Targeting and localized in vivo delivery of oligonucleotides

Abstract

This invention provides targeted conjugates and methods that improve the targeting and local in vivo delivery of oligonucleotides. [Solution] A composition containing a targeted oligonucleotide-HES conjugate is provided as a method for constructing and using the conjugate in therapeutic, diagnostic, and other applications. The oligonucleotide-HES complex contained in the targeted oligonucleotide-HES conjugate can cross membranes in a receptor-independent manner and can deliver oligonucleotides containing complementary sequences to the cytosol of living cells in vivo. The targeted oligonucleotide-HES conjugate has uses including the targeting and / or local delivery of antisense oligonucleotides, siRNAs, shRNAs, Dicer substrates, miRNAs, anti-miRNAs, and other nucleic acid sequences in living organisms.

Description

[Technical Field]

[0001] This disclosure relates to oligonucleotide therapies. In particular, this disclosure relates to targeted conjugates, including modified oligonucleotides and oligonucleotide mimetic compounds, that provide improved targeting and local in vivo delivery of oligonucleotides, as well as methods for preparing and using these conjugates. [Background technology]

[0002] Oligonucleotides are increasingly recognized as potential therapeutic agents for various human diseases. However, a major challenge in the development of therapeutic oligonucleotides is specific and efficient in vivo delivery to target cells, which is essential for successful clinical application. Targeted systems can significantly improve the efficiency and specificity of oligonucleotide delivery. However, effective delivery systems must successfully overcome numerous biological barriers to allow oligonucleotides to reach the site of action and access their biological targets. To achieve these objectives, several targeted delivery strategies based on various platform technologies and target-directed ligands have been developed, but each strategy has limitations that hinder its broad applicability to oligonucleotide therapies. Therefore, new targeted oligonucleotide delivery technologies are needed. [Overview of the project]

[0003] The present disclosure relates to targeted oligonucleotide-HES conjugate compounds comprising oligonucleotide complexes containing an H-type exciton structure (HES), and methods of making and using these compounds. The present disclosure is based in part on the inventors' important discovery that conjugating an oligo-HES complex to a targeting moiety increases the targeting and local delivery of the oligonucleotide contained in the target oligonucleotide-HES conjugate to intracellular and / or surrounding targeted nucleic acids that express cell surface antigens specifically bound by the targeting moiety of the conjugate. Targeted oligonucleotide-HES conjugates dramatically improve pharmacokinetic properties and selective tissue distribution compared to oligonucleotides alone and oligonucleotide-HES complexes, and enhance the intracellular uptake of the oligonucleotide in the conjugate in targeted cells and the microenvironment of the targeted cells.

[0004] In some embodiments, the present disclosure provides the following: [1] A conjugate comprising a targeting moiety conjugated to an oligonucleotide-HES complex, optionally wherein the oligonucleotide is a therapeutic oligonucleotide; [2] The conjugate according to [1], wherein the targeting moiety is conjugated directly to the oligonucleotide-HES complex or conjugated to the oligonucleotide-HES complex via a linker; [3] The conjugate according to [1] or [2], wherein the targeting moiety is conjugated to the oligonucleotide-HES complex via a linker; [4] Of formula (I) T-(L n -(oligo-HES) x ) p (I) A conjugate according to any one of [1] to [3] having the structure of (wherein, T is a targeting moiety that selectively binds to a target of interest; L is a linker, Oligo-HES is an oligonucleotide complex containing therapeutic oligonucleotides and H-type exciton structures (HES); n is either 0 or 1; x is 1 to 30, 1 to 20, 1 to 10, or 1 to 5; p is 1-30, 1-20, 1-10, or 1-5; [5] Formula (II) T-[L n -((Oligo2-SP) m -Oligo1-HES) s ] u (II) One of the following conjugates having the structure [1] to [4] (wherein the formula, T is a target-directed portion that selectively binds to the target of choice; L is a linker, SP is a linker, and in some cases, SP is a peptide or alkyl chain spacer consisting of 6 to 12 amino acid residues, such as a linear or branched C6, C10, or C18 alkyl chain; Oligo1-HES is an oligonucleotide complex containing oligonucleotide 1 (oligo1) and an H-type exciton structure (HES); Oligo 2 is an oligonucleotide that may be the same as or different from Oligo 1; n is either 0 or 1; m is either 0 or 1; s is either 1 or 2; u is 1, 2, 3, 4, or 5; [6] A conjugate from any one of [1] to [5] comprising an oligonucleotide-HES complex that specifically hybridizes to a nucleic acid sequence in vivo and modulates the level of a protein encoded or controlled by the nucleic acid; [7] A conjugate of a therapeutic oligonucleotide containing one, two, or three substitutions, deletions, or insertions compared to the corresponding reverse complementary strand of the nucleic acid sequence, [6]; [8] The therapeutic oligonucleotide is a conjugate of any one of [1] to [7], having a length of approximately 8 to 750 nucleotides; [9] A conjugate of any one of [1]-[8], wherein the therapeutic oligonucleotide is 18-25, 18-35, 18-40, or 18-45, 18-50, 18-60, 18-70, 18-80, 18-90, 18-100, 18-150, or 18-200 nucleotides in length;

[10] A single-stranded therapeutic oligonucleotide conjugate of any one of [1] to [9];

[11] A conjugate of any one of [1] to [9] in which the therapeutic oligonucleotide is double-stranded;

[12] Therapeutic oligonucleotides are conjugates of

[11] having lengths of 36–50, 36–60, 36–70, or 36–100 nucleotides;

[13] A therapeutic oligonucleotide comprising one or more modified nucleoside motifs selected from locked nucleic acid (LNA), alpha-LNA, 2'-fluoro(2'F), 2'-O(CH2)2OCH3(2'-MOE), 2'--deoxy-2'--fluoro-D-arabino nucleic acid (FANA), 2'-OCH3(2'-O-methyl)(2'OME), PNA, and morpholino, in any one of the conjugates of [1] to

[12] ;

[14] The modified nucleoside motif is a conjugate of

[13] in which the methylene (--CH2--)n group is an LNA or alpha-LNA bridging the 2' oxygen atom and the 4' carbon atom, and n is 1 or 2;

[15] Conjugates of

[13] or

[14] in which LNA or alpha-LNA has a methyl group at the 5' position;

[16] A conjugate from any one of [1] to

[15] containing one or more modified nucleoside links selected from phosphorothioates, phosphorodithioates, phosphoramides, 3'-methylenephosphonates, O-methylphosphoroamidiates, PNAs, and morpholino;

[17] A therapeutic oligonucleotide comprising one or more modified nucleic acid bases selected from C-5 propine and 5-methyl C, one of any of the conjugates from [1] to

[16] ;

[18] The therapeutic oligonucleotide conjugate is selected from the following nucleic acids: EGFR, HER2 / neu, ErbB3, cMet, p56lck, PDGFR, VEGF, VEGFR, FGF, FGFR, ANG1, ANG2, bFGF, TIE2, protein kinase C-alpha (PKC-alpha), p56lck PKA, TGF-β, IGFIR, P12, MDM2, BRCA, IGF1, HGF, PDGF, IGFBP2, IGF1R, HIF1 alpha, ferritin, transferrin receptor, TMPRSS2, IRE, HSP27, HSP70, HSP90, MITF, clatherin, PARP1 C-fos, C-myc, n-myc, C-raf, B-raf, A1, H-raf, Skp2, K-ras, N-ras, H-ras, pharensyltransferase, c-Src, Jun, Fos, Bcr-Abl, c-Kit, EphA2, PDGFB, ARF, NOX1, NF1, STAT3, E6 / E7, APC, WNT, betacatenin, GSK3b, PI3k, mTOR, Ak The method according to any one of [1] to

[17] , which specifically hybridizes to t, PDK-1, CDK, Mek1, ERK1, AP-1, P53, Rb, Syk, osteopontin, CD44, MEK, MAPK, NF-κβ (NF kappa beta), E cadherin, cyclin D, cyclin E, Bcl2, Bax, BXL-XL, BCL-W, MCL1, ER, MDR, telomerase, telomerase reverse transcriptase, DNA methyltransferase, histone deacetylase (e.g., HDAC1 and HDAC2), integrin, IAP, aurora kinase, metalloproteinase (e.g., MMP2, MMP3 and MMP9), proteasome, or metallothionein genes;

[19] The method according to any one of [1] to

[17] , wherein the conjugate therapeutic oligonucleotide specifically hybridizes to nucleic acids selected from: Survivin, HSPB1, EIF4E, PTPN1, RRM2, BCL2, PTEN, Bcr-abl, TLR9, HaRas, Pka-rIA, JNK2, IGF1R, XIAP, TGF-β2, c-myb, PLK1, K-RAS, KSP, PKN3, ribonucleotide reductases (e.g., ribonucleotide reductase R1 and ribonucleotide reductase R2), RecQ helicases (e.g., WRN, RecQL1, BLM, RecQL4, RecQ5, and RTS), MEM2, and TLR9;

[20] The oligonucleotide-HES complex comprises at least one fluorophore with excitation and / or emission in the range of 300–850 nm, one of the conjugates of [1]–

[19] ;

[21] The oligonucleotide-HES complex comprises two, three, four or more fluorophores capable of forming one or more HESs, one of any one of the conjugates from [1] to

[20] ;

[22] The oligonucleotide-HES complex comprises one of the conjugates [1] to

[21] , comprising 2, 3, 4 or more fluorophores accompanied by excitation and / or emission in the 300–850 nm range;

[23] The oligonucleotide-HES complex comprises at least one fluorophore selected from xanthene, indocarbocyanin, indodicarbocyanin, and coumarin, according to any one of [1] to

[22] ;

[24] The oligonucleotide-HES complex comprises at least one fluorophore selected from carboxyrhodamine 110, carboxytetramethylrhodamine, carboxyrhodamine-X, diethylaminocoumarin and N-ethyl-N'-[5-(N''-succinimidyloxycarbonyl)pentyl]indocarbocyanine chloride, N-ethyl-N'-[5-(N''-succinimidyloxycarbonyl))pentyl]-3,3,3',3'-tetramethyl-2',2'-indodicarbocyanine chloride dye, one of any one of [1] to

[23] ; in further embodiments, the oligo-HES complex is Rhodamine Green® carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green® carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green®-X succinimidyl ester or hydrochloride; Rhodol Green™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl) sulfonerhodamine or di(succinimidyl ester); 5-(and-6)-carboxynaphthofluorescein, 5-(and-6)-carboxynaphthofluorescein succinimidyl ester; 5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidyl ester; 6-carboxyrhodamine 6G succinimidyl ester; 5-(and-6)-carboxyrhodamine 6G succinimidyl ester; 5-Carboxy-2',4',5',7'-tetrabromosulfone fluorescein succinimidyl ester or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimidyl ester; 6-carboxytetramethylrhodamine succinimidyl ester; 5-(and-6)-carboxytetramethylrhodamine succinimidyl ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester;6-Carboxy-X-Rhodamine succinimidyl ester; 5-(and-6)-Carboxy-X-Rhodamine succinimidyl ester; 5-Carboxy-X-Rhodamine triethylammonium salt; Lissamine® Rhodamine B sulfonyl chloride; Malachite green isothiocyanate; Rhodamine Red®-X succinimidyl ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamide) succinimidyl hexanoate; Tetramethylrhodamine-5-isothiocyanate; Tetramethylrhodamine-6-isothiocyanate; Tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt; Texas Red®-X succinimidyl ester; Texas It contains a fluorophore selected from the group consisting of Red(registered trademark)-X succinimidyl ester; X-rhodamine-5-(and-6)-isothiocyanate; and carbocyanin;

[25] A therapeutic oligonucleotide is selected from siRNA, shRNA, miRNA, antagmir, Dicer substrates, and antisense, and is conjugated to one of any of [1] to

[24] ;

[26] A conjugate of any one of [1] to

[25] , wherein the therapeutic oligonucleotide is siRNA;

[27] A conjugate of any one of [1] to

[25] , wherein the therapeutic oligonucleotide is monoshRNA;

[28] A conjugate of any one of the following [1]-

[25] , wherein the therapeutic oligonucleotide is miRNA or antagmir (an inhibitor of miRNA);

[29] A conjugate of any one of [1] to

[25] in which the therapeutic oligonucleotide is a Dicer substrate;

[30] The therapeutic oligonucleotides are conjugates of two nucleic acid complementary nucleic acid chains, each having a length of 18-25, 18-30, 18-35, 18-40, 18-45, or 18-50 nucleotides and a 2-nucleotide 3' overhang;

[29]

[31] A therapeutic oligonucleotide that can induce RNA interference (RNAi) is one of the conjugates [1] to

[30] ;

[32] When hybridized to RNA, the therapeutic oligonucleotide is a substrate of RNAse H,

[25] conjugate;

[33] The therapeutic oligonucleotide is a gapmer,

[25] a conjugate;

[34] When hybridized to RNA, the therapeutic oligonucleotide is not a substrate of RNAse H,

[25] a conjugate;

[35] A conjugate of any one of [1] to

[25] , wherein the therapeutic oligonucleotide is an antisense oligonucleotide;

[36] Antisense oligonucleotides specifically hybridize to the target RNA,

[35] conjugate;

[37] Conjugate of antisense oligonucleotides that are DNA or DNA mimics,

[35] or

[36] ;

[38] Each nucleoside of a therapeutic antisense oligonucleotide comprises one of the conjugates

[35] -

[37] , including a modified sugar moiety with modification at the 2' position, a PNA motif, or a morpholino motif;

[39] The group of therapeutic antisense oligonucleotide sequences consisting of the following: (a) The sequence of mRNA within 30 nucleotides from the AUG start codon; (b) miRNA nucleotides 1-10; (c) Sequence of the 5'-untranslated region of mRNA; (d) Sequence in the 3'-untranslated region of mRNA; (e) intron / exon junctions of mRNA; (f) A sequence of a pre-miRNA or pri-miRNA that blocks miRNA processing when an oligonucleotide is bound to it; and (g) One of the conjugates

[35] -

[38] that specifically hybridizes to a target region of RNA selected from an intron / exon junction and the 1-50 nucleic acid base region at the 5' end of the RNA intron / exon junction;

[40] Any one of the

[35] -

[39] conjugates whose hybridization of oligonucleotides to their target mRNA sterically blocks the translation of the coding sequence or modulates the expression of the target mRNA and / or blocks nucleotide-binding proteins, thereby regulating the stability of the target mRNA;

[41] One of the conjugates

[35] -

[40] in which the antisense oligonucleotide-HES complex contains multiple antisense chains, and the antisense chains are crosslinked;

[42] A conjugate of

[41] comprising multiple antisense oligonucleotides linked by spacer arms (e.g., 6-30 amino acid peptides or linear alkyl groups (e.g., C6, C10, or C12) or polyethyloxyglycol (e.g., triethyloxyglycol, tetraethyloxyglycol, or hexaethyloxyglycol)) to another antisense oligonucleotide linked to the 5' or 3' terminal residue of the second chain, wherein the oligonucleotide-HES complex is linearly linked in series from the 5' to the 3' end, and the spacer arms are linked to another antisense oligonucleotide linked to the 5' or 3' terminal residue of the second chain;

[43] The conjugate according to any one of claims 1 to 42, wherein the linker arm (L or SP) is a linear alkyl of C6, C10, or C12, or a polyethyloxyglycol (e.g., triethyloxyglycol, tetraethyloxyglycol, or hexaethyloxyglycol);

[44] The oligo-HES complex comprises multiple therapeutic antisenses crosslinked with two or more antisense chains from 5' to 3' and two or more therapeutic antisenses from 3' to 5' in the opposite direction, and the oligo-HES complex comprises one of the conjugates of

[41] to

[43] comprising multiple therapeutic antisenses crosslinked with two or more antisense chains in the direction from 5' to 3' and then from 3' to 5' using a spacer SP;

[45] Conjugate of

[44] having two or more identical complementary sequences and / or two or more different complementary sequences in the antisense strand;

[46] A conjugate containing a linear or branched linker, one of any of [1] to

[45] ;

[47] A conjugate containing a peptide linker or SP spacer of 6 to 30 amino acid residues in length, one of the [1] to

[46] ;

[48] ​​Conjugates of

[46] or

[47] , in which the linker is a linear peptide with a length of 6 to 30 amino acid residues;

[49] A linker that can be cut off; one of the conjugates from

[46] to

[48] ;

[50] The linker has an amino acid sequence containing a protease cleavage site with P1-P1' residues and has a loop structure, and is a conjugate of

[49] ;

[51] A conjugate of

[49] or

[50] comprising an amino acid sequence that is a substrate of at least one protease, wherein the linker is cleavable;

[52] A cleavable linker comprising any one of the

[49] -

[51] conjugates, the amino acid sequence of at least one protease active in diseased tissue;

[53] The linkers that can be cleaved include metalloproteinases (e.g., meprin, neprilysin, PSMA, and BMP1); matrix metalloproteinases (e.g., MMP1-3, MMP7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27), thrombin, elastases (e.g., human neutrophil elastase), cysteine ​​proteases (e.g., regmine and clugipain), serine proteases (e.g., A conjugate comprising an amino acid sequence that is a substrate for at least one protease selected from cathepsin C, DECC1, FAP, matryptase-2, MT-SP1 / matryptase, and TTSPs such as TMPRSS2-4), urokinase (uPA), aspartate proteases (e.g., BACE and renin); aspartate cathepsin (e.g., cathepsin D), and threonine proteases, any one of

[49] -

[52] ;

[54] A linker that can be cut, (a) MMP9; (b) MMP14; (c) MMP1, MMP2, MMP3, MMP7, MMP8, MMP10, MMP11, MMP12, MMP13, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27; (d) Serine proteases (e.g., MT-SP1, uPA, and TMPRSS2); (e) Cysteine ​​protease; (f)(a)~(c) metalloproteinases; (g) Aspartyl protease; and (h) Threonine proteases; a conjugate from any one of

[49] to

[53] comprising an amino acid sequence that is a substrate of at least one protease selected from the above;

[55] The conjugate according to

[54] , comprising an amino acid sequence that is a substrate of MMP9 or MMP14, wherein the linker is cleavable;

[56] A conjugate of

[54] or

[55] in which a cleavable linker comprises an amino acid sequence that is a substrate for at least one enzyme of the immune complement system, such as u-plasminogen activator, tissue plasminogen activator, trypsin, and plasmin;

[57] A conjugate containing an amino acid sequence that is a substrate of a protease known or reported to colocalize with the target of the conjugate, any one of the

[49] -

[56] conjugates;

[58] The linker is cleavable under intracellular conditions (e.g., conditions within lysosomes, endosomes, or caveolae) and is a conjugate of

[49] ;

[59] Linker is pH sensitive,

[49] conjugate;

[60] The linker is cleavable under reducing conditions, the conjugate of

[49] ;

[61] A conjugate (of any of the conjugates) of any of the

[44] -

[56] , where the linker is a malonate linker;

[62] One of the conjugates

[46] -

[48] whose linker cannot be cut;

[63] A linker having one of the conjugates from

[46] to

[62] , wherein the linker has an H-dimer-forming fluorophore (e.g., a linker containing an H-dimer-forming fluorophore conjugated to an amino and / or carboxyl terminal residue);

[64] A linker having one of the conjugates from

[46] to

[63] having an H-dimer-forming fluorophore conjugated to an amino-terminal residue and a carboxyl-terminal residue;

[65] The linker is one of the conjugates

[46] to

[64] having sulfhydryl or amino functional groups at the amino and carboxyl terminals of the peptide;

[66] Any one of the conjugates from [1] to

[65] , wherein the target-directing portion of the conjugate is an aptamer, avimer, receptor-binding ligand, nucleic acid, biotin-avidin binding pair, peptide, protein, carbohydrate, lipid, vitamin, microbial component, hormone, receptor ligand (including Fc fusion proteins containing receptor ligands), antibody, antigen-binding portion of an antibody, alternative binding scaffold, or any derivative thereof;

[67] A conjugate of any one of [1] to

[66] , wherein the target-directing portion is an antibody, an antigen-binding portion of an antibody (e.g., Fab, and scFv), or a single-domain antibody;

[68] A conjugate of any one of [1] to

[67] , wherein the target-directing portion is an antibody, a humanized antibody, an antigen-binding fragment of an antibody, a single-chain antibody, a bispecific antibody, a synthetic antibody, or a PEGylated antibody;

[69] A conjugate of any one of [1] to

[68] in which the target-directing portion is an antibody;

[70] The conjugate according to

[69] , wherein the target-directing portion comprises a monospecific, bispecific, or multispecific antibody and / or a monovalent, bivalent, or polyvalent antibody;

[71] A conjugate of

[69] or

[70] in which the target-directing portion is an IgG1, IgG2, or IgG4 antibody;

[72] A conjugate of any one of

[69] -

[70] in which the target-directing portion is a therapeutic antibody;

[73] Antibodies include trastuzumab (HER2 / neu), pertuzumab (HER2 / neu), panitumumab (EGFR), nimotuzumab (EGFR), zaltumumab (EGFR), cetuximab (EGFR), (HER3), onarutuzumab (c-MET), patritumumab, cribatuzumab (MUC1), sofituzumab (MUC16), edrecolomab (EPCAM), adekatumumab (EPCAM), anetumab (MSLN), huDS6 (CA6), and rif Astuzumab (NAPI2B), sacituzumab (TROP2), PR1A3, humanized PR1A3 (CEA), humanized Ab2-3 (CEA), IMAB362 / claudiximab (Claudin 18.2), AMG595 (EGFRvIII), ABT806 (EGFRvIII), cibrotuzumab (FAP), DS-8895a variant 1 (EphA2), DS-8895a variant 2 (EphA2), anti-EphA2 (EphA2), MEDI-547 (E PhA2), nalnatumab (RON), RG7841 (LY6E), farletuzumab (FRA / folate receptor alpha), milbetuximab (FRA), J591 variant 1 (PSMA), J591 variant 2 (PSMA), lovalpituzumab (DLL3), PF-06647020 (PTK7), anti-PTK7 (PTK7), radilatuzumab (LIV1), silimutuzumab (ROR1), rituximab (CD20), ibritumomab tiuxetan (CD52 Conjugates selected from,

[72] , alemtuzumab (CD33), gemtuzumab ozogamicin (CD33), CT-011 (PD1), tositumomab (CD20), ipilimumab (CTLA4), tremelimumab (CP-675,206) (CTLA4), nivolumab (PD1), pembrolizumab (PD1), durvalumab (PDL1) anti-MAGE-A3, anti-NY-ESO-1, anti-ACE2, anti-hyaluronidase, or anti-neuraminidase;

[74] A conjugate of any one of the following [1]-

[68] , wherein the target-directing portion is an antibody, a single-chain antibody, a single-domain antibody, or an antigen-binding fragment of a bispecific antibody;

[75] A conjugate of any one of [1]-

[66] whose target-directing moiety is an alternative binding scaffold selected from affibody, nanobody, antikalin, finomer, DARPin, tetranectin, transbody, adonectin, affin, microbody, peptide aptamer, alterase, plastic antibody, filomer, stradobody, maxibody, epibody, Z-domain, D-domain, armadillo repeat protein, Knitz domain, avimer, atrimer, probody, immunobody, triomab, troybody, pepbody, waxibody, unibody, affimer, or duobody;

[76] A conjugate having a target-directing portion that specifically binds to a cell surface antigen of interest on or near a target cell or tissue, such as disease cells, cancer cells, immune cells, infected cells, or infectious agents, one of the [1] to

[75] ;

[77] A conjugate in which the target-directing portion specifically binds to any one of [1] to

[76] cell surface antigens, such as neoantigens, that are determined to originate from or be expressed in a specific target cancer (e.g., a tumor);

[78] A conjugate from [1] to

[77] whose target-directing moiety specifically binds to cell surface antigens that do not internalize the conjugate upon binding;

[79] A target-directing moiety that specifically binds to a cell surface antigen that internalizes the conjugate upon binding; one of the conjugates from [1] to

[75] ;

[80] A target-directing portion of one of the conjugates [1] to

[79] that specifically binds to tumor cell surface antigens;

[81] A conjugate from any of [1] to

[80] whose target-directing portion specifically binds to leukemia cells, lymphoma cells, pancreatic cancer cells, breast cancer cells, melanoma cells, lung cancer cells, head and neck cancer cells, ovarian cancer cells, bladder cancer cells, colorectal cancer cells, kidney cancer cells, liver cancer cells, prostate cancer cells, bone cancer cells, glioblastoma cells, or any one of the conjugates from [1] to

[80] ;

[82] The target-directed portion includes CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CD204, CD206, CD301, CAMPATH-1, HLD-DR, carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUC1, MUC15, folate-binding protein, A33, G250, prostate-specific membrane antigen (PSMA), ferritin, GD2, GD3, GM2, Ley, CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2 receptor, tenascin, metalloproteinase, endothialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6, HPV E7, EGFRvIII, HER2 / neu, MAGE A3, P53 non-mutant, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, P53 mutant, PR1, bcr-abl, tyrosinase, survivor, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin (MSLN), PSCA, MAGE A1, MAGE-A3, sLe (animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Regmine, Tie 3, Page 4, a conjugate from any one of [1] to

[81] that specifically binds to a cell surface antigen selected from VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, CMET, HER3, CA6, NAPI2B, TROP2, CLDN18.2, fibroblast-activating protein (FAP), RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, Fos-related antigen 1, VEGFR, endoglin, PDL, VTCN1, and VISTA;

[83] A conjugate whose target-directing portion specifically binds to any one of [1] to

[81] cell surface antigens selected from HER2, EGFR, CMET, HER3, MUC1, MUC16, EPCAM, MSLN, CA6, NAPI2B, TROP2, CEA, CLDN18.2, EGFRvIII, FAP, EphA2, RON, LY6E, FRA, PSMA, DLL3, PTK7, LIV1, ROR1, MAGE-A3, NY-ESO-1, endoglin, CD204, CD206, CD301, VTCN1, VISTA, GLP-3, CLDN6, CLDN16, UPK1B, STRA6, TMPRSS3, TMPRSS4, TMEM238, C1orf186, and LRRC15;

[84] A conjugate in which the target-directing portion specifically binds to a tumor microenvironment cell surface antigen (including a specific membrane-bound protease) from any one of [1] to

[83] ;

[85] A conjugate, any one of [1] to

[84] , in which the target-directing portion specifically binds to cell surface antigens expressed on endothelial cells or macrophages (e.g., VEGFR, TIE1, and TIE2), or to cell surface antigens expressed on tumor stromal cells, such as cancer-associated fibroblasts (CAFs), tumor-infiltrating T cells and other leukocytes, as well as to myeloid cells including mast cells, eosinophils, and tumor-associated macrophages;

[86] A conjugate in which the target-directing portion specifically binds to a cell surface antigen on an immune cell, one of the conjugates from [1] to

[85] ;

[87] The conjugate of

[86] in which the target-directing portion specifically binds to cell surface antigens on immune cells of lymphoid or myeloid origin, such as T cells, B cells, NK cells, NKT cells, or dendritic cells;

[88] The conjugate according to

[86] or

[87] , wherein the target-directing portion specifically binds to a cell surface antigen on an antigen-presenting cell;

[89] The conjugate according to

[88] , wherein the target-directing moiety specifically binds to an antigen selected from OX40L, 4-1BBL, MARCO, DC-SIGN, dectin-1, dectin-2, DEC-205, CLEC5A, CLEC9A, CLEC10A, CLEC12A, CD1A, CD16A, CD32A, CD32B, CD36, CD40, CD47, CD64, CD204, CD206, HVEM, PDL1, mannose scavenger receptor 1, and BDCA2;

[90] The conjugate according to

[86] or

[87] , wherein the target-directing moiety specifically binds to a cell surface antigen on an immune cell that is not an antigen-presenting cell;

[91] The conjugate according to any one of [1] to

[90] , wherein the target-directing moiety binds to a target of interest (e.g., a cell surface antigen) with an equilibrium dissociation constant (Kd) in the range of 0.5×10 -10 ~10×10 -6 determined by BIACORE® analysis;

[92] A method of regulating the nucleic acid or protein level in a cell, comprising contacting the cell with a therapeutically effective amount of any one of the conjugates of [1] to

[91] , wherein the oligonucleotide-HES complex: (a) a target-directing moiety that binds to a cell surface antigen on or near the cell; and (b) an oligonucleotide that specifically hybridizes to a nucleic acid and regulates the level of the nucleic acid and / or the protein encoded or controlled by the nucleic acid; the method as described above.

[93] The method according to

[92] , wherein the target-directing moiety of the T-oligo-HES conjugate specifically binds to a cell surface antigen on the contacted cell, and the cell expresses a cell surface protease that cleaves the cleavable linker of the T-oligo-HES conjugate to release the oligo-HES complex.

[94] The method according to

[92] or

[93] , wherein the regulated nucleic acid or protein is in a diseased cell, an infected cell, an infectious agent, or an immune cell;

[95] The method according to

[94] , wherein the regulated nucleic acid or protein is in a diseased cell.

[96] Modified nucleic acids or proteins are present in cancer cells, as in the

[95] method.

[97] A method in which the cancer cells are blood cancer cells or solid tumor cancer cells,

[96] .

[98] The method of

[97] in which cancer cells are leukemia cells, pancreatic cancer cells, breast cancer cells, melanoma cells, lung cancer cells, head and neck cancer cells, ovarian cancer cells, bladder cancer cells, colorectal cancer cells, kidney cancer cells, liver cancer cells, prostate cancer cells, bone cancer cells, glioblastoma cells, or any lymphoma, myeloma, blastoma, sarcoma, leukemia or carcinoma cells;

[99] A modified nucleic acid or protein is present in an infected cell or an infectious cell,

[94] by a method.

[0100] A method in which a modified nucleic acid or protein is present in an infected cell or infectious agent,

[99] .

[0101] Cells are infected with HIV, HTLV-1, Zika virus, dengue virus, influenza virus, Ebola virus, Marburg virus, Crimean-Congo hemorrhagic fever virus, Lassa fever virus, smallpox virus, SARS virus, Rift Valley fever virus, tuberculosis, anthrax, botulism, tularemia, plague, brucellosis, glanders, meridianus, Q fever, or alphaviruses, such as chikungunya virus, Sindbis virus, Semryqui forest virus, Western, Eastern and Venezuelan equine encephalitis virus, Ross River virus, COVID, or influenza, by the methods of

[99] or

[0100] ;

[0102] A modified nucleic acid or protein is present within immune cells,

[94] by the method described above.

[0103] The method of

[0102] wherein the immune cells are myeloid-derived lymphoid cells such as T cells, B cells, NK cells, NKT cells, or dendritic cells;

[0104] The therapeutic oligonucleotide is selected from siRNA, shRNA, miRNA, antagmir, Dicer substrate, and antisense by any one of the methods described in

[92] to

[0103] ;

[0105] One method of

[92] to

[0104] wherein the conjugate comprises a cleavable linker containing an amino acid sequence that is a substrate of at least one protease;

[0106] The linkers that can be cleaved include metalloproteinases (e.g., meprin, neprilysin, PSMA, and BMP1); matrix metalloproteinases (e.g., MMP1-3, MMP7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27), thrombin, elastases (e.g., human neutrophil elastase), cysteine ​​proteases (e.g., regmine and clugipain), serine proteases ( For example, the method of

[0105] , comprising an amino acid sequence that is a substrate for at least one protease selected from cathepsin C, DECC1, FAP, matryptase-2, MT-SP1 / matryptase, and TTSPs such as TMPRSS2-4, urokinase (uPA), aspartate proteases (e.g., BACE and renin); aspartate cathepsin (e.g., cathepsin D), and threonine proteases;

[0107] The target-directing portion of the conjugate is an antibody, an antigen-binding portion of an antibody (e.g., Fab, and scFv), or a single-domain antibody, one of the methods described in

[92] to

[0106] ;

[0108] A method for modulating nucleic acid or protein levels in a subject, comprising administering a therapeutically effective dose of one of the [1] to

[91] conjugates to a subject in need thereof, wherein the conjugate is: (a) A target-directed portion of a nucleic acid or protein that binds to a cell surface antigen on or near a cell that is regulated; and (b) an oligonucleotide that specifically hybridizes with a nucleic acid and modulates the levels of the nucleic acid and / or proteins encoded or controlled by the nucleic acid; and the method thereof;

[0109] nucleic acids or proteins (a) Overexpression or underexpression of nucleic acids in the subject, (b) The method of

[0108] , characterized by overexpression or underexpression of nucleic acid-encoded proteins in the subject.

[0110] The method of

[0108] or

[0109] , wherein a modified nucleic acid or protein is present in disease cells, infected cells, infectious agents, or immune cells;

[0111] The method of

[0108] , wherein a regulated nucleic acid or protein is present in diseased cells.

[0112] A modified nucleic acid or protein is present in cancer cells, according to the method described in

[0110] .

[0113] The method of

[0112] wherein the cancer cells are blood cancer cells or solid tumor cancer cells.

[0114] The method of

[0113] wherein the cancer cells are leukemia cells, pancreatic cancer cells, breast cancer cells, melanoma cells, lung cancer cells, head and neck cancer cells, ovarian cancer cells, bladder cancer cells, colorectal cancer cells, kidney cancer cells, liver cancer cells, prostate cancer cells, bone cancer cells, glioblastoma cells, or any lymphoma, myeloma, blastoma, sarcoma, leukemia or carcinoma cells;

[0115] The method of

[0110] , wherein a modified nucleic acid or protein is present within an infected cell or infectious agent.

[0116] The method of

[0115] in which a modified nucleic acid or protein is present within the infectious agent.

[0117] The method of

[0115] wherein the infected cells are immune cells of lymphoid or myeloid origin.

[0118] The method of

[0110] in which a regulated nucleic acid or protein is present in disease cells, or in immune cells.

[0119] The method of

[0118] in which the immune cells are lymphoid or myeloid cells such as T cells, B cells, NK cells, NKT cells, or dendritic cells;

[0120] The therapeutic oligonucleotide is selected from siRNA, shRNA, miRNA, antagmir, Dicer substrate, and antisense by any one of the methods described in

[0108] to

[0119] ;

[0121] One of the methods

[0108] to

[0120] , wherein the conjugate comprises a cleavable linker containing an amino acid sequence that is a substrate of at least one protease;

[0122] The linkers that can be cleaved include metalloproteinases (e.g., meprin, neprilysin, PSMA, and BMP1); matrix metalloproteinases (e.g., MMP1-3, MMP7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27), thrombin, elastases (e.g., human neutrophil elastase), cysteine ​​proteases (e.g., regmine and clugipain), serine proteases ( For example, the method of

[0121] , comprising an amino acid sequence that is a substrate for at least one protease selected from cathepsin C, DECC1, FAP, matryptase-2, MT-SP1 / matryptase, and TTSPs such as TMPRSS2-4, urokinase (uPA), aspartate proteases (e.g., BACE and renin); aspartate cathepsin (e.g., cathepsin D), and threonine proteases;

[0123] One of the methods described in

[0108] to

[0122] , wherein the conjugate target-directing portion is an antibody, an antigen-binding portion of an antibody (e.g., Fab, and scFv), or a single-domain antibody;

[0124] A method for treating a disease or disorder in a subject, comprising administering a therapeutically effective amount of one of [1] to

[91] conjugates to a subject in need thereof, wherein the oligonucleotide specifically hybridizes to a nucleic acid sequence in vivo and modulates the level of a protein encoded or controlled by the nucleic acid; the disease or disorder is: (a) Overexpression or underexpression of the target nucleic acid in the subject, (b) Overexpression or underexpression of a protein encoded by a target nucleic acid in a subject; the method;

[0125] The method of

[0124] wherein the conjugate comprises a target-directing moiety that binds to a cell surface antigen on or near a cell in which a nucleic acid or protein is regulated;

[0126] The method of

[0124] or

[0125] wherein the disease or disorder is a proliferative disorder or disorder, such as cancer, a disorder or disorder of the immune system, an inflammatory disorder or disorder, an infection, a neurological disorder or disorder, a cardiovascular disorder or disorder, a metabolic disorder or disorder, a skeletal disorder or disorder, or a skin or eye disorder or disorder;

[0127] The disease or disorder is a neurological disease or disorder such as cancer, an inflammatory disease or disorder, or a disease or disorder of the immune system, an infection, or a neurodegenerative disease or disorder, in any one of the ways described in

[0124] to

[0126] ;

[0128] The disease or disorder is cancer, in any one of the following ways:

[0124] to

[0127] ;

[0129] The method of

[0128] where the cancer is blood cancer or solid tumor cancer.

[0130] The cancer is leukemia, pancreatic cancer, breast cancer, melanoma, lung cancer, head and neck cancer, ovarian cancer, bladder cancer, colorectal cancer, kidney cancer, liver cancer, prostate cancer, bone cancer, brain tumor including glioblastoma; or any lymphoma, myeloma, blastoma, sarcoma, leukemia or carcinoma, as described in

[0129] ;

[0131] The method according to

[0124] , wherein the disease or disorder is cancer, an inflammatory disease or disorder, a disease or disorder of the immune system, or an infection;

[0132] The method of

[0131] wherein the disease is an inflammatory disease or disorder, or an autoimmune disease or disorder (e.g., rheumatoid arthritis);

[0133] If the disease or disorder is inflammatory,

[0132] method;

[0134] Method

[0133] for diseases or disorders that are infectious;

[0135] The infectious disease is HIV, HTLV-1, Zika fever, dengue fever, influenza, Ebola, Marburg disease, Crimean-Congo hemorrhagic fever, Lassa fever virus, smallpox, SARS, Rift Valley fever, tuberculosis, anthrax, botulism, tularemia, plague, brucellosis, glanders, meridian, Q fever, or alphavirus, such as chikungunya virus, Sindbis virus, Semryqui forest virus, Western, Eastern and Venezuelan equine encephalitis virus, Ross River virus, or COVID, as described in

[0134] ;

[0136] The method of

[0126] in which the disease or disorder is a neurological disease or disorder;

[0137] The method of

[0136] in which the neurological disorder or disability is a neurodegenerative disease, e.g., familial and sporadic amyotrophic lateral sclerosis (FALS and ALS, respectively), familial and sporadic Parkinson's disease, Huntington's disease (Huntington's chorea), familial and sporadic Alzheimer's disease, spinal muscular atrophy (SMA), multiple sclerosis, diffuse cortical atrophy, dementia, or Pick's disease;

[0138] The therapeutic oligonucleotide conjugate is selected from siRNA, shRNA, miRNA, antagmir, Dicer substrate, and antisense, one of the methods described in

[0124] to

[0137] ;

[0139] One of the methods

[0124] to

[0138] , wherein the conjugate comprises a cleavable linker containing an amino acid sequence that is a substrate of at least one protease;

[0140] The linkers that can be cleaved include metalloproteinases (e.g., meprin, neprilysin, PSMA, and BMP1); matrix metalloproteinases (e.g., MMP1-3, MMP7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27), thrombin, elastases (e.g., human neutrophil elastase), cysteine ​​proteases (e.g., regmine and clugipain), serine proteases ( For example, the method of

[0139] , comprising an amino acid sequence that is a substrate for at least one protease selected from cathepsin C, DECC1, FAP, matryptase-2, MT-SP1 / matryptase, and TTSPs such as TMPRSS2-4, urokinase (uPA), aspartate proteases (e.g., BACE and renin); aspartate cathepsin (e.g., cathepsin D), and threonine proteases;

[0141] One of the methods

[0124] to

[0140] wherein the target-directing portion of the conjugate is an antibody, an antigen-binding portion of an antibody (e.g., Fab, and scFv), or a single-domain antibody;

[0142] One of the methods

[0124] to

[0141] wherein the target-directing portion of the conjugate is an antibody or an antigen-binding fragment of an antibody;

[0143] The method of

[0142] wherein the target-directing portion is an antibody or a nanobody;

[0144] The method of

[0143] wherein the antibody is an IgG1, IgG2, or IgG4 antibody;

[0145] Method

[0143] or

[0144] , wherein the antibody is a therapeutic antibody;

[0146] One of the methods described in

[0124] to

[0145] , wherein the conjugate is administered simultaneously with, consecutively with, or separately from one or more other therapeutic agents.

