Inhibitory nucleic acids and methods of use thereof

Abstract

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

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 430,731, filed December 7, 2022, which is incorporated herein by reference in its entirety.

[0002] Incorporation by Reference of Electronically Submitted Material The Sequence Listing is provided herewith as Sequence Listing XML "BERK-480WO_SEQ_LIST" having a size of 12,658 bytes, created on December 1, 2023. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. [Background technology]

[0003] introduction Brachyury (also known as "TBXT") has been shown to be overexpressed in lung, breast, colon, prostate, and liver cancers, and chordoma. Brachyury has been associated with epithelial-mesenchymal transition in human tumors and is being investigated as a drug target for a wide range of cancers.

[0004] Chordoma, a malignant sarcoma arising from the notochord cells of the vertebrae, is rare (1:1,000,000), with 300 cases diagnosed annually in the United States. Chordoma tumors are slow-growing but difficult to treat due to the tumor's proximity to the spinal cord and its tendency to recur after surgery and radiation. There are no systemic targeted therapies for chordoma, and therefore, patients with advanced and metastatic disease face a poor prognosis.

[0005] There is a need in the art for compositions and methods for treating cancer. Summary of the Invention

[0006] overview The present disclosure provides inhibitory nucleic acids, compositions comprising inhibitory nucleic acids, and methods of using inhibitory nucleic acids to treat cancer. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a schematic diagram of the TBXT targeting mechanism of antisense oligonucleotide (ASO) libraries. [Figure 2] Figure 2 shows the effect of selected mix-mutated nucleic acid (LNA) ASOs on TBXT expression in UM-Chor1 cells. [Figure 3] Figure 3 shows the localization of anti-TBXT LNA ASO mixmers to the same TBXT exons. [Figure 4] Figure 4 shows the non-specific toxicity of anti-TBXT LNA ASO mixmer hits in U2OS cells. [Figure 5] FIG. 5 shows inhibition of TBXT expression by selected mixmer LNA ASOs in UM-Chor1 cells. [Figure 6-1] Figure 6 shows the non-specific toxicity of the anti-TBXT LNA ASO mix-matrix design in U2OS cells. [Figure 6-2] See description of Figure 6-1. [Figure 7] FIG. 7 shows inhibition of TBXT expression by non-toxic mixmer LNA ASO redesigns in UM-Chor1 cells. [Figure 8] FIG. 8 shows inhibition of TBXT expression in MUG-Chor1 cells by original and redesigned LNA ASOs. [Figure 9] Figure 9 shows inhibition of TBXT expression in JHC7 cells by original and redesigned LNA ASOs. [Figure 10] Figure 10 shows inhibition of TBXT expression in UCH-2 cells by original and redesigned LNA ASOs. [Figure 11] FIG. 11 shows an overview of the anti-TBXT mixmer LNA ASO screening. [Figure 12] Figures 12A-12B show inhibition of UM-Chor1 chordoma cell growth by anti-TBXT mixmer LNA ASO. [Figure 13] FIG. 13 provides the TBXT nucleotide sequence (SEQ ID NO:9). [Figure 14] FIG. 14 provides the TBXT amino acid sequence (SEQ ID NO:10). [Figure 15] Figures 15A-15B show the entry of anti-TBXT mixmer LNA ASO into chordoma cells without a lipid delivery vehicle. [Figure 16A] 16A-16D show exon 5 skipping in TBXT transcripts induced by anti-TBXT mixmer LNA ASO. [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 16D] See legend to Figure 16A. [Figure 17] Figures 17A-17B show induction of S-phase cell cycle arrest in chordoma cells by anti-TBXT mixmer LNA ASO. [Figure 18A] Figures 18A-18E show the tolerability of anti-TBXT mixmer LNA ASOs in mice. [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 18D] See legend to Figure 18A. [Figure 18E] See legend to Figure 18A. [Figure 19-1] Figures 19A-19D show eradication of patient-derived chordoma tumors in mice by anti-TBXT mixmer LNA ASO. [Figure 19-2] See description of Figure 19-1. DETAILED DESCRIPTION OF THE INVENTION

[0008] definition As used herein, "antisense oligonucleotide" ("ASO") refers to a nucleic acid sequence that is complementary to a DNA or RNA sequence.

[0009] "RNA" refers to a molecule containing at least one or more ribonucleotide residues. A "ribonucleotide" is a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranose moiety. As used herein, the term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, essentially pure RNA, synthetic RNA, and recombinantly produced RNA, and also includes modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. The nucleotides of an RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides, or chemically synthesized nucleotides or deoxynucleotides.

[0010] "MicroRNAs" (miRNAs) are single-stranded RNA molecules approximately 21-23 nt in length. Generally, miRNAs regulate gene expression. They are encoded by genes and transcribed from the DNA of those genes, but miRNAs are not translated into proteins. Each primary miRNA transcript is processed into a short stem-loop structure, which is then further processed into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their primary function is to downregulate gene expression.

[0011] As used herein, "interfering RNA" refers to a double-stranded or single-stranded RNA sequence that can directly or indirectly (i.e., upon translation) inhibit or downregulate gene expression by mediating RNA interference. Interfering RNA includes, but is not limited to, toenail interfering RNA ("siRNA") and short hairpin RNA ("shRNA"). "RNA interference" refers to the selective degradation of sequence-compatible messenger RNA transcripts.

[0012] As used herein, "shRNA" (short 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 double-stranded stem. After post-transcriptional processing, the short hairpin RNA is converted into a small interfering RNA by a cleavage event mediated by the enzyme Dicer, a member of the RNase III family.

[0013] "Small interfering RNA" or "siRNA," as used herein, refers to a small RNA molecule that can inhibit or downregulate gene expression by mediating RNA interference in a sequence-specific manner. Small RNAs can be, for example, about 18 to 21 nucleotides in length.

[0014] As used herein, "antagomir" refers to a small synthetic RNA that is complementary to a specific microRNA target, either with a mispairing at the cleavage site or with one or more base modifications to inhibit cleavage.

[0015] As used herein, the phrase "post-transcriptional processing" refers to mRNA processing that occurs after transcription, for example, mediated by the enzymes Dicer and / or Drosha.

[0016] 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, referring to the complete or partial prevention of a disease or its symptoms, and / or therapeutic, referring to the partial or complete cure of a disease and / or adverse effects resulting from a disease. "Treatment," as used herein, includes the treatment of disease in a mammal, e.g., a human, and includes (a) preventing the onset of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, i.e., causing regression of the disease.

[0017] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to an individual organism, e.g., a mammal, including, but not limited to, a mouse, a monkey, a human, and a non-human primate. In some cases, the "individual" is a human.

[0018] Before the present invention is further described, it is to be understood that this invention is not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and not for purposes of limitation, since the scope of the present invention will be limited only by the appended claims.

[0019] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.Although any method and material similar or equivalent to those described herein can also be used in carrying out or testing this invention, preferred methods and materials are described herein.All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials related to which the publication is cited.

[0021] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "an antisense oligonucleotide (ASO)" includes a plurality of such ASOs; a reference to "TBXT polypeptide" includes a reference to one or more TBXT polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude optional elements. Accordingly, this statement shall serve as an antecedent basis for use of exclusive terminology, such as "solely," "only," and the like, or for use of a "negative" limitation in reciting claim elements.

