Compositions and methods for inhibiting STAG1 expression and uses thereof

Abstract

Described herein is a composition and method for downregulating the expression of stromal antigen 1 (STAG1).The described composition comprises an antisense oligonucleotide (ASO) that has a sequence that is sufficiently complementary to a portion of STAG1 primary transcript.The described composition comprising ASO can be used for the treatment of cancer, for example, cohesin-deficient cancer and STAG2-deficient cancer. TIFF2025503956000016.tif102140

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application No. 63 / 303,192, filed January 26, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Technical Field The technology described herein relates to the use of antisense oligonucleotides for the downregulation of stromal antigen 1 for the treatment of cancer. [Background technology]

[0003] background Myeloid malignancies, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), constitute a heterogeneous group of clonal disorders of mutant hematopoietic stem cells. More than 30,000 new cases of MDS and more than 20,000 new cases of AML are diagnosed annually in the United States, with high mortality rates. Younger patients with MDS or AML may be candidates for intensive chemotherapy followed by allogeneic stem cell transplantation (the only potentially curative approach to MDS), but treatment options for older patients with these conditions are limited, with long-term survival of less than 5%. Thus, new treatments for these fatal diseases are urgently needed.

[0004] Genes encoding components of the cohesin complex are mutated in 11% of patients with MDS and 21% of patients with secondary AML. Cohesin mutations do not show hotspots and generally result in loss of function and / or haploinsufficiency. Mutations in cohesin would be expected to cause defects in chromosome segregation or genomic integrity, but mutations affecting these functions are likely to be lethal. Thus, diseases mediated by cohesin are characterized by the absence of aneuploidy or complex karyotypes, but with dysregulated gene expression that promotes oncogenic transformation.

[0005] Cohesin is a multi-subunit protein complex essential for sister chromatid cohesin, chromosome organization into looped domains, DNA damage repair, and transcriptional regulation. Cohesin is one of the most frequently mutated protein complexes in cancer, e.g., myeloid malignancies, with frequent somatic loss-of-function mutations in the core components of the cohesin ring. Importantly, cancer-associated mutations in cohesin rarely affect chromosome integrity, but instead selectively impair gene regulatory functions. However, how cohesin affects gene activity remains enigmatic and offers no clues for intervention. Cohesin mutations are associated with poor overall survival, and no treatments are currently known to have selective efficacy in cohesin-mutated cancers. Thus, there is a need to define the molecular targets and activities of cohesin and to identify targeted therapeutic approaches for the treatment of diseases involving cohesin mutations. Summary of the Invention

[0006] overview Cohesin mutations, which are common in myeloid malignancies, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), disrupt RNA splicing. As a result, cohesin-deficient cancers, such as STAG2-deficient cancers, are highly sensitive to inhibitors of mRNA splicing. The present inventors have found that one of the RNAs affected by splicing inhibitors in STAG2-deficient cells is the mis-spliced ​​STAG1 RNA. Given that STAG2-deficient cells, but not wild-type cells, depend on STAG1 paralogs for survival, treatments that interfere with the splicing and / or stability of STAG1 mRNA are found to selectively kill STAG2-deficient cells, such as STAG2-deficient cancer cells. Antisense oligonucleotides that target STAG1 RNA are described herein. Also described herein are methods of using such oligonucleotides, and / or compositions comprising them, to selectively kill STAG2-deficient cells, including but not limited to, myelodysplastic cells, AML cells, and other STAG2-deficient cancer cells that are STAG2-deficient. Antisense oligonucleotides targeting STAG1 expression are contemplated for use alone or in combination with other anti-cancer agents, including but not limited to, inhibitors of the DNA damage response, including but not limited to, PARP inhibitors.

[0007] In one aspect, described herein is a composition for downregulating expression of stromal antigen 1 (STAG1), the composition comprising an antisense oligonucleotide having a sequence sufficiently complementary to a portion of a STAG1 primary transcript to permit hybridization to the portion of the STAG1 primary transcript, wherein the oligonucleotide comprises at least one phosphorothioate backbone modification.

[0008] In one embodiment of this aspect and any other aspect described herein, the antisense oligonucleotide comprises an RNA oligonucleotide, a DNA oligonucleotide, or a combination of deoxyribonucleosides and ribonucleosides.

[0009] In another embodiment of this aspect or any other aspect described herein, the antisense oligonucleotide comprises at least one deoxyribonucleoside and at least one ribonucleoside.

[0010] In another aspect, described herein is a composition for downregulating expression of stromal antigen 1 (STAG1), the composition comprising an antisense oligonucleotide having a sequence sufficiently complementary to a portion of a STAG1 primary transcript to permit hybridization to a portion of the STAG1 primary transcript, wherein the oligonucleotide comprises at least one deoxyribonucleoside and at least one ribonucleoside.

[0011] In one embodiment of this aspect and any other aspect described herein, the antisense oligonucleotide comprises, in 5' to 3' order, 1-5 ribonucleosides, 6-10 deoxyribonucleosides, and 1-5 ribonucleosides. In another embodiment of this aspect and any other aspect described herein, the antisense oligonucleotide comprises, in 5' to 3' order, 1-5 ribonucleosides, 6-12 deoxyribonucleosides, and 1-5 ribonucleosides.

[0012] In another embodiment of this aspect or any other aspect described herein, one or more of the ribonucleosides includes a 2'-O-methoxyethyl (MOE) modification.

[0013] In another embodiment of this aspect or any other aspect described herein, the antisense oligonucleotide comprises at least one phosphorothioate backbone linkage.

[0014] In another embodiment of this aspect or any other aspect described herein, the antisense oligonucleotide comprises phosphorothioate linkages between each nucleoside.

[0015] In another embodiment of this aspect and any other aspect described herein, the antisense oligonucleotide comprises 15-22 nucleosides. In other embodiments, the antisense oligonucleotides useful in the methods and compositions described herein comprise, for example, 10-50 nucleosides, e.g., 10-45 nucleosides, 10-40 nucleosides, 10-35 nucleosides, 10-30 nucleosides, 10-29 nucleosides, 10-28 nucleosides, 10-27 nucleosides, , 10-26 nucleosides, 10-25 nucleosides, 10-24 nucleosides, 10-23 nucleosides, 10-22 nucleosides, 10-21 nucleosides, 10-20 nucleosides, 12-50 nucleosides, 12-45 nucleosides, 12-40 nucleosides, 12-35 nucleosides, 12-30 nucleosides, 12 ~29 nucleosides, 12-28 nucleosides, 12-27 nucleosides, 12-26 nucleosides, 12-25 nucleosides, 12-24 nucleosides, 12-23 nucleosides, 12-22 nucleosides, 12-21 nucleosides, 12-20 nucleosides, 15-45 nucleosides, 15-40 nucleosides, 15-35 nucleosides, 15-30 nucleosides, 15-29 nucleosides, 15-28 nucleosides, 15-27 nucleosides, 15-26 nucleosides, 15-25 nucleosides, 15-24 nucleosides, 15-23 nucleosides, 15-22 nucleosides, 15-21 nucleosides, or 15-20 nucleosides.

[0016] In another embodiment of this aspect and any other aspect described herein, the antisense oligonucleotide comprises one or more sugar modifications selected from 2'-fluoro, 2'-O-methyl, an LNA modification (2'-O,4'-methylene modification), a constrained ethyl nucleoside modification (2',4'-constrained 2'-ethyl nucleoside or 2'-O,4'-ethylene nucleoside), 5-methylcytosine (5-MeC), and a phosphorodiamidate morpholino (PMO) modification.

[0017] In another embodiment of this aspect and any other aspect described herein, the antisense oligonucleotide comprises a sequence that allows hybridization with exon 3 or 5 of the STAG1 RNA transcript. In another embodiment of this aspect and any other aspect described herein, the antisense oligonucleotide comprises a sequence that allows hybridization with exon 29.

[0018] In some embodiments, the ASO compositions described herein target elements of the primary transcript that affect mRNA splicing.

[0019] In another embodiment of this aspect or any other aspect described herein, the intra-exon splicing promoting sequence comprises a binding site for the RNA binding protein SRSF2.

[0020] In another embodiment of this aspect or any other aspect described herein, the antisense oligonucleotide comprises a sequence selected from those presented in Table 1, including SEQ ID NOs 1-81.

[0021] In another aspect, described herein is a pharmaceutical formulation comprising an antisense oligonucleotide described herein and a pharma- ceutically acceptable carrier.

[0022] In another aspect, described herein is a method of downregulating expression of STAG1 in a cell, the method comprising contacting the cell with an antisense oligonucleotide described herein.

[0023] In one embodiment of this aspect or any other aspect described herein, the cell is a cohesin mutant cell.

[0024] In another embodiment of this aspect or any other aspect described herein, the cell is a STAG2 mutant cell.

[0025] In another embodiment of this aspect and any other aspect described herein, the cell is a cancer cell or a myelodysplastic syndrome (MDS) cell. In one embodiment, MDS represents a precursor to acute myeloid leukemia (AML).

[0026] In another embodiment of this aspect or any other aspect described herein, the cell is a cancer cell selected from an acute myeloid leukemia (AML) cell, a glioblastoma cell, and a bladder cancer cell.

[0027] In another embodiment of this aspect or any other aspect described herein, the contacting reduces STAG1 mRNA in the cell by at least 50%.

[0028] In another aspect, described herein is a method of selectively killing cohesin mutant cells, the method comprising contacting the cells with an antisense oligonucleotide described herein.

[0029] In one embodiment of this aspect or any other aspect described herein, the cell is a STAG2 mutant cell.

[0030] In another embodiment of this aspect or any other aspect described herein, the cell is a cancer cell or a myelodysplastic syndrome cell.

[0031] In another embodiment of this aspect or any other aspect described herein, the cell is a cancer cell selected from an acute myeloid leukemia (AML) cell, a glioblastoma cell, and a bladder cancer cell.

[0032] In another embodiment of this aspect or any other aspect described herein, the contacting step reduces STAG1 mRNA in the cell by at least 50%. In another embodiment, the contacting step reduces STAG1 mRNA by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0033] In another aspect, described herein is a method for treating a cohesin mutant cancer, the method comprising administering to a subject in need thereof an antisense oligonucleotide described herein.

[0034] In one embodiment of this aspect or any other aspect described herein, the cohesin mutant cancer is STAG2 mutant.

[0035] In another embodiment of this aspect or any other aspect described herein, the cohesin mutated cancer is selected from AML, glioblastoma, and bladder cancer.

[0036] In another aspect, described herein is a method of treating myelodysplastic syndrome, the method comprising administering to a subject in need thereof an antisense oligonucleotide described herein.

[0037] In one embodiment of this aspect or any other aspect described herein, the myelodysplastic syndrome cells are cohesin mutant.

[0038] In another embodiment of this aspect or any other aspect described herein, the myelodysplastic syndrome cells are STAG2 mutant.

[0039] In another embodiment of the method of treating cancer or myelodysplastic syndrome described herein, the method further comprises administering an inhibitor of DNA damage response.In one embodiment, the inhibitor of DNA damage response is a poly(ADP)-ribose polymerase (PARP) inhibitor.

[0040] definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art to which this disclosure belongs. It should be understood that the present invention is not limited to the specific methodology, protocols, and reagents, etc. described herein, and thus may vary. The terminology used herein is intended only to describe specific embodiments, and is not intended to limit the scope of the present invention, which is defined solely by the claims.Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy,19th Edition,published by Merck Sharp & Dohme Corp.,2011(ISBN 978-0-911910-19-3); Robert S.Porter et al.(eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine,published by Blackwell Science Ltd.,1999-2012(ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference,published by VCH Publishers,Inc.,1995(ISBN 1-56081-569-8);Immunology by Werner Luttmann,published by Elsevier,2006;Janeway's Immunobiology,Kenneth Murphy,Allan Mowat,Casey Weaver (eds.),Taylor & Francis Limited,2014(ISBN 0815345305,9780815345305);Lewin's Genes XI,published by Jones & Bartlett Publishers,2014(ISBN-1449659055);Michael Richard Green and Joseph Sambrook,Molecular Cloning: A Laboratory Manual,4. thed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,USA(2012)(ISBN 1936113414);Davis et al.,Basic Methods in Molecular Biology,Elsevier Science Publishing,Inc.,New York,USA(2012)(ISBN 044460149X);Laboratory Methods in Enzymology:DNA,Jon Lorsch(ed.)Elsevier,2013(ISBN 0124199542);Current Protocols in Molecular Biology(CPMB),Frederick M.Ausubel(ed.),John Wiley and Sons,2014(ISBN 047150338X,9780471503385),Current Protocols in Protein Science(CPPS),John E.Coligan(ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of all of which are incorporated herein by reference in their entireties.

