Inhibitory nucleic acids and methods of use thereof

Abstract

The present disclosure provides inhibitory nucleic acids, compositions comprising the inhibitory nucleic acids, and methods of treating cancer using the inhibitory nucleic acids.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 430,731, filed December 7, 2022, the entire contents of which are incorporated herein by reference.

[0003] Incorporation by Reference of Electronically Submitted Materials

[0004] A sequence listing "BERK-480WO_SEQ_LIST" is attached as a sequence listing XML file, created on December 1, 2023, and is 12,658 bytes in size. The entire contents of this sequence listing XML are incorporated herein by reference.

[0005] introduction

[0006] Brachyury (also known as "TBXT") has been shown to be overexpressed in lung, breast, colon, prostate, and liver cancers, as well as chordomas. Brachyury has been linked to epithelial-mesenchymal transition in human tumors and is being investigated as a drug target for a wide range of cancers.

[0007] Chordomas are malignant sarcomas that arise from notochordal cells in the vertebrae and are rare (1:1,000,000), with 300 cases diagnosed annually in the United States. Chordoma tumors grow slowly; however, due to their proximity to the spinal cord, they are challenging to treat and have a tendency to recur after surgery and irradiation. There are no systemic targeted therapies for chordoma, and therefore patients with advanced and metastatic disease face a dismal prognosis.

[0008] There is a need in the art for compositions and methods for treating cancer.

[0009] Overview

[0010] The present disclosure provides inhibitory nucleic acids, compositions comprising the inhibitory nucleic acids, and methods of using the inhibitory nucleic acids to treat cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the TBXT targeting mechanism of the antisense oligonucleotide (ASO) library.

[0013] Figure 2 The effects of selected mixed-mer locked nucleic acid (LNA) ASOs on TBXT expression in UM-Chor1 cells are described.

[0014] Figure 3 The localization of mixed anti-TBXT LNA ASOs on the same TBXT exon is described.

[0015] Figure 4 Non-specific toxicity of anti-TBXT LNA ASO mixed-mer hits in U2OS cells is described.

[0016] Figure 5 The inhibition of TBXT expression in UM-Chor1 cells by selected mixed-mer LNA ASOs is described.

[0017] Figure 6 The nonspecific toxicity of anti-TBXT LNA ASO mixed polymer redesigns in U2OS cells is described.

[0018] Figure 7 The inhibition of TBXT expression in UM-Chor1 cells by redesigned non-toxic mixed-mer LNA ASOs is described.

[0019] Figure 8 The inhibition of TBXT expression in MUG-Chor1 cells by original and redesigned LNA ASOs is described.

[0020] Figure 9 The inhibition of TBXT expression in JHC7 cells by original and redesigned LNA ASOs is described.

[0021] Figure 10 The inhibition of TBXT expression in UCH-2 cells by original and redesigned LNA ASOs is described.

[0022] Figure 11 An overview of anti-TBXT mixed-mer LNA ASO screening is provided.

[0023] Figures 12A-12B Inhibition of UM-Chor1 chordoma cell growth by anti-TBXT mixed-mer LNA ASO is described.

[0024] Figure 13 A TBXT nucleotide sequence is provided (SEQ ID NO: 9).

[0025] Figure 14 The TBXT amino acid sequence is provided (SEQ ID NO: 10).

[0026] Figures 15A-15B The entry of anti-TBXT mixed-mer LNA ASOs into chordoma cells in the absence of a lipid delivery vehicle is described.

[0027] Figures 16A-16D Described is the skipping of exon 5 in TBXT transcripts induced by anti-TBXT mixed-mer LNA ASOs.

[0028] Figures 17A-17B described that anti-TBXT mixed-mer LNA ASO induces S-phase cell cycle arrest in chordoma cells.

[0029] Figures 18A-18E Tolerization of mice to an anti-TBXT mixed-mer LNA ASO is described.

[0030] Figures 19A-19D Eradication of patient-derived chordoma tumors in mice by anti-TBXT mixed-mer LNA ASOs is described.

[0031] definition

[0032] As used herein, an "antisense oligonucleotide" ("ASO") refers to a nucleic acid sequence that is complementary to a DNA or RNA sequence.

[0033] "RNA" refers to a molecule comprising at least one or more ribonucleotide residues. A "ribonucleotide" is a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranose moiety. As used herein, the term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides.

[0034] "MicroRNA" (miRNA) is a single-stranded RNA molecule of approximately 21-23 nt in length. In general, miRNA regulates gene expression. MiRNAs are encoded by genes from the DNA that transcribes them, but miRNAs are not translated into proteins. Each primary miRNA transcript is processed into a short stem-loop structure and then further processed into functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression.

[0035] As used herein, "interfering RNA" refers to any double-stranded or single-stranded RNA sequence that can directly or indirectly (i.e., by transformation) inhibit or downregulate gene expression by mediating RNA interference. Interfering RNA includes, but is not limited to, toenail interfering RNA ("siRNA") and small hairpin RNA ("shRNA"). "RNA interference" refers to the selective degradation of sequence-compatible messenger RNA transcripts.

[0036] As used herein, "shRNA" (small hairpin RNA) refers to an RNA molecule comprising an antisense region, a loop portion, and a sense region, wherein the sense region has complementary nucleotides that base-pair with the antisense region to form a duplex stem. Following post-transcriptional processing, the small hairpin RNA is converted into small interfering RNA by a cleavage event mediated by the enzyme Dicer, a member of the RNase III family.

[0037] As used herein, "small interfering RNA" or "siRNA" refers to any small RNA molecule that can inhibit or downregulate gene expression by mediating RNA interference in a sequence-specific manner. The length of the small RNA can be, for example, about 18 to 21 nucleotides.

[0038] As used herein, "antagomir" refers to a small synthetic RNA that has complementarity with a specific microRNA target and has a mismatch at the cleavage site or has one or more base modifications to inhibit cleavage.

[0039] As used herein, the phrase "post-transcriptional processing" refers to mRNA processing that occurs after transcription and is mediated by, for example, Dicer and / or Drosha enzymes.

[0040] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing a disease and / or adverse effects attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal (e.g., a human), and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0041] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to an individual organism, such as a mammal, including but not limited to a mouse, ape, human, and non-human primate. In some cases, the "individual" is a human.

[0042] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, as such embodiments may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0043] Where a range of values ​​is provided, it is to be understood that each intervening value between the upper and lower limits of the range (to one-tenth of the unit of the lower limit, unless expressly provided otherwise herein) and any other stated or intervening value within the stated range are encompassed within the present invention. The upper and lower limits of these smaller ranges may independently be included within the smaller range and are also encompassed within the present invention, subject to any specifically excluded limits within the stated range. Where the stated range includes one or both of the upper and lower limits, ranges excluding one or both of those included limits are also encompassed within the present invention.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials related to the cited publications.

[0045] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "antisense oligonucleotide (ASO)" includes a plurality of such ASOs, reference to a "TBXT polypeptide" includes reference to one or more TBXT polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that the claims can be drafted to exclude any optional element. Thus, this statement is intended to serve as an antecedent basis for the use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements or the use of a "negative" limitation.

[0046] In the specification sheets describing the present disclosure (especially in the context of the appended claims), the use of the terms "a", "an", and "the", and similar indicators, should be interpreted as covering both the singular and the plural, unless otherwise stated herein or clearly contradicted by the context. Unless otherwise stated, the terms "comprise", "have", "include", and "contain" should be interpreted as open-ended terms (i.e., meaning "including, but not limited to,"). Unless otherwise stated herein, the enumeration of ranges of numerical values ​​herein is intended only to serve as a shorthand method for referring to each separate numerical value within the range, and each separate numerical value is incorporated into this specification sheet as if it were individually enumerated herein. For example, if a range of 10-15 is disclosed, 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order, unless otherwise stated herein or clearly contradicted by the context. Any and all examples or exemplary language (e.g., "such as") provided herein are intended only to better illustrate embodiments of the present disclosure and are not intended to limit the scope of the present disclosure, unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosed embodiments.

[0047] As used herein, the term "about" used in conjunction with an amount means that the amount can vary by 10% of the amount described. For example, "about 100" refers to an amount between 90 and 110. When about is used in the context of a range, "about" used with respect to the lower amount of the range means that the lower amount includes an amount that is 10% less than the lower amount of the range, and "about" used with respect to the upper amount of the range means that the upper amount includes an amount that is 10% greater than the upper amount of the range. For example, from about 100 to about 1000 means that the range extends from 90 to 1100.

[0048] As used herein, the term "and / or" such as the phrase "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or" such as the phrase "A, B and / or C" is intended to encompass 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).

[0049] It should be understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0050] It should be understood that certain features of the present invention that are described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the present invention that are described in the context of a single embodiment for the sake of brevity may also be provided separately or in any suitable subcombination. All combinations of embodiments belonging to the present invention are specifically included in the present invention and are disclosed herein as if each combination were individually and clearly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically included in the present invention and are disclosed herein as if each such subcombination were individually and clearly disclosed herein.

[0051] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. In addition, the publication dates provided may differ from the actual publication dates, which may need to be independently confirmed.

[0052] Detailed description

[0053] The present disclosure provides inhibitory nucleic acids, compositions comprising the inhibitory nucleic acids, and methods of using the inhibitory nucleic acids to treat cancer.

[0054] Inhibitory nucleic acids

[0055] The present disclosure provides inhibitory nucleic acids that provide for a reduction in the levels of a TBXT polypeptide in a cell.

[0056] TBXT is also known in the art as "T-Box transcription factor T", "Brachyury protein", "SAVA" and "FTFT". Figure 14 The TBXT amino acid sequences shown in have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity.

