in vivo applications, including the detection and / or quantification of RIG-I and / or RIG-I function agonism.

[0232] The compositions described above are particularly useful in methods for treating or preventing various cancers or infections of the target. The compositions can be administered to a target, such as a human subject, by various methods that are partly dependent on the route of administration. The route may be, for example, intravenous injection or infusion (IV), subcutaneous injection (SC), intradermal injection (ID), intraperitoneal injection (IP), intramuscular injection (IM), intratumoral injection (IT), or intrathecal injection. The injection may be a bolus or a continuous infusion.

[0233] The nucleic acid compounds of the present invention can induce interferon production, such as type I interferon, within cells.

[0234] Accordingly, the present invention provides for the use of nucleic acid compounds of the present invention to prevent and / or treat diseases or conditions in which inducing IFN production is beneficial, such as infectious diseases, tumors / cancers, inflammatory diseases and disorders, and immune disorders.

[0235] Administration can be achieved, for example, by local injection, injection, or implantation. The implant may be made of porous, non-porous, or gelatinous material, including membranes or fibers such as sialastic membranes. The implant may be configured for continuous or periodic release of the composition to a target. See, for example, U.S. Patent Application Publication No. 20080241223; U.S. Patent No. 5,501,856; No. 4,863,457; and No. 3,710,795; EP No. 488401; and EP No. 430539, each of which disclosures are fully incorporated herein by reference. The composition may be delivered to a target by, for example, an implantable device based on a diffusive, erosive, or convective system, such as an osmotic pump, biodegradable implant, electrodiffusion system, electroosmotic system, vapor pressure pump, electrolytic pump, foaming pump, piezoelectric pump, erosion-based system, or electromechanical system.

[0236] In some embodiments, nucleic acid compounds are therapeutically delivered to a target by topical administration.

[0237] The preferred dose of the nucleic acid compounds described herein (which may treat or prevent the target cancer) may depend on a variety of factors, including, for example, the age, sex, and weight of the subject receiving treatment, and the specific inhibitory compound used. Other factors that may influence the dose administered to a subject include, for example, the type or severity of the cancer or infection. For example, a subject with metastatic melanoma may require a different dose of the nucleic acid compound than a subject with glioblastoma. Other factors may include, for example, other medical conditions affecting the subject simultaneously or prior to treatment, the subject's overall health, genetic predisposition, diet, timing of administration, excretion rate, drug combinations, and other additional treatments administered to the subject. It should also be understood that the specific dosage and treatment regimen for any particular subject may also depend on the judgment of the healthcare professional providing the treatment (e.g., a physician or nurse). Preferred dosages are described herein.

[0238] A pharmaceutical composition may contain a therapeutically effective amount of the nucleic acid compound described herein. Such an effective amount can be readily determined by those skilled in the art, in part, based on the effect of the administered nucleic acid compound, or, if multiple agents are used, the combined effect of the nucleic acid compound and one or more additional activators. The therapeutically effective amount of the nucleic acid compound described herein may also vary considerably depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the agonist (and one or more additional activators) to induce a desired response in the individual, such as a reduction in tumor growth. For example, a therapeutically effective amount of a nucleic acid compound can inhibit (reduce the severity of or eliminate the occurrence of) and / or prevent any one of the symptoms of a particular disorder known in the art or described herein. The therapeutically effective amount is also the amount in which any toxic or adverse effects of the composition are offset by the therapeutically beneficial effects.

[0239] A suitable human dose of any of the nucleic acid compounds described herein can be further evaluated, for example, in a Phase I dose-escalation study. See, for example, van Gurp et al. (2008) Am J Transplantation 8(8): 1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1): 523-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10): 3499-3500.

[0240] In some embodiments, the composition contains any of the nucleic acid compounds described herein and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or more) additional therapeutic agents such that the composition as a whole is therapeutically effective. For example, the composition contains a nucleic acid compound and an alkylating agent described herein, where the agonist and the agent are in concentrations that, when combined, are therapeutically effective in treating or preventing the target cancer (e.g., melanoma).

[0241] The toxicity and therapeutic efficacy of such compositions can be determined by known pharmacological procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be, for example, LD 50 (A lethal dose in 50% of the population) and ED 50 It can be used to determine the dose that is effective in 50% of the population. The dose-to-toxicity ratio is the therapeutic index, which is the LD50. 50 / ED 50 This can be expressed as a ratio. Nucleic acid compounds exhibiting a high therapeutic index are preferred. Compositions exhibiting toxic side effects may be used, but care should be taken to design a delivery system that targets such compounds to the site of the affected tissue, minimizing potential damage to normal cells and thereby reducing side effects.

[0242] Data obtained from cell culture assays and animal studies can be used to determine the dosage range for human use. For nucleic acid compounds described herein, the therapeutically effective dose can be initially estimated from cell culture assays. The dose is then determined in animal models, and the EC is determined in cell culture. 50 A range of circulating plasma concentrations can be obtained, including the agonist concentration that achieves semi-maximal inhibition of symptoms. Using such information, an effective dose in humans can be determined more accurately. Plasma levels can be measured, for example, by high-performance liquid chromatography. In some embodiments, for example, when local administration (e.g., to the eyes or joints) is desired, cell cultures or animal models can be used to determine the dose required to achieve a therapeutically effective concentration at the local site.

[0243] In some embodiments, this method can be carried out in combination with other treatments for cancer or infection. For example, the composition can be administered to the subject simultaneously with, before, or after, radiation, surgery, targeted or cytotoxic chemotherapy, chemoradiotherapy, hormone therapy, immunotherapy, gene therapy, cell transplantation therapy, precision medicine, genome editing therapy, or other drug therapies.

[0244] As described above, the compositions described herein (e.g., nucleic acid compound compositions) can be used to treat, but are not limited to, Kaposi's sarcoma, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, myeloblastic promyelocytic myelomonocytic monocytic erythroleukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt lymphoma, marginal zone B-cell lymphoma, polycythemia vera, and Hodgson's disease. Kin's disease, non-Hodgkin's disease, multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, osteosarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangiosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, lipid Adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminocarcinoma, fetal cancer, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and It can treat a variety of cancers, including central nervous system (CNS) cancers, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancers, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, carcinoma in situ, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; oral cancers (e.g., lips, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, rectal cancer; respiratory system cancers, sarcomas, skin cancers, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, and urinary tract cancers.

[0245] In some embodiments, the Disclosure provides a method for increasing the production of one or more cytokines mediated by RIG-I in cells, comprising contacting the cells with a nucleic acid compound provided by the Invention, wherein the agonist increases the production of RIG-I mediated cytokines in the cells.

[0246] In some embodiments, the present disclosure provides a method for increasing the expression of one or more interferon-stimulated genes via RIG-I in cells, comprising contacting the cells with a nucleic acid compound provided by the present invention, wherein the agonist increases the expression of one or more interferon-stimulated genes via RIG-I in the cells.

[0247] In some embodiments, the present disclosure provides a method for increasing RIG-I-dependent signaling in cells, comprising contacting the cells with a nucleic acid compound provided by the present invention, wherein the agonist increases RIG-I-dependent signaling in the cells.

[0248] In some embodiments, the Disclosure provides a method for stimulating an immune response in a subject, comprising administering to the subject an effective amount of a nucleic acid compound or a pharmaceutical composition provided by the Invention.

[0249] In some embodiments, the Disclosure provides a method for treating cancer in a subject or delaying its progression, comprising administering to the subject an effective amount of a nucleic acid compound or a pharmaceutical composition provided by the Invention.

[0250] In some embodiments, the Disclosure provides a method for reducing or inhibiting tumor growth in a subject that requires such reduction, comprising administering to the subject an effective amount of a nucleic acid compound or a pharmaceutical composition provided by the Invention.

[0251] In some embodiments, the Disclosure provides a method for stimulating an immune response, treating or delaying the progression of cancer, or inhibiting tumor growth in a subject in need thereof, comprising administering to the subject a nucleic acid compound or pharmaceutical composition provided by the Invention, wherein the compound or pharmaceutical composition increases the production of one or more cytokines via RIG-I in cells, increases the expression of one or more interferon-stimulated genes via RIG-I in cells, and / or increases RIG-I-dependent intracellular signaling in cells, thereby stimulating an immune response, treating or delaying the progression of cancer, or inhibiting tumor growth.

[0252] In some embodiments, the Disclosure provides methods for treating, improving, and / or preventing viral infections caused by RNA or DNA viruses in a subject, and / or improving, minimizing, reversing, and / or preventing persistent viral infections, and / or minimizing or preventing mortality and / or lethality resulting from viral infections, comprising administering a therapeutically effective amount of the nucleic acid compound of the Invention to the subject. In embodiments, the subject is a tumor-bearing subject. In embodiments, the administration induces type I interferon production in at least one cell of the subject.

[0253] In this embodiment, administration is performed before the subject is exposed to the virus. In this embodiment, administration is performed after the subject is exposed to the virus. In this embodiment, administration reduces, minimizes, and / or prevents viral replication in the subject. In this embodiment, the virus may be a positive- and negative-strand RNA virus or a DNA virus.

[0254] In any of the methods described herein, the administration reduces the recovery time for at least one complication of the viral infection, eliminates or minimizes the complication.

[0255] In any of the methods described herein, at least one complication is at least one of the following: weight loss, fever, cough, fatigue, muscle pain and / or general pain, nausea, vomiting, diarrhea, shortness of breath, loss of smell and / or taste, acute respiratory distress syndrome (ARDS), low blood oxygen levels, pneumonia, multiple organ failure, septic shock, heart failure, arrhythmia, cardiac inflammation, thrombosis, and death.

[0256] In this embodiment, the virus includes at least one of the following: hepatitis C virus, hepatitis B virus, influenza virus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), varicella stomatitis virus (VSV), cytomegalovirus (CMV), poliovirus, encephalomyocarditis virus (EMCV), human papillomavirus (HPV), and smallpox virus.

[0257] In one embodiment, the virus includes an orthomyxoviridae virus. In another embodiment, the orthomyxoviridae virus includes at least one of alpha-influenza virus, beta-influenza virus, delta-influenza virus, gamma-influenza virus, Isa virus, Togoto virus, and Kuaranja virus. In another embodiment, the alpha-influenza virus includes at least one of influenza A virus, influenza B virus, and influenza C virus.

[0258] In one embodiment, the virus includes a coronavirus. In another embodiment, the coronavirus includes at least one of MERS-CoV, SARS-CoV, and SARS-CoV-2.

[0259] In this embodiment, SARS-CoV-2 infection is caused by at least one variant strain of SARS-CoV-2. In this embodiment, SARS-CoV-2 includes at least one variant selected from B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta), B.1.429 / B.1.427 (epsilon), B.1.617.1 (kappa), B.1.525 (eta), B.1.526 (iota), P.3 (theta), P.2 (zeta), and B.1.1.529 (omicron).