[0147] A method for treating cancer in a subject, comprising administering a therapeutically effective amount of one of [1] to

[91] to a subject in need thereof, wherein the oligonucleotide specifically hybridizes with nucleic acids in cancer cells or tissues and modulates the levels of nucleic acids and / or proteins encoded or controlled by the nucleic acids, and the nucleic acids or proteins are as follows: (a) Overexpression or underexpression of nucleic acids in cancer cells, tissues, and / or subjects, (b) The method comprising overexpression or underexpression of nucleic acid-encoded proteins in cancer cells, tissues, and / or subjects;

[0148] The method of

[0147] wherein the target-directing portion of the conjugate specifically binds to cell surface antigens on or near cancer cells;

[0149] The method of

[0147] or

[0148] wherein the target-directing portion of the conjugate specifically binds to a cell surface antigen on cancer cells;

[0150] One of the methods

[0147] to

[0149] , wherein the target-directing portion of the conjugate specifically binds to cell surface antigens on cells near cancer cells (e.g., cells in the tumor microenvironment such as stromal cells, cancer-associated fibroblasts, immune cells, blood or lymphatic cells, endothelial cells, adipocytes, or neuroendocrine cells);

[0151] The cancer is a solid tumor, one of the methods described in

[0147] to

[0150] ;

[0152] Cancer is leukemia, pancreatic cancer, breast cancer, melanoma, lung cancer, head and neck cancer, ovarian cancer, bladder cancer, colorectal cancer, kidney cancer, liver cancer, prostate cancer, bone cancer, brain tumor including glioblastoma; or lymphoma, myeloma, blastoma, sarcoma, leukemia or carcinoma, in any one of the ways of

[0147] to

[0151] ;

[0153] The method for identifying blood cancer as described in

[0152] .

[0154] Method

[0153] for blood cancer being leukemia or lymphoma.

[0155] The target-directed portion of the conjugate targets CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CD204, CD206, CD301, CAMPATH-1, HLD-DR, carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUC1, MUC15, folate-binding protein, A33, G250, prostate-specific membrane antigen (PSMA), ferritin, GD2, GD3, GM2, Ley, CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2 receptor, tenascin, metalloproteinase, endothelial growth factor, avB3, WT1, LMP2, HPV E6, HPV E7, HIV GP120, HIV GP160, EGFRvIII, HER2 / neu, and MAGE. A3, P53 non-mutant, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, P53 mutant, PR1, bcr-abl, tyrosinase, survivor, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin (MSLN), PSCA, MAGE A1, MAGE-A3, sLe (animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Regmine, Tie 3. Page 4, one of the methods

[0147] to

[0154] for specifically binding to a cell surface antigen selected from VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, CMET, HER3, CA6, NAPI2B, TROP2, CLDN18.2, fibroblast-activating protein (FAP), RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, Fos-related antigen 1, VEGFR, endoglin, PDL, VTCN1, and VISTA;

[0156] One of the methods described in

[0147] to

[0154] , wherein the target-directing portion of the conjugate specifically binds to a cell surface antigen selected from DLL3, fibroblast-activating protein α (FAPα), NG2 (glial antigen-2), platelet-derived growth factor receptor-β (PDGFR-β), PD1, CD163, KIR, HMGB1, VEGFR3, LYVE1, CD31, CD34, P1GF, and VEGF;

[0157] One of the methods described in

[0147] to

[0154] , wherein the target-directing portion of the conjugate specifically binds to a cell surface antigen selected from HER2, EGFR, CMET, HER3, MUC1, MUC16, EPCAM, MSLN, CA6, NAPI2B, TROP2, CEA, CLDN18.2, EGFRvIII, FAP, EphA2, RON, LY6E, FRA, PSMA, DLL3, PTK7, LIV1, ROR1, MAGE-A3, NY-ESO-1, Endoglin, CD204, CD206, CD301, VTCN1, VISTA, GLP-3, CLDN6, CLDN16, UPK1B, STRA6, TMPRSS3, TMPRSS4, TMEM238, C1orf186, and LRRC15:

[0158] The therapeutic oligonucleotide conjugate is selected from siRNA, shRNA, miRNA, antagmir, Dicer substrate, and antisense, one of the methods described in

[0147] to

[0156] ;

[0159] The therapeutic oligonucleotide conjugate is selected from the following nucleic acids: EGFR, HER2 / neu, ErbB3, cMet, p56lck, PDGFR, VEGF, VEGFR, FGF, FGFR, ANG1, ANG2, bFGF, TIE2, protein kinase C-alpha (PKC-alpha), p56lck PKA, TGF-β, IGFIR, P12, MDM2, BRCA, IGF1, HGF, PDGF, IGFBP2, IGF1R, HIF1 alpha, ferritin, transferrin receptor, TMPRSS2, IRE, HSP27, HSP70, HSP90, MITF, clatherin, PARP1 C-fos, C-myc, n-myc, C-raf, B-raf, A1, H-raf, Skp2, K-ras, N-ras, H-ras, pharensyltransferase, c-Src, Jun, Fos, Bcr-Abl, c-Kit, EphA2, PDGFB, ARF, NOX1, NF1, STAT3, E6 / E7, APC, WNT, betacatenin, GSK3b, PI3k, mTOR, Akt, The method according to any one of

[0147] to

[0157] , which specifically hybridizes to PDK-1, CDK, Mek1, ERK1, AP-1, P53, Rb, Syk, osteopontin, CD44, MEK, MAPK, NF-κβ (NF kappa beta), E cadherin, cyclin D, cyclin E, Bcl2, Bax, BXL-XL, BCL-W, MCL1, ER, MDR, telomerase, telomerase reverse transcriptase, DNA methyltransferase, histone deacetylase (e.g., HDAC1 and HDAC2), integrin, IAP, aurora kinase, metalloproteinase (e.g., MMP2, MMP3 and MMP9), proteasome, or metallothionein genes;

[0160] The therapeutic oligonucleotide conjugate specifically hybridizes to nucleic acids selected from the following: Survivin, HSPB1, EIF4E, PTPN1, RRM2, BCL2, PTEN, Bcr-abl, TLR9, HaRas, Pka-rIA, JNK2, IGF1R, XIAP, TGF-β2, c-myb, PLK1, K-ras, KSP, PKN3, ribonucleotide reductases (e.g., ribonucleotide reductase R1 and ribonucleotide reductase R2), RecQ helicases (e.g., WRN, RecQL1, BLM, RecQL4, RecQ5, and RTS), MEM2, and TLR9, according to any one of the methods described in

[0147] to

[0157] ;

[0161] Any one of the methods

[0147] to

[0159] , wherein the conjugate comprises a cleavable linker containing an amino acid sequence that is a substrate of at least one protease;

[0162] The linkers that can be cleaved include metalloproteinases (e.g., meprin, neprilysin, PSMA, and BMP1); matrix metalloproteinases (e.g., MMP1-3, MMP7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27), thrombin, elastases (e.g., human neutrophil elastase), cysteine ​​proteases (e.g., regmine and clugipain), serine proteases ( For example, the method of

[0160] , comprising an amino acid sequence that is a substrate for at least one protease selected from cathepsin C, DECC1, FAP, matryptase-2, MT-SP1 / matryptase, and TTSPs such as TMPRSS2-4, urokinase (uPA), aspartate proteases (e.g., BACE and renin); aspartate cathepsin (e.g., cathepsin D), and threonine proteases;

[0163] One of the methods described in

[0147] to

[0161] , wherein the conjugate target-directing portion is an antibody, an antigen-binding portion of an antibody (e.g., Fab, and scFv), or a single-domain antibody;

[0164] One of the methods

[0147] to

[0162] wherein the target-directing portion of the conjugate is an antibody or an antigen-binding fragment of an antibody;

[0165] The method of

[0163] in which the target-directing portion is an antibody;

[0166] The method of

[0164] wherein the antibody is an IgG1, IgG2, or IgG4 antibody;

[0167] Method

[0164] or

[0165] , wherein the antibody is a therapeutic antibody;

[0168] The antibodies include trastuzumab (HER2 / neu), pertuzumab (HER2 / neu), panitumumab (EGFR), nimotuzumab (EGFR), zaltumumab (EGFR), cetuximab (EGFR), (HER3), onarutuzumab (c-MET), patritumab, cribatuzumab (MUC1), sofituzumab (MUC16), edrecolomab (EPCAM), adekatumumab (EPCAM), anetumab (MSLN), and huDS6 (CA). 6) Rifastuzumab (NAPI2B), Sacituzumab (TROP2), PR1A3, Humanized PR1A3 (CEA), Humanized Ab2-3 (CEA), IMAB362 / Claudiximab (Claudin 18.2), AMG595 (EGFRvIII), ABT806 (EGFRvIII), Cibrotuzumab (FAP), DS-8895a variant 1 (EphA2), DS-8895a variant 2 (EphA2), Anti-EphA2 (EphA 2) MEDI-547 (EphA2), nalnatumab (RON), RG7841 (LY6E), farletuzumab (FRA / folate receptor alpha), milbetuximab (FRA), J591 variant 1 (PSMA), J591 variant 2 (PSMA), lovalpituzumab (DLL3), PF-06647020 (PTK7), anti-PTK7 (PTK7), radilatuzumab (LIV1), sirumutuzumab (ROR1), rituximab (CD2 0) The method of

[0166] , selected from ibritumomab tiuxetan (CD52), alemtuzumab (CD33), gemtuzumab ozogamicin (CD33), CT-011 (PD1), tositumomab (CD20), ipilimumab (CTLA4), tremelimumab (CP-675,206) (CTLA4), nivolumab (PD1), pembrolizumab (PD1), durvalumab (PDL1) anti-MAGE-A3, and anti-NY-ESO-1;

[0169] The conjugate is administered simultaneously with, sequentially with, or separately from, one or more other anticancer drugs, in any one of the methods described in

[0146] to

[0167] .

[0170] Conjugates according to any one of [1] to

[91] for use in pharmaceuticals;

[0171] A conjugate defined in any one of [1] to

[91] for use in the treatment of a disease or disorder in the subject;

[0172] Conjugates described in any one of [1] to

[91] for use in the treatment of diseases or disorders selected from infectious diseases, cancer, proliferative disorders or disorders, neurological disorders or disorders, and inflammatory diseases or disorders, diseases or disorders of the immune system, diseases or disorders of the cardiovascular system, metabolic diseases or disorders, diseases or disorders of the skeletal system, and diseases or disorders of the skin or eyes;

[0173] A conjugate described in any one of [1] to

[91] for use in modulating a target nucleic acid or protein in a subject; in treating a disease or disorder characterized by overexpression or underexpression of a nucleic acid in a subject; in treating a disease or disorder characterized by overexpression or underexpression of a protein in a subject; or in treating a disease or disorder characterized by abnormal nucleic acid or protein expression in a subject.

[0005] Further other features and advantages of the compositions and methods described herein will become clearer from the following detailed description when read in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0006] [Figure 1]An exemplary synthesis of a T(antibody)-oligo-HES conjugate is shown. The structures of some click chemistry reagents are simplified for illustrative purposes. The linker is covalently bonded to the antibody via either the amino group or its sulfhydryl group, the latter (if via the sulfhydryl group) followed by reduction with a drug such as dithiothreitol or TCEP. To label the amino group, a functional group such as N-hydroxy-succinimidyl ester or other leaving groups can be used. To label the sulfhydryl of the antibody, a functional group such as maleimide or 3-arylpropioronitrile can be used. The linker arm typically has between 6 and 50 atoms. The functional group at the distal end of the linker, i.e., the end furthest from the antibody, can be either an azide or an alkyne, the latter preferably a carbon-carbon triple bond in a strained conformation, such as a cyclooctin ring with sp2 orbitals on both sides of the triple bond, e.g., dibenzocyclooctin. In click chemistry, either an alkyne or an azide can be used to conjugate a linker on an antibody to a peptide containing a conformation-dependent cleavage site that functions as a cleavage site for proteases on the cell surface. The peptide is conjugated distal to the antibody linkage to an oligonucleotide, which is either a single-stranded antisense or a double-stranded siRNA. Both the peptide containing the conformation-dependent cleavage site and the oligonucleotide are pre-derivatized with two fluorophores to form HES, specifically peptide HES and oligonucleotide HES. The structures of some click chemistry reagents have been simplified for illustrative purposes. [Figure 2]This shows an exemplary derivatization of an antibody covalently labeled with a linker arm. IgG2a was first reduced with TCEP, and then a 25-atom linker was added. Next, the linker, terminated with a dibenzocyclooctin group, was conjugated with an azide-containing fluorophore. After a reaction time of 1 hour, the solution was passed through a gel filtration column, and the conjugate was eluted immediately after the void volume. The peak at 280 nm is due to the antibody, and the peak at 641 nm indicates covalent bonding of the fluorophore. The complete synthesis of the T(e.g., antibody)-oligo-HES conjugate can be carried out in multiple steps, for example, by (a) first adding the linker, and then sequentially adding peptides and oligonucleotides containing conformation-dependent cleavage sites, or by (b) directly adding the linker, already conjugated to peptides and oligonucleotides containing conformation-dependent cleavage sites, to the antibody. Each step can be confirmed using complementary functional chemical groups containing reporter groups such as fluorophores. [Figure 3] This demonstrates the specificity of conformation-dependent cleavage sites. An 18-amino acid peptide containing the amino acid sequence of PLGIA (SEQ ID NO: 77) and covalently labeled with the same fluorophore near each end (resulting in an HES structure) is recognized by matrix metalloproteinase-9 (MMP-9) and cleaved between LG and IA. MMP-19 is added at a concentration of 2 μM to pH 7.5 buffer containing the PLGIA (SEQ ID NO: 77) peptide. Cleavage of the peptide increases the fluorescence intensity. This specificity is compared to a control peptide, i.e., an HES-supported peptide of the same length and labeling but without conformation-dependent cleavage sites. The fluorescence of the latter does not increase with the addition of MMP, which demonstrates the specificity of the PLGIA (SEQ ID NO: 77) sequence to MMP-19. [Figure 4]Figure A shows the fragmentation / cleavage of the PLGIA (SEQ ID NO: 77) peptide containing a conformation-dependent cleavage site. The retention time of the HES-PLGIA (SEQ ID NO: 77) peptide containing a conformation-dependent cleavage site was measured by HPLC. The retention time on a C18 column was measured under reversed-phase conditions, i.e., loaded with aqueous buffer and eluted with acetonitrile buffer after 38 minutes. Figure B shows the fragmentation / cleavage of the PLGIA (SEQ ID NO: 77) peptide containing a conformation-dependent cleavage site. The retention time of the HES-PLGIA (SEQ ID NO: 77) peptide containing a conformation-dependent cleavage site was measured by HPLC. The retention time on a C18 column was measured under reversed-phase conditions, i.e., loaded with aqueous buffer and eluted with acetonitrile buffer after 38 minutes. After exposure to MMP-9, the main peaks were located at approximately 30 and 31 minutes, with the peak almost completely disappearing at 38 minutes, consistent with the cleavage shown in Figure 3. [Figure 5] This shows the formation of an antibody linked to a peptide containing a conformation-dependent cleavage site. Rituximab, a monoclonal antibody that recognizes CD20 on B lymphocytes, was conjugated to a peptide containing a linker and a conformation-dependent cleavage site. The peak at 280 nm indicates the presence of the antibody. The conformation-dependent specificity of the peptide cleavage site due to the presence of HES is indicated by a more intense peak at 520 nm compared to the peak at 552 nm. In the absence of the two fluorophores that form an intramolecular H-dimer, the 552 peak is higher than the 520 peak. Therefore, the conformational specificity of the peptide is maintained even after covalent bond formation with the linker. [Figure 6] This study demonstrates the recognition of rituximab-oligo-HES conjugates by B cells. Raji cells, a CD20+ B lymphocyte cell line, were exposed at 4°C to rituximab labeled with a peptide containing linker arms and conformation-dependent cleavage sites. After washing and addition of viability dyes, the cells were examined by flow cytometry. Cells exposed to the modified rituximab conjugate recognized the antibody and bound to it without affecting cell viability. Therefore, chemical modification of the antibody by click chemistry did not impair the recognition function of the monoclonal antibody. [Modes for carrying out the invention]

[0007] This disclosure provides compositions and methods for the targeting and local in vivo delivery of oligonucleotides. Compositions containing targeted oligonucleotide-HES conjugates are provided as methods for constructing and using conjugates in therapeutic, diagnostic, and other applications. Oligonucleotide-HES complexes contained in targeted oligonucleotide-HES conjugates can cross membranes in a receptor-independent manner and deliver oligonucleotides containing complementary sequences to the cytosol of living cells in vivo. Targeted oligonucleotide-HES conjugates have uses including the targeting and / or local delivery of antisense oligonucleotides, siRNAs, shRNAs, Dicer substrates, miRNAs, anti-miRNAs, and other nucleic acid sequences in living organisms.

[0008] Definition: The meanings of certain terms listed herein are provided below or elsewhere in this disclosure:

[0009] The terms "nucleic acid" or "oligonucleotide" refer to at least two nucleotides linked together by a covalent bond. The nucleic acid oligonucleotides provided herein are preferably single-stranded or double-stranded and generally contain phosphodiester bonds, but may also contain, as outlined below, for example, phosphoramides (e.g., Beaucage et al., Tetrahedron 49(10): 1925 (1993)) and the references cited therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81: 579 (1977); Letsinger et al., Nucl. Acids Res. 14:3587 (1986); Sawai et al., Chem. Lett. 805 (1984); Letsinger et al., J. Am. Chem. Soc. 1 10:4470 (1988); and Pauwels et al., Chemica Scripta 26: 1419 See (1986), the entire contents of each of these are incorporated herein by reference), phosphorathioates (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Patent No. 5,644,048, the entire contents of each of these are incorporated herein by reference), phosphorodithioates (Briu et al., J. Am. Chem. Soc. 111:2321 (1989)), O-methylphosphoramidiate linkages (see, for example, Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see, for example, Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008) (1992);Nielsen, Nature 365:566 (1993);Carlsson et al.This includes nucleic acid / oligonucleotide analogs with alternative backbones, including, see Nature 380:207 (1996), the entire contents of each of these are incorporated herein by reference. Other nucleic acid / oligonucleotide analogs include those with a positive backbone (see, e.g., Dempcy et al., Proc. Natl, Acad. Sci USA 92:6097 (1995), the full contents of each of these are incorporated herein by reference); and non-ionic backbone (see, e.g., U.S. Patents Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew, Chem. Intl, Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13: 1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. YS Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Chaturvedi et al., Tetrahedron Lett. 37:743 (1996), the entire contents of each of these are incorporated herein by reference), as well as U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. YS Sanghui and P.This includes those having a non-ribose backbone as described by Dan Cook. Nucleic acids / oligonucleotides containing one or more carbocyclic sugars are also included in the definition of nucleic acids / oligonucleotides (see, for example, Jenkins et al., Chem. Soc. Rev. pp 169-176 (1995), the entire contents of each of these are incorporated herein by reference). Several nucleic acid / oligonucleotide analogs are described in Rawls, C & E News Jun. 2 1997, page 35, which is incorporated herein by reference. These modifications of the ribose-phosphate backbone can be made, for example, to facilitate the addition of addition moieties such as labels, or to increase the stability and half-life of such molecules in a physiological environment. The nucleic acid / oligonucleotide backbone of the oligonucleotides provided herein ranges from about 5 nucleotides to about 750 nucleotides. Preferred nucleic acids / oligonucleotides in the T-oligo-HES conjugates provided herein range in length from about 5 nucleotides to about 500 nucleotides, preferably from about 10 nucleotides to about 100 nucleotides. As used herein, the terms “about” or “approximately” refer to any number within 0.25%, 0.5%, 1%, 5%, or 10% of the reference number when used with a number.

[0010] The oligonucleotides in the oligo-HES complex of the T-oligo-HES conjugate are polymer structures of nucleosides and / or nucleotide monomers that can specifically hybridize to at least one region of a nucleic acid target. As described above, HES-oligonucleotides include, but are not limited to, naturally occurring compounds containing bases, sugars, and intersugar (backbone) links, naturally occurring modified monomers or parts thereof (e.g., oligonucleotide analogs or mimetic compounds) that function similarly to their naturally occurring counterparts, and combinations of these naturally occurring and naturally occurring monomers. As used herein, the terms “modified” or “modified” include any substitution and / or alteration from a starting or natural oligomeric compound such as an oligonucleotide. Modifications to oligonucleotides include substitution or alteration of internucleoside links, sugar moieties, or base moieties, such as those described herein and those otherwise known in the art.

[0011] As used herein, the term “antisense” refers to an oligonucleotide sequence described in the 5' to 3' direction, comprising the reverse complement of a corresponding region of a target nucleic acid, and / or capable of specifically hybridizing to a target nucleic acid under physiological conditions. Therefore, in some embodiments, the term “antisense” refers to an oligonucleotide comprising the reverse complement of a corresponding region of a small non-coding RNA, untranslated mRNA, and / or genomic DNA sequence. In certain embodiments, the antisense oligonucleotide in the T-oligo-HES conjugate provided herein, upon hybridization to a nucleic acid target, can induce or trigger a decrease in the expression of the target gene, the level of the target gene, or the level of the protein encoded by the target nucleic acid.

[0012] As used herein, "complementary" refers to the ability to form pairs between the monomeric component of an oligonucleotide and a nucleotide in a target nucleic acid (e.g., DNA, mRNA, and non-coding RNA such as miRNA (raiRNA)). For example, if a nucleotide at a certain position in an oligonucleotide can form a hydrogen bond with a nucleotide at the same position in a DNA / RNA molecule, then the oligonucleotide and DNA / RNA are considered complementary at that position.

[0013] In relation to this application, “hybridization” means the pairing of an oligonucleotide with a complementary nucleic acid sequence. Such pairing typically involves hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding, between the complementary nucleoside or nucleotide base (nucleic acid base) of the oligonucleotide and the target nucleic acid sequence (e.g., the oligonucleotide contains a reverse complementary nucleotide sequence for the corresponding region of the target nucleic acid). In certain embodiments, the oligonucleotide hybridizes specifically to the target nucleic acid. The terms “specifically hybridize” and “specifically hybridizable” are used interchangeably herein and indicate a sufficient degree of complementarity such that a stable and specific binding occurs between the oligonucleotide and the target nucleic acid (i.e., DNA or RNA). It is understood that an oligonucleotide does not need to be 100% complementary to its target nucleic acid sequence in order to be specifically hybridizable. In certain embodiments, an oligonucleotide is considered specifically hybridizable if the binding of the oligonucleotide to the target nucleic acid sequence interferes with the normal function of the target nucleic acid, resulting in a loss or alteration of utility or expression. In preferred embodiments, there is a degree of complementarity between the oligonucleotide and the target nucleic acid sufficient to avoid or minimize nonspecific binding of the oligonucleotide to undesirable non-target sequences under conditions where specific binding is desired (e.g., under physiological conditions in the case of in vivo assays or therapeutic procedures, and under the conditions under which the assay is performed in the case of in vitro assays). It is well within the technical capabilities of scientists in the oligonucleotide field to determine when the conditions are optimal for specific hybridization to the target nucleic acid with minimal nonspecific hybridization events. Therefore, in some embodiments, the oligonucleotide in the T-oligo-HES conjugate contains 1, 2, or 3 base substitutions compared to the corresponding complementary sequence of the region of the target DNA or RNA sequence it specifically hybridizes. In some embodiments, the non-complementary nucleic acid bases are located at the 5' or 3' end of the antisense oligonucleotide.In additional embodiments, non-complementary nucleic acid bases are located at internal positions within the oligonucleotide. If two or more non-complementary nucleic acid bases are present in the oligonucleotide, they may be continuous (i.e., linked), discontinuous, or both. In some embodiments, the oligonucleotides in the complex provided herein have at least 85%, at least 90%, or at least 95% sequence identity with respect to a target region in the target nucleic acid. In other embodiments, the oligonucleotides have 100% sequence identity with respect to a polynucleotide sequence in the target nucleic acid. Percent identity is calculated according to the number of bases that the oligonucleotide is identical to in the corresponding nucleic acid sequence being compared. This identity may extend across the entire length of the oligomeric compound (i.e., the oligonucleotide) or across a portion of the oligonucleotide (for example, the percentage identity of the oligonucleotide to the oligonucleotide can be determined by comparing nucleic acid bases 1-20 of a 27-mer with those of a 20-mer). The percentage identity between oligonucleotides and target nucleic acids can be systematically determined using alignment programs and BLAST programs (basic local alignment search tools) known in the art (see, for example, Altschul et al., J. Mol. Biol., 215:403-410 (1990); Zhang and Madden, Genome Res., 7:649-656 (1997)).

[0014] As used herein, the terms “target nucleic acid” and “target-encoding nucleic acid” are used to encompass any nucleic acid that can be targeted, including, but not limited to, DNA encoding a given molecular target (i.e., a protein or polypeptide), RNA transcribed from such DNA (including miRNA, pre-mRNA, and mRNA), and cDNA derived from such RNA. Exemplary DNA functions that may be interfered with include replication, transcription, and translation. The overall effect of such interference on the function of a target nucleic acid is the regulation of the expression of the target molecule. As used herein, “regulation” means either an increase (stimulation) or decrease (inhibition) in, for example, gene expression. Inhibition of gene expression by a decrease in RNA levels is a preferred form of regulation.

[0015] As used herein, the terms “pharmaceutically acceptable” or “physiologically acceptable,” and their grammatical variations, mean that, when referring to compositions, carriers, diluents, and reagents, the material is interchangeable and can be administered to or onto a subject (e.g., a mammal, such as a mouse, rat, rabbit, or human primate) without causing any undesirable physiological effects that are therapeutically contraindicated, such as nausea, dizziness, or stomach upset.

[0016] As used herein, “a pharmaceutical composition comprising an antisense oligonucleotide” refers to a composition comprising a T-oligo-HES conjugate and a pharmaceutically acceptable diluent. A suitable pharmaceutically acceptable diluent, for example, is phosphate-buffered saline.

[0017] "Stabilizing modification" or "stabilizing motif" means providing enhanced stability in the presence of a nuclease compared to the stability provided by a 2-deoxynucleoside linked by a phosphodiester nucleoside linkage. Therefore, such modifications provide oligonucleotides with "enhanced nuclease stability." Stabilizing modifications include at least a stabilizing nucleoside and a stabilizing nucleoside linkage group.

[0018] The term "subject" refers to any animal (e.g., mammal) that is a recipient of a particular treatment, including but not limited to humans, non-human primates, and rodents. Typically, the terms "subject" and "patient" are used interchangeably in this specification with respect to human subjects.

[0019] As used herein, the terms “administer” and “dosage” refer to adding a chemical substance, such as an oligonucleotide, to a subject in vivo or ex vivo. Therefore, administration encompasses both directly adding HES-oligonucleotides to a subject and contacting cells with a HES-oligonucleotide composition, followed by the introduction of the contacted cells into the subject. In one embodiment, cells removed from the subject are contacted with HES-oligonucleotides, and then the contacted cells are reintroduced into the subject. The term “contact” refers to adding a chemical substance, such as an oligonucleotide, to an in vivo organism, such as a mammal, plant, bacterium, or virus. In mammals, common contact routes include oral (by mouth), topical (skin), transmucosal (nose, cheek / sublingual, vagina, eye, and rectum), inhalation (lungs), intramuscular (muscle), and intravenous (vein). In bacteria and viruses, contact may be delivery into the cells or tissues of the host organism.

[0020] "Treatment" or "therapy" includes administering HES-oligonucleotides to prevent or delay the onset of symptoms, complications, or biochemical signs of a disease, condition, or disorder; alleviating symptoms; or halting or inhibiting further progression of a disease, condition, or disorder. Therapy may be prophylactic (to prevent or delay the onset of a disease, or to prevent the manifestation of its clinical or potential symptoms) or therapeutic suppression or alleviation of symptoms after the onset of a disease, condition, or disorder. Therapy may be carried out with a HES-oligonucleotide complex-containing composition alone or in combination with one, two, three or more additional therapeutic agents.

[0021] The term “therapeutic dose” refers to the amount of HES-oligonucleotide complex (“therapeutic agent”) or other drug that is effective in achieving a desired therapeutic outcome and / or “treating” a disease or disorder in a subject. The term “therapeutic dose” may also refer to the amount required to result in a delay in disease progression, an increase in survival time, and / or improvement in one or more indicators of disease or disease progression in a subject affected by the disease. For example, in the case of cancer, a therapeutic dose of HES-oligonucleotide complex may reduce angiogenesis and neovascularization; reduce the number of cancer cells; reduce tumor size; inhibit (i.e., delay or halt) the invasion of cancer cells into peripheral organs; inhibit (i.e., delay or halt) tumor metastasis; inhibit or delay tumor growth or tumorigenesis; stimulate an immune response against cancer cells; and / or alleviate one or more symptoms associated with cancer. In the case of infectious diseases, a therapeutically effective dose of HES-oligonucleotide complex may be associated with a reduction in the number of infectious agents (e.g., viral load) and / or improvement of one or more symptoms or conditions associated with the infection caused by the infectious agents. “Therapeutic dose” may also refer to the amount effective in the dosage and duration required to achieve the desired therapeutic outcome. The therapeutically effective dose of HES-oligonucleotide complex may vary depending on factors such as the disease state of the subject, age, sex, and weight, as well as the ability of the HES-oligonucleotide complex to induce the desired response in the subject. The therapeutically effective dose is also the amount in which the therapeutically beneficial effects of the HES-oligonucleotide complex outweigh any toxic or adverse effects.

[0022] "Therapeutic index" refers to the ratio of the dose of a HES-oligonucleotide complex that produces an undesirable effect to the dose that produces the desired effect. In relation to this disclosure, a HES-oligonucleotide complex exhibits an "improved therapeutic index" if its activity is maintained but the undesirable effect is reduced or absent. For example, a HES-oligonucleotide complex with an improved therapeutic index retains the ability to inhibit miRNA activity without producing undesirable effects such as immunostimulatory activity, or at least without producing undesirable effects that would prevent the administration of the complex.

[0023] As used herein, “therapeutic oligonucleotide” refers to an oligonucleotide that, when administered in sufficient doses, can achieve a desired therapeutic outcome and / or “treat” a disease or disorder in a subject or ex vivo. Such desired outcomes include, for example, delay in disease progression, increased survival time, and / or improvement of one or more indicators of disease, disease progression, or disease-related conditions in a subject suffering from the disease. Exemplary therapeutic oligonucleotides include siRNA, shRNA, Dicer substrates (e.g., dsRNA), miRNA, anti-miRNA, antisense oligonucleotides, decoys, aptamers, and plasmids capable of expressing siRNA, miRNA, ribozymes, antisense oligonucleotides, or protein-coding sequences. Oligonucleotides such as probes and primers that cannot achieve a desired therapeutic outcome are not considered therapeutic oligonucleotides for the purposes of this disclosure. On average, less than 1% of mRNA is a suitable target for antisense oligonucleotides. Numerous antisense oligonucleotides suitable for incorporation into HES-oligonucleotide complexes contained in the provided T-oligo-HES conjugates are described herein or otherwise known in the art. Similarly, suitable therapeutic oligonucleotides can be systematically designed using guidelines, algorithms, and programs known in the art (see, for example, Aartsma-Rus et al., Mol. Ther. 17(3):548-553 (2009) and Reynolds et al., Nat. Biotech. 22(3):326-330 (2004) and Zhang et al., Nucleic Acids Res. 31 e72 (2003), the contents of which are incorporated herein by reference in their entirety).Similarly, suitable therapeutic oligonucleotides can be systematically designed using commercially available programs (e.g., MysiRNA-Designer, AsiDesigner (Bioinformatics Research Center, KRIBB), siRNA Target Finder (Ambion), Block-iT RNAi Designer (Invitrogen), Gene specific siRNA selector (The Wistar Institute), siRNA Target Finder (GeneScript), siDESIGN Center (Dharmacon), SiRNA at Whitehead, siRNA Design (IDT), D: T7 RNAi Oligo Designer (Dudek P and Picard D.), sfold-software, and RNAstructure 4.5); programs available via the Internet, such as human splicing finder software (e.g., at ".umd.be / HSF / ") and Targetfmder (available at "bioit.org.cn / ao / targetfinder"); and commercial providers (e.g., Gene Tools, LLC). In some cases, oligonucleotides, oligonucleotide-HES, HES-oligonucleotides, or oligo-HES, and therapeutic oligonucleotides may be used interchangeably herein unless otherwise clearly indicated in the context.

[0024] As used herein, “therapeutic antibody” in relation to the T-oligo-HES conjugate provided herein refers to the target-directed portion, which is an antibody expected to bind to a therapeutic target molecule and result in disease relief or reduction of disease progression in vivo.

[0025] The terms “specifically binding” or “specific affinity” in relation to therapeutic antibodies or other target-directed moieties mean that a target-directed moiety, such as an antibody or antigen-binding antibody fragment, reacts to or associates with an epitope, protein, or target molecule more frequently, more quickly, for longer durations, with greater affinity, or in some combination of the above, than alternative substances containing proteins unrelated to the target epitope. Due to sequence identity between homologous proteins in different species, specific affinity may, in some embodiments, include a binder that recognizes proteins or targets in multiple species. Similarly, due to homology within a particular region of polypeptide sequences of different proteins, the terms “specific affinity” or “specifically binding” may include a binder that recognizes multiple proteins or targets. In certain embodiments, it is understood that a target-directed moiety that specifically binds to a first target may or may not specifically bind to a second target. Thus, “specific affinity” does not necessarily require exclusive binding, e.g., binding to a single target (although this may be included). Therefore, a target-directed moiety may, in certain embodiments, specifically bind to multiple targets. In certain embodiments, multiple targets may be bound by the same target-directing moiety. In some embodiments, the target-directing moiety (e.g., therapeutic antibody) is determined using BIACORE® analysis to be 0.5 × 10⁻¹⁶ -10 ~10×10 -6 It binds to a target (e.g., a cell surface antigen) based on an equilibrium dissociation constant (Kd) within a certain range.

[0026] As used herein, the term “infectious agent” refers to an agent that causes an infectious disease. Infectious agents belong to four main groups: viruses, bacteria, fungi, and parasites. Such infectious agents may be extracellular or intracellular (e.g., infected cells).

[0027] As used herein, the term “infection” refers to a disease caused by an infectious agent such as bacteria, viruses, parasites, or fungi. Infections can spread directly or indirectly (through vectors and / or reservoirs) from one organism (e.g., a human) to another.

[0028] T-Oligo-HES Conjugate: The provided T-oligonucleotide-HES (T-oligo-HES) conjugate comprises a target-directed moiety (T) conjugated to an oligonucleotide-HES complex (oligo-HES), the target-directed moiety being conjugated to the oligo-HES complex, optionally via a linker. In certain embodiments, the target-directed moiety is conjugated to the oligo-HES complex via a linker. In further specific embodiments, the target-directed moiety is conjugated to the oligo-HES complex via a cleavable linker. In certain embodiments, the oligonucleotide contained in the T-oligo-HES conjugate is a therapeutic oligonucleotide. In further specific embodiments, the conjugate contains a therapeutic oligonucleotide selected from siRNA, shRNA, miRNA, antagmir, dicer substrates, antisense oligonucleotides, and expression cassettes (e.g., plasmids) capable of expressing siRNA, miRNA, ribozymes, or antisense oligonucleotides.

[0029] In certain embodiments, the T-oligo-HES conjugate comprises a target-directed moiety conjugated to an oligo-HES complex via a linker, and the conjugate comprises a therapeutic oligonucleotide. In further specific embodiments, the T-oligo-HES conjugate comprises a target-directed moiety conjugated to an oligo-HES complex via a linker, and the conjugate comprises a therapeutic oligonucleotide selected from siRNA, shRNA, miRNA, antagmir, a dicer substrate, an antisense oligonucleotide, and an expression cassette (e.g., a plasmid) capable of expressing siRNA, miRNA, a ribozyme, or an antisense oligonucleotide.

[0030] In some embodiments, the present disclosure relates to formula (I) T-(L n -(Oligo-HES) x ) p (I) Provides a T-oligo-HES conjugate having the structure. (In the formula, T is a target-directed portion that selectively binds to the target of choice; L is the linker; Oligo-HES is an oligonucleotide complex containing therapeutic oligonucleotides and H-type exciton structures (HES); n is either 0 or 1; x is 1 to 30, 1 to 20, 1 to 10, or 1 to 5; p is 1-30, 1-20, 1-10, or 1-5).

[0031] In some embodiments, the T-oligo-HES conjugate having the structure of formula (I) comprises therapeutic oligonucleotides that specifically hybridize to nucleic acid sequences in vivo and modulate levels of proteins encoded or controlled by the nucleic acid.

[0032] In some embodiments, a T-oligo-HES conjugate having the structure of formula (I) comprises a therapeutic oligonucleotide containing one, two, or three substitutions, deletions, or insertions compared to the corresponding reverse complementary strand of the nucleic acid sequence.

[0033] In some embodiments, the present disclosure relates to formula (II) T-[L n -{((Oligo2-LL) m -Oligo1-HES) s} t ] u (II) Provides a T-oligo-HES conjugate having the structure. (In the formula, T is a target-directed portion that selectively binds to the target of choice; L is the linker; LL is a linker, and in some cases LL is an alkyl such as C6, C10, or C18 alkyl; Oligo1-HES is an oligonucleotide complex containing oligonucleotide 1 (oligo1) and an H-type exciton structure (HES); Oligo 2 is an oligonucleotide that may be the same as or different from Oligo 1; n is either 0 or 1; m is either 0 or 1; s is either 1 or 2; t is either 1 or 2; (u is 1, 2, 3, 4, or 5).

