Compositions and methods for inhibiting TIGIT gene expression

Abstract

Disclosed herein are compounds, compositions and methods for reducing TIGIT mRNA and protein expression. The methods can be used to treat, prevent or ameliorate TIGIT-related diseases, disorders and conditions.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 747,261 filed on October 18, 2018. All contents of the above application are incorporated herein by reference. Background Art

[0003] T cell immunoreceptor with Ig and ITIM domains (TIGIT, also known as VSIG9, WUCAM, and VSTM3) is an immune checkpoint receptor known to negatively regulate T cell function 1 TIGIT is expressed on some T cells and natural killer (NK) cells 1 And it can bind to the poliovirus receptor (PVR / CD155) on dendritic cells and macrophages 1 .

[0004] The interaction between TIGIT and PVR can negatively regulate T cell and NK cell function 1 Blocking the interaction between TIGIT and PVR / CD155 can reverse its inhibitory effect on NK cell cytotoxicity 2 In HIV patients, TIGIT expression is associated with disease progression due to its ability to cause T cell exhaustion. 3 Blockade of TIGIT and PD-1 (programmed death protein-1) with monoclonal antibodies restores HIV-specific CD8 + T cell response 3 .

[0005] TIGIT expression is highly correlated with other immune checkpoints such as PD-1 on TILs. TIGIT expression profiles are directly associated with immunosuppressive phenotypes. In preclinical settings, TIGIT knockout mice in mouse models showed reduced tumor burden and prolonged survival. 4 Furthermore, simultaneous blockade of TIGIT and PD-1 in melanoma patients resulted in tumor antigen-specific CD8 + T cell proliferation and CD8 + Tumor-infiltrating T cells (TIL) 5 .

[0006] In addition, TIGIT can also compete with the T cell co-stimulatory molecule CD226 for binding to CD155. 6 The data also found that TIGIT can hinder the dimerization of CD226 and eliminate the co-stimulatory activity of CD226 6 In conclusion, in disease settings such as cancer, viral infections, and inflammatory diseases / conditions, inhibition of TIGIT may provide therapeutic benefit by restoring T cell activity. Summary of the invention

[0007] Some embodiments provide antisense compounds, compositions and methods for treatment. In some embodiments, the compositions and methods for treatment include: administering a T cell immune receptor (TIGIT; also known as VSIG9, WUCAM and VSTM3) specific inhibitor with Ig and ITIM domains to an individual in need. In some embodiments, the TIGIT specific inhibitor is a nucleic acid. In some embodiments, the nucleic acid is an antisense compound targeting TIGIT as described herein. In some embodiments, the compounds, compositions and methods help prevent, treat, delay, slow progression and / or improve TIGIT-related diseases, disorders and conditions. In some embodiments, the diseases, disorders and conditions are cancer; inflammatory diseases such as but not limited to systemic lupus erythematosus, systemic sclerosis, ulcerative colitis, multiple sclerosis, rheumatoid arthritis, Crohn's disease, non-alcoholic fatty hepatitis and sepsis; viral infections such as AIDS (HIV), hepatitis B or hepatitis C.

[0008] Provided herein are compounds, compositions and methods for inhibiting TIGIT mRNA and / or protein expression. Also provided herein are methods for diagnosing, treating and / or preventing diseases and / or conditions that respond to inhibiting TIGIT mRNA and / or protein expression.

[0009] In some embodiments, the TIGIT-specific inhibitor is a synthetic oligonucleotide compound comprising 12 to 30 linked nucleotides, having at least 12 consecutive nucleobases complementary to an equal length portion of SEQ ID NO: 1. In some embodiments, the internucleotide linkages of the compound comprise phosphorothioate internucleotide linkages. In some embodiments, more than half but less than all of the internucleotide linkages are phosphorothioate internucleotide linkages. In some embodiments, all of the internucleotide linkages are phosphorothioate internucleotide linkages. In some embodiments, the phosphorothioate linkages can be a mixture of Rp and Sp enantiomers, or they can be manufactured as either of the stereoregular or substantially stereoregular Rp or Sp forms.

[0010] In some embodiments, the nucleobase of the compound is not further modified (ie, no modifications to the base or sugar moieties). In this regard, the nucleotides of the antisense oligonucleotide include naturally occurring nucleobases and sugars.

[0011] In some embodiments, the TIGIT-specific inhibitor is a synthetic oligonucleotide compound comprising 12 to 30 linked nucleotides, wherein the base sequence of the compound is at least 80% complementary to an equal length portion of SEQ ID NO: 1. In some embodiments, the internucleotide bonds of the compound include phosphorothioate internucleotide bonds. In some embodiments, more than half but less than all of the internucleotide bonds are phosphorothioate internucleotide bonds. In some embodiments, all internucleotide bonds are phosphorothioate internucleotide bonds. In some embodiments, the phosphorothioate bonds can be a mixture of Rp and Sp enantiomers, or they can be manufactured as either of the stereoregular or substantially stereoregular Rp or Sp forms. In some embodiments, the nucleobases of the compound are not further modified (i.e., the base or sugar moiety is not modified). In this regard, the nucleotides of the antisense oligonucleotide include naturally occurring nucleobases and sugars.

[0012] In some embodiments, the TIGIT-specific inhibitor is a synthetic oligonucleotide compound comprising 12 to 30 linked nucleotides, wherein the base sequence of the compound is at least 80% complementary to an equal length portion of nucleobases 121-780, 841-1440, 1650-2160 or 2700-2800 of SEQ ID NO: 1. In some embodiments, the internucleotide bonds of the compound include phosphorothioate internucleotide bonds. In some embodiments, more than half but less than all of the internucleotide bonds are phosphorothioate internucleotide bonds. In some embodiments, all internucleotide bonds are phosphorothioate internucleotide bonds. In some embodiments, the phosphorothioate bonds can be a mixture of Rp and Sp enantiomers, or they can be manufactured as either of the stereoregular or substantially stereoregular Rp or Sp forms. In some embodiments, the nucleobases of the compound are not further modified (i.e., the base or sugar moiety is unmodified). In this regard, the nucleotides of the antisense oligonucleotide include naturally occurring nucleobases and sugars.

[0013] Some embodiments provide compositions comprising a compound described herein and a pharmaceutically acceptable carrier or diluent.

[0014] In some embodiments, the present invention provides a method of inhibiting TIGIT mRNA or protein expression. In some embodiments, the method comprises: contacting a cell with at least one antisense compound or composition described herein. In some embodiments, the method comprises: administering at least one antisense compound or composition described herein.

[0015] In some embodiments, the inhibition of TIGIT expression occurs in cells or tissues. In some embodiments, the inhibition occurs in cells or tissues of animals. In some embodiments, the animal is a human. In some embodiments, the inhibition is a reduction in TIGIT mRNA levels. In some embodiments, the inhibition is a reduction in TIGIT protein levels. In some embodiments, both TIGIT mRNA and protein levels are reduced.

[0016] In some embodiments, the present invention provides a method for treating a disease, disorder or condition in an individual in need thereof, the disease, disorder or condition being associated with TIGIT expression and / or activity. In some embodiments, the method comprises administering at least one antisense compound or composition described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effects of TIGIT antisense analogs on the mouse CT26.CL25 cell line are illustrated.

[0018] Figure 2A and Figure 2B It was demonstrated that TIGIT antisense inhibited tumor growth in vivo in a dose-dependent manner.

[0019] Figure 3 illustrates that TIGIT antisense increases tumor infiltrating CD3 + T cells.

[0020] FIG. 4A to FIG. 4C This indicates that the anti-tumor effect of TIGIT antisense depends on NK and CD8 + T cells.

[0021] Figure 5 It is demonstrated that local injection of ASOs does not require chemical modification of oligonucleotides.

[0022] Fig. 6A and Figure 6B It was demonstrated that the combination therapy of TIGIT antisense and anti-PD-1 antibody synergistically inhibited tumor growth. DETAILED DESCRIPTION

[0023] It should be understood that the above general description and the following specific embodiments are only exemplary and explanatory and do not constitute limitations on the present disclosure. Here, unless otherwise expressly stated, the use of the singular includes the plural. Unless otherwise stated, "or" used herein means "and / or". Further, the use of the term "include" and other forms (such as "include (three singular forms)" and "include (past tense and past participle)") does not constitute a limitation.

[0024] Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, and treaties, are expressly incorporated herein by reference in their entirety for any purpose.

[0025] Unless a clear definition is provided herein, the terms, procedures and techniques related to analytical chemistry, synthetic organic chemistry and medicinal chemistry herein are well known and commonly used in the art. Standard techniques can be used for chemical synthesis and chemical analysis. Certain techniques and procedures can be found in the following documents: for example, "Carbohydrate Modification in Antisense Research", edited by Sangvi and Cook, American Chemical Society, Washington, 1994; "Remington Pharmaceutical Science", Mack Publishing Company, Easton, Pa., 21st edition, 2005; and "Antisense Drug Technology, Principles, Strategies and Applications", edited by Stanley T.Crooke, CRC Press, Boca Raton, Fla; and Sambrook et al., "Molecular Cloning, Laboratory Manual", Second Edition, Cold Spring Harbor Laboratory Press, 1989 (incorporated herein by reference for any purpose). Where permitted, all patents, applications, published applications and other publications and other data cited throughout this disclosure are incorporated herein by reference in their entirety.

[0026] Unless otherwise stated, the following terms have the following meanings:

[0027] As used herein, "nucleoside" refers to a compound comprising a nucleobase portion and a sugar portion. Nucleosides include, but are not limited to, naturally occurring nucleosides (such as those found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate portion.

[0028] The term "3", when used directly, generally refers to a region or position in a polynucleotide or oligonucleotide that is 3' to another region or position in the same polynucleotide or oligonucleotide (towards the 3' end of the nucleotides). The term "3' end" generally refers to the 3' terminus of the nucleotides that make up an oligonucleotide.