[0057] The inhibitory nucleic acids of the present disclosure comprise nucleotide sequences that bind to (hybridize with) a target TBXT nucleic acid. In some cases, the target TBXT nucleic acid is an mRNA encoding a TBXT polypeptide. In some cases, the target TBXT nucleic acid is a TBXT pre-mRNA. In some cases, the target TBXT nucleic acid comprises an intron 4 / exon 5 junction of a TBXT mRNA. The TBXT genomic nucleotide sequence is provided in NCBI Gene ID 6862. The target TBXT nucleotide sequence can be hybridized with Figure 13In some cases, the TBXT nucleotide sequence described in the present invention has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% nucleotide sequence identity. In some cases, the target TBXT nucleic acid comprises the nucleotide sequence CAGATCACAGCTCTTA (SEQ ID NO: 1). In some cases, the target TBXT nucleic acid comprises the nucleotide sequence AGATCACAGCTCTTAAA (SEQ ID NO: 2). In some cases, the target TBXT nucleic acid comprises the nucleotide sequence ATCACAGCTCTTAAAATT (SEQ ID NO: 3). In some cases, the target TBXT nucleic acid comprises the nucleotide sequence TTCAGATCAAGCTC (SEQ ID NO: 7). In some cases, the inhibitory nucleic acids of the present disclosure bind to (hybridize with) the TBXT target sequence CAGATCACAGCTCTTA (SEQ ID NO: 1). In some cases, the inhibitory nucleic acids of the present disclosure bind to (hybridize with) the TBXT target sequence AGATCACAGCTCTTAAA (SEQ ID NO: 2). In some cases, the inhibitory nucleic acids of the present disclosure bind to (hybridize with) the TBXT target sequence ATCACAGCTCTTAAAATT (SEQ ID NO: 3). In some cases, the inhibitory nucleic acids of the present disclosure bind to (hybridize with) the TBXT target sequence TTTTCAGATCACAGCTC (SEQ ID NO: 4). In some cases, the inhibitory nucleic acids of the present disclosure bind to (hybridize with) the TBXT target sequence TTTCAGATCACAGCTC (SEQ ID NO: 5). In some cases, the inhibitory nucleic acids of the present disclosure bind to (hybridize with) the TBXT target sequence TTTCAGATCACAGCTCT (SEQ ID NO: 6). In some cases, the inhibitory nucleic acids of the present disclosure bind to (hybridize with) the TBXT target sequence TTCAGATCACAGCTCT (SEQ ID NO: 7). In some cases, the inhibitory nucleic acids of the present disclosure bind to (hybridize with) the TBXT target sequence TTCAGATCACAGCTC (SEQ ID NO: 8).

[0058] The inhibitory nucleic acids of the present disclosure reduce the level of TBXT polypeptide in a cell (e.g., a target cell, such as a cancer, e.g., a chordoma) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, or more than 70% compared to the level of TBXT polypeptide in the cell in the absence of the inhibitory nucleic acid. In some cases, the inhibitory nucleic acids of the present disclosure inhibit splicing of TBXT transcripts. In some cases, the inhibitory nucleic acids of the present disclosure block translation of TBXT mRNA. In some cases, the inhibitory nucleic acids of the present disclosure increase cleavage of TBXT mRNA by RNase H.

[0059] In some cases, an inhibitory nucleic acid of the present disclosure inhibits cancer growth by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, or more than 70% compared to the level of cancer growth in the absence of the inhibitory nucleic acid.

[0060] Inhibitory nucleic acids for use in the present methods and compositions include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single-stranded or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified base / locked nucleic acid (LNA), antagomir, peptide nucleic acid (PNA) and other oligomeric compounds or oligonucleotide mimics that hybridize with at least a portion of a target nucleic acid (i.e., TBXT nucleic acid) and regulate its function. In some embodiments, inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interfering RNA (RNAi), short interfering RNA (siRNA); microinterfering RNA (miRNA); small molecule temporal RNA (stRNA); or short hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNA (saRNA) or a combination thereof. See, for example, WO 2010040112. In some cases, the inhibitory nucleic acid of the present disclosure is an ASO.

[0061] In some cases, the inhibitory nucleic acid is 10 to 50, 13 to 50, or 13 to 30 nucleotides in length. One of ordinary skill in the art will understand that this includes oligonucleotides having an antisense portion that is 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 nucleotides in length, or any range therein. In some cases, the inhibitory nucleic acid of the present disclosure is 15 nucleotides in length. In some cases, the inhibitory nucleic acid of the present disclosure is 12 to 30 or 13 to 30 nucleotides in length. One of ordinary skill in the art will understand that this includes inhibitory nucleic acids that are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0062] In some cases, an inhibitory nucleic acid of the present disclosure comprises a nucleotide sequence selected from the group consisting of:

[0063] +T*+A*A*G*+A*G*C*+T*G*T*+G*A*T*+C*+T*+G;

[0064] +T*+T*T*A*+A*G*A*+G*C*T*+G*T*G*+A*T*+C*+T;

[0065] +A*+A*T*T*+T*T*A*+A*G*A*+G*C*T*+G*T*+G*+A*+T;

[0066] +G*+A*+G*C*+T*G*T*+G*A*T*+C*+T*G*+A*A*+A*+A;

[0067] +G*+A*G*+C*+T*+G*T*+G*A*T*+C*T*G*+A*+A*+A;

[0068] +G*+A*G*C*+T*G*T*+G*A*+T*+C*T*+G*+A*+A*+A;

[0069] +A*+G*+A*G*C*+T*G*T*+G*A*T*+C*T*G*+A*+A*+A;

[0070] +A*+G*+A*G*C*+T*G*T*+G*A*T*+C*T*+G*A*+A*+A;

[0071] +G*+A*G*C*+T*G*+T*+G*A*T*+C*T*G*+A*+A; and

[0072] +A*+G*+A*G*C*+T*G*T*+G*A*T*+C*T*G*+A*+A,

[0073] Wherein * represents a phosphorothioate linkage, wherein a "+" preceding a nucleotide indicates that the nucleotide is an LNA. In some cases, the inhibitory nucleic acid is 15 nucleotides to 25 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides).

[0074] In some cases, inhibitory nucleic acids are chimeric oligonucleotides containing two or more chemically different regions, each region consisting of at least one nucleotide. These oligonucleotides typically contain at least one modified nucleotide region (which imparts one or more beneficial properties (e.g., increased nuclease resistance, increased uptake into cells, increased binding affinity to targets)) and a region that is a substrate for an enzyme capable of cutting RNA: DNA or RNA: RNA hybrids. Chimeric inhibitory nucleic acids of the present disclosure can be formed into a composite structure of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and / or oligonucleotide mimetics as described above. Such compounds are also referred to as hybrids or gapmers in the art.

[0075] In some embodiments, the inhibitory nucleic acid comprises at least one nucleotide modified at the 2' position of the sugar, such as a 2'-O-alkyl, 2'-O-alkyl-O-alkyl or 2'-fluoro modified nucleotide. In other cases, RNA modifications include 2'-fluoro, 2'-amino and 2'O-methyl modifications of the ribose of pyrimidines, abasic residues or the inverted bases at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides, and these oligonucleotides have been shown to have higher Tm (i.e., higher target binding affinity) than 2'-deoxy oligonucleotides for a given target.

[0076] Many nucleotide and nucleoside modifications have been shown to render the oligonucleotides into which they are incorporated more resistant to nuclease digestion than native oligodeoxynucleotides; these modified oligomers persist intact for longer periods of time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those containing modified backbones, e.g., phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl or cycloalkyl sugar linkages, or short chain heteroatom or heterocyclic sugar linkages. In some cases, the inhibitory nucleic acids are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2--NH--O--CH3, CH3--N(CH3)--O--CH2 (referred to as methylene(methylimino) or MMI backbones), CH2--O--N(CH3)--CH2, CH2--N(CH3)--N(CH3)--CH2, and O--N(CH3)--CH2--CH2 backbones, where the natural phosphodiester backbone is represented as O--P--O--CH), amide backbones (see De Mesmaeker et al., Ace. Chem. Res. 1995, 28: 366-374), morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506), peptide nucleic acid (PNA) backbones (in which the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone to which the nucleotides are directly or indirectly attached, see Nielsen et al., Science 1991, 254, 1497). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters; aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates including 3' alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those with reversed polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0077] The modified oligonucleotide backbones that do not contain phosphorus atoms have backbones formed from short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formyl and thioformyl backbones; methyleneformyl and thioformyl backbones; olefin-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazinyl backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S, and CH2 components.

[0078] One or more substituted sugar moieties may also be included, for example, one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3OCH3, OCH3, O(CH2)nCH3, O(CH2)nNH2 or O(CH2)nCH3, wherein n is 1 to about 10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O--, S-- or N-alkyl; O--, S-- or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleavage group; reporter group; intercalator; group for improving the pharmacokinetic properties of oligonucleotides; or group for improving the pharmacodynamic properties of oligonucleotides and other substituents with similar properties. Suitable modifications include 2'-methoxyethoxy [2'--O--CH2CH2OCH3, also known as: 2'-O-(2-methoxyethyl)] (Martin et al., Helv. Chim. Acta, 1995, 78, 486). Other modifications include 2'-methoxy (2'-O--CH3), 2'-propoxy (2'-OCH2CH2CH3) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions of the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of the 5' terminal nucleotide. Oligonucleotides can also be replaced with sugar mimetics (such as cyclobutyl) instead of the pentofuranosyl group.

[0079] Additionally or alternatively, the inhibitory nucleic acid may also include modifications or substitutions of nucleobases (often referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleotides include nucleobases that occur only rarely or transiently in natural nucleic acids, for example, hypoxanthine, 6-methyladenine, 5-methylpyrimidine, especially 5-methylcytosine (also known as 5-methyl-2'deoxycytosine, commonly referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentiobiosyl HMC, and synthetic nucleobases, for example, 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6-diaminopurine. Also included are "universal" bases known in the art, such as inosine. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-12° C. In some cases, the inhibitory nucleic acids of the present disclosure comprise one or more 5-Me-Cs.