[0034] In some embodiments, the T-oligo-HES conjugate having the structure of formula (II) comprises therapeutic oligonucleotides that specifically hybridize to nucleic acid sequences in vivo and modulate levels of proteins encoded or controlled by the nucleic acid.

[0035] In some embodiments, the T-oligo-HES conjugate having the structure of formula (II) comprises a therapeutic oligonucleotide containing one, two, or three substitutions, deletions, or insertions compared to the corresponding reverse complementary strand of the nucleic acid sequence.

[0036] Where any aspect or embodiment disclosed herein relates to a Markush group or other grouping of alternatives, the disclosure includes not only the entire enumerated group as a whole, but also each member of the group individually, all possible subgroups of a principal group, and principal groups in which one or more group members are absent. The disclosure also envisions the express exclusion of one or more of any group members.

[0037] The term "and / or" as used in phrases such as "A and / or B" in this specification is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, when the term "and / or" is used in a phrase such as "A, B, and / or C," it is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0038] H-type exciton structure (HES) A "chromophore" is a group, substructure, or molecule responsible for absorbing light. Typical chromophores each have a characteristic absorption spectrum.

[0039] A "fluorophore" is a chromophore that absorbs light at a characteristic wavelength and then re-emits light at a different wavelength, most typically characteristic of that wavelength. Fluorophores are well known to those skilled in the art and include, but are not limited to, xanthenes and xanthene derivatives (xanthenes include fluorescein and its derivatives), cyanines and cyanine derivatives (e.g., indocarbocyanine, indodicarbocyanine), coumarins and coumarin derivatives, and lanthanide ion series chelating agents. Fluorophores are distinguished from chromophores that absorb light but do not characteristically re-emit it.

[0040] A “H-type exciton structure” (HES) refers to two or more fluorophores in which transition dipoles are arranged in a parallel conformation, resulting in the splitting of excited singlet states; transitions between the ground state and higher excited states are permitted, while transitions between the ground state and lower excited states are prohibited. HES formation associated with certain fluorophores is known in the art, and this disclosure encompasses the attachment of these fluorophores to oligonucleotides (e.g., diagnostic and therapeutic oligonucleotides) by the methods described herein, and the use of the resulting oligo-HES complexes in T-oligo-HES conjugates. Examples of HES-forming fluorophores that can be included in T-oligo-HES conjugates and used by the methods provided herein include, but are not limited to, xanthenes and xanthene derivatives, cyanines and cyanine derivatives, coumarins, and chelating agents including the lanthanide ion series. In some embodiments, at least one fluorophore in the HES has maximum excitation and / or emission at 350–800 nm. In some embodiments, at least two fluorophores in the HES have maximum excitation and / or emission at 350–800 nm.

[0041] The terms “oligonucleotide-HES” complex, “oligo-HES” complex, and “HES-oligonucleotide” complex are used interchangeably herein and refer to a complex of one or more oligonucleotide chains (e.g., single-stranded, double-stranded, triple-stranded, or more-stranded linear or cyclic oligonucleotides containing the same, complementary, or different oligonucleotide sequences) that contain two or more fluorophores forming HES. As long as the assembled HES oligonucleotide contains one or more HES, the fluorophores of the HES oligonucleotide may be attached at the 5' and / or 3' terminal main chain phosphate and / or at another base within the oligonucleotide or in a different oligonucleotide. The fluorophores may be attached to the oligonucleotide via a linker, such as a flexible aliphatic chain. Oligonucleotide-HES complexes and their uses are further described in International Patent Application Publication No. WO2014201306A1, the contents of which are incorporated herein by reference in whole for all purposes.

[0042] Oligo-HES may contain one, two, three, four, or more HES molecules. Furthermore, HES molecules in HES oligonucleotides may contain two, three, four, or more identical or different fluorophores. See, for example, Toptygin et al., Chem. Phys. Lett. 277:430-435 (1997). In some embodiments, HES molecules are formed as a result of fluorophore aggregates between HES oligonucleotides. In some embodiments, HES molecules are formed as a result of fluorophore aggregates between oligonucleotides labeled individually with fluorophores capable of forming HES molecules.

[0043] The fluorophores in the oligo-HES complex contained in the provided T-oligo-HES conjugate can be any fluorophores in the complex that can form HES with homo- or hetero-homogeneous fluorophores in the complex. In some embodiments, the oligo-HES complex includes two fluorophores capable of forming an H-type exciton structure. In some embodiments, the oligo-HES complex includes at least one fluorophore with excitation and / or emission in the 300-850 nm range. In additional embodiments, the oligo-HES complex includes two, three, four or more fluorophores capable of forming an H-type exciton structure. In further embodiments, the oligo-HES complex in the T-oligo-HES conjugate includes two, three, four or more fluorophores with excitation and / or emission in the 300-850 nm range. In further embodiments, the oligo-HES complex contains about 2-20, about 2-10, about 2-6, or about 2-4 fluorophores capable of forming H-type exciton structures. In additional embodiments, the oligo-HES complex contains two, three, four, five or more fluorophores capable of forming one or more H-type exciton structures. In further embodiments, the oligo-HES complex contains two, three, four, five or more fluorophores with excitation and / or emission in the 300-850 nm range. Two or more fluorophores are said to quench each other in HES if their aggregate fluorescence is detectably smaller than the aggregate fluorescence of the fluorophores when they are separated, for example, at about 1 μM or less in solution. Compared to the spectra of individual fluorophores, the maximum value of the HES absorbance spectrum shows a shift in the maximum absorbance wavelength to a shorter wavelength, i.e., a blue shift. The fluorescence intensity of the H-type exciton structures or aggregates (here "HES") shows a lower intensity than the intensity of its components. The blue shift of the absorbance spectrum of the H-type exciton structure or aggregate, or the decrease in fluorescence intensity behavior, can be used as an indicator of the signal reporter portion.In preferred embodiments, two or more fluorophores in the oligo-HES complex of the T-oligo-HES conjugate are increased or quenched by at least 50%, preferably at least 70%, more preferably at least 80%, most preferably at least 90%, 95%, or even at least 99%. Examples of fluorophores that can form H-type exciton structures include, but are not limited to, xanthenes, indocarbocyanines, indodicarbocyanines, and coumarins. In particular embodiments, the HES of the oligo-HES complex contained in the T-oligo-HES conjugate provided herein contains at least one fluorophore which is xanthene, indocarbocyanine, indodicarbocyanine, or coumarin.

[0044] In some embodiments, the oligo-HES complex of the T-oligo-HES conjugate contains a fluorophore selected from carboxyrhodamine 110, carboxytetramethylrhodamine, carboxyrhodamine-X, diethylaminocoumarin, and N-ethyl-N'-[5-(N''-succinimidyloxycarbonyl)pentyl]indocarbocyanine chloride, N-ethyl-N'-[5-(N''-succinimidyloxycarbonyl)pentyl]-3,3,3',3'-tetramethyl-2',2'-indodicarbocyanine chloride dye, and Cy7 NHS ester.

[0045] In further embodiments, the oligo-HES complex contained in the T-oligo-HES conjugate is Rhodamine Green® carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green® carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green®-X succinimidyl ester or hydrochloride; Rhodol Green™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfonerhodamine or di(succinimidyl ester); 5-(and-6)-carboxynaphthofluorescein, 5-(and-6)-carboxynaphthofluorescein succinimidyl ester; 5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidyl ester; 6-carboxyrhodamine 6G succinimidyl ester; 5-(and-6)-carboxyrhodamine 6G succinimidyl ester; 5-carboxy-2',4',5',7'-tetrabromosulfonefluorescein succinimidyl ester or bis-(diisopropylethylammonium) salt 5-Carboxytetramethylrhodamine; 6-Carboxytetramethylrhodamine; 5-(and-6)-Carboxytetramethylrhodamine; 5-Carboxytetramethylrhodamine succinimidyl ester; 6-Carboxytetramethylrhodamine succinimidyl ester; 5-(and-6)-Carboxytetramethylrhodamine succinimidyl ester; 6-Carboxy-X-Rhodamine; 5-Carboxy-X-Rhodamine succinimidyl ester; 6-Carboxy-X-Rhodamine succinimidyl ester; 5-(and-6)-Carboxy-X-Rhodamine succinimidyl ester; 5-Carboxy-X-Rhodamine triethylammonium salt; Lissamine (trademark) Rhodamine B sulfonyl chloride; Malachite green isothiocyanate; Rhodamine Red(trademark)-X succinimidyl ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamide) succinimidyl hexanoate; tetramethylrhodamine-5-isothiocyanate;It contains a fluorophore selected from tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt; Texas Red®-X succinimidyl ester; Texas Red®-X succinimidyl ester; X-rhodamine-5-(and-6)-isothiocyanate; and carbocyanine.

[0046] In some embodiments, the oligo-HES complex contained in the T-oligo-HES conjugate contains hetero-HES composed of different fluorophores. In certain embodiments, the hetero-HES contains rhodamine or a rhodamine derivative and fluorescein or a fluorescein derivative or two carbocyanines. In further embodiments, the hetero-HES contains 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein; 5-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl) ether, -alanine-carboxamide, or succinimidyl ester; 5-carboxyfluorescein succinimidyl ester; 6-carboxyfluorescein succinimidyl ester, 5-(and-6)-carboxyfluorescein The present invention comprises fluorescein or a fluorescein derivative selected from: fluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 6-(fluorescein-5-carboxamide)hexanoic acid or succinimidyl ester; 6-(fluorescein-5 (and-6)-carboxamide)hexanoic acid or succinimidyl ester; fluorescein-5-EX succinimidyl ester; fluorescein-5-isothiocyanate; and fluorescein-6-isothiocyanate.

[0047] In some embodiments, at least one oligo-HES complex in the T-oligo-HES conjugate contains a fluorophore having maximum excitation and / or emission at 350-800 nm. In some embodiments, at least two oligo-HES complexes in the T-oligo-HES conjugate contain fluorophores having maximum excitation and / or emission at 350-800 nm. In some embodiments, all oligo-HES complexes in the T-oligo-HES conjugate contain fluorophores having maximum excitation and / or emission at 350-800 nm. In further embodiments, all fluorophores in the T-oligo-HES conjugate have maximum excitation and / or emission at 350-800 nm.

[0048] H-type exciton structures HES and fluorophores capable of forming HES are further described in International Patent Publication No. WO2014201306A1, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0049] Oligonucleotides As used herein, the terms “oligonucleotide” or “oligo” refer to oligomers or polymers of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or their mimics. This term includes oligonucleotides composed of naturally occurring nucleic acid bases, sugars, and nucleoside-intercellular (backbone) covalent bonds (i.e., “unmodified oligonucleotides”), as well as oligomeric compounds having nucleic acid bases, sugars, and / or nucleoside-intercellular links and / or similarly functioning DNA and / or RNA analogs (i.e., nucleic acid “mimetics” or “mimites”). Such mimetic oligonucleotides are often preferred over their native forms due to desirable properties such as improved affinity for nucleic acid targets and improved stability in the presence of nucleases. For example, as used herein, the term “oligonucleotide” includes morpholino (MNO), in which one or more ribose rings in the nucleotide backbone are replaced by morpholine rings, and phosphorodiamidate morpholino oligomers (PMO), in which one or more ribose rings in the nucleotide backbone are replaced by morpholine rings and negatively charged intersubunit linkages are replaced by uncharged phosphorodiamidate linkages. Similarly, the term oligonucleotide includes PNA, in which one or more sugar phosphate backbones of an oligonucleotide are replaced by amide-containing backbones. For the purposes of this specification, and as sometimes referred to in the art, modified oligonucleotides that do not have a phosphorus atom in the internucleoside backbone can also be considered oligonucleosides. Furthermore, oligonucleotides are sometimes called oligomers.

[0050] The delivery of oligonucleotides using the T-oligo-HES conjugate provided herein is sequence-independent, and therefore, the oligonucleotides contained in the T-oligo-HES conjugate vehicle may be any form of nucleic acid or mimetic known to be desirable for introduction into cells.

[0051] The oligonucleotides in the oligo-HES complex of the T-oligo-HES conjugate can be single-stranded, double-stranded, cyclic, or hairpin oligonucleotides. In some embodiments, the oligonucleotides are single-stranded DNA, RNA, or nucleic acid mimetic (e.g., oligonucleotides containing one or more modified nucleotides such as PMO, MNO, PNA, or 2'OME and LNA). In some embodiments, the oligonucleotides are double-stranded DNA, RNA, nucleic acid mimetic, DNA / nucleic acid mimetic, DNA-RNA, and RNA-nucleic acid mimetic.

[0052] The inventors have surprisingly discovered that complexes containing oligo-HES such as ssDNA and dsRNA exhibit superior sequence-independent intracellular delivery, requiring orders of magnitude less oligonucleotide administration than conventional oligonucleotide delivery vehicles. Examples of single-stranded nucleic acids included in the complexes and T-oligo-HES conjugates provided herein include, but are not limited to, antisense, siRNA, shRNA, ribozymes, miRNA, anti-miRNA, triple-helix-forming oligonucleotides, and aptamers.

[0053] In some embodiments, the oligonucleotide in the oligo-HES complex of the T-oligo-HES conjugate is single-stranded DNA (ssDNA). In preferred embodiments, at least a portion of the ssDNA oligonucleotide specifically hybridizes with target RNA to form an oligonucleotide-RNA duplex. In more preferred embodiments, the oligonucleotide-RNA duplex is susceptible to RNase cleavage mechanisms (e.g., RNase H). In some embodiments, the single-stranded oligonucleotide in the complex includes at least one modified backchain linkage, at least one modified sugar, and / or at least one modified nucleic acid base (e.g., as described herein). In some embodiments, the single-stranded oligonucleotide in the complex includes at least one modified backchain linkage, at least one modified sugar, and / or at least one modified nucleic acid base (e.g., as described herein) and can form an oligonucleotide-RNA duplex susceptible to RNase cleavage mechanisms. In certain embodiments, the single-stranded oligonucleotide is a gapmer (i.e., as described herein or otherwise known in the art). In additional embodiments, the T-oligo-HES conjugate contains an oligonucleotide comprising at least one modified backchain linkage, at least one modified sugar, and / or at least one modified nucleic acid base (e.g., as described herein) that reduces susceptibility to RNase cleavage mechanisms. In specific embodiments, the single-stranded oligonucleotide comprises at least one 2'OME, LNA, MNO, or PNA motif.

[0054] Examples of double-stranded DNA oligonucleotides included in the oligo-HES complex of the T-oligo-HES conjugates provided herein include, but are not limited to, dsRNAi and dicer substrates and other RNA interference reagents, as well as sequences corresponding to structural genes and / or regulatory and termination regions.

[0055] In some embodiments, the oligonucleotide contained in the oligo-HES complex of the T-oligo-HES conjugate is linear double-stranded RNA (dsRNA). In preferred embodiments, the dsRNA is susceptible to RNase cleavage mechanisms (e.g., Dicer and Drosha (RNase III enzyme)). In additional embodiments, the dsRNA can be inserted into the cellular RNA-induced silencing complex (RISC). In further embodiments, the RNA strand of the dsRNA can be used with the RISC complex to cleave an RNA target.

[0056] In additional embodiments, the oligonucleotides included in the oligo-HES complex of the T-oligo-HES conjugate are double-stranded oligonucleotides in which one or both oligonucleotides contain at least one modified backchain linkage, at least one modified sugar, and / or at least one modified nucleic acid base. In preferred embodiments, the double-stranded oligonucleotides are susceptible to RNase cleavage mechanisms (e.g., Dicer and Drosha (RNase III enzyme)). In additional embodiments, the double-stranded oligonucleotides can be inserted into the cellular RNA-induced silencing complex (RISC). In further embodiments, the oligonucleotide strands of the double-stranded oligonucleotides can be used with the RISC complex to cleave an RNA target.

[0057] In further embodiments, the oligonucleotides included in the oligo-HES complex of the T-oligo-HES conjugate are triple-stranded DNA / RNA chimeras. In some embodiments, the oligonucleotide complex contains at least one oligonucleotide comprising at least one modified backchain linkage, at least one modified sugar, and / or at least one modified nucleic acid base. In certain embodiments, at least one oligonucleotide in the complex comprises at least one 2'OME, LNA, MNO, or PNA motif.

[0058] The oligonucleotides in the T-oligo-HES conjugates provided herein are typically prepared in a linear form, but can be joined to form a ring or otherwise prepared, and may also include branched chains. Separate oligonucleotides can specifically hybridize to form double-stranded compounds, which may have blunt ends or may include overhangs at one or both ends. In certain embodiments, the double-stranded oligonucleotides contained in the oligo-HES complex of the T-oligo-HES conjugates provided herein (e.g., dsRNA and double-stranded oligonucleotides in which at least one of the oligonucleotide strands is a nucleic acid mimetic) are 21 to 25 nucleotides long and have 1, 2, or 3 nucleotide overhangs at either or both ends.

[0059] The oligonucleotides contained in the oligo-HES complex of the T-oligo-HES conjugates provided herein can be of various lengths, generally depending on the specific form of the nucleic acid or mimetic and its intended use. In some embodiments, the nucleic acid / oligonucleotides in the oligo-HES complex are in the range of about 5 to about 500 nucleotides, preferably about 10 to about 100 nucleotides in length.

[0060] In some embodiments, the oligo-HES complex of the T-oligo-HES conjugate contains an oligonucleotide comprising at least eight consecutive nucleic acid bases complementary to the target nucleic acid sequence. In various related embodiments, the oligonucleotide in the oligo-HES complex is about 8 to about 100 monomer subunits (as used herein interchangeably with the term "nucleotide") or about 8 to about 50 nucleotides in length.

[0061] In additional embodiments, the oligonucleotides in the oligo-HES complex of the T-oligo-HES conjugate are in the range of length from about 8 to about 30 nucleotides, about 15 to about 30 nucleotides, about 20 to about 30 nucleotides, about 18 to about 26 nucleotides, about 19 to about 25 nucleotides, about 20 to 25, or about 21 to 25 nucleotides.

[0062] In further embodiments, the oligonucleotides in the oligo-HES complex of the T-oligo-HES conjugate have a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 subunits (nucleotides). In certain embodiments, the oligonucleotides have a length of 19, 20, 21, 22, 23, 24, or 25 nucleotides.

[0063] In certain embodiments, the oligonucleotides in the oligo-HES complex of the T-oligo-HES conjugate contain double-stranded RNA oligonucleotides having a length between 21 and 25 nucleotides, with one or both ends having an overhang of 1, 2, or 3 nucleotides. In other embodiments, the oligonucleotides in the complex contain double-stranded oligonucleotides, with at least one of the oligonucleotide strands being a nucleic acid mimetic having a length between 21 and 25 nucleotides, and the double-stranded oligonucleotide having an overhang of 1, 2, or 3 nucleotides at either or both ends. Modified oligonucleotides

[0064] The oligonucleotides in the oligo-HES complex of the T-oligo-HES conjugates provided herein preferably comprise one or more modified nucleoside linkages, modified sugar moieties, and / or modified nucleic acid bases. Such modified oligonucleotides (i.e., mimics) are typically preferred over the natural forms due to desirable properties, including, for example, enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, and / or increased inhibitory activity.

[0065] Inter-modified nucleoside linking As used herein, the term "oligonucleotide" refers to oligonucleotides that retain a phosphorus atom in their internucleoside backbone, and oligonucleotides that do not have a phosphorus atom in their internucleoside backbone.

[0066] In some embodiments, the oligonucleotides in the oligo-HES complex of the T-oligo-HES conjugate include one or more modified nucleoside linkages. The modified nucleoside linkages in the oligonucleotides of the complexes and conjugates provided herein may include any form of nucleoside linkage known to provide enhanced nuclease stability to the oligonucleotide compared to, for example, that provided by phosphodiester nucleoside linkages. Oligonucleotides having modified nucleoside linkages include nucleoside linkages that retain a phosphorus atom and nucleoside linkages that do not contain phosphorus. In some embodiments, the oligonucleotide includes modified nucleoside linkages that alternate between modified and unmodified nucleoside linkages. In some embodiments, most of the nucleoside linkages in the oligonucleotide are modified. In further embodiments, all nucleoside linkages in the oligonucleotide are modified.

[0067] Preferred modified oligonucleotide backchains include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphodiesters, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkylphosphonates, such as 3'-alkylenephosphonates, 5'-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates, such as 3-aminophospholamidates and aminoalkylphospholamidates, thionophospholamidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages and their 2'-5 linkage analogs, and those having inverted polarity where one or more internucleoside linkages are 3'-3', 5'-5', or 2'-2' linkages. Preferred oligonucleotides having polarity reversal include a single 3'-3' linkage, i.e., a single inverted nucleoside residue (missing a nucleic acid base or having a hydroxyl group in its place), at the 3' end of the internucleotide linkage. Various salts, mixed salts, and free acid forms are also included.

[0068] In preferred embodiments, the oligonucleotide in the oligo-HES complex of the T-oligo-HES conjugate contains at least one phosphorothioate (PS) nucleoside linkage, in which one of the non-bridged oxygen atoms in the phosphodiester bond is replaced by sulfur. Oligonucleotides containing PS nucleoside linkages form a normal Watson-Crick base pair, activate RNase H, carry a negative charge for cell delivery, and exhibit other additional desired pharmacokinetic properties. In some embodiments, at least one modified nucleoside linkage is a phosphorothioate. In some embodiments, at least 2, 3, 4, 5, 10, or 15 of the nucleoside linkages contained in the oligonucleotide are phosphorothioate linkages. In some embodiments, at least 1-10, 1-20, or 1-30 of the modified nucleoside linkages are phosphorothioate linkages. In some embodiments, at least 2, 3, 4, 5, 10, or 15 of the modified nucleoside linkages are phosphorothioate linkages. In additional embodiments, the internucleoside links in the oligonucleotide are phosphorothioate nucleoside links.

[0069] In some embodiments, the oligonucleotides in the oligo-HES complex of the T-oligo-HES conjugate contain an 8-14 base PS-modified deoxynucleotide "gap" (i.e., MOE gapmer) flanked by 2-5 MOE nucleotides at either end. In some embodiments, the T-oligo-HES conjugate provided herein has an oligonucleotide containing an 8-14 base PS-modified deoxynucleotide "gap" (i.e., LNA gapmer) flanked by 2-5 LNA nucleotides at both ends. In additional embodiments, the oligo-HES complex has an oligonucleotide containing an 8-14 base PS-modified deoxynucleotide "gap" (i.e., tcDNA gapmer) flanked by 2-5 tricycloDNA nucleotides at either end.

[0070] Another preferred phosphorus-containing modified nucleoside linkage is the N3'-P5' phosphoramidate (NP), in which the 3'-hydroxyl group of the 2'-deoxyribose ring is replaced with a 3'-amino group. Oligonucleotides containing NP nucleoside linkages exhibit high affinity for complementary RNA and resistance to nucleases. Since phosphoramidates do not induce RNase H cleavage of target RNA, oligonucleotides containing these nucleoside linkages are applicable when RNA integrity must be maintained, such as in the regulation of oligonucleotide mRNA splicing. In some embodiments, at least 2, 3, 4, 5, 10, or 15 of the nucleoside linkages of the oligonucleotide contained in the T-oligo-HES conjugate are phosphoramidate linkages. In some embodiments, at least 1-10, 1-20, or 1-30 of the modified nucleoside linkages are phosphoramidate linkages. In some embodiments, at least 2, 3, 4, 5, 10, or 15 of the modified nucleoside linkages are phosphoramidate linkages. In additional embodiments, each nucleoside linkage in the antisense compound is a phosphoramidate nucleoside linkage.

[0071] Numerous modified nucleoside linkages and their synthesis methods are known in the art and are included in the modifications that may be present in oligonucleotides of T-oligo-HES conjugates. Examples of U.S. patents teaching the preparation of phosphorus-containing nucleoside linkages include U.S. Patents 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,194,599; and 5,264,4 No. 23; No. 5,276,019; No. 5,278,302; No. 5,286,717; No. 5,321,131; No. 5,399,676 No. 5,405,939; No. 5,489,677; No. 5,453,496; No. 5,455,233; No. 5,466,677; This includes, but is not limited to, Nos. 5,476,925; 5,519,126; 5,527,899; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,565,555; 5,602,240; 5,571,799; 5,587,361; 5,625,050; 5,646,269; 5,663,312; 5,672,697; 5,677,439; and 5,721,218, each of which is incorporated herein in whole by reference.

[0072] T-oligo-HES conjugates containing oligonucleotides that do not contain phosphorus atoms are also provided herein. Examples of such oligonucleotides include those containing backchains formed by short-chain alkyl or cycloalkyl nucleoside linkages, mixed nucleoside linkages of heteroatoms and alkyl or cycloalkyl groups, or one or more short-chain heteroatoms or heterocyclic nucleoside linkages. These modified backchains include, but are not limited to, oligonucleotides containing morpholino linkages (partially formed from the sugar moiety of a nucleoside); siloxane backchains; sulfide, sulfoxide and sulfone backchains; formacetyl and thioformacetyl backchains; methyleneformacetyl and thioformacetyl backchains; riboacetyl backchains; alkene-containing backchains; sulfamate backchains; methyleneimino and methylenehydrazino backchains; sulfonate and sulfonamide backchains; amide backchains; and others having mixed N, O, S and CH2 component moieties. Methods for producing oligonucleotides containing a main chain without phosphorus atoms are known in the art, as indicated by U.S. Patents No. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, and 5,541,307. This includes, but is not limited to, the methods and compositions disclosed in Patent Nos. 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,646,269, 5,663,312, 5,633,360, 5,677,437, 5,677,439, and 5,792,608, each of which is incorporated herein by reference in whole.

[0073] In some embodiments, the oligonucleotide in the T-oligo-HES conjugate contains one or more modified backbone links selected from 3'-methylenephosphonate, methylene(methylimino) (also known as MMI), morpholino, locked nucleic acid, and peptide nucleic acid links. The modified backbone links may be homogeneous or alternating with other links, particularly phosphodiester or phosphorothioate links, as long as RNAse H cleavage is not supported.

[0074] In some embodiments, the oligonucleotides in the T-oligo-HES conjugate contain oligonucleotides that are nucleic acid mimetic. The term mimetic, as applied to oligonucleotides, is intended to include oligonucleotides in which either a sugar or both a sugar and a nucleotide linkage are replaced by an alternative group.

[0075] In some embodiments, the T-oligo-HES conjugate contains oligonucleotides having one or more morpholino linkages. Due to the RNAse and nuclease resistance properties of morpholinos, they are particularly useful for regulating transcription in cells. Therefore, in some embodiments, complexes containing morpholino units are used to regulate gene expression. In some embodiments, the morpholino unit is phosphorodiamidate morpholino. In further embodiments, all monomer units of the oligonucleotide correspond to morpholino. In further embodiments, all monomer units of the oligonucleotide correspond to phosphorodiamidate morpholino. In certain embodiments, each monomer unit of the oligonucleotide corresponds to phosphorodiamidate morpholino (PMO). In additional embodiments, a T-oligo-HES conjugate containing a morpholino oligonucleotide (e.g., PMO) is used to modify mRNA splicing in a subject. In additional embodiments, a T-oligo-HES conjugate containing an oligonucleotide containing one or more morpholino nucleic acid bases, such as PMO, is used as an antisense agent.

[0076] In additional embodiments, the T-oligo-HES conjugate comprises an oligonucleotide, which is a peptide nucleic acid (PNA). A PNA is a nucleic acid mimetic in which the sugar phosphate backbone of the oligonucleotide is replaced by an amide-containing backbone. In certain embodiments, the phosphate backbone of the oligonucleotide is replaced by an aminoethylglycine backbone, and the nucleic acid base is directly or indirectly bound to the aza nitrogen atom of the amide portion of the backbone. Many PNAs and methods for producing PNAs are known in the art (see, for example, Nielsen et al., Science, 254: 1497-150 (1991), and U.S. Patents 5,539,082, 5,714,331 and 5,719,262, each of which is incorporated herein by reference in whole). PNA-containing oligonucleotides offer improved stability and favorable hybridization dynamics, have higher affinity for RNA than DNA compared to unsubstituted nucleic acids, and do not activate RNAse H-mediated degradation. Oligonucleotides that can be included in T-oligo-HES conjugates include PNA analogs, e.g., PNAs having modified backlines containing positively charged groups and / or one or more chiral stereocenters at the C2(α), e.g., D-amino acid, or C5(γ), e.g., L-amino acid (e.g., L-lysine) position of one or more monomeric units of the oligonucleotide.

[0077] The RNAse and nuclease resistance properties of PNA oligonucleotides are particularly useful for regulating intracellular RNA (e.g., mRNA and miRNA) via steric barrier mechanisms. In some embodiments, the oligonucleotide in the T-oligo-HES conjugate comprises at least one PNA oligonucleotide. In some embodiments, the oligonucleotide contains at least one PNA oligonucleotide and modulates gene expression by strand intrusion into chromosomal double-stranded DNA. In further embodiments, the T-oligo-HES conjugate contains at least one PNA oligonucleotide and modifies mRNA splicing in the subject. In additional embodiments, the T-oligo-HES conjugate contains an oligonucleotide, such as PMO, which contains at least one PNA oligonucleotide and acts as an antisense.

[0078] Similarly, due to the RNAse and nuclease resistance properties of morpholino-containing oligonucleotides, these oligonucleotides are useful for regulating intracellular RNA (e.g., mRNA and miRNA) via steric barrier mechanisms. In some embodiments, the T-oligo-HES conjugate contains an oligonucleotide comprising at least one morpholino oligonucleotide, such as PMO, which modulates gene expression by strand intrusion into chromosomal double-stranded DNA. In further embodiments, the oligonucleotide comprises at least one morpholino, such as PMO, which modifies mRNA splicing in the subject. In additional embodiments, the oligonucleotide comprises at least one morpholino oligonucleotide, such as PMO, which acts as an antisense.

[0079] Furthermore, due to the RNAse and nuclease resistance properties of bicyclic sugar-containing nucleotides, these oligonucleotides are useful for regulating intracellular RNA (e.g., mRNA and miRNA) via steric barrier mechanisms. In some embodiments, the T-oligo-HES conjugate contains an oligonucleotide comprising at least one bicyclic sugar containing a nucleotide. In some embodiments, the nucleotide-containing bicyclic sugar is locked nucleic acid (LNA). In further embodiments, the LNA has a 2'-hydroxyl group linked to the 3' or 4' carbon atom of the sugar ring. In further embodiments, the oligonucleotide comprises at least one locked nucleic acid (LNA) where a methylene(--CH2--)n group bridges the 2' oxygen atom and the 4' carbon atom, and n is 1 or 2. In some embodiments, the T-oligo-HES conjugate contains an oligonucleotide comprising at least one bicyclic sugar containing a nucleotide, such as LNA, to regulate gene expression by strand intrusion into chromosomal double-stranded DNA. In other embodiments, the oligonucleotide comprises at least one bicyclic sugar, such as LNA, to alter mRNA splicing in a subject. In additional embodiments, the oligonucleotide comprises at least one bicyclic sugar oligonucleotide, such as LNA, and acts as an antisense.

[0080] modified sugar moiety In some embodiments, the oligonucleotides in the T-oligo-HES conjugate comprise one or more nucleosides having one or more modified sugar moieties that are structurally distinct from naturally occurring or synthetic unmodified nucleic acid bases but functionally interchangeable. In further embodiments, the T-oligo-HES conjugate comprises oligonucleotides with modified sugars in each nucleoside (unit).

[0081] Examples of useful sugar modifications to oligonucleotides in T-oligo-HES conjugates include, but are not limited to, compounds containing sugar substituents selected from OH;F;O-, S-, or N-alkyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl.

[0082] Typical modified sugars include carbocyclic or acyclic sugars, sugars having substituents on one or more of their 2', 3', or 4' positions, sugars having substituents in place of one or more hydrogen atoms in the sugar, and sugars having a linkage between any two other atoms in the sugar. Useful 2'-sugar substituents in oligonucleotides include, but are not limited to, OH;F;O-, S-, or N-alkyl;O-, S-, or N-alkenyl;allyl, amino;azide;thio;O-allyl;O(CH2)2SCH3;O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. In certain embodiments, the oligonucleotide contains at least one 2'-sugar substituent selected from O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are 1 to about 10. Other preferred oligonucleotides contain at least one 2'-sugar substituent selected from C1-C10 lower alkyls, substituted lower alkyls, alkenyls, alkynyls, alkaryls, aralkyls, O-alkaryls or O-aralkyls, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heteroalkyls, heterocycloalkyls, heterocycloalkaryls, aminoalkylaminos, polyalkylaminos, substituted silyls, RNA cleavage groups, reporter groups, intercalators, groups for improving pharmacokinetic properties, or groups for improving the pharmacodynamic properties of oligonucleotide compounds, and other substituents having similar properties.

[0083] In certain embodiments, the oligonucleotide in the T-oligo-HES conjugate comprises at least one 2'-substituted sugar having a 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-MOE) substituent.

[0084] In some embodiments, the oligonucleotides in the T-oligo-HES conjugate are 2'-allyl (2'--CH2--CH--CH2), 2'-O-allyl (2'-O--CH2--CH--CH2), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-acetamide (2'-O--CH2C(--O)NR1R1), where each R1 independently comprises at least one 2'-modified nucleoside selected from H or C1-C1 alkyl.

[0085] In further embodiments, the oligonucleotide in the T-oligo-HES conjugate comprises at least one 2'-substituted sugar having a 2'-dimethylaminooxyethoxy(2'-O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE) substituent; a 2'-dimethylaminoethoxyethoxy(2'-O--CH2--O--CH2--N(CH2)2, also known as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE) substituent; or a 2'-O-methyl(2'-O--CH3) substituent. In further embodiments, the oligonucleotide in the T-oligo-HES conjugate comprises at least one 2'-substituted sugar having a 2'-fluoro(2'-F) substituent.

[0086] In some embodiments, the oligonucleotide in the T-oligo-HES conjugate comprises at least one bicyclic sugar. In certain embodiments, the oligonucleotide has at least one locked nucleic acid (LNA) in which a 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring. In certain embodiments, the oligonucleotide comprises at least one locked nucleic acid (LNA) in which a methylene(--CH2--)n group bridges the 2' oxygen atom and the 4' carbon atom, with n being 1 or 2. In another embodiment, the oligonucleotide comprises at least one bicyclic modified nucleoside having a bridge between a 4'-ribosyl ring atom and a 2'-ribosyl ring atom, where the bridge is selected from 4'-(CH2)-O-2'(LNA);4'-(CH2)-S-2;4'-(CH2)2-O-2'(ENA);4'-C(CH3)2-O-2';4'-CH(CH3)-O-2';4'-CH(CH2OCH3)-O-2';4'-CH2-N(OCH3)-2';4'-CH2-O--N(CH3)-2';4'-CH2-N(R)--O-2';4'-CH2-CH(CH3)-2' and 4'-CH2-C(--CH2)-2', where R is independently H, a C1-C12 alkyl, or a protecting group. In some embodiments, the oligonucleotide in the T-oligo-HES conjugate comprises at least one of the aforementioned sugar configurations, and additional motifs, e.g., α-L-ribofuranose, β-D-ribofuranose, or α-L-methyleneoxy(4'-CH2--O-2'). Further LNAs useful for the oligonucleotides provided herein and their preparations are known in the art. See, for example, U.S. Patents 6,268,490, 6,670,461, 7,217,805, 7,314,923, and 7,399,845; WO98 / 39352 and WO99 / 14226; and Singh et al., Chem. Commun. 4:455-456 (1998), the contents of which are incorporated herein by reference in their entirety.

[0087] In some embodiments, the oligonucleotide in the T-oligo-HES conjugate includes a chemically modified furanosyl (e.g., ribofuranose) ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (5' and 2' substituents, particularly at the 2' position, formation of bicyclic nucleic acids (BNAs) by bridging of non-geminal ring atoms, substitution of ribosyl ring oxygen atoms by S, N(R), or C(R1)(R)2 (R--H, C1-C12 alkyl or protecting groups), and combinations thereof. Examples of chemically modified sugars include 2'-F-5'-methyl-substituted nucleosides (see, for example, WO2008 / 101157 for other disclosed 5',2'-bis-substituted nucleosides), substitution of ribosyl ring oxygen atoms by S and further substitution at the 2' position (see, for example, US20050130923), or 5'-substitution of BNA (WO2007 / 134181, where LNA is substituted with, for example, a 5'-methyl or 5'-vinyl group).

[0088] Also provided herein are T-oligo-HES conjugates containing oligonucleotides having a modification similar to the above modification at the 3' position of the sugar on the 3' terminal nucleotide or a 2'-5' linked oligonucleotide and at least one nucleotide at the 5' position of the 5'-terminal nucleotide. Representative U.S. patents teaching the preparation of the 2'-modified nucleoside contained in the provided oligonucleotides include U.S. Patents Nos. 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, and 5,576,427. This includes, but is not limited to, Nos. 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, 5,700,920, and 5,792,747, each of which is incorporated herein by reference in its entirety.

[0089] In some embodiments, the T-oligo-HES conjugate contains an oligonucleotide comprising at least one heterocyclic or bicyclic nucleic acid. For example, in some embodiments, the oligonucleotide has at least one ENA motif (see, for example, WO01 / 49687, the contents of which are incorporated herein by reference in their entirety).

[0090] In additional embodiments, the T-oligo-HES conjugate contains an oligonucleotide comprising at least one substitution of a five-membered furanose ring by a six-membered ring. In at least one embodiment, the oligonucleotide has at least one cyclohexene nucleic acid (CeNA).

[0091] In some embodiments, the T-oligo-HES conjugate contains an oligonucleotide comprising at least one tricycloDNA (tcDNA). In additional embodiments, the oligonucleotide contains an 8-14 base PS-modified deoxynucleotide "gap" (i.e., tcDNA gapmer) flanked by 2-5 tricycloDNA nucleotides at either end.

[0092] In certain embodiments, the T-oligo-HES conjugate contains oligonucleotides comprising a phosphorothioate backbone, and / or heteroatom backbones, such as --CH2--NH--O--CH2--, --CH2--N(CH3)--O--CH2-- (also known as methylene (methylimino) or MMI backbone), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --O--N(CH3)--CH2--CH2--, and oligonucleotides comprising an amide backbone (see, for example, U.S. Patent No. 5,602,240). In additional embodiments, the T-oligo-HES conjugate contains oligonucleotides comprising a phosphorodiamidate backbone structure. In further embodiments, the T-oligo-HES conjugate contains an oligonucleotide comprising a phosphorodiamidate morpholino (i.e., PMO) backbone structure (see, for example, U.S. Patent No. 5,034,506, the contents of which are incorporated herein by reference in their entirety).