[0029] The term "5", when used directly, generally refers to a region or position in a polynucleotide or oligonucleotide that is 5' to another region or position in the same polynucleotide or oligonucleotide (towards the 5' end of the nucleotides). The term "5' end" generally refers to the 5' terminus of the nucleotides that make up an oligonucleotide.

[0030] As used herein, "chemical modification" refers to the chemical differences of a compound compared to its naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications, including sugar moiety modifications and nucleobase modifications and internucleoside bond modifications. For oligonucleotides, chemical modifications do not include differences in only the nucleobase sequence.

[0031] As used herein, "sugar moiety" refers to a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside. As used herein, "naturally occurring sugar moiety" refers to the furunculose found in naturally occurring RNA or the deoxyfuronculose found in naturally occurring DNA. As used herein, "modified sugar moiety" refers to a substituted sugar moiety or sugar surrogate.

[0032] As used herein, "nucleic acid" refers to a molecule composed of monomeric nucleotides. Nucleic acids include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acid (ssDNA), double-stranded nucleic acid (dsDNA), small interfering ribonucleic acid (siRNA) and microRNA (miRNA).

[0033] As used herein, "nucleotide" refers to a nucleoside further comprising a phosphate linking group. As used herein, "connected nucleosides" may or may not be connected by a phosphate bond, and therefore include but are not limited to "connected nucleotides". As used herein, "connected nucleosides" refer to nucleosides connected in a continuous sequence (i.e., without additional nucleosides between the connected objects).

[0034] As used herein, "nucleobase" refers to a group of atoms that can be linked to a sugar moiety to produce a nucleoside that can be incorporated into an oligonucleotide, and which is capable of bonding to a naturally occurring complementary nucleobase of another oligonucleotide or nucleic acid. Nucleobases can be naturally occurring or can be modified. As used herein, "nucleobase sequence" refers to the order of consecutive nucleobases, regardless of any sugar, bonding, or nucleobase modification.

[0035] As used herein, the term "unmodified nucleobase" or "naturally occurring nucleobase" refers to the 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) and uracil (U).

[0036] As used herein, a "modified nucleobase" refers to any nucleobase that is not a naturally occurring nucleobase.

[0037] As used herein, "modified nucleosides" refer to nucleosides that include at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides include modified sugar moieties and / or modified nucleobases.

[0038] As used herein, "oligonucleotide" refers to a compound comprising a plurality of connected nucleosides. In some embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA). In some embodiments, an oligonucleotide comprises only unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA). In some embodiments, an oligonucleotide comprises one or more modified nucleosides.

[0039] As used herein, "modified oligonucleotide" refers to an oligonucleotide comprising at least one modified nucleoside and / or at least one modified internucleoside linkage.

[0040] As used herein, "internucleoside linkage" refers to the covalent bond between consecutive nucleosides in an oligonucleotide. As used herein, "naturally occurring internucleoside linkage" refers to a 3' to 5' phosphodiester bond. As used herein, "modified internucleoside linkage" refers to any internucleoside linkage other than a naturally occurring internucleoside linkage.

[0041] As used herein, an "antisense compound" refers to a compound comprising or consisting of an oligonucleotide, at least a portion of which is complementary to a target nucleic acid capable of hybridizing thereto, thereby producing at least one antisense activity.

[0042] As used herein, "antisense activity" refers to any detectable and / or measurable change caused by the hybridization of an antisense compound to its target nucleic acid. In some embodiments, antisense activity includes regulating the amount and activity of target nucleic acid transcripts (such as mRNA). In some embodiments, antisense activity includes regulating the splicing of pre-mRNA.

[0043] As used herein, an "RNase H-based antisense compound" refers to an antisense compound that owes at least some of its antisense activity to hybridization of the antisense compound to a target nucleic acid and subsequent RNase H cleavage of the target nucleic acid.

[0044] As used herein, a "RISC-based antisense compound" refers to an antisense compound that attributes at least some of its antisense activity to the RNA-induced silencing complex (RISC).

[0045] As used herein, "expression" refers to the process by which a gene ultimately produces a protein, including but not limited to transcription, post-transcriptional modification (such as splicing, polyadenylation, 5' capping) and translation.

[0046] As used herein, "target nucleic acid" refers to a nucleic acid molecule to which an antisense compound will hybridize to produce the desired antisense activity. An antisense oligonucleotide has sufficient complementarity with its target nucleic acid to allow hybridization under physiological conditions.

[0047] As used herein, "nucleobase complementarity" or "complementarity" for nucleobases refers to the ability of a nucleobase to base pair with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In some embodiments, complementary nucleobases refer to the ability of a nucleobase of an antisense compound to base pair with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound can form a hydrogen bond with a nucleobase at a certain position of a target nucleic acid, then that nucleobase pair at the hydrogen bonding position between the oligonucleotide and the target nucleic acid is considered to be complementary. Nucleobases with certain modifications can maintain the ability to pair with corresponding nucleobases and therefore still have nucleobase complementarity.

[0048] As used herein, "non-complementary" with respect to nucleobases refers to a pair of nucleobases that do not form hydrogen bonds with each other.

[0049] As used herein, "complementarity" (e.g., connected nucleosides, oligonucleotides or nucleic acids) for oligomeric compounds refers to the ability of the oligomeric compound or its region to hybridize with another oligomeric compound or its region through core base complementarity. Complementary oligomeric compounds do not need to have core base complementarity on each nucleoside. Instead, some mispairings can be tolerated. In some embodiments, 70% of the core bases in the complementary oligomeric compound or region are complementary (70% complementary). In some embodiments, the complementary oligomeric compound or region is 80% complementary. In some embodiments, the complementary oligomeric compound or region is 90% complementary. In some embodiments, the complementary oligomeric compound or region is 95% complementary. In some embodiments, the complementary oligomeric compound or region is 100% complementary.

[0050] As used herein, "mismatch" refers to a situation in which a nucleobase of the first oligomeric compound cannot pair with a corresponding position of the second oligomeric compound when the first and second oligomeric compounds are aligned. Either or both of the first and second oligomeric compounds may be oligonucleotides.

[0051] As used herein, "hybridization" refers to the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). Although not limited to a specific mechanism, the most common pairing mechanism involves hydrogen bonding, and the hydrogen bonding between complementary nucleobases can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding.

[0052] As used herein, "specifically hybridizes" refers to the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than to another nucleic acid site.

[0053] As used herein, "fully complementary" to an oligonucleotide or portion thereof refers to each nucleobase of an oligonucleotide or portion thereof being capable of pairing with a nucleobase of a complementary nucleic acid or a contiguous portion thereof. Thus, any strand of a fully complementary region does not contain mismatched or unhybridized nucleobases.

[0054] As used herein, "complementarity percentage" refers to the percentage of nucleobases that are complementary to equal length portions of an oligomeric compound and a target nucleic acid. The percentage of complementarity is calculated by dividing the number of nucleobases that are complementary to the nucleobases at corresponding positions in the oligomeric compound and the target nucleic acid by the total length of the oligomeric compound.

[0055] As used herein, "percent identity" refers to the number of nucleobases in a first nucleic acid that are the same type of nucleobase as the corresponding position in a second nucleic acid (regardless of chemical modification) divided by the total number of nucleobases in the first nucleic acid.

[0056] As used herein, "pharmaceutically acceptable carrier or diluent" refers to any substance suitable for administration to an animal. In some embodiments, the pharmaceutically acceptable carrier or diluent is sterile saline. In some embodiments, the sterile saline is pharmaceutical grade saline.

[0057] The term "linear synthesis" generally refers to the synthesis starting from one end of the oligonucleotide and proceeding linearly to the other end. Linear synthesis allows the incorporation of identical or different monomer units (in terms of incorporated length, base composition and / or chemical modification) into an oligonucleotide.

[0058] As used herein, "about" means within + / - 10% of a numerical value. For example, if it is stated that "a marker can be increased by about 50%", it implies that the marker can be increased by 45% to 55%.

[0059] As used herein, "administered concomitantly" means that two agents are co-administered in any manner such that the pharmacological effects of both agents are manifested in the patient at the same time. Concomitant administration does not require that the two agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of the two agents do not have to be manifested at the same time. The effects only need to overlap for a period of time, and do not need to be the same in scope.

[0060] As used herein, "administering" or "dosing" refers to providing a pharmaceutical agent to an individual, including but not limited to: administration by a medical professional and self-administration. Administration of a pharmaceutical agent to an individual may be continuous, chronic, transient or intermittent. Administration may be parenteral or non-parenteral.

[0061] As used herein, "agent" refers to an active substance that can provide a therapeutic benefit when administered to an animal. "First agent" refers to a therapeutic compound of the invention. For example, the first agent can be an antisense oligonucleotide targeting TIGIT. "Second agent" refers to a second therapeutic compound of the invention (such as a second antisense oligonucleotide targeting TIGIT) and / or a non-TIGIT therapeutic compound.

[0062] As used herein, "effective amount" or "therapeutically effective amount" refers to an amount of active agent sufficient to achieve a desired physiological result in an individual in need of the agent. The effective amount will vary from individual to individual, depending on the health or physical condition of the individual to be treated, the taxonomic category of the individual to be treated, the formulation of the composition, the assessment of the individual's medical condition, and other relevant factors.

[0063] As used herein, "individual" or "subject" or "animal" refers to a human or non-human animal selected for treatment or therapy. As used herein, "individual in need thereof" refers to a human or non-human animal selected for such treatment or therapy.

[0064] As used herein, "inhibiting expression or activity" refers to reducing or blocking the expression or activity of RNA or protein, and does not necessarily mean completely eliminating the expression or activity.