[0080] It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and, in fact, more than one of the above modifications may be incorporated in a single oligonucleotide, or even in a single nucleoside within an oligonucleotide.

[0081] In some embodiments, the sugar and internucleoside linkages of the nucleotide units, i.e., the main chain, are replaced by novel groups. The base unit is kept for hybridization with a suitable nucleic acid target compound. A type of such oligomeric compound (an oligonucleotide mimic that has been shown to have excellent hybridization properties) is referred to as peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced by an amide-containing main chain, such as an aminoethylglycine main chain. The core base is retained and is directly or indirectly combined with the nitrogen-nitrogen atom of the amide portion of the main chain. Representative U.S. patents for teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, each of which is incorporated herein by reference. Further teachings of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0082] Inhibitory nucleic acids may also include one or more modifications or substitutions of nucleobases (often referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, 6-azocytosine, and 6-azothymine. and 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0083] In addition, nucleobases include those disclosed in U.S. Pat. No. 3,687,808, The Concise Encyclopedia of Polymer Science And Engineering, pp. 858-859, Kroschwitz, JI, ed., John Wiley & Sons, 1990, those disclosed by English et al., Angewandle Chemie, International Edition, 1991, 30, p. 613, and those disclosed by Sanghvi, YS, Chapter 15, Antisense Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B.ea., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of inhibitory nucleic acids. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, YS, Crooke, ST, and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are suitable for inclusion in inhibitory nucleic acids, for example, alone or in combination with 2′-O-methoxyethyl sugar modifications.

[0084] In some cases, the inhibitory nucleic acid is chemically linked to one or more moieties or conjugates that enhance oligonucleotide activity, cellular distribution, or cellular uptake. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties; cholic acid; thioethers, such as hexyl-S-tritylthiol; thiocholesterol; fatty chains, such as dodecandiol or undecyl residues; phospholipids; for example, di-hexadecyl-racem-glycerol or triethylammonium 1,2-di-O-hexadecyl-racem-glycerol-3-H-phosphonate; polyamines or polyethylene glycol chains; adamantaneacetic acid; palmitoyl moieties; octadecylamine moieties; or hexylamino-carbonyl-t oxycholesterol moieties.

[0085] These parts or conjugates can include conjugated groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Suitable conjugated groups include intercalators, importer molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugated groups include cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. Groups that enhance pharmacodynamic properties include groups that enhance uptake, enhance resistance to degradation, and / or enhance sequence-specific hybridization with target nucleic acids. Groups that enhance pharmacokinetic properties include groups that improve the uptake, distribution, metabolism, or excretion of inhibitory nucleic acids. Representative conjugated groups are disclosed in International Patent Application No. PCT / US92 / 09196, filed October 23, 1992, and U.S. Patent No. 6,287,860, which are incorporated herein by reference. Conjugated moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or polyethylene glycol chain, or adamantaneacetic acid, a palmitoyl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

[0086] The inhibitory nucleic acids used in the methods of the present invention are sufficiently complementary to all or part of a TBXT nucleic acid, i.e., they hybridize sufficiently well and with sufficient specificity to produce the desired effect. "Complementary" refers to the ability of two sequences, comprising naturally or non-naturally occurring bases or their analogs, to pair through hydrogen bonding. For example, if a base at one position in an inhibitory nucleic acid is capable of forming a hydrogen bond with a base at the corresponding position in a TBXT sequence, the bases are considered complementary to each other at that position. 100% complementarity is not required.

[0087] In the context of the present disclosure, hybridization refers to hydrogen bonding between complementary nucleoside or nucleotide bases, which can be Watson-Crick hydrogen bonding, Hoogsteen hydrogen bonding, or reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds. As used herein, "complementary" refers to the ability of two nucleotides to precisely pair together. An inhibitory nucleic acid and a TBXT nucleic acid are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that are capable of hydrogen bonding to each other. Thus, "specifically hybridizable" and "complementary" are terms used to indicate a sufficient degree of complementarity or precise pairing to allow stable and specific binding to occur between the inhibitory nucleic acid and the TBXT target sequence. For example, if a base at one position of an inhibitory nucleic acid is capable of forming a hydrogen bond with a base at the corresponding position of a TBXT nucleic acid molecule, then the bases are considered complementary at that position.

[0088] Typically, the inhibitory nucleic acid used in the methods described herein has at least 80% sequence complementarity with the target region within the target nucleic acid, for example, 90%, 95% or 100% sequence complementarity with the target region within the TBXT nucleic acid. For example, an antisense compound in which 18 of the 20 nucleobases of the antisense oligonucleotide are complementary to the target region and therefore specifically hybridize therewith will represent 90% complementarity. The percentage complementarity of the inhibitory nucleic acid to the target nucleic acid region can be determined routinely using a basic local alignment search tool (BLAST program) (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Inhibitory nucleic acids (e.g., ASOs) that hybridize to the TBXT target sequence can be identified by routine experiments. In general, the inhibitory nucleic acid must maintain specificity for its target, i.e., it must not directly bind to or directly significantly affect the expression level of transcripts other than the intended target.

[0089] Antonyms

[0090] As described above, in some cases, the inhibitory nucleic acids of the present disclosure are ASOs. ASOs are generally designed to block the expression of a DNA or RNA target by binding to the target and stopping expression at the transcriptional, translational, or splicing levels. The ASOs of the present disclosure are complementary nucleic acid sequences that are designed to hybridize to the TBXT target sequence under stringent conditions. Therefore, oligonucleotides are selected that are sufficiently complementary to the target, i.e., oligonucleotides that hybridize sufficiently and have sufficient specificity to produce the desired effect.

[0091] Modified bases / locked nucleic acids (LNA)

[0092] In some cases, the inhibitory nucleic acid of the present disclosure comprises one or more modified bonds or bases. Modified bases include phosphorothioates, methylphosphonates, peptide nucleic acids, or locked nucleic acid (LNA) molecules. For example, in some cases, the modified nucleotides are locked nucleic acid molecules, including [α]-L-LNA. LNA comprises a ribonucleic acid analog in which the ribose ring is "locked" by a methylene bridge between the 2'-oxygen and the 4'-carbon - that is, an oligonucleotide containing at least one LNA monomer (i.e., a 2'-O, 4'-C-methylene-β-D-ribofuranosyl nucleotide). LNA bases form standard Watson-Crick base pairs, but the locked configuration increases the rate and stability of the base pairing reaction (Jepsen et al., Oligonucleotides, 14, 130-146 (2004)). Compared to the affinity of DNA base pairing, LNA also has an enhanced affinity for base pairing with RNA. These properties make LNAs particularly suitable as probes for fluorescence in situ hybridization (FISH) and comparative genomic hybridization, as tools for miRNA knockdown, and as antisense oligonucleotides targeting mRNA or other RNA.

[0093] LNA molecules can include molecules comprising 10-30, for example 12-24, for example 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in each strand, wherein one of the strands is substantially identical to the TBXT target sequence, for example at least 80% (or more, for example, 85%, 90%, 95% or 100%) identical, for example with 3, 2, 1 or 0 mismatched nucleotides. LNA molecules can be chemically synthesized using methods known in the art.

[0094] Antagomir

[0095] In some cases, the inhibitory nucleic acid is an antagomir. An antagomir is a chemically modified antisense oligonucleotide that targets a TBXT target nucleotide sequence. For example, an antagomir for use in the methods described herein can include a nucleotide sequence that is sufficiently complementary to a TBXT target sequence of about 12 to 25 nucleotides or about 15 to 23 nucleotides to hybridize therewith.

[0096] Typically, antagomirs include a cholesterol moiety, for example, at the 3' end. In some embodiments, antagomirs have various modifications for RNase protection and pharmacological properties (such as enhanced tissue and cellular uptake). For example, in addition to the modifications of the antisense oligomers discussed above, antagomirs may also have one or more of complete or partial 2'-O-methylation of sugars and / or a phosphorothioate backbone. Phosphorothioate modifications provide protection against RNase activity, and their lipophilicity helps enhance tissue uptake. In some embodiments, antagomirs may include six phosphorothioate backbone modifications; two phosphorothioates are located at the 5' end and four at the 3' end. Antagomirs used in the present method may also be modified in terms of the length of the antagomir or the number of nucleotides that make up the antagomir. The antagomir must maintain specificity for its target, i.e., it must not directly bind to or directly significantly affect the expression level of transcripts other than the intended target. In some embodiments, the inhibitory nucleic acid is locked and includes a cholesterol moiety (e.g., a locked antagomir).

[0097] siRNA / shRNA

[0098] In some cases, the inhibitory nucleic acids of the present disclosure are interfering RNAs, including but not limited to small interfering RNAs ("siRNAs") or small hairpin RNAs ("shRNAs"). Methods of constructing interfering RNAs are well known in the art. For example, an interfering RNA can be assembled from two separate oligonucleotides, one of which is a sense strand and the other is an antisense strand, wherein the antisense strand and the sense strand are self-complementary (i.e., each strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the other strand; such as where the antisense strand and the sense strand form a duplex or double-stranded structure); the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (i.e., an undesired gene), and the sense strand comprises a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof. Alternatively, the interfering RNA is assembled from a single oligonucleotide in which the self-complementary sense and antisense regions are connected by one or more nucleic acid-based or non-nucleic acid-based linkers. The interfering RNA can be a polynucleotide having a duplex, an asymmetric duplex, a hairpin, or an asymmetric hairpin secondary structure, having a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence complementary to a nucleotide sequence in a separate target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The interferor can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed in vivo or in vitro to produce an active siRNA molecule capable of mediating RNA interference.