[0022] The use of the terms "a," "an," and "the" and similar referents with respect to the description of this disclosure (particularly with respect to the claims that follow) should be construed to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended (i.e., meaning "including, but not limited to"), unless otherwise noted. The recitation of ranges of values ​​herein, unless otherwise indicated herein, merely serves as a shorthand method for referring individually to each separate value within the range, and each separate value is incorporated herein as if individually set forth herein. For example, if a range of 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to further clarify aspects of the disclosure and do not limit the scope of the disclosure unless otherwise recited in the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of aspects of the disclosure.

[0023] As used herein, the term "about" when used in reference to a quantity indicates that the quantity may vary by 10% of the stated amount. For example, "about 100" means an amount of 90 to 110. When "about" is used in reference to a range, "about" when used in reference to the lower limit of a range means that an amount 10% lower than the lower limit of the range is included in the lower limit, and "about" when used in reference to the upper limit of a range means that an amount 10% higher than the upper limit of the range is included in the upper limit. For example, about 100 to about 1000 means that the range spans 90 to 1100.

[0024] As used herein, the term "and / or," e.g., 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," e.g., 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).

[0025] It is understood that the aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0026] It will be understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the aspects of the invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0027] 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 publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0028] Detailed Description The present disclosure provides inhibitory nucleic acids, compositions comprising inhibitory nucleic acids, and methods of using inhibitory nucleic acids to treat cancer.

[0029] inhibitory nucleic acid The present disclosure provides inhibitory nucleic acids that provide for a reduction in the level of TBXT polypeptide in a cell.

[0030] TBXT is also known in the art as "T-Box transcription factor T," "Brachyury protein," "SAVA," and "FTFT." A TBXT polypeptide can 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 to the TBXT amino acid sequence shown in Figure 14.

[0031] The inhibitory nucleic acids of the present disclosure comprise a nucleotide sequence that binds (hybridizes) to 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 the intron 4 / exon 5 junction of the TBXT mRNA. The TBXT genomic nucleotide sequence is provided in NCBI Gene ID 6862. The target TBXT nucleotide sequence may have 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 to the TBXT nucleotide sequence shown in FIG. 13. 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 TTCAGATCACAAGCTC (SEQ ID NO:7). In some cases, the inhibitory nucleic acids of the present disclosure bind (hybridize) to the TBXT target sequence CAGATCACAGCTCTTA (SEQ ID NO:1). In some cases, the inhibitory nucleic acids of the present disclosure bind (hybridize) to the TBXT target sequence AGATCACAGCTCTTAAA (SEQ ID NO:2). In some cases, the inhibitory nucleic acids of the present disclosure bind (hybridize) to the TBXT target sequence ATCACAGCTCTTAAAATT (SEQ ID NO:3). In some cases, the inhibitory nucleic acids of the present disclosure bind (hybridize) to the TBXT target sequence TTTTCAGATCACAGCTC (SEQ ID NO:4). In some cases, the inhibitory nucleic acids of the 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 (hybridize) to the TBXT target sequence TTTCAGATCACAGCTCT (SEQ ID NO:6). In some cases, the inhibitory nucleic acids of the present disclosure bind (hybridize) to the TBXT target sequence TTCAGATCACAAGCTC (SEQ ID NO:7). In some cases, the inhibitory nucleic acids of the present disclosure bind (hybridize) to the TBXT target sequence TTCAGATCACAAGCTCT (SEQ ID NO:8).

[0032] The inhibitory nucleic acids of the present disclosure reduce the level of TBXT polypeptide in a cell (e.g., a target cell, e.g., 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 the TBXT transcript. In some cases, the inhibitory nucleic acids of the present disclosure prevent translation of TBXT mRNA. In some cases, the inhibitory nucleic acids of the present disclosure increase cleavage of TBXT mRNA by RNAse H.

[0033] In some cases, the inhibitory nucleic acids of the present disclosure inhibit 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.

[0034] Inhibitory nucleic acids useful in the methods and compositions of the present invention 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 acids (LNA), antagomir, peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimics, which hybridize to at least a portion of the target nucleic acid (i.e., TBXT nucleic acid) and modulate its function. In some embodiments, inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides containing modified linkages, interfering RNA (RNAi), small interfering RNA (siRNA); microinterfering RNA (miRNA); small transient RNA (stRNA); or small 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.

[0035] In some cases, the inhibitory nucleic acid is 10-50, 13-50, or 13-30 nucleotides in length. Those skilled in the art will understand that this specifically refers to oligonucleotides having antisense portions that are 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 12-30 or 13-30 nucleotides in length. Those skilled in the art will understand that this specifically refers to inhibitory nucleic acids having a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0036] In some cases, the inhibitory nucleic acids of the present disclosure include: TIFF2026500230000001.tif58128.

[0037] where: * indicates a phosphorothioate bond, and a "+" preceding a nucleotide indicates that the nucleotide is an LNA. In some cases, the inhibitory nucleic acid has a length of 15 to 25 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides).

[0038] In some cases, the inhibitory nucleic acid is a chimeric oligonucleotide containing two or more chemically distinct regions, each composed of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confer one or more beneficial properties (e.g., increased nuclease resistance, increased cellular uptake, increased binding affinity to the target), and a region that is a substrate for an enzyme that can cleave RNA:DNA hybrids or RNA:RNA hybrids. The chimeric inhibitory nucleic acid of the present disclosure may be formed as a composite structure of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, and / or oligonucleotide mimetics, as described above. Such compounds are also referred to in the art as hybrids or gapmers.

[0039] In some embodiments, the inhibitory nucleic acid contains 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 on the ribose of pyrimidines, abasic residues or 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) for a given target than 2'-deoxyoligonucleotides.

[0040] A number of 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 oligonucleotides remain intact for longer periods of time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those containing modified backbones, such as phosphorothioates, phosphotriesters, methylphosphonates, short alkyl or cycloalkyl intersugar linkages, or short heteroatom or heterocyclic intersugar linkages. In some cases, inhibitory nucleic acids include oligonucleotides having phosphorothioate backbones, as well as heteroatom backbones, specifically CH2--NH--O--CH3, CH3--N(CH3)--O--CH2 (known as methylene (methylimino) or MMI backbones), CH2--O--N(CH3)--CH2, CH2--N(CH3)--N(CH3)--CH2, and O--N(CH3)--CH2--CH2 backbones (the native 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 (Summerton and Weller, U.S. Pat. No. 5,034,506); or oligonucleotides having a peptide nucleic acid (PNA) backbone (in which the phosphodiester backbone of an oligonucleotide is replaced with a polyamide backbone and the nucleotides are bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone; see Nielsen et al., Science 1991, 254, 1497).Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters; aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates, e.g., 3' alkylene phosphonates, and chiral phosphonates, phosphinates, phosphoramidates, e.g., 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with opposite polarity where pairs of adjacent nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0041] Modified oligonucleotide backbones that do not contain internal phosphorus atoms have backbones formed by 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, formacetyl and thioformacetyl backbones, methyleneformacetyl and methylenethioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 moieties.

[0042] One or more substituted sugar moieties may be included at the 2' position, for example, one of the following: OH, SH, SCH, F, OCN, OCHOCH, OCH, O(CH)CH, O(CH)NH, or O(CH)CH, where n is 1 to about 10; C-C lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl, or aralkyl; Cl; Br; CN; CF; OCF; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH; SOCH; ONO; NO; N; NH; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleaving 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-CH2CHOCH3, 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 on the oligonucleotide, specifically the 3' position of the sugar on the 3'-terminal nucleotide and the 5' position of the 5'-terminal nucleotide. Oligonucleotides can also have sugar mimetics, such as cyclobutyls, in place of the pentofuranosyl group.