[0260] In this embodiment, SARS-CoV-2 includes at least one mutant strain selected from A.1-A.6, B.3-B.7, B.9, B.10, B.13-B.16, B.2, B.1 lineage, P.1, P.2, P.3, and R.1.

[0261] In this embodiment, the B.1 system includes (but is not limited to) B.1, B.1.1, B.1.1.7, B.1.2, B.1.5~B.1.72, B.1.9, B.1.13, B.1.22, B.1.26, B.1.37, B.1.3~B.1.66, B.1.177, B.1.243, B.1. (including B.1.351, B.1.427, B.1.429, B.1.525, B.1.526, B.1.526.1, B.1.526.2, B.1.617, B.1.617.1, B.1.617.2, B.1.617.3, B.1.619, B.1.620, and B.1.621) including at least one of these.

[0262] In all of the methods described herein, the subjects have been suffering from long-term COVID-19.

[0263] As described herein, nucleic acid compounds are useful for treating tumors, wherein the tumors include cancers selected from biliary tract cancer, brain tumors, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, carcinoma in situ, leukemia, lymphoma, liver cancer, lung cancer, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, or kidney cancer.

[0264] In this embodiment, the tumor includes cancers selected from hairy cell leukemia, chronic myeloid leukemia, cutaneous T-cell leukemia, chronic myeloid leukemia, non-Hodgkin lymphoma, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, renal cell carcinoma, prostate cancer, bladder cell carcinoma, breast cancer, ovarian cancer, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, colon cancer, cervical dysplasia, and Kaposi's sarcoma (AIDS-related and non-AIDS-related).

[0265] Alternatively, polynucleotide molecules according to formula II, particularly antisense oligonucleotides or siRNA molecules, may be intended to downregulate, reduce, silence, or knock down the expression of a gene, especially when an endogenous gene is overexpressed, resulting in the overexpression of the encoded protein (or other gene product), and such overexpression contributes to cellular dysfunction; or when the gene is defective or dysfunctional, resulting in the encoded protein (or other gene product) being defective or dysfunctional, and therefore contributing to cellular dysfunction.

[0266] Therefore, in one embodiment, an aqueous liquid pharmaceutical formulation for use according to the present invention is provided, wherein the aqueous liquid pharmaceutical formulation reduces the endogenous expression of a protein (or other gene product). As used herein, the term “reduce” includes restoring endogenous gene expression, i.e., reducing it from a high value to a “normal” value, and impairing it, i.e., reducing it from a “normal” value to a low value or zero, for example, silencing.

[0267] Therefore, in one embodiment, the aqueous liquid pharmaceutical formulation for use according to the present invention, in which the polynucleotide molecule is a polynucleotide molecule that reduces the endogenous expression of a protein (or other gene product), is for use in the treatment of a disease or condition that is treated by reducing the endogenous expression of a protein (or other gene product).

[0268] In an alternative embodiment, the present invention provides a method for treating a disease or condition treated by reducing the endogenous expression of a protein (or other gene product), comprising administering a therapeutically or prophylactically effective amount of an aqueous liquid pharmaceutical formulation described herein to a subject in need thereof, wherein the polynucleotide molecule is a polynucleotide molecule that reduces the endogenous expression of a protein (or other gene product).

[0269] Furthermore, the present invention provides the use of an aqueous liquid pharmaceutical formulation described herein, in the manufacture of a pharmaceutical for use in the treatment of a disease or condition treated by reducing the endogenous expression of a protein (or other gene product), wherein the polynucleotide molecule is a polynucleotide molecule that reduces the endogenous expression of a protein (or other gene product).

[0270] In one embodiment, the disease or condition treated by reducing the endogenous expression of a protein (or other gene product) is an infectious disease. Preferably, the infectious disease is of bacterial, fungal, parasitic, or viral origin. In particular, the infectious disease is a viral infection or a disease associated with such a viral infection. Preferably, the virus infects the respiratory tract, and the disease associated with the viral infection is a respiratory disease.

[0271] In this embodiment, the nucleic acid compound or composition thereof according to the present invention is suitable for use as a pharmaceutical.

[0272] In embodiments, nucleic acid compounds or compositions thereof according to the present invention are used in the treatment of diseases or conditions that are treated by stimulating or activating the innate and / or adaptive immune systems, and / or by inducing innate and / or adaptive immune responses.

[0273] In embodiments, the present invention provides a method for treating a disease or condition treated by stimulation or activation of the innate and / or adaptive immune system, and / or induction of an innate and / or adaptive immune response, comprising administering a therapeutic or prophylactic effective amount of one or more nucleic acid compounds or compositions thereof according to the present invention to a subject in need thereof.

[0274] In any embodiment of the use or method described herein, the disease or condition is a viral infection or related to such viral infection. In the embodiment, the virus infects the respiratory system, and the disease related to the infection is a respiratory disease.

[0275] In any embodiment of the use or method described herein, the disease or condition is cancer.

[0276] In any of the methods described herein, the subjects may be immunodeficient and / or immunocompromised subjects.

[0277] In any of the methods described herein, the nucleic acid compounds of the present invention can be combined with antiviral agents such as protease inhibitors, polymerase inhibitors, integrase inhibitors, virus entry blockers, and antiviral antibodies.

[0278] (Combination of nucleic acid compounds and additional therapeutic agents) In some embodiments, the nucleic acid compounds described herein may be administered to a subject as monotherapy. Alternatively, the nucleic acid compounds may be administered to a subject as combination therapy with another treatment, for example, another treatment for cancer. For example, combination therapy may include administering one or more additional agents to a subject (e.g., a human patient) that provide a therapeutic benefit to a subject who has cancer or is at risk of developing cancer.

[0279] In some embodiments of the methods provided herein, a nucleic acid compound or pharmaceutical composition is administered in combination with one or more additional therapeutic agents, wherein one or more additional therapeutic agents are selected from the group consisting of: chemotherapy, targeted anticancer therapy, oncolytic drugs, cell death inducers, opsonizing agents (e.g., opsonized antibodies), cytotoxic agents, immune-based therapies, cytokines, activators or agonists of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, cellular immunotherapy, or combinations thereof.

[0280] In some embodiments, the nucleic acid compound or pharmaceutical composition is administered prior to or after the administration of one or more additional therapeutic agents, wherein the one or more additional therapeutic agents are administered simultaneously with, prior to, or after the administration of the agonist or pharmaceutical composition.

[0281] In some embodiments, one or more additional therapeutic agents are immune checkpoint inhibitors. In some embodiments, one or more additional therapeutic agents are PD-1 / PD-L1 antagonists, TIM-3 antagonists, VISTA antagonists, adenosine A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, CTLA-4 antagonists, IDO antagonists, KIR antagonists, LAG-3 antagonists, Toll-like receptor 3 (TLR3) agonists, Toll-like receptor 7 (TLR7) agonists, and Toll-like receptor 9 (TLR9) agonists.

[0282] (In combination with chemotherapy agents) Suitable chemotherapeutic agents for combination and / or co-administration with the compositions of the present invention include, for example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrancindione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogs or homologs. Further drugs include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechloretamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum(II) (DDP), procarbazolamine Examples include din, altoretamine, cisplatin, carboplatin, oxaliplatin, nedaplatin, satoraplatin, or triplatin tetranitrate), anthracyclines (e.g., daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomcin (formerly known as actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine), as well as temozolomide.

[0283] (In combination with PD-1 / PD-L1 antagonists) In some embodiments, nucleic acid compounds or pharmaceutical compositions thereof provided by this disclosure are combined with one or more PD-1 / PD-L1 antagonists that specifically bind to human PD-1 or PD-L1 and inhibit cellular processes mediated by the biological activity of PD-1 / PD-L1 and / or downstream pathways and / or human PD-1 / PD-L1 signaling or other human PD-1 / PD-L1-mediated functions (e.g., administered in combination).

[0284] Accordingly, provided herein are PD-1 / PD-L1 antagonists that directly or allosterically block, antagonistize, repress, inhibit, or reduce the biological activity of PD-1 / PD-L1, including downstream pathways and / or cellular processes mediated by PD-1 / PD-L1 signaling, such as receptor binding and / or induction of cellular responses to PD-1 / PD-L1. Also provided herein are PD-1 / PD-L1 antagonists that reduce the quantity or amount of human PD-1 or PD-L1 produced by cells or subjects.

[0285] In some embodiments, the Disclosure provides PD-1 / PD-L1 antagonists that bind to human PD-1 and interfere with, inhibit, or reduce the binding of PD-L1 to PD-1. In some embodiments, the PD-1 / PD-L1 antagonists bind to mRNA encoding PD-1 or PD-L1 and interfere with its translation. In some embodiments, the PD-1 / PD-L1 antagonists bind to mRNA encoding PD-1 or PD-L1 and cause degradation and / or turnover.

[0286] In some embodiments, PD-1 / PD-L1 antagonists inhibit PD-1 signaling or function. In some embodiments, PD-1 / PD-L1 antagonists block the binding of PD-1 to PD-L1, PD-L2, or both PD-L1 and PD-L2. In some embodiments, PD-1 / PD-L1 antagonists block the binding of PD-1 to PD-L1. In some embodiments, PD-1 / PD-L1 antagonists block the binding of PD-1 to PD-L2. In some embodiments, PD-1 / PD-L1 antagonists block the binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, PD-1 / PD-L1 antagonists specifically bind to PD-1. In some embodiments, PD-1 / PD-L1 antagonists specifically bind to PD-L1. In some embodiments, PD-1 / PD-L1 antagonists specifically bind to PD-L2.

[0287] In some embodiments, the PD-1 / PD-L1 antagonist inhibits the binding of PD-1 to its homologous ligand. In some embodiments, the PD-1 / PD-L1 antagonist inhibits the binding of PD-1 to PD-L1, to PD-1 to PD-L2, or to both PD-L1 and PD-L2. In some embodiments, the PD-1 / PD-L1 antagonist does not inhibit the binding of PD-1 to its homologous ligand.

[0288] In some embodiments, the PD-1 / PD-L1 antagonist is an isolated monoclonal antibody (mAb) or its antigen-binding fragment that specifically binds to PD-1 or PD-L1. In some embodiments, the PD-1 / PD-L1 antagonist is an antibody or its antigen-binding fragment that specifically binds to human PD-1. In some embodiments, the PD-1 / PD-L1 antagonist is an antibody or its antigen-binding fragment that specifically binds to human PD-L1. In some embodiments, the PD-1 / PD-L1 antagonist is an antibody or its antigen-binding fragment that binds to human PD-L1 and inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1 / PD-L1 antagonist is an antibody or its antigen-binding fragment that binds to human PD-1 and inhibits the binding of PD-L1 to PD-1.