[0093] Modified nucleic acid bases T-oligo-HES conjugates may also contain one or more oligonucleotides having one or more nucleic acid base modifications that are structurally distinct from naturally occurring or synthetic unmodified nucleic acid bases but are functionally interchangeable.

[0094] As used herein, the terms “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include synthetic and natural nucleic acid bases such as 5-methylcytosine (5-me-C). In some embodiments, the T-oligo-HES conjugate contains an oligonucleotide comprising at least one 5'-methylcytosine or C-5 propine. In some embodiments, each cytosine in the oligonucleotide is methylcytosine.

[0095] Modified nucleic acid bases are also referred to herein as heterocyclic base moieties and include other synthetic and natural nucleic acid bases, such as 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 and cytosine, 5-propynyl(--CC--CH3)uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azo Examples include uracil, cytosine and thymine, 5-uracil (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl 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, 3-deazaguanine and 3-deazaadenine.

[0096] The heterocyclic base moieties contained in oligonucleotides may also include those in which a purine or pyrimidine base is replaced by another heterocycle, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Nucleic acid bases particularly useful for increasing the binding affinity of the provided oligonucleotides include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.

[0097] Additional modified nucleic acid bases that may be present in oligonucleotides included in T-oligo-HES conjugates include tricyclic pyrimidines, e.g., phenoxazinecytidine (1H-pyrimido[5,4-b][1,4]benzoxazine-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiadin-2(3H)-one), and G-clamps, e.g., substituted phenoxazinecytidine. These include, but are not limited to, dins (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazine-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indole-2-one), pyridoindolecytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidine-2-one), or guanidinium G-clamps and analogues. Representative guanidino substituents are disclosed in U.S. Patent No. 6,593,466, which is incorporated herein by reference in whole. Representative acetamide substituents are disclosed in U.S. Patent No. 6,147,200, which is incorporated herein by reference in whole.

[0098] Numerous modified nucleic acid bases included in the oligonucleotides contained in the T-oligo-HES conjugates provided herein, and their synthesis methods, are known in the art. For example, these include: The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990; Englisch et al., Angewandte Chemie, International Edition, 30:613 (1993); Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302; Crooke, S. Ted., CRC Press. 1993; and U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,434,257, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, and 5,594,12 This includes modified nucleic acid bases disclosed in Patent No. 1, No. 5,596,091, No. 5,614,617, No. 5,645,985, No. 5,646,269, No. 5,681,941, No. 5,750,692, No. 5,830,653, No. 5,763,588, No. 6,005,096, No. 6,028,183, and No. 6,007,992, as well as U.S. Patent Application Publication No. 20030158403, each of which is incorporated herein by reference in whole. Chimeric oligonucleotides:

[0099] In some embodiments, the T-oligo-HES conjugate contains an oligonucleotide comprising one or more modified nucleoside linkages, a modified sugar moiety, and / or a modified nucleic acid base. In some embodiments, the oligonucleotide is a chimeric oligonucleotide (e.g., a chimeric oligomer compound). The terms "chimeric oligonucleotide" or "chimera" refer to a compound containing at least two chemically distinct regions (i.e., patterns and / or directions of chemically modified subunit motifs arranged along the length of the oligonucleotide), each composed of at least one monomer unit, i.e., a nucleotide or nucleoside in the case of a nucleic acid-based oligonucleotide compound. Chimeric oligonucleotides are also referred to, for example, hybrids (e.g., fusions) and gapmers. Representative U.S. patents teaching the preparation of such chimeric oligonucleotide structures include, but are not limited to, U.S. Patent Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is incorporated herein by reference in its entirety.

[0100] Chimeric antisense compounds typically contain at least one modified region to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity to target nucleic acids, and / or increased inhibitory activity. As an example, gapmers are chimeric oligonucleotides containing a sequence of nucleosides divided into three regions: two outer regions (wings) and a central region (gap). Gapmer designs typically contain a central region of about 5-10 consecutive 2'-deoxynucleotides, typically adjacent to one or two regions of a 2'-modified oligonucleotide that functions as a substrate for RNase H, increasing target RNA binding affinity but not supporting RNase H cleavage of the target RNA molecule. Therefore, when chimeras are used, comparable results can often be obtained using shorter oligonucleotides with substrate regions compared, for example, to phosphorothioate deoxyribonucleotides hybridizing to the same target region. Other chimeric oligonucleotides rely on regions that confer varying levels of binding affinity to their target across the length of the oligonucleotide, including, for example, regions of modified nucleosides that exhibit either increased or decreased affinity compared to other regions. So-called "MOE gapmers" have 2'-MOE modifications in the wings, often contain a complete PS backbone, and frequently include 5'MeC modifications on all cytosines.

[0101] Alternatively, in situations where RNAse H activity may be undesirable, such as the regulation of RNA processing, it may be preferable to use a design that employs uniformly modified oligonucleotides, for example, modified oligonucleotides that do not support RNAse H activity at each nucleotide or nucleoside position. As used in the present invention, the term “fully modified motif” means a sequence of sugar-modified nucleosides in which each nucleoside is modified to have the same modified sugar moiety. Suitable sugar-modified nucleosides for fully modified oligonucleotides include, but are not limited to, 2'-fluoro(2'F), 2'-O(CH2)2OCH3(2'-MOE), 2'-OCH3(2'-O-methyl), and bicyclic sugar-modified nucleosides. In one embodiment, the 3' and 5' terminal nucleosides remain unmodified. In a preferred embodiment, the modified nucleoside is one of 2'-MOE, 2'-F, 2'-O-Me, or a bicyclic sugar-modified nucleoside.

[0102] In some embodiments, the provided T-oligo-HES conjugate contains an oligonucleotide modified to have one or more stabilizing groups. In some embodiments, the stabilizing groups are attached to one or both ends of the oligonucleotide to enhance properties such as nuclease stability. In some embodiments, the stabilizing group is a cap structure. "Cap structure or terminal cap portion" means a chemical modification incorporated into either end of the oligonucleotide (see, for example, WO97 / 26270, the whole of which is incorporated herein by reference). These terminal modifications may protect the oligonucleotide having a terminal nucleic acid molecule from exonuclease degradation and / or aid in the delivery and / or localization of the oligonucleotide within the cell. The oligonucleotide may contain caps at the 5' end (5' cap), the 3' end (3' cap), or both the 5' and 3' ends. In the case of double-stranded oligonucleotides, the caps may be present at either or both ends of either strand. Cap structures are known in the art and include, for example, inverted deoxydebase caps. Further 3' and 5'-stabilizing groups that can be used to cap one or both ends of an oligonucleotide (e.g., antisense) compound to confer nuclease stability include those disclosed in WO03 / 004602, which are incorporated herein by reference in their entirety.

[0103] In some embodiments, the T-oligo-HES conjugate is a structure consisting of a 5'-cap and / or inverted abasic residue (part), 4',5'-methylene nucleotide; 1-(β-D-erythrofuranosyl) nucleotide; 4'-thionucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotide; α-nucleotide; modified nucleotide; phosphorodithioate linkage; threopentofuranosyl nucleotide; acyclic 3',4'-seconucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide Rheotide; 3-3'-inverted nucleotide moiety; 3'-3'-inverted debase moiety; 3'-2'-inverted nucleotide moiety; 3-2'-inverted debase moiety; 1,4-butanediol phosphate; 3'-phosphoramide; hexyl phosphate; aminohexyl phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate; or comprising a crosslinked or uncrosslinked methylphosphonate moiety (see, for example, WO97 / 26270, the whole thereof is incorporated herein by reference).

[0104] In some embodiments, the T-oligo-HES conjugate is a 3'-capped and / or 4',5'-methylene nucleotide; 1-(β-D-erythrofuranosyl) nucleotide; 4'-thionucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; α-nucleotide; modified base nucleotide; phosphorodithioate; s This includes leopentofuranosylnucleotides; acyclic 3',4'-seconucleotides; 3,4-dihydroxybutylnucleotides; 3,5-dihydroxypentylnucleotides, 5'-5'-inverted nucleotide moieties; 5'-5'-inverted debase moieties; 5'-phosphoramidates; 5'-phosphorothioates; 1,4-butanediol phosphates; 5'-aminos; and crosslinked and / or uncrosslinked 5'-phosphoramidates, phosphorothioates and / or phosphorodithioates, crosslinked or uncrosslinked methylphosphonates and 5'-mercapto moieties (see also the stabilizing groups disclosed in Beaucage et al., Tetrahedron 49:1925 (1993), which are incorporated herein by reference in their entirety).

[0105] In additional embodiments, the T-oligo-HES conjugate comprises a 5'-cap and one or more cationic tails. In further embodiments, the oligonucleotide is conjugated with at least one, two, three, four, or more positively charged amino acids, such as lysine or arginine. In specific embodiments, the oligonucleotide is PNA, with one or more lysine or arginine residues conjugated to the C-terminus of the molecule. In even more preferred embodiments, the oligonucleotide is PNA, comprising one to four lysine and / or arginine residues conjugated to each PNA linkage.

[0106] In additional relevant embodiments, the Disclosure provides T-oligo-HES conjugates and / or pharmaceutical compositions comprising T-oligo-HES conjugates further comprising one or more activators or therapeutic agents. In one embodiment, the activator or therapeutic agent is a nucleic acid. In various embodiments, the nucleic acid is a plasmid, immunostimulatory oligonucleotide, siRNA, shRNA, miRNA, anti-miRNA, Dicer substrate, decoy, aptamer, antisense oligonucleotide, or ribozyme.

[0107] oligonucleotide synthesis Oligonucleotides can be synthesized and / or modified by methods well established in the art. Oligomerization of modified and unmodified nucleosides is carried out, where appropriate, according to the literature concerning DNA-like compounds (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and / or RNA-like compounds (e.g., Scaringe, Methods 23:206-217 (2001) and Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36; Gallo et al., Tetrahedron 57:5707-5713 (2001)) (Current Protocols in Nucleic Acid Chemistry, Beaucage, SL et al.,(Edrs.), John Wiley & Sons, Inc., New York, NY, See also the USA, which is incorporated herein by reference in its entirety. Oligonucleotides are preferably synthesized chemically using appropriately protected reagents and commercially available oligonucleotide synthesizers. Suppliers of oligonucleotide synthesis reagents useful for the production of oligonucleotides include, but are not limited to, Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK).Alternatively, oligomers can be purchased from various oligonucleotide synthesis companies, such as Dharmacon Research Inc. (Lafayette, Colo), Qiagen (Germantown, MD), Proligo, and Ambion.

[0108] In certain embodiments, the preparation of oligonucleotides disclosed herein is carried out according to the procedures of the literature for DNA: Protocols for Oligonucleotides and Analogs, Agrawal, Ed., Humana Press, 1993, and / or RNA: Scaringe, Methods, 23:206-217 (2001); Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Smith, Ed., 1998, 1-36; Gallo et al., Tetrahedron 57:5707-5713 (2001). Additional methods for solid-phase synthesis can be found in U.S. Patents No. 4,415,732, No. 4,458,066, No. 4,500,707, No. 4,668,777, No. 4,725,677, No. 4,973,679, and No. 5,132,418, as well as reissued patent No. 34,069.

[0109] Regardless of the specific protocol used, the oligonucleotides contained in the provided T-oligo-HES conjugates can be prepared simply and systematically by well-known solid-phase synthesis techniques. Apparatus for such synthesis is available from several suppliers, including, for example, Gene Forge (Redwood City, Calif.). Suitable solid-phase techniques, including automated synthesis techniques, are described in *Oligonucleotides and Analogues, a Practical Approach*, F. Eckstein, Ed., Oxford University Press, New York, 1991. Any other means for such synthesis known in the art can be used additionally or alternatively (including liquid-phase synthesis).

[0110] The synthesis and preparation of bicyclic glycosylated monomers, adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil, as well as their oligomerization and nucleic acid recognition properties, are described (Koshkin et al., Tetrahedron, 54:3607-3630 (1998); WO98 / 39352 and WO99 / 14226), and the contents of each of these are incorporated herein by reference in their entirety. Other bicyclic glycosylated nucleoside analogs, such as the 4'-CH2--S-2' analog, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 8:2219-2222 (1998)). Preparations of other bicyclic sugar analogs containing oligodeoxyribonucleotide double helixes as substrates for nucleic acid polymerases are also described (WO98-DK393 19980914), and the contents of each of these are incorporated herein by reference in their entirety.

[0111] Techniques for linking fluorophores to oligonucleotides, such as those used according to the provided method, are well known in the art and can be used or systematically modified to prepare the HES-oligonucleotide complexes contained in the provided T-oligo-HES conjugate. See, for example, Connolly et al., Nucleic Acids Res. 13:4485-4502 (1985); Dreyer et al., Proc. Natl. Acad. Sci. 86:9752-9756 (1989); Nelson et al., Nucleic Acids Res. 17:7187-7194 (1989); Sproat et al., Nucleic Acids Res. 15:6181-6196 (1987) and Zuckerman et al., Nucleic Acids Res. 15:5305-5321 (1987), the contents of which are incorporated herein by reference in their entirety. Many fluorophores typically contain suitable reaction sites. Alternatively, fluorophores can be derivatized to provide reaction sites for bonding to another molecule. Fluorophores derivatized with functional groups for coupling to a second molecule are commercially available from various manufacturers. Derivatization may be by simple substitution of groups on the fluorophore itself, or by conjugation to a linker.

[0112] Fluorophores are optionally bonded to the 5' and / or 3' terminal main chain phosphate and / or other bases of the oligonucleotide via a linker. Various suitable linkers are known to those skilled in the art and / or discussed below. In some embodiments, the linker is a flexible aliphatic linker. In additional embodiments, the linker is a linear or branched saturated or unsaturated hydrocarbon chain of C1-C30. In some embodiments, the linker is a linear or branched saturated or unsaturated hydrocarbon chain of C2-C6. In additional embodiments, the hydrocarbon chain linker is substituted with one or more heteroatoms, aryls; or lower alkyls, hydroxyalkyls, or alkoxys.

[0113] In some embodiments, one or more fluorophores are incorporated into oligonucleotides during automated synthesis using one or more fluorophore-modified nucleosides, fluorophores and sugar / base / and / or linkage-modified nucleosides, and / or deoxynucleoside phosphoramidites.

[0114] In some embodiments, one or more fluorophores are incorporated into the oligonucleotide in a post-labeling reaction. Suitable post-labeling reactions are known in the Art and can be typically applied or modified to synthesize the oligonucleotide-HES complexes contained in the T-oligo-HES conjugates provided herein. In one embodiment, one or more fluorophores are incorporated into the oligonucleotide in a post-labeling reaction, in which case an amine- or thiol-modified nucleotide or deoxynucleotide in the synthesized oligonucleotide reacts with an amine- or thiol-reactive fluorophore, for example, succinimidyl ester fluorophores.

[0115] In further embodiments, one or more identical fluorophores are incorporated into an oligonucleotide in a single reaction, the single reaction comprising contacting the reaction form of the dye with an oligonucleotide containing a desired number of reactive groups capable of reacting with the fluorophore, in a suitable buffer, under conditions and for a sufficient time to achieve the incorporation of the fluorophore into the oligonucleotide. The reactive groups can be systematically incorporated into the oligonucleotide during synthesis using standard techniques and reagents known in the art.

[0116] Exemplary mechanism of action In some embodiments, the Disclosure provides a method for treating a disease or disorder characterized by the overexpression of a nucleic acid in a subject, comprising administering a HES-oligonucleotide complex to a subject that targets a nucleic acid containing or encoding a nucleic acid and acts to reduce the level of the nucleic acid in the subject and / or interfere with its function. In further embodiments, the Disclosure provides a method for treating a disease or disorder characterized by the overexpression of a protein in a subject, comprising administering a HES-oligonucleotide complex to a subject that targets a nucleic acid encoding a protein or contains an oligonucleotide that reduces the endogenous expression, processing, or function of the protein in the subject. In some embodiments, the nucleic acid is DNA, mRNA, or miRNA. In further embodiments, the oligonucleotide is selected from siRNA, shRNA, miRNA, antagmir (anti-miRNA), dicer substrates, antisense oligonucleotides, and plasmids capable of expressing siRNA, miRNA, ribozymes, and antisense oligonucleotides.

[0117] Antisense In some embodiments, the T-oligo-HES conjugate contains an antisense oligonucleotide. The terms “antisense oligonucleotide” or simply “antisense” are intended to include oligonucleotides corresponding to a single strand of nucleic acid (e.g., DNA, RNA, and nucleic acid mimetics such as PNA, morpholino (e.g., PMO), and modified nucleosides and / or internucleoside ligations) that bind to a homologous mRNA within a cell under treatment and modulate RNA function by, for example, altering the translocation of the target RNA to the protein translation site, altering the translation of proteins from the target RNA, altering the splicing of the target RNA (e.g., promoting exon skipping), altering catalytic activity associated with or that can be promoted by the target RNA, and targeting the mRNA for degradation by endogenous RNase H. In some embodiments, the antisense oligonucleotide alters cellular activity by specifically hybridizing with chromosomal DNA. The term antisense oligonucleotide also includes antisense oligonucleotides that may not be precisely complementary to the desired target gene. Therefore, the methods provided can be utilized when non-target-specific activity is observed in the antisense, or when an antisense sequence containing one or more mismatches with the target sequence is preferred for a particular use. The overall effect of such interference on the function of the target nucleic acid is the modulation of the target protein of interest. In relation to this disclosure, “modulation” means either an increase (stimulation) or decrease (inhibition) in the amount or level of gene or protein expression of a small non-coding RNA, a nucleic acid target, RNA or protein associated with the small non-coding RNA, or a downstream target of the small non-coding RNA (e.g., mRNA representing nucleic acid encoding a protein regulated by the small non-coding RNA). Inhibition is a preferred form of modulation, and small non-coding RNAs are preferred nucleic acid targets. Small non-coding RNAs that can modulate levels include miRNAs and miRNA precursors.In the context of this disclosure, “modulation of function” means alteration of the function or activity of a small non-coding RNA, or alteration of the function of a cellular component to which the small non-coding RNA is related or has downstream effects. In one embodiment, the modulation of function is the inhibition of the activity of the small non-coding RNA.

[0118] Antisense oligonucleotides are preferably linked nucleosides with a length of about 8 to about 80. In some embodiments, the antisense oligonucleotide is about 10 to about 50 nucleosides or about 13 to about 30 nucleotides. Antisense oligonucleotides provided herein include ribozymes, anti-miRNAs, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides that specifically hybridize to target nucleic acids and regulate their expression.

[0119] In some embodiments, the T-oligo-HES conjugate contains an antisense oligonucleotide having a length of about 15 to about 30 nucleosides (i.e., 15 to about 30 linked nucleosides), or a length of about 17 to about 25 nucleosides. In certain embodiments, the antisense oligonucleotide has a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides. In additional embodiments, the antisense oligonucleotide has a length of about 10 to about 50 nucleotides, more preferably about 15 to about 30 nucleotides. In further embodiments, the antisense oligonucleotide has a length of 4, 5, 6, or 7 nucleotides.

[0120] In additional embodiments, the oligonucleotide in the T-oligo-HES conjugate interferes with the transcription of the target RNA of interest. In some embodiments, the oligonucleotide interferes with the transcription of the target mRNA or miRNA by strand substitution. In other embodiments, the oligonucleotide interferes with mRNA transcription by forming a stable complex with a portion of the target gene by strand entry or triple-strand formation (triple-stranding oligonucleotides (THOs), including LNA as an example, e.g., see U.S. Patent Application Publication 2012 / 0122104, the entirety of which is incorporated herein by reference). In additional embodiments, the oligonucleotide in the T-oligo-HES conjugate interferes with the transcription of the target RNA (e.g., mRNA or miRNA) by interfering with the cell's transcription machinery. In some embodiments, the oligonucleotide in the oligo-HES complex is designed to specifically bind to the 5' end of mRNA or the region of the AUG start codon (e.g., within 30 nucleotides of the AUG start codon) to reduce translation. In some embodiments, the oligonucleotide component of the T-oligo-HES conjugate is designed to specifically hybridize to an intron / exon junction in RNA. In some embodiments, the oligonucleotide is designed to specifically bind to a 3' untranslated target sequence in RNA (e.g., mRNA). In further embodiments, the oligonucleotide is designed to specifically bind to nucleotides 1-10 of miRNA. In additional embodiments, the oligonucleotide is designed to specifically bind to a sequence in a pre-miRNA or primary miRNA (pri-miRNA) that, upon binding, blocks miRNA processing.

[0121] In other embodiments, the oligonucleotide in the T-oligo-HES conjugate either binds to a site on an essential RNA secondary structure or acts as a steric blocker that causes cleavage of the translated polypeptide. In some embodiments, the oligonucleotide is designed to interfere with intron excision, for example, by binding to or near the splice junction of a target mRNA. In some embodiments, the oligonucleotide is designed to interfere with intron excision or increase the expression of alternative splice variants.

[0122] RNase H is an endogenous enzyme that specifically cleaves the RNA portion of an RNA:DNA double helix. In some embodiments, antisense oligonucleotides induce RNase H activity upon binding to a target nucleic acid. In some embodiments, the oligonucleotide is DNA or a nucleic acid mimetic. Oligonucleotides induce RNase H activity and have certain advantages, for example, in that they reduce the target RNA by utilizing endogenous ribonucleases.

[0123] One antisense design for inducing RNase H activity is a gapmer motif design, which is a chimeric oligonucleotide having a central block composed of DNA with or without phosphorothioate modification, and nuclease-resistant 5' and 3' adjacent blocks, usually 2'-O-methyl RNA, but a wide range of 2'-modifications are used (see Crooke, Curr. Mol. Med. 4(5):465-487 (2004)). Other gapmer designs are described herein or otherwise known in the art.

[0124] In additional embodiments, the T-oligo-HES conjugate contains an antisense oligonucleotide designed to avoid activation of RNase H in cells. Oligonucleotides that do not induce RNase H activity have particular advantages, for example, in blocking transcription mechanisms (by steric blocking mechanisms) and altering the splicing of target RNA. In some embodiments, the oligonucleotide is designed to interfere with and / or modify intron excision by binding to or near the splice junction of target mRNA, for example. In additional embodiments, the oligonucleotide is designed to increase the expression of alternative splice variants of the message. In one preferred embodiment, the T-oligo-HES conjugate contains an oligonucleotide containing a morpholino (e.g., PMO) antisense oligonucleotide. In another preferred embodiment, the T-oligo-HES conjugate contains a PNA antisense oligonucleotide.

[0125] In certain embodiments, the T-oligo-HES conjugate contains an antisense oligonucleotide that targets at least a portion of the region up to 50 nucleic acid bases upstream of the intron / exon junction of the target mRNA. More preferably, the antisense oligonucleotide targets at least a portion of 20-24 or 30-50 nucleic acid bases upstream of the intron / exon junction of the target mRNA and preferably does not support RNAse H cleavage of the mRNA target upon binding. Preferably, the antisense oligonucleotide contains at least one modification that increases binding affinity to RNA targets (e.g., mRNA and miRNA) and increases nuclease resistance of the antisense compound.

[0126] In one embodiment, the T-oligo-HES conjugate contains an antisense oligonucleotide comprising at least one nucleoside having a 2' modification of its sugar moiety. In a further embodiment, the antisense oligonucleotide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleosides having a 2' modification of its sugar moiety. In a further embodiment, the antisense oligonucleotide comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides having a 2' modification of its sugar moiety. In a further embodiment, all nucleosides of the antisense oligonucleotide have a 2' modification of their sugar moiety. Preferably, the 2' modification is 2'-fluoro, 2'-OME, 2'-methoxyethyl (2'-MOE), or locked nucleic acid (LNA). In some embodiments, the modified nucleoside motif is LNA or alpha-LNA in which a methylene (--CH2--)n group bridges the 2' oxygen atom and the 4' carbon atom, and n is 1 or 2. In further embodiments, the LNA or alpha-LNA contains a methyl group at the 5' position. In some embodiments, the oligonucleotide includes a 2' modification and at least one internucleoside linkage. In certain embodiments, the antisense oligonucleotide includes at least one phosphorothioate internucleoside linkage. In one embodiment, the internucleoside linkages of the oligonucleotide alternate between phosphodiester backbone linkages and phosphorothioate backbone linkages. In another embodiment, all internucleoside linkages of the oligonucleotide are phosphorothioate linkages.

[0127] In additional preferred embodiments, the T-oligo-HES conjugate contains an antisense oligonucleotide comprising at least one 3'-methylenephosphonate linkage, LNA, peptide nucleic acid (PNA) linkage, or phosphorodiamidate morpholino linkage. In further embodiments, the antisense oligonucleotide contains at least one modified nucleic acid base. Preferably, the modified nucleic acid base is C-5 propyne or 5-methyl C.

[0128] In further embodiments, the T-oligo-HES conjugate contains an antisense oligonucleotide comprising one or more antisense strands complementary to different sequences of the target mRNA or target gene. In some embodiments, the antisense strands are linked in a linear or branched manner (e.g., a dendrimer). In further embodiments, the linked antisense strands induce a new secondary structure of the target mRNA and / or gene, thereby reducing or inhibiting proper transcription / translation of the target nucleotide.

[0129] The antisense oligonucleotide compounds contained in the T-oligo-HES conjugates provided herein can be synthesized systematically using techniques known in the art.

[0130] RNAi - Post-transcription gene silencing Short double-stranded RNA molecules and short hairpin RNA (shRNA), i.e., foldback stem-loop structures that give rise to siRNA, can induce RNA interference (RNAi). In some embodiments, the T-oligo-HES conjugate contains RNAi-inducing oligonucleotides. RNAi oligonucleotides contained in the T-oligo-HES conjugate include, but are not limited to, siRNA, shRNA, and dsRNA disorbers and / or dicer substrates. One or both strands of the siRNA, shRNA, and dsRNA preferably contain one or more modified nucleoside linkages, modified sugar moieties, and / or modified nucleic acid bases, as described herein or otherwise known in the art. These RNAi oligonucleotides have applications including, but are not limited to, interfering with the expression of a target gene(s) or polynucleotide(s) in a target. Therefore, in some embodiments, the oligonucleotides in the T-oligo-HES conjugate are used to specifically inhibit the expression of a target nucleic acid. In some embodiments, double-stranded RNA-mediated repression of gene and / or nucleic acid expression is achieved by administering a T-oligo-HES conjugate containing a dsRNA drosa substrate, a dsRNA dicer substrate, siRNA, or shRNA to the target and / or cells. Double-stranded RNA-mediated repression of gene and nucleic acid expression can be achieved by administering a T-oligo-HES conjugate containing dsRNA, siRNA, or shRNA to the target. siRNA may be a double-stranded RNA, or a hybrid molecule containing both RNA and DNA, for example, one RNA strand and one DNA strand.

[0131] In some embodiments, the T-oligo-HES conjugate comprises siRNA selected from RNA:RNA hybrids, DNA sense:RNA antisense hybrids, RNA sense:DNA antisense hybrids, and DNA:DNA hybrid double helixes, which are approximately 21–30 nucleotides long, and can associate with a cytoplasmic multiprotein complex known as RNAi-induced silencing complex (RISC). siRNA-supported RISCs mediate the degradation of homologous mRNA transcripts. The methods provided involve the use of RNAi molecules containing any of these different types of double-stranded molecules. In addition, it is understood that RNAi molecules can be introduced into cells in various forms. Thus, as used herein, RNAi molecules encompass any molecule that can induce an RNAi response in cells, including, but not limited to, double-stranded polynucleotides containing two separate strands, i.e., a sense strand and an antisense strand, such as small interfering RNA (siRNA); polynucleotides containing hairpin loops of complementary sequences that form a double-stranded region, such as shRNAi molecules; and expression vectors that express one or more polynucleotides that can form double-stranded polynucleotides alone or in combination with other polynucleotides.

[0132] In some embodiments, the T-oligo-HES conjugate contains an oligonucleotide that is double-stranded and has a length of 16–30 or 18–25 nucleotides. In additional embodiments, the dsRNA oligonucleotide is double-stranded and has a length of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 nucleotides. In certain embodiments, the dsRNA has a length of 21 nucleotides. In certain embodiments, the dsRNA has a 3' overhang of 0–7 nucleotides or a 5' overhang of 0–4 nucleotides. In certain embodiments, the dsRNA has a 3' overhang of 2 nucleotides. In further embodiments, the dsRNA contains two complementary RNA strands of 21 nucleotides in length with a 3' overhang of 2 nucleotides (i.e., containing a complementary region of 19 nucleotides between the sense strand and the antisense strand). In another embodiment, the dsRNA contains two complementary RNA strands, each 25 nucleotides long, with a 2-nucleotide 3' overhang (i.e., containing a 23-nucleotide complementary region between the sense strand and the antisense strand). In a particular embodiment, the overhang is a UU or dTdT3' overhang.

[0133] In some embodiments, the T-oligo-HES conjugate contains an siRNA oligonucleotide that is perfectly complementary to the corresponding reverse complementary strand of the target RNA. In other embodiments, the siRNA contains one or two substitutions, deletions, or insertions compared to the corresponding reverse complementary strand of the target RNA.

[0134] In additional embodiments, the T-oligo-HES conjugate contains an RNAi oligonucleotide, which is a short hairpin RNA. shRNA is a form of hairpin RNA containing a foldback stem-loop structure that gives rise to siRNA, and therefore similarly, can sequence-specifically reduce the expression of a target gene. Short hairpin RNAs are generally more stable than siRNAs and less susceptible to degradation in the cellular environment. The stem-loop structure of shRNAs varies in stem length, typically being 19–29 nucleotides. In certain embodiments, the T-oligo-HES conjugate contains shRNAs having stems of 19–21 or 27–29 nucleotides. In additional embodiments, the shRNAs have loop sizes between 4–30 nucleotides. While complete complementarity between the stem portion that specifically hybridizes to the target mRNA (antisense strand) and the mRNA is preferred, the shRNAs may, in some cases, contain mismatches between the two strands of the shRNA hairpin stem. For example, in some embodiments, the shRNA contains one or more GU pairs in the hairpin stem to stabilize the hairpin.

[0135] In one embodiment, the nucleic acid target of the RNAi oligonucleotide contained in the T-oligo-HES conjugate provided herein is selected by scanning the target RNA (e.g., mRNA or miRNA) for the appearance of AA dinucleotide sequences. Each AA dinucleotide sequence, combined with approximately 19 nucleotides adjacent to the 3', is a potential siRNA target site, and RNAi oligonucleotides can be systematically designed based thereon. In some embodiments, to avoid potential interference of the binding of the siRNP endonuclease complex by proteins that bind to the regulatory region of the target RNA, the RNAi oligonucleotide target site is not located within the 5' and 3' untranslated regions (UTR) or the region near the start codon (e.g., within approximately 75 bases of the start codon) of the target RNA.

[0136] RNAi oligonucleotides targeting specific polynucleotides can be readily prepared using reagents and procedures known in the art, or by standardized modifications. The structural characteristics of effective siRNA molecules have been identified. (Elshabir et al., Nature 411:494-498 (2001) and Elshabir et al., EMBO 20:6877-6888 (2001)). Therefore, those skilled in the art will understand that a wide variety of different siRNA molecules can be used to target specific genes or transcripts.

[0137] enzyme nucleic acid In some embodiments, the T-oligo-HES conjugate comprises an enzymatic oligonucleotide. Two preferred features of the enzymatic oligonucleotides in the provided conjugate are that they have a specific substrate-binding site complementary to one or more DNA or RNA regions of a target gene, and that they have a nucleotide sequence within or around the substrate-binding site that confers RNA cleavage activity to the oligonucleotide. In some embodiments, the enzymatic oligonucleotide is a ribozyme. A ribozyme is an RNA-protein complex having a specific catalytic domain that has endonuclease activity. Exemplary ribozyme oligonucleotides are formed from hammerhead, hairpin, hepatitis delta virus, group I intron or RNaseP RNA (associated with an RNA guide sequence) or neurospora VS RNA motifs.

[0138] The enzymatic oligonucleotides that may be included in the T-oligo-HES conjugates provided herein may contain modified nucleotides as described herein or otherwise known in the art, but it is important that such modifications do not lead to conformational changes that invalidate the catalytic activity of the enzymatic oligonucleotide. Methods for designing, manufacturing, testing and optimizing enzymatic oligonucleotides such as ribozymes are known in the art and are incorporated herein by reference (see, for example, WO91 / 03162; WO92 / 07065; WO93 / 15187; WO93 / 23569; WO94 / 02595, WO94 / 13688; EP 92110298; and U.S. Patent No. 5,334,711, each of which is incorporated herein by reference in whole).

[0139] Aptamers and decoys In some embodiments, the T-oligo-HES conjugate contains an aptamer and / or decoy. As used herein, an aptamer refers to a single-stranded nucleic acid molecule (such as DNA or RNA) that exhibits a specific sequence-dependent shape and specifically hybridizes to a target protein with high affinity and specificity. The aptamers of T-oligo-HES conjugates are generally less than 100 nucleotides, less than 75 nucleotides, or less than 50 nucleotides in length. As used herein, the term “aptamer” encompasses enantiomers (plural) (high-affinity L-enantiomer nucleic acids such as L-ribose or L-2'-deoxyribose units) that confer resistance to enzymatic degradation compared to D-oligonucleotides. In certain embodiments, the T-oligo-HES conjugate contains the aptamer Macugen (OSI Pharmaceuticals) or ARC1779 (Archemix, Cambridge, Mass.). In further embodiments, the T-oligo-HES conjugates provided herein contain oligonucleotides that compete with target proteins for binding to the aptamer Macugen (OSI Pharmaceuticals) or ARC1779 (Archemix, Cambridge, Mass.). In additional embodiments, the conjugates contain oligonucleotides that bind to Tat or Rev. In further embodiments, the conjugates contain oligonucleotides that bind to HIV-1 Tat, nucleocapsid, reverse transcriptase, integrase, or Rev. In additional embodiments, the conjugates contain oligonucleotides that bind to gp120, HCV NS3 protease, hepatitis C NS3m plague tyrosine phosphatase, the intracellular domain of receptor tyrosine kinase (e.g., EGFRvIII), or nucleolin (AML). Methods for producing and identifying aptamers are known in the art and can be systematically modified to identify aptamers having desired diagnostic and / or therapeutic properties, and these aptamers can be incorporated into the T-oligo-HES conjugates provided herein.For example, see Wlotzka et al., Proc. Natl. Acad. Sci. 99(13):8898-8902 (2002), which is incorporated herein by reference in its entirety.

[0140] As used herein, the term “decoy” refers to a short double-stranded nucleic acid (including single-stranded nucleic acids designed to “fold back” on their own) that mimics a site on a nucleic acid to which a factor, such as a protein, binds. Such decoys competitively inhibit and / or reduce the activity and / or function of the factor. Methods for constructing and identifying decoys are known in the Art and can be systematically modified to identify decoys with desired diagnostic and / or therapeutic properties, and these decoys can be incorporated into oligonucleotide-HES complexes of T-oligo-HES conjugates. See, for example, U.S. Patent No. 5,716,780, which is incorporated herein by reference in its entirety.

[0141] Small non-coding RNAs and antagonists (such as miRNAs and anti-miRNAs) As used herein, the term “small non-coding RNA” is used to encompass polynucleotide molecules ranging in length from 17 to 29 nucleotides, but is not limited to this definition. In one embodiment, the small non-coding RNA is miRNA (also known as miRNA, Mirs, miRs, mirs, and mature miRNA).

[0142] MicroRNAs (miRNAs), also known as "mature" miRNAs, are small (approximately 21-24 nucleotides in length) non-coding RNA molecules that have been identified as important regulators of development, cell proliferation, apoptosis, and differentiation. Examples of specific developmental processes in which miRNAs are involved include stem cell differentiation, neurogenesis, angiogenesis, hematopoiesis, and exocytosis (as outlined by Alvarez-Garcia and Miska, Development, 132:4653-4662 (2005)). miRNAs are known to be abnormally expressed in disease states; that is, certain miRNAs are present at high or low levels in diseased cells or tissues compared to healthy cells or tissues.

[0143] miRNAs are thought to originate from long endogenous primary miRNA transcripts (also known as pri-miRNA, pri-mir, pri-miR, or pri-pre-miRNA), which are often several hundred nucleotides long (Lee, et al., EMBO J., 21(17):4663-4670 (2002)). One mechanism by which miRNAs regulate gene expression is by binding to the 3' untranslated region (3'-UTR) of specific mRNAs. miRNA nucleotide (nt)RNA molecules incorporated into the RNA-induced silencing complex (RISC) mediate downregulation of gene expression through translation inhibition, transcript cleavage, or both. RISC is also involved in transcriptional silencing in the nuclei of a wide range of eukaryotes.

[0144] In some embodiments, this disclosure provides T-oligo-HES conjugates and methods for modulating low-molecular-weight non-coding RNA activity, including disease-state-related miRNA activity, among other things. Certain conjugates and compositions provided herein are particularly suitable for use in vivo due to improved delivery, potent activity, and / or improved therapeutic index.

[0145] This disclosure provides T-oligo-HES conjugates and methods for regulating small non-coding RNAs, including miRNAs. In certain embodiments, this disclosure provides T-oligo-HES conjugates and methods for regulating the level, expression, processing, or function of one or more small non-coding RNAs, such as miRNAs. Accordingly, in some embodiments, this disclosure encompasses compositions, such as pharmaceutical compositions, that include a T-oligo-HES conjugate having at least one oligonucleotide that specifically hybridizes with a small non-coding RNA, such as a miRNA.

[0146] In some embodiments, the T-oligo-HES conjugate includes oligonucleotides that specifically hybridize with or sterically interfere with one or more small non-coding RNAs, such as miRNAs, or nucleic acid molecules that encode them. In certain embodiments, the disclosure provides T-oligo-HES conjugates and methods useful for modulating the level, activity, or function of miRNAs, including those that are antisense mechanism-dependent and those that are not.

[0147] As used herein, the terms “target nucleic acid,” “target RNA,” “target RNA transcript,” or “nucleic acid target” are used to encompass any nucleic acid that can be targeted, including but not limited to RNA. In one embodiment, the target nucleic acid is a non-coding sequence including but not limited to miRNA and miRNA precursors. In a preferred embodiment, the target nucleic acid is miRNA, which may also be referred to as miRNA. If the oligonucleotide contains a substantially complementary sequence that is 100% complementary to the miRNA, then the oligonucleotide is “targeting miRNA.”