[0043] Inhibitory nucleic acids may additionally or alternatively contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases that are found only rarely or transiently in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidines, specifically 5-methylcytosine (also known as 5-methyl-2'deoxycytosine, often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentobiosyl HMC, as well as synthetic nucleobases such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine, or other hetero-substituted alkyl adenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, and 2,6-diaminopurine. "Universal" bases known in the art, such as inosine, may also be included. 5-Me-C substitutions increase nucleic acid duplex stability by 0.6-12. <0> It has been shown that 5-Me-C increases the C. In some cases, the inhibitory nucleic acids of the disclosure include one or more 5-Me-C.

[0044] 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 into a single oligonucleotide, or even at a single nucleoside within an oligonucleotide.

[0045] In some embodiments, both the sugar and internucleoside linkage, i.e., the backbone, of a nucleotide unit are replaced with novel groups. The base unit is maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an oligonucleotide is replaced with an amide-containing backbone, such as an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach 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 on PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0046] Inhibitory nucleic acids may also contain one or more nucleobase (often simply referred to in the art as "base") modifications or substitutions. 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 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine, and 6- These include azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted forms of adenine and guanine, 5-halo, specifically 5-bromo, 5-trifluoromethyl, and other 5-substituted forms of uracil and cytosine, 7-methylquanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine.

[0047] Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in 'The Concise Encyclopedia of Polymer Science and Engineering', pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, those disclosed in English et al., Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed in Sanghvi, YS, Chapter 15, 'Antisense Research and Applications', pages 289-302, Crooke, ST and Lebleu, B. ea., CRC Press, 1993. Some 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, such as 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine, with 5-methylcytosine substitutions increasing nucleic acid duplex stability by 0.6 to 1.2. <0> It has been shown to increase C by 2'-O-methoxyethyl sugar modifications (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., 'Antisense Research and Applications', CRC Press, Boca Raton, 1993, pp. 276-278), making it suitable for inclusion in inhibitory nucleic acids, for example, alone or in combination with 2'-O-methoxyethyl sugar modifications.

[0048] In some cases, inhibitory nucleic acid is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of oligonucleotide.Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties; cholic acid; thioethers, such as hexyl-S-tritylthiol; thiocholesterol; aliphatic chains, such as dodecanediol or undecyl residues; phospholipids; for example, di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate; polyamines or polyethylene glycol chains; adamantane acetic acid; palmityl moieties; octadecylamine moieties; or hexylamino-carbonyl-t-oxycholesterol moieties.

[0049] These moieties or conjugates can include functional groups, such as conjugate groups covalently bonded to primary or secondary hydroxyl groups. Suitable conjugate groups for use 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 conjugate 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 improve uptake, enhance resistance to degradation, and / or strengthen sequence-specific hybridization with target nucleic acids. Groups that enhance pharmacokinetic properties include groups that improve uptake, distribution, metabolism, or excretion of inhibitory nucleic acids. Representative conjugate 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. Conjugate moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acid, thioethers such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0050] Inhibitory nucleic acids useful in the methods of the present invention are sufficiently complementary to all or a portion of a TBXT nucleic acid, i.e., hybridize sufficiently well and with sufficient specificity, to confer the desired effect. "Complementary" refers to the ability to pair through hydrogen bonding between two sequences containing naturally occurring or non-naturally occurring bases or their analogs. For example, if a base at a position in the inhibitory nucleic acid can hydrogen bond with a base at the corresponding position in the TBXT sequence, then those bases are considered complementary to each other at that position. 100% complementarity is not required.

[0051] In the context of the present disclosure, hybridization refers to hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. "Complementary," as used herein, refers to the ability for precise pairing between two nucleotides. 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 can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms used to indicate a sufficient degree of complementarity or precise pairing for stable and specific binding to occur between an inhibitory nucleic acid and a TBXT target sequence. For example, if a base at a certain position in an inhibitory nucleic acid can hydrogen bond with a base at a corresponding position in a TBXT nucleic acid molecule, the bases are considered complementary to each other at that position.

[0052] Generally, inhibitory nucleic acids useful in the methods described herein have at least 80% sequence complementarity to the target region within the target nucleic acid, e.g., 90%, 95%, or 100% sequence complementarity to the target region within the TBXT nucleic acid. For example, an antisense compound in which 18 of the 20 nucleobases of an antisense oligonucleotide are complementary to the target region and therefore specifically hybridizes exhibits 90% complementarity. The percent complementarity between an inhibitory nucleic acid and a region of a target nucleic acid can be routinely determined using the 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 through routine experimentation. In general, an inhibitory nucleic acid must retain 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.

[0053] antisense As mentioned above, in some cases, the inhibitory nucleic acid of the present disclosure is ASO.ASO is typically designed to block the expression of DNA or RNA target by binding to the target and stopping expression at the level of transcription, translation or splicing.The ASO of the present disclosure is a complementary nucleic acid sequence designed to hybridize with TBXT target sequence under stringent conditions.Therefore, to achieve the desired effect, oligonucleotides are selected that are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity.

[0054] Modified bases / locked nucleic acids (LNA) In some cases, the inhibitory nucleic acid of the present disclosure contains one or more modified linkages or bases. Modified bases include phosphorothioates, methylphosphonates, peptide nucleic acids, or locked nucleic acid (LNA) molecules. For example, in some cases, the modified nucleotide is a locked nucleic acid molecule, such as [α]-L-LNA. LNA is a ribonucleic acid analog in which the ribose ring is "locked" by a methylene bridge between the 2'-oxygen and the 4'-carbon, i.e., an oligonucleotide containing at least one LNA monomer, i.e., one 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)). LNA also has an increased affinity for base pairing with RNA compared to DNA. These properties make LNAs particularly useful as probes for fluorescence in situ hybridization (FISH) and comparative genomic hybridization, as miRNA knockdown tools, and as antisense oligonucleotides for targeting mRNA or other RNAs.

[0055] LNA molecules can include molecules in which one strand contains 10 to 30, e.g., 12 to 24, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, to the TBXT target sequence, with, e.g., 3, 2, 1, or 0 mismatched nucleotides. LNA molecules can be chemically synthesized using methods known in the art.

[0056] Antagomir In some cases, the inhibitory nucleic acid is an antagomir. An antagomir is a chemically modified antisense oligonucleotide that targets a TBXT target nucleic acid sequence. For example, an antagomir for use in the methods described herein may contain a nucleotide sequence sufficiently complementary to hybridize to a TBXT target sequence of about 12 to 25 nucleotides or about 15 to 23 nucleotides.

[0057] Generally, antagomir contains a cholesterol moiety, for example, at the 3' end. In some embodiments, antagomir has various modifications for protection from RNase and pharmacological properties, such as enhanced uptake into tissues and cells. For example, in addition to the modifications described above for antisense oligos, antagomir can have one or more of full or partial 2'-O-methylation of the sugar and / or a phosphorothioate backbone. The phosphorothioate modification provides protection against RNase activity, and its lipophilicity contributes to enhanced uptake into tissues. In some embodiments, antagomir can contain six phosphorothioate backbone modifications (two phosphorothioates at the 5' end and four at the 3' end). An antagomir useful in the methods of the present invention may be modified in terms of its length or the number of nucleotides comprising the antagomir. An antagomir must retain 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 (eg, locked antagomir).