[0041] The term "hybridize" refers to the annealing of complementary nucleic acids, which occurs through nucleobase complementarity. Hybridization is governed by the base sequence involved; complementary nucleobases form hydrogen bonds; hybrid stability is determined by the identity of the base pairs (e.g., G:C base pairs are stronger than A:T base pairs) and the number of consecutive base pairs; the longer the stretch of complementary bases, the more stable the hybrid formed.

[0042] The term "mismatch" means a nucleobase of a first nucleic acid that is unable to base pair with a nucleobase at a corresponding position in a second nucleic acid.

[0043] As used herein, the term "nucleic acid" includes one or more of the following: polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or a modified version of a purine or pyrimidine base (including an abasic site). As used herein, the term "nucleic acid" also includes polymers of ribonucleosides or deoxyribonucleosides that are typically covalently linked by phosphodiester linkages between the subunits, but in some cases are covalently linked by phosphorothioates, methylphosphonates, etc. "Nucleic acid" includes single-stranded and double-stranded DNA, and also includes single-stranded and double-stranded RNA. In some embodiments, analogs with nucleic acid modifications are included, including but not limited to peptide nucleic acids (PNAs), bridged nucleic acids (BNAs), morpholinos, locked nucleic acids (LNAs), glycerol nucleic acids (GNAs), threose nucleic acids (TNAs), or other synthetic nucleic acids (XNAs) described in the art.

[0044] Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids may be DNA or hybrids, in which case they may contain combinations of deoxyribonucleotides and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids may be obtained by chemical synthesis or recombinant methods.

[0045] Nucleic acids generally contain phosphodiester bonds between nucleosides, but also include nucleic acid analogs that can have at least one different linkage, such as phosphoramidate linkage, phosphorothioate linkage, phosphorodithioate linkage, or O-methyl phosphoramidite linkage, as well as peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with normal backbones; those with nonionic backbones, and those with non-ribose backbones, such as those described in U.S. Patent Nos. 5,235,033 and 5,034,506, which are incorporated by reference. Nucleic acids that contain one or more non-naturally occurring or modified nucleotides are also included in one definition of nucleic acid. Modified nucleotide analogs can be located, for example, at the 5'-end and / or 3'-end of the nucleic acid molecule. Representative examples of nucleotide analogs can be selected from sugar-modified or backbone-modified ribonucleotides. However, it should be noted that ribonucleotides containing nucleobase-modified ribonucleotides, i.e., ribonucleotides containing non-naturally occurring nucleobases, such as uridine or cytidine modified at the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine; adenine and guanosine modified at the 8-position, e.g., 8-bromoguanosine; deazanucleotides, e.g., 7-deaza-adenine; O- and N-alkylated nucleotides, e.g., N6-methyladenine, instead of naturally occurring nucleobases, are also suitable. The 2'OH-group may be replaced with a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is C-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. Modification of the ribose phosphate backbone may be performed for various reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on biochips. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs can be made.

[0046] As used herein, the term "intra-exonic splicing-enhancing sequence" or "ESE" refers to a nucleic acid sequence motif of 6-8 bases within an exon that directs or enhances the accurate splicing of heterogeneous nuclear RNA (hnRNA) or pre-mRNA into messenger RNA (mRNA). ESEs are commonly present in both alternative and constitutive exons, where they act as binding sites for Ser / Arg-rich proteins (SR proteins), a family of conserved splicing factors involved in various steps of the splicing pathway (see, e.g., Gravely, RNA 6:1197-1211 (2000)). SR proteins bind to ESEs and recruit spliceosomal factors through protein-protein interactions mediated by serine-rich domains and / or by antagonizing the action of nearby splicing silencers. Different SR proteins have different RNA substrate sequence specificities, and numerous classes of ESE consensus motifs have been described (see, e.g., Cartegni et al., Nature Rev. Genet. 3:285-298 (2002); Gravely et al., supra; and Fairbrother et al., Science 297:1007-1013 (2002)). ESEs can be identified, for example, using a web resource called ESEfinder (see, e.g., Cartegni et al., Nucl. Acids Res. 31:3568-3571 (2003)).

[0047] As used herein, the term "selective killing" refers to a given treatment or process that results in the killing of cells that express or do not express one or more specific or target features, as the case may be, and substantially eliminates cells that do not have the feature. Thus, if a given treatment or process kills cells with mutated or defective STAG2 function, but does not kill cells with normal STAG2 function, the treatment or process selectively kills cells with mutated or defective STAG2. As used in this context, "substantial elimination" means that 30% or less of the killed cells do not have the target feature, or 25% or less, 20% or less, 15% or less, or 10% or less of the killed cells do not have the target feature, preferably 5% or less of the killed cells do not have the target feature; more preferably, 1% or less, and even more preferably, none of the cells lacking the target feature are killed.

[0048] As used herein, the term "PARP inhibitor" refers to a composition, substance, or molecule that blocks or interferes with the enzyme poly(ADP-ribose) polymerase (PARP). A variety of different PARP inhibitors are known, including, but not limited to, talazoparib, beleparib, pamiparib, olaparib, rucaparib, and niraparib.

[0049] The terms "increased", "increase", "enhance", and "activate" are all used herein to refer to a statistically significant amount of increase. In some embodiments, the terms "increased", "increase", "enhance", or "activate" can mean an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100%, or any increase of 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or more, compared to a reference level.

[0050] The term "ameliorate" or "improvement," when applied to a score on a standardized scale or rating, e.g., of disease symptoms or severity, means a statistically significant favorable change in the scale or rating on that scale.

[0051] The terms "reduce", "reduced", "reduction" or "inhibit" are all used herein to mean a statistically significant amount of reduction. In some embodiments, "reduce", "reduced", "reduction" or "inhibit" typically means a reduction of at least 10% compared to a reference level, for example, a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% compared to a reference level. As used herein, "reduction" or "inhibition" does not include complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level.

[0052] As used herein, "reference level" refers to a level or value for a given parameter to be compared to determine whether the level or value in a given sample or situation is significantly altered. A reference level can be a level in or from a sample that has not been treated to alter the parameter. Alternatively, a reference level can be a level in or from a normal or unaffected sample. Alternatively, a reference level can be a level in or from a sample obtained from a subject at a past time point, e.g., before a given treatment.

[0053] As used herein, a "suitable control" refers to an untreated, otherwise identical cell or population (e.g., a subject that has not been administered an agent described herein, or a subject that has been administered only a subset of an agent described herein compared to non-control cells).

[0054] The term "modulation" as applied to target gene expression refers to a change in level; i.e., an increase or decrease in expression (as these terms are defined herein). This modulation can be measured in a manner that is routine in the art, for example, by Northern blot assay, RNase protection assay, or reverse transcriptase PCR for the measurement of transcription or splicing products or mRNA, or by Western blot, ELISA, or immunoprecipitation assay for protein expression. The effect of the antisense oligonucleotides described herein on target gene expression may be determined as taught in the Examples herein, or, for example, in WO2020 / 191212, which is incorporated herein by reference. Modulation of a target gene, such as STAG1, is preferably sufficient to provide a measurable effect on cells that otherwise express the target gene. For example, if modulation reduces the expression of STAG1 in STAG2-deficient cells, the modulation can promote cell death.

[0055] As used herein, "exon" refers to the portion of a primary gene transcript that is included in the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term exon refers both to the DNA sequence within a gene and the corresponding sequence within the RNA transcript.

[0056] As used herein, "intron" refers to a nucleotide sequence within a gene or within the primary transcript of a gene that is removed by RNA splicing during maturation of the final RNA product. The term intron refers to both the DNA sequence within a gene and the corresponding sequence within the RNA transcript.

[0057] As used herein, the term "alternative splicing" refers to a regulated process during gene expression that results in a single gene encoding multiple proteins or products.In this process, certain exons of a gene may be included or excluded in the final processed messenger RNA (mRNA) produced from the gene.In one embodiment, the compositions and methods described herein do not modulate alternative splicing.

[0058] The term "therapeutically effective amount" refers to an amount of the ASO described herein, or a pharmaceutical composition comprising the ASO, sufficient to provide a specific beneficial effect when administered to a typical subject in need thereof. Effective amount as used herein also includes an amount sufficient to delay the onset of disease symptoms, change the course of disease symptoms (for example, but not limited to, slow the progression of disease symptoms), or reverse disease symptoms. Thus, it is not possible or practical to specify an exact "effective amount" for any given case and for every situation, but an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0059] In one embodiment, the compositions described herein, e.g., pharmaceutical compositions or formulations, further comprise a pharma- ceutically acceptable carrier. The term "pharma-ceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. The term "pharma-ceutically acceptable carrier" as used herein means a pharma-ceutically acceptable material, composition, or vehicle, e.g., a liquid or solid filler, diluent, excipient, solvent, medium, encapsulating material, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate, or zinc stearate, or stearic acid), or solvent encapsulating material that participates in maintaining the stability, solubility, or activity of the drug. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. The terms "excipient," "carrier," "pharmaceutically acceptable carrier," and the like are used interchangeably herein.

[0060] As used herein, "subject" refers to a human or animal. Generally, an animal is a vertebrate, such as a primate, rodent, livestock, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, marmots, polecats, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0061] Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. A non-human mammal may be advantageously used as a subject that represents an animal model of a disease. The subject may be male or female.

[0062] The subject may be a person who has been previously diagnosed with, or has been identified as having, a condition that requires treatment, or one or more complications associated with such a condition, and may optionally have already been treated for the condition, or one or more complications associated with the condition. Alternatively, the subject may be a person who has not previously been diagnosed with the condition, or one or more complications associated with the condition. For example, the subject may be a person who shows one or more risk factors for the condition, or one or more complications associated with the condition, or a subject who does not show risk factors.

[0063] As used herein, a "subject in need" of treatment for a particular condition may be a subject who has the condition, has been diagnosed with the condition, or is at increased risk of developing the condition.

[0064] The terms "statistically significant" or "significantly" refer to statistical significance, generally meaning a difference of 2 standard deviations (2 SD) or greater.

[0065] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective components that are essential to the methods or compositions, but do not exclude unspecified elements, whether essential or not.

[0066] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term allows for the presence of elements that do not materially affect the basic, novel, or functional characteristics of the embodiment. The term "consisting of" refers to compositions, methods, and their respective components described herein that exclude elements not recited in the description of the embodiment.