[0099] In some cases, the interfering RNA coding region encodes a self-complementary RNA molecule having a sense region, an antisense region, and a loop region. Such RNA molecules ideally form a "hairpin" structure when expressed and are referred to herein as "shRNA." The length of the loop region is typically about 2 to about 10 nucleotides. In some embodiments, the length of the loop region is about 6 to about 9 nucleotides. In some embodiments, the length of the sense region and the antisense region is about 15 to about 20 nucleotides. After post-transcriptional processing, the small hairpin RNA is converted into siRNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family. The siRNA is then able to inhibit the expression of genes to which it has homology.

[0100] siRNA-guided target RNA cleavage reactions have a high degree of sequence specificity. In general, siRNAs containing a nucleotide sequence identical to a portion of the target nucleic acid are used for inhibition. However, 100% sequence identity is not required between the siRNA and the target gene. Therefore, the present disclosure has the advantage of being able to tolerate sequence variations that may be expected due to genetic mutations, strain polymorphisms, or evolutionary divergence. For example, siRNA sequences with insertions, deletions, and single-point mutations relative to the target sequence have also been found to be effective for inhibition. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition. In general, siRNA must maintain specificity for its target, i.e., it must not directly bind to or directly significantly affect the expression levels of transcripts other than the intended target.

[0101] Preparation of inhibitory nucleic acids

[0102] Inhibitory nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques as described, for example, in Adams (1983) J. Am. Chem. Soc. 105: 661; Belousov (1997) Nucleic Acids Res. 25: 3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19: 373-380; Blommers (1994) Biochemistry 33: 7886-7896; Narang (1979) Meth. Enzymol. 68: 90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Pat. No. 4,458,066.

[0103] Pharmaceutical composition

[0104] The present disclosure provides compositions (including pharmaceutical compositions) comprising the inhibitory nucleic acids of the present disclosure. The inhibitory nucleic acids of the present disclosure may be referred to hereinafter as "agents" or "active agents."

[0105] In some cases, the composition is formulated with a pharmaceutically acceptable carrier. Pharmaceutical compositions and preparations can be administered parenterally (e.g., intravenously or intramuscularly), topically, orally, or by local administration (such as by intratumoral or peritumoral administration). Pharmaceutical compositions can be formulated in any manner and can be administered in a variety of unit dosage forms, depending on the condition or disease and the extent of the disease, the general medical condition of each patient, the preferred method of administration, etc. The technical details of the preparation and administration of drugs are described in detail in the scientific and patent literature, see, for example, Remington: The Science and Practice of Pharmacy, 21st edition, 2005.

[0106] Inhibitory nucleic acids can be used alone or as a component of a pharmaceutical preparation (composition). Inhibitory nucleic acids can be formulated for use in any convenient manner for human or veterinary use. Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweeteners, flavorings, and aromatics, preservatives, and antioxidants can also be present in the composition.

[0107] The preparations of the inhibitory nucleic acid of the present disclosure include preparations suitable for intradermal, inhalation, oral / nasal, topical, parenteral, rectal and / or intravaginal administration. The preparation can be conveniently presented in the form of a unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of the active ingredient (e.g., inhibitory nucleic acid of the present disclosure) that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated, the specific mode of administration (e.g., intradermal or inhalation). The amount of the active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount that produces a therapeutic effect, such as an antigen-specific T cell or humoral response compound.

[0108] Pharmaceutical preparations can be prepared according to any method known in the art of pharmaceutical manufacturing. Such medicines may contain sweeteners, flavorings, coloring agents, and preservatives. The preparations may be mixed with non-toxic, pharmaceutically acceptable excipients suitable for manufacture. The preparations may contain one or more diluents, emulsifiers, preservatives, buffers, excipients, etc., and may be provided in forms such as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.

[0109] Pharmaceutical preparations for oral administration can be formulated with appropriate and suitable dosages using pharmaceutically acceptable carriers well known in the art. Such carriers enable the drug to be formulated into unit dosage forms suitable for patient absorption, such as tablets, pills, powders, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc. Pharmaceutical preparations for oral use can be formulated into solid excipients, optionally grinding the resulting mixture, and processing the granular mixture after adding suitable additional compounds (if necessary) to obtain tablets or dragee cores. Suitable solid excipients are carbohydrates or protein fillers, including, for example, sugars, including lactose, sucrose, mannitol or sorbitol; starch from corn, wheat, rice, potato or other plants; cellulose, such as methylcellulose, hydroxypropyl methylcellulose or sodium carboxymethylcellulose; and gums, including gum arabic and tragacanth; and proteins, such as gelatin and collagen. Disintegrants or solubilizers, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or its salts, such as sodium alginate, can be added. Push-fit capsules can contain the active agent mixed with a filler or binder such as lactose or starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active agent can be dissolved or suspended in a suitable liquid such as a fatty oil, liquid paraffin, or liquid polyethylene glycol with or without a stabilizer.

[0110] Aqueous suspensions can contain an active agent (e.g., an inhibitory nucleic acid of the present disclosure) mixed with excipients suitable for making aqueous suspensions (e.g., for aqueous intradermal injection). Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic, and dispersants or wetting agents such as naturally occurring phospholipids (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain fatty alcohols (e.g., heptadecaethoxyhexadecanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives (such as ethyl or n-propyl p-hydroxybenzoate), one or more coloring agents, one or more flavoring agents, and one or more sweetening agents (such as sucrose, aspartame, or saccharin). The osmolarity of the formulation may be adjusted.

[0111] In some cases, oil-based drugs are used for the administration of inhibitory nucleic acids. Oil-based suspensions can be prepared by suspending the active agent in a vegetable oil (such as peanut oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin) or in a mixture of these oils. See, for example, U.S. Patent No. 5,716,928, which describes the use of essential oils or essential oil components to increase the bioavailability of hydrophobic drug compounds for oral administration and reduce inter- and intra-individual variability (see also U.S. Patent No. 5,858,401). Oil suspensions can contain thickeners such as beeswax, hard paraffin or cetyl alcohol. Sweeteners (such as glycerol, sorbitol or sucrose) can be added to provide a palatable oral formulation. These preparations can be preserved by adding antioxidants such as ascorbic acid. As an example of an injectable oil vehicle, see Minto (1997) J.Pharmacol.Exp.Ther.281:93-102.

[0112] Pharmaceutical preparations can also be in the form of oil-in-water emulsions. The oil phase can be a vegetable oil or mineral oil as described above, or a mixture of these oils. Suitable emulsifiers include naturally occurring gums (such as gum arabic and tragacanth), naturally occurring phospholipids (such as soy lecithin), esters or partial esters derived from fatty acids and hexitol anhydrides (such as sorbitan monooleate) and condensation products of these partial esters with ethylene oxide (such as polyoxyethylene sorbitan monooleate). Emulsions can also contain sweeteners and flavorings, such as in the preparation of syrups and elixirs. Such preparations can also contain a demulcent, a preservative, or a coloring agent. In alternative embodiments, injectable oil-in-water emulsions include paraffin oil, sorbitan monooleate, ethoxylated sorbitan monooleate, and / or ethoxylated sorbitan trioleate.

[0113] The pharmaceutical composition can be administered by intranasal, intraocular, and intravaginal routes, including suppositories, insufflations, powders, and aerosol formulations (for examples of steroid inhalers, see, e.g., Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107-111). Suppository formulations can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at body temperature and therefore melts in the body to release the drug. Examples of such materials are cocoa butter and polyethylene glycol.

[0114] In some cases, the pharmaceutical compositions can be delivered transdermally via a topical route and formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0115] In some cases, the pharmaceutical composition can be delivered as microspheres for slow release in vivo. For example, the microspheres can be administered by intradermal injection of the drug, which is slowly released subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7: 623-645; as a biodegradable and injectable gel formulation, see, for example, Gao (1995) Pharm. Res. 12: 857-863 (1995); or, as microspheres for oral administration, see, for example, Eyles (1997) J. Pharm. Pharmacol. 49: 669-674.

[0116] In some cases, the pharmaceutical composition can be administered parenterally, such as by intravenous (IV) administration or administration into the lumen of a body cavity or organ. These preparations can include solutions of active agents (e.g., inhibitory nucleic acids of the present disclosure) in pharmaceutically acceptable carriers. Acceptable vehicles and solvents that can be used are water and Ringer's solution (an isotonic sodium chloride). In addition, sterile, non-volatile oils can be used as solvents or suspension media. For this reason, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can also be used for the preparation of injections. These solutions are sterile and generally do not contain undesirable substances. These preparations can be sterilized by conventional, well-known sterilization techniques. The preparations can contain pharmaceutically acceptable auxiliary substances required for close physiological conditions, such as pH adjustment and buffering agents, toxicity regulators, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the active agent in these preparations can vary widely and will be selected based primarily on fluid volume, viscosity, body weight, etc., according to the specific mode of administration selected and the needs of the patient. For intravenous administration, the preparation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. The suspension can be prepared using those suitable dispersants or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a non-toxic parenteral acceptable diluent or solvent, such as 1,3-butanediol solution. Administration can be carried out by bolus injection or continuous infusion (e.g., introduced into a blood vessel substantially uninterruptedly over a specified period of time).

[0117] In some cases, the pharmaceutical composition can be lyophilized. The stable lyophilized composition comprising inhibitory nucleic acid can be prepared by lyophilizing a solution comprising a pharmaceutical composition of the present disclosure and a filler (e.g., mannitol, trehalose, raffinose and sucrose or a mixture thereof). The method for preparing a stable lyophilized formulation can include lyophilizing a solution of about 2.5 mg / mL nucleic acid, about 15 mg / mL sucrose, about 19 mg / mL NaCl and a sodium citrate buffer solution having a pH greater than 5.5 but less than 6.5. See, for example, U.S. Patent No. 20040028670.