[0148] As used herein, an oligonucleotide is "substantially complementary" to RNA, such as small non-coding RNA, if it can specifically hybridize to small non-coding RNA under physiological conditions. In some embodiments, an oligonucleotide is "miRNA-targeting" if it contains a substantially complementary sequence that includes 100% of at least eight consecutive nucleotides of the miRNA. In some embodiments, the T-oligo-HES conjugate contains an oligonucleotide that specifically hybridizes to miRNA and has a length in the range of about 8 to about 21 nucleotides, about 8 to about 18 nucleotides, or about 8 to about 14 nucleotides. In additional embodiments, the oligonucleotide specifically hybridizes to miRNA and has a length in the range of about 12 to about 21 nucleotides, about 12 to about 18 nucleotides, or about 12 to about 14 nucleotides. In certain embodiments, the oligonucleotide is a monomer subunit (nucleotide) having a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21. In certain embodiments, the oligonucleotide is a monomer subunit (nucleotide) having a length of 14, 15, 16, 17, or 18.

[0149] In certain embodiments, the T-oligo-HES conjugate contains an oligonucleotide that has full-length complementarity to the miRNA. In other embodiments, the oligonucleotide can still hybridize with the target miRNA and the function of the oligonucleotide is not substantially impaired, despite the complementarity length between the oligonucleotide and the target nucleic acid, and up to three "mismatches" between the oligonucleotide and the target miRNA. In other embodiments, the oligonucleotide includes cleavage or elongation of up to six nucleosides relative to the length of the target miRNA at either the 3' or 5' end of the oligonucleotide, or both the 3' and 5' ends. In certain embodiments, the oligonucleotide is cleaved by one or two nucleosides relative to the length of the target miRNA. As a non-limiting example, if the target miRNA is 22 nucleotides long, an oligonucleotide with substantially full-length complementarity may be 20 or 21 nucleotides long. In certain embodiments, the oligonucleotide is cleaved by one nucleotide at either the 3' or 5' end relative to the miRNA.

[0150] In some embodiments, the Disclosure provides a method for modulating small non-coding RNA, comprising contacting cells with a T-oligo-HES conjugate containing a sequence substantially complementary to the small non-coding RNA, a small non-coding RNA precursor (e.g., a miRNA precursor), or a nucleic acid encoding the small non-coding RNA. As used herein, the terms “small non-coding RNA precursor” and “miRNA precursor” are used to encompass any longer nucleic acid sequence from which the small (mature) non-coding RNA originates, and may include, but are not limited to, primary RNA transcripts, pri-small non-coding RNA, and pre-small non-coding RNA. For example, “miRNA precursor” encompasses any longer nucleic acid sequence from which the miRNA originates, and may include, but is not limited to, primary RNA transcripts, pri-miRNA, and pre-miRNA.

[0151] In some embodiments, the Disclosure provides compositions such as pharmaceutical compositions containing a T-oligo-HES conjugate that targets nucleic acids containing or encoding small non-coding RNA and acts to modulate the level or function of the small non-coding RNA. In further embodiments, the Disclosure provides compositions such as pharmaceutical compositions containing a T-oligo-HES conjugate that targets miRNA and acts to modulate the level of miRNA or interfere with its processing or function.

[0152] In some embodiments, the T-oligo-HES conjugate contains an oligonucleotide that specifically hybridizes to nucleotides 1-10 (i.e., the seed region) of the miRNA. In additional embodiments, the oligonucleotide specifically hybridizes to a sequence in the miRNA precursor (pre-miRNA) or primary miRNA (pri-miRNA) that, upon binding of the oligonucleotide, blocks miRNA processing.

[0153] In additional embodiments, the T-oligo-HES conjugate contains an oligonucleotide that targets nucleic acids containing or encoding small non-coding RNA and acts to reduce the level of the small non-coding RNA and / or interfere with its function within the cell.

[0154] In other embodiments, the composition contains a T-oligo-HES conjugate, which contains or codes for a small non-coding RNA, or an oligonucleotide that increases the endogenous expression, processing, or function of the small non-coding RNA (for example, by binding to a regulatory sequence of a gene encoding the non-coding RNA), thereby increasing the level of the small non-coding RNA and / or increasing its function within the cell.

[0155] Oligonucleotides contained in the T-oligo-HES conjugates provided herein can modulate the level, expression, or function of small non-coding RNAs by hybridizing to nucleic acids containing or encoding small non-coding RNA targets, thereby altering their normal function. For example, non-limited mechanisms by which oligonucleotides can reduce the activity (including level, expression, or function) of small non-coding RNAs include promoting the disruption of small non-coding RNAs by cleavage, sequestration, steric occlusion, and hybridization to small non-coding RNAs, preventing them from hybridizing to normal cellular targets and controlling their activity.

[0156] In another embodiment, the present disclosure provides a method for inhibiting the activity of a small non-coding RNA, comprising contacting a cell expressing a cell surface antigen with a T-oligo-HES conjugate comprising a target-directed moiety that specifically binds to the surface antigen, and an oligonucleotide that targets a nucleic acid containing or encoding the small non-coding RNA, thereby reducing the level of the small non-coding RNA in the cell and / or interfering with its function. In some embodiments, the oligonucleotide comprises a sequence substantially complementary to the nucleic acid containing or encoding the non-coding RNA. In certain embodiments, the small non-coding RNA is a miRNA.

[0157] In additional embodiments, the Disclosure provides a method for inhibiting the activity of small non-coding RNA, comprising administering to a subject a T-oligo-HES conjugate containing an oligonucleotide that targets a nucleic acid containing or encoding small non-coding RNA and acts to reduce the level of small non-coding RNA within the subject and / or interfere with its function. In some embodiments, the target nucleic acid is located in a cell expressing a cell surface antigen to which the target-directing moiety of the T-oligo-HES conjugate specifically binds. In some embodiments, the target nucleic acid is located in a cell close to the cell expressing the cell surface antigen to which the target-directing moiety of the T-oligo-HES conjugate specifically binds.

[0158] In some embodiments, the T-oligo-HES conjugate contains a non-coding RNA or an oligonucleotide having a sequence substantially complementary to a coding nucleic acid. In certain embodiments, the small non-coding RNA is a miRNA.

[0159] In additional embodiments, the Disclosure provides a method for increasing the activity of a small non-coding RNA, comprising contacting a cell with a T-oligo-HES conjugate containing an oligonucleotide that acts to increase the level of the small non-coding RNA and / or increase its function in the cell (e.g., by binding to a regulatory sequence of a gene encoding the non-coding RNA), either by containing or encoding the small non-coding RNA. In some embodiments, the oligonucleotide contains a sequence substantially identical to that of a nucleic acid containing or encoding the non-coding RNA. In some embodiments, the oligonucleotide shares 100% identity with at least 15 consecutive nucleotides and at least 20 consecutive nucleotides, or over the entire length, of the small non-coding RNA sequence. In certain embodiments, the small non-coding RNA is a miRNA.

[0160] In another embodiment, the Disclosure provides a method for increasing the activity of a small non-coding RNA, comprising administering a T-oligo-HES conjugate containing an oligonucleotide that contains or codes for the small non-coding RNA, or acts to increase the endogenous expression, processing, or function of the small non-coding RNA, to increase the level of the small non-coding RNA in the subject and / or to increase its function, to a subject requiring such action. In some embodiments, cells with increased activity of the small non-coding RNA express a cell surface antigen specifically bound by the target-directed moiety of the T-oligo-HES conjugate. In some embodiments, cells with increased activity of the small non-coding RNA are located near cells expressing the cell surface antigen to which the target-directed moiety of the T-oligo-HES conjugate specifically binds. In some embodiments, the oligonucleotide contains a sequence substantially identical to the nucleic acid containing or coding the non-coding RNA. In some embodiments, the oligonucleotide shares 100% identity with at least 15 consecutive nucleotides and at least 20 consecutive nucleotides, or over its entire length, of the small non-coding RNA sequence. In certain embodiments, the small non-coding RNA is a miRNA.

[0161] In additional embodiments, the T-oligo-HES conjugate contains an oligonucleotide sequence substantially identical to a nucleic acid containing or encoding a small non-coding RNA. In some embodiments, the oligonucleotide is a miRNA mimite. In some embodiments, the miRNA mimite is double-stranded. In further embodiments, the oligonucleotide is double-stranded and contains an oligonucleotide 18–23 units in length, which is blunt-ended or contains a miRNA mimite with one or more 3' overhangs of 1, 2, or 3 nucleotides. In additional embodiments, the oligonucleotide contains a single-stranded miRNA mimite 18–23 units in length. T-oligo-HES conjugates containing expression vectors that express these miRNA mimite are also included in this disclosure. In some embodiments, the oligonucleotide shares 100% identity with at least 15 consecutive nucleotides and at least 20 consecutive nucleotides, or over its entire length, of a small non-coding RNA sequence. In certain embodiments, the small non-coding RNA is a miRNA.

[0162] In some embodiments, the Disclosure provides a method for treating a disease or disorder characterized by overexpression of a small non-coding RNA in a subject, comprising administering a therapeutically effective amount of a T-oligo-HES conjugate to the subject that targets a nucleic acid containing or encoding a small non-coding RNA and acts to reduce the level of the small non-coding RNA in the subject and / or interfere with its function. In some embodiments, the cell surface antigen, which is specifically bound by the target-directing moiety of the conjugate, is expressed on the cell surface of the cell to which the nucleic acid activity is targeted, or on the surface of a nearby cell. In some embodiments, the oligonucleotide is an anti-miRNA (anti-miR). In additional embodiments, the anti-miRNA is double-stranded. In further embodiments, the oligonucleotide is double-stranded and contains an oligonucleotide 18–23 units in length, which is blunt-ended or contains anti-miRNA with one or more 3' overhangs of 1, 2, or 3 nucleotides. In additional embodiments, the oligonucleotide contains a single-stranded anti-miR 8–25 units in length. T-oligo-HES conjugates containing expression vectors expressing these anti-miRs are also included in this disclosure. In some embodiments, the oligonucleotide comprises a sequence substantially complementary to the overexpressed small non-coding RNA.

[0163] In further embodiments, the Disclosure provides a method for treating a disease or disorder characterized by the overexpression of miRNA in a subject, comprising administering to the subject a therapeutically effective amount of a T-oligo-HES conjugate containing an oligonucleotide that targets a nucleic acid containing or encoding miRNA and acts to reduce the level of miRNA in the subject and / or interfere with its function. In some embodiments, the cell surface antigen specifically bound by the target-directed moiety of the conjugate is expressed on the surface of the cell to which the nucleic acid activity is targeted. In some embodiments, the cell surface antigen specifically bound by the target-directed moiety of the conjugate is expressed on the surface of a cell near the cell to which the nucleic acid activity is targeted. In some embodiments, the oligonucleotide contains a sequence substantially complementary to the overexpressed miRNA.

[0164] A family of miRNAs can be characterized by the identity of the nucleotides at positions 2–8 of the miRNA, which is a region known as the seed sequence. Members of the miRNA family are referred to herein as “associated miRNAs.” Each member of the miRNA family shares the same seed sequence, which plays a crucial role in miRNA targeting and function. As used herein, the terms “seed sequence” or “seed region” refer to the nucleotides at positions 2–9 from the 5' end of a mature miRNA sequence. Examples of miRNA families, known in the art but not limited to, include the let-7 family (having 9 miRNAs), the miR-15 family (including miR-15a, miR-15b, miR15-16, miR-16-1, and miR-195), and the miR-181 family (including miR-181a, miR-181b, and miR-181c). In some embodiments, oligonucleotides in the oligo-HES complex specifically hybridize to the seed region of the miRNA, interfering with the processing or function of the miRNA. In some embodiments, oligonucleotides specifically hybridize to the seed region of miRNAs, interfering with the processing or function of multiple miRNAs. In further embodiments, at least two of the multiple miRNAs have a related seed sequence or are members of a miRNA superfamily.

[0165] The association between miRNA dysfunction associated with diseases, such as cancer, fibrosis, metabolic disorders, and inflammatory disorders, and the ability of miRNAs to affect the complete network of genes involved in general cellular processes, has made the selective modulation of miRNAs using anti-miRNAs and miRNA mimes a particularly attractive disease-modulating therapy. In some embodiments, the disclosure also provides a method for treating a disease or disorder characterized by protein overexpression in a subject, comprising administering a T-oligo-HES conjugate containing an oligonucleotide that targets a nucleic acid containing or encoding a small non-coding RNA that affects increased protein production, wherein the oligonucleotide acts to reduce the level of the small non-coding RNA in the subject and / or interfere with its function. In some embodiments, the cell surface antigen specifically bound by the target-directed moiety of the conjugate is expressed on the surface of the cell to which protein production is targeted. In some embodiments, the cell surface antigen specifically bound by the target-directed moiety of the conjugate is expressed on the surface of a cell near the cell to which protein production is targeted. In some embodiments, the oligonucleotide contains a sequence substantially complementary to the small non-coding RNA.

[0166] In some embodiments, the disclosure also provides a method for treating a disease or disorder characterized by protein overexpression in a subject, comprising administering a T-oligo-HES conjugate containing an oligonucleotide that targets a nucleic acid containing or encoding a miRNA that affects increased protein production, to a subject in need thereof, wherein the oligonucleotide acts to reduce the level of the miRNA in the subject and / or interfere with its function. In some embodiments, the cell surface antigen, specifically bound by the target-directed moiety of the conjugate, is expressed on the surface of the cell to which protein production is targeted. In some embodiments, the cell surface antigen, specifically bound by the target-directed moiety of the conjugate, is expressed on the surface of a cell near the cell to which protein production is targeted. In some embodiments, the oligonucleotide contains a sequence that is substantially complementary (specifically hybridizable) to the miRNA.

[0167] In some embodiments, the Disclosure provides a method for treating a disease or disorder characterized by underexpression of a small non-coding RNA in a subject, comprising administering to the subject a T-oligo-HES conjugate containing an oligonucleotide that contains or codes for the small non-coding RNA, or acts to increase the endogenous expression, processing, or function of the small non-coding RNA, thereby increasing the level of the small non-coding RNA in the subject and / or increasing its function. In some embodiments, the cell surface antigen, specifically bound by the target-directed moiety of the conjugate, is expressed on the surface of the cell to which the small non-coding RNA is targeted. In some embodiments, the cell surface antigen, specifically bound by the target-directed moiety of the conjugate, is expressed on the surface of a cell near the cell to which the small non-coding RNA is targeted. In some embodiments, the oligonucleotide contains a sequence that is substantially complementary (specifically hybridizable) to the overexpressed small non-coding RNA.

[0168] In further embodiments, the Disclosure provides a method for treating a disease or disorder characterized by the overexpression of miRNA in a subject requiring such treatment, comprising administering to the subject a T-oligo-HES conjugate containing an oligonucleotide that contains or encodes a small non-coding RNA, or acts to increase the endogenous expression, processing, or function of the small non-coding RNA, thereby increasing the level of the small non-coding RNA in the subject and / or increasing its function. In some embodiments, the cell surface antigen specifically bound by the target-directed moiety of the conjugate is expressed on the surface of the cell to which the small non-coding RNA is targeted. In some embodiments, the cell surface antigen specifically bound by the target-directed moiety of the conjugate is expressed on the surface of a cell near the cell to which the small non-coding RNA is targeted. In some embodiments, the oligonucleotide contains a sequence substantially complementary to the overexpressed miRNA.

[0169] In some embodiments, the Disclosure provides a method for treating a disease or disorder characterized by the overexpression of a protein in a subject, comprising administering to the subject a T-oligo-HES conjugate containing an oligonucleotide that contains or encodes a small non-coding RNA, or acts to increase the endogenous expression, processing, or function of the small non-coding RNA, thereby increasing the level of the small non-coding RNA in the subject and / or increasing its function. In some embodiments, the cell surface antigen specifically bound by the target-directed moiety of the conjugate is expressed on the surface of the cell to which the protein expression is targeted. In some embodiments, the cell surface antigen specifically bound by the target-directed moiety of the conjugate is expressed on the surface of a cell near the cell to which the protein expression is targeted. In some embodiments, the oligonucleotide contains a sequence substantially complementary to the small non-coding RNA.

[0170] In some embodiments, the Disclosure provides a method for treating a disease or disorder characterized by the overexpression of a protein in a subject, comprising administering to the subject a T-oligo-HES conjugate containing an oligonucleotide that contains or encodes a small non-coding RNA, or acts to increase the endogenous expression, processing, or function of a small non-coding RNA, to increase the level of the small non-coding RNA, and / or to increase its function in the subject. In some embodiments, the cell surface antigen, specifically bound by the target-directed moiety of the conjugate, is expressed on the surface of the cell to which the protein expression is targeted. In some embodiments, the cell surface antigen, specifically bound by the target-directed moiety of the conjugate, is expressed on the surface of a cell near the cell to which the protein expression is targeted. In some embodiments, the oligonucleotide contains a sequence that is substantially complementary (specifically hybridizable) to the miRNA.

[0171] In another embodiment, the present disclosure provides a method for inhibiting miRNA activity, comprising administering a T-oligo-HES conjugate having anti-miRNA activity, such as those described herein, to a subject requiring inhibition of miRNA activity. In some embodiments, the cell surface antigen specifically bound by the target-directing moiety of the conjugate is expressed on the surface of the cell to which the miRNA activity is targeted. In some embodiments, the cell surface antigen specifically bound by the target-directing moiety of the conjugate is expressed on the surface of a cell near the cell to which the miRNA activity is targeted.

[0172] In some embodiments, the T-oligo-HES conjugate contains oligonucleotides selected from siRNA, miRNA, dicer substrates (e.g., dsRNA), ribozymes, decoys, aptamers, antisense oligonucleotides, and plasmids capable of expressing siRNA, miRNA, or antisense oligonucleotides.

[0173] In some embodiments, the T-oligo-HES conjugate contains a chimeric oligonucleotide comprising an internal region containing at least 1, at least 2, at least 3, at least 4, at least 5, or all 2'-F modified oligonucleotides, and an external region containing at least one stability-enhancing modification. In one embodiment, the oligonucleotide comprises an internal region having a first 2'-modified nucleotide and an external region each containing a second 2'-modified nucleotide. In further embodiments, the oligonucleotide contains a gap region containing one or more 2'-fluoro modifications, and the wing region contains one or more 2'-methoxyethyl modifications. In one embodiment, the oligonucleotide in the T-oligo-HES conjugate is ISIS 393206 or ISIS 327985.

[0174] Targeting part The term “target-directed moiety” is used herein to refer to a molecule that provides the ability to specifically bind to a selected target, such as a cell surface antigen, cell, cell type, tissue, organ, region of the body, or compartment. Target-directed moieties can include a wide variety of entities, including naturally occurring molecules or recombinant or synthetic molecules. In some embodiments, the target-directed moiety is an aptamer, avimer, receptor-binding ligand, nucleic acid, biotin-avidin binding pair, peptide, protein, carbohydrate, lipid, vitamin, microbial component, hormone, receptor ligand (including Fc fusion proteins containing receptor ligands), antibody, antigen-binding moiety of an antibody, alternative binding scaffold, or a derivative of any of the foregoing.

[0175] In some embodiments, the target-directing moiety is an antibody, an antigen-binding moiety of an antibody (e.g., Fv, Fab, Fab', F(ab')2, dsFv, Fd, scFv, and diabody), or a single-domain antibody. In some embodiments, the target-directing moiety is a monoclonal antibody, a human antibody (e.g., a full-length human antibody), a humanized antibody, a bispecific antibody, a multispecific antibody, a synthetic antibody, or a PEGylated antibody. In some embodiments, the target-directing moiety is a full-length IgG1, IgG2, or IgG4 antibody.

[0176] In additional embodiments, the target-directed moiety is an alternative binding scaffold molecule. In some embodiments, the target-directed moiety is an alternative binding scaffold selected from affibody, nanobody (VHH), VNAR, antikalin, finomer, DARPin, tetranectin, transbody, adonectin, affin, microbody, peptide aptamer, alterase, plastic antibody, filomer, stradobody, maxibody, epibody, Z-domain, D-domain, armadillo repeat protein, Knitz domain, avimer, atrimer, probody, immunobody, triomab, troidbody, pepbody, waxibody, unibody, affimer, and duobody.

[0177] In various embodiments, the target-directing portion of the T-oligo-HES conjugate is an antigen-binding portion (fragment) of an antibody (e.g., Fab, scFv, and single-domain antibodies) that can specifically bind to a cell surface antigen. Antibody fragments such as Fab and scFv have advantages over full-length antibodies because they are less bulky and may lack the Fc domain, which can interfere with in vivo delivery.

[0178] In some embodiments, the target-directed portion of the T-oligo-HES conjugate is an activatable antibody. The activatable antibody is a protease-activated antibody designed to improve target-directed selectivity to a disease site (i.e., to the target antibody) by masking the antibody's binding site with an inhibitory domain that is cleaved and removed by a protease highly expressed at the disease site. In some embodiments, the target-directed portion of the T-oligo-HES conjugate is an activatable antibody, and the antibody is conjugated to at least one oligo-HES complex by a cleavable ligand. In some embodiments, the mask of the binding site of the activatable antibody is cleaved by the same protease as the at least one cleavable linker that conjugates the antibody to the oligo-HES complex in the conjugate. In some embodiments, the mask of the antigen-binding site of the activatable antibody is cleaved by a different protease than the at least one cleavable linker that conjugates the antibody to the oligo-HES complex in the conjugate. For example, in some embodiments, the mask of the antigen-binding site of an activatable antibody (e.g., an IgG antibody) is cleaved by a protease highly expressed at the disease site (e.g., a tumor), and at least one cleavable linker in the conjugate conjugating the antibody to an oligo-HES complex is cleaved by an enzyme of the immune complement system (e.g., u-plasminogen activator, tissue plasminogen activator, trypsin, or plasmin). The activatable antibody is described in International Patent Publication No. WO2016 / 179285, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0179] In some embodiments, the target-directed portion of the T-oligo-HES conjugate is a therapeutic antibody. In some embodiments, the term “therapeutic antibody” refers to an antibody that binds to a therapeutic target molecule and is expected to result in disease relief or a reduction in disease progression in vivo. In some embodiments, the therapeutic antibody specifically binds to a cell surface antigen. In further embodiments, the term “therapeutic antibody” means an antibody that has been or is being tested in clinical research for approval as a human therapeutic agent and can be administered to an individual for the treatment of a disease. In further embodiments, the “therapeutic antibody” is approved for administration as a human therapeutic agent by at least one regulatory body. In additional embodiments, the therapeutic antibody is an approved antibody.In further embodiments, the target-directed portion includes trastuzumab (HER2 / neu), pertuzumab (HER2 / neu), panitumumab (EGFR), nimotuzumab (EGFR), zaltumumab (EGFR), cetuximab (EGFR), (HER3), onarutuzumab (c-MET), patritumab, cribatuzumab (MUC1), sofituzumab (MUC16), edrecolomab (EPCAM), adekatumumab (EPCAM), and anetumab (MS). LN), huDS6 (CA6), rifastuzumab (NAPI2B), sacituzumab (TROP2), PR1A3, humanized PR1A3 (CEA), humanized Ab2-3 (CEA), IMAB362 / claudiximab (Claudin 18.2), AMG595 (EGFRvIII), ABT806 (EGFRvIII), cibrotuzumab (FAP), DS-8895a variant 1 (EphA2), DS-8895a variant 2 (EphA2), anti-Ep hA2 (EphA2), MEDI-547 (EphA2), nalnatumab (RON), RG7841 (LY6E), farletuzumab (FRA / folate receptor alpha), milbetuximab (FRA), J591 variant 1 (PSMA), J591 variant 2 (PSMA), lovalpituzumab (DLL3), PF-06647020 (PTK7), anti-PTK7 (PTK7), radilatuzumab (LIV1), sirumutuzumab (ROR1), rituximab Therapeutic antibodies selected from (CD20), ibritumomab tiuxetan (CD52), alemtuzumab (CD33), gemtuzumab ozogamicin (CD33), CT-011 (PD1), tositumomab (CD20), ipilimumab (CTLA4), tremelimumab (CP-675,206) (CTLA4), nivolumab (PD1), pembrolizumab (PD1), durvalumab (PDL1) anti-MAGE-A3, or anti-NY-ESO-1.

[0180] In certain embodiments, the target-directed portion of the T-oligo-HES conjugate is the therapeutic antibody trastuzumab.

[0181] In certain embodiments, the target-directed portion of the T-oligo-HES conjugate is the therapeutic antibody cetuximab.

[0182] In certain embodiments, the target-directed moiety of the T-oligo-HES conjugate is the therapeutic antibody rituximab.

[0183] In certain embodiments, the target-directed moiety of the T-oligo-HES conjugate is the therapeutic antibody bevacizumab (VEGF).

[0184] In certain embodiments, the target-directed moiety of the T-oligo-HES conjugate is the therapeutic antibody girentuximab (CAIX).

[0185] In certain embodiments, the target-directed moiety of the T-oligo-HES conjugate is the therapeutic antibody nivolumab.

[0186] In certain embodiments, the target-directed moiety of the T-oligo-HES conjugate is the therapeutic antibody pembrolizumab.

[0187] In some embodiments, the target of interest specifically bound by the target-directed moiety is a cell surface antigen on or near the target cell or tissue of interest. In certain embodiments, the target cell or tissue of interest is a diseased cell, cancer cell, immune cell, infected cell, or infectious agent. In some embodiments, the target of interest specifically bound by the target-directed moiety is a disease-associated antigen. In some embodiments, the target-directed moiety specifically binds to a cell surface antigen characteristic of cancer and / or a particular cell type (e.g., hyperproliferative cells). In some embodiments, the target-directed moiety specifically binds to a cell surface antigen associated with a disorder of the immune system.

[0188] In some embodiments, the target of interest specifically bound by the target-directed moiety is a surface antigen expressed on an infected cell. In some embodiments, the target-directed moiety specifically binds to infectious agents such as pathogens (e.g., bacterial cells such as tuberculosis, smallpox, and anthrax; viruses such as HIV; parasites such as malaria and leishmania; fungal infections; molds; and mycoplasmas). In further embodiments, the target of interest bound by the target-directed moiety is a bacterial antigen, viral antigen, fungal antigen, mycoplasma antigen, prion antigen, or parasitic antigen (e.g., those that infect mammals). In one embodiment, the target of the target-directed moiety is anthrax, hepatitis b, rabies, Nipah virus, West Nile virus, meningitis virus, or CMV. In additional embodiments, the target-directed moiety specifically binds to a pathogen.

[0189] In some embodiments, the target-directing moiety specifically binds to cell surface antigens that do not internalize a conjugate upon binding. In other embodiments, the target-directing moiety specifically binds to cell surface antigens that internalize a conjugate upon binding. In some embodiments, the target-directing moiety specifically binds to cell surface antigens that originate from or are determined to be expressed in a specific target cancer (e.g., a tumor), such as neoantigens.

[0190] In some embodiments, the target-directed moiety specifically binds to tumor cell surface antigens. The term “tumor cell surface antigen” (TSA) refers to antigens common to certain hyperproliferative disorders, such as cancer. In some embodiments, the target-directed moiety specifically binds to tumor cell surface antigens that are tumor-associated antigens (TAAs). TAAs are antigens found on both tumor cells and some normal cells. TAAs may be expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be present on normal cells at very low levels but expressed on tumor cells at much higher levels. Due to the dynamic nature of tumors, in some cases tumor cells may express antigens unique to them at certain stages, and at other stages they may also express antigens that are expressed on non-tumor cells. Therefore, including a particular marker as a TAA does not rule out the possibility that it may be considered a tumor-specific antigen. In some embodiments, the target-directed moiety specifically binds to tumor cell surface antigens that are tumor-specific antigens (TSAs). TSAs are antigens unique to tumor cells and do not occur on other cells in the body. In some embodiments, the target-directed portion specifically binds to tumor cell surface antigens expressed on the surface of cancer cells, including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer (such as NSCLC or SCLC), liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, multiple myeloma, glioblastoma, neuroblastoma, uterine cancer, cervical cancer, kidney cancer, thyroid cancer, bladder cancer, renal cancer, mesothelioma, and adenocarcinoma, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, and other cancers known in the art. In some embodiments, the target-directed portion specifically binds to cell surface antigens expressed on the surface of cells within the tumor microenvironment (for example, antigens such as VEGFR and TIE1 or TIE2 expressed on endothelial cells and macrophages, respectively, or antigens expressed on tumor stromal cells, such as cancer-associated fibroblasts (CAFs), tumor-infiltrating T cells and other leukocytes, as well as myeloid cells including mast cells, eosinophils, and tumor-associated macrophages (TAMs).

[0191] In some embodiments, the target-directing portion specifically binds to leukemia cells, lymphoma cells, pancreatic cancer cells, breast cancer cells, melanoma cells, lung cancer cells, head and neck cancer cells, ovarian cancer cells, bladder cancer cells, colorectal cancer cells, kidney cancer cells, liver cancer cells, prostate cancer cells, bone cancer cells, glioblastoma cells, and brain tumor cells; or to tumor cell surface antigens on lymphoma, myeloma, blastoma, sarcoma, leukemia, or carcinoma cells.

[0192] In some embodiments, the target-directed portion includes CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CD204, CD206, CD301, CAMPATH-1, HLD-DR, carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUC1, MUC15, folate-binding protein, A33, G250, prostate-specific membrane antigen (PSMA), ferritin, GD2, GD3, GM2, Ley, CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2 receptor, tenascin, metalloproteinase, endothelial, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6, HPV E7, EGFRvIII, HER2 / neu, and MAGE. A3, P53 non-mutant, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, P53 mutant, PR1, bcr-abl, tyrosinase, survivor, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin (MSLN), PSCA, MAGE A1, MAGE-A3, sLe (animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Regmine, Tie 3, Page 4, specifically binds to cell surface antigens selected from VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, CMET, HER3, CA6, NAPI2B, TROP2, CLDN18.2, fibroblast-activating protein (FAP), RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, Fos-related antigen 1, VEGFR, endoglin, PDL, VTCN1, and VISTA.

[0193] In additional embodiments, the target-directing moiety specifically binds to cell surface antigens selected from HER2, EGFR, CMET, HER3, MUC1, MUC16, EPCAM, MSLN, CA6, NAPI2B, TROP2, CEA, CLDN18.2, EGFRvIII, FAP, EphA2, RON, LY6E, FRA, PSMA, DLL3, PTK7, LIV1, ROR1, MAGE-A3, NY-ESO-1, endoglin, CD204, CD206, CD301, VTCN1, VISTA, GLP-3, CLDN6, CLDN16, UPK1B, STRA6, TMPRSS3, TMPRSS4, TMEM238, C1orf186, and LRRC15.

[0194] In some embodiments, the target-directing moiety specifically binds to cell surface antigens that internalize the conjugate after binding.In some embodiments, the target-directed moiety includes GONMB, TACSTD2 (TROP2), CEACAMS, EPCAM, folate receptors (e.g., folate receptor-α, folate receptor-β, or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylate cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), RAAG12, 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P-cadherin, and fibronectin. Extradomain B (ED-B), VEGFR2 (CD309), Tenascin, Collagen IV, Periostin, Endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, C D2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD 37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 It specifically binds to cell surface antigens selected from EphA receptor, EphB receptor, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4, EphB6, integrins (e.g., integrins avb3, avb5, or avb6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRα, PDGFRβ, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.

[0195] In some embodiments, the target-directed moiety specifically binds to tumor microenvironment cell surface antigens (including certain membrane-bound proteases). In further embodiments, the target-directed moiety specifically binds to cell surface antigens expressed on endothelial cells or macrophages (e.g., VEGFR, TIE1, and TIE2), or to cell surface antigens expressed on tumor stromal cells, such as cancer-associated fibroblasts (CAFs), tumor-infiltrating T cells, and other leukocytes, as well as myeloid cells, including mast cells, eosinophils, and tumor-associated macrophages.

[0196] In some embodiments, the target-directing moiety specifically binds to cell surface antigens selected from PD1, PD-L1, PD-L2, CTLA4 LAG3, TIM-3, TIGIT, VISTA, B7-H3, BTLA, A2aR, and CD73.

[0197] In additional embodiments, the target-directed moiety specifically binds to cell surface antigens on immune cells. In some embodiments, the immune cells are diseased, activated, leukemic, or normal. In further embodiments, the target-directed moiety specifically binds to cell surface antigens expressed on one or more immune cells, which may include, but are not limited to, myeloid lymphocytes, T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, antitumor macrophages (e.g., M1 macrophages), B cells, dendritic cells, or subsets thereof. In some embodiments, the target-directed moiety specifically binds to cell surface antigens expressed on lymphoid or myeloid immune cells, such as T cells, B cells, NK cells, NKT cells, or dendritic cells. In some embodiments, the cell surface antigens are found on megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or subsets thereof.

[0198] In further embodiments, the target-directing moiety specifically binds to cell surface antigens on antigen-presenting cells. In further embodiments, the target-directing moiety specifically binds to antigens selected from OX40L, 4-1BBL, MARCO, DC-SIGN, Dectin 1, Dectin 2, DEC-205, CLEC5A, CLEC9A, CLEC10A, CLEC12A, CD1A, CD16A, CD32A, CD32B, CD36, CD40, CD47, CD64, CD204, CD206, HVEM, PDL1, mannose scavenger receptor 1, and BDCA2.

[0199] Linker In some embodiments, the target oligo-HES conjugate includes one or more linkers.

[0200] In some embodiments, the T-oligo-HES conjugate includes a linker that connects a target-directed moiety to the oligonucleotides of the oligo-HES complex. In some embodiments, the linker connects 1 to 30, 1 to 20, 1 to 10, or 1 to 5 oligo-HES complexes to the target-directed moiety of the T-oligo-HES complex. In some embodiments, the T-oligo-HES conjugate includes multiple linkers that connect oligonucleotides to the target-directed moiety. In some embodiments, the T-oligo-HES conjugate includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 to 10, 1 to 15, or 1 to 20 linkers that connect oligonucleotides to the target-directed moiety. In some embodiments, the T-oligo-HES conjugate includes 1 to 30, 1 to 20, 1 to 10, or 1 to 5 linkers that connect oligonucleotides to the target-directed moiety. In some embodiments, one, two, three, four, or more of the linkers are different. In some embodiments, one, two, three, four, or more, or all of the linkers are the same. In some embodiments, one, two, three, four, or more, or all of the linkers connecting the oligonucleotide to the target-directed portion in the T-oligo-HES conjugate are the same. In some embodiments, one, two, three, four, or more, of the linkers connecting the oligonucleotide to the target-directed portion in the T-oligo-HES conjugate are different.

[0201] In additional embodiments, the T-oligo-HES conjugate contains linkers between oligonucleotides in the oligo-HES complex contained in the T-oligo-HES conjugate. In some embodiments, the T-oligo-HES conjugate contains multiple linkers between oligonucleotides in the oligo-HES complex of the conjugate. In some embodiments, the T-oligo-HES conjugate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-10, 1-15, or 1-20 linkers between oligonucleotides in the oligo-HES complex of the conjugate. In some embodiments, 1, 2, 3, 4, or more, or all of the linkers between oligonucleotides in the oligo-HES complex of the conjugate are the same. In some embodiments, 1, 2, 3, 4, or more, of the linkers between oligonucleotides in the oligo-HES complex of the conjugate are different.

[0202] In additional embodiments, the T-oligo-HES conjugate includes linkers connecting a target-directed moiety to the oligonucleotides of the conjugate, and the conjugate also includes linkers between the oligonucleotides in the oligo-HES complex of the conjugate. In further embodiments, the T-oligo-HES conjugate includes multiple linkers connecting a target-directed moiety to the oligonucleotides of the conjugate, and the conjugate also includes multiple linkers between the oligonucleotides in the oligo-HES complex of the conjugate.

[0203] In some embodiments, a vector is provided that encodes a provided target-directed moiety of an oligo-HES-conjugate ligated to one of the linkers described herein as a single nucleotide sequence, and such a target-directed moiety-linker complex can be prepared using this vector.

[0204] In some embodiments, the linker length allows the target-directing moiety and the T-oligo-HES conjugate to efficiently bind to the target cell surface antigen (e.g., cancer surface antigen, immune cell surface antigen, or infectious agent surface antigen as described herein). Preferably, the linker length optimizes the spacing between the target-directing moiety and the oligo-HES complex in the T-oligo-HES conjugate, thereby optimizing the linker function in a different way.

[0205] T-oligo-HES conjugates can contain various linker sequences, and each T-oligo-HES conjugate can contain different linkers with different compositions. For example, the linker connecting the oligonucleotide to the target-directing portion of the conjugate may be a linear peptide with a length of 15-30 amino acid residues, while the linker between oligonucleotides in the oligo-HES complex of the conjugate may be a linear or branched alkyl such as C2-6, C10, or C18 linear alkyl.

[0206] In various embodiments, the linker can be derived from a naturally occurring multi-domain protein or, for example, an empirical linker as described in Chichili et al., Protein Sci. 22(2):153-167 (2013), Chen et al., Adv Drug Deliv Rev. 65(10):1357-1369 (2013), the entire contents of which are incorporated herein by reference. In some embodiments, the linker is designed using linker design databases and computer programs such as those described in Chen et al., Adv Drug Deliv Rev. 65(10):1357-1369 (2013), and Crasto et al., Protein Eng. 13(5):309-312 (2000), the entire contents of which are incorporated herein by reference. In various embodiments, the linker can be functional. For example, without limitation, the linker can function to improve folding and / or stability, improve expression, improve pharmacokinetics, and / or improve the biological activity of the T-oligo-HES conjugate.

[0207] The linker(s) can be linear or branched. In certain embodiments, a heterobifunctional crosslinker that precludes undesirable homopolymer formation is used.

[0208] In some embodiments, the T-oligo-HES conjugate includes a peptide linker (e.g., a peptide linker that connects the target-directing moiety to the oligonucleotide of the oligo-HES complex). In some embodiments, the linker is less than 50 amino acid residues long. For example, the peptide linker may be less than 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 amino acid residues in length. In some embodiments, the peptide linker is 2-50, 2-40, 2-30, or 2-20, 2-15, or 2-10 amino acid residues in length. In some embodiments, the peptide linker is up to 15 or up to 30 amino acid residues in length. In some embodiments, the peptide linker is 5-50, 10-40, or 15-30 amino acid residues in length. In further embodiments, the linker is a linear peptide (for example, a linear peptide with a length of up to 15, up to 30, 5-50, 10-40, or 15-30 amino acid residues).