[0289] Several immune checkpoint antagonists that inhibit or disrupt the interaction between PD-1 and either or both of its ligands, PD-L1 and PD-L2, are in clinical development or are currently available to clinicians for the treatment of cancer.

[0290] Examples of anti-human PD-1 monoclonal antibodies or antigen-binding fragments thereof that may contain a PD-1 / PD-L1 antagonist in any of the compositions, methods, and uses provided herein include KEYTRUDA® (pembrolizumab, MK-3475, h409A11; see U.S. Patents 8,952,136, 8,354,509, 8,900,587, and EP2170959, all of which are incorporated herein by reference); Merck), OPDIVO (registered trademark) (nivolumab, BMS-936558, MDX-1106, ONO-4538; all of which are incorporated herein by reference in their entirety, see U.S. Patents No. 7,595,048, 8,728,474, 9,073,994, 9,067,999, EP1537878, U.S. Patents No. 8,008,449, 8,779,105, and EP2161336; Bristol Myers Squibb), MEDI0680 (AMP-514), BGB-A317 and BGB-108 (BeiGene), 244C8 and 388D4 (all of which are incorporated herein by reference in their entirety, see WO2016106159; Enumeral Examples include, but are not limited to, Biomedical, PDR001 (Novartis), and REGN2810 (Regeneron). Therefore, in some embodiments, the PD-1 / PD-L1 antagonist is pembrolizumab. In some embodiments, the PD-1 / PD-L1 antagonist is nivolumab.

[0291] Examples of anti-human PD-L1 monoclonal antibodies or antigen-binding fragments thereof that may contain a PD-1 / PD-L1 antagonist in any of the compositions, methods, and uses provided herein include BAVENCIO® (avelumab, MSB0010718C, see WO2013 / 79174, fully incorporated herein by reference; Merck / Pfizer), IMFINZI® (durvalumab, MEDI4736), TECENTRIQ® (atezolizumab, MPDL3280A, RG7446, see WO2010 / 077634, fully incorporated herein by reference; Roche), and MDX-1105 (BMS-936559, 12A4, both see U.S. Patent Nos. 7,943,743 and WO2013 / 173223, fully incorporated herein by reference). Examples include, but are not limited to, Medarex / BMS and FAZ053 (Novartis). Therefore, in some embodiments, the PD-1 / PD-L1 antagonist is avelumab. In some embodiments, the PD-1 / PD-L1 antagonist is durvalumab. In some embodiments, the PD-1 / PD-L1 antagonist is atezolizumab.

[0292] In some embodiments, the PD-1 / PD-L1 antagonist is a fusion protein containing an extracellular domain or PD-1 binding domain of PD-L1 or PD-L2 fused to a constant domain, such as the Fc region of an immunoglobulin molecule, which is an immunoadhesin that specifically binds to human PD-1 or human PD-L1. Examples of immunoadhesins that specifically bind to PD-1 are described in WO2010 / 027827 and WO2011 / 066342, both of which are fully incorporated herein by reference. In some embodiments, the PD-1 / PD-L1 antagonist is AMP-224 (also known as B7-DCIg), a PD-L2-FC fusion protein that specifically binds to human PD-1.

[0293] Those skilled in the art will understand that any PD-1 / PD-L1 antagonist that binds to PD-1 or PD-L1 and disrupts the PD-1 / PD-L1 signaling pathway is suitable for the compositions, methods, and applications disclosed herein.

[0294] In some embodiments, the PD-1 / PD-L1 antagonist is a small molecule, nucleic acid, peptide, peptide mime, protein, carbohydrate, carbohydrate derivative, or glycopolymer. Exemplary small molecule PD-1 inhibitors are described in Zhan et al. (2016) Drug Discov Today 21(6):1027-1036.

[0295] In some embodiments of the methods provided herein, a nucleic acid compound is combined with a PD-1 / PD-L1 antagonist, where the PD-1 / PD-L1 antagonist is selected from the group consisting of PDR001, KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), pizilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In some embodiments, the PD-1 / PD-L1 antagonist is selected from the group consisting of FAZ053, TENCENTRIQ® (atezolizumab), BAVENCIO® (avelumab), IMFINZI® (durvalumab), and BMS-936559.

[0296] (In combination with TIM-3 Antagonist) In some embodiments, the nucleic acid compounds or pharmaceutical compositions provided herein are combined with (for example, administered in combination with) a TIM-3 antagonist. The TIM-3 antagonist may be an antibody or its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the TIM-3 antagonist is selected from MGB453 (Novartis), TSR-022 (Tesaro), or LY3321367 (Eli Lilly).

[0297] (In combination with LAG-3 Antagonist) In some embodiments, the nucleic acid compounds or pharmaceutical compositions provided herein are combined with (e.g., administered in combination with) a LAG-3 antagonist. The LAG-3 antagonist may be an antibody or its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the LAG-3 inhibitor is selected from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb), TSR-033 (Tesaro), MK-4280 (Merck & Co), or REGN3767 (Regeneron).

[0298] (In combination with Toll-like receptor (TLR) agonists) In some embodiments, the nucleic acid compounds or pharmaceutical compositions provided herein are combined with (for example, administered in combination with) TLR agonists.

[0299] Toll-like receptors (TLRs) are a family of germline-encoded transmembrane proteins that promote pathogen recognition and activation of the innate immune system (Hoffmann et al. (1999) Science 284:1313-1318; Rock et al. (1998) Proc Natl Acad Sci USA 95:588-593). TLRs are pattern recognition receptors (PRRs) expressed by cells of the innate immune system. Examples of known ligands for TLRs include Gram-positive bacteria (TLR-2), bacterial endotoxins (TLR-4), flagellin proteins (TLR-5), bacterial DNA (TLR-9), double-stranded RNA and poly(I:C) (TLR-3), and yeast (TLR-2). In vivo activation of TLRs initiates an innate immune response involving specific cytokines, chemokines, and growth factors. While all TLRs can activate specific intracellular signaling molecules such as nuclear factor kappa beta (NF-κB) and mitogen-activated protein kinase (MAP kinase), the specific pairs of cytokines and chemokines released appear to be specific to each TLR. TLR7, 8, and 9 comprise a subfamily of TLRs located in the endosomal or lysosomal compartments of immune cells such as dendritic cells and monocytes. In contrast to TLR7 and 9, which are highly expressed in plasmacytoid dendritic cells (pDCs), TLR8 is primarily expressed in myeloid dendritic cells (mDCs) and monocytes. This subfamily mediates the recognition of microbial nucleic acids, such as single-stranded RNA.

[0300] The first TLR7 and TLR8 agonists identified were small, low molecular weight (less than 400 daltons) synthetic imidazoquinoline compounds that are similar to the purine nucleotides adenosine and guanosine. Many of these compounds have shown antiviral and anticancer properties. For example, the TLR7 agonist imiquimod (ALDARA®) has been approved by the U.S. Food and Drug Administration as a topical agent for the treatment of skin lesions caused by certain strains of human papillomavirus. Imiquimod may also be useful in the treatment of primary skin cancers and skin tumors such as basal cell carcinoma, keratocytoma, actinic keratosis, and Bowen's disease. The TLR7 / 8 agonist rexiquimod (R-848) is being evaluated as a topical agent for the treatment of human genital herpes.

[0301] The TLR agonists described herein can be any TLR agonist. For example, TLR agonists can include natural or synthetic TLR ligands, mutaines or derivatives of TLR ligands, peptide mimes of TLR ligands, small molecules that mimic the biological function of TLR ligands, or antibodies that stimulate TLR receptors. A TLR ligand is any molecule that binds to a TLR.

[0302] In some embodiments, the nucleic acid compounds or pharmaceutical compositions thereof provided by this disclosure are combined with a TLR agonist, where the TLR agonist is selected from the group consisting of TLR1 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists, TLR9 agonists, TLR10 agonists, and TLR11 agonists.

[0303] In some embodiments, the nucleic acid compounds provided by the present invention are combined with TLR3 agonists. TLR3 agonists are agonists that induce a signal response via TLR3. Representative TLR3 agonists include polyinosinic acid:polycytidic acid (Poly I:C), HILTONOL® (Poly ICLC), polyadenylate polyuridylic acid (Poly A:U), RIBOXXIM® (RGIC® 100), RIBOXXON® (RGIC® 50 bioconjugate), and RIBOXXOL® (RGIC® 50).

[0304] In some embodiments, the nucleic acid compound according to the present invention is combined with polyinosinic acid:polycytidic acid (Poly I:C). In some embodiments, the nucleic acid compound is combined with HILTONOL® (Poly ICLC). In some embodiments, the nucleic acid compound is combined with polyadenylic acid-polyuridylic acid (Poly A:U). In some embodiments, the nucleic acid compound is combined with RIBOXXIM® (RGIC® 100). In some embodiments, the nucleic acid compound is combined with RIBOXXON® (RGIC® 50 bioconjugate). In some embodiments, the nucleic acid compound is combined with RIBOXXOL® (RGIC® 50).

[0305] In some embodiments, the nucleic acid compounds provided by the present invention are combined with TLR7 agonists. A TLR7 agonist is an agonist that elicits a signal response via TLR7. Non-limiting examples of TLR7 agonists include single-stranded RNA (ssRNA), loxoribine (guanosine analogs derivatized at the N7 and C8 positions), imidazoquinoline compounds (e.g., imiquimod and reximod), or derivatives thereof. Further exemplary TLR7 agonists include, but are not limited to, GS-9620 (besatrimod), imiquimod (ALDARA®), and reximod (R-848).

[0306] In some embodiments, the nucleic acid compound provided by the present invention is combined with GS-9620 (besatrimod). In some embodiments, the nucleic acid compound is combined with imiquimod (ALDARA®). In some embodiments, the nucleic acid compound is combined with reximod (R-848).

[0307] In some embodiments, the nucleic acid compounds provided by the present invention are combined with a TLR9 agonist. A TLR9 agonist is an agonist that elicits a signal response via TLR9. Examples of TLR9 agonists include, but are not limited to, CpG oligodeoxynucleotides (CpG ODNs). In some embodiments, the CpG ODN is a class A CpG ODN (CpG-A ODN), a class B CpG ODN (CpG-B ODN), or a class C CpG ODN (CpG-B ODN).

[0308] In some embodiments, the nucleic acid compounds provided by the present invention are combined with CpG oligodeoxynucleotides (CpG ODNs). In some embodiments, the CpG ODNs are class A CpG ODNs (CpG-A ODNs). In some embodiments, the CpG ODNs are class B CpG ODNs (CpG-B ODNs). In some embodiments, the CpG ODNs are class C CpG ODNs (CpG-C ODNs).