[0058] siRNA / shRNA In some cases, the inhibitory nucleic acid of the present disclosure is an interfering RNA, for example, but not limited to, a small interfering RNA (" siRNA ") or a short hairpin RNA (" shRNA "). Methods for constructing an interfering RNA are well known in the art. For example, an interfering RNA can be assembled from two separate oligonucleotides, where one strand is a sense strand and the other is an antisense strand, and the antisense strand and the sense strand are self-complementary (i.e., each strand comprises a nucleotide sequence that is complementary to the nucleotide sequence of the other strand; for example, 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 the nucleotide sequence of a target nucleic acid molecule or a part thereof (i.e., an undesired gene), and the sense strand comprises a nucleotide sequence that corresponds to the target nucleic acid sequence or a part thereof. Alternatively, an interfering RNA can be assembled from a single oligonucleotide, where the self-complementary sense region and the antisense region are connected by a nucleic acid-based or non-nucleic acid-based linker. Interfering RNA can be the polynucleotide of double-stranded, asymmetric double-stranded, hairpin or asymmetric hairpin secondary structure, with self-complementary sense region and antisense region, wherein antisense region comprises the nucleotide sequence that is complementary to the nucleotide sequence of another target nucleic acid molecule or a part thereof, and sense region has the nucleotide sequence that corresponds to target nucleic acid sequence or a part thereof.Interference can also be the circular single-stranded polynucleotide of the stem, which comprises two or more loop structures and comprises self-complementary sense region and antisense region, wherein antisense region comprises the nucleotide sequence that is complementary to the nucleotide sequence of target nucleic acid molecule or a part thereof, and sense region has the nucleotide sequence that corresponds to target nucleic acid sequence or a part thereof, and circular polynucleotide can be processed in vivo or in vitro to produce the active siRNA molecule that can mediate RNA interference.

[0059] 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, when expressed, desirably form a "hairpin" structure and are referred to herein as "shRNAs." The loop region is generally about 2 to about 10 nucleotides in length. In some embodiments, the loop region is about 6 to about 9 nucleotides in length. In some embodiments, the sense and antisense regions are about 15 to about 20 nucleotides in length. After post-transcriptional processing, the short hairpin RNA is converted into siRNA by a cleavage event mediated by the enzyme Dicer, a member of the RNase III family. The siRNA can then inhibit expression of genes with which it shares homology.

[0060] The target RNA cleavage reaction induced by siRNA is highly sequence-specific.Generally, siRNA containing the same nucleotide sequence as a part of target nucleic acid is used for inhibition.However, 100% sequence identity between siRNA and target gene is not required.Therefore, the present disclosure has the advantage that it can tolerate sequence variations that can be expected due to genetic mutation, lineage polymorphism, or evolutionary divergence.For example, siRNA sequences with insertions, deletions, and single point mutations compared to target sequences have also been found to be effective for inhibition.Alternatively, siRNA sequences containing substitutions or insertions by nucleotide analogs can be effective for inhibition.Generally, siRNA must maintain its 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.

[0061] Preparation of inhibitory nucleic acids Inhibitory nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, for example, as described 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.

[0062] Pharmaceutical Compositions The present disclosure provides compositions, e.g., pharmaceutical compositions, comprising the inhibitory nucleic acids of the present disclosure. The inhibitory nucleic acids of the present disclosure are also referred to hereinafter as "drugs" or "active agents."

[0063] In some cases, the composition is formulated with a pharmaceutically acceptable carrier. Pharmaceutical compositions and formulations can be administered parenterally (e.g., intravenously or intramuscularly), topically, orally, or by local administration, for example, 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 overall medical condition of each patient, the resulting preferred method of administration, etc. Details regarding techniques for pharmaceutical formulation and administration are fully described in the scientific and patent literature; see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

[0064] The inhibitory nucleic acid can be administered alone or as a component of a pharmaceutical preparation (composition).The inhibitory nucleic acid can be formulated for administration in any convenient way for use in human medicine or veterinary medicine.Wetting agents, emulsifying agents, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants can also be present in the composition.

[0065] Formulations of the inhibitory nucleic acids of the present disclosure include those suitable for intradermal, inhalation, oral / nasal, topical, parenteral, rectal, and / or vaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of 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 treated and the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of compound that produces a therapeutic effect, e.g., an antigen-specific T cell response or humoral response.

[0066] Pharmaceutical preparations can be prepared according to any method known to those skilled in the art for the manufacture of pharmaceuticals. Such drugs can contain sweeteners, flavorings, colorings, and preservatives. Preparations can be mixed with non-toxic pharmaceutically acceptable excipients suitable for manufacturing. Preparations can contain one or more diluents, emulsifiers, preservatives, buffers, excipients, etc., and can be provided in the form of liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled-release preparations, tablets, pills, gels, patches, implants, etc.

[0067] Pharmaceutical preparations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate dosage amounts. Such carriers allow the formulation of medicines into unit dosage forms suitable for patient ingestion, such as tablets, pills, powders, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc. Pharmaceutical preparations for oral use can be formulated as solid excipients, optionally by grinding the resulting mixture and processing the granular mixture to obtain tablets or dragee cores, optionally after adding suitable additional compounds. Suitable solid excipients include carbohydrate or protein fillers, such as sugars, e.g., lactose, sucrose, mannitol, or sorbitol; starches derived from corn, wheat, rice, potato, or other plants; celluloses, e.g., methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; and gums, e.g., gum arabic and tragacanth; and proteins, e.g., gelatin and collagen. Disintegrants or solubilizers, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or its salts, such as sodium alginate, may be added. Push-fit capsules may 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 may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol, with or without a stabilizer.

[0068] Aqueous suspensions may contain the active agent (e.g., an inhibitory nucleic acid of the present disclosure) mixed with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injection. Such excipients include suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia, as well as dispersing or wetting agents, such as naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols (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). The aqueous suspensions may also contain one or more preservatives, for example, 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 preparations may be adjusted for osmotic pressure.

[0069] In some cases, oily pharmaceuticals are used for the administration of inhibitory nucleic acids.Oily suspensions can be prepared by suspending active agents in vegetable oils, such as peanut oil, olive oil, sesame oil, or coconut oil, or mineral oils, such as liquid paraffin; or mixtures thereof.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 orally administered hydrophobic pharmaceutical compounds and reduce inter- and intra-individual variability (see also U.S. Patent No. 5,858,401).Oily 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 preparation.These preparations can be preserved by adding antioxidants, such as ascorbic acid. For examples of injectable oil vehicles, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.

[0070] Pharmaceutical preparations may be in the form of an oil-in-water emulsion. The oil phase may be the aforementioned vegetable oil or mineral oil, or a mixture thereof. Suitable emulsifiers include naturally occurring gums, such as gum arabic and gum tragacanth, naturally occurring phosphatides, such as soybean 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 may also contain sweeteners and flavoring agents, for example, in the case of syrup and elixir formulations. Such formulations may also contain demulcents, preservatives, or coloring agents. In an alternative embodiment, the injectable oil-in-water emulsion contains paraffin oil, sorbitan monooleate, ethoxylated sorbitan monooleate, and / or ethoxylated esbitan trioleate.

[0071] Pharmaceutical compositions can be administered via intranasal, intraocular, and intravaginal routes, such as suppositories, insufflation, powders, and aerosol formulations (for examples of steroid inhalants, 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, thus melting and releasing the drug in the body. Such materials include cocoa butter and polyethylene glycol.

[0072] In some cases, pharmaceutical compositions formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols may be delivered by topical routes, transdermally.

[0073] In some cases, pharmaceutical compositions can be delivered as microspheres for sustained release in the body.For example, microspheres can be administered by subcutaneously slowly releasing drug through intradermal injection (see Rao (1995) J.Biomater Sci.Polym.Ed.7:623-645); as biodegradable and injectable gel formulation (see, for example, Gao (1995) Pharm.Res.12:857-863 (1995)); or as oral administration microspheres (see, for example, Eyles (1997) J.Pharm.Pharmacol.49:669-674).