[0118] Compositions and preparations can be delivered by using liposomes. By using liposomes, especially when the liposome surface carries a ligand specific to the target cell, or preferentially directs to a specific organ in other ways, it is possible to focus on delivering the active agent (e.g., the inhibitory nucleic acid of the present disclosure) to the target cell in vivo. See, for example, U.S. Patent Nos. 6,063,400 and 6,007,839; Al-Muhammed (1996) J.Microencapsul.13:293-306; Chonn (1995) Curr.Opin.Biotechnol.6:698-708; Ostro (1989) Am.J.Hosp.Pharm.46:1576-1587. As used herein, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in one or more bilayers. Liposomes are unilamellar or multilamellar vesicles having a membrane formed of a lipophilic material and an aqueous interior containing the composition to be delivered. Cationic liposomes are positively charged liposomes that are thought to interact with negatively charged DNA molecules to form stable complexes. pH-sensitive or negatively charged liposomes are thought to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0119] Liposomes can also include "sterically stabilized" liposomes, i.e., liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes having an extended circulation life relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Liposomes and their uses are further described in U.S. Patent No. 6,287,860.

[0120] The formulations of the present disclosure can be administered for preventive and / or therapeutic treatments. In some cases, for therapeutic applications, a composition is administered to a subject in need thereof (e.g., an individual at risk (e.g., at greater risk than the general population) or suffering from a disorder described herein) in an amount sufficient to cure, alleviate, or partially prevent the clinical manifestations of a disorder or its complications; this can be referred to as a therapeutically effective amount.

[0121] The amount of the pharmaceutical composition sufficient to achieve this is a therapeutically effective dose. The dosage regimen and amount effective for this application, i.e., the dosing regimen, will depend on a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the overall state of the patient's health, the patient's physical condition, age, etc. The mode of administration should also be considered when calculating the dosing regimen for a patient.

[0122] The dosing regimen also takes into account pharmacokinetic parameters well known in the art, i.e., absorption rate, bioavailability, metabolism, clearance rate, etc. of the active agent (see, e.g., Remington: The Science and Practice of Pharmacy, 21st edition, 2005). The prior art allows clinicians to determine a dosing regimen for each individual patient, active agent (e.g., inhibitory nucleic acid), and disease or condition being treated. The guidance provided for similar compositions used as drugs can be used as a guide for determining a dosing regimen (i.e., dosage schedule and dosage level).

[0123] Single or multiple administrations of the formulation can be given, depending on, for example, the dosage and frequency required and tolerated by the patient, the extent and amount of therapeutic effect produced following each administration (e.g., effect on blood glucose levels), etc. The formulation should provide a sufficient amount of the active agent (e.g., inhibitory nucleic acid) to effectively treat, prevent, or ameliorate the condition, disease, or symptom.

[0124] In an alternative embodiment, the daily amount of the pharmaceutical preparation for oral administration is 1 μg nucleic acid / kg body weight / day to 100mg nucleic acid / kg body weight / day. Compared with oral administration, lower dosage can be used to enter the bloodstream, enter the body cavity or enter the lumen of an organ. Significantly higher dosage can be used in local or oral administration or by powder, spray or inhalation. The actual method of preparing the preparation that can be parenteral or non-parenteral is known or obvious to those skilled in the art, and is described in more detail in publications such as Remington:The Science and Practice of Pharmacy, the 21st edition, 2005.

[0125] In some embodiments, the methods described herein may include co-administration with other agents or drugs (e.g., compositions for lowering blood glucose levels). For example, inhibitory nucleic acids can be co-administered with drugs for treating or reducing the risk of a disorder described herein.

[0126] Treatment

[0127] The present disclosure provides methods for inhibiting cancer proliferation in an individual and methods for treating cancer. The methods include administering an effective amount of an inhibitory nucleic acid of the present disclosure, or a pharmaceutical composition comprising an inhibitory nucleic acid of the present disclosure, to an individual in need thereof (e.g., an individual suffering from cancer).

[0128] In some cases, an effective amount of an inhibitory nucleic acid of the present invention is an amount that, when administered to an individual in need thereof at one or more doses, reduces the tumor mass / tumor volume of the individual. In some cases, an "effective amount" of an inhibitory nucleic acid is an amount that, when administered to an individual in need thereof at one or more doses, prolongs the survival of the individual. For example, in some cases, an "effective amount" of an inhibitory nucleic acid is an amount that, when administered to an individual in need thereof at one or more doses, prolongs the survival of the individual by at least 1 month, at least 2 months, at least 3 months, 3 months to 6 months, 6 months to 1 year, 1 year to 2 years, 2 years to 5 years, 5 years to 10 years, or more than 10 years, compared to the expected survival of an individual not administered the inhibitory nucleic acid.

[0129] In some cases, an "effective amount" of an inhibitory nucleic acid is an amount that, when administered in one or more doses to an individual in need thereof, whether as a monotherapy or as part of a combination therapy, reduces the individual's overall tumor burden, i.e., the amount of cancer in the body, or, alternatively, keeps the patient's overall tumor burden relatively stable for a sufficient period of time so that the patient has confirmed "stable disease" as determined by standard RECIST criteria. See, e.g., Aykan and (2020) World J. Clin. Oncol. 11:53.

[0130] In some cases, an effective amount of an inhibitory nucleic acid is an amount that, when administered in one or more doses to an individual in need thereof, whether as monotherapy or as part of a combination therapy, reduces tumor size by a sufficient amount, and for a sufficient period of time, for the patient to have a confirmed "partial response" as determined by standard Response Evaluation Criteria in Solid Tumors (RECIST) criteria.

[0131] In some cases, an effective amount of an inhibitory nucleic acid is an amount that, when administered in one or more doses to an individual in need thereof (e.g., an individual having a tumor), whether as a monotherapy or as part of a combination therapy, reduces tumor size by an amount sufficient, and for a period of time sufficient, for the patient to have a confirmed "complete response" as determined by standard RECIST criteria.

[0132] Cancers that can be treated by the methods disclosed herein include, but are not limited to, chordoma, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (of various tissues), bladder cancer (including transitional cell carcinoma (a malignant tumor of the bladder)), bronchogenic carcinoma, colon cancer, colorectal cancer, stomach cancer, lung cancer (including small cell carcinoma and non-small cell carcinoma of the lung), adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteoblastic cancer, epithelial cancer, and nasopharyngeal cancer. In some instances, the cancer is chordoma.

[0133] dose

[0134] The appropriate dosage of inhibitory nucleic acid can be determined by the attending physician or other qualified medical personnel based on various clinical factors. As is well known in the medical field, the dosage of any one patient depends on many factors, including the patient's body shape, body surface area, age, specific polypeptide to be administered or nucleic acid, the patient's sex, administration time and route, overall health status and other drugs administered simultaneously. The inhibitory nucleic acid of the present disclosure can be administered in an amount of 1ng / kg body weight to 20mg / kg body weight or higher (e.g., 0.1mg / kg body weight to 10mg / kg body weight, e.g., 1mg / kg body weight to 5mg / kg body weight or 5mg / kg body weight to 10mg / kg body weight, 10-15mg / kg or higher) per dose; however, dosages lower than or higher than this exemplary range are foreseeable.

[0135] The frequency of administration of inhibitory nucleic acid can change according to any one of various factors, but in general will be used once a week, used once every two weeks, used once every three weeks, used once every four weeks, used once monthly, or used with a frequency lower than once a month, for example, used once every five weeks, used once every six weeks, used once every two months, used once every three months, etc., but can also be used more frequently than once a week, for example, twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod) or once a day (qd). In some cases, inhibitory nucleic acid is used once every three weeks. When disease progression or unacceptable toxicity occur, usually should stop using.

[0136] The duration of administration of the inhibitory nucleic acid can vary, depending on any of a variety of factors, such as patient response, etc. For example, the inhibitory nucleic acid can be administered over a period ranging from 1 month to about 2 months, from about 2 months to about 4 months, from about 4 months to about 6 months, from about 6 months to about 8 months, from about 8 months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or longer. In some cases, the inhibitory nucleic acid will continue to be administered for at least as long as the patient continues to obtain a clinically determined benefit, which can be at least many months to several years.

[0137] Route of administration

[0138] Suitable routes of administration include oral, rectal, nasal, pulmonary, topical, subcutaneous, intramuscular, intraperitoneal, intravenous, intradermal, intrathecal, epidural, intracranial, intraspinal, intratumoral, and peritumoral. In some cases, the route of administration is intramuscular. In some cases, the route of administration is intravenous. In some cases, the route of administration is intracranial. In some cases, the route of administration is intraspinal. In some cases, the route of administration is intratumoral.

[0139] Combination therapy

[0140] The present disclosure contemplates the use of inhibitory nucleic acids of the present disclosure in combination with one or more additional agents (e.g., one or more additional active therapeutic agents) or other preventive or therapeutic modalities. In such combination therapies, the various active agents typically have different mechanisms of action. Such combination therapies may be particularly advantageous because they can reduce the dosage of one or more agents, thereby reducing or eliminating adverse reactions associated with one or more of the agents; in addition, such combination therapies can have a synergistic therapeutic or preventive effect on the underlying disease, disorder, or condition.

[0141] In some cases, the present disclosure provides methods for treating individual cancers including: a) administering an inhibitory nucleic acid of the present disclosure; and b) administering at least one additional therapeutic agent or therapeutic treatment. Suitable additional therapeutic agents include, but are not limited to, small molecule cancer chemotherapeutics and immune checkpoint inhibitors. Suitable additional therapeutic treatments include, for example, irradiation, surgery (e.g., surgical resection of a tumor), and the like.

[0142] As used herein, "combination" is meant to include therapies that can be administered separately, e.g., formulated separately for separate administration (e.g., as may be provided in a kit), and therapies that can be administered together in a single formulation (i.e., a "co-formulation").