[0309] (Other combinations) In some embodiments, the nucleic acid compounds or pharmaceutical compositions provided by the present invention are combined with (for example, administered in combination with) a VISTA antagonist, an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO antagonist, or a KIR antagonist.

[0310] In some embodiments, the nucleic acid compounds or pharmaceutical compositions provided by the present invention are combined with an agonist comprising a polypeptide (e.g., an antibody or its antigen-binding moiety) that specifically binds to CD137(4-1BB) (e.g., administered in combination).

[0311] In some embodiments, the nucleic acid compounds or pharmaceutical compositions provided by the present invention are combined with an agonist comprising a polypeptide (e.g., an antibody or its antigen-binding moiety) that specifically binds to CD134(OX40) (e.g., administered in combination).

[0312] The nucleic acid compounds described herein can replace or enhance therapies that have been or are currently being administered. For example, treatment with a nucleic acid compound may discontinue or reduce the administration of one or more additional activators, for example, allowing them to be administered at lower levels or dosages. In some embodiments, the administration of the previous therapy can be maintained. In some embodiments, the previous therapy is maintained until the level of the nucleic acid compound reaches a level sufficient to produce a therapeutic effect. Two therapies can be administered in combination.

[0313] Monitoring a subject (e.g., a human patient) for improvement in cancer as defined herein means evaluating the subject for changes in disease parameters, such as a decrease in tumor growth. In some embodiments, the evaluation is performed at least one hour after administration, for example, at least 2, 4, 6, 8, 12, 24, or 48 hours later, or at least 1, 2, 4, 10, 13, or 20 days later, or at least 1, 2, 4, 10, 13, or 20 weeks later. The subject may be evaluated during one or more of the following periods: before the start of treatment; during treatment; or after the administration of one or more elements of treatment. The evaluation may include assessing the need for further treatment, for example, whether the dosage, frequency of administration, or duration of treatment should be changed. This may also include assessing the need to add or discontinue a selected treatment modality, for example, adding or discontinuing any of the cancer treatments described herein.

[0314] In some embodiments, the nucleic acid compounds described herein are administered to modulate a patient's T cell response, for example, by increasing T cell activation and / or proliferation. Enhancement of T cell proliferation, IFN production and secretion, and / or T cell cytolytic activity may be beneficial to patients who need it to treat a disease or condition. Accordingly, in some embodiments, the nucleic acid compounds of the present invention are administered to patients who need it to induce or increase T cell activation, enhance T cell proliferation, induce IFN production and / or secretion, and / or induce a cytolytic T cell response.

[0315] In some embodiments, the nucleic acid compounds described herein can be used in methods for detecting and / or quantifying human RIG-I in biological samples. Therefore, the nucleic acid compounds described herein can be used to diagnose diseases (e.g., cancer) in patients, determine their prognosis, and / or determine their progression.

[0316] In some embodiments, the nucleic acid compounds described herein can be used in combination with vaccines to enhance their immunogenicity. Such vaccines include, but are not limited to, existing and emerging vaccines against infectious diseases such as influenza virus, respiratory syncytial virus, rotavirus, Ebola virus, poliovirus, smallpox virus, cowpox virus, monkeypox virus, mumps, hepatitis B virus, and bacteria including tetanus and tuberculosis, and parasites such as malaria. Table of compounds (connector element (L) is underlined) [Table 1]

[0317] (definition) Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but only selected methods and materials are described.

[0318] As used herein, the following terms have the meanings associated with them in this section.

[0319] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the article. For example, “an element” means one or more elements.

[0320] As used herein, the term “about” when referring to measurable values ​​such as quantity or temporal duration is intended to encompass variations of 20% or 10%, more preferably 5%, even more preferably +1%, and still more preferably 0.1% from a specified value, and such variations are therefore appropriate for carrying out the disclosed method.

[0321] Agonist: As used herein, the term “agonist” is used in its broadest sense and encompasses any molecule or compound that partially or completely promotes, induces, increases, and / or activates the biological activity of any natural polypeptide disclosed herein. Agonist molecules according to this disclosure include nucleic acids (e.g., oligonucleotides, polynucleotides), antibodies or antigen-binding fragments, fragments or amino acid sequence variants of natural polypeptides, peptides, oligonucleotides, lipids, carbohydrates, and small organic molecules. In some embodiments, activation in the presence of an agonist is observed in a dose-dependent manner. In some embodiments, the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% higher than the signal measured using a negative control under comparable conditions. Methods for identifying agonists suitable for use in the methods of the present disclosure are also disclosed herein. Examples of such methods include, but are not limited to, binding assays such as enzyme-linked immunosorbent assays (ELISA), Forte Bio® systems, fluorescence polarization (FP) assays, and radioimmunoassays (RIA). These assays determine the ability of an agonist to bind to a target polypeptide (e.g., a receptor or ligand), and thus demonstrate the agonist's ability to promote, enhance, or activate the polypeptide's activity. The efficacy of an agonist can also be determined using functional assays, e.g., the agonist's ability to activate or promote the function of a polypeptide. For example, a functional assay may involve contacting a polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities typically associated with the polypeptide. The potency of an agonist is usually determined by EC 50Defined by the value (the concentration required to activate 50% of the agonist response). EC 50 The lower the value, the more potent the agonist is, and the lower the concentration required to activate the maximum biological response.

[0322] To improve: As used herein, the term “improve” means any therapeutically beneficial outcome in the treatment of a disease condition, such as cancer, including prevention, reduction of its severity or progression, remission, or cure.

[0323] Amino Acids: As used herein, the term “amino acids” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., hydrogen, a carboxyl group, an amino group, and a carbon atom bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic structure as naturally occurring amino acids. Amino acid mimes refer to compounds that have a different structure from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

[0324] Amino acids may be referred to herein by either their commonly known three-letter code or the one-letter code recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted one-letter code.

[0325] Amino acid substitution: As used herein, “amino acid substitution” refers to the substitution of at least one existing amino acid residue in a given amino acid sequence (the amino acid sequence of the starting polypeptide) with a second, different “substitutive” amino acid residue. “Amino acid insertion” refers to the incorporation of at least one additional amino acid into a given amino acid sequence. Insertions typically consist of the insertion of one or two amino acid residues, but larger “peptide insertions,” e.g., insertions of about 3 to about 5 or up to about 10, 15, or 20 amino acid residues, are also possible. The inserted residues may be naturally occurring or non-natural, as discussed above. “Amino acid deletion” refers to the removal of at least one amino acid residue from a given amino acid sequence.

[0326] Base composition: As used herein, the term “base composition” refers to the proportion of all nucleotides in a nucleic acid (e.g., RNA) consisting of guanine (or hypoxanthine) + cytosine and / or uracil (or thymine) + adenine nucleobases.

[0327] Base pairing: As used herein, the term “base pairing” refers to two nucleobases on opposite, complementary polynucleotide chains, or regions of the same chain, that interact through the formation of a specific hydrogen bond. As used herein, the term “Watson-Crick base pairing,” used interchangeably with “complementary base pairing,” refers to a set of base pairing rules in which, in the DNA molecule, purines always bind to pyrimidines, such that the nucleobase adenine (A) forms a complementary base pair with thymine (T), and guanine (G) forms a complementary base pair with cytosine (C). In the RNA molecule, thymine is replaced by uracil (U), which, like thymine (T), forms a complementary base pair with adenine (A). Complementary base pairs are linked by hydrogen bonds, the number of hydrogen bonds differing between base pairs. As is well known in the art, guanine (G)-cytosine (C) base pairs are linked by three hydrogen bonds, and adenine (A)-thymine (T) or uracil (U) base pairs are linked by two hydrogen bonds.

[0328] Base pair interactions that do not follow these rules can occur in natural, unnatural, and synthetic nucleic acids and are referred to herein as “non-Watson-Crick base pairing” or “non-standard base pairing” and universal nucleobases. “Fluctuating base pairing” refers to the pairing of two nucleobases in an RNA molecule that does not follow the Watson-Crick base pairing rules. For example, inosine is a nucleoside that is structurally similar to guanosine but lacks a 2-amino group. Inosine can form two hydrogen bonds with each of the four natural nucleobases (Oda et al. (1991) Nucleic Acids Res 19:5263-5267) and is often used by researchers as a “universal” base, meaning that inosine can base pair with all naturally occurring or standard bases. The four main fluctuating base pairs are the guanine-uracil (GU) base pair, the hypoxanthine-uracil (IU) base pair, the hypoxanthine-adenine (IA) base pair, and the hypoxanthine-cytosine (IC) base pair. To maintain consistency in nucleic acid nomenclature, "I" is used for hypoxanthine because it is the nucleobase of inosine; otherwise, the nomenclature follows the names of the nucleobase and its corresponding nucleoside (e.g., "G" for both guanine and guanosine, and for deoxyguanosine). The thermodynamic stability of fluctuating base pairs is equivalent to that of Watson-Crick base pairs. Fluctuating base pairs play a role in the formation of the secondary structure of RNA molecules.

[0329] Biologically active: As used herein, the term “biologically active” refers to the characteristic of any substance that is active in a biological system and / or organism. For example, a substance that, when administered to an organism, has a biological effect on that organism is considered biologically active and therefore has biological activity. In certain embodiments, if a nucleic acid is biologically active, the entire nucleic acid and the portion of that nucleic acid that shares at least one biological activity are usually referred to as the “biologically active” portion.

[0330] Blunt ends: As used herein, the terms “blunt ends” and “blunt-ended” refer to the terminal structure of a double-stranded or double-stranded nucleic acid in which both complementary strands, including the double helix, are terminated by a base pair at at least one end. Thus, neither strand, including the double helix, extends further from the end than the other strand.

[0331] As used herein, the term “cancer” is defined as a disease characterized by the rapid and uncontrolled proliferation of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain tumors, lymphoma, leukemia, and lung cancer.

[0332] "Complementary" refers to the broad concept of sequence complementarity between regions of two nucleic acid chains or between two regions of the same nucleic acid chain. It is known that an adenine residue in the first nucleic acid region can form a specific hydrogen bond ("base-pair") with a residue in the second nucleic acid region that is antiparallel to the first region, provided that the residue is thymine or uracil. Similarly, it is known that a cytosine residue in the first nucleic acid chain can base-pair with a residue in the second nucleic acid chain that is antiparallel to the first chain, provided that the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged antiparallel, at least one nucleotide residue in the first region can base-pair with a residue in the second region. In one embodiment, the first region includes a first portion and the second region includes a second portion, so that when the first and second portions are arranged in an antiparallel configuration, at least about 50%, preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion can form base pairs with the nucleotide residues of the second portion. In one embodiment, all of the nucleotide residues of the first portion can form base pairs with the nucleotide residues of the second portion.