[0209] In some embodiments, the T-oligo-HES conjugate includes a protease substrate-therapeutic oligo-conjugate linking arm (Cargo-Projection Arm) between a target-directed ligand (e.g., an antibody) and a protease substrate providing a rigid linker arm. In some embodiments, the linker arm comprises the sequence 5'-CUCUCCTTCTAGCCTCCGCTAGTCAAAAUU-3' (SEQ ID NO: 80). In further embodiments, the linker arm comprises the sequence 5'-[C6 aminolinker arm]-CUCUCCTTCTAGCCTCCGCTAGTCAAAAUU[C6 aminolinker arm]-3' (SEQ ID NO: 80). In further embodiments, the nucleotide of the linker arm comprises the sequence 5'-[C6 aminolinker arm]-CUCUCCTTCTAGCCTCCGCTAGTCAAAAUU[C6 aminolinker arm]-3' (SEQ ID NO: 80), which is a phosphorothioate backbone in each oligo binding. In a further embodiment, the linker arm nucleotide is of the sequence 5'-[C6 aminolinker arm]-CUCUCCTTCTAGCCTCCGCTAGTCAA x A x A x U x U x It contains [C6 aminolinker arm]-3' (SEQ ID NO: 80), which is the phosphorothioate backbone in each oligo bond. XThis indicates a 2'OMe modification on the 2'OH group of the sugar. In some embodiments, the oligolinker arm further includes a sense chain having the sequence 5'-ATTTTGACTAGCGGAGGCTAGAAGGAGA-3' (SEQ ID NO: 81). While we do not wish to be bound by theory, the presence of this sense chain to the linker arm, i.e., the cargo projection arm between the antibody and the protease substrate, increases the rigidity of the arm, thereby causing the protease substrate to project toward the target cell membrane, so that the protease expressed on the cell surface can recognize and cleave the substrate bound to the target ligand (such as an antibody). This releases a therapeutic oligonucleotide from the antibody having half of the protease substrate peptide.

[0210] In some embodiments, the T-oligo-HES conjugate includes a severable linker. In some embodiments, the linker of the conjugate is non-severable. In some embodiments, the T-oligo-HES conjugate includes one or more severable linkers and one or more non-severable linkers.

[0211] In certain embodiments, the T-oligo-HES conjugate contains a cleavable linker comprising an amino acid sequence that is a substrate for at least one protease, such as an extracellular protease. In some embodiments, the cleavable linker has an amino acid sequence comprising a protease cleavage site having a loop structure and containing P1-P1' residues. Suitable amino acid sequences that are substrates for the protease of interest can be systematically identified using any of a variety of known techniques. For example, peptide substrates can be identified using the methods described in U.S. Patent No. 7,666,817, U.S. Patent No. 8,563,269, and International Publication No. WO2014 / 026136, the contents of which are incorporated herein by reference in their entirety (see also Boulware et al. Biotechnol Bioeng. 106(3): 339-346 (2010)).

[0212] In certain embodiments, the linker is a peptide containing a conformation-dependent cleavage site. This conformation-dependent cleavage protease substrate is thought to provide a distinct secondary structure that results in increased specificity and sensitivity compared to linear sequence protease cleavage sites. The latter allows the substrate to be cleaved with a small amount of target protease.

[0213] In further specific embodiments, the linker is rigid and includes cargo projection arms, as further described herein, which are intended to physically expose the protease substrate away from the antibody relative to the target cell surface where the protease is localized.

[0214] In certain embodiments, the T-oligo-HES conjugate contains a cleavable linker comprising an amino acid sequence that is a substrate of at least one protease active in diseased tissue. For example, in some embodiments, the T-oligo-HES conjugate contains a cleavable linker comprising an amino acid sequence that is a substrate of at least one protease active and / or colocalizes with cells expressing a cell surface antigen that is specifically bound by the target-directing moiety of the T-oligo-HES conjugate (e.g., in tumors and / or the tumor microenvironment). In some embodiments, the cleavable linker comprises an amino acid sequence that is a substrate of at least one protease that is upregulated or is thought to be upregulated in inflammation. In some embodiments, the cleavable linker comprises an amino acid sequence that is a substrate of at least one protease that is upregulated or is thought to be upregulated or otherwise unregulated in autoimmunity.

[0215] In some embodiments, the cleavable linker comprises an amino acid sequence that is a substrate for at least one protease that is upregulated or otherwise unregulated in cancer. In certain embodiments, the T-oligo-HES conjugate includes metalloproteinases (e.g., meprin, neprilysin, PSMA, and BMP1); matrix metalloproteinases (e.g., MMP1-3, MMP7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27); thrombin; elastases (e.g., human neutrophil elastase); cysteine ​​proteases (e.g., regmine and clugipain); serine proteases (e.g., For example, it contains a cleavable linker comprising an amino acid sequence that is a substrate for at least one protease selected from cathepsin C, DESC1, FAP, matryptase-2, MT-SP1 / matryptase, and type II transmembrane serine proteases (TTSPs) such as TMPRSS2-4, urokinase (uPA), aspartate proteases (e.g., BACE and renin); aspartate cathepsin (e.g., cathepsin D); and threonine proteases.

[0216] In some embodiments, the cleavable linker contains an amino acid sequence that is a substrate for at least one protease selected from hepsin (HPN), furin, matryptase, matryptase-2, gelatinase (e.g., gelatinase A (MMP 2), and progelatinase B (MMP 9) and progelatinase A), TMPRSS2, TMPRSS3, TMPRSS4 (CAP2), fibroblast-activating protein (FAP), kallikrein-related peptidases (KLK family), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14.

[0217] In some embodiments, the cleavable linker comprises an amino acid sequence that is a substrate of at least one ADAM having a thrombospondin motif (ADAMTS) selected from ADAMTS1, ADAMTS4, and ADAMTS5.

[0218] In some embodiments, the T-oligo-HES conjugate contains a cleavable linker comprising an amino acid sequence that is a substrate for at least one protease selected from (a) MMP9; (b) MMP14; (c) MMP1, MMP2 (e.g., gelatinase A), MMP3, MMP7, MMP8, MMP10, MMP11, MMP12, MMP13, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27; (d) serine proteases (e.g., MT-SP1 and uPA); (e) cysteine ​​proteases; (f) metalloproteases including (a) to (c); (g) aspartyl proteases; and (h) threonine proteases. In some embodiments, the T-oligo-HES conjugate contains a cleavable linker comprising an amino acid sequence that is a substrate of at least one MMMP. In some embodiments, the cleavable linker comprises an amino acid sequence that is a substrate of MMP9 or MMP14.

[0219] In some embodiments, the T-oligo-HES conjugate is ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMMDEC1, ADAMTS1, ADAMTS4, ADAMTS5); cathepsin E); caspases (e.g., caspase 1-10, and caspase 14); cysteine ​​cathepsins (e.g., cathepsin B, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, and cathepsin X / Z / P); KLK (e.g., KLK4-8, KLK10, KLK It contains a cleavable linker comprising an amino acid sequence that is a substrate for at least one enzyme or protease selected from: 11, KLK13, and KLK14); metalloproteinases (e.g., meprin, neprilysin, PSMA, and BMP1); cathepsin A, chymase, coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, and FXIIa); granzyme B; guanidinobenzoatase; HtrAl; lactoferrin; malapsin; NS3 / 4A; PACE4; plasmin; PSA, tPA; tryptase; DPP-4; and hepsin.

[0220] Suitable amino acid sequences for substrates containing protease cleavage sites are known in the art and can be included in the sequences of cleavable linkers provided herein. In some embodiments, the T-oligo-HES conjugate contains a cleavable linker comprising the amino acid sequences of protease substrates disclosed in International Patent Application Publication No. WO2019018828A1 (see, e.g., paragraphs 105-117, and Tables 4, 6, 68, and the substrate sequences corresponding to sequence identifier numbers 393 (356-423, 680-698, 713, 714, 789-808, and 1037)), and WO2016 / 179285 (see, e.g., pages 40-47), the contents of which are incorporated herein by reference in their entirety for all purposes.

[0221] In some embodiments, the T-oligo-HES conjugate contains a linker that is cleaved by enzymes of the immune complement system (complement cascade), such as u-plasminogen activator, tissue plasminogen activator, trypsin, and plasmin, but not limited to these. The target-directed portion of these conjugates preferably contains an antibody capable of activating complement, and the antibody in the conjugate retains both the ability to bind to an antigen and the ability to activate the complement cascade. According to one embodiment, the oligo-HES complex is conjugated to an antibody via a linker that is susceptible to cleavage by complement. Thus, when these conjugates bind to an antigen in the presence of complement, the linker is cleaved, and the oligo-HES complex is released from the conjugate. Thus, the T-oligo-HES conjugate activates the complement cascade and releases the oligo-HES complex at the target site. In another embodiment, the oligo-HES complex is attached / conjugated via a linker that is susceptible to cleavage by enzymes having proteolytic activity, such as u-plasminogen activator, tissue plasminogen activator, plasmin, or trypsin.

[0222] In some embodiments, the T-oligo-HES conjugate includes a linker that can be cleaved by a cleavage agent present in the intracellular environment (e.g., within lysosomes, endosomes, or caveolae). This linker may be a peptidyl linker that can be cleaved by intracellular peptidase or protease enzymes, including, but not limited to, lysosomal or endosomal proteases. In some embodiments, the linker is a peptide with a length of at least two or three amino acid residues. The cleavage agents may include cathepsins B and D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug within the target cell (see, for example, Dubowchik and Walker, Pharm. Therapeutics 83:67-123 (1999)). The most typical is the peptidyl linker. In certain embodiments, the peptide linker can be cleaved by cathepsin-B, which is highly expressed in cancer tissue. In some embodiments, the linker comprises the sequence Phe-Leu or Gly-Phe-Leu-Gly (SEQ ID NO: 9), or a cathepsin B cleavable amino acid sequence as disclosed in U.S. Patent No. 6,214,345, which is incorporated herein. In certain embodiments, the linker is a Val-Cit linker or a Phe-Lys linker (see, for example, U.S. Patent No. 6,214,345).

[0223] In some embodiments, the T-oligo-HES conjugate contains a pH-sensitive cleavable linker, i.e., a linker that is sensitive to hydrolysis at a specific pH value. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, acid-unstable linkers that are hydrolyzable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) can be used. (See, for example, U.S. Patents 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, Pharm. Therapeutics 83:67-123 (1999); and Neville et al., Biol. Chem. 264:14653-14661 (1989)). Such linkers are relatively stable under neutral pH conditions, such as in blood, but unstable below pH 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to the target-directed moiety via an acylhydrazone bond) (see, for example, U.S. Patent No. 5,622,929).

[0224] In some embodiments, the T-oligo-HES conjugate contains a linker (e.g., a disulfide linker) that can be cleaved under reducing conditions. Various disulfide linkers are known in the art, including, for example, SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl)-α-methyl-α-(2-pyridyldithio)toluene), SPDB, and SMPT. (See, for example, Thorpe et al., Cancer Res. 47:5924-5931 (1987); Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel ed., Oxford U. Press, 1987). See also U.S. Patent No. 4,880,935.)

[0225] In other embodiments, the T-oligo-HES conjugate contains a malonate linker (Johnson et al., Anticancer Res. 15:1387-93 (1995)), a maleimide-benzoyl linker (Lau et al., Bioorg-Med-Chem. 3(10):1299-1304 (1995)), or a 3'-N-amide analog (Lau et al., Bioorg-Med-Chem. 3(10):1305-1312 (1995)).

[0226] In some embodiments, the T-oligo-HES conjugate contains a linker having an H-dimer-forming fluorophore conjugated to an amino-terminal or carboxyl-terminal residue. In some embodiments, the linker has an H-dimer-forming fluorophore conjugated to an amino-terminal and a carboxyl-terminal residue. In some embodiments, the linker has a sulfhydryl or amino functional group at the amino-terminal and carboxyl-terminal of the peptide.

[0227] In some embodiments, the T-oligo-HES conjugate may be designed so that the oligo-HES complex is delivered to the target but not released. This can be achieved by attaching the oligo-HES complex directly to the target-directed portion or via an inseparable linker. In some embodiments, the T-oligo-HES conjugate contains an inseparable linker. In some embodiments, the T-oligo-HES conjugate contains an oligo-HES complex directly linked to the target-directed portion.

[0228] These uncleavable linkers may include amino acids, peptides, D-amino acids, or other organic compounds that can be modified to include functional groups that can be later used for attachment to AB by the method described herein.

[0229] Preferably, the linker of the T-oligo-HES conjugate is substantially insensitive to a normal plasma environment. As used herein, "substantially insensitive to a normal plasma environment" in relation to the linker means that, when the targeted oligo-HES conjugate compound is present in the plasma of a normal subject, about 20% or less, typically about 15% or less, more typically about 10% or less, and even more typically about 5% or less, about 3% or less, or about 1% or less of the linker of the targeted oligo-HES conjugate compound in the sample will be cleaved. Whether the linker is substantially insensitive to a normal plasma environment can be determined, for example, by incubating the targeted oligo-HES conjugate with plasma for a predetermined time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free oligo-HES complex in the plasma.

[0230] In some embodiments, higher specific activity (or a higher ratio of drug to AB) can be achieved by attaching single-site linkers to multiple sites on AB. These multiple sites can be introduced to AB by one of two methods. First, multiple aldehyde groups and / or sulfhydryl groups can be generated within the same AB. Second, a "branched-chain linker" having multiple functional sites for subsequent linker attachment can be attached to the aldehyde or sulfhydryl of AB. The functional sites of the branched-chain linker or multi-site linker may be aldehyde or sulfhydryl groups, or any chemical site to which the linker can attach. Combining these two approaches, i.e., attaching multiple sites, may yield even higher specific activity. In addition, drugs can attach to AB via disulfide bonds (e.g., disulfide bonds on cysteine ​​molecules). Many tumors naturally release high levels of glutathione (a reducing agent), which reduces disulfide bonds, and the drug can then be released at the delivery site. In some embodiments, the reducing agent modifying the CM also modifies the linker of the conjugated activatable antibody.

[0231] Manufacturing method: This disclosure utilizes several methods for conjugating an oligo-HES complex to a target-directed moiety, for example, (a) attachment of the target-directed moiety (e.g., an antibody) to a carbohydrate moiety, (b) attachment of the target-directed moiety (e.g., an antibody) to a sulfhydryl group, (c) attachment of the target-directed moiety (e.g., an antibody) to an amino group, and (d) attachment of the target-directed moiety (e.g., an antibody) to a carboxylate group. According to this disclosure, the target-directed moiety may be covalently bonded to the oligo-HES complex via an intermediate linker having at least two reactive groups, one of which reacts with the target-directed moiety and the other which reacts with the oligo-HES complex. The linker, which may contain any compatible organic compound, can be selected such that the reaction with the target-directed moiety and the oligo-HES complex does not adversely affect the reactivity and selectivity of the target-directed moiety or the activity of the oligo-HES complex.

[0232] Suitable linkers for reaction with oxidized polypeptide target-directing moieties such as oxidized antibodies or oxidized antibody fragments include those containing amines selected from the group consisting of primary amines, secondary amines, hydrazines, hydrazides, hydroxylamines, phenylhydrazines, semicarbazides, and thiosemicarbazide groups. Such reactive functional groups may be present as part of the linker structure or can be introduced by suitable chemical modifications of linkers that do not contain such groups.

[0233] Linkers suitable for reaction with target-directed moieties of reducing polypeptides, such as reducing antibodies or reducing antibody fragments, include linkers having certain reactive groups that can react with the sulfhydryl groups of the reducing antibody or fragment. Such reactive groups include, but are not limited to, reactive haloalkyl groups (e.g., including haloacetyl groups), p-merclique benzoate groups, and groups capable of Michael-type addition reactions (e.g., maleimide and groups of the type described by Mitra and Lawton, 1979, J. Amer. Chem. Soc. 101: 3097-3110).

[0234] Suitable linkers for attachment to unoxidized or unreducible polypeptides, such as antibodies or antibody fragments, include linkers having certain functional groups that can react with primary amino groups present on unmodified lysine residues of the target-directing moiety. Such reactive groups include, but are not limited to, NHS carboxylic acids or carbonates, sulfo-NHS carboxylic acids or carbonates, 4-nitrophenyl carboxylic acids or carbonates, pentafluorophenyl carboxylic acids or carbonates, acylimidazoles, isocyanates, and isothiocyanates. In some embodiments, suitable linkers include linkers having certain functional groups that can react with carboxylic acid groups present on aspartate or glutamate residues of the target-directing moiety, activated with a suitable reagent. Suitable activating reagents include EDC with or without NHS or sulfo-NHS, and other dehydrating agents used for carboxamide formation. In these cases, functional groups present on the suitable linkers include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides.

[0235] The oligo-HES complex can be attached to the linker either before or after attachment of the linker to the target-directed moiety. In certain applications, it may be desirable for the linker to initially generate a target-directed moiety-linker intermediate that does not contain the oligo-HES complex. Depending on the specific application, the oligo-HES complex can be covalently bonded to the linker.

[0236] Treatment method: This disclosure also provides T-oligo-HES conjugates and methods for modulating nucleic acids and proteins encoded or controlled by oligonucleotides in the conjugates. In certain embodiments, this disclosure provides compositions and methods for modulating the level, expression, processing, or function of mRNA, small non-coding RNA (e.g., miRNA), genes, or proteins in vivo in targeted or localized cells(s).

[0237] In some embodiments, the disclosure provides a method for targeting and delivering oligonucleotides to cells in vivo by administering a T-oligo-HES conjugate containing an oligonucleotide to a target requiring it. In certain embodiments, the oligonucleotide is a therapeutic oligonucleotide.

[0238] Accordingly, in some embodiments, the present disclosure provides a pharmaceutical composition comprising, for example, (a) at least one oligonucleotide capable of hybridizing with a target nucleic acid sequence under physiological conditions, and (b) a T-oligo-HES conjugate having a target-directing moiety that specifically binds to a cell surface antigen on or within the cellular microenvironment containing the target nucleic acid.

[0239] In some embodiments, the present disclosure provides methods for targeting and / or locally delivering oligonucleotides to a target requiring such delivery. In certain embodiments, the method comprises administering a T-oligo-HES conjugate to a target requiring such delivery, the conjugate comprising a therapeutically effective amount of oligonucleotide sufficient to modulate a target RNA (e.g., mRNA and miRNA) or target gene, and a target-directing moiety that specifically binds to a cell surface antigen on or within the cellular microenvironment of a cell containing the target RNA or target gene.

[0240] According to one embodiment, the disclosure provides a method for modulating a target nucleic acid in a subject, comprising administering a T-oligo-HES conjugate to the subject, wherein the oligonucleotide of the conjugate comprises a sequence substantially complementary to the target nucleic acid that specifically hybridizes to the nucleic acid to modulate its level or interfere with its processing or function, and the conjugate comprises a target-directed moiety that specifically binds to a cell surface antigen on or within the cellular microenvironment of a cell containing the target nucleic acid. In some embodiments, the target nucleic acid is RNA, and in further embodiments, the RNA is mRNA or miRNA. In further embodiments, the oligonucleotide reduces the level of the target RNA in one or more cells or tissues of the subject by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%. In some embodiments, the target nucleic acid is DNA.

[0241] According to one embodiment, the disclosure provides a method for modulating a protein in a subject, comprising administering a T-oligo-HES conjugate to the subject, wherein the oligonucleotide of the conjugate comprises a sequence substantially complementary to a nucleic acid that encodes a protein or affects the transcription, translation, production, processing, or function of a protein, and a target-directed moiety that specifically binds to a cell surface antigen on a cell or in the cellular microenvironment to which the protein is modulated. In some embodiments, the oligonucleotide specifically hybridizes to RNA. In further embodiments, the RNA is mRNA or miRNA. In additional embodiments, the oligonucleotide reduces the level of the protein or target RNA in one or more cells or tissues of the subject by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%. In some embodiments, the oligonucleotide specifically hybridizes to DNA.

[0242] In certain embodiments, the oligonucleotide in the T-oligo-HES conjugate is selected from siRNA, shRNA, miRNA, anti-miRNA, dicer substrate (e.g., dsRNA), aptamer, decoy, antisense oligonucleotide, and plasmids capable of expressing siRNA, miRNA, or antisense oligonucleotide. In some embodiments, the oligonucleotide specifically hybridizes to RNA or an RNA-coding sequence. In other embodiments, the oligonucleotide specifically hybridizes to an RNA-coding DNA sequence or its regulatory sequence.

[0243] In additional embodiments, nucleic acid or protein expression is regulated in a subject by administering to the subject requiring it a T-oligo-HES conjugate containing an antisense oligonucleotide and a target-directed moiety that specifically binds to a cell surface antigen on the cell or within the cellular microenvironment where the nucleic acid or protein is regulated. In certain embodiments, the antisense oligonucleotide in the T-oligo-HES conjugate is a substrate of RNAse H when bound to the target RNA. In some embodiments, the antisense oligonucleotide is a gapmer. As used herein, “gapmer” refers to an antisense compound having a central region (also referred to as a “gap” or “gap segment”) located between two external adjacent regions (also referred to as “wings” or “wing segments”). The regions are distinguished by the type of sugar moiety that constitutes each different region. Types of sugar moieties used to distinguish gapmer regions include, in some embodiments, β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides include, for example, 2'-MOE, 2'-fluoro and 2'-O-CH3, etc.), and bicyclic sugar-modified nucleosides (such bicyclic sugar-modified nucleosides include, for example, LNA® or ENA®).

[0244] In some embodiments, each wing of the gapmer oligonucleotide contains the same number of subunits. In other embodiments, one wing of the gapmer oligonucleotide contains a different number of subunits than the other wing of the gapmer. In one embodiment, the wings of the gapmer oligonucleotide independently have 1 to about 5 nucleosides, of which 1, 2, 3, 4, or 5 wing nucleosides are sugar-modified nucleosides. In one embodiment, the central or gap region contains 8 to 25 β-D-ribonucleosides or β-D-deoxyribonucleosides (i.e., nucleosides of length 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, or 25). In further embodiments, the central or gap region contains 17–24 nucleotides (i.e., nucleosides of length 17, 18, 19, 20, 21, 22, 23, or 24). In some embodiments, the gapmer oligonucleotide includes phosphodiester nucleotide linkages, phosphorothioate nucleotide linkages, or a combination of phosphodiester nucleotide linkages and phosphorothioate nucleotide linkages. In certain embodiments, the central region of the gapmer oligonucleotide contains at least 2, 3, 4, 5, or 10 modified nucleosides, modified nucleoside linkages, or a combination thereof. In certain embodiments, the central region of the gapmer oligonucleotide contains at least 10 β-D-2'-deoxy-2'-fluororibofuranosyl nucleosides. In some embodiments, each nucleoside in the central region of the oligonucleotide is a β-D-2'-deoxy-2'-fluororibofuranosyl nucleoside. In one embodiment, the gapmer oligonucleotide is perfectly complementary to the target RNA in terms of length complementarity. In one embodiment, one or both wings of the gapmer contain at least one 2'-modified nucleoside. In one embodiment, one or both wings of the gapmer contain one, two, or three 2'-MOE-modified nucleosides. In one embodiment, one or both wings of the gapmer contain one, two, or three 2'-OCH3-modified nucleosides.In another embodiment, one or both wings of the gapmer contain one, two, or three LNA or alpha-LNA nucleosides. In some embodiments, the LNA or alpha-LNA in the wings of the gapmer contain one or more methyl groups in (R) or (S) configuration at the 6'(2',4'-restricted-2'-O-ethylBNA,S-cEt) or 5'(-5'-Me-LNA or -5'-Me-alpha-LNA) position of the LNA, or contain a carbon atom substituted in place of the 2'-oxygen atom in the LNA or alpha-LNA. In further embodiments, the LNA or alpha-LNA in the gapmer contain a stereobulk moiety (e.g., a methyl group) at the 5' position. In further embodiments, the gap contains at least one 2'-fluoromodified nucleoside. In additional embodiments, the wings are each 2 or 3 nucleosides long, and the gap region is 19 nucleotides long. In additional embodiments, the gapmer has at least one 5-methylcytosine.

[0245] In another embodiment, the nucleoside of the central region (gap) contains a uniform sugar moiety distinct from one or both sugar moieties of the outer wing regions. In one non-limiting example, the gap is uniformly composed of a first 2'-modified nucleoside, and each wing is uniformly composed of a second 2'-modified oligonucleoside. For example, in one embodiment, the central region contains 2'-F modified nucleotides adjacent to each end of an outer region, each having two 2'-MOE modified nucleotides (2'-MOE / 2'-F / 2'-MOE). In a particular embodiment, the gapmer is ISIS 393206. In another embodiment, the central region contains 2'-F modified nucleotides adjacent to each end of an outer region, each having two 2'-MOE modified nucleotides (2'-MOE / 2'-F / 2'-MOE). In a particular embodiment, each outer region has two LNA or αLNA modified nucleotides in the wings of the gapmer. In further embodiments, the LNA or alpha-LNA modified nucleotide contains one or more methyl groups in an (R) or (S) configuration at the 6'(2',4'-restricted-2'-O-ethylBNA,S-cEt) or 5'(-5'-Me-LNA or -5'-Me-alpha-LNA) position of the LNA, or contains a carbon atom substituted in place of the 2'-oxygen atom in the LNA or alpha-LNA.

[0246] In another embodiment, the disclosure provides the use of T-oligo-HES conjugates in the manufacture of compositions for the treatment of a disease or disorder. In another embodiment, the disclosure provides the use of T-oligo-HES conjugates in the manufacture of compositions for the treatment of one or more conditions associated with miRNA or the miRNA family.

[0247] According to one embodiment, the method comprises the step of administering or exposing a subject to a therapeutically effective amount of a T-oligo-HES conjugate provided herein, sufficient to modulate the expression of a target gene or RNA (e.g., mRNA and miRNA), thereby treating one or more conditions or symptoms associated with a disease or disorder. Exemplary compounds provided herein effectively modulate the expression, activity, or function of a gene, mRNA, or small non-coding RNA target. In preferred embodiments, the small non-coding RNA target is a miRNA, pre-miRNA, or polycistronic or monocistronic pri-miRNA. In additional embodiments, the small non-coding RNA target is a single member of the miRNA family. In further embodiments, two or more members of the miRNA family are selected for modulation.

[0248] In additional embodiments, the present disclosure provides a method for inhibiting the activity of a target nucleic acid in a subject, comprising administering to the subject a T-oligo-HES conjugate comprising an oligonucleotide that targets a nucleic acid containing or encoding a nucleic acid, and acts to reduce the level of the nucleic acid and / or interfere with its function within a cell, wherein the conjugate further comprises a target-directed moiety that specifically binds to a cell surface antigen on the cell or in the cellular microenvironment in which the activity of the target nucleic acid is inhibited.

[0249] In certain embodiments, the target nucleic acid is a small non-coding RNA such as a miRNA. In some embodiments, the oligonucleotide contains a sequence substantially complementary to the target nucleic acid.

[0250] In some embodiments, the Disclosure provides a method for reducing the expression of a target RNA in a subject where it is necessary to reduce the expression of said target RNA, the method comprising administering an antisense T-oligo-HES conjugate to said subject. In certain embodiments, the oligonucleotide in the conjugate is a substrate of RNAse H when bound to the target mRNA. In some embodiments, the oligonucleotide is a gapmer. As disclosed herein, the oligonucleotide in the T-oligo-HES conjugate provided herein exhibits an increased serum half-life. In certain embodiments, the serum half-life of the oligonucleotide in the T-oligo-HES conjugate provided herein is greater than 10 minutes. In additional embodiments, the serum half-life of the oligonucleotide in the T-oligo-HES conjugate provided herein is greater than 20, 30, 40, 50, 60, 90, 120, 180, or 200 minutes. In additional embodiments, the serum half-life of the oligonucleotide in the T-oligo-HES conjugate provided herein is 30–300 minutes, 30–200 minutes, or 30–120 minutes. In certain embodiments, the serum half-life of the oligonucleotide in the T-oligo-HES conjugate provided herein is 1.5–4 times, 2–4 times, or 3–4 times the serum half-life of the naked oligonucleotide (i.e., the oligonucleotide component without HES) in serum alone. In other embodiments, the serum half-life of the oligonucleotide in the T-oligo-HES conjugate provided herein is at least 1, 2, 3, or 4 hours longer than the serum half-life of the naked oligonucleotide in serum alone. Techniques and methods for determining serum half-life are generally known in the art.

[0251] In additional embodiments, the disclosure provides a method for reducing the expression of a target RNA in a subject requiring a reduction in the expression of the target RNA, comprising administering to the subject a T-oligo-HES conjugate containing an antisense oligonucleotide to the subject, wherein the antisense sequence specifically hybridizes to the target RNA. In certain embodiments, the T-oligo-HES conjugate contains an antisense oligonucleotide that is a substrate of RNAse H when bound to the target RNA. In additional embodiments, the antisense oligonucleotide is a gapmer. In some embodiments, the oligonucleotide is 18 to 24 nucleotides long and comprises a gap region having 11 or more consecutive 2'-deoxyribonucleotides, as well as a first wing region and a second wing region adjacent to the gap region, each of the first and second wing regions independently having 1 to 8 2'-O-2-methoxyethyl)ribonucleotides.

[0252] In another embodiment, the antisense oligonucleotide is not a substrate of RNAse H when bound to target RNA (e.g., mRNA and miRNA). In some embodiments, the oligonucleotide includes at least one modified sugar moiety with a modification at the 2' position. In some embodiments, each nucleoside of the oligonucleotide includes a modified sugar moiety with a modification at the 2' position. In some embodiments, the oligonucleotide includes at least one PNA motif. In further embodiments, all monomeric units of the oligonucleotide correspond to PNA. In other embodiments, the oligonucleotide includes at least one morpholino motif. In some embodiments, the morpholino is phosphorodiamidate morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to phosphorodiamidate morpholino (e.g., PMO). In some embodiments, the oligonucleotide sequence is specifically hybridizable to a sequence within 30 nucleotides of the AUG start codon of the target RNA. In additional embodiments, the oligonucleotide in the oligo-HES complex sequence is specifically hybridizable to a sequence in the 5' untranslated region of the target RNA. In some embodiments, the oligonucleotide in the oligo-HES complex is designed to target the 3' untranslated sequence in RNA (e.g., mRNA). In further embodiments, the oligonucleotide in the oligonucleotide-HES complex is designed to target the 3' untranslated sequence in RNA bound by miRNA (i.e., the miRNA 3'UTR target site in mRNA).One such example is a “miR-mask” or “target protector,” which is a single-stranded 2'-O-methyl-modified (or other chemically modified) antisense oligonucleotide that is perfectly complementary to the expected miRNA-binding site in the 3'-UTR of a specific target mRNA, covering the miRNA’s access to the miRNA-binding site on the target mRNA (see, e.g., Choi et al., Science 318:271 (2007); Wang, Methods Mol. Biol. 676:43 (2011)). In further embodiments, the oligonucleotide in the oligonucleotide-HES complex is designed to mimic the 3' untranslated sequence in the mRNA bound by the miRNA. One such example is a “miRNA sponge,” i.e., a competitive miRNA inhibitor transgene expressing multiple tandem binding sites of an endogenous miRNA, which stably interacts with the corresponding miRNA and prevents the association of the endogenous target mRNA with the target miRNA. In additional embodiments, the nucleic acid is mRNA, and the oligonucleotide sequence is specifically hybridizable to a target region of RNA selected from the group consisting of an intron / exon junction of the target RNA, or a 1-50 nucleic acid base region at the 5' end of the intron / exon junction of the target RNA. In some embodiments, the target region is selected from the group consisting of 1-15 nucleic acid bases at the 5' end of the intron / exon junction, 20-24 nucleic acid bases at the 5' end of the intron / exon junction, and 30-50 nucleic acid bases at the 5' end of the intron / exon junction. In further embodiments, the T-oligo-HES conjugate contains an oligonucleotide that specifically hybridizes to nucleotides 1-10 (i.e., the seed region) of miRNA, or specifically hybridizes to a sequence in a miRNA precursor (pre-miRNA) or primary miRNA (pri-miRNA) that blocks miRNA processing when the oligonucleotide is bound.

[0253] In another embodiment, the disclosure provides a method for inhibiting protein production, comprising administering a T-oligo-HES conjugate to a subject that targets a protein-coding nucleic acid or reduces the endogenous expression, processing, or function of the protein in the subject. In a further embodiment, the administered conjugate includes a target-directed moiety that specifically binds to a cell surface antigen on or within the cellular microenvironment of a cell whose protein production is inhibited. In some embodiments, the oligonucleotide includes a sequence substantially complementary to the protein-coding nucleic acid.

[0254] In some embodiments, the disclosure provides a method for reducing the amount of target cellular RNA or corresponding protein in cells by contacting cells expressing target RNA with a T-oligo-HES conjugate having an oligonucleotide sequence that specifically hybridizes to the target RNA, wherein the amount of target RNA or corresponding protein is reduced. In further embodiments, the administered conjugate includes a target-directed moiety that specifically binds to a cell surface antigen on the cell or in the cellular microenvironment that reduces the amount of target cellular RNA or protein. In some embodiments, the RNA is mRNA or miRNA. In additional embodiments, the oligonucleotide is selected from siRNA, shRNA, miRNA, anti-miRNA, dicer substrate (e.g., dsRNA), decoy, aptamer, decoy, antisense oligonucleotide, and plasmids capable of expressing siRNA, miRNA, anti-miRNA, ribozyme, or antisense oligonucleotide.

[0255] In certain embodiments, the oligonucleotide in the oligonucleotide-HES complex is an antisense oligonucleotide. In one embodiment, the antisense oligonucleotide is a substrate of RNAse H when bound to the target RNA. In additional embodiments, the antisense oligonucleotide is a gapmer. In some embodiments, the oligonucleotide is 18 to 24 nucleotides long and comprises a gap region having 11 or more consecutive 2'-deoxyribonucleotides, as well as a first wing region and a second wing region adjacent to the gap region, each of the first and second wing regions independently having 1 to 8 2'-O-2-methoxyethyl)ribonucleotides. In certain embodiments, the oligonucleotide contains 12 to 30 linked nucleosides.

[0256] In another embodiment, the oligonucleotide is not a substrate of RNAse H when bound to target RNA (e.g., mRNA and miRNA). In some embodiments, the oligonucleotide includes at least one modified sugar moiety with a modification at the 2' position. In some embodiments, each nucleoside of the oligonucleotide includes a modified sugar moiety with a modification at the 2' position. In some embodiments, the oligonucleotide includes at least one PNA motif. In further embodiments, all monomeric units of the oligonucleotide correspond to PNA. In other embodiments, the oligonucleotide includes at least one morpholino motif. In further embodiments, the oligonucleotide includes at least one phosphorodiamidate morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to phosphorodiamidate morpholino (PMO). In some embodiments, the oligonucleotide sequence specifically hybridizes to a sequence within 30 nucleotides of the AUG start codon of the target RNA. In some embodiments, the oligonucleotide in the oligo-HES complex is designed to target the 3' untranslated sequence in RNA (e.g., mRNA). In further embodiments, the oligonucleotide in the oligo-HES complex is designed to target the 3' untranslated sequence in the RNA bound by the miRNA. In additional embodiments, the target RNA is mRNA, and the oligonucleotide sequence specifically hybridizes to a target region of mRNA selected from the group consisting of the intron / exon junction of the target RNA and the 1-50 nucleic acid base region on the 5' side of the intron / exon junction of the target RNA. In some embodiments, the target region is selected from the group consisting of 1-15 nucleic acid bases on the 5' side of the intron / exon junction, 20-24 nucleic acid bases on the 5' side of the intron / exon junction, and 30-50 nucleic acid bases on the 5' side of the intron / exon junction.In further embodiments, the T-oligo-HES conjugate contains an oligonucleotide that specifically hybridizes to nucleotides 1-10 (i.e., the seed region) of miRNA, or to a sequence in a pre-miRNA or pri-miRNA that blocks miRNA processing upon binding of the oligonucleotide.

[0257] In some embodiments, oligonucleotides can induce RNA interference (RNAi). In some embodiments, the oligonucleotide is siRNA, shRNA, or a Dicer substrate. In some embodiments, the oligonucleotide is siRNA having a length of 18 to 35 nucleotides. In some embodiments, the oligonucleotide is shRNA having a stem of 19 to 29 nucleotides and a loop size between 4 and 30 nucleotides. In further embodiments, the siRNA or shRNA oligonucleotide contains one or more modified nucleosides, modified nucleoside linkages, or combinations thereof. In some embodiments, the oligonucleotide is a Dicer substrate containing two nucleic acid strands, each having a length of 18 to 25 nucleotides and containing a 2-nucleotide 3' overhang. In certain embodiments, the Dicer substrate is a double-stranded nucleic acid containing a length of 21 nucleotides and containing a 2-nucleotide 3' overhang. In further embodiments, one or both strands of the Dicer substrate contain one or more modified nucleosides, modified nucleoside linkages, or combinations thereof.

[0258] In additional embodiments, the disclosure provides a method for reducing the expression of a target RNA in a subject requiring a reduction in the expression of that target RNA, comprising administering a T-oligo-HES conjugate having an oligonucleotide sequence that specifically hybridizes to the target RNA, thereby reducing the expression of the target RNA in the cells or tissue of the subject. In further embodiments, the administered conjugate includes a target-directed moiety that specifically binds to a cell surface antigen on the cell or within the cellular microenvironment where the expression of the target RNA is reduced. In some embodiments, the RNA is mRNA or miRNA. In additional embodiments, the oligonucleotide is selected from plasmids capable of expressing siRNA, shRNA, miRNA, anti-miRNA, dicer substrates, aptamers, decoys, antisense oligonucleotides, siRNA, miRNA, ribozymes, and antisense oligonucleotides.

[0259] In certain embodiments, the oligonucleotide in the oligo-HES complex is an antisense oligonucleotide. In one embodiment, the antisense oligonucleotide is a substrate of RNAse H when bound to target RNA (e.g., mRNA and miRNA). In additional embodiments, the antisense oligonucleotide is a gapmer. In some embodiments, the oligonucleotide is 18 to 24 nucleotides long and comprises a gap region having 11 or more consecutive 2'-deoxyribonucleotides, as well as a first wing region and a second wing region adjacent to the gap region, each of the first and second wing regions independently having 1 to 8 2'-O-2-methoxyethyl)ribonucleotides. In certain embodiments, the oligonucleotide contains 12 to 30 linked nucleosides.