[0143] In some cases, the inhibitory nucleic acid of the present disclosure and at least one additional agent can be administered or applied sequentially, for example, wherein one agent is administered before one or more other agents. In other cases, the inhibitory nucleic acid of the present disclosure and at least one additional agent are administered simultaneously, for example, wherein two or more agents are administered simultaneously or approximately simultaneously; the two or more drugs can be present in two or more separate formulations, or combined into a single formulation (i.e., a co-formulation). Regardless of whether two or more agents are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of the present disclosure.

[0144] Suitable additional therapeutic agents include, for example, cancer chemotherapeutics, immune checkpoint inhibitors, immunotherapeutics, and the like.

[0145] Subjects

[0146] Subjects suitable for treatment with the methods of the present disclosure include individuals who have been diagnosed with cancer, individuals who have received cancer treatment but have not responded to treatment, and individuals who have received cancer treatment and initially responded but subsequently became refractory to treatment and / or whose disease progressed while on prior treatment.

[0147] Examples of non-limiting aspects of the present disclosure

[0148] The various aspects of the present subject matter described above, including embodiments, may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the present disclosure are provided below. As will be apparent to those skilled in the art upon reading this disclosure, each individually numbered aspect may be used or combined with any preceding or following individually numbered aspect. This is intended to provide support for all such combinations of aspects and is not limited to the combinations of aspects explicitly provided below:

[0149] Aspect 1. An inhibitory nucleic acid comprising a nucleotide sequence complementary to a target nucleotide sequence in a TBXT transcript, wherein the inhibitory nucleic acid comprises one or more locked nucleic acids (LNAs).

[0150] Aspect 2. The inhibitory nucleic acid of aspect 1, wherein the inhibitory nucleic acid has a length of about 15 nucleotides to about 30 nucleotides.

[0151] Aspect 3. The inhibitory nucleic acid of aspect 1, wherein the inhibitory nucleic acid has a length of about 15 nucleotides to about 20 nucleotides.

[0152] Aspect 4. The inhibitory nucleic acid of any of aspects 1-3, wherein the nucleotide sequence complementary to the nucleotide sequence in the TBXT transcript is complementary to the nucleotide sequence at the intron 4 / exon 5 junction.

[0153] Aspect 5. The inhibitory nucleic acid of any of aspects 1-3, wherein the target TBXT nucleotide sequence is selected from the group consisting of:

[0154] CAGATCACAGCTCTTA(SEQ ID NO:1);

[0155] AGATCACAGCTCTTAAA(SEQ ID NO:2);

[0156] ATCACAGCTCTTAAAATT(SEQ ID NO:3);

[0157] TTTTCAGATCACAGCTC(SEQ ID NO:4);

[0158] TTTCAGATCACAGCTC(SEQ ID NO:5);

[0159] TTTCAGATCACAGCTCT(SEQ ID NO:6);

[0160] TTCAGATCACAGCTC (SEQ ID NO: 7); and

[0161] TTCAGATCACAGCTCT (SEQ ID NO:8).

[0162] Aspect 6. The inhibitory nucleic acid of any of aspects 1-5, wherein the inhibitory nucleic acid comprises one or more phosphorothioate linkages.

[0163] Aspect 7. The inhibitory nucleic acid of any one of aspects 1 to 6, wherein the inhibitory nucleic acid comprises a nucleotide sequence selected from the group consisting of:

[0164] +T*+A*A*G*+A*G*C*+T*G*T*+G*A*T*+C*+T*+G;

[0165] +T*+T*T*A*+A*G*A*+G*C*T*+G*T*G*+A*T*+C*+T;

[0166] +A*+A*T*T*+T*T*A*+A*G*A*+G*C*T*+G*T*+G*+A*+T;

[0167] +G*+A*+G*C*+T*G*T*+G*A*T*+C*+T*G*+A*A*+A*+A;

[0168] +G*+A*G*+C*+T*+G*T*+G*A*T*+C*T*G*+A*+A*+A;

[0169] +G*+A*G*C*+T*G*T*+G*A*+T*+C*T*+G*+A*+A*+A;

[0170] +A*+G*+A*G*C*+T*G*T*+G*A*T*+C*T*G*+A*+A*+A;

[0171] +A*+G*+A*G*C*+T*G*T*+G*A*T*+C*T*+G*A*+A*+A;

[0172] +G*+A*G*C*+T*G*+T*+G*A*T*+C*T*G*+A*+A; and

[0173] +A*+G*+A*G*C*+T*G*T*+G*A*T*+C*T*G*+A*+A,

[0174] wherein * indicates a phosphorothioate linkage, and wherein a "+" immediately preceding a nucleotide indicates that the nucleotide is an LNA.

[0175] Aspect 8. A composition comprising:

[0176] a) the inhibitory nucleic acid of any one of aspects 1-7; and

[0177] b) pharmaceutically acceptable excipients.

[0178] Aspect 9. The pharmaceutical composition of Aspect 8, wherein the pharmaceutically acceptable excipient comprises one or more lipids.

[0179] Aspect 10. The pharmaceutical composition of Aspect 8, wherein the pharmaceutically acceptable excipient comprises poly(amidoamine), poly(propyleneimine), or poly(L-lysine).

[0180] Aspect 11. A lipid nanoparticle comprising:

[0181] a) the inhibitory nucleic acid of any one of aspects 1-7; and

[0182] b) pharmaceutically acceptable excipients.

[0183] Aspect 12. A method of treatment comprising administering to an individual in need thereof an effective amount of the inhibitory nucleic acid of any one of aspects 1-7, the pharmaceutical composition of any one of aspects 8-10, or the lipid nanoparticle of aspect 11.

[0184] Aspect 13. A method of inhibiting cancer proliferation in an individual, the method comprising administering to the individual an effective amount of the inhibitory nucleic acid of any one of aspects 1-7, the pharmaceutical composition of any one of aspects 8-10, or the lipid nanoparticle of aspect 11.

[0185] Aspect 14. The method of Aspect 13, wherein the cancer is lung cancer, breast cancer, colon cancer, prostate cancer or liver cancer.

[0186] Aspect 15. The method of aspect 13, wherein the cancer is chordoma.

[0187] Aspect 16. A method of treating cancer in an individual, comprising administering to the individual an effective amount of the inhibitory nucleic acid of any one of aspects 1-7, the pharmaceutical composition of any one of aspects 8-10, or the lipid nanoparticle of aspect 11.

[0188] Aspect 17. The method of Aspect 16, wherein the cancer is lung cancer, breast cancer, colon cancer, prostate cancer or liver cancer.

[0189] Aspect 18. The method of aspect 16, wherein the cancer is chordoma.

[0190] Aspect 19. The method of any one of aspects 16-18, wherein said administration comprises an intravenous, intramuscular, intratumoral or peritumoral administration route.

[0191] Aspect 20. The method of any one of aspects 16-19, further comprising administering one or more additional therapeutic treatments.

[0192] Aspect 21. The method of Aspect 20, wherein the one or more additional therapeutic treatments comprise cancer chemotherapy, radiation, or surgery. Example

[0193] The following examples are presented so as to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the invention and are not intended to limit the scope of what the inventors believe to be their invention nor to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.) but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pairs; kb, kilobase; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acid; kb, kilobase; bp, base pairs; nt, nucleotide; im, intramuscular (m); ip, intraperitoneal (m); sc, subcutaneous (m); etc.

[0194] Example 1:

[0195] method

[0196] Cell culture

[0197] Chordoma cell lines were obtained from ATCC and maintained in a medium containing a 4:1 ratio of IMDM:RPMI plus 10% FBS and non-essential amino acids and glutamine supplementation. All cell lines were grown on tissue culture plastic except UCH-2, which was grown on type I collagen-coated flasks. The U2OS cell line was obtained from the UC Berkeley Cell Culture Facility and maintained in DMEM high glucose + 10% FBS.

[0198] antisense oligonucleotides

[0199] ASO design was performed by a contracted third party and ordered from IdT Technologies. Lyophilized ASOs were resuspended in water at 200 μM and stored at -20°C. ASOs were further diluted in cell culture PBS for use in the assay. A scrambled control mixed-polymer LNA ASO (#406, sequence: +C*G*+T*T*+A*G*+A*+T*T*+A*+C* / iMe-dC / *G*+C*+G) was used.

[0200] ASO transfection

[0201] Cells were plated at 13,158 cells / cm2 and allowed to adhere for at least 24 hours or until 70% confluence was reached. ASOs were then diluted in Opti-Mem and transfected using Lipofectamine 3000 according to the manufacturer's instructions. Final ASO concentrations in the culture medium ranged from 0.1 nM to 50 nM. Cells were incubated for 48 to 96 hours, at which point the culture medium was removed, the cells were washed in PBS, and the plates were frozen at -80°C.

[0202] Western blotting

[0203] Cell lysates were collected in 1x Laemmli buffer plus beta-mercaptoethanol. Samples were separated according to molecular weight using a Bio-Rad MiniProteanTetra gel electrophoresis system. Proteins were transferred to the membrane using a Bio-Rad Trans-Blot Turbo transfer system and then blocked in 5% milk for 1 hour. The membrane was incubated overnight in a human Brachyury primary antibody (R&D Systems, AF2085) or a histone H3 primary antibody (Cell Signaling Technologies, 9715). The membrane was washed in TBS-Tween and then incubated in anti-goat or anti-rabbit secondary antibodies for 1 hour, respectively. After washing, the membrane was treated with SuperSignal West PicoPlus chemiluminescent substrate and then imaged on an iBright 1500 blot imager.

[0204] Nonspecific toxicity assay

[0205] U2OS cells were grown at a density of 7,813 cells / cm 2 Cells were plated at a density of 100 μg / mL in white opaque 96-well plates and allowed to adhere for at least 24 hours or until 70% confluence was reached. ASOs were transfected in triplicate at 50 nM as described above, and the plates were incubated for 6 days, with medium refreshed on day 3. An equal volume of Cell Titer Glo 2.0 (Promega) was added to each well according to the manufacturer's instructions, and luminescence was measured on a Biotek Synergy LX plate reader. Luminescence readings were normalized to controls containing no ASO or scrambled ASOs, and data were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test.