[0067] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Suitable methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to denote a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example." [Brief description of the drawings]

[0068] (Figure 1) Figure 1 shows the composition and structure of cohesin. Cohesin is a ring-shaped complex consisting of SMC1A, SMC3, RAD21, and STAG1 or STAG2. SMC contains two coiled-coil stretches separated by a flexible globular "hinge" domain. When Smc1A and Smc3 proteins fold at the hinge domain, the NTP-binding motifs and DA boxes in their amino- and carboxy-terminal globular domains join to form a functional ATPase. Smc1A and Smc3 interact through the hinge, while the kleisin subunit Rad21 bridges their head domains and associates with the SA. The outer diameter of the resulting ring-shaped complex is estimated to be approximately 50 nm and can hold two 10 nm chromatin fibers. The ring is closed by the mutually exclusive paralogs STAG1 or STAG2 proteins. (Figure 2) Figure 2 shows a mutation matrix of cohesin subunits in AML illustrating the cohesin subunits SMC1A, SMC3, RAD21, STAG1, and STAG2. (FIG. 3A) FIGS. 3A-C show that an AML cell model with a genetically modified STAG2 KO can recapitulate selective dependency on STAG1. (Figure 3B) See legend to Figure 3A. (Figure 3C) See legend to Figure 3A. (Figure 4) Figures 4A-B show that STAG2 KO renders cells hypersensitive to reduced STAG1 levels. (Figure 5) Figure 5 shows that cohesin mutations render AML cells hypersensitive to splicing inhibitors, e.g., H3B-8800. (Figure 6) Figure 6 shows modifications to the backbone or sugar positions of antisense oligonucleotides (ASO). Chemical modifications that can impart drug-like properties to oligonucleotides include 2'O-methoxyethyl (MOE), 2'fluoro (F), 2'-O-methyl (OMe), constrained ethyl nucleosides (cET), also known as locked nucleic acids or 2'-O,4'-methylene nucleosides (LNA), 2',4' constrained 2'-ethyl nucleosides or 2'-O,4'-ethylene nucleosides, and phosphorodiamidate morpholinos (PMO). These modifications can increase stability against nucleases and increase affinity for, for example, proteins or RNA. (Figure 7) Figure 7 shows a single-stranded phosphorothioate (PS) modified MOE gapmer ASO (yellow spheres indicate PS and pink spheres indicate MOE modification). A gapmer is an ASO that has a central region or gap with DNA features flanked on either side by modified RNA bases. The short central DNA stretch forms an RNA-DNA hybrid with the RNA target. This RNA-DNA hybrid can recruit RNAase H to degrade the target RNA. (Figure 8A) Figures 8A-B show the uptake of fluorescently labeled ASO in HEK 293T cells. (Figure 8B) See legend to Figure 8A. (Figure 9A) Figures 9A-B show the uptake of fluorescently labeled ASO in the U937 AML cell line. (Figure 9B) See legend to Figure 9A. (Figure 10A) Figures 10A-10B show that ASOs targeting regions of STAG1 reduce STAG1 expression. (Figure 10B) See legend to Figure 10A. (Figure 11) Figures 11A-11B show a reduction in STAG1 protein expression following treatment with ASO targeting STAG1. (Figure 12) Figure 12 shows truncations of SEQ ID NO:62 STAG1 ASO. Similar truncations of any of the other ASOs in Table 1 are also contemplated, for example, to assess potential effects on optimal ASO function. TIFF2025503956000002.tif107138 (Figure 13) Figure 13 shows the optimization of ASO length and sequence. E5-12 and E5-11 were the ASOs with the greatest effect, and the remaining ASOs were synthesized as derivatives of those sequences. (Figure 14) Figure 14 shows HiBit readout using STAG1-HiBit expressing HEK 293T cells after ASO targeting STAG1. (Figure 15) Figure 15 shows STAG1 protein levels after treatment with ASO targeting STAG1. (Figure 16) Figures 16A-B show that optimized ASO E5-14 causes selective killing in STAG2 KO AML cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] explanation The compositions and methods described herein relate to the treatment of cancer. Cohesin is one of the most frequently mutated protein complexes in cancer, such as myeloid malignancies, and somatic loss-of-function mutations occur frequently in the core components of the cohesin ring. Cohesin-deficient cancers, such as STAG2-deficient cancers, are highly sensitive to inhibitors of mRNA splicing. The inventors have found that one of the RNAs affected by splicing inhibitors in STAG2-deficient cells is the mis-spliced ​​STAG1 RNA. If STAG2-deficient cells, but not wild-type cells, depend on STAG1 paralogs for survival, treatments that interfere with the splicing and / or stability of STAG1 mRNA are found to selectively kill STAG2-deficient cells, such as STAG2-deficient cancer cells.

[0070] Described herein are antisense oligonucleotides that target STAG1 RNA, including but not limited to, antisense oligonucleotides that can hybridize to a portion of STAG1 transcript that affects mRNA splicing efficiency.Described herein are also methods of using such oligonucleotides and / or compositions that contain them to selectively kill STAG2-deficient cells, including but not limited to, myelodysplastic cells, AML cells, and other STAG2-deficient cancer cells that are deficient in STAG2.The antisense oligonucleotides that target STAG1 expression are intended for use alone or in combination with other anticancer agents, including but not limited to, inhibitors of DNA damage response, including but not limited to, PARP inhibitors.

[0071] The following considerations are provided to enable one of ordinary skill in the art to make and use the technology of the present invention.

[0072] Cohesin is a multisubunit protein complex that forms a ring-like structure around DNA, with three structural subunits, SMC1A, SMC3, and RAD21, bound to either STAG1 or STAG2 proteins (Figure 1). Cohesin is one of the most frequently mutated protein complexes in cancer (see, for example, Leiserson et al., Nat. Genet. (2015) doi:10.1038 / ng.3168; Lawrence et al., Nature (2014) doi:10.1038 / nature12912; and Losada et al., Nat. Rev. cancer (2014) doi:10.1038 / nrc3743). Furthermore, the cohesin subunit STAG2 is one of only 12 human genes that are significantly mutated in four or more different types of human cancer (Lawrence et al., supra; Cucco & Musio, Am. J. Med. Genet. Part C: Seminars in Medical genetics (2016) doi:10.1002 / ajmg.c.31492), and is frequently mutated in myeloid malignancies, glioblastoma, breast cancer, bladder cancer, melanoma, and Ewing's sarcoma (Leiserson et al., supra; Lawrence et al., supra; Cucco & Musio, id., Viny & Levine, Curr. Opin. Hematol. 25:61-66 (2018)). Despite this incidence, there are no targeted therapeutic approaches available to treat diseases involving cohesin mutations.

[0073] Cohesin mutant cells include cells in which one or more subunits of the cohesin complex are mutated such that cohesin function is negatively affected or defective. As used herein, the term "cohesin defective" means that the activity or expression of the cohesin complex or its subunits is at least 20% reduced compared to wild type, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more reduced compared to wild type expression or activity. Subunits of the cohesin complex include SMC3, SMC1A, RAD21, and mutually exclusive paralogs STAG1 or STAG2 (stromal antigen 2).

[0074] As used herein, the term "cohesin mutant cells" refers to cells that have a genotype that differs from an initial genotype due to changes in the DNA of any of the genes encoding cohesin subunits, e.g., RAD21, SMC1A, SMC3, and / or STAG1 / STAG2. Cohesin mutant cells are defective with respect to expression or function of one or more of these subunits, i.e., expression or function of one or more of these subunits is absent or reduced (as this term is defined herein), such that cohesin function is reduced (as this term is defined herein).

[0075] STAG2 is the most frequently mutated cohesin subunit in AML (Figure 2) and is also frequently mutated in solid tumors. STAG2 resides on the X chromosome, and thus mutations in males (or females with an active X allele) often lead to a complete loss of STAG2 protein and its replacement within the cohesin complex by its paralog STAG1. Importantly, this establishes an absolute dependency of STAG2 mutant cells on the expression of STAG1, and both RNAi and CRISPR / Cas9 screens highlight STAG1 as a selective critical dependency in STAG2 mutant cells. Cancer cells that acquire mutations in STAG2 become hypersensitive to loss of the cohesin subunit STAG1, a functionally redundant paralog of STAG2 (Viny et al., supra; Antony et al., Blood (2018) doi:10.1182 / blood-2018-99-117965; Tothova et al., JCI Insight 6:1-16 (2021); and Jann & Tothova, Blood 138:649-661 (2021)). Importantly, STAG1 is dispensable in cells with wild-type STAG2 (Viny et al., Cell Stem Cell 25:682-696.e8 (2019)).

[0076] As used herein, the term "stromal antigen 2 (STAG 2) mutant cells" refers to cells that have a genotype that differs from the original genotype due to DNA changes in the gene encoding STAG2. STAG2 mutant cells have reduced STAG2 function (as this term is defined herein) compared to STAG2 wild-type cells. In some embodiments, STAG2 mutant cells have less than 50% of the STAG2 expression or function of wild-type cells, such as less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or no STAG2 expression or function at all. STAG2 expression can be measured using standard methods. STAG2 function can be measured, for example, using a splicing reporter construct that depends on STAG2 for proper splicing.

[0077] The present inventors have discovered that cohesin mutations, which are common in myeloid malignancies, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), disrupt RNA splicing and render cells hypersensitive to broad-spectrum splicing inhibitors. Through detailed investigation of gene activity and splicing profiles in cohesin-mutated AML cells, several genes were discovered whose alternative splicing events are exacerbated by splicing inhibitors. Notably, one of these is STAG1, a component of the cohesin complex. The present inventors have identified antisense oligonucleotides (ASOs) that selectively modulate the splicing and / or stability of STAG1 mRNA to precisely target the vulnerability of cohesin-mutated cells and induce lethality.

[0078] STAGE1:STAG1 or stromal antigen 1 encodes a member of the SCC3 family, a component of cohesin, a multi-subunit protein complex that provides sister chromatid cohesin along the length of chromosomes from DNA replication through prophase and prometaphase, and then dissociates in preparation for separation at anaphase. STAG1 sequences are known for numerous species (e.g., human STAG1 (NCBI Gene ID: 10274), mRNA (e.g., NM_005862.3), and polypeptide (e.g., NP_005853.2)). Antibodies for specific detection of STAG1 are available, for example, from Abcam (see anti-SA1 antibody [SUSI63B], ab241544). Candidate ASOs for reducing STAG1 expression are presented in Table 1, including SEQ ID No 1-81. STAG1 has a total of 39 exons, and NM_005862.3 contains 34 exons.

[0079] In some embodiments, the STAG1 nucleic acid comprises or is derived from a human STAG1 pre-mRNA having a nucleic acid sequence of NC_000003.12, based on the reference GRCh38.p14 primary assembly and the range 136336236 to 136752378, which contains 416143 base pairs.

[0080] In some embodiments, the STAG1 mRNA sequence is the following sequence NM_005862.3 (SEQ ID No. 98): Containing or derived from human STAG1 having TIFF2025503956000003.tif8158TIFF2025503956000004.tif243158TIFF2025503956000005.tif214158

[0081] With reference to the STAG1 genomic sequence NC_000003.12, the locations of the 34 exons that are spliced ​​to form this transcript are as follows: TIFF2025503956000006.tif80133.

[0082] In some embodiments, the STAG1 polypeptide has the following amino acid sequence NP_005853.2 (SEQ ID No. 82): Containing or derived from human STAG1 having TIFF2025503956000007.tif99158.

[0083] STAGE2 :STAG2 or stromal antigen 2 encodes a subunit of the cohesin complex that controls the separation of sister chromatids during cell division. Targeted inactivation of this gene results in chromatid cohesin defects and aneuploidy, suggesting that genetic disruption of cohesin is a cause of aneuploidy in human cancers. Alternatively spliced ​​transcript variants have been found for this gene that encode different isoforms. STAG2 sequences are known for many species (e.g., human STAG2 (NCBI Gene ID: 10735), mRNA (e.g., NM_001042749.2), and polypeptide (e.g., NP_001036214.1)). Antibodies for the specific detection of STAG2 are available, for example, from Abcam (see anti-SA2 antibody [EPR17865], ab201451). STAG2 has a total of 40 exons, and NM_001042749.2 contains 35 exons.

[0084] In some embodiments, the STAG2 nucleic acid comprises or is derived from a human STAG2 pre-mRNA having a nucleic acid sequence of NG_033796.2, based on reference RefSeqGene (LRG_782) on chromosome X, and a range of 5001 to 147097 containing 142097 base pairs.

[0085] In some embodiments, the STAG2 mRNA sequence is the following sequence NM_001042749.2 (SEQ ID No. 83): TIFF2025503956000008.tif142158TIFF2025503956000009.tif243158TIFF2025503956000010.tif104158

[0086] In some embodiments, the STAG2 polypeptide has the following amino acid sequence: NP_001036214.1 (SEQ ID No. 99); Containing or derived from human STAG2 having TIFF2025503956000011.tif99158.

[0087] STAG2 mutant cells include cells in which the STAG2 gene is mutated relative to the wild-type STAG2 gene such that STAG2 expression or activity is lost or substantially deficient (e.g., reduced by 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% relative to the wild-type). Loss of STAG2 renders the cells dependent on STAG1 for survival, making diseases involving STAG2-deficient cells amenable to treatment with STAG1 inhibitors.