[0333] Covalently linked: As used herein, “covalently linked” (or, “conjugated,” “linked,” “joined,” “fused,” or “tethered” means, when used in reference to two or more parts, that these parts are physically related or linked to one another, either directly or through one or more additional parts acting as linkers, by any means including chemical conjugation, recombinant techniques, or enzymatic activity, to the extent that these parts are stable enough to maintain a physically linked state under the conditions in which the structure is used, e.g., physiological conditions)

[0334] As used herein, the term “fragment” applied to nucleic acids refers to a subsequence of a larger nucleic acid. A “fragment” of nucleic acid can be at least about 5 nucleotides in length; for example, at least about 10 to about 100 nucleotides; at least about 100 to about 500 nucleotides; at least about 500 to about 1000 nucleotides; at least about 1000 to about 1500 nucleotides; or about 1500 to about 2500 nucleotides; or about 2500 nucleotides (and any integer value in between).

[0335] As used herein, “homology,” “homology,” or “identity,” “identity,” as used herein, refers to comparisons between amino acid sequences and nucleic acid sequences. When referring to nucleic acid molecules, “homology,” “identity,” or “identity percentage” refers to the percentage of nucleotides in the target nucleic acid sequence that have been matched with identical nucleotides by a sequence analysis program. Homology can be readily calculated by known methods. Nucleic acid sequences and amino acid sequences can be compared using computer programs that align similar nucleic acid or amino acid sequences and, as a result, define their differences. In preferred methods, the BLAST program (NCBI) and its parameters are used, and ExPaSy is used to align sequence fragments of genomic DNA sequences. However, equivalent alignment evaluations can be obtained with any standard alignment software.

[0336] As used herein, “homologous” refers to the similarity of subunit sequences between two polymer molecules, e.g., two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. Two DNA molecules are homologous in that position if the subunit positions in both are occupied by the same subunit, for example, if the positions in each of the two DNA molecules are occupied by adenine. The homology between two sequences is a direct function of the number of matching or homologous positions. For example, if half of the positions of two composite sequences (e.g., five positions in a polymer with a length of 10 subunits) are homologous, these two sequences are 50% homologous, and if 90% of the positions, e.g., nine out of ten are matching or homologous, these two sequences share 90% homology. As an example, the DNA sequences 5'-ATTG-3' and 5'-AATC-3' share 50% homology.

[0337] The term "hybridization" refers to the process by which two single-stranded nucleic acids are non-covalently joined to form a double-stranded nucleic acid; triple-stranded hybridization is also theoretically possible. Complementary sequences in nucleic acids pair up to form a double helix. The resulting double-stranded nucleic acid is a "hybrid." Hybridization can occur, for example, between two complementary or partially complementary sequences. A hybrid may have a double-stranded region and a single-stranded region. A hybrid can be DNA:DNA, RNA:DNA, or DNA:RNA. Hybrids can also be formed between modified nucleic acids. One or both nucleic acids may be immobilized on a solid support. Hybridization techniques can be used to detect and isolate specific sequences, measure homology, or define other features of one or both strands.

[0338] The stability of the hybrid depends on various factors, including the length of complementarity, the presence of mismatches within the complementary region, temperature, and salt concentration during the reaction. Hybridization is typically carried out under stringent conditions, for example, at a salt concentration of 1 M or less and a temperature of at least 25°C. For example, conditions such as 5×SSPE (750 mM NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7.4) or 100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween-20, and a temperature of 25–50°C are suitable for allele-specific probe hybridization. In a particularly preferred embodiment, hybridization is carried out at 40–50°C. Acetylated BSA and herring sperm DNA can be added to the hybridization reaction. Suitable hybridization conditions for microarrays are described in the Gene Expression Technical Manual and GeneChip Mapping Assay Manual, available from Affymetrix (Santa Clara, Calif.).

[0339] When two oligonucleotides anneal under conditions where only oligonucleotides that are at least about 75%, preferably at least about 90% or at least about 95%, complementary to each other, the first oligonucleotide anneals with the second oligonucleotide in a "high stringency" manner. The stringency of the conditions used to cause two oligonucleotides to anneal is a function of several factors, among others, particularly temperature, the ionic strength of the annealing medium, incubation period, oligonucleotide length, GC content of the oligonucleotides, and, where known, the degree of expected non-homologousity between the two oligonucleotides. Methods for adjusting the stringency of annealing conditions are known (see, for example, Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0340] Needing: As used herein, “needing prevention,” “needing treatment,” or “needing it” means a subject who, in the judgment of an appropriate healthcare professional (e.g., a physician, nurse, or nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from the treatment in question (e.g., treatment with a compound or composition containing a RIG-I agonist).

[0341] Where used herein, “Educational Materials” includes publications, records, diagrams, or any other medium of expression that can be used to communicate the usefulness of the compounds, compositions, vectors, or delivery systems of the present invention in kits for alleviating various diseases or disorders listed herein. Optionally, or instead, educational materials may describe one or more methods for alleviating diseases or disorders in mammalian cells or tissues. Educational materials for the kits of the present invention may, for example, be attached to a container containing the specified compounds, compositions, vectors, or delivery systems, or shipped together with the container containing the specified compounds, compositions, vectors, or delivery systems. Alternatively, educational materials may be shipped separately from the container, with the intention that the educational materials and compounds be used in conjunction by the recipient.

[0342] As used herein, “isolate” refers to nucleic acids obtained from an organism or from a sample obtained from an organism. Nucleic acids can be analyzed at any time after they are obtained (e.g., before and after laboratory culture, before and after amplification).

[0343] As used herein, the term “label” refers to luminescent labels, light-scattering labels, or radioactive labels. Examples of fluorescent labels include, but are not limited to, commercially available fluorescein phosphoamidites such as Fluoreprime (Pharmacia), Fluoredite (Millipore), and FAM (ABI). See U.S. Patent No. 6,287,778.

[0344] The term "mismatch" refers to a nucleic acid whose sequence is not fully complementary to a particular target sequence. A mismatch may contain one or more bases. As used herein, "nucleic acid" refers not only to naturally occurring molecules such as DNA and RNA, but also to various derivatives and analogues.

[0345] Modified: As used herein, “modified” or “modified” refers to a change in state or structure resulting from modification of a polynucleotide, such as RNA. Polynucleotides can be modified in a variety of ways, including chemically, structurally, and / or functionally. For example, the RNA molecules of this disclosure can be modified by the incorporation of non-natural bases or sequence motifs, including functional sequences or secondary structures that provide biological activity. In one embodiment, RNA is modified, in the case of natural ribonucleotides A, U, G, and C, by the introduction of non-natural or chemically modified bases, nucleosides, and / or nucleotides.

[0346] As used herein, the term “nucleotide base” refers to a substituted or unsubstituted aromatic ring(s). In some embodiments, the aromatic ring(s) contains at least one nitrogen atom. In some embodiments, the nucleotide base can form a Watson-Crick and / or Hoogstine hydrogen bond with a suitably complementary nucleotide base. Exemplary nucleotide bases and their analogues include the naturally occurring nucleotide bases adenine, guanine, cytosine, 6-methylcytosine, uracil, thymine, and analogues of naturally occurring nucleotide bases, e.g., 7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, N 6 -Delta 2-isopentenyl adenine (6iA), N 6 -Delta 2-isopentenyl-2-methylthioadenine (2 ms6iA), N 2 -Dimethylguanine (dmG), 7-methylguanine (7mG), inosine, nebularin, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine, isoguanine, 7-deazaguanine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiouracil, 0 6 -methylguanine, N 6 -Methyladenine, 0 4Examples of exemplary nucleotide bases include, but are not limited to, methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, pyrazolo[3,4-D]pyrimidines (see, for example, U.S. Patents 6,143,877 and 6,127,121 and PCT application publication WO 01 / 38584), indoles such as etenoadenine, nitroindole and 4-methylindole, and pyrroles such as nitropyrrole. Specific exemplary nucleotide bases are described, for example, in Fasman's Literature, 1989, Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca Raton, Fla. and the references cited therein.

[0347] As used herein, the term “nucleotide” refers to sugars such as ribose, arabinose, xylose, and pyranose, and compounds containing a nucleotide base linked to the C-1' carbon of their sugar analogues. The term nucleotide also includes nucleotide analogues. Sugars may be substituted or unsubstituted. Substituted ribose sugars include those in which one or more of the carbon atoms, for example, the 2'-carbon, are the same or different Cl, F, -R, -OR, -NR2, or halogen groups (where each R is independently H, C1-C6 alkyl, or C5-C). 14 Examples of ribose include, but are not limited to, ribose substituted with one or more of the following (which are aryl): 2'-(C1-C6)alkoxyribose, 2'-(C5-C 14 ) Aryloxyribose, 2',3'-didehydroribose, 2'-deoxy-3'-haloribose, 2'-deoxy-3'-fluororibose, 2'-deoxy-3'-chlororibose, 2'-deoxy-3'-aminoribose, 2'-deoxy-3'-(C1-C6)alkylribose, 2'-deoxy-3'-(C1-C6)alkoxyribose, and 2'-deoxy-3'-(C5-C 14Examples include, but are not limited to, aryloxyribose, ribose, 2'-deoxyribose, 2',3'-dideoxyribose, 2'-halolibose, 2'-fluororibose, 2'-chlororibose, and 2'-alkylribose, e.g., 2'-O-methyl, 4'-anomeric nucleotides, 1'-anomeric nucleotides, 2'-4'- and 3'-4' linkages, and other "locked" or "LNAs," bicyclic sugar modifications (e.g., PCT application publications WO 98 / 22489, WO 98 / 39352; and WO 99 / 14226). The term "nucleic acid" usually refers to large polynucleotides.

[0348] The term "oligonucleotide" typically refers to a short polynucleotide, usually about 50 nucleotides or less. If a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it will be understood that this also includes RNA sequences (i.e., A, U, G, C) where "U" replaces "T".

[0349] As used herein, the term “overhang” refers to a terminal non-base-pairing nucleotide resulting from the extension of one strand or region beyond the end of the complementary strand that forms the double helix with the first strand or region. One or more polynucleotides capable of forming a double helix via hydrogen bonding can have overhangs. A single-stranded region that extends beyond the 3'-terminus and / or 5'-terminus of a double helix is ​​called an overhang.

[0350] As described herein, the compounds of this disclosure may contain “optionally substituted” moieties. Generally, the term “substituted” means that one or more hydrogens of a given moiety are substituted with preferred substituents, whether preceded by the phrase “optionally.” Unless otherwise indicated, an “optionally substituted” group may have preferred substituents at each of its substituted positions, and if multiple positions in any given structure can be substituted with multiple substituents selected from the given group, the substituents may be the same or different at each position. The substituent combinations envisioned under this disclosure preferably result in the formation of stable or chemically feasible compounds.