[0074] In some cases, pharmaceutical compositions may be administered parenterally, for example, by intravenous (IV) administration or administration into a body cavity or lumen of an organ. These formulations may contain a solution of an active agent (e.g., an inhibitory nucleic acid of the present disclosure) in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that may be used include water and Ringer's solution, isotonic sodium chloride. Additionally, sterile, fixed oils may be used as a solvent or suspending medium. For this purpose, any bland, fixed oil, such as synthetic monoglycerides or diglycerides, may be used. Additionally, fatty acids, such as oleic acid, may also be used in the preparation of injectable solutions. These solutions are sterile and generally free of undesirable substances. These formulations may be sterilized by conventional, well-known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the active agent in these formulations can vary widely and is selected primarily based on the volume, viscosity, body weight, etc. of the liquid, depending on the particular mode of administration selected and the patient's needs. Formulations for intravenous administration can be sterile injectable preparations, such as sterile injectable aqueous or oily suspensions. The suspensions can be formulated using appropriate dispersing or wetting agents and suspending agents. The sterile injectable preparations can also be suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions of 1,3-butanediol. Administration can be by bolus injection or continuous infusion (e.g., substantially uninterrupted introduction into the blood vessels for a specified period of time).

[0075] In some cases, the pharmaceutical composition can be a lyophilized product. A stable lyophilized composition containing an inhibitory nucleic acid can be produced by lyophilizing a solution containing the pharmaceutical composition of the present disclosure and a bulking agent, such as mannitol, trehalose, raffinose, and sucrose, or a mixture thereof. The process for preparing a stable lyophilized formulation can include lyophilizing a solution containing about 2.5 mg / mL of nucleic acid, about 15 mg / mL of sucrose, about 19 mg / mL of NaCl, and a sodium citrate buffer solution having a pH greater than 5.5 and less than 6.5. See, for example, US20040028670.

[0076] Compositions and formulations can be delivered by using liposomes.By using liposomes, the delivery of active agents (such as the inhibitory nucleic acids of the present disclosure) can be focused to target cells in vivo, especially when the liposome surface carries a ligand specific to target cells or is otherwise preferentially directed to a specific organ.See, for example, U.S. Patent No. 6,063,400; 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" refers to a vesicle composed of amphiphilic lipids arranged as a bilayer. Liposomes are unilamellar or multilamellar vesicles with a membrane made of lipophilic material and an inner aqueous phase 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 liposomes or negatively charged liposomes are thought to capture DNA rather than form complexes with DNA.Both cationic and non-cationic liposomes have been used to deliver DNA to cells.

[0077] Liposomes may also include "sterically stabilized" liposomes, i.e., liposomes containing one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the lipid moiety forming the liposome vesicle contains 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.

[0078] The preparation of the present disclosure can be administered for preventive treatment and / or therapeutic treatment.In some cases, the composition for therapeutic application is administered to the subject in need thereof (for example, the individual who has the risk of the disorder described herein (for example, the risk higher than the general population) or has the disorder described herein) in an amount sufficient to cure, alleviate or partially stop the clinical manifestation of disorder or its complications; this can be called therapeutically effective amount.

[0079] The amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose. Effective administration schedules and amounts, i.e., dosing regimens, for this use depend on a variety of factors, such as the stage of the disease or condition, the severity of the disease or condition, the patient's general health, the patient's physical condition, age, etc. In calculating the dosing regimen for a patient, the mode of administration is also taken into consideration.

[0080] The dosing regimen also takes into account pharmacokinetic parameters well known in the art, i.e., the rate of absorption, bioavailability, metabolism, clearance, etc., of the active agent (see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005). The state of the art allows physicians to determine the dosing regimen for each individual patient, active agent (e.g., inhibitory nucleic acid), and disease or condition being treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance for determining the dosing regimen, i.e., the dosing schedule and dosage level.

[0081] For example, single or multiple administrations of the formulation may be given, depending on the dosage and frequency required and tolerated by the patient, the extent and amount of therapeutic effect (e.g., effect on blood glucose levels) occurring after each administration, etc. The formulation should provide a sufficient amount of the active agent (e.g., inhibitory nucleic acid) to effectively treat, prevent, or ameliorate a condition, disease, or symptom.

[0082] In an alternative embodiment, the daily dosage of a pharmaceutical formulation for oral administration is about 1 μg to about 100 mg of nucleic acid per kilogram of body weight per day. In contrast to oral administration, lower dosages may be used for the bloodstream, body cavities, or organ lumens. Substantially higher dosages may be used for topical or oral administration, or for administration by powder, spray, or inhalation. Actual methods for preparing formulations suitable for parenteral or oral administration are known or apparent to those skilled in the art and are described in more detail in publications such as Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

[0083] In some embodiments, the methods described herein can include co-administration with other drugs or medications, such as compositions for lowering blood glucose levels. For example, the inhibitory nucleic acids can be co-administered with drugs for treating or reducing the risk of a disorder described herein.

[0084] Treatment method The present disclosure provides methods for inhibiting the growth of cancer and methods for treating cancer in an individual, comprising administering to an individual in need thereof (e.g., an individual with cancer) 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.

[0085] In some cases, an effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, reduces tumor burden / tumor volume in the individual. 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, increases the survival time of the individual. For example, 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, increases the survival time 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 individual's expected survival time in the absence of administration of the inhibitory nucleic acid.

[0086] 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, either 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 keeps the patient's overall tumor burden relatively stable for a period of time sufficient for the patient to have confirmed "stable disease" as determined by standard RECIST criteria. See, e.g., Aykan and Ozatli (2020) World J. Clin. Oncol. 11:53.

[0087] In some cases, an effective amount of an inhibitory nucleic acid is one that, when administered in one or more doses to an individual in need thereof as a monotherapy or as part of a combination therapy, causes a reduction in tumor size by an amount and for a sufficient duration for the patient to have a confirmed "partial response" as determined by standard Response Evaluation Criteria in Solid Tumors (RECIST) criteria.

[0088] In some cases, an effective amount of an inhibitory nucleic acid is one that, when administered in one or more doses to an individual in need thereof (e.g., an individual with a tumor) as a monotherapy or as part of a combination therapy, causes a reduction in tumor size by an amount sufficient and for a duration sufficient for the patient to have a confirmed "complete response" as determined by standard RECIST criteria.

[0089] 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 type of skin cancer), squamous cell carcinoma (various tissues), bladder cancer, e.g., transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon cancer, colorectal cancer, gastric cancer, lung cancer, including small cell lung cancer and non-small cell lung cancer, 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 carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteosarcoma, epithelial carcinoma, and nasopharyngeal carcinoma. In some cases, the cancer is chordoma.

[0090] Dosage The appropriate dosage of an inhibitory nucleic acid can be determined by the attending physician or other qualified medical professional based on various clinical factors. As is well known in the medical field, the dosage for a single patient depends on many factors, including the patient's size, body surface area, age, the specific polypeptide or nucleic acid being administered, the patient's sex, the time and route of administration, general health, and other drugs being administered concomitantly. The inhibitory nucleic acids of the present disclosure can be administered in amounts of 1 ng / kg to 20 mg / kg body weight or more per dose, e.g., 0.1 mg / kg to 10 mg / kg body weight, e.g., 1 mg / kg to 5 mg / kg, or 5 mg / kg to 10 mg / kg body weight; 10 to 15 mg / kg or more, although doses lower or higher than this exemplary range are also contemplated.