[0088] In such embodiments, early diagnosis of STAG2-deficient MDS and treatment with antisense oligonucleotides targeting STAG1 as described herein may provide benefits compared to treatment initiated when the disease has progressed to AML.

[0089] In the methods described herein, as described herein above, inhibition of STAG1 expression is effective to selectively kill cells lacking STAG2, which is frequently mutated in various cancers. STAG2 expression can be investigated, for example, by measuring the level of (spliced) RNA via RT-PCR using primers spanning one or more introns. The level of STAG2 expression can also be determined, for example, by protein assays, such as STAG2 immunoassays known to those skilled in the art (for example, but not limited to, Western blot, ELISA, immunoprecipitation, etc.), mass spectrometry, or other methods known to those skilled in the art. The level of STAG2 RNA or protein in cancer cells can be compared to a suitable control, for example, the level in normal cells of the same or similar lineage. Absence of STAG2 expression, or a reduction of at least 70%, 80%, 90% or more compared to the control, is considered STAG2 deficient (as this term is used herein). Whether a patient's cancer has STAG2 mutation or disruption can also be determined by clinical exome sequencing, i.e., targeted RNA sequencing of RNA derived from the patient's cancer cells.Changes in amino acid coding sequence, whether frameshift or other missense mutations or amino acid changes, i.e., mutations that interfere with or change splice sites, or mutations that introduce premature stop codons, can indicate that a given cancer cell is STAG2 deficient, among others.

[0090] Antisense oligonucleotides As described herein, target gene expression can be reduced by administering antisense oligonucleotides complementary to selected regions of target transcripts. Antisense oligonucleotides that target splicing of STAG1 transcripts are of particular interest. Interference with splicing can reduce the amount of correctly or completely spliced ​​mRNA encoding STAG1, thereby reducing STAG1 protein expression. Non-limiting examples of ASOs that can interfere with splicing include those that hybridize or overlap with the 5' splice site in the primary transcript and those that hybridize or overlap with intra-exonic splicing-promoting sequence elements. In some embodiments, the ASO overlaps with the intra-exonic splicing-promoting sequence on the STAG1 RNA transcript by at least one nucleotide, and preferably the overlap is by 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, or more. In some embodiments, the ASO compositions described herein target RNA for degradation, for example, via RNAse H-mediated degradation.Non-limiting examples include the gapmers described herein, which comprise RNA-DNA-RNA chimeric oligos, which have a tendency to promote RNAse H activity on hybridized target RNA.In some embodiments, the ASOs described herein target elements that affect RNA splicing and target RNA transcripts for degradation.Such a combination approach can provide improved knockdown of target gene expression.Thus, in another embodiment of this aspect and any other aspect described herein, hybridization of antisense oligonucleotide overlaps with 5' splice site or intra-exonic splicing-promoting sequence (ESE) on STAG1 RNA transcript.ESEs are present in both alternative and constitutive exons, where they provide binding sites for Ser / Arg-rich proteins (SR proteins), a family of conserved splicing factors involved in multiple steps of the splicing pathway. Different SR proteins have different substrate specificities, and numerous classes of ESE consensus motifs have been described (e.g., Blencowe, Trends Biochem. Sci. 25:106-110 (2000); Cartegni et al., Nat. Rev. Genet. 3:285-298, Graveley, RNA 6:1197-1211 (2000), and Fairbrother et al., Science 297:1007-1013 (2002)). A web resource for identifying intra-exon splice-promoting sequences is described by Cartegni et al., Nucl. Acids res. 31:3568-3571 (2003).

[0091] In some embodiments, the intra-exonic splicing-promoting sequence comprises a binding site for the RNA-binding protein SRSF2. As used herein, the term "binding site for the RNA-binding protein SRSF2" refers to an RNA sequence to which the splicing regulator serine-arginine (SR) protein 2 (SRSF2) can bind. SRSF2 is an SR protein that recognizes and binds to certain ESEs. SRSF2 encodes a 221 amino acid protein that is the only nuclear-retained member of the SR protein family (14, 15). It contains two functional domains, including an RNA-binding motif and a highly phosphorylated serine-arginine-rich domain. Proline 95 (P95) is in the linker region between the RNA-binding motif and the SR-rich region (16). SRSF2 binds to cis-elements on pre-mRNA transcripts that functionally redefine putative exon-intron boundaries. The sequences recognized and bound by SRSF2 have been investigated using SELEX; ​​see, for example, Tacke & Manley, EMBO J. 14:3540-3551 (1995) and Lui et al., Mol. Cell. Biol. 20:1063-1071 (2000). These publications also describe SRSF2 binding assays. By considering the RNA sequences thus identified, together with the approaches known in the art or described herein for identifying ESEs, it is possible to determine whether a given SRSF2 binding sequence in RNA is likely to be involved in splicing, and an in vitro assay of SRSF2 binding can be carried out to confirm whether SRSF2 binds to a given RNA sequence.

[0092] As used herein, "antisense oligonucleotide" refers to a synthetic single-stranded nucleic acid molecule that is complementary to a sequence on an RNA transcript, such as a 5' splice site, an exon, an intron, a protein-binding motif, or other transcript sequence element. An oligonucleotide that is sufficiently complementary (as this term is defined herein) to a target is selected to produce a desired effect. For example, an antisense oligonucleotide can include at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or more bases that are complementary to a portion of the STAG1 transcript. Non-limiting examples are provided in Table 1.

[0093] As used herein, the term "RNA oligonucleotide" refers to a polymer of nucleosides containing the sugar ribose as a building block and typically covalently linked by intersubunit phosphodiester linkages, and in some cases, for modified RNAs, by phosphorothioates, methylphosphonates, etc.

[0094] As used herein, the term "DNA oligonucleotide" refers to a polymer of nucleosides containing the sugar deoxyribose as a building block, typically covalently joined by phosphodiester linkages between the subunits, and in some cases, for modified DNAs, by phosphorothioates, methylphosphonates, etc.

[0095] As used herein, the term "sequence sufficiently complementary to a portion of the STAG1 primary transcript to permit hybridization" refers to a nucleic acid sequence, e.g., an antisense oligonucleotide sequence, that is at least partially complementary to a target sequence, e.g., STAG1, and has sufficient complementarity to form a duplex with the target sequence through Watson-Crick base pairing under physiological conditions. The degree of complementarity required for a given nucleic acid to hybridize or form a duplex with another nucleic acid under physiological conditions depends on the length of the nucleic acid and the specific nucleotide composition (e.g., %GC content vs. %AT or AU content). Calculation of the free energy of binding between a nucleic acid and its complementary strand, or a molecule with at least partial complementarity, can provide a prediction of whether a given sequence will hybridize to another sequence under given conditions. Determination of binding free energies for nucleic acid molecules is well known in the art (e.g., Turner et al, 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, / . Am. Chem. Soc. 109:3783-3785).Additionally, calculations and / or predictions of hybridization energies may be determined using software tools or modeling known in the art, including, but not limited to, S-Fold, available on the World Wide Web at sfold.wadsworth.org; PFRED, available on the World Wide Web at ncbi.nlm.nih.gov / pms / articles / PMC7822268; OligoEvaluator, from Sigma, available on the World Wide Web at "oligoevaluator.com / oligocalcservlet; OligoAnalyzer, from IDT, available on the World Wide Web at idtdna.com / pages / tools / oligoanalyzer (see, e.g., Wang et al., 2022, Plos One. 17(5) and Tulpan et al., 2010 BMC Bioinformatics 105).

[0096] In some embodiments, the binding or hybridization of antisense oligonucleotides can stop the expression of targets at the transcription, translation, or splicing levels. These sequences hybridize well enough and with sufficient specificity in the cellular environment to give the desired effect. An oligonucleotide sequence that is fully complementary to a target sequence can be 85%, 90%, 95% or more complementary to the sequence corresponding to the target sequence. In some embodiments, a sequence that is fully complementary (as this term is used herein) contains 1, 2, 3, or 4 or less mismatched nucleotides that are not complementary to the target sequence. In another embodiment, a sequence is 100% complementary to a target sequence.

[0097] In some embodiments of any of the aspects, the antisense oligomer consists of 8 to 40 nucleobases. In some embodiments of any of the aspects, the antisense oligomer consists of 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 40 nucleobases, 10 to 3 ... ~30 nucleobases, 10-25 nucleobases, 10-20 nucleobases, 10-15 nucleobases, 11-40 nucleobases, 11-35 nucleobases, 11-30 nucleobases, 11-25 nucleobases, 11-20 nucleobases, 11-15 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12-15 nucleobases.

[0098] In some embodiments of any of the aspects, the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID Nos. 1-81.

[0099] As used herein, the term "nucleotide" refers to an organic molecule that serves as a monomer unit to form the nucleic acid polymers deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Conventional naturally occurring nucleotides are the building blocks of nucleic acids and are composed of three subunit molecules: a nitrogenous base, a pentose sugar, and at least one phosphate group. Nucleosides have a nitrogenous base and a five-carbon carbohydrate, which is ribose in the case of ribonucleosides and deoxyribose in the case of deoxynucleosides. In standard nomenclature, the addition of one or more phosphate groups to a nucleoside results in a nucleotide. Nucleotides may be modified. Modifications to nucleotides, or oligonucleotides composed of them, may improve, for example, stability, strength of hybridization, and / or characteristics of delivery or uptake into cells. Acceptable modifications are those that maintain the ability to hybridize to sequences in RNA transcripts and interfere with protein expression.

[0100] It should be understood that antisense oligonucleotides applicable in the compositions and methods described herein can include 100% DNA, 100% RNA, any combination of ribonucleosides (RNA) and deoxyribonucleosides (DNA), oligos that are unmodified with respect to the sugar, nucleobase, and phosphate backbone, as well as oligos that are modified with respect to the sugar, nucleobase, and / or backbone linkages.

[0101] It is often beneficial to include chemical modifications in oligonucleotides to alter activity. Chemical modifications can alter oligonucleotide activity, for example, by increasing the affinity of antisense oligonucleotides to target RNA, increasing nuclease resistance, and / or altering the pharmacokinetics of oligonucleotides. The use of chemistry that increases the affinity of oligonucleotides to their targets can allow for the use of shorter oligonucleotides. The antisense oligonucleotides described herein can also contain one or more nucleosides with modified sugar moieties. The furanosyl sugar ring of the nucleoside can be modified in a number of ways, including but not limited to, the addition of a substituent, bridging two non-geminal ring atoms to form bicyclic nucleic acids (BNAs), and replacing the ring oxygen at the 4' position with an atom or group such as -S-, -N(R)-, or -C(R1)(R2). Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of antisense oligonucleotides to their targets and / or increase nuclease resistance. A representative list of preferred modified sugars includes, but is not limited to, bicyclic modified sugars (BNAs), such as LNAs and ENAs (4'-(CH2)2-O-2' bridges); and substituted sugars, specifically 2'-substituted sugars having 2'-F, 2'-OCH2, or 2'-O(CH2)2-OCH3 substituents. The sugars may be exchanged, among others, for sugar mimetic groups. Methods for the preparation of modified sugars are well known to those of skill in the art. Suitable compounds may include one of the following at the 2' position: OH; F; O-alkyl, S-alkyl, or N-alkyl; O-alkenyl, S-alkenyl, or N-alkenyl; O-alkynyl, S-alkynyl, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl.Also suitable are O((CH2)nO)mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10. Other oligonucleotides contain one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents with similar properties. One modification includes 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., an alkoxyalkoxy group. Further modifications include 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-(CH2)2-O-(CH2)2-N(CH3)2. Other modifications include 2'-methoxy (2'-O-CH), 2'-aminopropoxy (2'-OCHCHCHNH), 2'-allyl (2'-CH-CH-CH), 2'-O-allyl (2'-O-CH-CH-CH), and 2'-fluoro (2'-F). The 2' modifications can be in the arabino (up) or ribo (down) positions. One 2'-arabino modification is 2'-F.Similar modifications can be made at other positions on the oligonucleotide, specifically the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Antisense oligonucleotides can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576, These include, but are not limited to, Nos. 427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; and 6,147,200.