[0351] The term "substituent" refers to a group that is "substituted" at any atom on an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group. Preferred substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO3H, sulfate, phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo, thioxo, imino(alkyl, aryl, aralkyl), and S(O). n Alkyl (where n is 0-2), S(O) n Aryl (where n is 0-2), S(O) n Heteroaryl (where n is 0-2), S(O) nExamples include heterocyclyls (where n is 0-2), amines (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and combinations thereof), esters (alkyl, aralkyl, heteroaralkyl), amides (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), sulfonamides (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), unsubstituted aryls, unsubstituted heteroaryls, unsubstituted heterocyclyls, and unsubstituted cycloalkyls. In one embodiment, the substituents on the group are independently any single substituent or any subset of the substituents described above.

[0352] The term "halo" refers to any radical of fluorine, chlorine, bromine, or iodine.

[0353] The term "alkyl" refers to a hydrocarbon chain that contains a specified number of carbon atoms and may be linear or branched. For example, C1-C 12 The term "alkyl" indicates that the group may contain 1 to 12 carbon atoms (including those at both ends). The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are substituted by a halo, and includes alkyl moieties in which all hydrogens are substituted by a halo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may optionally have O, N, or S inserted. The term "aralkyl" refers to an alkyl moiety in which alkyl hydrogen atoms are substituted by aryl groups. Aralkyl groups also include groups in which multiple hydrogen atoms are substituted by aryl groups. Examples of "aralkyl" groups include benzyl, 9-fluorenyl, benzhydryl, and trityl groups.

[0354] The term "alkenyl" refers to a linear or branched hydrocarbon chain characterized by containing 2 to 8 carbon atoms and having one or more double bonds. Typical examples of alkenyls include, but are not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl, and 3-octenyl groups. The term "alkynyl" refers to a linear or branched hydrocarbon chain characterized by containing 2 to 8 carbon atoms and having one or more triple bonds. Some typical examples of alkynyls are ethynyl, 2-propynyl, 3-methylbutynyl, and propargyl. 2 and sp 3 The carbon atoms can optionally function as bonding sites for alkenyl and alkynyl groups, respectively.

[0355] The terms "alkylamino" and "dialkylamino" refer to the -NH(alkyl) and -NH(alkyl)2 radicals, respectively. The term "aralkylamino" refers to the -NH(aralkyl) radical. The term "alkoxy" refers to the -O-alkyl radical, and the terms "cycloalkoxy" and "aralkoxy" refer to the -O-cycloalkyl radical and -O-aralkyl radical, respectively. The term "siloxy" refers to the R3SiO- radical. The term "mercapto" refers to the SH radical. The term "thioalkoxy" refers to the -S-alkyl radical.

[0356] The term "alkylene" refers to a divalent alkyl group (i.e., -R-), such as -CH2-, -CH2CH2-, and -CH2CH2CH2-. The term "alkylenedioxo" refers to a divalent species of the structure -ORO- (where R represents alkylene).

[0357] The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, where any ring atom may be substituted. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, anthracenyl, and pyrenyl.

[0358] As used herein, the term "cycloalkyl" includes saturated cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12 carbon atoms, where any ring atom may be substituted. Cycloalkyl groups described herein may also include fused rings. A fused ring is a ring sharing a common carbon-carbon bond or a common carbon atom (e.g., a spiro-fused ring). Examples of cycloalkyl moieties include, but are not limited to, cyclohexyl, adamantyl, norbornyl, and decalin.

[0359] The term "heterocyclyl" refers to a non-aromatic 3-10 member monocyclic, 8-12 member bicyclic, or 11-14 member tricyclic ring system having 1-3 heteroatoms in the case of a monocyclic, 1-6 heteroatoms in the case of a bicyclic, or 1-9 heteroatoms in the case of a tricyclic, where the heteroatoms are selected from O, N, or S (for example, in the case of a monocyclic, bicyclic, or tricyclic, a carbon atom and 1-3, 1-6, or 1-9 N, O, or S heteroatoms, respectively), where any ring atom may be substituted. The heterocyclyl groups described herein may also include fused rings. A fused ring is a ring that shares a common carbon-carbon bond or a common carbon atom (for example, a spiro-fused ring). Examples of heterocyclyls include, but are not limited to, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino, pyrrolidinyl, and pyrrolidinyl.

[0360] As used herein, the term "cycloalkenyl" includes a partially unsaturated, non-aromatic, cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon group having 5 to 12 carbon atoms, preferably 5 to 8 carbon atoms, where any of the ring atoms may be substituted. The cycloalkenyl groups described herein may also include fused rings. A fused ring is a ring that shares a common carbon-carbon bond or a common carbon atom (e.g., a spiro-fused ring). Examples of cycloalkenyl moieties include, but are not limited to, cyclohexenyl, cyclohexadienyl, or norbornenyl.

[0361] The term "heterocycloalkenyl" refers to a partially saturated, non-aromatic 5-10 member monocyclic, 8-12 member bicyclic, or 11-14 member tricyclic ring system having 1-3 heteroatoms in the case of a monocyclic, 1-6 heteroatoms in the case of a bicyclic, or 1-9 heteroatoms in the case of a tricyclic, where the heteroatoms are selected from O, N, or S (for example, in the case of monocyclic, bicyclic, or tricyclic rings, a carbon atom and 1-3, 1-6, or 1-9 N, O, or S heteroatoms, respectively), where any ring atom may be substituted. The heterocycloalkenyl groups described herein may also include fused rings. A fused ring is a ring sharing a common carbon-carbon bond or a common carbon atom (e.g., a spiro-fused ring). Examples of heterocycloalkenyls include, but are not limited to, tetrahydropyridyl and dihydropyran.

[0362] The term "heteroaryl" refers to aromatic 5-8 member monocyclic, 8-12 member bicyclic, or 11-14 member tricyclic ring systems having 1-3 heteroatoms in the case of monocyclic, 1-6 heteroatoms in the case of bicyclic, or 1-9 heteroatoms in the case of tricyclic, where the heteroatoms are selected from O, N, or S (for example, in the case of monocyclic, bicyclic, or tricyclic, a carbon atom and 1-3, 1-6, or 1-9 N, O, or S heteroatoms, respectively), where any of the ring atoms may be substituted. Heteroaryl groups described herein may also include fused rings that share a common carbon-carbon bond.

[0363] The term "oxo" refers to an oxygen atom that forms a carbonyl group when bonded to carbon, an N-oxide when bonded to nitrogen, and a sulfoxide or sulfone when bonded to sulfur.

[0364] The term "acyl" refers to alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituents, any of which may be further substituted by substituents.

[0365] Patient: As used herein, the term “patient” includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0366] As used herein, the term “polynucleotide” is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, as used herein, nucleic acids and polynucleotides are used interchangeably. Those skilled in the art have general knowledge that nucleic acids are polynucleotides that can be hydrolyzed to monomeric “nucleotides.” Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning and amplification techniques, as well as by synthetic means. As used herein, “oligonucleotide” usually refers to short polynucleotides less than 100 base pairs in length.

[0367] Medicinally acceptable: As used herein, “medically acceptable” means a compound, material, composition, and / or dosage form suitable for use in contact with human and animal tissues, organs, and / or bodily fluids, within the bounds of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications that do not justify a reasonable benefit / risk ratio.

[0368] Pharmacopoeciable carriers: As used herein, the term “pharmacopoeciable carriers” means, and includes, any and all physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc. The compositions may contain pharmacopoeciable salts, such as acid addition salts or base addition salts (see, for example, Berge et al. (1977) J Pharm Sci 66:1-19).

[0369] Phosphate: As used herein, the term “phosphate” means a salt or ester of phosphoric acid. A polyphosphate is a salt or ester of a polymer oxyanion formed from tetrahedral PO4(phosphate) structural units linked together by sharing an oxygen atom. As used herein, the term “diphosphate” refers to a polyphosphate containing two phosphate structural units. As used herein, the term “triphosphate” refers to a polyphosphate containing three phosphate structural units. In some embodiments, the disclosure provides RIG-I agonists containing a diphosphate moiety linked to the 5' end, or derivatives or analogs thereof. In some embodiments, the disclosure provides RIG-I agonists containing a triphosphate moiety linked to the 5' end, or derivatives or analogs thereof.

[0370] Preventive: As used herein, the term “preventive” as used in relation to a condition means the administration of a composition that reduces the frequency of symptoms of a medical condition in a subject or delays the onset of such symptoms compared to a subject that does not receive the composition.

[0371] Purified: As used herein, the terms “purified” or “isolated” applied to any of the proteins (antibodies or fragments) described herein refer to polypeptides that have been isolated or purified from naturally associated components (e.g., proteins or other naturally occurring biological or organic molecules), such as other proteins, lipids, and nucleic acids within the prokaryotes expressing the protein. Typically, a polypeptide is purified if it accounts for at least 60% by weight (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99) of the total protein in the sample.

[0372] RIG-I agonist: As used herein, the term "RIG-I agonist" refers to a nucleic acid compound (e.g., RNA) that binds to a RIG-I receptor and partially or completely promotes, induces, enhances, and / or activates biological activity, response, and / or downstream pathways mediated by RIG-I signaling or other RIG-I-mediated functions. Examples of RIG-I agonists are provided herein.

[0373] Subjects: As used herein, the term “subjects” includes humans and non-human animals. For example, the methods and compositions of the present invention can be used to treat subjects with immune disorders. The term “non-human animals” includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc.

[0374] Therapeutic Effective Dose: As used herein, the terms “therapeutic effective dose” or “therapeutic effective amount” or similar terms are intended to mean the amount of an agent (e.g., the synthetic RIG-I agonist of the present invention) that elicits a desired biological or medical response, such as curing or at least partially cessating a condition or disease and its complications in a patient who is already suffering from the disease (e.g., improvement of one or more symptoms of cancer). The amount effective for this use will depend on the severity of the disease being treated and the overall state of the patient’s own immune system.

[0375] To treat: As used herein, the terms “to treat,” “to treat,” and “treatment” refer to therapeutic or preventive measures as described herein. A “treatment” method utilizes administering the nucleic acid compounds of the Disclosure to a subject in need of such treatment in order to prevent, cure, delay, reduce the severity of, or improve one or more symptoms of a disorder or recurrent disorder, or to extend the survival of the subject beyond the survival expected in the absence of such treatment.