[0206] Chordoma growth inhibition assay

[0207] Cells were plated and transfected as described previously, but in large-format 12-well plates. After incubation at 72°C, cells were trypsinized, counted, and counted at 1,563 cells / cm 2Cells were reseeded. The cells were grown for 21 days, with culture medium refreshed every 3-4 days. At each time point, an equal volume of CellTiter Glo 2.0 was added to each well, and cell viability was assessed as described for nonspecific toxicity testing. Data were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test.

[0208] result

[0209] A library of 147 LNA ASOs (75 gapmers; 72 mixed-mers) was designed; this library may be referred to as the "original" library hereinafter. The LNA ASOs bind along the human Brachyury (TBXT) pre-mRNA (NCBI Gen ID: 6862). Figure 1 Schematic depiction of the TBXT-targeting mechanism of the ASO library. After screening in the chordoma cell line UM-Chor1, 17 mixed-mer ASOs efficiently abolished TBXT expression. Figure 2 All 17 ASOs were located in the same exon of TBXT pre-mRNA. Figure 3 .Use of the TBXT-independent cell line U2OS to remove ASOs exhibiting nonspecific toxicity resulted in 9 remaining on-targets. Figure 4 Dose-response testing identified the most potent target spanning the intron 4 / exon 5 junction of the TBXT pre-mRNA, suggesting that the exon skipping mechanism is lethal for this protein. Figure 5 ; Figure 3 .

[0210] Figure 1 The TBXT-targeting mechanism of the ASO library. ASOs are designed to base pair at various sites along the TBXT pre-mRNA. Gapmer ASOs induce RNase H-mediated mRNA cleavage, while mixed-mer ASOs sterically inhibit splicing or translation. The end result is an absent or nonfunctional Brachyury protein product.

[0211] Figure 2 Mixed-mer LNA ASOs were selected to effectively inhibit TBXT expression. UM-Chor1 cells were transfected with 25 nM of each ASO. After 48 hours, cell lysates were probed for Brachyury by Western blotting. The absence of a 50 kDa protein indicated that mixed-mers #2, 5, 6, 7, 10, 11, 12, 15, 18, 28, 29, 31, 32, 69, 70, 71, and 72 were on target. This experiment was repeated once with identical results.

[0212] Figure 3All anti-TBXT LNA ASO mixtures target the same exon. Comparison of the target sites of 17 ASOs that inhibit TBXT protein revealed that all "targets" bind to and around exon 5 of the pre-mRNA associated with TBXT transcript variant 1 (NCBI accession number: NM_003181).

[0213] Figure 4 Non-specific toxicity of the anti-TBXT LNA ASO mixed polymer. The TBXT-independent U2OS cell line was transfected with 50 nM of each ASO. On day 6, Cell Titer Glo was added, and luminescence was measured and normalized to a control without ASO. Three replicates were measured and compared to the control without ASO using a one-way ANOVA and Dunnett's multiple comparison test. A significant decrease in luminescence indicates a decrease in ATP and, therefore, reduced cell viability.

[0214] Figure 5 Mixed-polymer LNA ASOs were selected for potent inhibition of TBXT expression. UM-Chor1 cells were transfected with each ASO at the indicated concentrations. After 48 hours, cell lysates were probed for Brachyury by Western blotting. Dose-response testing demonstrated potent TBXT inhibition as low as 10 nM for ASOs #2, 5, and 7.

[0215] A second library of 33 LNA ASOs spanning the intron 4 / exon 5 junction of TBXT was designed; this library will be referred to as the "redesigned" library hereinafter. Nonspecific toxicity testing identified 7 non-toxic hits ( Figure 6 ), which was shown to inhibit TBXT protein in UM-Chor1 chordoma cells. Figure 7 Dose-response testing confirmed that the redesigned compounds had equivalent TBXT inhibitory potency to ASO#2, 5, and 7. The targets generated by both the original and redesigned compounds were validated in three additional chordoma cell lines, MUG-Chor1, JHC7, and UCH-2, and demonstrated broad TBXT protein inhibition. Figures 8-10 For a schematic overview of ASO screening, see Figure 11 The 10 anti-TBXT LNA ASO targets (3 original plus 7 redesigned) were then tested for their ability to inhibit chordoma cell growth, as Brachyury has been shown to be essential for chordoma proliferation. All 10 ASOs were able to significantly inhibit the growth of UM-Chor1 cells (Figure 12). See Table 1 for sequences associated with the 10 anti-TBXT LNA ASO targets.

[0216] Figure 6Nonspecific toxicity of the anti-TBXT LNA ASO mixed polymer redesign. The TBXT-independent U2OS cell line was transfected with 50 nM of each ASO. On day 6, Cell Titer Glo was added, and luminescence was measured and normalized to a control without ASO. Three replicates were measured and compared to the control without ASO using a one-way ANOVA and Dunnett's multiple comparison test. A significant decrease in luminescence indicates a decrease in ATP and, therefore, reduced cell viability.

[0217] Figure 7 All non-toxic mixed-mer LNA ASO redesigns effectively inhibited TBXT expression. UM-Chor1 cells were transfected with 25 nM of each ASO. After 48 hours, cell lysates were probed for Brachyury by Western blotting. The absence of a 50 kDa protein indicated that mixed-mers #129, 135, 136, 137, 138, 139, and 141 were on target.

[0218] Figure 8 All original and redesigned mixed-mer LNA ASOs effectively inhibited TBXT expression in MUG-Chor1 cells. MUG-Chor1 cells were transfected with 25 nM of each ASO. After 96 hours, cell lysates were probed for Brachyury by Western blotting. The absence of a 50 kDa protein indicates that mixed-mers #2, 5, 7, 129, 135, 136, 137, 138, 139, and 141 are capable of inhibiting TBXT in multiple chordoma cell lines.

[0219] Figure 9 All original and redesigned mixed-mer LNA ASOs effectively inhibited TBXT expression in JHC7 cells. JHC7 cells were transfected with 25 nM of each ASO. After 96 hours, cell lysates were probed for Brachyury by Western blot. The absence of a 50 kDa protein indicates that mixed-mers #2, 5, 7, 129, 135, 136, 137, 138, 139, and 141 are capable of inhibiting TBXT in multiple chordoma cell lines.

[0220] Figure 10 All original and redesigned mixed-mer LNA ASOs effectively inhibited TBXT expression in UCH-2 cells. UCH-2 cells were transfected with 25 nM of each ASO. After 96 hours, cell lysates were probed for Brachyury by Western blotting. The absence of a 50 kDa protein indicates that mixed-mers #2, 5, 7, 129, 135, 136, 137, 138, 139, and 141 are capable of inhibiting TBXT in multiple chordoma cell lines.

[0221] Figure 11 .Overview of anti-TBXT mixed-mer LNA ASO screening. 72 mixed-mer ASOs were tested for their ability to inhibit TBXT protein in the UM-Chor1 chordoma cell line. 17 hits emerged, 8 of which were eliminated due to non-specific toxicity. 9 ASOs were tested in a dose-response study, and the 3 most potent ASOs were able to identify the optimal binding site on TBXT pre-mRNA. ASO redesign targeting this optimal site produced 33 new anti-TBXT mixed-mer LNA ASOs. 26 ASO redesigns eliminated non-specific toxicity, and the remaining 7 showed potent inhibition of TBXT. The combination of 7 redesigned hits with 3 original design hits was validated in three independent chordoma cell lines, resulting in a total of 10 hits.

[0222] Figures 12A-12B .Anti-TBXT mixed-polymer LNA ASOs inhibit chordoma cell growth. UM-Chor1 cells were transfected with targeted Lipofectamine anti-TBXT ASOs. After 72 hours, each treatment was trypsinized, counted, and replated in triplicate in consistent quantities. (A) At the indicated time points, CellTiter Glo was added and luminescence was measured. (B) Luminescence values ​​at t = 14 days were normalized to the control without ASO. Three replicates were measured and compared to a scrambled mixed-polymer control by one-way ANOVA and Dunnett's multiple comparison test. A significant decrease in luminescence indicates a decrease in ATP and, therefore, reduced cell viability.

[0223] Figures 15A-15B Mixed-mer #139 enters chordoma cells in the absence of a lipid delivery vehicle that inhibits TBXT expression. UM-Chor1 cells were plated, and ASO #139 (B) or a scrambled control ASO (A) was added directly to the culture medium at the indicated concentrations. The cells were incubated for 12 days, with the ASO and culture medium refreshed every 3-4 days. Cell lysates were then probed for Brachyury by Western blot. The absence of a 50 kDa protein with increasing concentrations of mixed-mer #139 indicates that the anti-TBXT ASO is able to passively enter chordoma cells.

[0224] Figures 16A-16D. ASOs targeting the intron 4 / exon 5 junction of TBXT induce exon skipping and lethal frameshift mutations. UM-Chor1 cells were transfected with lipofectamine at 25nM of mixed polymers #2, 15, 139 or a scrambled control mixed polymer (A) and incubated for 72 hours. RNA was extracted, and the sequences covering the ASO binding site were PCR amplified using the indicated primers (B) and then visualized on an acrylamide gel (C). The 165 base pair band in all anti-TBXT ASOs indicated the deletion of exon 5. The primer products were excised and sequenced, and the sequencing data confirmed that TBXT exon 5 was accurately skipped, resulting in a premature stop codon. A schematic diagram (D) showing the generation of exon skipping and frameshift mutations induced by anti-TBXT ASOs is shown.