[0091] The frequency of administration of the inhibitory nucleic acid can vary depending on any of a variety of factors, but generally speaking, it is administered once a week, once every two weeks, once every three weeks, once every four weeks, once a month, or less frequently than once a month, such as once every five weeks, once every six weeks, once every two months, once every three months, etc., and may also be administered more frequently than once a week, such as 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 every day (qd). In some cases, the inhibitory nucleic acid is administered once every three weeks. Generally, administration should be discontinued upon disease progression or unacceptable toxicity.

[0092] The duration of administration of the inhibitory nucleic acid can vary depending on any of a variety of factors, such as patient response. For example, the inhibitory nucleic acid can be administered for a period of 1 month to about 2 months, about 2 months to about 4 months, about 4 months to about 6 months, about 6 months to about 8 months, about 8 months to about 1 year, about 1 year to about 2 years, or about 2 years to about 4 years, or longer. In some cases, the inhibitory nucleic acid continues to be administered for at least the period during which the patient continues to receive a clinically determined benefit, which can be at least several months to several years.

[0093] Route of administration 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.

[0094] Combination therapy The present disclosure contemplates the use of the inhibitory nucleic acid of the present disclosure in combination with one or more additional drugs (for example, one or more additional active therapeutic agents) or other preventive or therapeutic modalities.In such combination therapy, various active agents often have different mechanisms of action.Such combination therapy can be particularly advantageous because it allows for the reduction of the dose of one or more drugs, thereby reducing or eliminating the adverse effects associated with one or more drugs; Moreover, such combination therapy can have a synergistic therapeutic or preventive effect on the underlying disease, disorder or condition.

[0095] In some cases, the disclosed methods for treating cancer in an individual include (a) administering an inhibitory nucleic acid of the present disclosure; and (b) administering at least one additional therapeutic agent or treatment. Suitable additional therapeutic agents include, but are not limited to, small molecule anti-cancer chemotherapeutic agents and immune checkpoint inhibitors. Suitable additional therapeutic treatments include, for example, radiation, surgery (e.g., surgical removal of a tumor), etc.

[0096] As used herein, "combination" is intended to include therapies that may be administered separately, e.g., formulated separately for separate administration (e.g., as may be provided in a kit), and therapies that may be administered together in a single formulation (i.e., a "combination").

[0097] In certain cases, the inhibitory nucleic acid of the present disclosure and at least one additional agent are administered or applied sequentially, for example, 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, two or more agents are administered simultaneously or nearly simultaneously; the two or more agents may be present in two or more separate formulations or may be combined into a single formulation (i.e., a combination formulation). For the purposes of this disclosure, two or more agents are considered to be administered in combination, regardless of whether they are administered sequentially or simultaneously.

[0098] Suitable additional therapeutic agents include, for example, anti-cancer chemotherapeutic agents, immune checkpoint inhibitors, immunotherapeutic agents, and the like.

[0099] subject Suitable subjects for treatment by the methods of the present disclosure include individuals who have been diagnosed with cancer, individuals who have received treatment for cancer but have failed to respond to the treatment, and individuals who have received treatment for cancer and initially responded to the treatment but have subsequently become refractory and / or whose disease has progressed during previous treatment.

[0100] Examples of Non-Limiting Aspects of the Disclosure The above-mentioned aspects (e.g., embodiments) of the subject matter of the present invention can be useful alone or in combination with one or more other aspects or embodiments.Without limiting the above description, some non-limiting aspects of the present disclosure are provided below.As will be clear to those skilled in the art by referring to this disclosure, each individually numbered aspect can be used with or combined with any of the individually numbered aspects before and after it.This is to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below.

[0101] Aspect 1. An inhibitory nucleic acid comprising a nucleotide sequence complementary to a target nucleotide sequence within a TBXT transcript, the inhibitory nucleic acid comprising one or more locked nucleic acids (LNAs). Aspect 2. The inhibitory nucleic acid of Aspect 1, having a length of about 15 nucleotides to about 30 nucleotides. Aspect 3. The inhibitory nucleic acid of Aspect 1, having a length of about 15 nucleotides to about 20 nucleotides. Aspect 4. The inhibitory nucleic acid of any one of Aspects 1-3, wherein the nucleotide sequence complementary to a nucleotide sequence within the TBXT transcript is complementary to a nucleotide sequence within the intron 4 / exon 5 junction. Aspect 5. The target TBXT nucleotide sequence 4. The inhibitory nucleic acid of any one of Aspects 1 to 3, selected from TIFF2026500230000002.tif48128. Aspect 6. The inhibitory nucleic acid of any one of Aspects 1-5, comprising one or more phosphorothioate linkages. Aspect 7. the inhibitory nucleic acid TIFF2026500230000003.tif61128, * 7. The inhibitory nucleic acid of any one of Aspects 1 to 6, wherein a "+" immediately preceding a nucleotide indicates that the nucleotide is an LNA. Aspect 8. (a) an inhibitory nucleic acid of any one of Aspects 1-7; and (b) a pharmaceutically acceptable excipient; A composition comprising: Aspect 9. The pharmaceutical composition of Aspect 8, wherein the pharmaceutically acceptable excipient comprises one or more lipids. Aspect 10. The pharmaceutical composition of Aspect 8, wherein the pharmaceutically acceptable excipient comprises poly(amidoamine), poly(propyleneimine), or poly(L-lysine). Aspect 11. (a) an inhibitory nucleic acid of any one of Aspects 1-7; and (b) a pharmaceutically acceptable excipient; A lipid nanoparticle comprising: 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. Aspect 13. A method of inhibiting cancer growth 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. Aspect 14. The method of Aspect 13, wherein the cancer is lung cancer, breast cancer, colon cancer, prostate cancer, or liver cancer. Aspect 15. The method of Aspect 13, wherein the cancer is chordoma. 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. Aspect 17. The method of Aspect 16, wherein the cancer is lung cancer, breast cancer, colon cancer, prostate cancer, or liver cancer. Aspect 18. The method of Aspect 16, wherein the cancer is chordoma. Aspect 19. The method of any one of Aspects 16-18, wherein the administering step comprises an intravenous, intramuscular, intratumoral, or peritumor route of administration. Aspect 20. The method of any one of Aspects 16-19, further comprising administering one or more additional therapeutic treatments. Aspect 21. The method of Aspect 20, wherein the one or more additional therapeutic treatments include cancer chemotherapy, radiation, or surgery. [Example]

[0102] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. 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. Standard abbreviations may be used, such as bp, base pairs; kb, kilobases; pl, picoliters; s or sec, seconds; min, minutes; h or hr, hours (hours); aa, amino acids; kb, kilobases; bp, base pairs; nt, nucleotides; im, intramuscular (intramuscular); ip, intraperitoneal (intraperitoneal); sc, subcutaneous (subcutaneous); etc.

[0103] Example 1: method cell culture Chordoma cell lines were obtained from ATCC and maintained in a medium containing IMDM:RPMI in a 4:1 ratio, supplemented with 10% FBS and non-essential amino acids and glutamine. All cell lines were grown on tissue culture plastic, except for UCH-2, which was grown in flasks coated with type I collagen. The U2OS cell line was obtained from the UC Berkeley Cell Culture Facility and maintained in DMEM high glucose + 10% FBS.

[0104] antisense oligonucleotides 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. For assays, ASOs were further diluted in cell culture PBS. A scrambled control mixmer LNA ASO (#406, sequence: +C * G * +T * T *+A * G * +A * +T * T * +A * +C * / iMe-dC / * G * +C * +G).