[0376] Conventional notation is used herein to describe polynucleotide sequences: the left end of a single-stranded polynucleotide sequence is the 5' end. The DNA strand having the same sequence as mRNA is called the "coding strand"; the sequence on the DNA strand located 5' from the reference point on the DNA is called the "upstream sequence"; and the sequence on the DNA strand located 3' from the reference point on the DNA is called the "downstream sequence".

[0377] Those skilled in the art will understand that all nucleic acid sequences described in this specification in the forward direction are also useful in the reverse direction and in complementary directions of the forward and reverse directions in the compositions and methods of the present invention, and are described in the same manner as if they were explicitly described herein.

[0378] As used herein, the terms “ribonucleotide” and “ribonucleic acid (RNA)” refer to a modified or unmodified nucleotide or polynucleotide comprising at least one ribonucleotide unit. A ribonucleotide unit comprises an oxygen atom attached to the 2'-position of a ribosyl moiety, which has a nitrogen base attached to the 1'-position of the ribosyl moiety by an N-glycoside linkage, and a portion that either enables or prevents linkage to another nucleotide.

[0379] As used herein, the term “target” refers to a molecule having an affinity for a given molecule. Targets may be naturally occurring molecules or man-made molecules. They may also be available in their unmodified state or as aggregates with other species. Targets can be covalently or noncovalently bound to a binding member, either directly or via a specific binding agent. Examples of examples that can be used in this invention include, but are not limited to, proteins, peptides, oligonucleotides, and nucleic acids.

[0380] Where used herein, "mutant" refers to a nucleic acid sequence or peptide sequence that differs in sequence from a reference nucleic acid sequence or reference peptide sequence, but retains the essential properties of the reference molecule. Sequence changes in nucleic acid mutants may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or they may result in amino acid substitutions, additions, deletions, fusions, and cleavages. Mutants of nucleic acids or peptides may be naturally occurring, such as allele mutants, or they may be mutants not known to exist naturally. Mutants of nucleic acids and peptides that do not exist naturally can be produced by mutagenesis techniques or by direct synthesis.

[0381] Scope: Throughout this disclosure, various aspects of the invention may be presented in range form. It should be understood that descriptions in range form are merely for convenience and brevity and should not be interpreted as restrictive limitations that lack flexibility to the scope of the invention. Accordingly, range descriptions should be considered to specifically disclose not only the individual numbers within that range, but also all possible subranges. For example, a range description such as 1 to 6 should be considered to specifically disclose not only the individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6, but also subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, and 3 to 6. This applies regardless of the breadth of the range.

[0382] While this disclosure is described with reference to specific embodiments, it should be understood by those skilled in the art that various modifications can be made and equivalents can be substituted without departing from the true intent and scope of this disclosure. Furthermore, many modifications can be made to adapt specific circumstances, materials, substance compositions, processes, or process steps to the purpose, intent, and scope of this disclosure. All such modifications are intended to be within the scope of this disclosure. [Examples]

[0383] (Examples) This disclosure will be better understood by referring to the following examples. However, these should not be construed as limiting the scope of this disclosure. It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes taking them into account are proposed to those skilled in the art and should be included within the spirit and scope of this application and the appended claims.

[0384] (Synthesis of compounds:) The nucleic acid compounds of the present invention can be synthesized by standard solid-phase synthesis. Various modifications can be introduced by methods known in the art. For example, site-directed phosphorothioate nucleotide linkages (PS) can be introduced into the solid-phase assembly using either 3H-1,2-benzodithiol-3-one 1,1-dioxide or a sulfurizing reagent such as 3-((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thion (DDTT). The incorporation of diphosphate or triphophate groups into oligonucleotides can be incorporated into the solid-phase assembly using protocols described in the literature.

[0385] For the synthesis of amino-linked oligonucleotides of the present invention, the corresponding phosphoramidite building block is incorporated between the oligonucleotide assemblies in the solid-phase assembly.

[0386] In one embodiment, amino-modified oligonucleotides are prepared by automated solid-phase synthesis using standard phosphoramidite chemistry in conjunction with corresponding nucleoside phosphoramidite building blocks. Therefore, appropriate protected nucleoside phosphoramidite building blocks are sequentially added to a 2'-O-TBDMS-protected N-benzoyl-cytosine nucleoside bound to a CPG support using a synthetic cycle involving detritylation, coupling, oxidation, and capping. For the incorporation of the amino-terminal portion, the following modified phosphoramidite building blocks were used. [ka]

[0387] Oligonucleotide assembly was continued until the final G-nucleoside building block was added to the 5'-terminus. Tert-butyl hydroperoxide was used for the oxidation of each internucleotide phosphite linkage. Next, the oligonucleotides bound to the support were diphosphated on a synthesizer using the fully automated procedure described in Zlatev et al.'s literature (Solid-Phase Chemical Synthesis of 5'-Triphosphate DNA, RNA, and Chemically Modified Oligonucleotides. Current Protocols in Nucleic Acid Chemistry (2012) 1.28.1-1.28.16). In this protocol, the full-length 5'-hydroxyl oligonucleotides were first converted to the corresponding 5'-H-phosphonate mono-esters by reacting them with diphenyl phosphite in pyridine. Subsequently, the intermediate H-phosphonate was activated in the presence of imidazole and bromo-trichloromethane to obtain an activated 5'-phosphorimidazolide, which was then reacted with tributylammonium monophosphate to form the desired diphosphate compound. The reagents for the diphosphorylation procedure (1M diphenyl phosphite in pyridine, 0.1M triethylammonium bicarbonate (TEAB) in water / acetonitrile, 1M imidazole / 1M N,O-bis(trimethylsilyl)acetamide in CBrCl3 / acetonitrile / triethylamine, and 0.25M tributylammonium monophosphate in dimethylformamide / acetonitrile) were prepared as described in the literature by Zlatev et al.

[0388] (Cutting and deprotection of oligos bound to the support) After completing the oligonucleotide assembly, the oligos bound to the support in the column were treated with a 1:1 mixture of methylamine (40% in water) and ammonium hydroxide (28-30%, JT Baker) (enough to cover and wet the entire support), and incubated at room temperature for 15 minutes. The solution was then collected, and this procedure was repeated two more times. Finally, the column was washed with AMA and placed in the same vial. The resulting solution was then incubated at 65°C for 15 minutes with shaking to deprotect the base, frozen at -80°C for 1 hour, and dried in a SpeedVac. Ethanol was added to the dried oligos, and the material was dried again in a SpeedVac to remove all remaining trace amounts of water.

[0389] (2'-O-TBDMS deprotection) The 2'-OH protecting group was removed by treatment with 1 M tetrabutylammonium fluoride (TBAF) in THF at room temperature for 36 hours with shaking. It is important that the reaction mixture contains no trace amounts of water; otherwise, deprotection will be incomplete. The use of a desiccator or a jar with a desiccant is strongly recommended.

[0390] After incubation with TBAF, an equal volume of 2 M NaOAc was added to the mixture, and the resulting solution was concentrated to half its volume using SpeedVac. The mixture was then extracted three times with 1.6 volumes of siRNA, evaporated for 15 minutes using SpeedVac to remove trace amounts of siRNA, and precipitated with ethanol to obtain a completely deprotected oligonucleotide. The deprotected oligonucleotide was further purified by RP-HPLC and ion pair chromatography, and the solution was lyophilized to obtain an off-white solid compound.

[0391] Subsequently, amino-functionalized oligonucleotides can be used in the synthesis of various conjugates as described herein and below. The compounds can be characterized by mass spectrometry, NMR, and other spectral methods.

[0392] (Synthesis of compound 1(5'-ppGGAUCGAUCGAUCGUU-(NH2C6CH2OH)CGCGAUCGAUCGAUCC-3')) (Alternative representation for Compound 1:) [ka] Compound 1 is assembled using a standard phosphoramidite chemical solid support. Compound 1 is assembled from the 3'-terminus of the oligonucleotide sequence. A trifunctional phosphoramidite reagent is used to introduce a connector segment as shown in Scheme 1 below, and the oligonucleotide sequence is assembled to the last nucleotide at the 5'-terminus. Finally, the introduction of a diphosphoryl group (DP) is carried out via the initial formation of a 5'-H-phosphonate and subsequent reaction with tributylammonium monophosphate. After removal of the base and phosphate protecting groups with ammoniacal methylamine, 2'-O-TBDMS is removed with NEt3-HF. The purification steps include desalting, ion exchange chromatography, ultrafiltration, and lyophilization. A second purification using RP-HPLC can be performed. [ka]

[0393] (Synthesis of biotin conjugate (compound 2):) [ka] To prepare the oligo-biotin conjugate, loop-modified oligonucleotides were reacted with the activated biotin ester (sulfo-NHS-LC biotin) in an aqueous acetonitrile solution or another solvent. After the reaction, the crude conjugate was purified by preparative HPLC, desalted, and freeze-dried to obtain the pure oligo-biotin conjugate. The HPLC and mass spectrometry data of the conjugate are shown in Figure 1.

[0394] (Preparation of IR-dye conjugate (compound 3):) For the preparation of the oligo-pigment conjugate, loop-modified oligonucleotides were reacted with the activated NHS ester of the pigment in an aqueous acetonitrile solution or another solvent. After the reaction, the crude conjugate was purified by preparative HPLC, desalted, and freeze-dried to obtain the pure oligo-biotin conjugate. The HPLC and mass spectrometry data of the conjugate are shown in Figure 2. [ka]

[0395] (Preparation of folic acid conjugate (compound 4)) [ka] Compound 1 (10 micromoles) was reacted with folic acid N-hydroxysuccinimide (folic acid NHS) in DMF solvent and stirred at room temperature for 24 hours. Folic acid conjugate compound 4 was obtained by post-treatment and purification by HPLC chromatography.

[0396] (Preparation of β-sitosterol conjugate (compound 5)) [ka] Compound 1 (10 micromoles) was reacted with the active carbonylimidazolide ester of 3-hydroxy-beta-sitosterol in DMF solvent and stirred at room temperature for 24 hours. Workup and purification by HPLC chromatography yielded beta-sitosterol conjugate 5.

[0397] (Preparation of oleic acid conjugate (compound 6)) [ka] 1.5 mmol of N-hydroxysuccinimide oleic acid is reacted with 191 mmol of the compound in the presence of 10% aqueous potassium carbonate. The contents are stirred for 24-48 hours until the reaction is complete and the starting material (oligo compound) is no longer present. The reaction mixture is subjected to organic extraction to remove the oleic acid intermediate, and the resulting oleic acid conjugate compound 6 is obtained by RP-HPLC purification and lyophilization.