[0065] As used herein, a "pharmaceutical" refers to a substance that provides a therapeutic benefit when administered to an individual. For example, in some embodiments, an antisense oligonucleotide targeting TIGIT is a pharmaceutical agent. As used herein, a "pharmaceutical composition" or "composition" refers to a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition can include one or more active agents and a pharmaceutical carrier (such as a sterile aqueous solution).

[0066] As used herein, a "region" or "target region" is defined as a portion of a target nucleic acid having at least one identifiable structure, function or feature. For example, a target region may include a 3'UTR, a 5'UTR, an exon, an intron, an exon / intron junction, a coding region, a translation initiation region, a translation termination region, or other defined nucleic acid regions. The structurally defined regions of TIGIT can be obtained by accession numbers in sequence databases such as NCBI, and this information is incorporated herein by reference. In some embodiments, a target region may include a sequence from a 5' target site of a target fragment within the target region to a 3' target site of another target fragment within the target region.

[0067] As used herein, "treat," "treatment," or "treating" refers to the administration of a compound described herein to alter or improve a disease, disorder, or condition.

[0068] "Portion" refers to a specified number of consecutive nucleobases (i.e., linked nucleobases) of a nucleic acid. In some embodiments, a portion is a specified number of consecutive nucleobases of a target nucleic acid. In some embodiments, a portion is a specified number of consecutive nucleobases of an antisense compound.

[0069] Some embodiments provide compounds and methods for reducing TIGIT mRNA and protein expression. In some embodiments, the compound is a TIGIT-specific inhibitor for treating, preventing or ameliorating TIGIT-related diseases. In some embodiments, the compound is an antisense oligonucleotide targeting TIGIT.

[0070] In some embodiments, antisense compounds targeting human TIGIT nucleic acid are provided. In some embodiments, the human TIGIT nucleic acid is the sequence shown in GENBANK Accession No. NM_173799.3 (incorporated herein as SEQ ID NO: 1).

[0071] 1 cgtcctatct gcagtcggct actttcagtg gcagaagagg ccacatctgc ttcctgtagg

[0072] 61 ccctctgggc agaagcatgc gctggtgtct cctcctgatc tgggcccagg ggctgaggca

[0073] 121 ggctcccctc gcctcaggaa tgatgacagg cacaatagaa acaacgggga acatttctgc

[0074] 181 agagaaaggt ggctctatca tcttacaatg tcacctctcc tccacccacgg cacaagtgac

[0075] 241 ccaggtcaac tggggagcagc aggaccagct tctggccatt tgtaatgctg acttggggtg

[0076] 301 gcacatctcc ccatccttca aggatcgagt ggccccaggt cccggcctgg gcctcaccct

[0077] 361 ccagtcgctg accgtgaacg atacagggga gtacttctgc atctatcaca cctaccctga

[0078] 421 tgggacgtac actgggagaa tcttcctgga ggtcctagaa agctcagtgg ctgagcacgg

[0079] 481 tgccaggttc cagattccat tgcttggagc catggccgcg acgctggtgg tcatctgcac

[0080] 541 agcagtcatc gtggtggtcg cgttgactag aaagaagaaa gccctcagaa tccattctgt

[0081] 601 ggaaggtgac ctcaggagaa aatcagctgg acaggaggaa tggagcccca gtgctccctc

[0082] 661 acccccagga agctgtgtcc aggcagaagc tgcacctgct gggctctgtg gagagcagcg

[0083] 721 gggagaggac tgtgccgagc tgcatgacta cttcaatgtc ctgagttaca gaagcctggg

[0084] 781 taactgcagc ttcttcacag agactggtta gcaaccagag gcatcttctg gaagatacac

[0085] 841 ttttgtcttt gctattatag atgaatatat aagcagctgt actctccatc agtgctgcgt

[0086] 901 gtgtgtgtgt gtgtgtatgt gtgtgtgtgt tcagttgagt gaataaatgt catcctcttc

[0087] 961 tccatcttca tttccttggc cttttcgttc tattccattt tgcattatgg caggcctagg

[0088] 1021 gtgagtaacg tggatcttga tcataaatgc aaaattaaaa aatatcttgacctggtttta

[0089] 1081 aatctggcag ttgagcaga tcctatgtct ctgagagaca cattcctcataatggccagc

[0090] 1141 atttgggct acaaggtttt gtggttgatg atgaggatgg catgactgcagagccatcct

[0091] 1201 catctcattt tttcacgtca ttttcagtaa cttcactca ttcaaaggcaggttataagt

[0092] 1261 aagtcctggt agcagcctct atggggagat ttgagagtga ctaaatcttggtatctgccc

[0093] 1321 tcaagaactt acagttaaat ggggagacaa tgttgtcatg aaaggtattatagtaagga

[0094] 1381 gagaaggaga catacacagg ccttcaggaa gagacgacag tttggggtgaggtagttggc

[0095] 1441 ataggcttat ctgtgatgaa gtggcctggg agcaccaagg ggatgttgaggctagtctgg

[0096] 1501 gaggagcagg agttttgtct agggaacttg taggaaattc ttggagctgaaagtcccaca

[0097] 1561 aagaaggccc tggcaccaag ggagtcagca aacttcagat tttattctctgggcaggcat

[0098] 1621 ttcaagttc cttttgctgt gacatactca tccattagac agcctgatacaggcctgtag

[0099] 1681 cctcttccgg ccgtgtgtgc tggggaagcc ccaggaaacg cacatgcccacacgggc

[0100] 1741 caagtcgtag catttgggcc ttgatctacc ttttctgcat caatacactcttgagcctttt

[0101] 1801 gaaaaaagaa cgtttcccac taaaaagaaa atgtggattt ttaaatagggactcttcct

[0102] 1861 aggggaaaaa ggggggctgg gagtgataga gggtttaaaa aataaacaccttcaaactaa

[0103] 1921 cttcttcgaa cccttttatt cactccctga cgactttgtg ctggggttggggtaactgaa

[0104] 1981 ccgcttattt ctgtttaatt gcattcaggc tggatcttag aagacttttatccttccacc

[0105] 2041 atctctctca gaggaatgag cggggaggtt ggatttactg gtgactgattttctttg

[0106] 2101 ggccaaggaa ctgaaagaga atgtgaagca aggttgtgtc ttgcgcatggttaaaaataa

[0107] 2161 agcattgtcc tgcttcctaa gacttagact ggggttgaca attgttttagcaacaagaca

[0108] 2221 attcaactat ttctcctagg atttttatta ttattatttt ttcacttttctaccaaatgg

[0109] 2281 gttacatagg aagaatgaac tgaaatctgt ccagagctcc aagtcctttggagaaagat

[0110] 2341 tagatgaacg taaaaatgtt gttgtttgct gtggcagttt acagcatttttcttgcaaaa

[0111] 2401 ttagtgcaaa tctgttggaa atagaacaca attcacaaat tggaagtgaactaaaatgta

[0112] 2461 atgacgaaaa gggagtagtg ttttgatttg gaggaggtgt atattcggcagaggttggac

[0113] 2521 tgagagttgg gtgttattta acataattat ggtaattggg aaacatttataaacactatt

[0114] 2581 gggatggtga taaaatacaa aagggcctat agatgttaga aatgggtcaggttactgaaa

[0115] 2641 tgggattcaa tttgaaaaaa attttttaa atagaactca ctgaactagattctcctctg

[0116] 2701 agaaccagag aagaccattt catagttgga ttcctggaga catgcgctatccaccacgta

[0117] 2761 gccactttcc acatgtggcc atcaaccact taagatgggg ttagtttaaatcaagatgtg

[0118] 2821 ctgttataat tggtataagc ataaaatcac actagattct ggagatttaatatgaataat

[0119] 2881 aagaatacta tttcagtagt tttggtatat tgtgtgtcaa aaatgataatattttggatg

[0120] 2941 tattgggtga aataaaatat taacattaaa aaaaaaaa (SEQ ID NO: 1)

[0121] In some embodiments, antisense compounds targeting mouse TIGIT nucleic acid are provided. In some embodiments, the mouse TIGIT nucleic acid is the sequence shown in GENBANK Accession No. EU675311.1 (incorporated herein as SEQ ID NO: 52).

[0122] 1 gccagtttca gttggaggag aggccacatc cactttgctg taggcctctg gttagaagca

[0123] 61 tgcatggctg gctgctcctg gtctgggtcc aggggctgat acaggctgcc ttcctcgcta

[0124] 121 caggagccac agcaggcacg atagatacaa agaggaacat ctctgcagag gaaggtggct

[0125] 181 ctgtcatctt acagtgtcac ttctcctctg acacagctga agtgacccaa gtcgactgga

[0126] 241 agcagcagga ccagcttctg gccatttata gtgttgacct ggggtggcat gtcgcttcag

[0127] 301 tcttcagtga tcgggtggtc ccaggcccca gcctaggcct caccttccag tctctgacaa

[0128] 361 tgaatgacac gggagagtac ttctgtacct atcatacgta tcctggtggg atttacaagg

[0129] 421 ggagaatatt cctgaaggtc caagaaagct cagtggctca gttccagact gccccgcttg

[0130] 481 gaggaaccat ggctgctgtg ctgggactca tttgcttaat ggtcacagga gtgactgtac

[0131] 541 tggctagaaa gaagtctatt agaatgcatt ctatagaaag tggccttggg agaacagaag

[0132] 601 cggagccaca ggaatggaac ctgaggagtc tctcatcccc tggaagccct gtccagacac

[0133] 661 aaactgcccc tgctggtccc tgtggagagc aggcagaaga tgactatgct gacccacagg

[0134] 721 aatactttaa tgtcctgagc tacagaagcc tagagagctt cattgctgta tcgaagactg

[0135] 781 gctaacgaca gctctctatc cctctcccta tgtctctctc tctgtctctc tctgtctctc

[0136] 841 tctgtctctg tctctgtctc tgtctctctc tctctctctc tctctctctc tgtgtgtgtg

[0137] 901 tgtgtgtatg tgtgtataca tcattaatgt tcattaacac taactgcata tggtggagga

[0138] 961 ccaggaaata aaagtttgtg ttgctaataa aattaagtgc taactt (SEQ ID NO: 52)

[0139] Some embodiments provide a compound targeting TIGIT, wherein the compound comprises 12 to 30 linked nucleosides. In some embodiments, the compound consists of 15 to 30, 18 to 24, 19 to 22, 13 to 25, 14 to 25, or 15 to 25 linked nucleosides. In some embodiments, the compound comprises at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 linked nucleosides. In some embodiments, the compound consists of 20 linked nucleosides. In some embodiments, the compound consists of 22 linked nucleosides.