[0102] Other useful RNA derivatives are those that incorporate modified carbohydrate moieties, such as nucleotides with 2'O-alkylated residues, or 2'-O-methylribosyl derivatives, and 2'-O-fluororibosyl derivatives, or 2'-O,4'-constrained 2'-ethyl nucleosides, or 2'-O,4'-ethylene nucleosides. RNA bases can also be modified. Any modified base that is useful for inhibiting or interfering with the expression of a target sequence can be used. For example, halogenated bases, such as 5-bromouracil and 5-iodouracil, can be incorporated. Bases can also be alkylated, such as 7-methylguanosine can be incorporated in place of guanosine residues. Non-natural bases that provide successful inhibition can also be incorporated. Preferred siRNA modifications include 2'-deoxy-2'-fluorouridine, or locked nucleic acid (LNA) nucleotides, and RNA duplexes that contain either phosphodiester linkages or a variety of numbers of phosphorothioate linkages. Such modifications are known to those of skill in the art and are described, for example, in Braasch et al., Biochemistry, 42:7967-7975, 2003; Morita et al., Bioorg Med Chem Lett. 12(1); 2002.

[0103] In one aspect of the technology described herein, oligomeric compounds include modified nucleosides to induce 3'-endo sugar conformation. Nucleosides can incorporate modifications to the heterocyclic base, sugar moiety, or both to induce desired 3'-endo sugar conformation. These modified nucleosides are used to mimic RNA-like nucleosides so that certain properties of oligomeric compounds can be enhanced while maintaining the desired 3'-endo conformation geometry.

[0104] The monomers of the oligonucleotides described herein are coupled together via a linking group. Suitably, each monomer is linked to the 3' adjacent monomer via a linking group. The term "linking group" or "internucleoside linkage" refers to a group that can covalently couple two consecutive nucleoside monomers together. Specific preferred examples include a phosphate group (forming a phosphodiester between adjacent nucleoside monomers) and a phosphorothioate group (forming a phosphorothioate linkage between adjacent nucleoside monomers). Suitable linking groups include those described in WO 2007 / 031091 (incorporated herein by reference), for example, the linking groups described in the first paragraph on page 34 of WO 2007 / 031091. Phosphorothioate backbone modifications are well known in the art (see, for example, Hyjek-Skladanowska et al., 2020 J.Am Chem.142(16),7456-7468). In various embodiments, it is preferred to modify the linking group from the normal phosphodiester to one that is more resistant to nuclease attack, such as phosphorothioate or boranophosphate, two of which are cleavable by RNase H, allowing for RNase-mediated antisense inhibition of target gene expression. In some embodiments, suitable sulfur (S)-containing linking groups provided herein are preferred. In various embodiments, phosphorothioate linking groups are preferred.

[0105] ASOs that target splicing elements of STAG1 transcripts to interfere with normal splicing or promote defective splicing are described herein, but ASOs that target STAG1 transcripts, for example, for RNAse H-mediated degradation, are also contemplated.Thus, in certain embodiments, phosphorothioate linkages are used to link monomers together in the flanking regions of gapmers that include modified nucleotides (e.g., LNA) or ribonucleotide mimics flanking both ends of the antisense DNA sequence and stimulate RNAse H-mediated degradation of target RNA.In various embodiments, the flanking or gap regions of gapmers include linkage groups other than phosphorothioate, for example, phosphodiester linkages, particularly when, for example, the use of nucleoside analogs protects linkage groups in the flanking regions from endonuclease degradation, for example, when the flanking regions include LNA monomers. It has been recognized that the inclusion of phosphodiester linkages, e.g., one or two linkages, in oligonucleotides having phosphorothioate backbones (typically in the gapmer flanking regions), particularly those having phosphorothioate linkages between or adjacent to nucleoside analog monomers, can modify the bioavailability and / or biodistribution of the oligonucleotide (see WO 2008 / 053314, incorporated herein by reference).

[0106] In some embodiments, such as those mentioned above, where appropriate and unless otherwise indicated, all of the remaining linking groups can be either phosphodiester or phosphorothioate, or a mixture thereof, hi some embodiments, all of the internucleoside linking groups are phosphorothioate.

[0107] Target Disease or Disorder The ASO of targeting STAG1 as described herein can be used to treat any disease, disorder or cancer that involves cells with lack of STAG2.STAG2 mutation inactivation is common in, for example, MDS, AML, glioblastoma, urothelial carcinoma and Ewing's sarcoma.In some embodiments of any one of the aspects described herein, disease or disorder is MDS or leukemia.

[0108] It is clear that less than 100% reduction in STAG1 mRNA is effective for killing cohesin mutant or STAG2 mutant cells. In some embodiments, the contacting step reduces STAG1 mRNA by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0109] Myelodysplastic syndromes are a heterogeneous group of closely related clonal hematopoietic disorders that originate from early hematopoietic cells in the bone marrow. Such disorders are characterized by cellular bone marrow with impaired morphology and maturation (myelodysplasia), and peripheral blood cytopenias due to ineffective blood cell production. In other words, maturing blood cells often die in the bone marrow before reaching full maturity and entering the blood, which causes low blood cell concentrations. In patients suffering from myelodysplastic syndromes, there may also be an accumulation of extremely immature bone marrow cells called leukemic blasts. Patients with myelodysplastic syndromes experience symptoms including, but not limited to, fatigue, shortness of breath, abnormal paleness (pallor), frequent or abnormal bruising or bleeding under the skin, tiny red spots under the skin, and frequent infections.

[0110] Diagnosis of myelodysplastic syndromes includes blood tests to determine the number of red blood cells, white blood cells, and platelets and to look for abnormal changes in the size, shape, and appearance of blood cells, as well as a bone marrow biopsy and aspiration to remove a small amount of liquid bone marrow and test for blood cell characteristics. Successful treatment of MDS may be indicated by the restoration of bone marrow and blood cell counts to normal, or normalization of blood cell characteristics, as well as molecular remission and the absence of further signs or symptoms of the disease.

[0111] MDS, formerly known as pre-leukemia, can lead to acute myeloid leukemia (AML). AML is a cancer of the blood and bone marrow. The disease progresses rapidly and primarily affects white blood cells called myeloid cells, which normally develop into mature blood cells, such as red blood cells, white blood cells, and platelets. AML results from uncontrolled blood cell production, in which the bone marrow produces immature cells that develop into leukemic white blood cells called myeloblasts. These abnormal cells are unable to function properly and grow up to crowd out healthy cells.

[0112] Early signs and symptoms of AML include fever, bone pain, lethargy and fatigue, shortness of breath, pale skin, frequent infections, easy bruising, and abnormal bleeding. AML can be diagnosed through blood tests that determine the number of white blood cells, red blood cells, and platelets. Patients with AML frequently have too many white blood cells or not enough red blood cells or platelets. In addition, the presence of blast cells, immature cells that are normally found in the bone marrow but do not circulate in the blood, is another indicator of AML. Other methods of diagnosis include bone marrow examination, lumbar puncture, and subsequent laboratory testing for blood cell characteristics and gene mutations. Successful treatment of AML is indicated by the return of bone marrow and blood cell counts to normal values, or a decrease in circulating blast cells, as well as molecular remission and the absence of further signs or symptoms of the disease.

[0113] Pharmaceutical Composition, Administration, and Efficacy Methods known in the art for the delivery of oligonucleotides or nucleic acids to cells in vivo can be adapted for use in the methods and compositions described herein. Although the uptake of free oligonucleotides described herein can be efficient, in some embodiments, the oligonucleotides described herein can be covalently linked to conjugated moieties to aid in the delivery of oligonucleotides through cell membranes. An example of a conjugated moiety that aids in the delivery of oligonucleotides through cell membranes is a lipophilic moiety, such as cholesterol. In various embodiments, the oligonucleotides described herein are formulated with lipid formulations that form liposomes, such as Lipofectamine 2000™ or Lipofectamine RNAiMAX™, both of which are commercially available from Invitrogen. In some embodiments, the oligonucleotides described herein are formulated with a mixture of one or more lipid-like non-naturally occurring small molecules ("lipidoids"). A library of lipidoids can be synthesized by conventional synthetic chemistry methods, and various amounts and combinations of lipidoids can be assayed to develop a vehicle for effectively delivering oligonucleotides of a particular size to targeted tissues by a selected route of administration. Libraries and compositions of suitable lipidoids can be found, for example, in Akinc et al. (2008) Nature Biotech. 26:561-569 (2008), which is incorporated herein by reference. In the context of this disclosure, "cellular uptake" refers to the delivery and internalization of oligonucleotide compounds into cells. Oligonucleotide compounds can be internalized, for example, into cells grown in culture (in vitro), cells taken from an animal (ex vivo), or tissues after administration to an animal (in vivo).

[0114] Exemplary formulations that can be used to administer oligonucleotides and / or dsRNA according to the present invention are described below.

[0115] The ASOs described herein can be formulated in membranous molecular assemblies, such as liposomes or micelles, for delivery. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, such as one or more bilayers. Liposomes include unilamellar and multilamellar vesicles with a membrane formed from a lipophilic material and an internal aqueous phase. The aqueous portion contains the ASO. The lipophilic material separates the internal aqueous phase from the external aqueous phase, which typically does not contain the ASO, but in some cases may. Liposomes are useful for transporting and delivering active ingredients to the site of action. Because the liposome membrane is structurally similar to biological membranes, when the liposomes are applied to tissues, the liposome bilayer fuses with the bilayer of the cell membrane. As the fusion of the liposome with the cell proceeds, the internal aqueous contents, including the ASOs described herein, are delivered into the cell. In some cases, the liposomes are also specifically targeted, for example, to direct the conjugate to a particular cell type.

[0116] Liposomes containing ASOs as described herein can be prepared by a variety of methods. In one example, the lipid components of the liposome are dissolved in a detergent so that the lipid components form micelles. For example, the lipid components can be amphiphilic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholic acid, CHAPS, octylglucoside, deoxycholic acid, and lauroyl sarcosine. ASOs are then added to the micelles containing the lipid components. After aggregation, the detergent is removed, for example, by dialysis, to obtain a liposome preparation.

[0117] If necessary, carrier compounds can be added during the agglutination reaction to aid in agglutination, for example, by controlled addition.For example, the carrier compounds can be polymers other than nucleic acids (for example, spermine or spermidine).The pH can also be adjusted to favor agglutination.

[0118] Further description of methods for making stable polynucleotide or oligonucleotide delivery vehicles that incorporate polynucleotide / cationic lipid complexes as a structural component of the delivery vehicle is described, for example, in WO 96 / 37194. Liposome formation is described in detail in the following publications: Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Patent No. 4,897,355; U.S. Patent No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. The method may also include one or more aspects of the exemplary methods described in (Mayer, et al. Endocrinol. 115:757, 1984). Techniques commonly used to prepare lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw-extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986, which is incorporated by reference in its entirety). Microfluidization may be used when consistently small (50-200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984, which is incorporated by reference in its entirety).

[0119] Liposomes that are pH-sensitive or negatively charged capture nucleic acid molecules rather than forming complexes with them. Both nucleic acid molecules and lipids have similar charges, so repulsion occurs rather than complex formation. Nevertheless, some nucleic acid molecules are captured in the internal aqueous phase of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding thymidine kinase gene to cultured cell monolayers. The expression of exogenous genes was detected in target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274, incorporated by reference in its entirety).

[0120] One major type of liposome composition includes phospholipids other than naturally occurring phosphatidylcholine. Neutral liposome composition can be formed from, for example, dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome composition is generally formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are mainly formed from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), for example, soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0121] Other examples of methods for introducing liposomes into cells in vitro include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0122] Cationic liposomes can also be used.Cationic liposomes have the advantage of being able to fuse with cell membrane.Non-cationic liposomes cannot fuse with plasma membrane efficiently, but can be taken up by macrophages in vivo and can be used to deliver siRNA to macrophages.

[0123] Further advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs; liposomes can protect siRNA encapsulated in the inner compartment from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p.245). Important considerations in the preparation of liposome formulations are lipid surface charge, vesicle size, and the aqueous phase volume of the liposomes.