[0398] (RIG-I agonist activity of the compound of the present invention) RIG-I and MDA-5 stimulation will be tested by evaluating IRF3 activation in HEK293 cells expressing human RIG-I or MDA-5 genes. The test samples will be: [ka] Poly (I:C); 5'-pppdsRNA-positive control; and IFNα Includes:

[0399] The activity of the test product will be tested as a potential agonist in human RIG-I and MDA-5 expressing cells. The test product will be evaluated at one concentration in MDA-5 expressing cells and compared to the control ligand. Furthermore, the activity of the test product will be evaluated at five concentrations in RIG-I expressing cells.

[0400] The secreted luciferase reporter is under the control of a promoter induced by an IRF transcription factor. This reporter gene enables monitoring of signaling through the RIG-I and MDA-5 genes based on IRF3 activation. In a 96-well plate (total volume 200 μL) containing appropriate cells, 20 μL of the test sample or positive control ligand is added to each well. After 16–24 hours of incubation, IRF pathway activation is monitored using a luciferase detection assay. Luciferase activity is assayed from the supernatant of induced cells, and relative luminescence units (RLUs) are detected using a Promega GloMax luminometer. Figure 3 shows the results as relative luminescence units (RLUs). The RIG compounds (i.e., compound X and compound 1) did not activate MDA-5 (data not shown).

[0401] IRF stimulation can be tested by evaluating activation in THP1-Dual cells, a human monocytic cell line that naturally expresses many pattern recognition receptors (PRRs). The compounds of the present invention can be evaluated at one or several concentrations and compared with a control ligand. This process can be carried out in a triple series. The results can be provided as relative luminescence units (RLUs).

[0402] IRF stimulation can be tested by evaluating activation in A549 cells, a lung epithelial cell line that naturally expresses many pattern recognition receptors (PRRs). The compounds of the present invention can be evaluated at one or several concentrations and compared with a control ligand. This process can be carried out in a triple series. The results can be provided as relative luminescence units (RLUs).

[0403] (Selectivity assay:) To evaluate the selectivity of the compound of the present invention as a RIG-I agonist, the compound can be evaluated in multiple cell lines expressing Toll-like receptor (TLR) or NOD-like receptor (NLR) stimulation. The test can be performed by evaluating NF-κB activation in HEK293 cells expressing a given TLR or NLR. The activity of the compound of the present invention can be tested against seven human TLRs (TLR2, 3, 4, 5, 7, 8, and 9), two human NLRs (NOD1 and NOD2), eight mouse TLRs (2, 3, 4, 5, 7, 8, 9, and 13), and two mouse NLRs (NOD1 and NOD2) as potential agonists. The activity of the compound of the present invention can be tested at a single concentration and compared with a control ligand. These steps can be performed in a triple series.

[0404] STING stimulation can be tested by evaluating activation in THP1-Dual cells, a human monocytic cell line that naturally expresses many pattern recognition receptors (PRRs), including human STING. STING stimulation in THP1-Dual cells can be tested by evaluating IRF activation. The compounds of the present invention can be evaluated at one or more concentrations and compared with a control ligand. This process can be carried out in a triple series. The results can be provided as relative luminescence units (RLUs).

[0405] (Evaluation of apoptosis via RIG-I) The compounds of the present invention can be evaluated using Luminex to assess cytokine / chemokine production in mouse and human tumor cell lines, myeloid cell lines, and / or primary immune cells (e.g., PBMCs, myeloid macrophages, and / or TILs), and cell death can be measured by flow cytometry or Incucyte imaging using CytoTox-Glo® and / or caspase / annexin V / ICD markers. Various transfection reagents (e.g., Lipofectamine® RNAiMAX and JET-PEI) can be used to facilitate the delivery of the compounds to the cytosol. The tests can be performed using RIG-I knockout cells or by gene silencing against RIG-I using siRNA or shRNA to demonstrate functional dependence. Detection of pro-inflammatory cytokines / chemokines may include reading the NF-κB pathway (e.g., by an NFκB reporter and IL6, TNFα ELISA).

[0406] (Antiviral assay:) The compound can be tested for antiviral activity against viruses such as human rhinovirus, parainfluenza, influenza, coronavirus, or RSV using appropriate cell lines such as HeLa, MDCK, Vero76, and A549 cells. Antiviral activity can be evaluated at different concentrations ranging from 0.01 to 10 μg / mL using a cytopathic assay, and cytotoxicity can be evaluated by a neutral red assay. EC 50 and CC 50 The value can be evaluated, and the selectivity index can be used to determine the CC. 50 / EC 50 It can be calculated using ratios.

[0407] (Dose response to RIG-I activation) (IRF signaling detection in the A549 dual cell assay system) To evaluate the compound's ability to activate interferon regulatory factor (IRF) signaling, we used A549-Dual® cells (InvivoGen, Toulouse, France) expressing the Lucia luciferase gene, which encodes secreted luciferase, under the control of the ISG54 minimal promoter in combination with five IFN stimulus-response elements. First, the A549-Dual® cells were harvested and placed in pre-warmed fresh growth medium in a 2.8 × 10⁶ cell volume. 5 The cells were resuspended at a concentration of cells / mL. A 20 μL mixture of the compound and reconstituted LyoVec (InvivoGen) was applied to 180 μL of cell suspension in a 96-well plate. After incubation in a CO2 incubator at 37°C for 18–24 hours, 20 μL of the supernatant was transferred to a 96-well white (opaque) plate. The luminescent reporter signal, as an activation marker for the IRF pathway, was mixed with 50 μL of QUANTIU-Luc® 4 reagent and detected using a multimode plate reader (CLARIOstar®, BMG LABTECH, Aylesbury, UK). 5' ppp-dsRNA (InvivoGen) was also used as an assay control. The results are shown in Figure 5.

[0408] (Induction of CXCL-10) A gas-liquid interface (ALI) system was prepared using a culture of lung epithelial cells endogenously expressing RIG-I. Activation of these lung epithelial cells with the RIG-I ligand leads to the induction of interferon and interferon-stimulating genes such as CXCL10. CXCL10 production can be measured by ELISA. ALI cultures were treated with compound 1 and a conjugate compound for 30 minutes, and after 24 hours of exposure, the cultures were harvested and the supernatant was subjected to ELISA, resulting in good induction of CXCL10. As shown in Figure 6, compounds 1 and 2 showed good CXCL10 induction as aqueous solutions compared to the untreated control, but the addition of a surfactant such as polysorbate to the aqueous solution appeared to promote compound uptake and CXCL10 induction. The induction of CXCL10 clearly demonstrates that the tested compounds can activate RIG-I and induce the IFN signaling cascade.

Claims

1. A nucleic acid compound capable of inducing interferon production, comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are complementary to each other and hybridize to form a double-stranded portion, the number of base pairs of the double-stranded portion being an integer in the range of 8 to 19; and wherein the 3' end of the first nucleotide sequence is conjugated to one end of a connector element, and the other end of the connector element is conjugated to the 5' end of the second nucleotide sequence; and wherein the furthest 5' nucleotide of the first nucleic acid sequence comprises a 5' diphosphate or triphotate portion or a derivative or analog thereof.

2. The nucleic acid compound according to claim 1, wherein the nucleic acid compound has the structure of formula I. 【Chemistry 1】 (In the formula, 5'-P z -(N) b N-3' represents the first nucleic acid sequence; 5'-N(N) b' -3' represents the second nucleic acid sequence; In each case, P is independently a phosphate or an analogue thereof; z is either 2 or 3; In each case, N is any nucleotide or modified nucleotide or its analogue or derivative; b and b' are independently 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; 5'-(E) y (E)-L-(E)(E) y' -3' represents the connector element, where, E is, independently, any nucleotide, modified nucleotide, or debase at each instance; y and y' are independently between 0 and 9, where y+y' is equal to 0 and 8; L is structure 【Chemistry 2】 (Here, X and X' are independently O or S; Y and Y' are independently OR'', SR'', or NRR'; V and V' are independently O, S, or NRR'; q is between 1 and 20; k is between 1 and 20; t is between 1 and 20; M is selected from aliphatic, substituted aliphatic, aryl, substituted aryl, heteroalkyl, heterocyclyl, or substituted heterocyclyl; W is any reactive or conjugation group; and (d is either 0 or 1) (It is a non-nucleotide segment that has [a certain characteristic].)

3. The nucleic acid compound according to claim 2, wherein z is 2.

4. The nucleic acid compound according to claim 3, wherein at least one P is a phosphate analog.

5. The nucleic acid compound according to claim 3, wherein both P are phosphate analogs.

6. The nucleic acid compound according to claim 2, wherein z is 3.

7. The nucleic acid compound according to claim 6, wherein at least one P is a phosphate analog.

8. The nucleic acid compound according to claim 6, wherein at least two P are phosphate analogs.

9. The nucleic acid compound according to claim 6, wherein all three Ps are phosphate analogs.

10. If the phosphate analog exists, the structure 【Transformation 3】 (Here, Y is O or S, or CH-R (where R = alkyl, aralkyl, heteroaryl, or cycloalkylamine (e.g., piperazine)), X is either O or S, and Z is OH, SH, NHR' (where R' is H, alkyl, aralkyl, and heteroaryl). A nucleic acid compound according to any one of claims 1 to 9, comprising:

11. The nucleic acid compound according to any one of claims 1 to 10, wherein the nucleotides of the first and second nucleotide sequences are ribonucleic acid (RNA).

12. The nucleic acid compound according to any one of claims 1 to 11, wherein b and b' are independently 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; preferably 11, 12, 13, 14, 15, 16, 17, or 18; preferably 13, 14, 15, 16, 17, or 18.

13. The aforementioned compound, 【Chemistry 4】 A nucleic acid compound according to any one of claims 1 to 12, selected from the above.

14. The aforementioned compound, 【Transformation 5】 The nucleic acid compound according to claim 13.

15. A pharmaceutical composition comprising a nucleic acid compound according to any one of claims 1 to 14 and a pharmaceutically acceptable excipient.

16. A nucleic acid compound according to any one of claims 1 to 14 or a composition according to claim 15, for use as a pharmaceutical.

17. A nucleic acid compound according to any one of claims 1 to 14 or a composition according to claim 15, for use in the treatment of a disease or condition treated by stimulation or activation of the natural and / or adaptive immune system and / or induction of a natural and / or adaptive immune response.

18. A method for treating a disease or condition treated by stimulation or activation of the natural and / or adaptive immune system and / or induction of a natural and / or adaptive immune response, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a nucleic acid compound according to any one of claims 1 to 14 or a composition according to claim 15.

19. The use or method according to claim 17 or 18, wherein the disease or condition is caused by a viral infection or is associated with such a viral infection.

20. The use or method according to claim 19, wherein the virus infects the respiratory tract, and the disease associated with the infection is a disease of the respiratory tract.

21. The use or method according to claim 17 or claim 18, wherein the disease or condition is cancer.

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