[0140] Some embodiments provide a compound targeting TIGIT, wherein the compound consists of 12 to 30 linked nucleosides, and the nucleobase sequence of the compound includes at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 or 22 consecutive nucleobases of any nucleobase sequence of SEQ ID NO: 1. In some embodiments, the nucleobase sequence of the compound includes at least 12 consecutive nucleobases of any nucleobase sequence of SEQ ID NO: 1.

[0141] Some embodiments provide a compound targeting TIGIT, wherein the compound comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 or 22 consecutive nucleobases that are complementary to an equal length portion of SEQ ID NO: 1 or any one of SEQ ID NO: 27-51.

[0142] Some embodiments provide compounds targeting TIGIT fragments, wherein the compound includes at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 or 22 consecutive nucleobases that are complementary to an equal length portion of any target fragment shown in Table 1. In this table, "starting site" refers to the 5' most nucleotide of the target fragment, and "termination site" refers to the 3' most nucleotide of the target fragment. The range of the target fragment can be from the starting site to the termination site of each sequence listed in the table. Alternatively, the range of the target fragment can start from the starting site of one sequence and end at the termination site of another sequence. For example, as shown in Table 1, the range of the target fragment can be 151-172, i.e., the starting site to the termination site of SEQ ID NO: 27. In another example, as shown in Table 1, the range of the target fragment can be 319-440, i.e., the starting site of SEQ ID NO: 29 to the termination site of SEQ ID NO: 32.

[0143] Some embodiments provide a compound targeting TIGIT, wherein the base sequence of the compound is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to SEQ ID NO: 1. Some embodiments provide a compound targeting TIGIT, wherein the base sequence of the compound is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to any target fragment as shown in Table 1.

[0144] In some embodiments, the gene silencing compound targets any position within nucleobases 121-300, 301-600, 601-780, 900-1250, 1251-1440, or 1920-2400 of SEQ ID NO:1.

[0145] In some embodiments, the gene silencing compound targets nucleobases 100 to 300, 150 to 250, 175 to 225, 180 to 220, 50 to 250, 100 to 250, 150 to 350, 150 to 300, 300 to 500, 350 to 450, 375 to 425, 385 to 425, 250 to 450, 300 to 450, 300 to 550, 350 to 550, or 350 to 500, 300 to 800, 500 to 800, 550 to 750, 800 to 1150, 900 to 1250, 1150 to 1250, or 1250 to 1300. Any location in the area of ​​1400-1500, 1200-1350, 1225-1325, 1240-1300, 1050-1350, 1100-1350, 1200-1450, 1200-1400, 1400-1700, 1650-1800, 1650-2160, 1650-2200, 1600-1900, 1900-2200, 1900-2400, 2160-2500, 2300-2600, 2700-2800, or 2500-2978.

[0146] In some embodiments, the gene silencing compound targets any position within nucleobases 100 to 300, 150 to 250, 175 to 225, 180 to 220, 50 to 250, 100 to 250, 150 to 350, or 150 to 300 of SEQ ID NO:1.

[0147] In some embodiments, the gene silencing compound targets any position within the nucleobases 300 to 500, 350 to 450, 375 to 425, 385 to 425, 250 to 450, 300 to 450, 300 to 550, 350 to 550, or 350 to 500, 300 to 800, 500 to 800, 550 to 750, 800 to 1150, 900 to 1250 of SEQ ID NO: 1.

[0148] In some embodiments, the gene silencing compound targets any position within nucleobases 1150 to 1400, 1200 to 1350, 1225 to 1325, 1240 to 1300, 1050 to 1350, 1100 to 1350, 1200 to 1450, 1200 to 1400 of SEQ ID NO: 1.

[0149] In some embodiments, the gene silencing compound targets any position within nucleobases 1400 to 1700, 1650 to 1800, 1650 to 2160, 1650 to 2200, 1600 to 1900, 1900 to 2200, 1900 to 2400, 2160 to 2500, 2300 to 2600, 2700 to 2800, or 2500 to 2978 of SEQ ID NO: 1.

[0150] In some embodiments, the gene silencing compound targets any position within the nucleobase 300 to 800 region of SEQ ID NO:1.

[0151] In some embodiments, the gene silencing compound targets any position within the region of nucleobases 300 to 450 of SEQ ID NO:1.

[0152] In some embodiments, the gene silencing compound targets any position within the region of nucleobases 300 to 550 of SEQ ID NO:1.

[0153] In some embodiments, the gene silencing compound targets any position within the region of nucleobases 900 to 1250 of SEQ ID NO:1.

[0154] In some embodiments, the gene silencing compound targets any position within the nucleobase region 1600 to 1900 of SEQ ID NO:1.

[0155] In some embodiments, the gene silencing compound targets any position within the region of nucleobases 1650 to 2200 of SEQ ID NO:1.

[0156] In some embodiments, the gene silencing compound targets any position within the region of nucleobases 1650 to 2160 of SEQ ID NO:1.

[0157] In some embodiments, the gene silencing compound targets any position within the region of nucleobases 1900 to 2200 of SEQ ID NO:1.

[0158] In these embodiments, gene silencing compounds targeting the region of SEQ ID NO: 1 within the designated region (ie, a sub-region within the designated region) are also included.

[0159] Some embodiments provide a compound targeting TIGIT, wherein the compound consists of 12 to 30 linked nucleosides, and the nucleobase sequence of the compound includes at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 or 22 consecutive nucleobases of any nucleobase sequence of SEQ ID NO: 2-26. In some embodiments, the nucleobase sequence of the compound includes at least 12 consecutive nucleobases of any one of the nucleobase sequences in SEQ ID NO: 2-26. In some embodiments, the compound consists of any one of SEQ ID NO: 2-26.

[0160] Some embodiments provide an antisense compound targeting TIGIT as described herein, wherein the compound is single-stranded.

[0161] Some embodiments provide an antisense compound targeting TIGIT as described herein, wherein the oligonucleotide is unmodified.

[0162] Some embodiments provide antisense oligonucleotides targeting TIGIT as described herein, wherein the oligonucleotide includes at least one modified internucleoside bond. In some embodiments, the modified internucleoside bond is a phosphorothioate internucleoside bond. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 internucleoside bonds of the modified oligonucleotide are phosphorothioate internucleoside bonds. In some embodiments, the modified internucleotide bond is a phosphorothioate internucleotide bond. In some embodiments, more than half but less than all of the internucleotide bonds are phosphorothioate internucleotide bonds. In some embodiments, all internucleotide bonds are phosphorothioate internucleotide bonds. In some embodiments, the phosphorothioate bonds can be a mixture of Rp and Sp enantiomers, or they can be manufactured as any one of stereoregular or substantially stereoregular Rp or Sp forms.

[0163] Some embodiments provide an antisense oligonucleotide targeting TIGIT as described herein, wherein the oligonucleotide comprises at least one modified internucleotide linkage, and wherein the nucleotides of the oligonucleotide comprise naturally occurring nucleobases and sugars.

[0164] Some embodiments provide an antisense oligonucleotide targeting TIGIT as described herein, wherein the oligonucleotide comprises at least one modified internucleotide linkage, and wherein at least one nucleotide of the oligonucleotide comprises a modified nucleobase, a modified sugar, or a combination thereof.

[0165] In some embodiments, the modified nucleobase is selected from the group consisting of universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases, as defined herein. 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil (6-azo uracil), 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include: tricyclic pyrimidines, such as benzoxazine cytidine ([5,4-b][1,4]benzoxazine-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazine-2(3H)-one); G-clamps, such as substituted phenoxazine cytidine (such as 9-(2-aminoethoxy)-H-pyrimidinyl[5,4-b][1,4]benzoxazine-2(3H)-one), carbazole cytidine (2H-pyrimidinyl[4,5-b]indol-2-one), and pyridindol cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidinyl-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is substituted with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. In some embodiments, the modified nucleobase is 5-methylcytosine.

[0166] In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar includes 2'-O-methoxyethyl, constrained ethyl, 3'-fluoro-HNA or 4'-(CH2) n -O-2' bridge, wherein n is 1 or 2.

[0167] Some embodiments provide a compound comprising a modified oligonucleotide targeting TIGIT, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides, and the modified oligonucleotide includes: (a) a gap fragment consisting of linked deoxynucleosides; (b) a 5' wing fragment consisting of linked nucleosides; (c) a 3' wing fragment consisting of linked nucleosides; and the gap fragment is located between the 5' wing fragment and the 3' wing fragment, and each nucleoside of each wing fragment contains a modified sugar.

[0168] Some embodiments provide a compound comprising a modified oligonucleotide targeting TIGIT, wherein the modified oligonucleotide consists of 22 linked nucleosides, and the modified oligonucleotide includes: (a) a gap fragment consisting of 12 linked deoxynucleosides; (b) a 5' wing fragment consisting of 5 linked nucleosides; (c) a 3' wing fragment consisting of 5 linked nucleosides; and the gap fragment is located between the 5' wing fragment and the 3' wing fragment, each nucleoside of each wing fragment contains a 2'-O-methoxyethyl sugar, at least one internucleoside bond is a phosphorothioate bond, and each cytosine residue is a 5-methylcytosine.