[0105] ASO transfection Cells were seeded at 13,158 cells / cm2 and allowed to adhere for at least 24 hours or until they reached 70% confluency. ASOs were then diluted in Opti-Mem and transfected via Lipofectamine 3000 according to the manufacturer's instructions. Final ASO concentrations in the medium ranged from 0.1 nM to 50 nM. Cells were incubated for 48–96 hours, after which the medium was removed, the cells were washed with PBS, and the plates were frozen at -80°C.

[0106] Western blot Cell lysates were collected in 1x Laemmli buffer plus β-mercaptoethanol. Samples were separated by molecular weight using a Bio-Rad Mini Protean Tetra gel electrophoresis system. Proteins were transferred to membranes using a Bio-Rad Trans-Blot Turbo Transfer System and then blocked with 5% milk for 1 hour. Membranes were incubated overnight in either human Brachyury primary antibody (R&D Systems, AF2085) or histone H3 primary antibody (Cell Signaling Technologies, 9715). Membranes were washed with TBS-Tween and then incubated in anti-goat or anti-rabbit secondary antibodies, respectively, for 1 hour. After washing, membranes were treated with SuperSignal West Pico Plus chemiluminescent substrate and then imaged on an iBright 1500 blot imager.

[0107] Nonspecific toxicity assay U2OS cells were plated in white opaque 96-well plates at 7,813 cells / cm 2 Cells were seeded at 100°C and allowed to adhere for at least 24 hours or until they reached 70% confluency. 50 nM ASO was transfected in triplicate as described above, and plates were incubated for 6 days, with medium changed 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 using a Biotek Synergy LX plate reader. Luminescence readings were normalized to ASO-free or scrambled ASO controls, and data were analyzed via one-way analysis of variance followed by Dunnett's multiple comparison test.

[0108] Chordoma growth inhibition assay Cells were seeded and transfected as described above, but in larger-format 12-well plates. After 72 hours of incubation, each treatment was trypsinized, counted, and replated at 1,563 cells / cm2. Cells were grown for 21 days, with medium changes every 3–4 days. At each time point, an equal volume of Cell Titer Glo 2.0 was added to each well, and cell viability was assessed as described for the nonspecific toxicity test. Data were analyzed via one-way analysis of variance followed by Dunnett's multiple comparison test.

[0109] result We designed a library of 147 LNA ASOs (75 gapmers and 72 mixmers). This library is hereafter referred to as the "original" library. The LNA ASOs bind along the human Brachyury (TBXT) pre-mRNA (NCBI Gen ID: 6862). Figure 1 shows a schematic of the TBXT targeting mechanism of the ASO library. When screened in the chordoma cell line UM-Chor1, 17 mixmer ASOs effectively disrupted TBXT expression (Figure 2). All 17 ASOs localized to the same exon of the TBXT pre-mRNA (Figure 3). Using the TBXT-independent cell line U2OS to eliminate ASOs exhibiting nonspecific toxicity, nine hits remained (Figure 4). A dose-response study determined that the most potent hit spanned the intron 4 / exon 5 junction of the TBXT pre-mRNA, suggesting a lethal exon skipping mechanism for the protein. Figure 5; Figure 3.

[0110] Figure 1. 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, whereas mixmer ASOs sterically inhibit splicing or translation. The end result is a lack of Brachyury protein product or a nonfunctional Brachyury protein product.

[0111] Figure 2. Selected mixmer LNA ASOs effectively inhibit TBXT expression. UM-Chor1 cells were transfected with 25 nM of each ASO. After 48 h, cell lysates were probed for Brachyury via Western blot. The absence of a 50 kDa protein indicates that mixmers #2, 5, 6, 7, 10, 11, 12, 15, 18, 28, 29, 31, 32, 69, 70, 71, and 72 were hits. This experiment was repeated once more with the same results.

[0112] Figure 3. All anti-TBXT LNA ASO mixer hits localize to the same exon. Comparison of the target sites of 17 ASOs capable of inhibiting TBXT protein reveals that all "hits" bind to and around exon 5 of the pre-mRNA associated with TBXT transcript variant 1 (NCBI accession #NM_003181).

[0113] Figure 4. Nonspecific toxicity of anti-TBXT LNA ASO mixer hits. The TBXT-independent cell line U2OS was transfected with 50 nM of each ASO. After 6 days, Cell Titer Glo was added, and luminescence was measured and normalized to the ASO-free control. Three replicates were measured and compared to the ASO-free control by one-way ANOVA and Dunnett's multiple comparison test. A significant decrease in luminescence indicates a decrease in ATP and, therefore, cell viability.

[0114] Figure 5. Selected mixmer LNA ASOs potently inhibit TBXT expression. UM-Chor1 cells were transfected with the indicated concentrations of each ASO. After 48 h, cell lysates were probed for Brachyury via Western blot. Dose-response studies show effective TBXT inhibition up to 10 nM in ASOs #2, 5, and 7.

[0115] A second library of 33 LNA ASOs spanning the intron 4 / exon 5 junction of TBXT was designed. This library is hereafter referred to as the "redesign" library. Nonspecific toxicity testing identified seven nontoxic hits (Figure 6), which were confirmed to inhibit TBXT protein in UM-Chor1 chordoma cells (Figure 7). Dose-response testing determined that the redesign had comparable TBXT inhibitory potency to ASOs #2, #5, and #7. Hits from both the original and redesigned designs were then validated in three additional chordoma cell lines: MUG-Chor1, JHC7, and UCH-2, demonstrating broad potency in inhibiting TBXT protein (Figures 8-10). For a schematic of the ASO screening, see Figure 11. Because Brachyury has been shown to be important for chordoma proliferation, the 10 anti-TBXT LNA ASO hits (3 original + 7 redesigned) were then tested for their ability to inhibit chordoma cell growth. All 10 ASOs were able to significantly inhibit the growth of UM-Chor1 cells (Figure 12). See Table 1 for sequences related to the 10 anti-TBXT LNA ASO hits.

[0116] Figure 6. Nonspecific toxicity of the anti-TBXT LNA ASO mix-matrix design. The TBXT-independent cell line U2OS was transfected with 50 nM of each ASO. After 6 days, Cell Titer Glo was added, and luminescence was measured and normalized to the ASO-free control. Three replicates were measured and compared to the ASO-free control by one-way ANOVA and Dunnett's multiple comparison test. A significant decrease in luminescence indicates a decrease in ATP and, therefore, cell viability.

[0117] Figure 7. All non-toxic mixmer LNA ASO redesigns effectively inhibit TBXT expression. UM-Chor1 cells were transfected with 25 nM of each ASO. After 48 h, cell lysates were probed for Brachyury via Western blot. The lack of a 50 kDa protein indicates that mixmers #129, 135, 136, 137, 138, 139, and 141 are hits.

[0118] Figure 8. All original and redesigned mixmer LNA ASO hits effectively inhibit TBXT expression in MUG-Chor1 cells. MUG-Chor1 cells were transfected with 25 nM of each ASO. After 96 h, cell lysates were probed for Brachyury via Western blot. The lack of a 50 kDa protein indicates that mixmers #2, 5, 7, 129, 135, 136, 137, 138, 139, and 141 can inhibit TBXT in multiple chordoma cell lines.

[0119] Figure 9. All original and redesigned mixmer LNA ASO hits effectively inhibit TBXT expression in JHC7 cells. JHC7 cells were transfected with 25 nM of each ASO. After 96 h, cell lysates were probed for Brachyury via Western blot. The lack of a 50 kDa protein indicates that mixmers #2, 5, 7, 129, 135, 136, 137, 138, 139, and 141 can inhibit TBXT in multiple chordoma cell lines.