[0260] In another embodiment, the antisense oligonucleotide is not a substrate of RNAse H when bound to target RNA (e.g., mRNA and miRNA). In some embodiments, the oligonucleotide includes at least one modified sugar moiety with a modification at the 2' position. In some embodiments, each nucleoside of the oligonucleotide includes a modified sugar moiety with a modification at the 2' position. In some embodiments, the oligonucleotide includes at least one PNA motif. In further embodiments, all monomeric units of the oligonucleotide correspond to PNA. In other embodiments, the oligonucleotide contains at least one morpholino motif. In some embodiments, the morpholino is phosphorodiamidate morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to phosphorodiamidate morpholino (PMO). In some embodiments, the oligonucleotide sequence specifically hybridizes to a sequence within 30 nucleotides of the AUG start codon of the target RNA. In additional embodiments, the oligonucleotide sequence specifically hybridizes to a sequence in the 5' untranslated region of the target RNA. In some embodiments, the oligonucleotide in the oligo-HES complex is designed to target the 3' untranslated sequence in RNA (e.g., mRNA). In further embodiments, the oligonucleotide is designed to target the 3' untranslated sequence in RNA bound by miRNA. In additional embodiments, the target RNA is mRNA, and the oligonucleotide sequence specifically hybridizes to a target region of the target mRNA selected from the group consisting of the intron / exon junction of the target RNA and the 1-50 nucleic acid base region on the 5' side of the intron / exon junction of the target RNA. In some embodiments, the target region is selected from the group consisting of the 1-15 nucleic acid bases on the 5' side of the intron / exon junction, the 20-24 nucleic acid bases on the 5' side of the intron / exon junction, and the 30-50 nucleic acid bases on the 5' side of the intron / exon junction.In further embodiments, the T-oligo-HES conjugate contains an oligonucleotide that specifically hybridizes to nucleotides 1-10 (i.e., the seed region) of miRNA, or to a sequence in a pre-miRNA or pri-miRNA that blocks miRNA processing upon binding of the oligonucleotide.

[0261] In some embodiments, oligonucleotides can induce RNA interference (RNAi). In some embodiments, the oligonucleotide is siRNA, shRNA, or a Dicer substrate. In some embodiments, the oligonucleotide is siRNA having a length of 18 to 35 nucleotides. In some embodiments, the oligonucleotide is shRNA having a stem of 19 to 29 nucleotides and a loop size between 4 and 30 nucleotides. In further embodiments, the siRNA or shRNA oligonucleotide contains one or more modified nucleosides, modified nucleoside linkages, or combinations thereof. In some embodiments, the oligonucleotide is a Dicer substrate containing two nucleic acid strands, each having a length of 18 to 25 nucleotides and containing a 2-nucleotide 3' overhang. In certain embodiments, the Dicer substrate is a double-stranded nucleic acid containing a length of 21 nucleotides and containing a 2-nucleotide 3' overhang. In further embodiments, one or both strands of the Dicer substrate contain one or more modified nucleosides, modified nucleoside linkages, or combinations thereof.

[0262] In some embodiments, T-oligo-HES conjugates are administered to target oligonucleotides that specifically hybridize to target nucleic acids (e.g., genes, mRNA, or miRNA) to provide a proliferative advantage to tumor cells or enhance microbial replication. In other embodiments, T-oligo-HES conjugates are administered to provide targeted and / or localized delivery of antisense, siRNA, shRNA, dicer substrates, or miRNAs that target mRNA sequences encoding proteins (e.g., protein variants) believed to be involved in disease. Thus, in some embodiments, the disclosure provides targeted and / or localized in vivo delivery systems for delivering specific nucleic acid sequences to living cells to silence genes in organisms suffering, for example, from pathological conditions resulting from abnormal gene expression.

[0263] In some embodiments, the disclosure provides a method for reducing the amount of a polypeptide of interest in cells, comprising contacting cells expressing a nucleic acid encoding the polypeptide or its complement with a T-oligo-HES conjugate having an oligonucleotide sequence that specifically hybridizes to the polypeptide-encoding DNA or mRNA, thereby reducing the expression of the polypeptide of interest. In further embodiments, the administered conjugate includes a target-directed moiety that specifically binds to a cell surface antigen on or within the cellular microenvironment of the cell in which the amount of polypeptide is reduced. In further embodiments, the oligonucleotide is selected from siRNA, shRNA, miRNA, anti-miRNA, dicer substrate, antisense oligonucleotide, plasmid capable of expressing siRNA, miRNA, ribozyme, and antisense oligonucleotide, wherein the oligonucleotide specifically hybridizes to the nucleic acid encoding the polypeptide or its complement, thereby reducing the expression of the polypeptide. In certain embodiments, the oligonucleotide comprises 12 to 30 linked nucleosides. In some embodiments, the complex comprises double-stranded RNA (dsRNA). In some embodiments, the oligonucleotide comprises at least one modified oligonucleotide. In further embodiments, the oligonucleotide comprises at least one modified oligonucleotide motif selected from 2' modifications (e.g., 2'-fluoro, 2-OME, and 2-methoxyethyl (2-MOE)), locked nucleic acids (LNA and alpha-LNA), PNA motifs, and morpholino motifs.

[0264] In certain embodiments, the oligonucleotide in the T-oligo-HES conjugate is an antisense sequence and, when bound to the target RNA, is a substrate of RNAse H. In additional embodiments, the antisense oligonucleotide is a gapmer. In some embodiments, the gapmer is an antisense oligonucleotide that is a chimeric oligonucleotide. In some embodiments, the chimeric oligonucleotide includes a 2'-deoxynucleotide central gap region located between the 5' and 3' wing segments. The wing segments contain a nucleoside containing at least one 2'-modified sugar. The wing segments have a nucleoside containing at least one 2' sugar moiety selected from a 2'-O-methoxyethyl sugar moiety or a bicyclic nucleic acid sugar moiety. In some embodiments, the gap segment may be a 2'-deoxynucleotide of length 10, and each of the wing segments may be a 2'-O-methoxyethyl nucleotide of length 5. The chimeric oligonucleotide may be uniformly composed of phosphorothioate nucleoside linkages. Furthermore, each cytosine in the chimeric oligonucleotide may be 5'-methylcytosine.

[0265] In another embodiment, the antisense oligonucleotide is not a substrate of RNAse H when hybridized to RNA. In some embodiments, each nucleoside of the oligonucleotide contains a modified sugar moiety with a modification at the 2' position. In some embodiments, the oligonucleotide contains at least one PNA motif. In further embodiments, all monomeric units of the oligonucleotide correspond to PNA. In other embodiments, the oligonucleotide contains at least one morpholino motif. In some embodiments, the morpholino is phosphorodiamidate morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to phosphorodiamidate morpholino (PMO). In some embodiments, the oligonucleotide sequence specifically hybridizes to a sequence within 30 nucleotides of the AUG start codon of the target RNA. In additional embodiments, the oligonucleotide sequence specifically hybridizes to a sequence in the 5' untranslated region of the target RNA. In some embodiments, the oligonucleotide in the oligo-HES complex is designed to target the 3' untranslated sequence in RNA (e.g., mRNA). In further embodiments, the oligonucleotide is designed to target the 3' untranslated sequence in the RNA bound by the miRNA. In additional embodiments, the oligonucleotide sequence specifically hybridizes to a target region of the target mRNA, selected from the group consisting of the intron / exon junction of the target RNA and the 1-50 nucleic acid base region on the 5' side of the intron / exon junction of the target RNA. In some embodiments, the target region is selected from the group consisting of 1-15 nucleic acid bases on the 5' side of the intron / exon junction, 20-24 nucleic acid bases on the 5' side of the intron / exon junction, and 30-50 nucleic acid bases on the 5' side of the intron / exon junction.In further embodiments, the T-oligo-HES conjugate contains an oligonucleotide that specifically hybridizes to nucleotides 1-10 (i.e., the seed region) of miRNA, or to a sequence in a pre-miRNA or pri-miRNA that blocks miRNA processing upon binding of the oligonucleotide.

[0266] In further embodiments, the oligonucleotide can induce RNA interference (RNAi). In some embodiments, the oligonucleotide is siRNA, shRNA, or a Dicer substrate. In some embodiments, the oligonucleotide is siRNA having a length of 18 to 35 nucleotides. In some embodiments, the oligonucleotide is shRNA having a stem of 19 to 29 nucleotides and a loop size between 4 and 30 nucleotides. In further embodiments, the siRNA or shRNA oligonucleotide contains one or more modified nucleosides, modified nucleoside linkages, or combinations thereof. In some embodiments, the oligonucleotide is a Dicer substrate containing two nucleic acid strands, each having a length of 18 to 25 nucleotides and containing a 2-nucleotide 3' overhang. In certain embodiments, the Dicer substrate is a double-stranded nucleic acid containing a length of 21 nucleotides and containing a 2-nucleotide 3' overhang. In further embodiments, one or both strands of the Dicer substrate contain one or more modified nucleosides, modified nucleoside linkages, or combinations thereof.

[0267] In additional embodiments, the Disclosure provides a method for increasing the activity of a target nucleic acid in a subject, comprising administering a T-oligo-HES conjugate containing an oligonucleotide that acts to increase the activity of a target nucleic acid, including or encoding the nucleic acid, or to increase the endogenous expression, processing, or function of the nucleic acid (e.g., by binding to a regulatory sequence of a nucleic acid encoding gene), and to increase the level of the nucleic acid within the cell and / or to increase its function. In further embodiments, the administered conjugate includes a target-directed moiety that specifically binds to a cell surface antigen on the cell or in the cellular microenvironment where the activity of the nucleic acid is increased. In some embodiments, the oligonucleotide contains a sequence substantially identical to that of the nucleic acid, including or encoding the nucleic acid.

[0268] In another embodiment, the disclosure provides a method for increasing protein production, comprising administering a T-oligo-HES conjugate to a subject that encodes a protein or increases the endogenous expression, processing, or function of a protein in the subject. In some embodiments, the oligonucleotide comprises a sequence substantially identical to that of the nucleic acid encoding the protein. In some embodiments, the oligonucleotide shares 100% identity with at least 15 consecutive nucleotides and at least 20 consecutive nucleotides, or over its entire length, of the endogenous nucleic acid sequence encoding the protein.

[0269] In some embodiments, the Disclosure provides a method for treating a disease or disorder characterized by the overexpression of a nucleic acid in a subject, comprising administering to the subject a T-oligo-HES conjugate containing an oligonucleotide that targets a nucleic acid, or a nucleic acid encoding a nucleic acid, and acts to reduce the level of the nucleic acid in the subject and / or interfere with its function. In further embodiments, the administered conjugate includes a target-directed moiety that specifically binds to a cell surface antigen on a cell or in the cellular microenvironment where the nucleic acid is overexpressed. In some embodiments, the nucleic acid is DNA, mRNA, or miRNA, and the conjugate includes a target-directed moiety that specifically binds to a cell surface antigen on a cell or in the cellular microenvironment where protein production is inhibited. In additional embodiments, the oligonucleotide is selected from plasmids capable of expressing siRNA, shRNA, miRNA, anti-miRNA, dicer substrates, antisense oligonucleotides, siRNA, miRNA, ribozymes, and antisense oligonucleotides.

[0270] In certain embodiments, the nucleic acid is RNA, and the oligonucleotide in the oligo-HES complex is an antisense oligonucleotide. In one embodiment, the antisense oligonucleotide is a substrate of RNAse H when hybridized to RNA. In additional embodiments, the antisense oligonucleotide is a gapmer. In some embodiments, the oligonucleotide is 18 to 24 nucleotides long and comprises a gap region having 11 or more consecutive 2'-deoxyribonucleotides, as well as a first wing region and a second wing region adjacent to the gap region, each of the first and second wing regions independently having 1 to 8 2'-O-2-methoxyethyl)ribonucleotides. In certain embodiments, the oligonucleotide contains 12 to 30 linked nucleosides. In some embodiments, the oligonucleotide contains a sequence substantially complementary to the nucleic acid.

[0271] In another embodiment, the oligonucleotide is not a substrate of RNAse H when bound to nucleic acid. In some embodiments, each nucleoside of the oligonucleotide contains a modified sugar moiety with a modification at the 2' position. In some embodiments, the oligonucleotide contains at least one PNA motif. In further embodiments, all monomeric units of the oligonucleotide correspond to PNA. In other embodiments, the oligonucleotide contains at least one morpholino motif. In some embodiments, the morpholino is phosphorodiamidate morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to phosphorodiamidate morpholino (PMO). In some embodiments, the oligonucleotide sequence specifically hybridizes to a sequence within 30 nucleotides of the AUG start codon of the target RNA. In additional embodiments, the oligonucleotide sequence specifically hybridizes to a sequence in the 5' untranslated region of the target RNA. In some embodiments, the oligonucleotide in the oligo-HES complex is designed to target the 3' untranslated sequence in RNA (e.g., mRNA). In further embodiments, the oligonucleotide is designed to target the 3' untranslated sequence in the RNA bound by the miRNA. In additional embodiments, the nucleic acid is mRNA, and the oligonucleotide sequence specifically hybridizes to a target region of mRNA selected from the group consisting of the intron / exon junction of the target RNA and the 1-50 nucleic acid base region on the 5' side of the intron / exon junction of the target RNA. In some embodiments, the target region is selected from the group consisting of 1-15 nucleic acid bases on the 5' side of the intron / exon junction, 20-24 nucleic acid bases on the 5' side of the intron / exon junction, and 30-50 nucleic acid bases on the 5' side of the intron / exon junction.In further embodiments, the T-oligo-HES conjugate contains an oligonucleotide that specifically hybridizes to nucleotides 1-10 (i.e., the seed region) of miRNA, or to a sequence in a pre-miRNA or pri-miRNA that blocks miRNA processing upon binding of the oligonucleotide.

[0272] In further embodiments, the oligonucleotide can induce RNA interference (RNAi). In some embodiments, the oligonucleotide is siRNA, shRNA, or a Dicer substrate. In some embodiments, the oligonucleotide is siRNA having a length of 18 to 35 nucleotides. In some embodiments, the oligonucleotide is shRNA having a stem of 19 to 29 nucleotides and a loop size between 4 and 30 nucleotides. In further embodiments, the siRNA or shRNA oligonucleotide contains one or more modified nucleosides, modified nucleoside linkages, or combinations thereof. In some embodiments, the oligonucleotide is a Dicer substrate containing two nucleic acid strands, each having a length of 18 to 25 nucleotides and containing a 2-nucleotide 3' overhang. In certain embodiments, the Dicer substrate is a double-stranded nucleic acid containing a length of 21 nucleotides and containing a 2-nucleotide 3' overhang. In further embodiments, one or both strands of the Dicer substrate contain one or more modified nucleosides, modified nucleoside linkages, or combinations thereof.

[0273] In further embodiments, the disclosure provides a method for treating a disease or disorder characterized by protein overexpression in a subject, comprising administering to the subject a T-oligo-HES conjugate containing an oligonucleotide that targets a protein-coding nucleic acid or reduces the endogenous expression, processing, or function of the protein in the subject, the conjugate further comprising a target-directed moiety that specifically binds to a cell surface antigen on a cell or in the cellular microenvironment where the protein is overexpressed. In some embodiments, the nucleic acid is DNA, mRNA, or miRNA. In additional embodiments, the oligonucleotide is selected from plasmids capable of expressing siRNA, shRNA, miRNA, anti-miRNA, dicer substrates, aptamers, decoys, antisense oligonucleotides, siRNA, miRNA, ribozymes, and antisense oligonucleotides. In some embodiments, the oligonucleotide shares 100% identity with at least 15 consecutive nucleotides and at least 20 consecutive nucleotides, or over its entire length, of an endogenous nucleic acid sequence encoding a protein.

[0274] In certain embodiments, the targeted nucleic acid is RNA, and the oligonucleotide in the oligo-HES complex is an antisense oligonucleotide. In one embodiment, the antisense oligonucleotide is a substrate of RNAse H when hybridized to RNA. In additional embodiments, the antisense oligonucleotide is a gapmer. In some embodiments, the oligonucleotide is 18 to 24 nucleotides long and comprises a gap region having 11 or more consecutive 2'-deoxyribonucleotides, as well as a first wing region and a second wing region adjacent to the gap region, each of the first and second wing regions independently having 1 to 8 2'-O-2-methoxyethyl)ribonucleotides. In certain embodiments, the oligonucleotide contains 12 to 30 linked nucleosides. In some embodiments, the oligonucleotide contains a sequence substantially complementary to the nucleic acid.

[0275] In another embodiment, the oligonucleotide is not a substrate of RNAse H when bound to target RNA (e.g., mRNA and miRNA). In some embodiments, the oligonucleotide includes at least one modified sugar moiety with a modification at the 2' position. In some embodiments, each nucleoside of the oligonucleotide includes a modified sugar moiety with a modification at the 2' position. In some embodiments, the oligonucleotide includes at least one PNA motif. In further embodiments, all monomeric units of the oligonucleotide correspond to PNA. In other embodiments, the oligonucleotide includes at least one morpholino motif. In some embodiments, the morpholino is phosphorodiamidate morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to phosphorodiamidate morpholino (PMO). In some embodiments, the oligonucleotide sequence specifically hybridizes to a sequence within 30 nucleotides of the AUG start codon of the target RNA. In additional embodiments, the oligonucleotide sequence can specifically hybridize to a sequence within the 5' untranslated region of the target RNA. (For example, within 30 nucleotides of the AUG start codon) This reduces translation. In some embodiments, the oligonucleotide in the oligo-HES complex is designed to target the 3' untranslated sequence in RNA (e.g., mRNA). In further embodiments, the oligonucleotide is designed to target the 3' untranslated sequence in RNA bound by miRNA. In additional embodiments, the nucleic acid is mRNA, and the oligonucleotide sequence specifically hybridizes to a target region of mRNA encoding a protein selected from the group consisting of the intron / exon junction of the target RNA and the 1-50 nucleic acid base region on the 5' side of the intron / exon junction of the target RNA.In some embodiments, the target region is selected from the group consisting of 1 to 15 nucleic acid bases on the 5' side of the intron / exon junction, 20 to 24 nucleic acid bases on the 5' side of the intron / exon junction, and 30 to 50 nucleic acid bases on the 5' side of the intron / exon junction. In further embodiments, the T-oligo-HES conjugate contains an oligonucleotide that specifically hybridizes to nucleotides 1 to 10 (i.e., the seed region) of the miRNA, or to a sequence in a pre-miRNA or primary miRNA that blocks miRNA processing when the oligonucleotide is bound.

[0276] In further embodiments, the oligonucleotide can induce RNA interference (RNAi). In some embodiments, the oligonucleotide is siRNA, shRNA, or a Dicer substrate. In some embodiments, the oligonucleotide is siRNA having a length of 18 to 35 nucleotides. In some embodiments, the oligonucleotide is shRNA having a stem of 19 to 29 nucleotides and a loop size between 4 and 30 nucleotides. In further embodiments, the siRNA or shRNA oligonucleotide contains one or more modified nucleosides, modified nucleoside linkages, or combinations thereof. In some embodiments, the oligonucleotide is a Dicer substrate containing two nucleic acid strands, each having a length of 18 to 25 nucleotides and containing a 2-nucleotide 3' overhang. In certain embodiments, the Dicer substrate is a double-stranded nucleic acid containing a length of 21 nucleotides and containing a 2-nucleotide 3' overhang. In further embodiments, one or both strands of the Dicer substrate contain one or more modified nucleosides, modified nucleoside linkages, or combinations thereof.

[0277] In some embodiments, the disclosure provides a method for treating (e.g., mitigating) a disease or disorder characterized by abnormal expression of a protein in a subject, comprising administering to the subject a T-oligo-HES conjugate containing an oligonucleotide that specifically hybridizes to the mRNA encoding the protein and alters the splicing of the target RNA (e.g., promotes exon skipping). In further embodiments, the administered conjugate includes a target-directed moiety that specifically binds to a cell surface antigen on a cell or in the cellular microenvironment where the protein expression is abnormal. In some embodiments, each nucleoside of the oligonucleotide contains at least one modified sugar moiety with a modification at the 2' position. In certain embodiments, the modified oligonucleotide is a 2'OME or a 2'Allyl. In additional embodiments, the modified oligonucleotide is LNA, alpha-LNA (e.g., LNA or alpha-LNA containing a stereobulk moiety (e.g., a methyl group) at the 5' position). In some embodiments, the oligonucleotide contains at least one PNA motif. In further embodiments, all monomeric units of the oligonucleotide correspond to PNA. In other embodiments, the oligonucleotide contains at least one morpholino motif. In some embodiments, the morpholino is phosphorodiamidate morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to phosphorodiamidate morpholino (PMO). In some embodiments, the oligonucleotide sequence specifically hybridizes to a sequence within 30 nucleotides of the AUG start codon of the target RNA. In additional embodiments, the oligonucleotide sequence specifically hybridizes to a sequence in the 5' untranslated region of the target RNA. In some embodiments, the oligonucleotide in the oligo-HES complex is designed to target the 3' untranslated sequence in RNA (e.g., mRNA). In further embodiments, the oligonucleotide is designed to target the 3' untranslated sequence in RNA bound by miRNA.In additional embodiments, the oligonucleotide sequence can specifically hybridize to a target region of target mRNA selected from the group consisting of the intron / exon junction of the target RNA and the 1-50 nucleic acid base region at the 5' end of the intron / exon junction of the target RNA. In some embodiments, the target region is selected from the group consisting of 1-15 nucleic acid bases at the 5' end of the intron / exon junction, 20-24 nucleic acid bases at the 5' end of the intron / exon junction, and 30-50 nucleic acid bases at the 5' end of the intron / exon junction.

[0278] In certain embodiments, the disease or disorder is Duchenne muscular dystrophy (DMD). In some embodiments, the oligonucleotide specifically hybridizes to a message splicing-promoting mRNA sequence to "skip" exons 44, 45, 50, 51, 52, 53, or 55 of the dystrophin gene. In certain embodiments, the oligonucleotide specifically hybridizes to a message splicing-promoting mRNA sequence to "skip" exon 51 of the dystrophin gene. In certain embodiments, the oligonucleotide in the T-oligo-HES conjugate is AVI-4658 (AVI Biopharma). In other embodiments, the oligonucleotide in the T-oligo-HES conjugate competes for dystrophin mRNA to bind to AVI-4658. In certain embodiments, the oligonucleotide in the T-oligo-HES conjugate is eteplirsen or drisapersen. In other embodiments, oligonucleotides in the T-oligo-HES conjugate compete for dystrophin mRNA that binds to eteplirsene or drysapersene.

[0279] Further embodiments of the present disclosure provide a method comprising selecting a subject diagnosed with a disease or disorder, administering a therapeutically effective amount of a T-oligo-HES conjugate containing an oligonucleotide that specifically hybridizes to a nucleic acid sequence thought to associate with or encode a protein associated with the disease or disorder or an associated condition, and monitoring the progression of the disease in the subject. In further embodiments, the administered conjugate includes a target-directed moiety that specifically binds to a cell surface antigen on or within the cellular microenvironment of a nucleic acid-containing cell.

[0280] In some embodiments, the nucleic acid is DNA, mRNA, or miRNA. In additional embodiments, the oligonucleotide is selected from siRNA, shRNA, miRNA, anti-miRNA, dicer substrate, aptamer, decoy, and antisense oligonucleotides, plasmids capable of expressing siRNA, miRNA, ribozymes, and antisense oligonucleotides. In some embodiments, the oligonucleotide shares 100% identity with at least 15 consecutive nucleotides of the nucleic acid sequence, at least 20 consecutive nucleotides, or over its entire length.

[0281] In certain embodiments, the nucleic acid is RNA, and the oligonucleotide in the oligo-HES complex is an antisense oligonucleotide. In one embodiment, the antisense oligonucleotide is a substrate of RNAse H when hybridized to RNA. In additional embodiments, the antisense oligonucleotide is a gapmer. In some embodiments, the oligonucleotide is 18 to 24 nucleotides long and comprises a gap region having 11 or more consecutive 2'-deoxyribonucleotides, as well as a first wing region and a second wing region adjacent to the gap region, each of the first and second wing regions independently having 1 to 8 2'-O-2-methoxyethyl)ribonucleotides. In certain embodiments, the oligonucleotide contains 12 to 30 linked nucleosides. In some embodiments, the oligonucleotide contains a sequence substantially complementary to the nucleic acid.

[0282] In another embodiment, the oligonucleotide is not a substrate of RNAse H when bound to target RNA (e.g., mRNA and miRNA). In some embodiments, the oligonucleotide includes at least one modified sugar moiety with a modification at the 2' position. In some embodiments, all nucleosides of the oligonucleotide include a modified sugar moiety with a modification at the 2' position. In some embodiments, the oligonucleotide includes at least one PNA motif. In further embodiments, all monomeric units of the oligonucleotide correspond to PNA. In other embodiments, the oligonucleotide includes at least one morpholino motif. In some embodiments, the morpholino is phosphorodiamidate morpholino. In additional embodiments, all monomeric units of the oligonucleotide correspond to morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to phosphorodiamidate morpholino (PMO). In some embodiments, the oligonucleotide sequence specifically hybridizes to a sequence within 30 nucleotides of the AUG start codon of the target RNA. In additional embodiments, the oligonucleotide sequence specifically hybridizes to a sequence in the 5' untranslated region of the target RNA. In some embodiments, the oligonucleotide in the oligo-HES complex is designed to target the 3' untranslated sequence in RNA (e.g., mRNA). In further embodiments, the oligonucleotide is designed to target the 3' untranslated sequence in RNA bound by miRNA. In additional embodiments, the oligonucleotide specifically hybridizes to a target region of mRNA selected from the group consisting of the intron / exon junction of the target RNA and the 1-50 nucleic acid base region on the 5' side of the intron / exon junction of the target RNA. In some embodiments, the target region is selected from the group consisting of 1-15 nucleic acid bases on the 5' side of the intron / exon junction, 20-24 nucleic acid bases on the 5' side of the intron / exon junction, and 30-50 nucleic acid bases on the 5' side of the intron / exon junction.In additional embodiments, the T-oligo-HES conjugate contains an oligonucleotide that specifically hybridizes to nucleotides 1-10 (i.e., the seed region) of the miRNA, or to a sequence in a pre-miRNA or pri-miRNA that blocks miRNA processing upon binding of the oligonucleotide.

[0283] In further embodiments, the oligonucleotide can induce RNA interference (RNAi). In some embodiments, the oligonucleotide is siRNA, shRNA, or a Dicer substrate. In some embodiments, the oligonucleotide is siRNA having a length of 18 to 35 nucleotides. In some embodiments, the oligonucleotide is shRNA having a stem of 19 to 29 nucleotides and a loop size between 4 and 30 nucleotides. In further embodiments, the siRNA or shRNA oligonucleotide contains one or more modified nucleosides, modified nucleoside linkages, or combinations thereof. In some embodiments, the oligonucleotide is a Dicer substrate containing two nucleic acid strands, each having a length of 18 to 25 nucleotides and containing a 2-nucleotide 3' overhang. In certain embodiments, the Dicer substrate is a double-stranded nucleic acid containing a length of 21 nucleotides and containing a 2-nucleotide 3' overhang. In further embodiments, one or both strands of the Dicer substrate contain one or more modified nucleosides, modified nucleoside linkages, or combinations thereof.

[0284] In another embodiment, the disclosure provides a method for slowing disease progression in subjects suffering from a disease or disorder associated with protein overexpression, comprising administering a T-oligo-HES conjugate having an oligonucleotide that specifically hybridizes to protein-encoding DNA or mRNA to the subject to reduce polypeptide expression. In a further embodiment, the administered conjugate includes a target-directed moiety that specifically binds to a cell surface antigen on or within the cellular microenvironment of cells overexpressing the protein. In additional embodiments, the oligonucleotide is selected from plasmids capable of expressing siRNA, shRNA, miRNA, anti-miRNA, dicer substrates, antisense oligonucleotides, siRNA, miRNA, ribozymes, and antisense oligonucleotides. In some embodiments, the oligonucleotide shares 100% identity with at least 15 consecutive nucleotides and at least 20 consecutive nucleotides, or over its entire length, of the protein-encoding DNA or mRNA.

[0285] In certain embodiments, the nucleic acid is mRNA, and the oligonucleotide in the oligo-HES complex is an antisense oligonucleotide. In one embodiment, the antisense oligonucleotide is a substrate of RNAse H when hybridized to RNA. In additional embodiments, the antisense oligonucleotide is a gapmer. In some embodiments, the oligonucleotide is 18 to 24 nucleotides long and comprises a gap region having 11 or more consecutive 2'-deoxyribonucleotides, as well as a first wing region and a second wing region adjacent to the gap region, each of the first and second wing regions independently having 1 to 8 2'-O-2-methoxyethyl)ribonucleotides. In certain embodiments, the oligonucleotide contains 12 to 30 linked nucleosides. In some embodiments, the oligonucleotide contains a sequence substantially complementary to the nucleic acid.

[0286] In another embodiment, the oligonucleotide is not a substrate of RNAse H when bound to target RNA (e.g., mRNA and miRNA). In some embodiments, the oligonucleotide includes at least one modified sugar moiety with a modification at the 2' position. In some embodiments, each nucleoside of the oligonucleotide includes a modified sugar moiety with a modification at the 2' position. In some embodiments, the oligonucleotide includes at least one PNA motif. In further embodiments, all monomeric units of the oligonucleotide correspond to PNA. In other embodiments, the oligonucleotide includes at least one morpholino motif. In some embodiments, the morpholino is phosphorodiamidate morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to morpholino. In further embodiments, all monomeric units of the oligonucleotide correspond to phosphorodiamidate morpholino (PMO). In some embodiments, the oligonucleotide sequence specifically hybridizes to a sequence within 30 nucleotides of the AUG start codon of the target RNA. In additional embodiments, the oligonucleotide sequence can specifically hybridize to a sequence in the 5' untranslated region of the target RNA. In some embodiments, the oligonucleotide in the oligo-HES complex is designed to target the 3' untranslated sequence in RNA (e.g., mRNA). In further embodiments, the oligonucleotide is designed to target the 3' untranslated sequence in RNA bound by miRNA. In additional embodiments, the nucleic acid is mRNA, and the oligonucleotide sequence specifically hybridizes to a target region of mRNA selected from the group consisting of the intron / exon junction of the target RNA and the 1-50 nucleic acid base region on the 5' side of the intron / exon junction of the target RNA. In some embodiments, the target region is selected from the group consisting of 1-15 nucleic acid bases on the 5' side of the intron / exon junction, 20-24 nucleic acid bases on the 5' side of the intron / exon junction, and 30-50 nucleic acid bases on the 5' side of the intron / exon junction.

[0287] In further embodiments, the oligonucleotide can induce RNA interference (RNAi). In some embodiments, the oligonucleotide is siRNA, shRNA, or a Dicer substrate. In some embodiments, the oligonucleotide is siRNA having a length of 18 to 35 nucleotides. In some embodiments, the oligonucleotide is shRNA having a stem of 19 to 29 nucleotides and a loop size between 4 and 30 nucleotides. In further embodiments, the siRNA or shRNA oligonucleotide contains one or more modified nucleosides, modified nucleoside linkages, or combinations thereof. I...

Claims

1. A conjugate comprising a target-directed moiety conjugated to an oligonucleotide-HES complex, wherein the oligonucleotide is a therapeutic oligonucleotide.

2. The conjugate according to claim 1, wherein the target-directing portion is directly conjugated to the oligonucleotide-HES complex or conjugated to the oligonucleotide-HES complex via a linker.

3. The conjugate according to claim 1 or 2, wherein the target-directing portion is conjugated to the oligonucleotide-HES complex via a linker.

4. Equation (I) T-(L ｎ - (Oligo-HES) ｘ ) ｐ (I) A conjugate having the structure of any one of claims 1 to 3 (In the formula, T is a target-directed portion that selectively binds to the target of choice; L is the linker; Oligo-HES is an oligonucleotide complex containing therapeutic oligonucleotides and H-type exciton structures (HES); n is either 0 or 1; x is 1 to 30, 1 to 20, 1 to 10, or 1 to 5; p is 1-30, 1-20, 1-10, or 1-5).

5. Formula (II) T-[L ｎ - ((Oligo 2-SP) ｍ - Oligo 1 - HES) ｓ ] ｕ (II) A conjugate having the structure of any one of claims 1 to 4 (In the formula, T is a target-directed portion that selectively binds to the target of choice; L is the linker; SP is a linker, and in some cases, SP is a peptide or alkyl chain spacer consisting of 6 to 12 amino acid residues, such as a linear or branched alkyl of C6, C10, or C18; Oligo1-HES is an oligonucleotide complex containing oligonucleotide 1 (Oligo1) and an H-type exciton structure (HES); Oligo 2 is an oligonucleotide that may be the same as or different from Oligo 1; n is either 0 or 1; m is either 0 or 1; s is either 1 or 2; (u is 1, 2, 3, 4, or 5).

6. The conjugate according to any one of claims 1 to 5, wherein the oligonucleotide-HES complex comprises a therapeutic oligonucleotide that specifically hybridizes to a nucleic acid sequence in vivo and modulates the level of a protein encoded or controlled by the nucleic acid.

7. The conjugate according to claim 6, wherein the therapeutic oligonucleotide contains one, two, or three substitutions, deletions, or insertions compared to the corresponding reverse complementary strand of the nucleic acid sequence.

8. The conjugate according to any one of claims 1 to 7, wherein the therapeutic oligonucleotide has a length of about 8 nucleotides to about 750 nucleotides.

9. The conjugate according to any one of claims 1 to 8, wherein the therapeutic oligonucleotide has a length of 18-25, 18-35, 18-40, or 18-45, 18-50, 18-60, 18-70, 18-80, 18-90, 18-100, 18-150, or 18-200 nucleotides.

10. The conjugate according to any one of claims 1 to 9, wherein the therapeutic oligonucleotide is single-stranded.

11. The conjugate according to any one of claims 1 to 9, wherein the therapeutic oligonucleotide is double-stranded.

12. The conjugate according to claim 11, wherein the therapeutic oligonucleotide has a length of 36 to 50, 36 to 60, 36 to 70, or 36 to 100 nucleotides.

13. The conjugate according to any one of claims 1 to 12, wherein the therapeutic oligonucleotide contains one or more modified nucleoside motifs selected from locked nucleic acid (LNA), alpha-LNA, 2'-fluoro(2'F), 2'-O(CH2)2OCH3(2'-MOE), 2'--deoxy-2'--fluoro-D-arabino nucleic acid (FANA), 2'-OCH3(2'-O-methyl)(2'OME), PNA, and morpholino.

14. The conjugate according to claim 13, wherein the modified nucleoside motif is an LNA or alpha-LNA in which a methylene(--CH2--)n group bridges a 2' oxygen atom and a 4' carbon atom, and n is 1 or 2.

15. The conjugate according to claim 13 or 14, wherein the LNA or alpha-LNA contains a methyl group at the 5' position.

16. A conjugate according to any one of claims 1 to 15, comprising one or more modified nucleoside links selected from phosphorothioate, phosphorodithioate, phosphoramide, 3'-methylenephosphonate, O-methylphosphoamidiate, PNA, and morpholino.

17. The conjugate according to any one of claims 1 to 16, wherein the therapeutic oligonucleotide contains one or more modified nucleic acid bases selected from C-5 propine and 5-methyl C.

18. The therapeutic oligonucleotide of the conjugate is selected from the following nucleic acids: EGFR, HER2 / neu, ErbB3, cMet, p56lc, PDGFR, VEGF, VEGFFR, FGF, FGFR, ANG1, ANG2, bFGF, TIE2, protein kinase C-alpha (PKC-alpha), p56lc PKA, TGF-β, IGFIR, P12, MDM2, BRCA, IGF1, HGF, PDGF, IGFBP2, IGF1R, HIF1alpha, ferritin, transferrin receptor, TMPRSS2, IRE, HSP27, HSP70, HSP90, MITF, clatherin, PARP1C-fos, C-myc, n-myc, C-ra f, B-raf, A1, H-raf, Skp2, K-ras, N-ras, H-ras, phalensyltransferase, c-Src, Jun, Fos, Bcr-Abl, c-Kit, EphA2, PDGFB, ARF, NOX1, NF1, STAT3, E6 / E7, APC, WNT, betacatenin, GSK3b, PI3k, mTOR, A The method according to any one of claims 1 to 17, which specifically hybridizes to kt, PDK-1, CDK, Mek1, ERK1, AP-1, P53, Rb, Syk, osteopontin, CD44, MEK, MAPK, NF-κβ (NF kappa beta), E cadherin, cyclin D, cyclin E, Bcl2, Bax, BXL-XL, BCL-W, MCL1, ER, MDR, telomerase, telomerase reverse transcriptase, DNA methyltransferase, histone deacetylase (e.g., HDAC1 and HDAC2), integrin, IAP, aurora kinase, metalloproteinase (e.g., MMP2, MMP3 and MMP9), proteasome, or metallothionein gene.

19. The method according to any one of claims 1 to 17, wherein the therapeutic oligonucleotide of the conjugate specifically hybridizes with nucleic acids selected from the following: Survivin, HSPB1, EIF4E, PTPN1, RRM2, BCL2, PTEN, Bcr-abl, TLR9, HaRas, Pka-rIA, JNK2, IGF1R, XIAP, TGF-β2, c-myb, PLK1, K-ras, KSP, PKN3, ribonucleotide reductases (e.g., ribonucleotide reductase R1 and ribonucleotide reductase R2), RecQ helicases (e.g., WRN, RecQL1, BLM, RecQL4, RecQ5, and RTS), MEM2, and TLR9.

20. The conjugate according to any one of claims 1 to 19, wherein the oligonucleotide-HES complex comprises at least one fluorophore that is excited and / or emits light in the range of 300 to 850 nm.

21. The conjugate according to any one of claims 1 to 20, wherein the oligonucleotide-HES complex comprises two, three, four or more fluorophores capable of forming one or more HESs.

22. The conjugate according to any one of claims 1 to 21, wherein the oligonucleotide-HES complex comprises two, three, four or more fluorophores that are excited and / or emit light in the range of 300 to 850 nm.

23. The conjugate according to any one of claims 1 to 22, wherein the oligonucleotide-HES complex comprises at least one fluorophore selected from xanthene, indocarbocyanin, indodicarbocyanin, and coumarin.

24. The conjugate according to any one of claims 1 to 23, wherein the oligonucleotide-HES complex comprises at least one fluorophore selected from carboxyrhodamine 110, carboxytetramethylrhodamine, carboxyrhodamine-X, diethylaminocoumarin, and N-ethyl-N'-[5-(N''-succinimidyloxycarbonyl)pentyl]indocarbocyanine chloride and N-ethyl-N'-[5-(N''-succinimidyloxycarbonyl))pentyl]-3,3,3',3'-tetramethyl-2',2'-indodicarbocyanine chloride dye.

25. The conjugate according to any one of claims 1 to 24, wherein the therapeutic oligonucleotide is selected from siRNA, shRNA, miRNA, antagmir, a dicer substrate, and antisense.

26. The conjugate according to any one of claims 1 to 25, wherein the therapeutic oligonucleotide is siRNA.

27. The conjugate according to any one of claims 1 to 25, wherein the therapeutic oligonucleotide is shRNA.

28. The conjugate according to any one of claims 1 to 25, wherein the therapeutic oligonucleotide is miRNA or antagmir (an inhibitor of miRNA).