[0169] Some embodiments provide a compound comprising a modified oligonucleotide targeting TIGIT, wherein the modified oligonucleotide consists of 22 linked nucleosides, and the nucleobase sequence of the modified oligonucleotide includes at least 8 consecutive nucleobases of any one of SEQ ID NOs: 2-26, and the modified oligonucleotide includes: (a) a gap fragment consisting of 12 linked deoxynucleosides; (b) a 5' wing fragment consisting of 5 linked nucleosides; (c) a 3' wing fragment consisting of 5 linked nucleosides; and the gap fragment is located between the 5' wing fragment and the 3' wing fragment, each nucleoside of each wing fragment contains a 2'-O-methoxyethyl sugar, at least one internucleoside bond is a phosphorothioate bond, and each cytosine residue is a 5-methylcytosine.

[0170] In some embodiments, the target nucleic acid is RNA. In some embodiments, the target nucleic acid is non-coding RNA. In some embodiments, the target nucleic acid encodes a protein. In some embodiments, the target nucleic acid is selected from mRNA, pre-mRNA, microRNA, non-coding RNA, including small non-coding RNA and promoter guide RNA.

[0171] In some embodiments, antisense oligonucleotide provided by the invention has a sequence complementary to the target nucleic acid. These antisense compounds can hybridize with the target nucleic acid to produce at least one antisense activity. In some embodiments, antisense compounds specifically hybridize to one or more target nucleic acids. In some embodiments, the core base sequence of specific hybridization antisense compounds includes such a region: the region is fully complementary to the target nucleic acid (so that hybridization and antisense activity can be produced) and the complementarity of any non-target sequence is insufficient (thereby avoiding or reducing the non-specific hybridization with non-target nucleic acid sequences when specific hybridization is needed (for example, under physiological conditions for in vivo or treatment, and under conditions of testing in the case of in vitro tests). In some embodiments, oligonucleotides have selectivity between target molecules and non-target molecules, even if target molecules and non-target molecules all include target sequences. In such embodiments, selectivity can come from the relative accessibility of the target region of a nucleic acid molecule relative to another.

[0172] In some embodiments, the disclosure provides antisense compounds, which include oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the oligonucleotide. In some embodiments, the oligonucleotide is 99% complementary to the target nucleic acid. In some embodiments, the oligonucleotide is 95% complementary to the target nucleic acid. In some embodiments, these oligonucleotides are 90% complementary to the target nucleic acid.

[0173] In some embodiments, these oligonucleotides are 85% complementary to the target nucleic acid. In some embodiments, these oligonucleotides are 80% complementary to the target nucleic acid. In some embodiments, the antisense compounds include a region that is fully complementary to the target nucleic acid and is 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In these embodiments, the length of the fully complementary region is 6 to 14 core bases.

[0174] In some embodiments, the oligonucleotide comprises a hybridization region and a termination region. In these embodiments, the hybridization region is composed of 12-30 connected nucleosides and is completely complementary to the target nucleic acid. In some embodiments, the hybridization region comprises 1 mismatch relative to the target nucleic acid. In some embodiments, the hybridization region comprises 2 mismatches relative to the target nucleic acid. In some embodiments, the hybridization region comprises 3 mismatches relative to the target nucleic acid. In some embodiments, the termination region is composed of 1-4 termination nucleosides. In some embodiments, the termination nucleosides are located at the 3' end. In some embodiments, one or more termination nucleosides are not complementary to the target nucleic acid.

[0175] Antisense mechanisms include any mechanism involving hybridization of an oligonucleotide to a target nucleic acid, wherein the hybridization results in a biological effect. In some embodiments, such hybridization results in degradation or occupancy of the target nucleic acid, and is accompanied by inhibition or stimulation of cellular mechanisms, such as translation, transcription, or splicing of the target nucleic acid.

[0176] One antisense mechanism involving degradation of target RNA is RNase H-mediated antisense. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that "DNA-like" single-stranded antisense compounds can induce RNase H activity in mammalian cells. Therefore, activation of RNase H results in cleavage of the RNA target, thereby greatly improving the efficiency of DNA-like oligonucleotide-mediated inhibition of gene expression.

[0177] The synthetic antisense compounds of the present invention can be prepared by methods recognized in the art, such as phosphoramidate or H-phosphonate chemistry that can be performed manually or by an automated synthesizer. The synthetic antisense compounds of the present invention can also be modified in a variety of ways without compromising their ability to hybridize with mRNA.

[0178] In some embodiments, the oligonucleotide-based compounds of the invention are synthesized by linear synthesis methods.

[0179] Another way to synthesize is "parallel synthesis", where the synthesis proceeds outward from a central linker. As described in U.S. Pat. No. 5,912,332, a linker attached to a solid support can be used for parallel synthesis. Alternatively, a universal solid support (e.g., phosphate-attached controlled pore glass) can be used.

[0180] At the end of the synthesis carried out by linear synthesis or parallel synthesis scheme, if the nucleoside of modification is incorporated, the compound based on oligonucleotide of the present invention can be conveniently deprotected with concentrated ammonia solution or deprotected according to the instructions of the phosphoramidite supplier. The compound product based on oligonucleotide is preferably purified by reversed-phase HPLC, trityl removed, desalted and dialyzed.

[0181] In some embodiments, the oligonucleotides of the antisense compounds according to the present invention are selected from the non-limiting list of oligonucleotides shown in Table 1. Unless otherwise specified, the target sequence and antisense oligonucleotides are relative to the human TIGIT nucleic acid sequence shown in SEQ ID NO: 1. The oligonucleotides shown in Table 1 have phosphorothioate (PS) bonds, but may also include phosphodiester bonds. Those skilled in the art will recognize that other bonds based on phosphodiester or non-phosphodiester moieties may be included.

[0182] Table 1

[0183]

[0184]

[0185] Some embodiments provide a composition comprising an antisense oligonucleotide compound as described herein and a pharmaceutically acceptable carrier or diluent. Some embodiments provide a composition comprising two or more compounds as described herein and a pharmaceutically acceptable carrier or diluent. The two or more compounds can be complementary to the same target region and / or fragment or different target regions and / or fragments of TIGIT.

[0186] In some embodiments, the present invention provides a composition comprising a compound of the present invention and a second agent, wherein the second agent includes but is not limited to: one or more vaccines, antigens, antibodies, cytotoxic drugs, chemotherapeutic drugs (traditional chemotherapy and modern targeted therapy), radiation, kinase inhibitors, allergens, antibiotics, agonists, antagonists, antisense oligonucleotides, ribozymes, RNAi molecules, siRNA molecules, miRNA molecules, nucleic acid aptamers, proteins, gene therapy vectors, DNA vaccines, adjuvants, co-stimulatory molecules or combinations thereof.

[0187] Some embodiments provide compositions and methods, the methods comprising administering an antisense compound or composition as described herein to an animal. In some embodiments, the antisense compound is administered to prevent, treat, improve or slow the progression of cancer, inflammatory diseases and viral infections, such as melanoma, head and neck squamous cell carcinoma, small cell or non-small cell lung cancer, hepatocellular carcinoma, breast cancer, cervical cancer, gastric cancer, colorectal cancer, renal cancer, esophageal squamous cell carcinoma, Merkel cell carcinoma, skin squamous cell carcinoma, microsatellite instability high cancer or bladder urothelial carcinoma, inflammatory diseases such as systemic lupus erythematosus, systemic sclerosis, multiple sclerosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, non-alcoholic fatty hepatitis and sepsis, and viral infections such as HIV, hepatitis B or hepatitis C.

[0188] Some embodiments provide antisense compounds, compositions and methods as described herein for treating TIGIT-related diseases, disorders or conditions. In some embodiments, TIGIT levels are elevated in an animal. In some embodiments, the compounds targeting TIGIT are used to treat, prevent, slow progression or improve a disease, disorder or condition. In some embodiments, the compositions and methods for treatment include administering a TIGIT-specific inhibitor to an individual in need.

[0189] Some embodiments provide compositions and methods for reducing TIGIT levels. Some embodiments provide compositions and methods for reducing TIGIT mRNA or protein expression in an animal, comprising administering to the animal an antisense compound or composition described herein to reduce TIGIT mRNA or protein expression in the animal.

[0190] In some embodiments, a method of treating a disease or disorder is provided, wherein reducing the expression of TIGIT contributes to the treatment of the disease or disorder, comprising administering a compound or composition described herein to an animal to reduce the expression of TIGIT mRNA or protein in the animal. In some embodiments, the animal is a human.

[0191] Some embodiments provide compositions and methods for preventing, treating, delaying, slowing progression and / or improving TIGIT-related diseases, disorders and conditions in subjects in need. In some embodiments, these diseases, disorders and conditions include cancer, inflammatory diseases and viral infections. Some such cancers include, but are not limited to: melanoma, head and neck squamous cell carcinoma, small cell and non-small cell lung cancer, hepatocellular carcinoma, gastroesophageal cancer, breast cancer, cervical cancer, gastric cancer, colorectal cancer, renal cancer, esophageal squamous cell carcinoma, Merkel cell carcinoma, cutaneous squamous cell carcinoma, microsatellite instability high cancer or urothelial bladder and urinary tract and renal cell carcinoma. Some embodiments provide compositions and methods for preventing, treating, delaying, slowing progression and / or improving cancer.

[0192] In some embodiments, the regulation of TIGIT expression occurs in cells, tissues and organs. In some embodiments, the regulation occurs in cells, tissues or organs of animals. In some embodiments, the regulation is a reduction in TIGIT mRNA levels. In some embodiments, the regulation is a reduction in TIGIT protein levels. In some embodiments, both TIGIT mRNA and protein levels are reduced. This reduction can occur in a time-dependent or dose-dependent manner.