[0120] Figure 10. All original and redesigned mixmer LNA ASO hits effectively inhibit TBXT expression in UCH-2 cells. UCH-2 cells were transfected with 25 nM of each ASO. After 96 h, cell lysates were probed for Brachyury via Western blot. The lack of a 50 kDa protein indicates that mixmers #2, 5, 7, 129, 135, 136, 137, 138, 139, and 141 can inhibit TBXT in multiple chordoma cell lines.

[0121] Figure 11. Overview of anti-TBXT mixmer LNA ASO screening. 72 mixmer ASOs were tested for their ability to inhibit TBXT protein in the UM-Chor1 chordoma cell line. 17 hits emerged, of which 8 were eliminated due to nonspecific toxicity. Nine ASOs were tested in dose-response studies, and the three most potent ASOs allowed for the identification of optimal binding sites on TBXT pre-mRNA. ASO redesign focused on these optimal sites generated 33 new anti-TBXT mixmer LNA ASOs. 26 ASO redesigns were eliminated due to nonspecific toxicity, leaving seven that showed potent inhibition of TBXT. The seven redesign hits, combined with the three original design hits, were validated in three independent chordoma cell lines, yielding a total of 10 hits.

[0122] Figures 12A-12B. Anti-TBXT mixmer LNA ASO inhibits chordoma cell growth. UM-Chor1 cells were transfected with anti-TBXT ASO hits using Lipofectamine. After 72 h, each treatment was trypsinized, counted, and re-seeded in triplicate at matching numbers. (A) At the indicated time points, Cell Titer Glo was added and luminescence was measured. (B) Luminescence values ​​at t = 14 days were normalized to the ASO-free control. Three replicates were measured and compared to the scrambled mixmer control by one-way ANOVA and Dunnett's multiple comparison test. A significant decrease in luminescence indicates a decrease in ATP and, therefore, cell viability.

[0123] Figures 15A-15B. Mixmer #139 enters chordoma cells without a lipid delivery vehicle and inhibits TBXT expression. UM-Chor1 cells were seeded, and ASO #139 (B) or a scrambled control ASO (A) was added directly to the culture medium at the indicated concentrations. Cells were incubated for 12 days, with ASO and medium changes every 3-4 days. Cell lysates were then probed for Brachyury via Western blot. The lack of a 50 kDa protein with increasing concentrations of mixmer #139 indicates that the anti-TBXT ASO can passively enter chordoma cells.

[0124] Figures 16A-16D. ASOs directed against the intron 4 / exon 5 junction of TBXT induce exon skipping and lethal frameshift mutations. UM-Chor1 cells were transfected with 25 nM of mixmers #2, 15, 139, or a scrambled control mixmer (A) using Lipofectamine and incubated for 72 h. RNA was extracted, and sequences encompassing the ASO binding site were PCR-amplified using the indicated primers (B) and then visualized on an acrylamide gel (C). A 165-base pair band in all anti-TBXT ASOs indicated the loss of exon 5. Primer products were excised and sequenced, and the sequencing data confirmed the accurate skipping of TBXT exon 5, resulting in a premature stop codon. A schematic of the generation of exon skipping and frameshift mutations induced by anti-TBXT ASOs (D) is shown.

[0125] Figures 17A-17B. Mixmer #139 induces S-phase cell cycle arrest in chordoma cells. UM-Chor1 cells were transfected with 25 nM anti-TBXT ASO #139 or a scrambled control mixmer via Lipofectamine. Cells were incubated for 48 h, then expanded and cultured for an additional 4 days (A) or 7 days (B) with medium changes every 2–3 days. Cells were fixed, stained with propidium iodide, and analyzed via flow cytometry to estimate the proportion of cells in each phase of the cell cycle. A free cell cycle control derived from actively dividing cells was included as an additional control. The decrease in G1-phase cells accompanied by an increase in S-phase cells upon treatment with anti-TBXT #139 indicated S-phase arrest, suggesting that TBXT inhibition induces a replicative stress checkpoint.

[0126] Figures 18A-18E. Anti-TBXT ASO#139 was well tolerated in mice. Six-week-old female nude mice were randomly assigned to treatment groups (3 / group) by body weight and treated via sc injection of 40 or 60 mg / kg of anti-TBXT ASO#139. A saline vehicle group was included as a negative control. Treatment continued weekly or biweekly as indicated for 14 days, and body weights were measured daily and reported as percent change from 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). Neither body weight nor liver / renal toxicity markers of treated mice were significantly different from saline controls (NS), indicating good tolerability of ASO#139.

[0127] Figures 19A-19D. Treatment with anti-TBXT ASO #139 selectively eradicates patient-derived chordoma tumors in mice. Six-week-old female nude mice were sc-implanted with 5x5mm CF466 patient-derived chordoma tumor fragments. Tumors were 100-200mm 3When the tumor reached 100 mg / mL, mice were randomized into groups (7 / group) and treatment began. Anti-TBXT ASO #139 was injected sc every other week at 30 or 60 mg / kg, with a saline vehicle group serving as a negative control. The positive control arm received 20 mg / kg afatinib. Body weight (B) and tumor dimensions (A, C) were measured twice weekly, and treatment continued for 6 weeks. Body weight was reported as a percentage change from baseline. Tumors and livers were collected at the end of the study and snap-frozen for quantification of ASO concentrations. 10-20 mg tumor samples were homogenized, and ASO #139 was quantified via Splint® qPCR using custom probes and primers for the target ASO. The mean cycle threshold from the ASO standard curve was calculated as the logarithm of the concentration. 10 The data were plotted against the linear portion of the curves. Regressions were fitted to the linear portions of the curves, and ASO concentrations in tissue lysate samples were interpolated from these curves (D). In this pilot study, 2 / 7 mice treated with 30 mg / kg ASO#139 experienced complete tumor regression (C), indicating at least partial efficacy. ASO levels were detected in the tumors of the remaining treated mice, demonstrating robust accumulation of anti-TBXT ASO in solid tumors following systemic administration.

[0128] Table 1. Sequences associated with anti-TBXT mixmer LNA ASO hits. Each ASO is a modified single-stranded DNA / LNA mixmer oligonucleotide. The phosphodiester backbone (" * The target sequence represents the binding sequence spanning the intron 4 / exon 5 junction of TBXT pre-mRNA transcript variant 1 (NCBI accession #NM_003181).

[0129] [Table 1]

[0130] The target sequences in Table 1 are, from top to bottom, SEQ ID NOs: 1 to 8, respectively.

[0131] While the present invention has been described with reference to specific embodiments thereof, it should 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. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step(s), to the objective, 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 within a TBXT transcript, the inhibitory nucleic acid comprising one or more locked nucleic acids (LNAs).

2. 10. The inhibitory nucleic acid of claim 1, having a length of about 15 nucleotides to about 30 nucleotides.

3. 10. The inhibitory nucleic acid of claim 1, having a length of about 15 nucleotides to about 20 nucleotides.

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

5. The target TBXT nucleotide sequence The inhibitory nucleic acid of any one of claims 1 to 3, selected from:

6. 6. The inhibitory nucleic acid of any one of claims 1 to 5, comprising one or more phosphorothioate linkages.

7. the inhibitory nucleic acid comprising a nucleotide sequence selected from * The inhibitory nucleic acid of any one of claims 1 to 6, wherein "+" immediately before a nucleotide indicates that the nucleotide is an LNA.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Images