29. The conjugate according to any one of claims 1 to 25, wherein the therapeutic oligonucleotide is a Dicer substrate.

30. The conjugate according to claim 29, wherein the therapeutic oligonucleotides each contain two nucleic acid complementary nucleic acid chains having a length of 18-25, 18-30, 18-35, 18-40, 18-45, or 18-50 nucleotides and having a 2-nucleotide 3' overhang.

31. The conjugate according to any one of claims 1 to 30, wherein the therapeutic oligonucleotide can induce RNA interference (RNAi).

32. The conjugate according to claim 25, wherein the therapeutic oligonucleotide, when hybridized with the RNA, is a substrate of RNAse H.

33. The conjugate according to claim 25, wherein the therapeutic oligonucleotide is a gapmer.

34. The conjugate according to claim 25, wherein the therapeutic oligonucleotide, when hybridized with the RNA, is not a substrate of RNAse H.

35. The conjugate according to any one of claims 1 to 25, wherein the therapeutic oligonucleotide is an antisense oligonucleotide.

36. The conjugate according to claim 35, wherein the antisense oligonucleotide specifically hybridizes with the target RNA.

37. The conjugate according to claim 35 or 36, wherein the antisense oligonucleotide is DNA or a DNA mimetic.

38. The conjugate according to any one of claims 35 to 37, wherein each nucleoside of the therapeutic antisense oligonucleotide comprises a modified sugar moiety including modification at the 2' position, a PNA motif, or a morpholino motif.

39. The therapeutic antisense oligonucleotide sequences consist of the following groups: (a) The sequence of mRNA within 30 nucleotides from the AUG start codon; (b) nucleotides 1-10 of miRNA; (c) Sequence in the 5'-untranslated region of mRNA; (d) Sequence in the 3'-untranslated region of mRNA; (e) mRNA intron / exon junctions; (f) A sequence of a pre-miRNA or primary miRNA that, when bound to the oligonucleotide, blocks miRNA processing; and (g) a conjugate according to any one of claims 35 to 38, which specifically hybridizes to a target region of RNA selected from an intron / exon junction and a 1 to 50 nucleic acid base region on the 5' side of an RNA intron / exon junction.

40. The conjugate according to any one of claims 35 to 39, wherein hybridization of the oligonucleotide to its target mRNA sterically blocks translation of the coding sequence, or regulates the expression of the target mRNA and / or blocks nucleotide-binding proteins, thereby regulating the stability of the target mRNA.

41. The conjugate according to any one of claims 35 to 40, wherein the antisense oligonucleotide-HES complex comprises a plurality of antisense chains, and the antisense chains are crosslinked in some cases.

42. The conjugate according to claim 41, wherein the oligonucleotide-HES complex is linearly linked in series with a spacer arm from the 5' end to the 3' end, and comprises a plurality of antisense oligonucleotides linked to another antisense oligonucleotide linked to the 5' or 3' terminal residue of the second chain by a spacer arm (for example, a 6-30 amino acid residue peptide or a linear alkyl (for example, C6, C10, or C12) or polyethyloxyglycol (for example, triethyloxyglycol, tetraethyloxyglycol, or hexaethyloxyglycol)).

43. The conjugate according to any one of claims 1 to 42, wherein the linker arm (L or SP) is a C6, C10, or C12 linear alkyl group, or a polyethyloxyglycol (e.g., triethyloxyglycol, tetraethyloxyglycol, or hexaethyloxyglycol).

44. The conjugate according to any one of claims 41 to 43, wherein the oligo-HES complex comprises a plurality of therapeutic antisenses crosslinked with two or more antisense chains from the 5' side to the 3' side and two or more therapeutic antisenses in the opposite direction from the 3' side to the 5' side, and the oligo-HES complex comprises a plurality of therapeutic antisenses crosslinked with two or more antisense chains in the direction from the 5' side to the 3' side and then from the 3' side to the 5' side using a spacer SP.

45. The conjugate according to claim 44, wherein the antisense strand has two or more identical complementary sequences and / or two or more different complementary sequences.

46. A conjugate according to any one of claims 1 to 45, comprising a linear or branched linker.

47. A conjugate according to any one of claims 1 to 46, comprising a peptide linker or SP spacer having a length of 6 to 30 amino acid residues.

48. The conjugate according to claim 46 or 47, wherein the linker is a linear peptide having a length of 6 to 30 amino acid residues.

49. The conjugate according to any one of claims 46 to 48, wherein the linker is severable.

50. The conjugate according to claim 49, wherein the linker has an amino acid sequence including a protease cleavage site containing a P1-P1' residue and has a loop structure.

51. The conjugate according to claim 49 or 50, wherein the cleavable linker comprises an amino acid sequence that is a substrate for at least one protease.

52. The conjugate according to any one of claims 49 to 51, wherein the cleavable linker comprises an amino acid sequence that is a substrate of at least one protease active in diseased tissue.

53. The cleavable linker is a metalloproteinase (e.g., meprin, neprilysin, PSMA, and BMP1); a matrix metalloproteinase (e.g., MMP1-3, MMP7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27), thrombin, elastase (e.g., human neutrophil elastase), a cysteine ​​protease (e.g., regmine and clugipain), a serine protease (e.g., catechin A conjugate according to any one of claims 49 to 52, comprising an amino acid sequence that is a substrate for at least one protease selected from: psin C, and TTSPs such as DECC1, FAP, matryptase-2, MT-SP1 / matryptase, and TMPRSS2-4; urokinase (uPA); aspartate proteases (e.g., BACE and renin); aspartate cathepsin (e.g., cathepsin D); and threonine proteases.

54. The aforementioned severable linker (a) MMP9; (b) MMP14; (c) MMP1, MMP2, MMP3, MMP7, MMP8, MMP10, MMP11, MMP12, MMP13, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27; (d) Serine proteases (e.g., MT-SP1, uPA, and TMPRSS2); (e) Cysteine ​​protease; (f) Metalloproteinases including (a) to (c); (g) Aspartyl protease; and (h) threonine proteases; a conjugate according to any one of claims 49 to 53, comprising an amino acid sequence that is a substrate of at least one protease selected from the above.

55. The conjugate according to claim 54, wherein the cleavable linker comprises an amino acid sequence that is a substrate of MMP9 or MMP14.

56. The conjugate according to claim 54 or 55, wherein the cleavable linker comprises an amino acid sequence that is a substrate for at least one enzyme of the immune complement system, such as u-plasminogen activator, tissue plasminogen activator, trypsin, or plasmin.

57. The conjugate according to any one of claims 49 to 56, wherein the cleavable linker comprises an amino acid sequence that is a substrate of a protease known or reported to colocalize with the target of the conjugate.

58. The conjugate according to claim 49, wherein the linker is cleavable under intracellular conditions (e.g., conditions within a lysosome, endosome, or caveolae).

59. The conjugate according to claim 49, wherein the linker is pH sensitive.

60. The conjugate according to claim 49, wherein the linker is cleavable under reducing conditions.

61. The conjugate (of a conjugate) according to any one of claims 44 to 56, wherein the linker is a malonate linker.

62. The conjugate according to any one of claims 46 to 48, wherein the linker is incutable.

63. The conjugate according to any one of claims 46 to 62, wherein the linker has an H-dimer-forming fluorophore (for example, a linker containing an H-dimer-forming fluorophore conjugated to an amino-terminal residue and / or a carboxyl-terminal residue).

64. The conjugate according to any one of claims 46 to 63, wherein the linker has an H-dimer-forming fluorophore conjugated to the amino-terminal residue and the carboxyl-terminal residue.

65. The conjugate according to any one of claims 46 to 64, wherein the linker has a sulfhydryl or amino functional group at the amino terminus and the carboxyl terminus of the peptide.

66. The conjugate according to any one of claims 1 to 65, wherein the target-directing portion of the conjugate is an aptamer, an avimer, a receptor-binding ligand, a nucleic acid, a biotin-avidin binding pair, a peptide, a protein, a carbohydrate, a lipid, a vitamin, a microbial component, a hormone, a receptor ligand (including an Fc fusion protein containing a receptor ligand), an antibody, an antigen-binding portion of an antibody, an alternative binding scaffold, or any derivative thereof.

67. The conjugate according to any one of claims 1 to 66, wherein the target-directing portion is an antibody, an antigen-binding portion of an antibody (e.g., Fab and scFv), or a single-domain antibody.

68. The conjugate according to any one of claims 1 to 67, wherein the target-directing portion is an antibody, a humanized antibody, an antigen-binding fragment of an antibody, a single-chain antibody, a bispecific antibody, a synthetic antibody, or a PEGylated antibody.

69. The conjugate according to any one of claims 1 to 68, wherein the target-directing portion is an antibody.

70. The conjugate according to claim 69, wherein the target-directing portion is a monospecific, bispecific, or multispecific antibody and / or a monovalent, bivalent, or polyvalent antibody.

71. The conjugate according to claim 69 or 70, wherein the target-directing portion is an IgG1, IgG2, or IgG4 antibody.

72. The conjugate according to any one of claims 69 to 70, wherein the target-directing portion is a therapeutic antibody.

73. The aforementioned antibodies include trastuzumab (HER2 / neu), pertuzumab (HER2 / neu), panitumumab (EGFR), nimotuzumab (EGFR), zaltumumab (EGFR), cetuximab (EGFR), (HER3), onarutuzumab (c-MET), patritumab, cribatuzumab (MUC1), sofituzumab (MUC16), edrecolomab (EPCAM), adekatumumab (EPCAM), anetumab (MSLN), huDS6 (CA6), and rifas. Tuzumab (NAPI2B), Sacituzumab (TROP2), PR1A3, Humanized PR1A3 (CEA), Humanized Ab2-3 (CEA), IMAB362 / Claudiximab (Claudin 18.2), AMG595 (EGFRvIII), ABT806 (EGFRvIII), Cibrotuzumab (FAP), DS-8895a variant 1 (EphA2), DS-8895a variant 2 (EphA2), Anti-EphA2 (EphA2), MEDI-547 (EphA 2) Narunatumab (RON), RG7841 (LY6E), Farletuzumab (FRA / folate receptor alpha), Milbetuximab (FRA), J591 variant 1 (PSMA), J591 variant 2 (PSMA), Robalpituzumab (DLL3), PF-06647020 (PTK7), Anti-PTK7 (PTK7), Radilatuzumab (LIV1), Silumutuzumab (ROR1), Rituximab (CD20), Ibritumomab tiuxetan (CD52), Allergen The conjugate according to claim 72, selected from mutuzumab (CD33), gemtuzumab ozogamicin (CD33), CT-011 (PD1), tositumomab (CD20), ipilimumab (CTLA4), tremelimumab (CP-675,206) (CTLA4), nivolumab (PD1), pembrolizumab (PD1), durvalumab (PDL1) anti-MAGE-A3, anti-NY-ESO-1, anti-ACE2, anti-hyaluronidase, or anti-neuraminidase;

74. The conjugate according to any one of claims 1 to 68, wherein the target-directing portion is an antigen-binding fragment of an antibody, a single-chain antibody, a single-domain antibody, or a bispecific antibody.

75. The conjugate according to any one of claims 1 to 66, wherein the target-directing portion is an alternative binding scaffold selected from affibody, nanobody, antikalin, finomer, DARPin, tetranectin, transbody, adonectin, affin, microbody, peptide aptamer, alterase, plastic antibody, phylomer, stradobody, maxibody, epibody, Z-domain, D-domain, armadillo repeat protein, Knitz domain, avimer, atrimer, probody, immunobody, triomab, troybody, pepbody, waxibody, unibody, affimer, or duobody.

76. The conjugate according to any one of claims 1 to 75, wherein the target-directing portion specifically binds to a cell surface antigen on or near a target cell or tissue, such as diseased cells, cancer cells, immune cells, infected cells, or infectious agents.

77. The conjugate according to any one of claims 1 to 76, wherein the target-directing portion specifically binds to a cell surface antigen(s) that originates from or is determined to be expressed in a specific target cancer (e.g., a tumor), such as a neoantigen.

78. The conjugate according to any one of claims 1 to 77, wherein the target-directing portion specifically binds to cell surface antigens that do not internalize the conjugate upon binding.

79. The conjugate according to any one of claims 1 to 75, wherein the target-directing portion specifically binds to a cell surface antigen that internalizes the conjugate upon binding.

80. The conjugate according to any one of claims 1 to 79, wherein the target-directing portion specifically binds to tumor cell surface antigens.

81. The conjugate according to any one of claims 1 to 80, wherein the target-directing portion specifically binds to brain tumor cells including leukemia cells, lymphoma cells, pancreatic cancer cells, breast cancer cells, melanoma cells, lung cancer cells, head and neck cancer cells, ovarian cancer cells, bladder cancer cells, colorectal cancer cells, kidney cancer cells, liver cancer cells, prostate cancer cells, bone cancer cells, and glioblastoma cells; or to tumor cell surface antigens on lymphoma, myeloma, blastoma, sarcoma, leukemia, or carcinoma cells.

82. The target-directed portion includes CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CD204, CD206, CD301, CAMPATH-1, HLD-DR, carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUC1, MUC15, folate-binding protein, A33, G250, prostate-specific membrane antigen (PSMA), ferritin, GD2, GD3, GM2, Ley, CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2 receptor, tenascin, metalloproteinase, endothelial, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6, HPV E7, EGFRvIII, HER2 / neu, MAGE A3, P53 non-mutant, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, P53 mutant, PR1, bcr-abl, tyrosinase, survivorbin, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin (MSLN), PSCA, MAGE A1, MAGE-A3, sLe (animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Regmine, Tie 3. A conjugate according to any one of claims 1 to 81, which specifically binds to a cell surface antigen selected from Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, CMET, HER3, CA6, NAPI2B, TROP2, CLDN18.2, fibroblast-activating protein (FAP), RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, Fos-related antigen 1, VEGFR, endoglin, PDL, VTCN1, and VISTA.

83. The target-directing portion is HER2, EGFR, CMET, HER3, MUC1, MUC16, EPCAM, MSLN, CA6, NAPI2B, TROP2, CEA, CLDN18.2, EGFRvIII, FAP, EphA2, RON, LY6E, FRA, PSMA, DLL3, PTK7, LIV1, ROR1, MAGE-A3, NY-ESO-1, E A conjugate according to any one of claims 1 to 81, which specifically binds to a cell surface antigen selected from endogrin, CD204, CD206, CD301, VTCN1, VISTA, GLP-3, CLDN6, CLDN16, UPK1B, STR6, TMPRSS3, TMPRSS4, TMEM238, C1orf186, and LRRC15.

84. The conjugate according to any one of claims 1 to 83, wherein the target-directing portion specifically binds to a tumor microenvironment cell surface antigen (including a specific membrane-immobilized protease).

85. The conjugate according to any one of claims 1 to 84, wherein the target-directing portion specifically binds to cell surface antigens expressed on endothelial cells or macrophages (e.g., VEGFR, TIE1, and TIE2), or to cell surface antigens expressed on tumor stromal cells, such as cancer-associated fibroblasts (CAF), tumor-infiltrating T cells and other leukocytes, as well as to bone marrow cells including mast cells, eosinophils, and tumor-associated macrophages.

86. The conjugate according to any one of claims 1 to 85, wherein the target-directing portion specifically binds to cell surface antigens on immune cells.

87. The conjugate according to claim 86, wherein the target-directing portion specifically binds to cell surface antigens on lymphoid or myeloid immune cells such as T cells, B cells, NK cells, NKT cells, or dendritic cells.

88. The conjugate according to claim 86 or 87, wherein the target-directing portion specifically binds to a cell surface antigen on an antigen-presenting cell.

89. The conjugate according to claim 88, wherein the target-directing portion specifically binds to an antigen selected from OX40L, 4-1BBL, MARCO, DC-SIGN, Dectin 1, Dectin 2, DEC-205, CLEC5A, CLEC9A, CLEC10A, CLEC12A, CD1A, CD16A, CD32A, CD32B, CD36, CD40, CD47, CD64, CD204, CD206, HVEM, PDL1, mannose scavenger receptor 1, and BDCA2.

90. The conjugate according to claim 86 or 87, wherein the target-directing portion specifically binds to a cell surface antigen on an immune cell that is not an antigen-presenting cell.

91. The target-directing portion binds to the target of interest (e.g., cell surface antigen) with an equilibrium dissociation constant (Kd) in the range of 0.5×10 －１０ to 10×10 －６ , determined using BIACORE® analysis, conjugate according to any one of claims 1 to 90.

92. A method for regulating intracellular nucleic acid or protein levels, comprising contacting the cells with a therapeutically effective amount of a conjugate described in any one of claims 1 to 91, wherein the oligonucleotide-HES complex is: (a) A target-directing portion that binds to cell surface antigens on or near the cell; and (b) an oligonucleotide that specifically hybridizes with the nucleic acid and modulates the levels of the nucleic acid and / or proteins encoded or controlled by the nucleic acid; the method comprising:

93. The method according to claim 92, wherein the target-directing portion of the T-oligo-HES conjugate specifically binds to a cell surface antigen on the contacted cell, and the cell expresses a cell surface protease that cleaves the cleavable linker of the T-oligo-HES conjugate and releases the oligo-HES complex.

94. The method according to claim 92 or 93, wherein the regulated nucleic acid or protein is present in disease cells, infected cells, infectious agents, or immune cells.

95. The method according to claim 94, wherein the regulated nucleic acid or protein is present in diseased cells.

96. The method according to claim 95, wherein the regulated nucleic acid or protein is present in cancer cells.

97. The method according to claim 96, wherein the cancer cells are hematological cancer cells or solid tumor cancer cells.

98. The method according to claim 97, wherein the cancer cells are leukemia cells, lymphoma cells, pancreatic cancer cells, breast cancer cells, melanoma cells, lung cancer cells, head and neck cancer cells, ovarian cancer cells, bladder cancer cells, colorectal cancer cells, prostate cancer cells, brain tumor cells including glioblastoma cells, sarcoma cells, or any carcinoma cells.

99. The method according to claim 94, wherein the regulated nucleic acid or protein is located within an infected cell or infectious agent.

100. The method according to claim 99, wherein the regulated nucleic acid or protein is located within the infectious agent.

101. The method according to claim 99 or 100, wherein the cells are infected with HIV, HTLV-1, Zika virus, dengue virus, influenza virus, Ebola virus, Marburg virus, Crimean-Congo hemorrhagic fever virus, Lassa fever virus, smallpox virus, SARS virus, Rift Valley fever virus, tuberculosis, anthrax, botulism, tularemia, plague, brucellosis, glanders, meridianus, Q fever, or alphaviruses, such as chikungunya virus, Sindbis virus, Semryqui forest virus, Western, Eastern and Venezuelan equine encephalitis viruses, Ross River virus, COVID, or influenza.

102. The method according to claim 94, wherein the regulated nucleic acid or protein is present in an immune cell.

103. The method according to claim 102, wherein the immune cells are myeloid-derived lymphoid cells such as T cells, B cells, NK cells, NKT cells, or dendritic cells.

104. The method according to any one of claims 92 to 103, wherein the therapeutic oligonucleotide is selected from siRNA, shRNA, miRNA, antagmir, dicer substrate, and antisense.

105. The method according to any one of claims 92 to 104, wherein the conjugate comprises a cleavable linker containing an amino acid sequence that is a substrate of at least one protease.

106. The cleavable linker is a metalloproteinase (e.g., meprin, neprilysin, PSMA, and BMP1); a matrix metalloproteinase (e.g., MMP1-3, MMP7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27), thrombin, elastase (e.g., human neutrophil elastase), cysteine ​​protease (e.g., regmine and clugipain), serine protease ( The method according to claim 105, comprising, for example, an amino acid sequence that is a substrate for at least one protease selected from cathepsin C, and TTSPs such as Decc1, FAP, matryptase-2, MT-SP1 / matryptase, and TMPRSS2-4; urokinase (uPA); aspartate proteases (e.g., BACE and renin); aspartate cathepsin (e.g., cathepsin D); and threonine proteases.

107. The method according to any one of claims 92 to 106, wherein the target-directing portion of the conjugate is an antibody, an antigen-binding portion of an antibody (e.g., Fab and scFv), or a single-domain antibody.

108. A method for modulating nucleic acid or protein levels in a subject, comprising administering a therapeutically effective amount of a conjugate according to any one of claims 1 to 91 to a subject in need thereof, wherein the conjugate is: (a) A target-directing moiety of the nucleic acid or protein that binds to a cell surface antigen on or near the cell that is regulated; and (b) an oligonucleotide that specifically hybridizes with the nucleic acid and modulates the levels of the nucleic acid and / or proteins encoded or controlled by the nucleic acid; the method comprising:

109. The nucleic acid or protein (a) Overexpression or underexpression of the nucleic acid in the subject, (b) Overexpression or underexpression of the protein encoded by the nucleic acid in the subject; the method according to claim 108.

110. The method according to claim 108 or 109, wherein the regulated nucleic acid or protein is present in disease cells, infected cells, infectious agents, or immune cells.

111. The method according to claim 108, wherein the regulated nucleic acid or protein is present in diseased cells.

112. The method according to claim 110, wherein the regulated nucleic acid or protein is present in cancer cells.

113. The method according to claim 112, wherein the cancer cells are blood cancer cells or solid tumor cancer cells.

114. The method according to claim 113, wherein the cancer cells are leukemia cells, lymphoma cells, pancreatic cancer cells, breast cancer cells, melanoma cells, lung cancer cells, head and neck cancer cells, ovarian cancer cells, bladder cancer cells, colorectal cancer cells, prostate cancer cells, brain tumor cells including glioblastoma cells, sarcoma cells, or any carcinoma cells.

115. The method according to claim 110, wherein the regulated nucleic acid or protein is located within an infected cell or infectious agent.

116. The method according to claim 115, wherein the regulated nucleic acid or protein is located within the infectious agent.

117. The method according to claim 115, wherein the infected cells are immune cells of lymphoid or myeloid origin.

118. The method according to claim 110, wherein the regulated nucleic acid or protein is present in an immune cell.

119. The method according to claim 118, wherein the immune cells are lymphoid or myeloid cells such as T cells, B cells, NK cells, NKT cells, or dendritic cells.

120. The method according to any one of claims 108 to 119, wherein the therapeutic oligonucleotide is selected from siRNA, shRNA, miRNA, antagmir, dicer substrate, and antisense.

121. The method according to any one of claims 108 to 120, wherein the conjugate comprises a cleavable linker containing an amino acid sequence that is a substrate of at least one protease.

122. The cleavable linker is a metalloproteinase (e.g., meprin, neprilysin, PSMA, and BMP1); a matrix metalloproteinase (e.g., MMP1-3, MMP7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27), thrombin, elastase (e.g., human neutrophil elastase), cysteine ​​protease (e.g., regmine and clugipain), serine protease ( The method according to claim 121, comprising, for example, an amino acid sequence that is a substrate for at least one protease selected from cathepsin C, and TTSPs such as Decc1, FAP, matryptase-2, MT-SP1 / matryptase, and TMPRSS2-4; urokinase (uPA); aspartate proteases (e.g., BACE and renin); aspartate cathepsin (e.g., cathepsin D); and threonine proteases.

123. The method according to any one of claims 108 to 122, wherein the conjugate target-directing portion is an antibody, an antigen-binding portion of an antibody (e.g., Fab and scFv), or a single-domain antibody.

124. A method for treating a disease or disorder in a subject, comprising administering a therapeutically effective amount of a conjugate according to any one of claims 1 to 91 to a subject in need thereof, wherein the oligonucleotide specifically hybridizes to a nucleic acid sequence in vivo and modulates the level of a protein encoded or controlled by the nucleic acid; the disease or disorder is (a) Overexpression or underexpression of the nucleic acid in the subject, (b) Overexpression or underexpression of the protein encoded by the nucleic acid in the subject; the method.

125. The method according to claim 124, wherein the conjugate includes a target-directed portion that binds to a cell surface antigen on or near the cell in which the nucleic acid or protein is regulated.

126. The method according to claim 124 or 125, wherein the disease or disorder is a proliferative disorder, such as cancer, a disease or disorder of the immune system, an inflammatory disease or disorder, an infectious disease, a neurological disorder, a cardiovascular disorder, a metabolic disorder, a skeletal disorder, or a skin or eye disorder.

127. The method according to any one of claims 124 to 126, wherein the disease or disorder is a neurological disease or disorder such as cancer, an inflammatory disease or disorder, an immune system disorder or disorder, an infectious disease, or a neurodegenerative disease or disorder.

128. The method according to any one of claims 124 to 127, wherein the disease or disorder is cancer.

129. The method according to claim 128, wherein the cancer is a blood cancer or a solid tumor cancer.

130. The method according to claim 129, wherein the cancer is leukemia, pancreatic cancer, breast cancer, melanoma, lung cancer, head and neck cancer, ovarian cancer, bladder cancer, colorectal cancer, kidney cancer, liver cancer, prostate cancer, bone cancer, brain tumor including glioblastoma; or any lymphoma, myeloma, blastoma, sarcoma, leukemia or carcinoma.

131. The method according to claim 124, wherein the disease or disorder is cancer, an inflammatory disease or disorder, a disease or disorder of the immune system, or an infectious disease.

132. The method according to claim 131, wherein the disease is an inflammatory disease or disorder, or an autoimmune disease or disorder (for example, rheumatoid arthritis).

133. The method according to claim 132, wherein the disease or disorder is inflammation.

134. The method according to claim 133, wherein the disease or disorder is an infectious disease.

135. The method according to claim 134, wherein the infection is HIV, HTLV-1, Zika fever, dengue fever, influenza, Ebola, Marburg disease, Crimean-Congo hemorrhagic fever, Lassa fever virus, smallpox, SARS, Rift Valley fever, tuberculosis, anthrax, botulism, tularemia, plague, brucellosis, glanders, meridian, Q fever, or an alphavirus, such as chikungunya virus, Sindbis virus, Semryqui forest virus, Western, Eastern and Venezuelan equine encephalitis viruses, Ross River virus, or COVID.

136. The method according to claim 126, wherein the disease or disorder is a neurological disease or disorder.

137. The method according to claim 136, wherein the neurological disease or disorder is a neurodegenerative disease, for example, familial and sporadic amyotrophic lateral sclerosis (FALS and ALS, respectively), familial and sporadic Parkinson's disease, Huntington's disease (Huntington's chorea), familial and sporadic Alzheimer's disease, spinal muscular atrophy (SMA), multiple sclerosis, diffuse cerebral cortical atrophy, dementia, or Pick's disease.

138. The method according to any one of claims 124 to 137, wherein the therapeutic oligonucleotide of the conjugate is selected from siRNA, shRNA, miRNA, antagmir, a dicer substrate, and antisense.

139. The method according to any one of claims 124 to 138, wherein the conjugate comprises a cleavable linker containing an amino acid sequence that is a substrate of at least one protease.

140. The cleavable linker is a metalloproteinase (e.g., meprin, neprilysin, PSMA, and BMP1); a matrix metalloproteinase (e.g., MMP1-3, MMP7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27), thrombin, elastase (e.g., human neutrophil elastase), cysteine ​​protease (e.g., regmine and clugipain), serine protease ( The method according to claim 139, comprising, for example, an amino acid sequence that is a substrate for at least one protease selected from cathepsin C, and TTSPs such as Decc1, FAP, matryptase-2, MT-SP1 / matryptase, and TMPRSS2-4; urokinase (uPA); aspartate proteases (e.g., BACE and renin); aspartate cathepsin (e.g., cathepsin D); and threonine proteases.

141. The method according to any one of claims 124 to 140, wherein the target-directing portion of the conjugate is an antibody, an antigen-binding portion of an antibody (e.g., Fab and scFv), or a single-domain antibody.

142. The method according to any one of claims 124 to 141, wherein the target-directing portion of the conjugate is an antibody or an antigen-binding fragment of an antibody.

143. The method according to claim 142, wherein the target-directing portion is an antibody or a nanobody.

144. The method according to claim 143, wherein the antibody is an IgG1, IgG2, or IgG4 antibody.

145. The method according to claim 143 or 144, wherein the antibody is a therapeutic antibody.

146. The method according to any one of claims 124 to 145, wherein the conjugate is administered simultaneously, consecutively, or separately with one or more other therapeutic agents.

147. A method for treating cancer in a subject, comprising administering a therapeutically effective amount of a conjugate according to any one of claims 1 to 91 to a subject in need thereof, wherein the oligonucleotide specifically hybridizes with nucleic acids in the cancer cells or tissue, modulates the levels of nucleic acids and / or proteins encoded or controlled by the nucleic acids, and the nucleic acids or proteins (a) Overexpression or underexpression of the nucleic acid in the cancer cells, tissues, and / or subjects, (b) Overexpression or underexpression of the protein encoded by the nucleic acid in the cancer cells, tissues, and / or subjects; the method.

148. The method according to claim 147, wherein the target-directing portion of the conjugate specifically binds to a cell surface antigen on or near the cancer cell.

149. The method according to claim 147 or 148, wherein the target-directing portion of the conjugate specifically binds to the cell surface antigen on the cancer cell.

150. The method according to any one of claims 147 to 149, wherein the target-directing portion of the conjugate specifically binds to cell surface antigens on cells near the cancer cells (for example, cells in the tumor microenvironment such as stromal cells, cancer-associated fibroblasts, immune cells, blood or lymphatic cells, endothelial cells, adipocytes, or neuroendocrine cells).

151. The method according to any one of claims 147 to 150, wherein the cancer is a solid tumor.

152. The method according to any one of claims 147 to 151, wherein the cancer is leukemia, pancreatic cancer, breast cancer, melanoma, lung cancer, head and neck cancer, ovarian cancer, bladder cancer, colorectal cancer, kidney cancer, liver cancer, prostate cancer, bone cancer, brain tumor including glioblastoma; or lymphoma, myeloma, blastoma, sarcoma, leukemia or carcinoma.

153. The method according to claim 152, wherein the cancer is a blood cancer.

154. The method according to claim 153, wherein the blood cancer is leukemia or lymphoma.

155. The target-directed portion of the conjugate is CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CD204, CD206, CD301, CAMPATH-1, HLD-DR, carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUC1, MUC15, folate-binding protein, A33, G250, prostate-specific membrane antigen (PSMA), ferritin, GD2, GD3, GM2, Ley, CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2 receptor, tenascin, metalloproteinase, endothelial growth factor, avB3, WT1, LMP2, HPV E6, HPV E7, HIV GP120, HIV GP160, EGFRvIII, HER2 / neu, MAGE A3, P53 non-mutant, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, P53 mutant, PR1, bcr-abl, tyrosinase, Survivin, PSA, hTERT, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin (MSLN), PSCA, MAGE A1, MAGE-A3, sLe (animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Regmine, Tie 3. The method according to any one of claims 147 to 154, which specifically binds to a cell surface antigen selected from Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, CMET, HER3, CA6, NAPI2B, TROP2, CLDN18.2, fibroblast-activating protein (FAP), RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, Fos-related antigen 1, VEGFR, endoglin, PDL, VTCN1, and VISTA.

156. The method according to any one of claims 147 to 154, wherein the target-directing portion of the conjugate specifically binds to a cell surface antigen selected from DLL3, fibroblast-activating protein α (FAPα), NG2 (glial antigen-2), platelet-derived growth factor receptor-β (PDGFR-β), PD1, CD163, KIR, HMGB1, VEGFR3, LYVE1, CD31, CD34, P1GF, and VEGF.

157. The target-direction portion of the conjugate is HER2, EGFR, CMET, HER3, MUC1, MUC16, EPCAM, MSLN, CA6, NAPI2B, TROP2, CEA, CLDN18.2, EGFRvIII, FAP, EphA2, RON, LY6E, FRA, PSMA, DLL3, PTK7, LIV1, ROR1, MAGE-A3, NY-ES The method according to any one of claims 147 to 154, which specifically binds to a cell surface antigen selected from O-1, endoglin, CD204, CD206, CD301, VTCN1, VISTA, GLP-3, CLDN6, CLDN16, UPK1B, STR6, TMPRSS3, TMPRSS4, TMEM238, C1orf186, and LRRC15.

158. The method according to any one of claims 147 to 156, wherein the therapeutic oligonucleotide of the conjugate is selected from siRNA, shRNA, miRNA, antagmir, a dicer substrate, and antisense.

159. The therapeutic oligonucleotide of the conjugate is selected from the following nucleic acids: EGFR, HER2 / neu, ErbB3, cMet, p56lc, PDGFR, VEGF, VEGFFR, FGF, FGFR, ANG1, ANG2, bFGF, TIE2, protein kinase C-alpha (PKC-alpha), p56lc PKA, TGF-β, IGFIR, P12, MDM2, BRCA, IGF1, HGF, PDGF, IGFBP2, IGF1R, HIF1alpha, ferritin, transferrin receptor, TMPRSS2, IRE, HSP27, HSP70, HSP90, MITF, clatherin, PARP1C-fos, C-myc, n-myc, C-raf B-raf, A1, H-raf, Skp2, K-ras, N-ras, H-ras, pharensyltransferase, c-Src, Jun, Fos, Bcr-Abl, c-Kit, EphA2, PDGFB, ARF, NOX1, NF1, STAT3, E6 / E7, APC, WNT, betacatenin, GSK3b, PI3k, mTOR, Akt The method according to any one of claims 147 to 157, which specifically hybridizes to PDK-1, CDK, Mek1, ERK1, AP-1, P53, Rb, Syk, osteopontin, CD44, MEK, MAPK, NF-κβ (NF kappa beta), E cadherin, cyclin D, cyclin E, Bcl2, Bax, BXL-XL, BCL-W, MCL1, ER, MDR, telomerase, telomerase reverse transcriptase, DNA methyltransferase, histone deacetylase (e.g., HDAC1 and HDAC2), integrin, IAP, aurora kinase, metalloproteinase (e.g., MMP2, MMP3 and MMP9), proteasome, or metallothionein gene.

160. The method according to any one of claims 147 to 157, wherein the therapeutic oligonucleotide of the conjugate specifically hybridizes with nucleic acids selected from the following: Survivin, HSPB1, EIF4E, PTPN1, RRM2, BCL2, PTEN, Bcr-abl, TLR9, HaRas, Pka-rIA, JNK2, IGF1R, XIAP, TGF-β2, c-myb, PLK1, K-ras, KSP, PKN3, ribonucleotide reductases (e.g., ribonucleotide reductase R1 and ribonucleotide reductase R2), RecQ helicases (e.g., WRN, RecQL1, BLM, RecQL4, RecQ5, and RTS), MEM2, and TLR9.

161. The method according to any one of claims 147 to 159, wherein the conjugate comprises a cleavable linker containing an amino acid sequence that is a substrate of at least one protease.

162. The cleavable linker is a metalloproteinase (e.g., meprin, neprilysin, PSMA, and BMP1); a matrix metalloproteinase (e.g., MMP1-3, MMP7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27), thrombin, elastase (e.g., human neutrophil elastase), cysteine ​​protease (e.g., regmine and clugipain), serine protease ( The method according to claim 160, comprising, for example, an amino acid sequence that is a substrate for at least one protease selected from cathepsin C, and TTSPs such as Decc1, FAP, matryptase-2, MT-SP1 / matryptase, and TMPRSS2-4; urokinase (uPA); aspartate proteases (e.g., BACE and renin); aspartate cathepsin (e.g., cathepsin D); and threonine proteases.

163. The method according to any one of claims 147 to 161, wherein the conjugate target-directing portion is an antibody, an antigen-binding portion of an antibody (e.g., Fab and scFv), or a single-domain antibody.

164. The method according to any one of claims 147 to 162, wherein the target-directing portion of the conjugate is an antibody or an antigen-binding fragment of an antibody.

165. The method according to claim 163, wherein the target-directing portion is an antibody.

166. The method according to claim 164, wherein the antibody is an IgG1, IgG2, or IgG4 antibody.

167. The method according to claim 164 or 165, wherein the antibody is a therapeutic antibody.

168. The aforementioned antibodies include trastuzumab (HER2 / neu), pertuzumab (HER2 / neu), panitumumab (EGFR), nimotuzumab (EGFR), zaltumumab (EGFR), cetuximab (EGFR), (HER3), onarutuzumab (c-MET), patritumab, cribatuzumab (MUC1), sofituzumab (MUC16), edrecolomab (EPCAM), Adekatumumab (EPCAM), Anetumab (MSLN), huDS6 (CA6), Rifastuzumab (NAPI2B), Sacituzumab (TROP2), PR1A3, Humanized PR1A3 (CEA), Humanized Ab2-3 (CEA), IMAB362 / Claudiximab (Claudin 18.2), AMG595 (EGFRvIII), ABT806 (EGFRvIII) Cibrotuzumab (FAP), DS-8895a variant 1 (EphA2), DS-8895a variant 2 (EphA2), anti-EphA2 (EphA2), MEDI-547 (EphA2), nalnatumab (RON), RG7841 (LY6E), farletuzumab (FRA / folate receptor alpha), milbetuximab (FRA), J591 variant 1 (PSMA The method according to claim 166, selected from J591 variant 2 (PSMA), lovalpituzumab (DLL3), PF-06647020 (PTK7), anti-PTK7 (PTK7), radilatuzumab (LIV1), sirumutuzumab (ROR1), rituximab (CD20), ibritumomab tiuxetan (CD52), alemtuzumab (CD33), gemtuzumab ozogamicin (CD33), CT-011 (PD1), tositumomab (CD20), ipilimumab (CTLA4), tremelimumab (CP-675,206) (CTLA4), nivolumab (PD1), pembrolizumab (PD1), durvalumab (PDL1) anti-MAGE-A3, and anti-NY-ESO-1.

169. The method according to any one of claims 146 to 167, wherein the conjugate is administered simultaneously, consecutively, or separately with one or more other anticancer drugs.

170. A conjugate according to any one of claims 1 to 91 for use in pharmaceuticals.

171. A conjugate as defined in any one of claims 1 to 91, for use in the treatment of a disease or disorder in a subject.

172. A conjugate according to any one of claims 1 to 91 for use in the treatment of a disease or disorder selected from infectious diseases, cancer, proliferative disorders or disorders, neurological diseases or disorders, and inflammatory diseases or disorders, diseases or disorders of the immune system, diseases or disorders of the cardiovascular system, metabolic diseases or disorders, diseases or disorders of the skeletal system, and diseases or disorders of the skin or eyes.

173. A conjugate according to any one of claims 1 to 91 for use in regulating a target nucleic acid or protein in a subject; in treating a disease or disorder characterized by overexpression or underexpression of nucleic acids in a subject; in treating a disease or disorder characterized by overexpression or underexpression of proteins in a subject; or in treating a disease or disorder characterized by abnormal nucleic acid or protein expression in a subject.

Images