[0193] In some embodiments, the subject or animal is a human.

[0194] In any method of the invention, the compounds of the invention, administered alone or in combination with other agents, may be administered by any suitable route, including, but not limited to, intramuscular, parenteral, mucosal, oral, sublingual, intratumoral, transdermal, topical, inhaled, intrathecal, intranasal, aerosol, intraocular, intratracheal, rectal, vaginal, by gene gun, skin patch, or in the form of eye drops or mouthwashes. In any method of the invention, the compounds of the invention, administered alone or in combination with other agents, may be administered directly to tissues and organs, such as, but not limited to, bladder, liver, lung, kidney, or lung. In some embodiments, the compounds of the invention are administered alone or in combination with other agents by intramuscular administration. In some embodiments, the gene silencing compounds of the invention are administered alone or in combination with other agents by mucosal administration. In some embodiments, the gene silencing compounds of the invention are administered alone or in combination with other agents by oral administration. In some embodiments, the gene silencing compounds of the invention are administered alone or in combination with other agents by intratumoral administration.

[0195] In some embodiments, the antisense compound is administered parenterally. In further embodiments, the parenteral administration is subcutaneous administration.

[0196] In some embodiments, an antisense compound or composition is co-administered with a second agent or therapy. In some embodiments, an antisense compound or composition is administered concomitantly with a second agent.

[0197] In some embodiments, the second agent includes, but is not limited to, one or more vaccines, antigens, antibodies, cytotoxic drugs, chemotherapeutic drugs (traditional chemotherapy and modern targeted therapy), kinase inhibitors, allergens, antibiotics, agonists, antagonists, antisense oligonucleotides, ribozymes, RNAi molecules, siRNA molecules, miRNA molecules, nucleic acid aptamers, peptides, targeted therapeutic agents, proteins, gene therapy vectors, DNA vaccines, adjuvants, costimulatory molecules, or combinations thereof. Agents or therapies can be administered together or concomitantly. Agents or therapies can be administered sequentially or subsequently.

[0198] Some embodiments provide the use of antisense compounds targeting TIGIT for reducing TIGIT levels in an animal. Some embodiments provide the use of antisense compounds targeting TIGIT for treating, preventing or ameliorating TIGIT-related diseases, disorders or conditions.

[0199] Some embodiments provide the use of an antisense compound targeting TIGIT in the preparation of a medicament for reducing TIGIT levels in an animal. Some embodiments provide the use of an antisense compound in the preparation of a medicament for treating, preventing or ameliorating a TIGIT-related disease, disorder or condition.

[0200] The administration of the therapeutic composition of the compound of the present invention can be carried out using known methods, using an effective amount and continuing for a certain period of time to effectively alleviate the symptoms of the disease or surrogate markers. For example, the effective amount of the compound of the present invention for treating a disease and / or disorder can be the amount required to alleviate or reduce symptoms or delay or improve the disease and / or disorder. In the case of administering a composition that regulates gene expression, the effective amount of the compound of the present invention is an amount sufficient to achieve the required regulation compared to the gene expression when the compound of the present invention is not present. For any particular application, the effective amount may vary due to factors such as the disease or condition being treated, the specific compound administered, the body size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can determine the effective amount of a particular compound empirically without the need for unnecessary experiments.

[0201] When systemically administered, the preferred therapeutic composition is administered with a dose sufficient to achieve a blood level of the compound of the present invention of about 0.0001 micromolar to about 10 micromolar. For topical administration, concentrations much lower than this concentration may be effective, and much higher concentrations may be tolerated. Preferably, the total dose of the compound of the present invention is from about 0.001 mg / patient / day to about 200 mg / kg body weight / day. In some embodiments, the total dose may be 0.08, 0.16, 0.32, 0.48, 0.32, 0.64, 1, 10 or 30 mg / kg body weight, administered daily, twice a week or weekly. It may be desirable to administer one or more therapeutic compositions of the present invention of a therapeutically effective amount simultaneously or sequentially as a single treatment cycle to an individual.

[0202] The method of this aspect of the invention is helpful for model studies of gene expression. The method can also be used for the prevention or treatment of human or animal diseases. For example, the method is helpful for pediatric and veterinary applications of the inhibition of gene expression.

[0203] Some embodiments provide a kit for treating, preventing, or ameliorating a disease, disorder, or condition described herein, wherein the kit comprises: (i) a TIGIT-specific inhibitor described herein; and optionally, (ii) a second agent or therapy described herein. The kit of the present invention may also include instructions for using the kit to treat, prevent, or ameliorate a disease, disorder, or condition described herein.

[0204] Example

[0205] The following examples illustrate some embodiments of the present disclosure and are non-limiting.In addition, although specific embodiments are provided, the inventors have anticipated the general application of these specific embodiments.

[0206] Example 1 - Synthesis of antisense oligonucleotides

[0207] All oligonucleotides were synthesized on a 10 μmol scale using β-cyanophosphoramidite chemistry on a solid support using an automated DNA / RNA synthesizer (Mermade6, BioAutomation, Texas). Phosphoramidites of dA, dC, dG, and T were sequentially linked in the desired order on an automated DNA / RNA synthesizer. Crude oligonucleotides were treated with ammonium hydroxide concentrate at 55°C overnight to deprotect and cleave from the solid support. Crude oligonucleotides were purified by preparative anion exchange HPLC. Purified oligonucleotides were purified at C 18 Desalting in the column and dialyzing overnight with large volume sterile water. Oligonucleotide solution is filtered with sterile filter membrane (0.2 μm or 0.45 μm HT Tuffryn Membrane, Pall Corporation) and lyophilized to obtain the final product. All oligonucleotides are identified by IE-HPLC (Waters 600, Waters 486 260nm adjustable absorbance detector, Empower software) and MALDI-TOF mass spectrometer (Waters MALDI-ToF mass spectrometer carrying 337nm N2 laser) purity and molecular weight. The purity range of full-length oligonucleotides is 95-98% by ion exchange HPLC, and the remaining oligonucleotides lack one or two nucleotides. All oligonucleotides are measured endotoxin levels by Limulus analyzer (Bio-Whittaker), and control endotoxin levels <0.1EU / mg.

[0208] Example 2 - In vitro screening of TIGIT antisense oligonucleotides

[0209] To identify potential human TIGIT antisense, antisense oligonucleotides (ASOs) targeting TIGIT mRNA were screened in human HeLa and / or NTERA-2 cell lines (ATCC, Manassas, VA) and their effects on human TIGIT mRNA expression were evaluated compared with scrambled control oligonucleotides. 5 Cells were seeded into 12-well tissue culture plates and cultured overnight at 37°C, 5% CO2. On the day of transfection, fresh culture medium was added to each well. Antisense oligonucleotides at concentrations of 25 and 50 nM were prepared in 50 μl of serum-free culture medium and mixed with 3 μl of (Thermo Fisher Scientific, Waltham, MA). The mixture was incubated at room temperature for 10 minutes and then added to the culture plate. The plates were then incubated at 37°C, 5% CO2 for 48 hours. Total RNA was extracted using RNeasy Mini (Qiagen, Germantown, MD) according to the manufacturer's instructions. RNA concentration was determined by UV spectrophotometer at 260 / 280 nm wavelength. For cDNA synthesis, 1 μg of total RNA was transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Human TIGIT mRNA expression levels were determined by real-time quantitative PCR. Briefly, approximately 75 μg of cDNA was mixed with 10 μl of TaqMan TM Rapid Advanced Master Mix (Thermo Fisher Scientific) and 1 μl human TIGIT gene expression probe (Hs00545087_m1, Thermo Fisher Scientific) or 1 μl human HPRT1 gene expression probe (Hs02800695_m1, Thermo Fisher Scientific) were mixed. TM Real-time quantitative PCR was performed using a real-time PCR system (Thermo Fisher Scientific), and the relative expression of the TIGIT gene was calculated using StepOne software version 2 (Thermo Fisher Scientific). The results are shown in Table 2.

[0210] Table 2.

[0211]

[0212]

[0213] Mouse TIGIT antisense oligonucleotide (mTIGIT ASO) (5'-GCTGTCGTTAGCCAGTCTTCGATAC-3') (SEQ ID NO: 53) was used to analyze its effect on mouse TIGIT mRNA expression in mouse CT26.CL25 cell line (ATCC). 5×10 5 Cells were seeded into 12-well tissue culture plates and cultured overnight at 37°C, 5% CO2. On the day of transfection, fresh culture medium was added to each well. Antisense oligonucleotides at concentrations of 0.21, 0.62, 1.85, 5.56, 16.67, and 50 nM were prepared in 50 μl of serum-free culture medium and mixed with 3 μl of (Thermo Fisher Scientific) was mixed with 50 μl of serum-free medium. The mixture was incubated at room temperature for 10 minutes and then added to the culture plate. The plate was then cultured at 37°C, 5% CO2 for 48 hours. Total RNA was extracted using RNeasy Mini (Qiagen) according to the manufacturer's instructions. RNA concentration was determined at 260 / 280 nm wavelength by UV spectrophotometer. For cDNA synthesis, 1 μg of total RNA was transcribed using HighCapacity cDNA (Thermo Fisher Scientific) according to the manufacturer's instructions. Mouse TIGIT mRNA expression levels were determined by real-time quantitative PCR. Briefly, approximately 75 μg of cDNA was mixed with 10 μl of Fast TaqMan assay buffer (Thermo Fisher Scientific) and 1 μl of mouse TIGIT gene expression probe (Mm03807522_m1, Thermo Fisher Scientific) or 1 μl of mouse HPRT1 gene expression probe (Mm01324427_m1, Thermo Fisher Scientific). Real-time quantitative PCR was performed using a StepOnePlus PCR instrument (Thermo Fisher Scientific), and the relative expression of the TIGIT gene was calculated using StepOne software version 2 (Thermo Fisher Scientific). Mouse TIGIT antisense oligonucleotide (mTIGITASO) homologue showed potent inhibition of mouse TIGIT mRNA in the mouse CT26.CL25 cell line ( Figure 1 ).