[0124] A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with the negatively charged lipids of tissue culture cell membranes and result in delivery of siRNA (see, e.g., Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355, which are incorporated by reference in their entireties, for a description of DOTMA and its use with DNA).

[0125] The DOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with phospholipids to form vesicles that complex with DNA. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to live tissue culture cells, including positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. When liposomes with sufficient positive charges are used, the net charge of the resulting complex is also positive. The positively charged complexes thus prepared spontaneously attach to negatively charged cell surfaces and fuse with the plasma membrane, effectively delivering functional nucleic acids to, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moieties are linked by ester linkages rather than ether linkages.

[0126] Other reported cationic lipid compounds include those conjugated to a variety of moieties, e.g., carboxyspermine conjugated to one of two types of lipids, including compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0127] Another cationic lipid conjugate includes lipid derivatization with cholesterol ("DC-Chol") formulated in liposomes in combination with DOPE (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine with DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991, which is incorporated by reference in its entirety). For certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0128] Liposomes containing the oligonucleotides and / or ASOs described herein can be made highly deformable. Such deformability can allow liposomes to penetrate pores smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposome. Transfersomes can be made by adding surface edge activators, typically surfactants, to standard liposome compositions. Transfersomes containing the oligonucleotides and / or ASOs described herein can be delivered, for example, subcutaneously, by infection to deliver ASOs to keratinocytes in the skin. To cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter of less than 50 nm, under the influence of a suitable transdermal gradient. Furthermore, due to lipid properties, these transfersomes can self-optimize (e.g., adapt to the shape of the skin pores), self-repair, reach targets frequently without fragmentation, and are often self-loading.

[0129] Other formulations that may be amenable to the present invention are described in U.S. Provisional Application Nos. 61 / 018,616, filed January 2, 2008; 61 / 018,611, filed January 2, 2008; 61 / 039,748, filed March 26, 2008; 61 / 047,087, filed April 22, 2008, and 61 / 051,528, filed May 8, 2008. PCT Application No. PCT / US2007 / 080331, filed October 3, 2007, also describes formulations that may be amenable to the present invention.

[0130] Another example of liposome is lipid nanoparticle (LNP). As used herein, the term "LNP" refers to stable nucleic acid-lipid particle. LNP contains cationic lipid, non-cationic lipid, and lipid that prevents particle aggregation (e.g., PEG-lipid conjugate). LNP shows extended circulation life after intravenous (iv) injection and accumulates at distant sites (e.g., sites physically separated from the administration site), making it very useful for systemic application.

[0131] The ASOs described herein may be used alone or in combination with other treatments, such as chemotherapy, radiation, cancer immunotherapy, or a combination thereof. Such treatments may directly target tumors (e.g., by inhibiting tumor cell proteins or killing highly mitotic cells) or may act indirectly, for example, to induce or enhance antitumor immune responses. Anticancer treatments that damage DNA to a lesser extent than chemotherapy may also have efficacy. Examples of such treatments include radiation therapy, immunotherapy, hormone therapy, and gene therapy. Such treatments include, but are not limited to, the use of antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, and the like, where the nucleotide sequence of such compounds is related to the DNA and / or RNA nucleotide sequence of genes associated with the initiation, progression, and / or pathology of tumors or cancer. For example, oncogenes, growth factor genes, growth factor receptor genes, cell cycle genes, DNA repair genes, and the like may be used in such treatments.

[0132] The radiation used in radiation therapy may be ionizing radiation. Radiation therapy may be gamma radiation, X-rays, or protons. Examples of radiation therapy include, but are not limited to, external beam radiation therapy, interstitial implants of radioisotopes (I-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and / or total abdominal pelvic radiation therapy. For a general overview of radiation therapy, see Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al., eds., JB Lippencott Company, Philadelphia. Radiation therapy may be administered as external beam radiation or teletherapy, where radiation is directed from a remote source. Radiation treatment may also be administered as internal therapy or brachytherapy, where a radioactive source is placed close to cancer cells or tumor masses inside the body. Also encompassed is the use of photosensitizers, such as hematoporphyrin and its derivatives, vertoporphin (BPD-MA), phthalocyanines, photosensitizer Pc4, demethoxy-hypocrellin A; and photodynamic therapy, including administration of 2BA-2-DMHA.

[0133] Immunotherapy may include, for example, the use of cancer vaccines and / or sensitized antigen-presenting cells. Immunotherapy may include passive immunity for short-term host defense, achieved by administering preformed antibodies against cancer or disease antigens (e.g., administering monoclonal antibodies against tumor antigens, optionally linked with chemotherapeutic agents or toxins). Immunotherapy may also focus on the use of epitopes of cancer cell lines that are recognized by cytotoxic lymphocytes.

[0134] Hormonal therapeutic treatments may include, for example, hormone agonists, hormone antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, as well as steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogens, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).

[0135] DNA damage response inhibitors are widely used anti-cancer drugs that have strong activity against tumor cells with various DNA damage response protein defects.The inhibitors of the proteins of this pathway target genes including but not limited to PARP, DNA-PK, WEE1, CHK1 / 2, ATR, or ATM.Inhibitors of DNA damage response are known in the art (see, for example, Carlsen et al., 2022 Sec.Radiation Oncology 12, which is incorporated herein in its entirety).

[0136] In a preferred embodiment, the ASO is used in combination with one or more PARP inhibitors.

[0137] A variety of different PARP inhibitors are known, and any of them are contemplated for use in combination with the antisense oligonucleotides targeting STAG1 described herein for the treatment of MDS or cancer, such as cohesin mutant cancer or STAG2 mutant cancer.In one embodiment, the inhibitor of DNA damage response is selected from talazoparib, beleparib, pamiparib, olaparib, rucaparib and niraparib.Although the combination of the antisense oligonucleotides targeting STAG1 with PARP inhibitors or other inhibitors of DNA damage response may provide therapeutic benefits that may be additive and / or synergistic, it is also contemplated that the antisense oligonucleotides described herein can be administered in combination with other anticancer therapeutic agents, such as, but not limited to, chemotherapeutic agents.

[0138] PARP has essential roles in enhancing DNA repair, regulating RNA transcription, mediating cell death, and controlling immune responses. PARP inhibitors effectively target cells with reduced capacity for homologous recombination repair. Treatment with double-strand break (DSB) repair inhibitors can sensitize cells with intact homologous recombination machinery to PARP inhibitors, enhance the efficacy of PARP inhibitors in homologous recombination-deficient cancers, and avoid cases of acquired resistance to PARP inhibitors. Examples of PARP inhibitors include Iniparib (BSI) 201; 4-iodo-3-nitrobenzamide), olaparib (AZD-2281; KU-59436; 4-[(3-[(4-cyclopropylcarbonyl)piperazin-4-yl]carbonyl)-4-fluorophenyl]methyl(2H)phthalazin-1-one), rucaparib (AG014699, PF-01367338; 2-{4-[(methylamino)methyl]phenyl}-1,3,4,5-tetrahydro-6H-azepino[5,4,3-cd]indol-6-one), veliparib (ABT-888; 2-((R)-2-methylpyrrolidin-2-yl)-1H-benzimidazole-4-carboxamide); CEP-8983; CEP-9722; M K-4827 (Niraparib; 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide); BMN-673 (LT-673; (8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-8,9-dihydro-2H-pyrido[4,3,2-de]phthalazin-3(7H)-one); LT-674; LT-628; 3-aminobenzamide (INO-1001; 3-AB); PD128763 (3,4-dihydro-5-methyl-1(2H)-isoquinolinone); NU1025 (8-hydroxy-2-methyl-4(3H)-quinazolinone); DR 2313 (1,5,7,8-tetrahydro-2-methyl-4H-thiopyrano[4,3-d]pyrimidin-4-one);UPF 1069 (5-(2-oxo-2-phenylethoxy)-3,4-dihydroisoquinolin-1(2H)-one);EB 47 (5'-deoxy-5'-[4-[2-[(2,3-dihydro-1-oxo-1H-isoindol-4-yl)amino]-2-oxoethyl]-1-piperazinyl]-5'-oxoadenosine dihydrochloride); E7016 (benzopyrano[4,3,2-de]phthalazin-3(2H)-one, 10-[(4-hydroxy-1-piperidinyl)methyl]-); 4-HQN (4-(1H)-quinazolinone); ABT-767, as well as Griffin et al 1998, J. Med. Chem. 41, 5247; Skalitzky et al. 2003, J. Med. Chem. 46:210-213; Zaremba et al. 2007, Anti-Cancer Agents, each of which is incorporated herein by reference in its entirety. in Medicinal Chemistry 7,515;Lewis et al.2007,Curr Opin.Investigational Drugs 8,1061;Guha 2011,Nature Biotechnology 29,373-374;Rouleau et al.2010,Nature Reviews Cancer 10,293-301;Miknyoczki et al.,2007 Mol Cancer Ther,6(8),2290-2302;Pellicciari et al.2008,Chem.Med.Chem 3,91;Jones et al.,2009,J Med Chem,52(22),7170-7185;Mason et al.2008,Invest New Drugs,26(1),1-5;Ferraris et al. al.,2010,J.Med.Chem.53 4561;US Patent Applications:US2006 / 0229289;US20070259937;US20120309717;US20130011365;US Patents:US8372987;US8362030;US8236802;US8217070;US8183250;US8088760;International Patent Applications:WO 01 / 85686;WO 00 / 42040;WO 00 / 39070;WO 00 / 39104;WO 99 / 11623;WO 99 / 11628;WO 99 / 11622;WO 99 / 59975;WO 99 / 11644;WO 99 / 11945;WO 99 / 11649; and WO 99 / 59973.

[0139] Additionally, other anti-cancer agents that may be used in combination with the ASOs described herein include alkylating agents, such as thiotepa and CYTOXAN™; cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelanamines, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (specifically, bullatacin and bullatacinone); camptothecins (e.g., the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (specifically, cryptophycin 1 and cryptophycin 8). ); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); erytherobin; pancratistatin; sarcodictyin; spongiostatin; nitrogen mustards, e.g., chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednisolone, nimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, specifically, calicheamicin γ1I and calicheamicin ω11); dynemicins, such as dynemicin A; bisphosphonates, such as clodronic acid; esperamicins;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, e.g., mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potofilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogs, e.g., denopterin, methotrexate. , pteropterin, trimetrexate;purine analogues, for example, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;pyrimidine analogues, for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens, for example, calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;antiadrenal drugs, for example, aminoglutethimide, mitotane, trilostane;folic acid supplements, for example, For example, furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diazicon; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids, for example maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitraelin; pentostatin; phenamet; pirarubicin;losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK; polysaccharide complexes (JHS Natural Products™, Eugene, Oreg.); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (specifically, T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL™ paclitaxel (Bristol-Meyers Squibb Oncology, Princeton, NJ), ABRAXANE™ Cremophor Free, an albumin-modified nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE™ doxetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR™, gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE™, vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronic acid; irinotecan (CAMPTOSAR™, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin); topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin (including oxaliplatin treatment regimen (FOLFOX™)); lapatinib (TYKERB™);Included are inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (TARCEVA™)), and VEGF-A, which reduce cell proliferation, as well as pharma- ceutically acceptable salts, acids, or derivatives of any of the above. Additionally, methods of treatment may further include the use of radiation;

[0140] A pharmaceutical or therapeutic composition comprising a therapeutic agent for the treatment of cancer may contain a physiologically acceptable carrier in which the therapeutic agent is dissolved or dispersed as an active ingredient. In a preferred embodiment, the pharmaceutical composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes. As used herein, the terms "pharmaceutical acceptable", "physiologically acceptable", and grammatical variations thereof are used interchangeably when referring to compositions, carriers, diluents, and reagents, and indicate that such materials can be administered to a mammal without producing undesirable physiological effects, such as nausea, dizziness, stomach upset, and the like. A pharmaceutical acceptable carrier does not promote the development of an immune response against the agent mixed therewith, unless desired. The preparation of pharmacological or pharmaceutical compositions containing active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared to be injectable as either liquid solutions or suspensions, although solid forms suitable for dissolving or suspending in liquid prior to use may also be prepared. The preparation may be emulsified or presented as a liposomal composition. The active ingredient may be mixed with an appropriate amount of excipient that is pharma- ceutically acceptable and compatible with the active ingredient and is used in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances that enhance the effectiveness of the active ingredient, such as wetting or emulsifying agents, pH buffering agents, and the like. The therapeutic composition containing the therapeutic agent for the treatment of cancer or other diseases or disorders involving STAG2-deficient cells may contain pharma-ceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed by the free amino groups of the polypeptide) formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, tartaric acid, mandelic acid, and the like.Salts may be obtained from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like formed with the free carboxyl groups.