[0225] Figures 17A-17B .Mixed polymer #139 induces S phase cell cycle arrest in chordoma cells. UM-Chor1 cells were transfected with 25nM anti-TBXT ASO #139 or a scrambled control mixed polymer. The cells were incubated for 48 hours, then expanded and cultured for another 4 days (A) or 7 days (B), with the culture medium refreshed every 2-3 days. The cells were fixed and stained with propidium iodide and then analyzed by flow cytometry to estimate the percentage of cell populations in each phase of the cell cycle. A free cell cycle control from happily dividing cells was included as an additional control. After treatment with anti-TBXT #139, a decrease in cells in G1 and an increase in cells in S indicated S phase arrest, indicating that TBXT inhibition induces the replication stress checkpoint.

[0226] Figures 18A-18E Mice tolerated anti-TBXT ASO#139 well. Six-week-old female nude mice were randomly divided into treatment groups (3 / group) by weight and treated with 40 or 60 mg / kg anti-TBXT ASO#139 by subcutaneous injection. A saline vehicle group was included as a negative control. As indicated, treatment continued weekly or biweekly for 14 days, with body weights measured daily and reported as percentage change relative to baseline (A). Serum was collected at the end of the study for measurement of creatinine (B), alanine aminotransferase (C), aspartate aminotransferase (D), and blood urea nitrogen (E). Body weights and markers of liver / kidney toxicity in treated mice were not significantly different (NS) from those in the saline control group, indicating high tolerance to ASO#139.

[0227] Figures 19A-19DTreatment with anti-TBXT ASO #139 selectively eradicates patient-derived chordoma tumors in mice. Six-week-old nude female mice were subcutaneously implanted with 5x5 mm CF466 patient-derived chordoma tumor fragments. When tumors reached 100-200 mm 3 At the end of the study, mice were randomly divided into 7 groups (7 / group) and treatment was started. Anti-TBXT ASO#139 was injected subcutaneously at 30 or 60 mg / kg once every two weeks, with a saline vehicle group as a negative control. The positive control arm received 20 mg / kg afatinib. Body weight (B) and tumor size (A, C) were measured twice a week, and treatment continued for six weeks. Body weight is reported as a percentage change relative to baseline. Tumors and livers were collected at the end of the study and quickly frozen to quantify ASO concentrations. 10-20 mg tumor samples were homogenized and ASO#139 was quantified by SplintR qPCR using custom probes and primers for the target ASO. The average cycle threshold value of the ASO standard curve was plotted against the log of the concentration. 10 Plot. The linear portion of the curve was regressed and the ASO concentrations in tissue lysate samples were interpolated from these curves (D). 2 / 7 mice treated with 30 mg / kg ASO #139 experienced complete tumor regression (C), indicating at least partial efficacy in this pilot study. ASO levels were detected in the tumors of the remaining treated mice, demonstrating robust accumulation of anti-TBXT ASOs in solid tumors following systemic administration.

[0228] Table 1. Target-related sequences in anti-TBXT mixed-mer LNA ASOs. Each ASO is a modified single-stranded DNA / LNA mixed-mer oligonucleotide. The phosphodiester backbone has been 100% replaced with a phosphorothioate backbone (indicated by the "*" between each nucleotide below the sequence). DNA monomers are mixed with LNA monomers (nucleotides preceded by a "+" indicate LNA). The target sequence represents the binding sequence, which spans the intron 4 / exon 5 junction of TBXT pre-mRNA transcript variant 1 (NCBI accession number: NM_003181).

[0229] Table 1

[0230] name sequence Target sequence 2 mixed anti-TBXT +T*+A*A*G*+A*G*C*+T*G*T*+G*A*T*+C*+T*+G CAGATCACAGCTCTTA 5 mixed anti-TBXT +T*+T*T*A*+A*G*A*+G*C*T*+G*T*G*+A*T*+C*+T AGATCACAGCTCTTAAA 7 mixed anti-TBXT +A*+A*T*T*+T*T*A*+A*G*A*+G*C*T*+G*T*+G*+A*+T ATCACAGCTCTTAAAATT 129 mixed anti-TBXT +G*+A*+G*C*+T*G*T*+G*A*T*+C*+T*G*+A*A*+A*+A TTTTCAGATCACAGCTC 135 mixed anti-TBXT +G*+A*G*+C*+T*+G*T*+G*A*T*+C*T*G*+A*+A*+A TTTCAGATCACAGCTC 136 mixed anti-TBXT +G*+A*G*C*+T*G*T*+G*A*+T*+C*T*+G*+A*+A*+A TTTCAGATCACAGCTC 137 mixed anti-TBXT +A*+G*+A*G*C*+T*G*T*+G*A*T*+C*T*G*+A*+A*+A TTTCAGATCACAGCTCT 138 mixed anti-TBT +A*+G*+A*G*C*+T*G*T*+G*A*T*+C*T*+G*A*+A*+A TTTCAGATCACAGCTCT 139 mixed anti-TBXT +G*+A*G*C*+T*G*+T*+G*A*T*+C*T*G*+A*+A TTCAGATCACAGCTC 141 mixed anti-TBXT +A*+G*+A*G*C*+T*G*T*+G*A*T*+C*T*G*+A*+A TTCAGATCACAGCTCT

[0231] The target sequences in Table 1 are SEQ ID NOs: 1-8 from top to bottom.

[0232] Although the present invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. Furthermore, many modifications may be made to adapt a particular situation, material, composition of matter, process, or one or more process steps to the purpose, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

1. An inhibitory nucleic acid comprising a nucleotide sequence complementary to a target nucleotide sequence in a TBXT transcript, wherein the inhibitory nucleic acid comprises one or more locked nucleic acids (LNAs).

2. The inhibitory nucleic acid of claim 1, wherein the inhibitory nucleic acid has a length of about 15 nucleotides to about 30 nucleotides.

3. The inhibitory nucleic acid of claim 1, wherein the inhibitory nucleic acid has a length of about 15 nucleotides to about 20 nucleotides.

4. The inhibitory nucleic acid of any one of claims 1-3, wherein the nucleotide sequence that is complementary to a nucleotide sequence in a TBXT transcript is complementary to a nucleotide sequence at the intron 4 / exon 5 junction.

5. The inhibitory nucleic acid of any one of claims 1-3, wherein the target TBXT nucleotide sequence is selected from the group consisting of: CAGATCACAGCTCTTA(SEQ ID NO:1); AGATCACAGCTCTTAAA(SEQ ID NO:2); ATCACAGCTCTTAAAATT(SEQ ID NO:3); TTTTCAGATCACAGCTC(SEQ ID NO:4); TTTCAGATCACAGCTC(SEQ ID NO:5); TTTCAGATCACAGCTCT(SEQ ID NO:6); TTCAGATCACAGCTC (SEQ ID NO: 7); and TTCAGATCACAGCTCT (SEQ ID NO:8).

6. The inhibitory nucleic acid of any one of claims 1-5, wherein the inhibitory nucleic acid comprises one or more phosphorothioate linkages.

7. The inhibitory nucleic acid of any one of claims 1 to 6, wherein the inhibitory nucleic acid comprises a nucleotide sequence selected from the group consisting of: +T*+A*A*G*+A*G*C*+T*G*T*+G*A*T*+C*+T*+G; +T*+T*T*A*+A*G*A*+G*C*T*+G*T*G*+A*T*+C*+T; +A*+A*T*T*+T*T*A*+A*G*A*+G*C*T*+G*T*+G*+A*+T; +G*+A*+G*C*+T*G*T*+G*A*T*+C*+T*G*+A*A*+A*+A; +G*+A*G*+C*+T*+G*T*+G*A*T*+C*T*G*+A*+A*+A; +G*+A*G*C*+T*G*T*+G*A*+T*+C*T*+G*+A*+A*+A; +A*+G*+A*G*C*+T*G*T*+G*A*T*+C*T*G*+A*+A*+A; +A*+G*+A*G*C*+T*G*T*+G*A*T*+C*T*+G*A*+A*+A; +G*+A*G*C*+T*G*+T*+G*A*T*+C*T*G*+A*+A; and +A*+G*+A*G*C*+T*G*T*+G*A*T*+C*T*G*+A*+A, wherein * indicates a phosphorothioate linkage, and wherein a "+" immediately preceding a nucleotide indicates that the nucleotide is an LNA.

8. A composition comprising: a) an inhibitory nucleic acid according to any one of claims 1 to 7; and b) pharmaceutically acceptable excipients.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable excipient comprises one or more lipids.

10. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable excipient comprises poly(amidoamine), poly(propyleneimine), or poly(L-lysine).

11. A lipid nanoparticle comprising: a) an inhibitory nucleic acid according to any one of claims 1 to 7; and b) pharmaceutically acceptable excipients.

12. A method of treatment comprising administering to an individual in need thereof an effective amount of the inhibitory nucleic acid of any one of claims 1-7, the pharmaceutical composition of any one of claims 8-10, or the lipid nanoparticle of claim 11.

13. A method of inhibiting cancer proliferation in a subject, the method comprising administering to the subject an effective amount of the inhibitory nucleic acid of any one of claims 1-7, the pharmaceutical composition of any one of claims 8-10, or the lipid nanoparticle of claim 11.

14. The method of claim 13, wherein the cancer is lung cancer, breast cancer, colon cancer, prostate cancer, or liver cancer.

15. The method of claim 13, wherein the cancer is chordoma.

16. A method of treating cancer in an individual, the method comprising administering to the individual an effective amount of the inhibitory nucleic acid of any one of claims 1-7, the pharmaceutical composition of any one of claims 8-10, or the lipid nanoparticle of claim 11.

17. The method of claim 16, wherein the cancer is lung cancer, breast cancer, colon cancer, prostate cancer, or liver cancer.

18. The method of claim 16, wherein the cancer is chordoma.

19. The method of any one of claims 16-18, wherein the administration comprises an intravenous, intramuscular, intratumoral, or peritumoral route of administration.

20. The method of any one of claims 16-19, further comprising administering one or more additional therapeutic treatments.

21. The method of claim 20, wherein the one or more additional therapeutic treatments comprise cancer chemotherapy, radiation, or surgery.

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