[0214] Example 3 - In vivo experiments evaluating the anti-tumor activity of TIGIT inhibition

[0215] To determine whether knockdown of TIGIT by ASO would affect T cell and NK cell function, the hTIGIT mouse homologue alone or in combination with an anti-PD-1 antibody was evaluated in mouse tumor models.

[0216] The mouse CT26.CL25 tumor model was used to analyze the in vivo effect of TIGIT inhibition in mice. 6-8 week old female BALB / c mice were injected subcutaneously on both sides with 3x10 6 Mouse colon cancer CT26.CL25 (ATCC) cells were used to establish solid tumors. On day 4 after tumor implantation, when the tumor volume reached 50 to 150 mm 3 Treatment was started between 1 and 2 weeks. Tumor-bearing mice were randomly divided into 3 treatment groups (n=10 mice / group). Control oligomer (15 mg / kg), 5 or 15 mg / kg of mTIGITASO in 100 μl PBS were administered intratumorally (it) to the right tumor on days 4, 7, 11, 13 and 15, for a total of 5 injections. Caliper measurements were taken every two weeks and the formula: Volume = (width) 2× length / 2 to calculate tumor volume. Tumor-bearing mice treated with mTIGIT ASO showed dose-dependent tumor growth inhibition in both injected and uninjected tumors (Figure 2). Tumor samples from the 15 mg / kg mTIGIT ASO and control oligomer-treated groups were collected on day 15 and stored in 10% formaldehyde buffer. Tissue sections were prepared by Mass Histology Service (Worcester, MA) and CD3 was stained with anti-mouse CD3 antibody. + T cell staining. Inhibition of TIGIT expression in mice resulted in a decrease in tumor-infiltrating CD3 T cells compared to tumors injected with control oligomers. + There was an increase in T lymphocytes (indicated by white arrows) (Figure 3).

[0217] It has been reported that TIGIT is mainly expressed in NK cells and some T cells. 1 Considering that TIGIT functions as a negative regulator of NK and T cell immune function 1,2 In order to evaluate the effects of TIGIT ASO on NK and CD8 + The role of T cell function in NK or CD8 + The study was conducted in a mouse tumor model with T cell depletion. 6 Mouse colon cancer CT26.CL25 cells were injected subcutaneously into the right flank of BALB / c mice to establish solid tumors. On days 1, 4, 8, and 12, 50 μl of Ultra-LEAF TM Purified anti-Asialo-GM1 antibody (n=10, Biolegend, San Diego, California) or 25 mg / kg anti-mouse CD8 monoclonal antibody (n=8, clone YTS169.4; order number BE0117, BioXcell, West Lebanon, New Hampshire) was used to activate NK cells or CD8 + T cell depletion. Tumor-bearing mice without T cell depletion (n=10), NK or CD8 + Depleted tumor-bearing mice were treated with intratumoral injections of 15 mg / kg on days 5, 8, 11, 12, and 13, for a total of 5 injections. NK or CD8 + Depletion of these cells did not affect tumor growth. Tumor-bearing mice were injected intratumorally with 15 mg / kg control oligomer. Caliper measurements were taken every two weeks using the formula: volume = (width) 2 × length / 2 to calculate the tumor volume.

[0218] Two days after the last mTIGIT ASO or control oligomer treatment, plasma samples were collected and interferon-γ levels were measured by ELISA. ELISA analysis was performed using the Mouse Interferon-γ Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Figure 4A ) and CD8 + cell( Figure 4B This further supports that mTIGIT ASO targets TIGIT gene expression (TIGIT is expressed in NK cells and some T cells, including CD8 + Furthermore, mTIGIT ASO treatment resulted in increased production of IFN-γ, a key cytokine in NK cells ( Figure 4C ). Through NK cell depletion rather than CD8 + T cell depletion abolished the increase in IFN-γ production caused by TIGIT inhibition ( Figure 4C ). This further supports that mTIGIT ASO inhibits TIGIT expression and activates NK cells.

[0219] Based on the above, and without wishing to be bound by any particular theory, it is believed that unmodified ASOs have an advantage over modified ASOs for local administration because the charge of the unmodified ASOs may result in a longer residence time at the local site. To evaluate this hypothesis, the 5' and 3' ends of the oligonucleotides of five mTIGIT ASOs were simultaneously modified to obtain 2'-O-methyl ribonucleotides and their antitumor activity was compared with that of the original version. BALB / c female mice aged 6-8 weeks were injected subcutaneously in the right flank with 3×10 6 Mouse colon cancer CT26.CL25 (ATCC) cells were used to establish solid tumors. On the 4th day after tumor transplantation, when the tumor volume reached 50 to 150 mm 3 Treatment was started between 1 and 2 weeks. Tumor-bearing mice were randomly divided into 3 treatment groups (n=10 mice / group). Control oligomers (15 mg / kg), 15 mg / kg of mTIGIT ASO or 15 mg / kg of mTIGIT 2'-OMe ASO (where 5 mTIGIT 2'-OMe ASOs in 100 μl of PBS were administered to the tumor by intratumoral injection (it), injected on days 4, 7, 11, 13 and 15, for a total of 5 injections). Caliper measurements were taken every two weeks and the formula: volume = (width) 2 × length / 2 to calculate tumor volume. Although 2'-O-methyl modification still has some antitumor activity, its potency is greatly reduced ( Figure 5), indicating that unmodified antisense oligomers may be more suitable for intratumoral administration compared with 2'-O-methyl or 2'-O-methoxyethyl modified antisense oligomers.

[0220] Compared with checkpoint inhibitor monotherapy, since inhibition of TIGIT can reactivate NK and T cells, combination with other checkpoint inhibitors may provide therapeutic advantages in treating cancer. When mTIGIT and PD-1 were simultaneously targeted in mouse tumor models, synergistic tumor growth inhibition was observed (Figure 6).

[0221] References

[0222] 1. Yu X et al., 2009. The surface protein TIGIT suppresses T cellactivation by promoting the generation of mature immunoregulatory dendritic cells. Nat Immunol. 10:48–57.

[0223] 2. Stanietsky N et al., 2009. The interaction of TIGIT with PVR andPVRL2 inhibits human NK cell cytotoxicity. Proc Natl Acad Sci US A. 106:17858–63.

[0224] 3. Chew GM et al., 2016. TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV andSIV Infection. PLoS Pathogens. 12:e1005349.

[0225] 4.Guillerey C et al.,2018.TIGIT immune checkpoint blockade restoresCD8 +T cell immunity against multiple myeloma. Blood 2018:blood-2018-01-825265; doi:https: / / doi.org / 10.1182 / blood-2018-01-825265.

[0226] 5.Chauvin J et al.,2015.TIGIT and PD-1 impair tumor antigen-specificCD8 + T cells in melanoma patients. J Clin Invest.125:2046–2058.