[0141] Physiologically acceptable carriers are well known in the art.Exemplary liquid carriers are sterile aqueous solutions that contain no material in addition to active ingredient and water, or contain buffers, such as sodium phosphate, saline, or both, such as phosphate-buffered saline, at physiological pH values.In addition, aqueous carriers may contain multiple buffer salts, such as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes.Liquid compositions may contain liquid phases in addition to water, or without water.Examples of such additional liquid phases are glycerin, vegetable oils, such as cottonseed oil, and water-oil emulsions.The amount of active agent used in the methods described herein that is effective in treating cancer or other diseases or disorders involving STAG2-deficient cells depends on the nature of the disorder or condition, and can be determined by standard clinical techniques.

[0142] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion.The preparations for injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers, optionally with the addition of preservatives.The compositions can be suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulation agents, for example, suspending agents, stabilizing agents, and / or dispersing agents.

[0143] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions.

[0144] Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[0145] Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free aqueous solution, before use.

[0146] In some embodiments, the therapeutic agent can be delivered in an immediate release form. In other embodiments, the therapeutic agent can be delivered in a controlled or sustained release system. Controlled or sustained release pharmaceutical compositions can have a common goal of improving drug therapy compared to the results achieved by non-controlled or non-sustained release corresponding compositions. The advantages of controlled or sustained release compositions include extended activity of the therapeutic agent, less frequent administration, and increased compliance. In addition, controlled or sustained release compositions can favorably affect the time of onset of action or other characteristics, such as blood levels of the therapeutic agent, thus reducing the occurrence of adverse side effects. Controlled or sustained release of active ingredients can be stimulated by various conditions, including but not limited to changes in pH, changes in temperature, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0147] The appropriate dosage range for a given therapeutic agent depends on its potency and includes an amount that is large enough to produce the desired effect, for example, the reduction of at least one symptom of cancer.The dosage of the therapeutic agent should not be so large as to cause unacceptable or life-threatening adverse side effects, or should be used under close supervision by medical professionals.In general, dosage varies according to the type of anticancer agent, as well as the age, condition, and sex of the patient.Dosage can be determined by those skilled in the art, and may be adjusted by individual physicians in the event of any complications.

[0148] Typically, the dosage of a given therapeutic agent may range from 0.001 mg / kg body weight to 5 g / kg body weight. In some embodiments, the dosage range is 0.001 mg / kg body weight to 1 g / kg body weight, 0.001 mg / kg body weight to 0.5 g / kg body weight, 0.001 mg / kg body weight to 0.1 g / kg body weight, 0.001 mg / kg body weight to 50 mg / kg body weight, 0.001 mg / kg body weight to 25 mg / kg body weight, 0.001 mg / kg body weight to 10 mg / kg body weight, 0.001 mg / kg body weight to 5 mg / kg body weight, 0.001 mg / kg body weight to 1 mg / kg body weight, 0.001 mg / kg body weight to 0.1 mg / kg body weight, or 0.001 mg / kg body weight to 0.005 mg / kg body weight. Alternatively, in some embodiments, the dosage range is 0.1 g / kg body weight to 5 g / kg body weight, 0.5 g / kg body weight to 5 g / kg body weight, 1 g / kg body weight to 5 g / kg body weight, 1.5 g / kg body weight to 5 g / kg body weight, 2 g / kg body weight to 5 g / kg body weight, 2.5 g / kg body weight to 5 g / kg body weight, 3 g / kg body weight to 5 g / kg body weight, 3.5 g / kg body weight to 5 g / kg body weight, 4 g / kg body weight to 5 g / kg body weight, 4.5 g / kg body weight to 5 g / kg body weight, 4.8 g / kg body weight to 5 g / kg body weight. In one embodiment, the dosage range is 5 μg / kg body weight to 30 μg / kg body weight. Alternatively, the dosage range is titrated to maintain a serum level of 5 μg / mL to 30 μg / mL.

[0149] Currently available treatments, including experimental treatments, for cancer or symptoms thereof, and their dosages, routes of administration, and recommended uses are known in the art and / or described in references such as the Physician's Desk Reference (60th ed., 2017). For experimental treatments, appropriate dosages can be estimated based on dose-response modeling in animal models or in silico modeling of drug effects.

[0150] The administration of the doses described above or utilized by those skilled in the art can be repeated for a limited and well-defined period. In some embodiments, the doses are given once a day or multiple times a day, for example, but not limited to, three times a day. Typically, the dosing schedule is informed by the half-life of the drug and the minimum therapeutic concentration of the drug in blood, serum, or localized in a given biological tissue. In a preferred embodiment, the doses described above are administered daily for several weeks or months. The duration of treatment depends on the subject's clinical progression and continued responsiveness to treatment. A continuous lower maintenance dose is contemplated after the initial higher therapeutic dose.

[0151] A therapeutically effective amount is an amount of drug sufficient to produce a statistically significant measurable change in a given symptom of cancer. Such an effective amount can be measured in clinical trials and animal studies for a given drug. For example, a reduction in a given symptom of cancer can be an indication of the appropriate therapeutic effectiveness of the drug.

[0152] Agents useful in the methods and compositions described herein may be administered topically, intravenously (by bolus or continuous infusion), orally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, optionally delivered by peristaltic means, or by other means known to those skilled in the art. Agents may also be administered systemically, if desired.

[0153] Therapeutic compositions containing at least one therapeutic agent may be conventionally administered in unit doses. The term "unit dose," when used in reference to a therapeutic composition, refers to physically discrete units suitable as single administration for a subject, each unit containing a predetermined amount of a therapeutic agent calculated to produce a desired therapeutic effect, together with the required physiologically acceptable diluent, i.e., carrier or vehicle.

[0154] The composition is administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.The amount and timing of administration depend on the subject to be treated, the ability of the subject's system to utilize the active ingredient, and the degree of desired therapeutic effect.The drug can be targeted, for example, by targeting moiety such as antibody, or by targeted liposome technology.

[0155] The exact amount of active ingredient that needs to be administered depends on the judgment of the practitioner and is specific to each individual.However, the suitable dosage range for systemic application is disclosed herein and depends on the route of administration.The suitable schedule for administration is also variable, but typically is an initial administration followed by repetition of the dose at one or more intervals by subsequent injection or other administration.Alternatively, continuous intravenous infusion sufficient to maintain blood concentration in the range specified for in vivo treatment is contemplated.

[0156] In some embodiments, a combination of anti-cancer therapeutic agents is used in treating cancer in a subject diagnosed as described herein.

[0157] In some embodiments, therapeutically effective agents are administered to a subject at the same time by combination therapy. As used herein, the term "simultaneously" is not limited to administration of two or more agents at exactly the same time, but means that they are administered to a subject in an order and time interval that allows them to act together (e.g., synergistically to provide increased benefits than if administered otherwise). For example, a combination of therapeutic agents may be administered at the same time, or may be administered sequentially in any order at different times; however, if not administered at the same time, they should preferably be administered close enough in time to provide the desired therapeutic effect synergistically. The agents may be administered separately in any suitable form and by any suitable route. It is understood that when each of the therapeutic agents of the combination is not administered in the same pharmaceutical composition, they may be administered to a subject in need thereof in any order. For example, a first therapeutic agent may be administered to a subject in need thereof prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of a second therapeutic agent (or vice versa). In other embodiments, the delivery of one therapeutic agent ends before the delivery of the other agent / treatment begins. In some embodiments in either case, the treatment becomes more effective due to the combined administration. For example, the therapeutic agents used in combination are more effective than seen with either agent alone. In some embodiments, delivery is such that a reduction in symptoms or other parameters associated with the disorder is greater than observed with either therapeutic agent alone. The effect of such a combination may be partially additive, fully additive, or greater than additive.The drugs and / or other therapeutic agents, procedures, or modalities may be administered during an active phase of the disease or during a persistent or less active phase of the disease.

[0158] When administered in combination, one or more of the therapeutic agents can be administered in an amount or dosage that is higher, lower, or the same as the amount or dosage of a given agent used individually as a monotherapy.In certain embodiments, the amount or dosage of the first therapeutic agent administered when administered in combination with a second therapeutic agent is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of the first agent when used individually.In other embodiments, the amount or dosage of the first therapeutic agent, when administered in combination with a second therapeutic agent, produces a desired effect (e.g., improving cognitive function), and is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of the first (or second) agent required to achieve the same therapeutic effect when administered alone.

[0159] The effectiveness of a given treatment for cancer can be determined by a skilled physician. However, if any one or all of the signs or symptoms of cancer are beneficially altered or other clinically accepted symptoms or markers of disease are improved or alleviated, for example, by at least 10%, after treatment with a therapeutic agent for cancer, the treatment is considered to be "effective treatment" (as the term is used herein). Efficacy can also be measured by the individual's lack of progression (i.e., the progression of disease is stopped or at least slowed down), as assessed by the stabilization of disease or the need for medical intervention. Methods for measuring these indicators are known to those skilled in the art and / or are described herein. Treatment includes treating disease in an individual or animal (some non-limiting examples include humans or mammals) and includes (1) inhibiting the disease, e.g., arresting or slowing the progression of cancer; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of disease onset or preventing secondary diseases / disorders associated with infection.

[0160] An effective amount for the treatment of a disease means an amount that is sufficient to provide effective treatment (as that term is defined herein) for that disease when administered to a mammal in need thereof. The effectiveness of an agent can be determined by assessing physical indicators of the disease, such as, for example, anemia, white blood cell level or identity, pain, fatigue, fever, and the like.

[0161] Treatment with the methods provided herein can reduce or eliminate one or more symptoms associated with cancer, such as fatigue, pain, tumor size, tumor growth, etc. In one embodiment, the cancer is prostate cancer, and one or more symptoms associated with prostate cancer include difficulty urinating, increased frequency of urination, pelvic pain or discomfort, decreased force of urination, difficulty starting or stopping urine flow, blood in semen, and bone pain.

[0162] The technology provided herein may be further defined by the following numbered paragraphs:

[0163] Table 1. Examples of ASOs targeting STAG1 expression TIFF2025503956000012.tif224164TIFF2025503956000013.tif239164TIFF2025503956000014.tif239164TIFF2025503956000015.tif110164Legend: 2'MOE modifications are underlined. PS bonds are * As shown by: EXAMPLES

[0164] Example 1: STAG2 is the most frequently mutated cohesin subunit in AML (Figure 2) and is also frequently mutated in solid tumors. Indeed, STAG2 is one of only 12 human genes that are significantly mutated in four or more different types of human cancer. 2 , and are frequently mutated in cancers including glioblastoma, breast cancer, bladder cancer, melanoma, and Ewing's sarcoma 2-4 STAG2 resides on the X chromosome, and thus mutations in males (or in females with an active X allele) often lead to a complete loss of the STAG2 protein and its replacement within the cohesin complex by its paralog STAG1. Loss of STAG2 makes STAG1 an essential protein. Importantly, this establishes an absolute dependency of STAG2 mutant cells on STAG1 expression, and both RNAi and CRISPR / Cas9 screens highlight STAG1 as a selective critical dependency in STAG2 mutant cells (MS#1, 13). Data from a report using next-generation sequencing in AML samples show the cohesin subunits SMC1A, SMC3, RAD21, STAG1, and STAG2 to have frequent mutations. Collectively, the core components of the cohesin complex are mutated in 14% of patients with de novo AML. Missense mutations include in-frame indels, and truncating mutations include nonsense, frameshift, and splice-site mutations. The AML genome harbors fewer mutations than most adult cancers, of which 13% correspond to cohesin-associated genes. STAG2 is the most frequently mutated cohesin subunit in AML.

[0165] Example 2: We used an AML cell model with a genetically modified STAG2 KO that could recapitulate the selective dependency on STAG1. STAG2 KO was generated using CRISPR / Cas9 (Figure 3A). We used genome-scale CRISPR screening in WT or STAG2 KO U937 cell lines using Avana's sgRNA library targeting a total of 20,000 protein-coding genes with 4 unique sgRNAs per gene and including 1000 non-targeting sgRNA controls (Figure 3B). Volcano plots illustrating differential dependence in STAG2 mutant cells compared to WT cells (Figure 3C) show combined data for five STAG2 mutant cell lines (U937 STAG2-KO2, STAG2-KO3, KOC5, KOD5C, KOG8B) and six STAG2-WT cell lines (U937 WT-1, WT-2, NCB1, NCB12, NCB2A, NCC4). Each set of genes that exhibit greater dependence in STAG2 mutants than in WT cells at an FDR of less than 5% is indicated by a highlighted dot. STAG2 mutant cells were highly dependent on STAG1 (Figure 3C). STAG1 knockout induces no phenotype in WT U937 cells or animal models.

[0166] Example 3: We showed that STAG2 KO renders cells hypersensitive to reduction of STAG1 levels. Guide RNAs targeting STAG1 for CRISPR KO had variable efficacy in control cells, reducing STAG1 protein levels by 47%, 71%, and 81%, respectively, as measured by Western blot using STAG1 antibody. Actin served as a loading control. Efficacy was determined as the % of non-homologous end joining across the gRNA target site (Figure 4A). Measurement of cell viability 2-6 days after knockdown of STAG1 using STAG1 sgRNA showed that in STAG2 functional cells, none of the STAG1 gRNAs affected viability, whereas in STAG2 KO cells, knockdown of STAG1 greatly reduced cell viability, indicating that in the absence of STAG2, reduction of STAG1 by approximately 50-75% greatly impacts the viability of these cells (Figure 4B).

[0167] Example 4: We show that cohesin mutations make AML cells hypersensitive to splicing inhibitors, such as H3B-8800. Measurement of STAG1 exon 5 inclusion rate in control cells or STAG2 KO cells after treatment with H3B-8800 shows that skipping STAG1 exon 5 shifts the coding sequence out of frame and causes RNA degradation by nonsense-mediated RNA decay. Data show that STAG2 KO leads to increased STAG1 exon 5 skipping after treatment with H3B-8800. These data show that splicing inhibitors have increased efficacy in STAG2 KO cells.

[0168] Example 5: Knowing that reduction of STAG1 mRNA selectively kills STAG2 mutant cells, we aimed to develop an ASO that would cause mis-splicing and / or reduction of STAG1 mRNA. We designed an ASO with phosphorothioate (PS) backbone linkages to allow for cell entry by "free uptake". Furthermore, MOE modification increases the stability of the ASO to nucleases and binding to the RNA target. We developed gapmers, in which modified RNA bases are flanked on both sides of a central region or "gap" with DNS characteristics. A short central DNA stretch forms an RNA-DNA hybrid with the RNA target. This recruits RNaseH to degrade the target RNA.

[0169] Example 6: We demonstrate the uptake of fluorescently labeled ASOs in HEK 293T cells. Fluorescently labeled gapmer ASOs containing PS linkages and MOE base modifications were added to the medium of HEK 293T cells at concentrations ranging from 0 μM to 10 μM. Fluorescence was observed in the cells, indicating successful free uptake of the ASOs (Figure 8A). Measurement of the average fluorescent signal per cell 7 days after ASO transfection or free uptake indicates successful uptake of ASOs added to the medium of HEK 293T cells at concentrations ranging from 0.1 μM to 10 μM (Figure 8B).

[0170] Example 7: We demonstrate the uptake of fluorescently labeled ASO in the U937 AML cell line. Fluorescently labeled ASO was added to the medium of U937 AML cells at concentrations ranging from 0 μM to 10 μM. Fluorescence was observed within the cells, indicating successful free uptake of the ASO (Figure 9A). Measurement of the average fluorescent signal per cell 5-7 days after addition of fluorescently labeled ASO to the medium of U937 AML cells indicates successful free uptake of the ASO at concentrations ranging from 0.1 μM to 10 μM (Figure 9B).

[0171] Example 8: We show that ASOs targeting regions of STAG1 reduce STAG1 expression. ASOs were tiled in STAG1 exons, specifically exons that showed mis-splicing in cells treated with H3B-8800. ASOs targeting these regions were designed to promote exon skipping and subsequent degradation of the STAG1 transcript. ASOs were designed against exons 3, 5, and 29. Measurement of STAG1 expression in HEK 293T cells after 7 days of treatment with 10 uM ASOs showed the greatest reduction in STAG1 expression, with a 50-75% reduction in expression, when ASOs 3-5, 5-11, and 5-12 were used (Figure 10B).

[0172] Example 9: We show the reduction of STAG1 protein expression after treatment with ASO targeting STAG1. STAG1 protein levels measured by Western blot. HEK 293T cells were treated with ASO (E3-5, E5-11, and E5-12) or negative control at a concentration of 10 μM for 7 days, and histone H3 was used as a loading control, showing that ASO successfully reduced STAG1 protein levels in HEK 293T cells (Figure 11A). Figure 11B shows STAG1 protein levels measured by Western blot. U937 AML cells were treated with ASO (E3-5, E5-11, and E5-12) or negative control at a concentration of 10 μM for 7 days. GAPDH was used as a loading control. The data show that ASO successfully reduced STAG1 protein levels in AML cell lines.

[0173] We use HiBit readout using STAG1-HiBit expressing HEK 293T cells to show the efficacy of ASO targeting STAG1. After 4 days of treatment with 10 uM ASO, we measured the relative STAG1-HiBit levels. E5-14 was the most potent ASO.

[0174] Example 10: We show that STAG1 protein level measured by Western blot is reduced by ASO targeting STAG1. K562 AML cells were treated with ASO (E5-11, E5-13, E5-14, and E5-12) or negative control at a concentration of 10 μM for 4 days. GAPDH was used as a loading control. The data show that ASO successfully reduces STAG1 protein level in AML cell lines, with E5-14 showing the greatest efficacy. ASO also successfully increases H3 protein level in AML cell lines.

[0175] Example 11: We show that the optimized ASO E5-14 causes selective lethality in STAG2 KO AML cells. STAG2 protein levels measured by Western blot show that STAG2 knockout cells do not have STAG2 protein, but STAG1 and actin protein levels are not affected (Figure 14A). Measurement of cell viability after 12 days of treatment with increasing concentrations of control or E5-14 shows that in STAG2 functional cells, ASO E5-14 does not affect viability, but in STAG2 KO cells, E5-14 ASO greatly reduces cell viability (Figure 14B). In conclusion, free uptake provides consistent and long-lasting delivery of ASO in AML cells. We designed an ASO that achieves a substantial reduction in STAG1 mRNA and protein in 293T cells.

Claims

1. A composition for downregulating expression of stromal antigen 1 (STAG1), the composition comprising an antisense oligonucleotide having a sequence sufficiently complementary to a portion of a STAG1 primary transcript to permit hybridization to a portion of the STAG1 primary transcript, the oligonucleotide comprising at least one phosphorothioate backbone modification.

2. The composition of claim 1 , wherein the antisense oligonucleotide comprises an RNA oligonucleotide, a DNA oligonucleotide, or a combination of deoxyribonucleosides and ribonucleosides.

3. The composition of claim 1 or claim 2, wherein the antisense oligonucleotide comprises at least one deoxyribonucleoside and at least one ribonucleoside.

4. A composition for downregulating expression of stromal antigen 1 (STAG1), the composition comprising an antisense oligonucleotide having a sequence sufficiently complementary to a portion of a STAG1 primary transcript to enable hybridization to a portion of the STAG1 primary transcript, the oligonucleotide comprising at least one deoxyribonucleoside and at least one ribonucleoside.

5. The composition of any one of claims 1 to 4, wherein the antisense oligonucleotide comprises, in 5' to 3' order, 1 to 5 ribonucleosides, 6 to 12 deoxyribonucleosides, and 1 to 5 ribonucleosides.

6. The composition of any one of claims 1 to 5, wherein one or more of the ribonucleosides comprises a 2'-O-methoxyethyl (MOE) modification.

7. The composition of any one of claims 4 to 6, wherein the antisense oligonucleotide comprises at least one phosphorothioate backbone linkage.

8. The composition of any one of claims 1 to 7, wherein the antisense oligonucleotide comprises phosphorothioate linkages between each nucleoside.

9. The composition of any one of claims 1 to 8, wherein the antisense oligonucleotide comprises 15 to 22 nucleosides.

10. The composition of any one of claims 1 to 9, wherein the antisense oligonucleotide comprises one or more sugar modifications selected from 2'-fluoro, 2'-O-methyl, an LNA modification, a cEt modification, and a PMO modification.

11. The composition of any one of claims 1 to 10, wherein the antisense oligonucleotide comprises a sequence that allows hybridization with exon 3 or 5 of the STAG1 RNA transcript.

12. The composition of any one of claims 1 to 10, wherein hybridization of the antisense oligonucleotide overlaps with a 5' splice site or an intra-exon splicing promoting sequence on the STAG1 RNA transcript.

13. The composition of claim 12, wherein the intra-exon splicing promoting sequence comprises a binding site for the RNA-binding protein SRSF2.

14. The composition of any one of claims 1 to 13, wherein the antisense oligonucleotide comprises a sequence selected from SEQ ID NOs: 1 to 81.

15. A pharmaceutical formulation comprising the composition of any one of claims 1 to 14 and a pharma- ceutically acceptable carrier.

16. A method for downregulating expression of STAG1 in a cell, comprising contacting said cell with a composition according to any one of claims 1 to 15.

17. The method of claim 16, wherein the cell is a cohesin mutant cell.

18. The method of claim 16 or 17, wherein the cell is a stromal antigen 2 (STAG2) mutant cell.

19. The method of any one of claims 16 to 18, wherein the cell is a cancer cell or a myelodysplastic syndrome cell.

20. The method of any one of claims 16 to 19, wherein the cell is a cancer cell selected from an acute myeloid leukemia (AML) cell, a glioblastoma cell, and a bladder cancer cell.

21. The method of any one of claims 16 to 20, wherein said contacting step reduces STAG1 mRNA in said cell by at least 50%.

22. 16. A method for selectively killing cohesin mutant cells, comprising contacting said cells with a composition according to any one of claims 1 to 15.

23. The method of claim 22, wherein the cell is a STAG2 mutant cell.

24. 24. The method of claim 22 or 23, wherein the cell is a cancer cell or a myelodysplastic syndrome cell.

25. The method of any one of claims 22 to 24, wherein the cell is a cancer cell selected from an acute myeloid leukemia (AML) cell, a glioblastoma cell, and a bladder cancer cell.

26. The method of any one of claims 22 to 25, wherein said contacting step reduces STAG1 mRNA in said cell by at least 50%.

27. 16. A method for treating a cohesin mutant cancer comprising administering to a subject in need thereof the composition of any one of claims 1 to 15.

28. 28. The method of claim 27, wherein the cohesin mutant cancer is STAG2 mutant.

29. 29. The method of claim 27 or 28, wherein the cohesin mutant cancer is selected from AML, glioblastoma, and bladder cancer.

30. 16. A method of treating myelodysplastic syndrome, comprising administering to a subject in need thereof the composition of any one of claims 1 to 15.

31. The method of claim 30, wherein the myelodysplastic syndrome cells are cohesin mutant.

32. The method of claim 30 or 31, wherein the myelodysplastic syndrome cells are STAG2 mutant.

33. The method of any one of claims 27 to 32, further comprising administering an inhibitor of the DNA damage response.

34. 34. The method of claim 33, wherein the inhibitor of DNA damage response is a poly(ADP)-ribose polymerase (PARP) inhibitor.

35. 35. The method of claim 33 or 34, wherein the inhibitor of DNA damage response is selected from talazoparib, veleparib, pamiparib, olaparib, rucaparib, and niraparib.

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