[0227] 6. Pauken KE and Wherry EJ. 2014. TIGIT and CD226: tipping the balance between co-stimulatory and coinhibitory molecules to augment the cancerimmunotherapy toolkit. Cancer Cell 26:785-787. Sequence Listing <110> SCHNEUNG KANG COMPANY <120> Compositions and methods for inhibiting TIGIT gene expression <130> PUSCNN213383T <140> 201980067732.3 <141> 2019-10-17 <150> 62 / 747,261 <151> 2018-10-18 <160> 53 <170> PatentIn version 3.5 <210> 1 <211> 2978 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 1 cgtcctatct gcagtcggct actttcagtg gcaagaagagg ccacatctgc ttcctgtagg 60 ccctctgggc agaagcatgc gctggtgtct cctcctgatc tgggcccagg ggctgaggca 120 ggctcccctc gcctcaggaa tgatgacagg cacaatagaa acaacggga acatttctgc 180 agaaaggt ggctctatca tcttacaatg tcacctctcc tccaccacgg cacaagtgac 240 ccaggtcaac tgggagcagc aggaccagct tctggccatt tgtaatgctg acttggggtg 300 gcacatctcc ccatccttca aggatcgagt ggccccaggt cccggcctgg gcctcaccct 360 ccagtcgctg accgtgaacg atacagggga gtacttctgc atctatcaca cctaccctga 420 tgggacgtac actgggagaa tcttcctgga ggtcctagaa agctcagtgg ctgagcacgg 480 tgccaggttc cagattccat tgcttggagc catggccgcg acgctggtgg tcatctgcac 540 agcagtcatc gtggtggtcg cgttgactag aaaagaaa gccctcagaa tccattctgt 600 ggaaggtgac ctcaggagaa aatcagctgg acaggagaa tggagcccca gtgctccctc 660 acccccagga agctgtgtcc aggcagaagc tgcacctgct gggctctgtg gagagcagcg 720 gggagaggac tgtgccgagc tgcatgacta cttcaatgtc ctgagttaca gaagcctggg 780 taactgcagc ttcttcacag agactggtta gcaaccagag gcatcttctg gaagatacac 840 ttttgtcttt gctattatag atgaatatat aagcagctgt actctccatc agtgctgcgt 900 gtgtgtgtgt gtgtgtatgt gtgtgtgtgt tcagttgagt gaataaatgt catcctcttc 960 tccatcttca tttccttggc cttttcgttc tattccattt tgcattatgg caggcctagg 1020 gtgagtaacg tggatcttga tcataaatgc aaaattaaaa aatatcttga cctggtttta 1080 aatctggcag tttgagcaga tcctatgtct ctgagagaca cattcctcat aatggccagc 1140 attttgggct acaaggtttt gtggttgatg atgaggatgg catgactgca gagccatcct 1200 catctcattt tttcacgtca ttttcagtaa ctttcactca ttcaaaggca ggttataagt 1260 aagtcctggt agcagcctct atggggagat ttgagagtga ctaaatcttg gtatctgccc 1320 tcaagaactt acagttaaat ggggagacaa tgttgtcatg aaaaggtatt atagtaagga 1380 gagaaggaga catacacagg ccttcaggaa gagacgacag tttggggtga ggtagttggc 1440 ataggcttat ctgtgatgaa gtggcctggg agcaccaagg ggatgttgag gctagtctgg 1500 gaggagcagg agttttgtct agggaacttg taggaaatttc ttggagctga aagtcccaca 1560 aagaaggccc tgcaccag gggtcagca aacttcagat tttctct gggcaggcat 1620 ttcaagtttc cttttgctgt gatactca tccattagac agcctgatac aggcctgtag 1680 cctcttccgg ccgtgtgtgc tggggaagcc cacaggaacg cacatgccca cacagggagc 1740 caagtcgtag cattgggcc ttgatctacc ttttctgcat caatacactc tgagccttt 1800 gaaaaaagaa cgttcccac taaaaaagaa atgtggatt ttaaaatagg gactctcct 1860 agggaaaaa ggggggctgg gagtgataga gggtttaaaaaaaacc ttcaactaa 1920 cttcttcgaa cccttttatt cactccctga cgactttgtg ctggggttgg ggtactgaa 1980 ccgcttattt ctgtttaatt gcattcaggc tggatcttag agacttta tccttccacc 2040 atctctctca gaggaatgag cggggaggtt ggatttactg gtgactgat ttcttcatg 2100 ggccaaggaa ctgaaagaga atgtgaagca aggttgtgtc ttgcgcatgg ttaaaaataa 2160 agcattgtcc tgcttcctaa gacttagact ggggttgaca attgttttag caacaagaca 2220 attcaactat ttctcctagg atttttatta ttattatttt ttcacttttc taccaaatgg 2280 gttacatagg aagaatgaac tgaaatctgt ccagagctcc aagtcctttg gaagaaagat 2340 tagatgaacg taaaaatgtt gttgtttgct gtggcagttt acagcatttt tcttgcaaaa 2400 ttagtgcaaa tctgttggaa atagaacaca attcacaaat tggaagtgaa ctaaaatgta 2460 atgacgaaaa gggagtagtg ttttgatttg gaggaggtgt atattcggca gaggttggac 2520 tgagagttgg gtgttattta acataattat ggtaattggg aaaacatttat aaacactatt 2580 gggatggtga taaaatacaa aagggcctat agatgttaga aatgggtcag gttactgaaa 2640 tgggattcaa tttgaaaaaa attttttaa atagaactca ctgaactaga ttctcctctg 2700 agaaccagag aagaccattt catagttgga ttcctggaga catgcgctat ccaccacgta 2760 gccactttcc acatgtggcc atcaaccact taagatgggg ttagtttaaa tcaagatgtg 2820 ctgttataat tggtataagc ataaatcac actagattct ggagatttaa tatgaataat 2880 aagaatacta tttcagtagt tttggtatat tgtgtgtcaa aaatgataat attttggatg 2940 tattgggtga aataaaatat taacattaaa aaaaaaaa 2978 <210> 2 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 2 gttccccgtt gtttctattg tg 22 <210> 3 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 3 gtcacttgtg ccgtggtgga gg 22 <210> 4 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 4 acctggggcc actcgatcct tg 22 <210> 5 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 5 gcccaggccg ggacctgggg cc 22 <210> 6 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 6 gtatcgttca cggtcagcga ct 22 <210> 7 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 7 ttctcccagt gtacgtccca tc 22 <210> 8 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 8 gaccaccagc gtcgcggcca tg 22 <210> 9 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 9 ttctagtcaa cgcgaccacc ac 22 <210> 10 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 10 tagtcatgca gctcggcaca gt 22 <210> 11 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 11 acacacacac acgcagcact ga 22 <210> 12 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 12 aaatggaata gaacgaaaag gc 22 <210> 13 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 13 aagatccacg ttactcaccc ta 22 <210> 14 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 14 aattactgaa aatgacgtga aa 22 <210> 15 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 15 accccaaact gtcgtctctt cc 22 <210> 16 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 16 ccagcacaca cggccggaag ag 22 <210> 17 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 17 tgtgggcatg tgcgtttcct gg 22 <210> 18 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 18 ggcccaaatg ctacgacttg gc 22 <210> 19 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 19 tctttttagt gggaaacgtt ct 22 <210> 20 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 20 aataaaaggg ttcgaagaag tt 22 <210> twenty one <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> twenty one agcacaaagt cgtcagggag tg 22 <210> twenty two <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> twenty two agaaataagc ggttcagtta cc 22 <210> twenty three <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> twenty three ccaacctccc cgctcattcc tc 22 <210> twenty four <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> twenty four aaccatgcgc aagacacaac ct 22 <210> 25 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 25 aacattttta cgttcatcta at 22 <210> 26 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 26 gctacgtggt ggatagcgca tgtc 24 <210> 27 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 27 cacaatagaa acaacgggga ac 22 <210> 28 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 28 cctccaccac ggcacaagtg ac 22 <210> 29 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 29 caaggatcga gtggccccag gt 22 <210> 30 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 30 ggccccaggt cccggcctgg gc 22 <210> 31 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 31 agtcgctgac cgtgaacgat ac 22 <210> 32 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 32 gatgggacgt acactgggag aa 22 <210> 33 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 33 catggccgcg acgctggtgg tc 22 <210> 34 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 34 gtggtggtcg cgttgactag aa 22 <210> 35 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 35 actgtgccga gctgcatgac ta 22 <210> 36 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 36 tcagtgctgc gtgtgtgtgtgt gt 22 <210> 37 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 37 gccttttcgt tctattccat tt 22 <210> 38 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 38 tagggtgagt aacgtggatc tt 22 <210> 39 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 39 tttcacgtca ttttcagtaa tt 22 <210> 40 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 40 ggaagagacg acagtttggg gt 22 <210> 41 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 41 ctcttccggc cgtgtgtgct gg 22 <210> 42 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 42 ccaggaaacg cacatgccca ca 22 <210> 43 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 43 gccaagtcgt agcatttggg cc 22 <210> 44 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 44 agaacgtttc ccactaaaaa ga 22 <210> 45 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 45 aacttcttcg aaccctttta tt 22 <210> 46 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 46 cactccctga cgactttgtg ct 22 <210> 47 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 47 ggtaactgaa ccgcttattt ct 22 <210> 48 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 48 gaggaatgag cggggaggtt gg 22 <210> 49 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 49 aggttgtgtc ttgcgcatgg tt 22 <210> 50 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 50 attagatgaa cgtaaaaatg tt 22 <210> 51 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 51 gacatgcgct atccaccacg tagc 24 <210> 52 <211> 1006 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 52 gccagtttca gttggaggag aggccacatc cactttgctg taggcctctg gttagaagca 60 tgcatggctg gctgctcctg gtctgggtcc aggggctgat acaggctgcc ttcctcgcta 120 caggagccac agcaggcacg atagatacaa agaggaacat ctctgcagag gaaggtggct 180 ctgtcatctt acagtgtcac ttctcctctg acacagctga agtgacccaa gtcgactgga 240 agcagcagga ccagcttctg gccatttata gtgttgacct ggggtggcat gtcgcttcag 300 tcttcagtga tcgggtggtc ccaggcccca gcctaggcct caccttccag tctctgacaa 360 tgaatgacac gggagagtac ttctgtacct atcatacgta tcctggtggg atttacaagg 420 ggagaatatt cctgaaggtc caagaaagct cagtggctca gttccagact gccccgcttg 480 gaggaaccat ggctgctgtg ctgggactca tttgcttaat ggtcacagga gtgactgtac 540 tggctagaaa gaagtctatt agaatgcatt ctatagaaag tggccttggg agaacagaag 600 cggagccaca ggaatggaac ctgaggagtc tctcatcccc tggaagccct gtccagacac 660 aaactgcccc tgctggtccc tgtggagagc aggcagaaga tgactatgct gacccacagg 720 aatactttaa tgtcctgagc tacagaagcc tagagagctt cattgctgta tcgaagactg 780 gctaacgaca gctctctatc cctctcccta tgtctctctc tctgtctctc tctgtctctc 840 tctgtctctg tctctgtctc tgtctctctc tctctctctc tctctctctc tgtgtgtgtg 900 tgtgtgtatg tgtgtataca tcattaatgt tcattaacac taactgcata tggtggagga 960 ccaggaaata aaagtttgtg ttgctaataa aattaagtgc taactt 1006 <210> 53 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 53 gctgtcgtta gccagtcttc gatac 25

Claims

1. A synthetic modified oligonucleotide compound, the nucleotide sequence of which is shown in SEQ ID NO:

6.

2. A composition comprising the compound according to claim 1 and a pharmaceutically acceptable carrier.

3. The composition according to claim 2, wherein The composition also comprises one or more vaccines, antigens, cytotoxic drugs, chemotherapeutic drugs, kinase inhibitors, allergens, antibiotics, agonists, antagonists, antisense oligonucleotides, RNAi molecules, siRNA molecules, miRNA molecules, nucleic acid aptamers, proteins, gene therapy vectors, adjuvants, or combinations thereof.

4. The composition according to claim 3, wherein The vaccine is a DNA vaccine.

5. The composition according to claim 3, wherein The protein is selected from the group consisting of an antibody, a ribozyme and a co-stimulatory molecule.

6. A method for inhibiting the expression of TIGIT mRNA or TIGIT protein in vitro, wherein: The method comprises contacting a cell with the compound of claim 1.

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